Download PW 22 - myobase

Myology 2008, May 26-30; Marseille
(suite des communications)
PW 22:
Cardiomyopathies and
experimental models
PW22-271
A GENETIC VARIANT OF THE HISTIDINE-RICH CALCIUM BINDING PROTEIN
CONFERS SUSCEPTIBILITY TO ARRHYTHMOGENESIS AND SUDDEN CARDIAC
DEATH IN DCM PATIENTS
ARVANITIS DA1, SANOUDOU D1, KOLOKATHIS F2, VAFIADAKI E1, THEODORAKIS
G3, KONTROGIANNI-KONSTANTOPOULOS A4, PARASKEVAIDIS IA2,
ADAMOPOULOS S2, DORN II GW 5, KREMASTINOS DTh2, KRANIAS EG5
(1) Biomedical Research Foundation, Academy of Athens, Athens, GREECE. (2)
University of Athens, Medical School, “Attikon” University Hospital, Athens, GREECE.
(3) Onassis Cardiac Surgery Center, Athens, GREECE. (4) University of Maryland
Baltimore, Baltimore, USA. (5) University of Cincinnati, Cincinnati, USA.
Abnormal Ca-cycling in the cardiomyocyte is a hallmark of dilated cardiomyopathy
(DCM). Furthermore, DCM patients are at substantial risk for sudden cardiac death
To contact the author::
due to malignant ventricular arrhythmias. The histidine-rich calcium binding protein
[email protected] (HRC) is a low affinity, high capacity, calcium handling protein that has been
.
implicated in the regulation of both sarcoplasmic reticulum calcium release and
uptake. To address whether HRC genetic variants may be associated with DCM
development and progression, we screened 123 non-ischemic DCM patients and 96
healthy individuals by single strand conformation polymorphism analysis and direct
sequencing. Six genetic variants were identified L35L, S43N, S96A, E202_E203insE,
D261del and an in frame insertion of 51 amino acid residues at H321. A statistically
significant correlation was observed between the S96A polymorphism and the
occurrence of life-threatening ventricular arrhythmic events, defined as sustained
ventricular tachycardia, ventricular fibrillation or sudden cardiac death, in 28 patients
(22.8%). During a follow-up period of 4.02 ± 2.4 years, the risk for ventricular
arrhythmias was higher in the S96A homozygous patients. Using multivariate Cox
regression analysis, the S96A polymorphism was the only significant arrythmogenesis
predictor in DCM patients, with a hazard risk of 4.1 (95% CI: 2.0 to 8.2; p<0.001). In
conclusion, our findings demonstrate that the S96A genetic variant of HRC is
associated with life-threatening ventricular arrhythmias in nonischemic DCM and may
serve as an independent predictor of susceptibility to arrhythmogenesis in the setting
of dilated cardiomyopathy.
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PW22-272
To contact the author::
[email protected].
EVALUATION OF AN INNOVATIVE THERAPEUTIC APPROACH AND NEW TOOL
FOR EARLY DIAGNOSIS OF CARDIOMYOPATHIES.
MONGUE-DIN H1, LIU JM2, SALMON A1, FISZMAN MY1, WDZIECZAK-BAKALA J2,
FROMES Y1
(1) Institut de Myologie, INSERM U582, Paris, FRANCE. (2) ICSN,CNRS, Gif-surYvette, FRANCE.
The chronically failing heart is characterized by alterations in tissue structure,
particularly fibrous tissue formation, responsible for the loss of myocardial compliance.
Furthermore, rhythm disturbances are commonly observed. The underlying
arrhythmogenic mechanisms are multiple, but myocardial fibrosis is frequently
observed. Heart rate variability (HRV) reflects the functional status of the autonomic
nervous system and its effects on sinus node. Considering autonomic dysfunction
playing an important role in the pathogenesis and prognosis of congestive heart
failure, HRV analysis may help to identify those who are at risk of cardiac death.
Antifibrotic drugs may be considered for the treatment of heart failure.
These experiments aimed at studying the diagnostic potential of HRV analysis to
detect early modifications in cardiac electrical activity and at investigating the potential
benefits of an antifibrotic treatment in CHF147 cardiomyopathic hamsters.
HRV analysis was based on quantitative analysis of time series and allowed
determining an early dysfunction in cardiac electrical activity in CHF147
cardiomyopathic hamsters. Pharmacological assays, coupled with HRV analysis
allowed identifying a high sympathetic activity. Western blot and RT-PCR studies
confirmed the neurovegetative imbalance. In a second set of experiment CHF147
hamsters were treated during 42 days with 3 different doses of Ac-SDKP or placebo,
using osmotic minipumps. Weight and size of the ventricles were comparable in all
groups. Ventricular fibrosis was quantified on Sirius red stained cryostat sections.
Whereas fibrotic scar tissue remained comparable, interstitial fibrosis was significantly
less pronounced in treated groups. This difference appeared to be more important for
the lowest dose of Ac-SDKP. Histological results were confirmed by
immunohistochemistery.
Thus, HRV analysis appears to be an interesting tool for early diagnosis of
cardiomyopathies, detecting decreased heart rate variability and activation of the
sympathetic tone. Moreover, an antifibrotic treatment based on low doses of Ac-SDKP
might be of therapeutic interest in cardiomyopathies.
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PW22-273
To contact the author::
[email protected].
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CARDIAC MYOSIN-BINDING PROTEIN C MODULATES THE TUNING OF THE
MOLECULAR MOTOR IN THE HEART
LECARPENTIER Y1, VIGNIER N2, OLIVERO P3, GUELLICH A4, CARRIER L5,
COIRAULT C6
(1) Inserm U689, Cardiovascular Research Center, Paris, FRANCE. (2) Inserm
U582, Institut de Myologie, Paris, FRANCE. (3) Inserm U689, Cardiovascular
Research Center, Paris, FRANCE. (4) Inserm U689, Cardiovascular Research
Center, Paris, FRANCE. (5) Institute of Experimental and Clinical Pharmacology and
Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY.
(6) Inserm U582, Institut de Myologie, Paris, FRANCE.
The precise role of cardiac myosin binding protein C (cMyBP-C) on actomyosin
interaction (AMI) remains unknown. We hypothesized that the lack of cMyBP-C
impaired cardiac AMI. Experiments were performed on 16 weeks old cMyBP-C-/- (KO)
and age-matched wild-type (WT) mice (n=20/group). In vitro mechanical and
energetics properties were performed on left ventricular (LV) papillary muscles and
Huxley’s equations were used to characterize AMI. In vitro motility assays were
performed using myosin purified from LV. Myosin-based sliding velocities of actin
filaments were analyzed at baseline, after pretreatment of the myosin solution with 10
umol of the catalytic subunit of PKA and/or in the presence of increasing amount of
alpha-actinin, an actin-binding protein that acts as an internal load thereby providing
an index of relative isometric force. Western-blot analysis was used to quantify
cMyBP-C and phosphorylated cMyBP-C. The probability for myosin to be weakly
bound to actin was higher in KO than in WT (p<0.05), whereas the number of strongly
bound, high-force generated state cross-bridges was lower in KO (p<0.001). The
unitary force per AMI was lower in KO than in WT (p<0.01). At baseline, myosin-based
velocities of actin were slower in KO than in WT (1.65±0.01 vs. 1.98±0.01 um/s,
p<0.01). The minimum amount of -actinin needed to completely arrest the thin
filament motility was significantly higher in WT than in KO (p<0.001). As expected,
cMyBP-C was present in WT myosin solution whereas cMyBP-C was not detected in
KO. In WT, PKA induced a 1.6-fold increased in cMyBP-C phosphorylation associated
-actinin required to arrest thin filament
motility (p<0.001). PKA did not modify sliding velocity in WT. In KO, PKA had no effect
on myosin sliding. We conclude that cMyBP-C regulates AMI by limiting inefficient
cross-bridge formation and by enhancing the power stroke step.
PW22-274
To contact the author::
[email protected]
r.
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ATTEMPTS TO RESCUE A DILATED CARDIOMYOPATHY INDUCED BY
CARDIAC-SPECIFIC DISRUPTION OF THE SRF GENE IN THE MOUSE
DECAUX JF1, LAMOTTE L1, ESCOUBET B2, TOUVRON M1, ROSENTHAL N3,
LESSARD J4, MERICSKAY M5, LI Z5, DAEGELEN D1, TUIL D1
(1) INSERM U567, CNRS UMR 8104, Université Paris Descartes, Paris, FRANCE.
(2) Faculté de Médecine Xavier-Bichat, Université Paris 7, Paris, FRANCE. (3)
Mouse Biology Unit Monterotondo, Rome, ITALY. (4) University of Cincinnati,
Cincinnati, USA. (5) CNRS UMR 7079, Université Paris 6, Paris, FRANCE.
Serum response factor (SRF) is a transcription factor controlling the expression of
many extracellular signal-regulated genes as well as genes encoding sarcomeric
contractile proteins. Many SRF target genes are involved in familial hypertrophic
cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). By using the tamoxifen
inducible Cre/loxP system, we have previously shown that SRF is crucial for adult
cardiac function and integrity. Triggering in mice cardiac-restricted knock out of SRF
led to impaired left ventricular function with reduced contractility, subsequently
progressing to DCM without hypertrophic compensation. Under these conditions, all
mutant mice died from heart failure 10 weeks after tamoxifen injections. Functional
and structural heart defects were preceded by early alterations in the cardiac gene
-actin, IGF-1, muscle creatine kinase and
calcium-handling genes.
To evaluate the importance of altered IGF-actin genes expression in
the progression of DCM and compensate for their loss, we mated our mice model of
inducible SRF disruption with transgenic mice overexpressing either IGF-1 or cardiac
-actin specifically in the heart. Cardiac-specific IGF-actin
overexpression both attenuates the progression of DCM due to the loss of SRF.
Cardiac IGF-1 overexpression improves functional parameters and the survival of the
animals until almost 4 months. In the same way, an almost normal cardiac function
and a life extension beyond 6 months were associated to
-actin
compensatory expression. Histological and molecular analysis of these new models
are in progress.
PW22-275
To contact the author::
[email protected].
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ISLET-1 CARDIAC PROGENITORS DETECTION IN MOUSE
KHATTAR P1, DECOSTRE V1, CATELAIN C1, SCHWARTZ K1, FISZMAN M1,
BONNE G1, VILQUIN JT1
(1) Inserm U582 - Institut de Myologie, UPMC Univ Paris 06, UMR S582, IFR14,
Paris, FRANCE.
Purpose: Islet-1+ cells are cardiogenic and angiogenic progenitors participating to
heart formation. The first aim was to locate and quantify these cells in view of potential
pre-clinical developments. The second aim was to compare their presence and
localisation in normal animals and in a model of dilated cardiomyopathy, the LmnaH222P mouse carrying a lamin A/C gene mutation.
Methods: We set up a time-course study in C57BL/6J, SVJ129, KI-LmnaH22P/H222P
mice and wild type littermates. Animals were sacrificed at days 1, 5, 10, 15, 20, 25,
then every month (from 3rd to 12th) and Islet-1+ cells detected by
immunohistofluorescence on consecutive cryostat sections of whole heart.
Results: Islet-1+ cells are gathered in one cluster in C57BL/6J animals. Cells are
located in the proximal (basal) portion encompassing 8 to 20% of the organ height in
outflow tract, atria, ventricles, septa. Approximately 150 to 300 cells are observed in
each heart, from birth to 7 month. In the KI-Lmna mice (C57BL/6J x SVJ129) and wildtype SVJ129, cells are more restricted to atria and septa, and persist beyond 10
months. Importantly, a second cluster located in a more proximal (basal) position
contains numerous cells (800-1800) of smaller size, and is no longer observed after
the age of 4 months. The cells in this second cluster express sarcomeric actin and are
intricated within the resident cardiac tissue. No significant differences are observed
between the KI-LmnaH222P/H222P mice and their wild-type littermates. Ki67 expression is
not detected, suggesting that islet1+ cells are not proliferating.
Conclusions: The presence and localisation of Islet-1+ cells in the heart of young
adult mice is linked to the genetic background. However, their low amounts and
specific localisation make Islet-1+ cells unlikely to soon become practical tools in a
therapeutic perspective.
Supported by grants from Leducq Foundation (CaPTAA network) and AFM.
PW22-276
To contact the author::
[email protected]
r.
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MICRORNA SIGNATURE IN CARDIAC COMMITTED HUMAN EMBRYONIC STEM
CELLS
NURY D1, BARBRY P2, PUCÉAT M3
(1) INSERM UMR861, evry, FRANCE. (2) IPMC CNRS, nice, FRANCE. (3)
INSERM UMR861, evry, FRANCE.
Genetic cardiomyopathies are predominant among rare diseases. They often
originate from mutations in early cardiac transcription factors. Human
Embryonic stem (HES) cells represent a key developmental model which
recapitulate early cardiogenesis. MicroRNAs are emerging as key regulators
of transcriptional pathways, acting as inhibitors of translation initiation.
Lineage specification of Human Embryonic stem cells is a tightly regulated
process in which miRNAs are likely to play a key role. Using a Chip
technology, we compared the expression profile of miRNAs in two distinct
HES cell lines (HUES-1 and HUES24). HES cells were then committed
toward a cardiac lineage using 10 ng/ml BMP2 for 48hrs. Comparison of HES
cells and cardiac specified cells revealed a differential expression of 110
miRNAs (45 were up-regulated and 55 downregulated following BMP2
treatment). Chip data were validated by stem-loop real time PCR. The
pattern of miRNA expression was then correlated with the one of mesodermal
gene expression induced by the morphogen in both cell lines.
Profile of miRNAs under basal or BMP2 conditions will help to comprehend
early cardiogenesis under normal or pathological (i.e genetic disease)
conditions.
This work is supported by the French National Research Agency (grant
Blanc06-28138290).
PW22-277
To contact the author::
[email protected].
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CORONARY ARTERY PATTERNING IN TBX1 NULL EMBRYOS.
THÉVENIAU-RUISSY M1, DANDONNEAU M2, MIQUEROL L3, KELLY R4
(1) Developmental Biology Institute, Marseille, FRANCE. (2) Developmental Biology
Institute, Marseille, FRANCE. (3) Developmental Biology Institute, Marseille,
FRANCE. (4) Developmental Biology Institute, Marseille, FRANCE.
TBX1, encoding a T-box containing transcription factor, is the major
candidate gene for del22q11.2 or DiGeorge syndrome, characterized by craniofacial
and cardiovascular defects including tetralogy of Fallot and common arterial trunk.
Mice lacking Tbx1 have severe defects in the development of pharyngeal
derivatives including cardiac progenitor cells of the second heart field contributing to
the arterial pole of the heart. The outflow tract of mutant embryos is short and narrow
and fails to septate resulting in a common arterial trunk. A series of crosses using
transgene markers of the second heart field and coronary artery endothelial cells
reveal that Tbx1 mutant hearts form in the absence or severe reduction of a specific
subpopulation of progenitor cells normally giving rise to subpulmonary myocardium.
The Tbx1 mutant ventricular outlet thus has an aorta-like morphology with three valve
leaflets. This defect is associated with anomalous coronary artery patterning. Both
right and left coronary ostia form predominantly at the right/ventral sinus in mutant
hearts; as a result, proximal coronary arteries course across the normally coronary
free ventral region of the heart. We have identified Semaphorin3c as a Tbx1dependent gene expressed in subpulmonary myocardium.
Our results provide new insights into the association between conotruncal
defects and coronary artery anomalies and implicate second heart field derived cells
in coronary artery patterning.
PW22-278
GLYCOSAMINOGLYCANS IN THE EXTRACELLULAR MATRIX REMODELLING
DURING AGEING AND CARDIAC DISORDER
HUYNH M1, GARCIA-FILIPE S1, MORIN C1, BARBIER-CHASSEFIÈRE V1, BESSE S1,
NARASSIMPRAKASH H1, JENISKENS G2, MARTELLY I1, PAPY-GARCIA D1
(1) Laboratory CRRET, CNRS UMR-7149, Université Paris 12 / Paris-Est, Créteil,
FRANCE. (2) Department of Biochemistry, University Medical Centre, NCMLS,
Nijmegen, THE NETHERLANDS.
Despite
To contact the author::
[email protected].
the
increasing
evidences
demonstrating
the
diverse
roles
of
glycosaminoglycans (GAGs) in many fundamental biological processes, most studies
directed to understand the extracellular matrix implication in cardiac dysfunction
focuses mainly in protein components. Age-associated changes in heart structure and
function represent the major risk factor in heart failure syndromes. This may be
associated to changes in the myocardium intrinsic cardioprotective response to harm.
In this study, we were interested to know how the physiological ageing process
influences the content and composition of sulfated GAGs in myocardium and how GAG
changes can influence important protein functions associated to the response to
myocardial isquemia. Sulfated GAGs were isolated from heart left ventricle of healthy
rats at different ages. GAGs were quantified according to Barbosa et al 2003. The
isolated GAGs were subjected to heparin binding competition assays with some
heparin binding growth factors as FGF-2, HARP and VEGF. Extracted GAGs were also
tested in a functional assay based on their capacity to potentiate the mitogenic activity
of FGF-2. We found that the amount of total sulfated GAG increased gradually with
ageing. This increase concerns both, heparan sulfate and chondroïtin sulfate.
Contrariously, we observed a clear decrease in these myocardial GAGs abilities to
bind to FGF-2, but not to HARP, in aged animals. This low capacity of GAGs extracted
from aged hearts to bind FGF-2 was confirmed in a FGF-2 mitogenic activity assay.
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Immunohistological studies of GAGs on aged and young myocardium are in course. In
conclusion, we demonstrated for the first time that the quantity and the quality of the
sulfated GAGs in myocardium change during ageing, and that these modifications can
differently affect the biological activity of locally expressed growth factors. This might
stand for important physiological implications. Acknowledgments: This work was kindly
financed by the AFM.
PW22-279
To contact the author::
dsanoudou@bioacadem
y.gr.
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SIGNIFICANT IMPAIRMENTS IN ION TRANSPORT AND CARDIAC
CYTOARCHITECTURE IN THE “HUMANIZED” PHOSPHOLAMBAN MOUSE
MODEL
ARVANITIS DA1, DONG M2, ZHAO W 2, PAPALOUKA V1, KRANIAS EG2, WANG
HS2, SANOUDOU D1
(1) Biomedical Research Foundation of the Academy of Athens, Athens, GREECE.
(2) University of Cincinnati, Cincinnati, USA.
Phospholamban (PLN), the reversible inhibitor of SERCA2, is a key regulator or
calcium homeostasis and cardiac function, and it has been directly implicated in the
development of dilated cardiomyopathy. Its amino acid sequence is highly conserved
across species except for humans where Asn is replaced by Lys at amino acid
position 27. To evaluate the significance of this single nucleotide difference we
induced cardiac-specific insertion of the human-PLN in the null background. The
“humanized” PLN expressing transgenic (TG) mouse hearts presented increased
inhibition of SERCA2, abnormal calcium handling, fibrosis, and hypertrophy. Using
microarrays, we identified the global molecular pathways implicated in these
processes, including ion transport, muscle contraction, cell cycle and proteolysis. The
observed changes in key sodium, potassium and calcium plasma membrane pumps
were confirmed at the protein level and suggested an ongoing electrical remodeling
process with direct implications in cardiac function. In support of this findings, ex vivo
Langedorff perfusion of intact hearts further revealed decreased rates of contraction
and relaxation in TGs. Furthermore, patch clamp analysis of isolated cardiac
myocytes unveiled significant alterations of their electrophysiological properties.
Specifically, the cardiac myocyte action potential duration was significantly prolonged,
the transient outward current (Ito) was decreased and the sodium/calcium exchanger
activity was increased in TG compared to wild-type mice. In conclusion, “human-PLN”
directly affects calcium cycling and contractility, which in turn triggers electrical
remodeling through differential expression of key ion channels.
PW 23:
Hereditary neuropathies
PW23-280
PATHOPHYSIOLOGICAL MECHANISMS IN LAMIN A/C ASSOCIATED CHARCOTMARIE-TOOTH DISEASE (CMT2B1)
POITELON Y1, BAUDOT C1, HAMADOUCHE T1, LEVY N1, DELAGUE V1
(1) Inserm UMR_S 910, Génétique Médicale & Génomique Fonctionelle, Faculté de
Médecine de la Timone, Marseille, FRANCE.
Lamins, a class of intermediate filaments, are major components of the nuclear lamina,
a filamentous network underlying the inner face of the nuclear membrane. A-type
To contact the author::
Yannick.Poitelon@univm Lamins are encoded by the same gene: LMNA, regulated by the AP1 complex (c-Fos
ed.fr.
& c-Jun). Up to date, eleven pathologies, described as laminopathies, have been
associated with mutations in LMNA. One of these, Charcot-Marie-Tooth disease
(CMT), type 2B1, is an autosomal recessive form of axonal CMT caused by the
c.892C>T transition in LMNA exon 5. (p.Arg298Cys). In order to progress towards
understanding of the pathophysiological mechanisms underlying CMT2B1, we studied
two different models for the disease: human cells from patients homozygous for the
c.892C>T mutation, and a Knock-In LmnaR298C/R298C mouse model.
Gene expression studies performed on human microfluidic plates (Low Density Arrays)
evidenced significant decrease in expression levels of several genes, including LMNA.
These observations were confirmed in mouse brain, skeletal and cardiac muscle,
sciatic nerve and spinal cord at the transcriptional level, as well as on lymphoblastoid
human cell lines at the protein level.
The p.Arg298Cys mutation lies within a coil-coiled domain, an important functional
domain for intermediate filament polymerization. In silico predictions are in favor of a
potential destabilizing effect of the mutation. Moreover, previous publications have
shown that Lamins interact with c-Fos, a Leucine Zipper transcription factor, through
their coiled-coil domain.We therefore propose a two-hit pathophysiological mechanism
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model:
- The pArg298Cys mutation might destabilize complexes between A-type Lamins and
transcription factors: the latter might be either components of the AP1 complex, or
some nerve specific transcription factors, which remain to be identified.
- LMNA seems to be autoregulated through an A_type Lamins – AP1 complex, which
might be disrupted at the DNA level by the presence of the mutation.
We are actually conducting further experiments in order to confirm these hypotheses.
PW23-281
To contact the author::
[email protected].
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ABNORMAL INTERACTION OF MUTANT HSP22 (HSPB8) WITH THE RNA
HELICASE DDX20 (GEMIN3, DP103) IN DISTAL HEREDITARY MOTOR
NEUROPATHY AND CHARCOT-MARIE-TOOTH DISEASE.
SUN X1, FONTAINE JM1, SIMON S2, HOPPE A1, CARRA S3, DE GUZMAN C1,
MARTIN J4, VICART P5, LANDRY J6, WELSH M1, BENNDORF R7
(1) University of Michigan, Ann Arbor, USA. (2) CGMC-UMR5534-Stress, Chaperons
et Mort Cellulaire, Lyon, FRANCE. (3) University Medical Center Groningen,
Groningen, NETHERLANDS ANTILLES. (4) Loyola University Chicago, Maywood,
USA. (5) Université Paris 7 - EA300, Paris, FRANCE. (6) Université de Laval,
Québec, CANADA. (7) Center for Clinical and Translational Research, Columbus,
USA.
Eight mutations in the small heat shock proteins (sHSP) Hsp22 and Hsp27 have
been associated with the motor neuron diseases (MND) distal hereditary motor
neuropathy and Charcot-Marie-Tooth disease. Hsp22 and Hsp27 interact with
each other, suggesting that these two etiologic factors may act in the same
pathway. In an effort to learn about the role of Hsp22 in MND, we screened a
human cDNA library by the yeast two-hybrid method for potential binding
proteins. One identified protein was the RNA helicase Ddx20, a core component
of the survival-of-motor neuron (SMN) complexes. This interaction was verified
by independent methods including FRET. Both mutant Hsp22 forms showed
abnormally increased binding to Ddx20. Interestingly, Ddx20 itself binds to the
SMN protein, and mutations in the SMN1 gene cause spinal muscular atrophy,
another MND. Thus, these protein interaction data have linked the etiologic
factors Hsp22, Hsp27, and SMN, and mutations in any of these genes cause the
various forms of MND. SMN complexes are involved in RNP processing. The
mutant Hsp22/Ddx20 interaction was sensitive to treatment with RNase
suggesting involvement of RNA in this interaction and a potential role of sHSPs
in RNP processing.
PW23-282
To contact the author::
[email protected].
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MOLECULAR EXPLORATION OF AXONAL CMT IN A LARGE SERIES OF
PATIENTS : FOCUS ON MFN2 AND GDAP1 GENES.
BONELLO-PALOT N1, LATOUR P2, MARTINI N1, MAYENÇON M2, PÉCHEUX C1,
MÉGARBANÉ A3, ATTARIAN S4, POUGET J4, LÉVY N5, BERNARD R1
(1) Département de Génétique médicale Laboratoire de Biologie Moléculaire CHU
Timone, Marseille, FRANCE. (2) Neurogénétique Centre de Biologie Groupement
Hospitalier Est CHU, Lyon, FRANCE. (3) Unité de Génétique Médicale, Faculté de
Médecine, Université Saint-Joseph de Beyrouth, Paris, FRANCE. (4) Service des
maladies neuromusculaires CHU Timone, Marseille, FRANCE. (5) Inserm UMR 910
Faculté de Médecine, Marseille, FRANCE.
Charcot-Marie-Tooth neuropathies (CMT), also known as hereditary motor and
sensory neuropathies (HMSN), are a group of genetically and clinically heterogeneous
diseases of the peripheral nervous system. 40 genes and more than 60 loci have
been identified to date. The French CMT diagnosis network has defined 6 types of
strategies for molecular exploration, linked to 6 phenotypic sub-types of CMT. We
focused our study on type 2 CMT (axonal) which phenotypic presentation is
characterised by relative preservation of nerve conduction velocity (>38m/s in the
median nerve), with decrease in compound motor action potentials providing evidence
of axonal loss. We studied in particular MFN2 (Mitofusine 2) and GDAP1 (Ganglioside
Induced Differentiation-Associated Protein 1) genes because of several points of
particular interest. First, their mutational spectrum is large and only recently described,
making molecular interpretation of news variants and subsequent genetic counselling
difficult. Second they are both implicated in mitochondrial dynamic. Third their place
within the exploration strategy diagram is not completely solved to date. We aimed to
estimate the frequency of mutations in MFN2 and GDAP1 genes in a big cohort of
CMT patients collected in France and to evaluate the potential pathogenicity of new
mutations. The frequency of mutation in MFN2 gene is about 11% (25/226) and in
GDAP1 gene is about 6.7% (9/135) in our study. Indeed, 75% (6/8) of GDAP1
sequence variations and 72% (18/25) of MFN2 sequence variations, we found weren’t
reported to date and raised to a lot of difficulties in genetic counselling. We report in
particular and discuss a missense mutation in GDAP1 that seems to segregate with
the disease in both dominant and recessive mode of inheritance.
PW23-283
To contact the author::
cecile.baudot@univmed.
fr.
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FGD4, ENCODING THE RHOGEF FRABIN, IS IMPLICATED IN CHARCOT MARIE
TOOTH TYPE 4H
BAUDOT C1, POITELON Y1, HAMADOUCHE T1, JACQUIER A2, BOCCACCIO I1,
CHOUERY E3, CHAOUCH M4, KASSOURI N4, JABBOUR R5, GRID D6,
MÉGARBANÉ A3, HAASE G2, LÉVY N1, DELAGUE V1
(1) U 910 Génétique Médicale et Génomique Fonctionnelle, Marseille, FRANCE. (2)
IBDML, Marseille, FRANCE. (3) Université Saint-Joseph, Beirut, LEBANON. (4)
Centre Hospitalier Universitaire Ben Aknoun, Alger, ALGERIA. (5) American
University of Beirut Medical Center, Beirut, LEBANON. (6) Généthon III, Evry,
FRANCE.
Charcot-Marie-Tooth (CMT) disorders are a clinically and genetically heterogeneous
group of hereditary neuropathies characterized by chronic distalweakness and
sensory loss. CMT4H is an autosomal recessive demyelinating subtype recently
mapped by us at chromosome 12p11.21-q13.11, in two consanguineous families of
Mediterranean origin. In both families, we identified mutations in FGD4, encoding
FGD4/FRABIN, a Rho GDP/GTP Exchange Factor (Rho GEF) specific to RhoGTPase
Cdc42. Both mutations affect the same codon: the p.Met298fsX8 (c.893T>G) in the
Lebanese and the p.Met298Thr (c.893T>C) in the Algerians.When overexpressing
wild-type and truncated (p.Met298fsX8) forms of Frabin in rat primary motoneurons
and rat RT4 Schwannomacells, we observed that the truncated form of Frabin induced
significantly fewer microspikes than the wild-type. At the transcriptional level, we
showed a 60% reduction in FGD4 mRNA levels in Lebanese patient’s fibrobasts,
indicating that the truncated mRNA might be degraded by NMD. Moreover, a broad
transcriptional study in different human tissues led us to characterize 17 alternative
transcripts for FGD4. Interestingly, some of them are deleted of several exons and the
corresponding protein lacks one or more functional domains. In consequence, we
propose that FRABIN might have different roles in different tissues, depending on the
functional domains present on the protein.
In conclusion, FRABIN is the first RhoGEF to be identified in CMT disease. Several
mechanisms and pathways leading to the pathology remain to be elucidated.
However, three main hypothesis might be proposed: 1) loss of Cdc42/Rac1 activation,
leading to a disorganization of the cytoskeleton, and perturbation of movements
and/or migration 2) disruption of JNK pathway, affecting myelination process 3)
implication of FRABIN in the activation of mitochondrial GTPases mutated in CMT
(MFN2…), and perturbation of mitochondrial dynamics.
We show here some primary results, further experiments are under process in order
to validate our hypothesis.
PW23-284
A NOVEL PERIAXIN MUTATION CAUSES LATE ONSET AND SLOW
PROGRESSIVE CHARCOT-MARIE-TOOTH DISEASE
NOUIOUA S1, BERNARD R2, HAMADOUCHE T3, VALLAT JM4, LEVY N2, TAZIR M1
(1) Service de Neurologie, CHU Mustapha, Algiers, ALGERIA. (2) Département de
Génétique Médicale, Laboratoire de Génétique Moléculaire, Hôpital d'Enfants de la
Timone, Marseille, FRANCE. (3) Laboratoire de Biologie Moléculaire, Institut Pasteur,
Algiers, ALGERIA. (4) Service de Neuropathologie, Hôpital Dupuytren, Limoges,
FRANCE.
Autosomal recessive forms of Charcot-Marie –Tooth disease are clinically and
genetically heterogeneous. One locus, termed CMT4F, showing similarities to
To contact the author::
Déjerine- Sottas syndrome, was mapped to 19q13.3 in a large consanguineous
[email protected] Lebanese family and a mutation in the periaxin gene (PRX) was identified in this
.
family. In the recent years, only seven other mutations were described in this gene.
Here, we report a large family with four adolescent and adult patients harbouring a
novel homozygous c.1090C>T (p. Arg364Stop) mutation in exon 7 of the PRX gene.
The clinical phenotype is characterized by a relatively late onset demyelinating
sensory motor neuropathy with spine deformities (kyphoscoliosis) and feet
deformities. Sensory ataxia was also present in the 2 youngest patients. In contrast
with the majority of the CMT4F patients already reported, evolution is slowly
progressive and the 4 patients have a mild motor disability. Nerve biopsy shows
severe loss of myelinated and unmyelinated fibers and some myelin outfoldings.
This is the first Algerian CMT4F family reported, and the causative PRX mutation is
responsible for a moderate CMT phenotype with some intrafamilial variability.
_0MS0G0OLK
PW23-285
To contact the author::
[email protected].
_0MS0G0OLK
PHENOTYPICAL FEATURES OF 14 MOROCCAN FAMILIES WITH AUTOSOMAL
RECESSIVE CHARCOT-MARIE-TOOTH DISEASE ASSOCIATED WITH MUTATIONS
IN THE GDAP1 GENE
BIROUK N1, BOUHOUCHE A2, BELAÏDI H1, BENOMAR A2, AZZEDDINE H3,
DUBOURG O4, MAISONOBE T4, YAHYAOUI M2, LE GUERN E3, OUAZZANI R1
(1) Service de Neurophysiologie Clinique, Rabat, MOROCCO. (2) Service de
Neurologie et Neurogénétique, Rabat, MOROCCO. (3) INSERM U289, Hôpital de la
Salpetrière, Paris, FRANCE. (4) Laboratoire de Neuropathologie Raymond Escourolle,
Hôpital de la Salpêtrière, Paris, FRANCE.
Mutations in GDAP1 gene located in 8q13 chromosome have
been identified in families with either axonal or demyelinating form
of autosomal recessive Charcot-Marie-Tooth (CMT) disease.
Twenty five patients belonging to 14 Moroccan consanguineous
families were examined clinically and electrophysiologically. In one
patient, a peroneal nerve biopsy was performed. Linkage to 8q13
was then demonstrated and a mutation in the coding region of the
GDAP1 gene was identified by direct sequencing.
Neuropathy was evident during early childhood, walking was
delayed in 5 cases and onset of symptoms occurred before 6
years in the others. The phenotype was very severe: foot
deformities and disability involving the hands and feet developed
towards the end of the first decade and followed by involvement of
proximal muscles in the lower limbs leading to loss of autonomy in
16 cases. Only three patients had hoarse voice. Thirteen patients
belonging to 7 families were homozygous for the S194X mutation
of GDAP1. They all had axonal form of CMT according to
electrophysiological and morphological findings. Six patients
belonging o 3 families had either homozygous P78L mutation or
compound heterozygous P78L/S194X mutations. Most of them
had markedly reduced motor nerve conduction velocity ranging
from 11 to 34 m/s consisting with a demyelinating form of CMT.
For the remaining 6 patients belonging to 3 families, GDAP1
mutation has not been identified yet.
The main phenotype characteristics of GDAP1 mutations are:
early onset in childhood, severity and important foot deformities.
The homozygous S194X mutation is the most frequent in our
families and was related to axonal form of CMT suggesting a
probable founder effect and a possible phenotype/genotype
correlation.
PW23-286
GAIT ANALYSIS IN CHARCOT MARIE TOOTH (CMT) DISEASE: PRELIMINARY
STUDY
BOULAY C1, POMERO V2, JACQUEMIER M2, CUSTAUD M3, PAGNI S2,
VIEHWEGER E2, GLARD Y2, JACOPIN S2, LAUNAY F2, JOUVE JL2, BOLLINI G2,
CHABROL B1
(1) Centre de Référence des Maladies neuromusculaires de l'enfant (Pr. Chabrol),
CHU Timone enfants, Marseille, FRANCE. (2) Laboratoire d'analyse du mouvement,
service de chirurgie orthopédique pédiatrique (Pr. Bollini), CHU Timone enfants,
Marseille, FRANCE. (3) Lecante Orthosud, Marseille, FRANCE.
The gait analysis provided different kinematic patterns of walking in the Charcot Marie
To contact the author::
[email protected].
Tooth disease (CMT). The dynamic electromyography (EMG) described the muscles
pattern for each type of gait in CMT.
The classical gait pattern in CMT is characterized by a drop foot during the swing
phase: the dorsal flexor muscles of ankle were weak in relation to the plantar flexor
muscles. But the drop foot was often absent and in this case it was the plantar flexor
muscles of ankle which were weak in relation to the dorsal flexor muscles. Also the
gait analysis described a delayed and/or increased peak of dorsal flexor ankle, during
the stance phase: the heel lift (“third rocker”) is delayed. These data are studied in a
sample of 10 subjects.
Thus the dynamic EMG revealed the balance between agonist and antagonist ankle
muscles and the reliability of the gait pattern and the foot deformity. It performs the
importance of this functional exam. These neurophysiological and dynamic data
facilitate the therapeutic strategy (orthosis, physical therapy, surgery) and the
functionnal evaluation.
_0MS0G0OLK
PW23-287
PHENOTYPIC VARIABILITY IN GIANT AXONAL NEUROPATHY
NOUIOUA S1, MAGY L2, HAMADOUCHE T3, RAUTENSTRAUSS B4, GRID D5,
ASSAMI S6, VALLAT JM7, TAZIR M8
(1) Service de Neurologie, CHU Mustapha, Algiers, ALGERIA. (2) Service de
Neuropathologie, Hôpital Dupuytren, Limoges, FRANCE. (3) Laboratoire de Biologie
Moléculaire, Institut Pasteur d'Algérie, Algiers, ALGERIA. (4) MGZ-Medizinisch
Genetisches Zentrum, München, GERMANY. (5) Genethon, Evry, FRANCE. (6)
Service de Neurologie, CHU Mustapha, Algiers, ALGERIA. (7) Service de
Neuropathologie, Hôpital Dupuytren, Limoges, FRANCE. (8) Service de Neurologie,
CHU Mustapha, Algiers, ALGERIA.
Giant axonal neuropathy (GAN) is a recessively inherited neurological disorder
To contact the author::
affecting both central and peripheral nervous system. The main pathological hallmark
[email protected] of the condition is abnormal accumulation of intermediate filaments in giant axons and
.
other cell types. Mutations in the GAN gene encoding Gigaxonin are responsible for
the phenotype.
We report clinicopathological, neurophysiological and genetic data from 15 patients
belonging to 7 families with giant axons observed on nerve biopsy.
In 6 families, we identified 3 different disease-causing homozygous mutations in the
GAN gene. The c.1429C>T (R477X) mutation which seems prevalent, was observed
in 4 unrelated families originating from eastern Algeria. This mutation was responsible
for 3 different phenotypes: 5 patients from 2 families had the classical GAN clinical
picture with kinky red-hair, sensory motor axonal neuropathy, moderate mental
retardation and cerebellar syndrome. The third family patient had congenital
neuropathy with arthrogryposis and the fourth family patient had a pyramidal
paraparesia with facial diplegia. All the patients of these 4 families had a diffuse
cerebral dysmyelination on MRI.
The 2 other families linked to the GAN gene had a CMT-like presentation. One family
with 4 patients had a c. 431G>A (R138H) mutation, and the second family patient had
a c.505G>A (E169K) mutation. In the seventh family also with a CMT-like phenotype
but no mutation in the GAN gene, we identified a c.2710C>T (R904X) mutation in the
SH3TC2 gene implicated in CMT4C.
In conclusion, the phenotypic consequences of GAN mutations can be variable. The
same mutation c.1429C>T (R477X), found in 4 families from the same geographical
area suggesting a founder effect, is responsible for different phenotypes. CMT-like
presentation with characteristic giant axons may be linked to mutations in the GAN
gene but also in other genes, especially the SH3TC2 gene (CMT4C) and the NEFL
gene (CMT2E).
_0MS0G0OLK
PW23-288
To contact the author::
[email protected].
CELLULAR MODELS FOR GIANT AXONAL NEUROPATHY: DEVELOPMENT OF
A NEW DIAGNOSTIC TOOL AND ASSESSMENT OF THE ROLE OF TUBULIN
CHAPERONES IN VIMENTIN AGGREGATION
CLEVELAND D1, YAMANAKA K2, BOMONT P3
(1) Ludwig Institute for Cancer Research, University of California San Diego, La Jolla,
USA. (2) RIKEN Brain Science Institute, Saitama, JAPAN. (3) INMED - Inserm U901;
Aix Marseille Université, Marseille, FRANCE.
Mutations in the gene encoding gigaxonin are causative for the fatal, early-onset
recessive neurodegenerative disorder Giant Axonal Neuropathy (GAN). The crucial
role of gigaxonin in neuronal maintenance, first assessed by alterations in the
motor/sensory tracts of the peripheral nervous system and the impairment of the
central nervous systems was subsequently confirmed in the GAN mouse model.
Characterized by a generalized aggregation of Intermediate Filaments (IFs), GAN
points to the essential role of cytoskeleton architecture in neuronal function, and
especially to the implication of IF disorganization in neurodegeneration. Identification
of gigaxonin as the substrate adaptor of a new Cul3-ubiquitin ligase E3 and three of its
partners (the microtubule associated proteins MAP1B, MAP8 and the tubulin
chaperone TBCB) allows now to dissect the mechanisms of neurodegeneration and
IFs organization in GAN.
We studied here cellular models of GAN A) to develop an alternative diagnostic tool
(to nerve biopsies) for GAN patients and B) to assess the role of microtubules (MTS)
and tubulin chaperones in IF aggregation.
A) Using our new gigaxonin specific monoclonal antibodies and protein extracts from
lymphoblast cell lines derived from GAN patients, multiple disease causing mutants
are shown to be unstable, demonstrating that GAN is caused by the loss of function of
gigaxonin.
B) We assessed the implication of MTs and tubulin chaperones in vimentin
organization in transfection experiments but also in the GAN cellular model: the
patients’s skin derived primary fibroblasts. This allowed us to demonstrate that neither
MTs instability nor TBCB overabundance are able to reproduce the vimentin
aggregates so characteristic of GAN patients.
_0MS0G0OLK
PW23-289
AHNAK EXPRESSION BY MYELINATING SCHWANN CELLS REVEALS CAJAL
BANDS IN VIVO AND IS INVOLVED IN THEIR MORPHOGENESIS AND LAMININ
SUBSTRATE ADHESION IN VITRO
VON BOXBERG Y1, SALIM C1, ALTERIO J1, FÉRÉOL S1, NOTHIAS F1
(1) UPMC Paris06-CNRS UMR 7101 "NSI", Paris, FRANCE.
Relatively little is known about the precise molecular mechanisms regulating the
differentiation-associated morphological changes during Schwann cell (SC)
To contact the author::
development and regeneration/remyelination after injury, implying specific interactions
[email protected] with the local environment that ultimately converge on a reorganization of the
ssieu.fr.
cytoskeleton. We investigated the role in SC differentiation and function of the giant
phosphoprotein AHNAK, which we show for the first time to be expressed at high
levels in developing and mature SC.
A detailed study of the cellular and subcellular distribution of AHNAK during
development of the rat sciatic nerve was performed on the light and electron
microscope levels. During the first post-natal month, AHNAK distribution shifts
from adaxonal compartments to abaxonal and outer-mesaxonal SC membranes in
contact with basement membrane, and ultimately delineates the so-called “Cajal
bands”, exhibiting a staining pattern complementary to that of periaxin. Highly
expressed in non-confluent cultured primary SC seeded on laminin, AHNAK is
downregulated in confluent cells, mainly concentrated around the nucleus.
Furthermore, we noted that the SC laminin receptor beta-dystroglycan exhibits a
similar distribution pattern as AHNAK on both confluent and non-confluent cultured
SC. AHNAK silencing in cultured SC via siRNA transfection was found to affect the
morphology and adhesive properties of SC. This is likely related to our observation
that ahnak-siRNA transfection leads to a reduction of beta-dystroglycan
expression levels, and dislocation of the receptor from the plasma membrane.
Taken together, these results strongly suggest a role of AHNAK in SC interaction
with laminin, an important component of the basement membrane surrounding
myelinating SC.
Loss or mutation of basal lamina components (e.g. laminin-2), or of laminin receptors
(integrins and dystroglycan), is known to cause severe demyelinating pathologies
affecting the sensori-motor system. Elucidating the role of AHNAK in SC may thus
contribute to our understanding of SC differentiation, myelin formation, and myelin
maintenance.
_0MS0G0OLK
PW23-290
To contact the author::
tarikhamadouche@hotm
ail.com.
_0MS0G0OLK
MOLECULAR STUDY OF AUTOSOMAL RECESSIVE HEREDITARY MOTOR AND
SENSORY NEUROPATHIES IN A PANEL OF 150 ALGERIAN FAMILIES
HAMADOUCHE T1, DELAGUE V2, DE SANDRE-GIOVANNOLI A3, BERNARD R3,
GÉNIN E4, BENHASSINE T1, TAZIR M5, CHAOUCH M6, NOUIOUA S5, KASSOURI
N6, AMER EL KHEDDOUD W 6, VALLAT JM7, GRID D8, LÉVY N3
(1) Laboratory of Molecular Biology, Pasteur Institute of Algeria, Algiers, ALGERIA.
(2) INSERM UMR 910, Faculty of Medicine la Timone, Marseille, FRANCE. (3)
Department of Medical Genetics, Hospital la Timone, Marseille, FRANCE. (4)
INSERM UMR S535, Paul Brousse Hospital, Villejuif, FRANCE. (5) Department of
Neurology, CHU Mustapha Bacha, Algiers, ALGERIA. (6) Department of Neurology,
CHU Ben Aknoun, Algiers, ALGERIA. (7) Department of Neurology, CHU Limoges,
Limoges, FRANCE. (8) Généthon III, Evry, FRANCE.
Hereditary motor and sensory neuropathies (HMSN), commonly referred to as
Charcot-Marie-Tooth disease (CMT), are among the most common inherited
neurological diseases, with an overall prevalence of about 1-4/10,000. While all
modes of inheritance have been reported, clinical, anatomopathological and
particularly genetic heterogeneity have also already been underlined for this disease
(more than 50 loci and 30 genes identified to date).
The aim of this work was to investigate Algerian families for whom a clinical diagnosis
of hereditary motor and sensory neuropathy has been suspected. Considering the
high rate of consanguinity in Algeria, we focussed our analysis on families affected
with autosomal recessive forms of the disease.
The use of the strategy of homozygosity mapping, as well as a set of molecular tools,
allowed us to explore a panel of 150 families and to characterize for some of them the
molecular defect responsible of the observed phenotype, as well as the identification
of a new locus whose gene has been further characterized.
Thus, we could establish a precise molecular diagnosis for about 1/3 of the families,
by characterizing mutations in several genes, MTMR2 (CMT4B1), GDAP1 (CMT4A),
PRX (CMT4F), SH3TC2 (CMT4C), GAN (GAN) and LMNA (CMT2B1), whereas one
new locus/gene could be identified (CMT4H/FGD4). Although molecular
investigations are in progress for the remaining famlies, our results greatly reinforce
the genetic heterogeneity already reported for CMT disease, and suggest that many
other loci/genes have to be discovered.
This study allowed us to estimate the relative frequency of autosomal recessive forms
of CMT disease in our panel, to outline a certain geographical distribution for some
CMT subtypes and to delineate the occurrence of recurrent mutations or founder
effects. These observations are all important informations that will guide us to
establish an efficient approach for diagnosis of autosomal recessive types of CMT
disease in Algeria.
PW23-291
To contact the author::
alexandre.mezghrani@ig
f.cnrs.fr.
MECHANISM OF EPISODIC ATAXIA TYPE 2 AND CORRECTION BY STOPCODON READ-THROUGH STRATEGY
MEZGHRANI A1, BARBARA G1, MONTEIL A1, LORY P1, NARGEOT J1
(1) Institut de Génétique Fonctionnelle, MONTPELLIER, FRANCE.
Aminoglycycosides compounds can read-through to premature
termination codon mutations and appear an interesting
therapeutic approach for some genetic diseases. Indeed, clinical
studies have been initiated for cystic fibrosis and muscular
dystrophy diseases. The channelopathy episodic ataxia type-2
(EA2) is an autosomal dominant disorder related to mutations in
the pore-forming Cav2.1 subunit of P/Q type calcium channels.
In addition, P/Q type channels are also the major actors of
neurotransmitter release at the neuromuscular junctions. Most of
the EA2 mutations are nonsense and lead to the expression of
truncated channels that can abolish channel activity via a
dominant–negative mechanism (Mezghrani et al, 2008). We
have use the EA2 model as a prototype of neuronal and
neuromuscular disease in order to evaluate the aminoglycosideinduced read-through approach. Indeed, the EA2 mutant
(R1279X) exerts a specific dominant-negative effect on the
Cav2.1 channel. Using this mutant, we used cytometry-based
assay and patch clamp electrophysiology to compare different
aminoglycoside compounds for their efficacy to restore
functional calcium channels. These data may open new
perspectives for future therapeutic strategies for neuronal and
neuromuscular diseases.
_0MS0G0OLK
Mezghrani A, Monteil A, Watschinger K, Sinnegger-Brauns MJ, Barrère C,
Bourinet E, Nargeot J, Striessnig J and Lory P. A destructive interaction
mechanism accounts for dominant-negative effects of misfolded mutants of
voltage-gated Ca2+ channels. J Neurosci. 2008, in press.
PW23-292
To contact the author::
[email protected].
CA-BINDING PROTEINS IN THE SEARCH FOR NEW MODELS OF GLYCINE
RECEPTOR POTENTIATION IN HYPEREKPLEXIA
MUKHTAROV M1, BULDAKOVA S2, BREGESTOVSKI P3
(1) Institut de Neurobiologie de la Méditerranée (INMED), INSERM U901, Marseille,
FRANCE. (2) Institut de Neurobiologie de la Méditerranée (INMED), INSERM U901,
Marseille, FRANCE. (3) Institut de Neurobiologie de la Méditerranée (INMED), INSERM
U901, Marseille, FRANCE.
Hyperekplexia is a genetic neurological human disease, which is
accompanied by muscular hypertonia, hyperreflexia, and hypokinesia.
Hyperekplexia originates from a dysfunction of glycine receptor (GlyR)
channels, which mediate fast inhibitory synaptic transmission in spinal cord,
brainstem, retina and other areas of the nervous system of vertebrates.
Several point mutations of the GlyR gene result in decreased activation of
GlyR channels, leading to a reduction of inhibitory drive through glycinergic
synapses and, consequently, to the development of motor disorders.
Previously our team discovered a new mechanism for GlyR modulation:
potentiation by intracellular calcium (Ca2+) involving a Ca2+-binding protein
(Fucile et al., 2000). This mechanism could be exploited to increase activity of
GlyR channels in glycinergic synapses in normal and pathological conditions.
Here we analyzed the mechanisms of Ca2+-induced potentiation in
neurons and in heterologous systems. We find out that neuronal Ca 2+-binding
protein (NECAB) is involved in Ca2+-dependent regulation of GlyRs.
Overexpression of NECAB caused: (i) a decrease the apparent affinity to
glycine and shift to the right of “dose-response” curve of glycine-induced
currents in CHO, HEK-293 cells and in spinal neurons; (ii) a shortening of the
decay time of glycinergic currents in spinal neurons.
These observations suggest that the development of peptides mimicking
the GlyR sequence responsible for binding with NECAB might be important for
modulation inhibitory drive through glycinergic synapses. Such peptides,
interacting with NECAB, should prevent its binding to the GlyR, thus leading to
a functional up-regulation of GlyR channels. Such an approach is a promising
avenue to develop pharmacological compounds upregulating GlyRs function in
hyperekplexia models.
_0MS0G0OLK
Fucile S., De Saint Jan D., de Carvalho L.P., Bregestovski P., 2000. Fast
potentiation of glycine receptor channels by intracellular calcium in neurons and
transfected cells. Neuron 28: 571-583.
ACKNOWLEDGEMENTS This work was supported by French Association
against Myopathies (AFM) for M.Mukhtarov.
PW 24:
Pharmacological
therapies –
Target identification/
clinical trials
PW24-293
MODIFICATION OF PROTEOTOXICITY ASSOCIATED TO NEUROMUSCULAR
DISEASES: GENETIC ANALYSIS IN C. ELEGANS
PASCO M1, CATOIRE H1, TOURETTE C1, PARKER A1, NERI C1
(1) Inserm, Paris, FRANCE.
To contact the author::
[email protected].
The goal of our study is to identify signalling pathways and networks that may modify
proteotoxicity associated to neuromuscular diseases. The rationale of our study is that
genetic/biological modifiers of proteotoxicity may constitue a useful source of disease
targets and markers. We use a combination of genetic and pharmacological
manipulations to test whether genes and compounds may modify the cytotoxicity
produced by disease protein expression in transgenic nematodes. Results will be
presented showing that disease protein expression produces cellular and behavioral
defects in C. elegans transgenics and allows conserved modulators of proteotoxicity
to be identified, some of them which may be manipulated by pharmacological means.
Our data suggest that C. elegans genetics may be a useful translational research
component and starting point to screen for neuromuscular disease targets by
searching for in vivo modifiers of proteotoxicity.
_0MS0G0OLK
PW24-294
INHIBITION OF PROTEASOME ACTIVITY PROMOTES THE CORRECT
LOCALIZATION OF DISEASE-CAUSING ALPHA-SARCOGLYCAN MUTANTS IN A
HETEROLOGOUS CELL SYSTEM EXPRESSING BETA-, GAMMA-, AND DELTASARCOGLYCAN
SANDONÀ D1, GASTALDELLO S1, FRANZOSO S1, FANIN M2, ANGELINI C2, VIDAL
J3, BASSE N4, REBOUD-RAVAUX M4, BETTO R5
(1) Department of Biomedical Sciences, University of Padova, Padova, ITALY. (2)
Department of Neurological Sciences, University of Padova, Padova, ITALY. (3)
Chimie et Photonique Moléculaires, CNRS - Université de Rennes 1, Rennes,
FRANCE. (4) Equipe Enzymologie Moléculaire et Fonctionnelle, CNRS - Université
Paris 6, Paris, FRANCE. (5) CNR Institute of Neuroscience, Padova, ITALY.
Sarcoglycanopathies are progressive muscle wasting disorders caused by genetic
defects of four proteins, -, -, -, and -sarcoglycan, elements of a key
transmembrane complex of striated muscle. The proper assembly of the sarcoglycan
To contact the author::
complex represents a critical issue of sarcoglycanopathies, as several mutations
[email protected] severely perturb tetramer formation. Misfolded proteins are generally discarded
.
through the cell’s quality-control system that, however, could lead to the removal of
functional polypeptides. To explore whether it is possible to rescue sarcoglycan
mutants by preventing their degradation, we generated a heterologous cell system
constitutively expressing three ( , , and ) of the four sarcoglycans. In these cells
(
-HEK), the lack of -sarcoglycan prevented complex formation and cell surface
localization, while the presence of -sarcoglycan allowed maturation and targeting of
the tetramer. On the contrary, transfection of
-HEK cells with disease-causing sarcoglycan mutants led to a dramatic reduction of mutated proteins, compared to wild
type, and the absence of the complex from cell surface. This result closely reproduces
the observations made in muscle patients. Proteasomal inhibition reduced the
degradation of mutants and facilitated the assembly and targeting of the sarcoglycan
complex to the plasma membrane. However, as expected for a critical process
necessary for cell survival, the prolonged inhibition of proteasome severely
compromised cell viability. Importantly, when the treatment included the inhibition of
caspase activity, the cytotoxic effect of proteasome inhibitor was almost completely
abolished. Finally, in a trial application of this approach to a skeletal muscle explant
isolated from an LGMD-2D patient, incubation with the FDA-approved proteasome
inhibitor Velcade (bortezomib) rescued the expression of mutant -sarcoglycan to the
cell membrane. The present data provide important insights for the development of
pharmacological therapies for sarcoglycanopathies. Funded by AFM (grant # 12988)
and University of Padova.
_0MS0G0OLK
PW24-295
SPECIFIC INHIBITION OF PROTEIN QUALITY CONTROL SYSTEM AS A
THERAPEUTIC APPROACH FOR TREATMENT OF SARCOGLYCANOPATHIES.
SOHEILI T1, GICQUEL E1, BARRAULT L1, BARTOLI M1, RICHARD I1
(1) Genethon/CNRS, Evry, FRANCE.
To contact the author::
[email protected].
Sarcoglycanopathies are recessive muscular disorders caused by defects in a group
of transmembrane proteins, known as sarcoglycans, and part of the dystrophinassociated complex. Mutations in the , ,  and  sarcoglycan genes lead to a similar
phenotype and are referred as limb-girdle muscular dystrophies type 2D, 2E, 2C and
2F (LGMD2D, 2E, 2C and 2F) respectively. Dysfunction of one of the sarcoglycan
destabilizes the whole sarcoglycan complex, leading to a partial or complete
disappearance of the other sarcoglycans at the membrane. To date, no treatment
exists for these diseases.
The most frequently reported mutation in the -sarcoglycan gene is the substitution of
an arginine in position 77 by a cysteine (-R77C). We demonstrated that this mutation
encodes a misfolded protein that fails to be delivered to its proper sarcolemmal
localization due to blockade in the endoplasmic reticulum (ER). The quality control
system of the ER is an important monitoring mechanism in the protein maturation
process, which ensures export of properly folded proteins from the ER. Incorrectly or
incompletely folded proteins are retained in the ER for refolding or translocated in the
cytosol for degradation by the ER associated proteasome (ERAD).
_0MS0G0OLK
PW24-296
We hypothesized that, by blocking the ER quality control system, we should be able to
rescue the misfolded -sarcoglycan protein at the cell membrane. Consistently,
results of our experiments using an heterologous cellular model of sarcoglycan
complex formation showed that the -mannosidase I inhibitors prevent -R77C
degradation by proteasome/ERAD and restore correct localization of the protein.
Furthermore, this membrane targeting allows the assembly of the sarcoglycan
complex. Consequently, we were able to rescue other sarcoglycan mutations using
the same approach. Overall, these results suggests a therapeutic approach for
LGMD2 patients carrying mutations that impair sarcoglycan trafficking.
CHAPERONE MIMICS BASED STRATEGY TO TREAT NEUROMUSCULAR
DISEASES
SIMON S1, ARRIGO AP1
(1) CGMC-UMR5534-Université Claude Bernard Lyon1, Lyon, FRANCE.
To contact the author::
[email protected].
Small stress proteins are characterized by a common alpha-crystallin domain. Among
these proteins, Hsp27 and B-Crystallin are oligomeric molecular chaperones that are
highly expressed in pathological conditions such as those generated by
neuromuscular diseases. These proteins share the ability to protect cells against the
toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In
addition, they have anti-apoptotic properties. The missense mutation R120G in human
B Crystallin, which strongly decreases the chaperone activity of this protein, is
associated in vivo with autosomal dominant myopathy, cardiomyopathy, and cataract
(Vicart et al., 1998). In muscle cells, R120G mutant forms aggregates that contain
desmin, the major chaperone substrate of B-Crystallin. Consequently, the structural
organization of muscle cells is disorganized. Overexpression of Hsp27 or Hsp22 (an
other small stress protein) results in the formation of complex oligomeric stuctures
containing R120 mutant. As a consequence, the toxicity induced by R120G mutant is
reduced, probably because of the partial refolding and restoration of the activity of this
mutant. This implies that it may be possible to restore the function of R120G mutant
by chaperone mimics that interfere with the aggregative process of this deleterious
protein. Recent approaches using peptides aptamers that specifically target this
protein support this assumption.
Since the expression of small stress proteins has implications in pathologies as
diverses as neurodegeneration, myopathies, asthma, cataracts and cancers,
approaches towards therapeutic strategies will be discussed.
Arrigo, A. P., Simon, S., Gibert, B., Kretz-Remy, C., Nivon, M., Czekalla, A., Guillet,
D., Moulin, M., Diaz-Latoud, C., and Vicart, P. (2007). Hsp27 (HspB1) and alphaBcrystallin (HspB5) as therapeutic targets. FEBS Lett.
Vicart, P., Caron, A., Guicheney, P., Li, Z., Prevost, M. C., Faure, A., Chateau, D.,
Chapon, F., Tome, F., Dupret, J. M., et al. crystallin chaperone gene causes a
desmin-related myopathy. Nat Genet 20, 92-95.
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PW24-297
MYOSTATIN AND NOTCH SIGNALLING PATHWAYS: PARTNERS IN THE
CONTROL OF POST-NATAL MYOGENESIS?
VERNUS B1, CARNAC G2, TASSISTRO V1, KOECHLIN C1, MORNET D2, HUGON
G2, BONNIEU A1
(1) Inra, UMR 866-Différenciation Cellulaire et Croissance, Montpellier, FRANCE. (2)
Inserm, ERI 25-Muscle et Pathologies, Montpellier, FRANCE.
Myostatin is an endogenous, negative regulator of muscle growth determining both
To contact the author::
muscle fiber number and size. Recently, inhibition of this pathway has emerged as a
[email protected] promising therapy for muscle wasting although more details of myostatin regulation
.
and its mechanisms of actions need to be clarified. Despite the importance of
myostatin signalling for proper myogenic differentiation, little is known about the
interaction of the myostatin signalling pathway with other major signalling pathways
involved in myogenesis. To begin to address this, we investigated the possibility of a
signal integration between the myostatin and Notch signalling pathways, two negative
regulators of myogenic differentiation. In a previous study using a potent inhibitor of
presenilin-dependent cleavage of Notch, DAPT, we induced myotube hypertrophy in
primary human myoblasts. Here we show that this myotube hypertrophy-induced by
Notch inhibition was associated with downregulation of myostatin expression.
Consistent with these data we further show that both pathways are integrated in the
transcriptional regulation of Notch and myostatin responsive genes. These results
suggest that targeting myostatin through manipulating Notch signalling could have
important roles in regulation of muscle mass. We are now currently investigating the
relevance of this interaction for adult myogenesis.
_0MS0G0OLK
PW24-298
GPCR REPERTOIRE APPROACH TO IDENTIFY SPECIFIC PHARMACOLOGICAL
TARGETS IN SKELETAL MUSCLE
MOORE-MORRIS T1, VARRAULT A1, LE DIGARCHER A1, JOURNOT L1, NARGEOT
J1, COUETTE B1
(1) IGF, Montpellier, FRANCE.
To contact the author::
[email protected].
Maintaining or even enhancing skeletal muscle mass is critical not only in
inherited muscular disorders but also in the context of aging and in various disease
states associated with muscle loss. The aim of any treatment for muscle wasting is to
restore, maintain, or improve muscle size and strength. Many therapeutic trials have
been undertaken to identify and validate new targets for treating muscular
dystrophies. Compounds used include anabolic steroids, growth factors and
interestingly agonists of G protein-coupled receptors (GPCRs) such as the beta(2)Adrenergic receptor (AR). The later have been put forward as a promising new target
for treating muscle wasting. However, their potential is limited because of the high
doses of beta(2)-AR agonists required which have several deleterious effects such as
cardiac hypertrophy.
We have produced a repertoire of the GPCRs in mouse heart, revealing new
cardiac receptors. Our data is validated by the identification of all the well known
receptors and atypical or new receptors such as mGluR1 whose presence we
evidenced at the protein and functional level. We have repeated this approach on
proliferating and differentiating myoblasts and are currently also creating GPCR
repertoires for normal and atrophied skeletal muscle. Our aim is to identify receptors
actively involved in skeletal muscle atrophy that are weakly represented or absent in
the heart.
The work put forward in this proposal should enable the identification of new
targets and molecules of interest for the treatments of skeletal muscle loss, owing to
the GPCR repertoire approach. Indeed, GPCRs are well known for being highly
accessible pharmacological targets.
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PW24-299
CROSS TALK BETWEEN GLUCOCORTICOID AND WNT/BETA CATENIN
SIGNALING PATHWAYS IN SCHWANN CELLS ON THE LEVEL OF MYELIN
GENES
MAKOUKJI J1, TROUSSON A1, FONTE C2, GRENIER J1, SCHUMACHER M2,
MASSAAD C2
(1) CNRS UPR2228 University Paris Descartes, Paris, FRANCE. (2) Inserm UMR
788 Univeristy Paris-Sud, Le Kremlin-Bicetre, FRANCE.
To contact the author::
[email protected].
Glucocorticoids play a major role in the nervous system and promote myelination.
Their action is mediated by the glucocorticoid receptor (GR) that recruits
coactivators(CBP or p300). We investigated the role of CBP and p300 in Schwann
cells. We showed that neither CBP nor p300 enhanced GR transcriptional activation
and unexpectedly, p300 acted as a corepressor. Functional and pull-down assays
showed that beta catenin is the coactivator replacing CBP in the GR transcriptional
complex, indicating that glucocorticoids may act by means of unusual partners in
Schwann cells. Beta catenin is involved in Wnt signaling pathway which plays a role in
development and diseases.
We have then evaluated the physiological significance of our findings, by studying
the regulation of myelin genes expression by glucocorticoids and Wnt/beta catenin.
We showed that P0 and PMP22 genes (two major peripheral myelin genes) are
stimulated by Wnt pathway, moreover, the combination of of glucocorticoids and Wnt
signaling have a synergistic effect (i.e. 13-fold stimulation of P0 gene expression). We
have then studied the mechanism of regulation of myelin genes by Wnt signaling by
using either siRNA targeting beta catenin and LEF/TCF, or dominant-negative forms
of the receptor frizzled and Disheveled. We found that Wnt/beta catenin pathway is
essential for both basal and stimulated activities of myelin genes. Finally, we have
studied the cross-talk between the GR and Wnt/beta catenin pathways and found that
glucocorticoid enhances the expression of beta catenin, which is able to bind to GR
and TCF. Our findings highlight the importance of Wnt/beta catenin and glucocorticoid
in the expression of myelin genes, and opens a new strategy in the treatment of
demyelinating disease such as Charcot-Marie-Tooth by modulating the action of
Wnt/beta catenin and glucocorticoid pathways.
_0MS0G0OLK
PW24-300
LONG-TERM BLINDED PLACEBO-CONTROLLED STUDY SHOWING
PHENOTYPIC CORRECTION OF DYSTROPHIN DEFICIENCY IN THE MDX MOUSE
BY SNT-MC17/IDEBENONE
BUYSE G1, VAN DER MIEREN G1, ERB M2, D'HOOGE J1, HERIJGERS P1,
VERBEKEN E1, JARA A3, VAN DEN BERGH A1, MERTENS L1, COURDIER-FRUH I2,
BARZAGHI P2, MEIER T2
(1) University Hospitals K.U. Leuven, Leuven, BELGIUM. (2) Santhera
Pharmaceuticals, Liestal, SWITZERLAND. (3) Biostatistical Center K.U. Leuven,
Leuven, BELGIUM.
Background - Duchenne muscular dystrophy (DMD) is a severe and still incurable
disease, with heart failure as a major cause of death. The identification of a diseaseTo contact the author::
gunnar.buyse@uzleuven
.be.
modifying therapy may require early-initiated and long-term administration, but such
type of therapeutic trial is not evident in humans. We have performed such a trial of
SNT-MC17/idebenone in the mdx mouse model of DMD, based on the drug’s potential
to improve mitochondrial respiratory chain function and reduce oxidative stress. The
mouse model allowed presymptomatic initiation and veritable long-term administration
of treatment, as well as the use of gold standard in vivo invasive pressure-volume
measurements for assessing cardiac contractility.
Methods & Results - 200 mg/kg bodyweight of either SNT-MC17/idebenone or placebo
was given from age 4 weeks until 10 months in mdx and wild-type mice. All evaluators
were blinded to mouse type and treatment groups. Compared to wild-type mice,
placebo-treated mdx mice showed cardiac hypertrophy, diastolic dysfunction, reduced
contractile reserve with systolic failure and 58% mortality during low-dose dobutamine
stress, cardiac inflammation and fibrosis, and reduced voluntary wheel running
performance. Idebenone treatment significantly corrected cardiac diastolic dysfunction
and significantly prevented mortality from cardiac pump failure induced by dobutamine
stress testing, significantly reduced cardiac inflammation and fibrosis, and significantly
improved voluntary running performance in mdx mice.
Conclusions - We have identified a novel potential therapeutic strategy for human
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DMD, as SNT-MC17/idebenone was cardioprotective and improved exercise
performance in the dystrophin-deficient mdx mouse. These animal data encourage
investigation of SNT-MC17/idebenone in human DMD. Our data also illustrate that the
mdx mouse provides unique opportunities for long-term controlled prehuman
therapeutic studies.
PW24-301
SNT-MC17/IDEBENONE IMPROVES CARDIAC AND RESPIRATORY FUNCTION
IN DUCHENNE MUSCULAR DYSTROPHY: RESULTS OF A 12 MONTH DOUBLEBLIND RANDOMIZED CONTROLLED TRIAL
BUYSE G1, MERTENS L1, VAN DEN HAUWE M1, THIJS D1, DE GROOT I2, SCHARA
U3, CEULEMANS B4, MEIER T5, GOEMANS N1
(1) University Hospitals K.U. Leuven, Leuven, BELGIUM. (2) UMC St Radboud,
Nijmegen, THE NETHERLANDS. (3) Lukaskrankenhaus GmbH, Neuss, GERMANY.
(4) University Hospital Antwerp, Antwerp, BELGIUM. (5) Santhera Pharmaceuticals
Ltd, Liestal, SWITZERLAND.
Objective
-
To
evaluate
efficacy and
tolerability of
treatment
with
SNT-
MC17/idebenone compared to placebo in children with Duchenne muscular dystrophy
To contact the author::
gunnar.buyse@uzleuven
.be.
(DMD). Background - Idebenone supports mitochondrial respiratory chain function
and reduces oxidative stress, pathways that are involved in DMD pathogenesis. In a
controlled study we have shown that presymptomatic-initiated and long-term treatment
with SNT-MC17/idebenone is cardioprotective and improves exercise performance in
the homologous dystrophin-deficient mdx mouse. Design/Methods - 21 DMD patients
(8-16 yr) with cardiac dysfunction were enrolled in a phase II double-blind randomized
placebo-controlled trial. Comedication with glucocorticoids was allowed at stable
dosage; use of ACE-inhibitors was excluded. Thirteen patients received SNT-MC17
(450 mg daily) for 52 weeks, 8 patients were randomized to the placebo group. The
primary endpoint was the change from baseline in peak systolic radial strain of the left
ventricular (LV) inferolateral wall, the region of the heart which is most affected in
DMD. Secondary outcome measures included other cardiac parameters, respiratory
function and upper limb muscle strength tests. Results - All subjects completed the
study, and SNT-MC17 showed good safety and tolerability. Compared to patients on
placebo, patients on SNT-MC17 showed significant improvement in peak systolic
radial strain of the LV inferolateral wall (p= 0.030). Peak systolic longitudinal strain of
the LV lateral wall improved in patients on SNT-MC17 and deteriorated in patients on
placebo (p=0.039). Whereas SNT-MC17 treatment was associated with improvement
in respiratory peak flow, patients on placebo deteriorated (p= 0.039). Changes in
forced vital capacity and maximal inspiratory pressures were not statistically
significant between the groups. No significant differences were detected for
quantitative upper limb muscle strength. Conclusions - This is the first indication of
clinical efficacy with SNT-MC17/idebenone on functional cardiac and respiratory
parameters in DMD. The results provide the basis and guidance for phase II/III studies
with SNT-MC17/idebenone in DMD.
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PW24-302
RATIONALE FOR THE USE OF BEZAFIBRATE IN THE TREATMENT OF VERY
LONG CHAIN ACYLCOA DEHYDROGENASE DEFICIENCY.
GOBIN-LIMBALLE S2, DJOUADI F1, AUBEY F1, OLPIN S3, ANDRESEN B.S4,
FUKAO T5, WANDERS R.J6, KIM JJ7, BASTIN J1
(1) CNRS UPR 9078, Université Paris Descartes, Paris, FRANCE. (2) Service de
Genetique, Hôpital Necker, Paris, FRANCE. (3) Department of Clinical Chemistry,
Sheffield Children's Hospital,, Sheffield, UNITED-KINGDOM. (4) Research Unit for
Molecular Medicine, Aarhus University Hospital, Aarhus, DENMARK. (5) Department
of Pediatrics, Gifu University, School of Medicine, Gifu, JAPAN. (6) Academic Medical
Center, University of Amsterdam, Amsterdam, THE NETHERLANDS. (7) Medical
College of Wisconsin,, Milwaukee, USA.
Very-Long-Chain-AcylCoA (VLCAD) deficiency is one of the more common
mitochondrial ß-oxidation defect, without treatment to date, with three distinct
phenotypes including neonatal-onset severe cardiomyopathy, liver failure in infancy,
or adolescent-onset myopathy with exercise intolerance, myalgia and rhabdomyolysis.
Because of the severity of symptoms, newborn screening of VLCAD deficiency is
performed in several countries. Molecular studies have revealed >80 VLCAD gene
missense mutations, with generally unpredictable effects on enzyme activity, and for
which genotype/phenotype correlations are globally unclear. We sought to determine
if, via activation of the PPAR (Peroxisome Proliferator Activated Receptors) signaling
pathway, bezafibrate could be effective to stimulate residual metabolic capacities in
this disorder. Palmitate oxidation tests were performed in a panel of patient fibroblasts
from the three phenotypes, representing 36 genotypes and 45 missense mutations.
About two thirds of the cell lines exhibited a marked increase in Fatty Acid Oxidation
(FAO) in response to bezafibrate, whereas treatment was ineffective in the remaining
cell lines. Similar increases in VLCAD mRNA were found in all cell lines, and the
differences in FAO could then be ascribed to variable increases in VLCAD residual
enzyme activity in response to the drug. Unresponsive genotypes were all found to
correspond to severe clinical presentations whereas cells that responded to
bezafibrate were from patients with the myopathic form of the disorder. Cross-analysis
of genotypes allowed to characterize groups of individual severe or mild missense
mutations accounting for the response to the drug.
This pharmacogenetics study provides a new way for functional analysis of VLCADdeficient genotypes, demonstrates the potential of bezafibrate in the correction of the
more common myopathic form of the disorder, and provides a rationale for the
selection of patients who might respond to bezafibrate in a future clinical trial, based
on the association of in vitro tests and molecular data.
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PW24-303
THE PSYCHOLOGICAL IMPACT OF PARTICIPATION IN A PLACEBO
CONTROLLED TRIAL OF ENZYME REPLACEMENT THERAPY ON PATIENTS WITH
LATE-ONSET POMPE DISEASE
HERSON A1, GALLAIS B1, MICHON CC1, DOPPLER V1, PAYAN C1, HERSON S2,
EYMARD B1, GARGIULO M1, LAFORÊT P1
(1) Institut of Myology, Pitié-Salpêtrière, Paris, FRANCE. (2) Internal Medicine service,
Pitié-Salpêtrière, Paris, FRANCE.
To contact the author::
[email protected].
Objective: To prospectively evaluate the psychological impact of the participation in a
clinical trial on patients with late-onset Pompe Disease.
Method: From the moment of the inclusion and until the end of the trail at 18 months: we
assessed every 3 months the psychological status of 10 patients who have been
included in a placebo-controlled study of enzyme replacement therapy.
Assessment: a) Open interviews were conducted by psychologists to qualitatively assess
the impact of the clinical trial on the patient’s personal, marital and socio-professional
life. b) Mood and anxiety were assessed with Self-report scales: Beck Depression
Inventory, Beck Hopelessness Scale (B.H.S) and State and Trait Anxiety Inventory of
Spielberger (S.T.A.I. I and II). c) Impact of the clinical trial and coping were evaluated
with Impact of Event Scale (I.E.S) and the IPC scale (Internal, Powerful others, Chance)
of Levenson. d) Quality of life and social adjustment were assessed with Whoqol-26 and
Social Adjustment Scale in self report (S.A.S S-R).
Preliminary and partial results: Throughout the trial mean scores of depression and
anxiety-state improved. Quality of life and social adjustment were conserved. Patients
privileged coping strategies focused on internal factor.
Discussion: According to the preliminary results, there is no negative impact of
participation in clinical trial on psychological status, moreover some psychological
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dimensions improved. We can explain that by two features: a) The medical team offered
an enough secure and supportive environment during the trial. b) Participation in a trial
has an influence on patient’s subjective position in front of their disease: it gives them a
sense of having an active part in medical progress. For patients who are now under
treatment (8 patients), the psychological assessment has to be continued in front of
weight of treatment, involving a “medicalisation of their existence”.
PW24-304
MONITORING OF CHANGES IN FRIEDREICH'S ATAXIA DURING A PERIOD OF 8
YEARS IN 49 PATIENTS IN REUNION ISLAND
MIGNARD C1, CHARLIN C1, MIGNARD D2, ROELENS P2
(1) Centre de Référence des maladies neuro musculaires, Saint Pierre(REUNION),
FRANCE. (2) SCP Mignard-Roelens-Tabailloux, Saint Pierre(REUNION), FRANCE.
To contact the author::
[email protected].
_0MS0G0OLK
In Reunion Island, we followed fifty ataxic patients every six months
during eight years, from November 1999 to July 2007, using the same
protocol. 1 The work protocol: Patients were included regardless of the
stage of their disease. They were reviewed for a neurological
examination, including a Kurtzke and an ataxia WFN scale, a biological
and a cardiological assessment with ultrasound and holter monitor. A
few patients were placed under Idebenone. They all received
physiotherapy.2 Patients: All patients had a diagnosis confirmed by
molecular biology. At the beginning of the study. 6 had a heart disease.
37 patients could still move, with or without support. 33 patients were
treated for more than two years. 3 Results : Natural evolution studied in
14 untreated patients: 5 aggravations, 9 stabilisations or
improvements. Under treatment, studied in 33 patients: 23
aggravations, 10 stabilisations or improvements from a neurological
standpoint: a/ Stabilisations: Among the patients most affected and
throughout the study period, 11 patients have similar neurological signs
at the end of the study, as those presented at the beginning of the
study. b/ Improvements: 6 : often at a light stage of their illness. c/
Aggravations: 25 patients have worsened despite taking regular
Idébénone. These exacerbations occur more often in patients with mild
stage of the disease.From a cardiological standpoint: 6 patients had
cardiomyopathy at the beginning of the study. At the end of the study,
there are 9 patients. Out of the 6 patients already affected at the
beginning of the study, two patients died, 4 have seen no worsening of
their cardiac disease. Three people ended up with cardiac disease.
Conclusion The evolution of the Friedreich's ataxia is spontaneously
slow. The treatment stabilizes or improves a good number of cardiac
disease and neurological, but many patients worsen regardless the
stage of disease.
PW 25:
Pharmacological
therapies –
Evaluation and animal
models
PW25-305
THERAPEUTIC EFFICACY AND MECHANISM OF ACTION OF NITRIC OXIDERELEASING NON STEROIDAL ANTI-INFLAMMATORY DRUGS IN MUSCULAR
DYSTROPHY
SCIORATI C1, AZZONI E1, ONGINI E2, MONOPOLI A2, BRUNELLI S1, COSSU G1,
CLEMENTI E3
(1) Stem Cell Research Institute-San Raffaele Scientific Institute, Milano, ITALY. (2)
NicOx Research Institute, Bresso, ITALY. (3) E. Medea Scientific Institute and
Department of Preclinical Sciences, University of Milano, Milano, ITALY.
Duchenne muscular dystrophy is a relatively common disease that affects skeletal
muscle leading to progressive paralysis and death. There is currently no resolutive
To contact the author::
therapy. We have developed a novel strategy based on the combination of nitric oxide
[email protected].
(NO), which has beneficial effects in skeletal muscle, with non steroidal antiinflammatory drugs (NSAIDs). To this end we used a new class of NO-releasing
NSAIDs (NO-NSAIDs). We report the results of long term (one-year) oral treatment in
the mouse model for limb girdle muscular dystrophy (-sarcoglycan null mice) with
two such NO-NSAIDs, nitroibuprofen and nitroparacetamol. Both drugs significantly
ameliorated the morphological, biochemical and functional phenotype in the absence
of secondary effects, efficiently slowing down disease progression and were
significantly more effective than the corticosteroid prednisolone analyzed in parallel.
NO-NSAIDs acted by reducing inflammation, preventing muscle damage and
preserving the number and function of satellite cells. To assess the mechanisms of
NO-NSAID action we dissected the contribution of NO and NSAID activities, by
analyzing the effects of the NSAID ibuprofen and of isosorbide dinitrate that release
NO with a pharmacokinetic profile similar to that of NO-NSAIDs. The NO-NSAIDs
were significantly more effective than either isosorbide dinitrate or ibuprofen. Of
importance, the NO-NSAIDs were more effective therapeutic agents also with respect
to the combination of isosorbide dinitrate and ibuprofen, suggesting that NO-NSAIDs
have properties additional to NO release and NSAID activity.
_0MS0G0OLK
The new therapeutic strategy we propose is not selective for a subset of
mutations, provides ground for immediate clinical experimentation with NO-NSAIDS,
which are approved for use in humans.
.
PW25-306
To contact the author::
olivier.dorchies@pharm.
unige.ch.
GREEN TEA POLYPHENOLS DISPLAY POTENT ANTI-FIBROTIC ACTIVITY ON
PRIMARY CULTURES OF DYSTROPHIC MUSCLE CELLS
DORCHIES O1, COMYN S1, RUEGG U1
(1) University of Geneva, Geneva, SWITZERLAND.
Muscular dystrophies are characterized by fibrosis, a process leading to abnormal
accumulation of materials of fibroblastic origin in the skeletal muscles as necrosisregeneration cycles take place. Fibrosis results from alterations in a multifactorial
balance involving cytokines and growth factors, expression of their receptors at the
cells’ surface, oxidative stress, production of extracellular matrix components, release
of matrix-metalloproteinases (MMPs) and of their endogenous inhibitors TIMPs (tissue
inhibitors of MMPs), all collectively regulating extracellular matrix turnover and
fibroblast invasiveness. Fibrosis severely impairs skeletal and cardiac muscle
functions both in patients suffering from Duchenne muscular dystrophy (DMD) and in
the mdx mouse, the most commonly used model for DMD. Published data indicate
that green tea polyphenols (GTP) exhibit anti-fibrotic properties in several cell types
and tissues, and pentoxifylline (PTX), a well-known inhibitor of TNF release, has
been proposed to reduce fibrosis in the dystrophic mdx mouse. Treatment of primary
1,
and PDGF-BB. When needed, fibrosis was induced in the cultures with TNF or
TGB1 (30ng/mL). GTP dose-dependently decreased both basal and TNF or
TGB1fibroblasts), collagen, TGF receptor type 2, and CTGF (connective tissue growth
factor, a key mediat
expression of pro- and active forms of MMP-2 and MMP-9 were not changed with
GTP and PTX. However, the MMP-2 and MMP-9 gelatinase activity in the
supernatants was significantly decreased and correlated with a marked elevation of
TIMP-1 levels. Overall, PTX was less potent than GTP on these endpoints. Our
results indicate that GTP profoundly alter the expression and/or the activity of proteins
involved in the fibrogenic process.
_0MS0G0OLK
PW25-307
OXIDATIVE INJURY OF MYOBLASTS: SEARCH FOR A PROTECTIVE EFFECT
BY A GLYCOSAMINOGLYCAN MIMETIC (OTR4120)
VAN ZOGGEL J1, ALABANESE P1, JACOBS MS1, COURTY J1, PAPY-GARCIA D1,
MORIN C1, MARTELLY I1
(1) CRRET Laboratory, Université Paris-Est, Faculté de Sciences et Technologie,
Paris 12, Créteil, FRANCE.
Overproduction of reactive oxygen species has been implicated in cellular defect that
may lead to apoptosis. Skeletal muscle cells are frequently submitted to oxidative
To contact the author::
stress upon excessive exercise or in disease. Although cells withstand oxidative
[email protected].
exposure by developing antioxidant defences, their resistance capacity could be
sometimes overcome, resulting in cell entering apoptosis. It is therefore important to
investigate mechanisms occurring in skeletal muscle cells in response to oxidative
stress. Among defence mechanisms, it has been suggested that glycosaminoglycans
(GAGs) could exert a protective effect. Therefore, we have explored how myoblasts
react in vitro to oxidative stress and whether treatment with a synthetic GAG mimetic
(namely OTR4120) would protect them against oxidative injury.
An oxidative stress induced by oxygen peroxide (H 202, 10pmol/cell, 30min) was
applied on proliferating myoblasts (C2.7 cell line). Cell survival was decreased by
about 70% within 24 hrs. Compared to untreated cells, oxidative stress induced
apoptosis assessed either by the presence of apoptotic features of nuclei stained with
diaminophenyl indol, or by the presence of subG1-DNA observed by Flow cytometry
or by an increase in caspases-3, 9 and 8 activities at 48hrs. This was accompanied by
a two times increase in the amount of total sulphated GAGs produced by stressed
cells compared to untreated cells, without change in the overall GAG composition.
However, as shown by immunocytology, these GAGs seemed to remain in the
intracellular compartment instead of being addressed to the cellular membrane.
Treatment with the GAG mimetic OTR4120 (1.0 µg/ml) added just after the oxidative
_0MS0G0OLK
stress period partially prevented such features associated with apoptosis, in part
restored GAG localisation at membranes and allowed cells to differentiate after 72hrs.
Possible mechanisms of action of OTR4120 are being further explored.
These data support the working hypothesis that GAG mimetics could protect
myoblasts from oxidative stress injury.
PW25-308
MELATONIN PREVENTS OXIDATIVE-STRESS MEDIATED MITOCHONDRIAL
PERMEABILITY TRANSITION AND DEATH VIA MAINTENANCE /
ENHANCEMENT OF REDUCED PYRIDINE NUCLEOTIDES AND GLUTATHIONE
IN MOUSE SKELETAL MUSCLE CELLS
HIBAOUI Y1, ROULET E1, RUEGG UT1
(1) Laboratory of Pharmacology, Geneva Lausanne School of Pharmaceutical
Sciences, University of Geneva, Quai Ernest Ansermet 30, CH-1211 Geneva 4,
SWITZERLAND.
Oxidative stress-induced mitochondrial dysfunction has been shown to play a
To contact the author::
crucial role in the pathogenesis of a wide range of diseases including muscle
youssef.hibaoui@pharm. disorders. Protecting mitochondrial function, therefore, is vital for cells to survive.
unige.ch.
In this study, we demonstrate that melatonin, the main secretory product of the pineal
gland, readily rescued mitochondria from oxidative stress-induced dysfunction and
effectively prevented subsequent apoptotic/necrotic events and death in C57BL/6J
myotubes. In particular, melatonin potently prevented myotube death induced by tertbutylhydroperoxide (t-BHP) in a concentration-dependent manner (10-4-10-6 M). This
protective effect was more potent than that of N-acetyl-L-cystein, a well known
antioxidant that increases cellular pools of free-radical scavengers. Moreover,
melatonin maintained plasma membrane integrity (t-BHP-induced membrane
blebbing) after t-BHP exposure and prevented t-BHP-induced fissions of the long
mitochondrial filaments and inhibited mitochondrial swelling that was clearly visible
after t-BHP treatment. To determine if the mitochondrial protection provided by
melatonin was due to the inhibition of the formation of reactive oxygen species (ROS),
intracellular ROS levels were measured using fluorescence imaging. Application of tBHP produced a rapid and significant increase in free-radical generation in myotubes.
This effect was concentration dependently prevented by pretreatment of the myotubes
with melatonin. Considering that t-BHP cytotoxicity was also prevented by cyclosporin
A, a mitochondrial permeability transition pore (mPTP) inhibitor, we investigated the
effect of melatonin on mPTP. Melatonin prevented t-BHP-induced mitochondrial
depolarization and protected the pyridine nucleotides and glutathione (two regulators
of mPTP opening under conditions of oxidative stress) against t-BHP-induced stress.
Using isolated mitochondria, we found that melatonin (10-8-10-6 M) desensitized the
mPTP to Ca2+ and prevented t-BHP-induced mitochondrial swelling, pyridine
nucleotides and glutathione oxidation, and enhanced mitochondrial function.
In conclusion, our findings suggest that inhibition of the mPTP may essentially
contribute to the protective effect of melatonin against oxidative stress in myotubes.
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PW25-309
''BENEFICIAL'' EFFECTS OF GINKGO BILOBA ON MOTOR ENDPLATES (NMJ),
NEUROMUSCULAR TRANSMISSION AND MUSCLE CONTRACTILITY:
THERAPEUTICAL APPROACH OF SARCOPENIA IN RAT.
KOENIG H1, BAUCHÉ S2, MOLGO J3, ROUCHE A2, DE LA PORTE S3, PIGNOL B4,
CHRISTEN Y5, HANTAÏ D2, KOENIG J2, KOENIG J6
(1) Institut Pierre et Marie Curie et CNRS UMR 7091,Hôpital Pitié, Paris, FRANCE.
(2) Institut Pierre et Marie Curie et INSERM U582, Hôpital Salpétrière, Paris,
FRANCE. (3) CNRS UPR 9040, NBCM, Institut A Fessard, Gif sur Yvette, FRANCE.
(4) IPSEN, Les Ulis, FRANCE. (5) Ipsen-Beaufour, Paris, FRANCE. (6) Université
Bordeaux 2, Bordeaux, FRANCE.
The aging process in mammals, including humans, is associated with a decline in
neuromuscular function and performance. The main aspect of this decline, known as
To contact the author::
sarcopenia, is the reduction in skeletal muscle mass, related to a loss of muscle
[email protected]. strength. As no pharmacological treatment was reported at present, we analyzed the
effects of a Ginkgo biloba extract (EGb 761, IPSEN), which exhibits biochemical and
pharmacological effects, mostly beneficial, in the nervous system, among other
tissues. Recently, it was also shown that EGb 761 modifies the expression of many
aged rat muscles genes (group of J.Mallet, CNRS UMR 7091). After a 2 months oral
treatment, the 2 years old rats showed that EGb 761 had “rejuvenating”effects on
aged muscles 1) the ratio muscle/rat weight increased, close to young rat values 2)
muscle creatine kinase content decreased as compared to control muscles 3) the
highly fragmented NMJ of aged control muscles recovered a more juvenile pattern 4)
as a consequence, the axonal sprouting was reduced 4) the number of terminal
Schwann cells doubled 5) the percentage of degenerating NMJ was decreased 6) in
connection with these adaptative morphological responses to the treatment, EGb761
increased the evoked quantal transmitter release (Ach) and 6) as a result, the muscle
contractile strength was enhanced. However, the muscle fibers diameter and the ratio
of type 1 vs type 2 fibers was not modified in the treated slow and fast muscles.
Similarities and differences between a slow-twitch muscle (Soleus) and a fast twitch
muscle (EDL) will be emphasized.
In conclusion, the muscles of rats treated with an herbal extract, Ginkgo biloba, show
several “rejuvenated” patterns of neuromuscular junctions and of the major functions
of muscle cell physiology, which could provide a pharmacological treatment of
sarcopenia in humans.
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PW25-310
To contact the author::
[email protected].
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RECOVERY OF SKELETAL MUSCLE MASS AND PHENOTYPE AFTER
EXTENSIVE MUSCLE INJURY IN RATS: NO POSITIVE EFFECTS OF CREATINE
SUPPLEMENTATION
KOULMANN N1, CRASSOUS B1, RICHARD-BULTEAU H1, DELDICQUE L2,
SERRURIER B1, PASDELOUP M1, FRANCAUX M2, BIGARD X1
(1) Département des facteurs humains, Centre de Recherches du Service de santé
des Armées, La Tronche, FRANCE. (2) Département d'éducation physique et de
réadaptation, Université catholique de Louvain, Louvain-la-Neuve, BELGIUM.
Recent studies have shown that creatine supplementation (Cr) may enhance muscle
functional capacity in patients with neuromuscular diseases, disuse atrophy or
muscular dystrophies. Because it has been shown in culture cells that the fusion of
myogenic satellite cells is largely enhanced while Cr is added to the culture medium
during the differentiation phase, we hypothesized that Cr-supplementation may have
beneficial effects during the early steps of regeneration following muscle injury, then
may accelerate the recovery of both muscle mass and phenotype.
Degeneration of left soleus muscle was induced by notexin injection in rats
supplemented or not with Cr both in powder food and drink. At days 1, 3, 7, 14, 21, 28,
35 and 42 after injury, we studied in regenerated compared with contralateral intact
muscles, muscle weight and protein levels of the Proliferator Cell Nuclear Antigen
(PCNA) as a marker of initial cell proliferation, and expression of MRFs as markers of
differentiation. We also studied the myosin heavy chain (MHC) profile and activities of
citrate synthase (CS) and lactate dehydrogenase isozymes (LDH), as markers of the
maturation of muscle phenotype.
Cr-supplementation allowed to progressively recover the creatine content
within regenerated muscles. However, we observed, without differences between Crtreated and non-treated rats, that: 1) regenerated muscles did not recover weight
values similar to intact muscles 42 days after injury; 2) PCNA and MRFs expression
strongly and early increased in regenerated muscles; 3) the MHC profile of
regenerated muscles was recovered 35 days after injury; 4) a full recovery of CS
activity was observed from day 14, while the specific H-LDH activity remained lower
than in intact muscles until 42 days.
In contrast with results from in vitro experiments, Cr-supplementation had no
beneficial effect in vivo on the time course of recovery of skeletal muscle mass and
phenotype after notexin-induced injury.
PW25-311
To contact the author::
jlachey@acceleronphar
ma.com.
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TREATMENT WITH A SOLUBLE ACTIVIN RECEPTOR TYPE IIB ATTENUATES
MUSCLE WEAKNESS IN YOUNG MDX MICE.
LACHEY J1, PULLEN A1, WONG V1, PEARSALL RS1, SEEHRA J1
(1) Acceleron Pharma, Cambridge, USA.
Dystrophin-deficient, or mdx, mice contain a mutation in the dystrophin gene and are
therefore the genetic homolog of Duchenne muscular dystrophy.
While the
consequences of dystrophin deficiency in mice are less severe than what is seen in
DMD patients, mdx mice exhibit a period of muscle degeneration that manifests as
muscle weakness before subsequent regeneration restores strength to wild-type
levels. Activin type llB receptors (ActRllB) are one of the receptors responsible for
TGF signaling and are capable of binding myostatin as well as other negative
muscle mass regulators in the TGF superfamily. Treatment with a soluble ActRllB
molecule blocks the ability of these ligands to signal and results in increased lean
tissue mass. Here we describe the ability of a soluble ActRIIB fusion protein (RAP031) to increase lean tissue mass and restore strength in young mdx mice. RAP-031
treatment significantly increases body weight in both C57BL/10 and mdx mice. NMR
scans revealed increased lean tissue gain accompanied the higher body weights.
RAP-031 treated C57BL/10 mice gained 35.2% and the RAP-031 treated mdx group
gained 48.3% more lean tissue mass than their respective control cohorts. Further,
the effect of RAP031 treatment on strength was assessed. Vehicle treated mdx mice
grip strength scores were 15.7% lower than the vehicle C57BL/10 cohort thereby
illustrating the muscle weakness associated with dystrophin deficiency. In contrast,
the RAP-031 mdx mice improved their grip strength compared to the mdx vehicle
group, and attained grip strength measurements which surpassed C57BL/10 vehicle
mice and reached the level of the RAP-031 C57BL/10 grip strength scores (vehicle
mdx: 0.140 ± 0.01 KgF; RAP-031 mdx: 0.199 ± 0.02 KgF; vehicle C57BL/10: 0.166 ±
0.03; RAP-031 C57BL/10: 0.205 ± 0.02 KgF). These data support the idea that
inhibition of ActRllB signaling could have important clinical applications in Duchenne
muscular dystrophy.
PW25-312
CREATINE AND L-ARGININE TREATMENTS IMPROVE SARCOPLASMIC
RETICULUM FUNCTION AND CONTRACTILE PERFORMANCE OF DIAPHRAGME
FROM MDX MICE.
LAFOUX A1, BERTRAND J2, GERVIER P1, HUCHET-CADIOU C1
(1) UMR CNRS 6204 "Biocatalyse, Biotechnologie et Biorégulation",
Nantes, FRANCE. (2) UMR 6187 "Institut de Physiologie et
Biologie Cellulaires", Poitiers, FRANCE.
In duchenne’s muscular dystrophy, that is characterized by a progressive skeletal
muscle fiber necrosis, the membrane-stabilizing protein dystrophin is missing, and this
To contact the author::
[email protected].
leads to altered total Ca2+ content in muscle fibers. This study investigated the
potential therapeutic benefits of pharmacological tools in reversing the Ca 2+
sequestration function of the sarcoplasmic reticulum and improving the functionnal
capacity of skeletal muscle of mdx mice. Creatine (CrM: 2,15 mg/mL/day) and Larginine (L-Arg: 3,75 mg/mL/day) were administred per os to male mdx mice (12
weeks) during 4 weeks. Then, the effectiveness of the treatment was investigated on
the contractile protein properties, the sarcoplasmic reticulum Ca 2+ uptake and the
expression of SERCA1 and calsequestrin in diaphragm muscle of mdx mice. The data
demonstrate that after treatments, the maximal Ca 2+ activated tension developped by
skinned fibers were increased approxymately by 30% (Tmax mdx= 46.8 ± 4.5 mN.mm2;
CrM= 60.3 ± 5.7 mN.mm-2; L-Arg= 61.0 ± 2.8 mN.mm-2). The study of sarcoplasmic
function, assessed in skinned fibers and vesicle preparations, shows that the Ca2+
uptake was improved in mdx diaphragm muscle after both pharmacological treatments
(Ca2+ uptake mdx= 20.3 ± 1.1 nmole Ca2+.s-1.mg-1; CrM= 22.1 ± 0.6 nmole Ca2+.s1.mg-1;
L-Arg= 26.4 ± 1.1 nmole Ca2+.s-1.mg-1). Furthemore, the overall force of the
musculature was tested in living animals using the grip strenght test, the wire test and
the rotarod. The results show that the locomotor activity was enhanced and that mdx
mice were more resistant to fatigue. During the grip test, the force developped by the
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four limbs was ameliorated in mdx mice after both treatments (Strength mdx= 4.0 ±
0.1 g.g-1; CrM= 4.2 ± 0.2 g.g-1; L-Arg= 4.7 ± 0.2 g.g-1). In conclusion, the data show
that creatine and L-arginine treatments significantly normalized many functionnal and
biochemical parameters by acting on events that are related to Ca 2+ homeostasis.
PW25-313
To contact the author::
olivier.dorchies@pharm.
unige.ch.
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TAMOXIFEN IMPROVES THE STRUCTURE AND THE FUNCTION OF SKELETAL
MUSCLE IN MDX MICE
DORCHIES O1, REUTENAUER J1, VUADENS O1, COMYN S1, RUEGG U1
(1) University of Geneva, Geneva, SWITZERLAND.
Data from the literature indicate that tamoxifen (Tam), a selective estrogen receptor
modulator (SERM) used in the treatment of certain types of breast cancers, displays
antioxidant, anti-apoptotic and anti-fibrotic properties and stabilises biological
membranes. We hypothetised that Tam might improve both the structure and the
function of dystrophic muscles. When applied to primary cultures of dystrophic
myotubes, Tam and 4-hydroxy-Tam (10-8 to 10-6M) were not toxic, reduced collagen
content slightly, and exhibited a limited antioxidant activity against hydrogen peroxide.
Mdx dystrophic mice, the most common model for Duchenne muscular dystrophy,
were treated for about 15 months following weaning with 0.01% tamoxifen mixed into
the food pellets (Tam intake approx. 10mg/kg/day). Tam-treated mdx mice showed a
significant decrease in body weight, likely due to the important reduction in white
adipose tissue compared to untreated mice. The mass of the triceps surae (composed
of soleus, plantaris and gastrocnemius muscles) was lowered to normal values, while
the EDL and the diaphragm showed significant hypertrophy compared with both
normal and untreated dystrophic animals. Using the horizontal wire test, the Tamtreated mdx performed as well as the normal mice and 3 times better than the
untreated mdx mice. Isometric muscle force of the triceps surae was recorded.
Remarkably, specific phasic and tetanic twitch tensions were increased by 100% and
70%, respectively, compared with untreated mdx, resulting in values higher than those
of normal animals. The rates of contraction and relaxation were much slower than in
the untreated mdx, and the force-frequency curve was significantly shifted to the left,
suggesting a marked change toward a slower phenotype. In the Tam-treated mdx, the
resistance to repetitive tetanisations was improved by 44% and creatine kinase levels
were lowered by 50% compared with the untreated mdx. On-going histological
analysis suggests a normalisation of the mean fiber diameter.
PW25-314
To contact the author::
[email protected].
BENEFICIAL EFFECT OF PENTADECAPEPTIDE BPC 157 ON DENERVATED
MUSCLE IN RATS
MIHOVIL I1, RADIC B1, BRCIC L2, BRCIC I2, DRMIC D1, VUKOJA I1, ILIC S1, BOBAN
BLAGAIC A1, SEIWERTH S2, SIKIRIC P1
(1) Department of Pharmacology, University of Zagreb Medical School, Zagreb,
CROATIA. (2) Institute of Pathology, University of Zagreb Medical School, Zagreb,
CROATIA.
AIM: Since it was previously reported that pentadecapeptide BPC 157, applied without
a carrier, improved the healing of transected quadriceps muscle, as well as muscle
healing in rats with muscle crush injury, we believe it could also be an effective
treatment for denervated muscles.
METHODS: The left opturatorius nerve of Wistar Albino rats was transected through
opturatorius canal. Animals received either 10 ug/kg of BPC 157
(GEPPPGKPADDAGLV, M.W. 1419, manufactured by Diagen, Ljubljana, Slovenia) or
5 ml/kg of 0.9% NaCl, intraperitoneally every day. Functional evaluation (muscle
function index and walking pattern) and physical examination of hind limbs were
performed every month. After 1 year animals were sacrificed, muscles gracilis were
isolated and weighted, followed by patohistological and morphometrical analyses
using ISSA software (Vamstec, Croatia)..
RESULTS: One year after m. gracilis denervation measurements showed difference in
muscle function index values, being statistically better in BPC 157 treated animals
(p<0.05), as well as higher muscle weight in the latter group (p<0,017 vs controls),
and without statistical difference of those parameters in comparison of BPC treated
animals and healthy animals. BPC 157 treated animals demonstrated practically no
difference in muscle fibers diameters compared to healthy animals, while control
group revealed significantly shorter diameter, being only 70% of healthy animals
values. Besides, cross sections of control muscles revealed many smaller muscle
fibers with centralized nuclei, while morphologically there were also no significant
difference between BPC 157 treated animals and healthy ones.
CONCLUSION: Pentadecapeptide BPC 157 prevented muscle atrophy and preserved
muscle function after denervation.
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PW25-315
MEDICAL FOOD IN MDX MICE: GENISTEIN AND FLAVOCOXID AMELIORATE
MUSCLE PATHOLOGY AND FUNCTION.
MESSINA S1, MAZZEO A1, BITTO A2, AGUENNOUZ M1, MIGLIORATO A1, DE
PASQUALE MG1, SQUADRITO F2, VITA G1
(1) Department of Neuroscience, Psychiatry and Anaesthesiology, University of
Messina, Messina, ITALY. (2) Department of Clinical and Experimental Medicine and
Pharmacology, University of Messina, Messina, ITALY.
Abstract
To contact the author::
[email protected].
Soy isoflavones have been reported to have antioxidant bioactivities, scavenging free
radicals and increasing antioxidant protein expression, and also to inhibit the
transcription factor NF-kB. We showed in previous studies that the inhibition of the
transcription factor NF-kB throught drugs with also antioxidant properties, have
beneficial effects in mdx mice. The drugs used are not available for clinical studies.
We tested whether genistein and flavocoxid, supplements with known antioxidant and
antinflammatory properties readily available for clinical use, could have a beneficial
effect on muscle function, morphology and biochemical pattern in mdx mice.
Five-week old mdx mice received for five weeks either genistein (2 mg/kg i.p. daily),
flavocoxid (5 mg/kg i.p. daily) or vehicle. Flavonoids treatment 1) increased forelimb
strength ( p<0.05) and strength normalized to weight (p<0.05) and decreased fatigue
(p<0.05); 2) reduced serum creatine-kinase levels (p<0.01); 3) increased GPX activity
and reduced markers of oxidative stress (p<0.05); 4) blunted NF-B DNA-binding
activity (p<0.05); 5) reduces muscle necrosis (p<0.01) and enhances regeneration
(p<0.05) with an augmented number of myogenin-positive satellite cells and
myonuclei, and of developmental myosin heavy chain-positive fibers.
Our results suggest that these flavonoids might have a beneficial effect on muscle
function and morphology in mdx mice. Further studies are needed to investigate the
biochemical substrates of such encouraging preliminary results taking into account
that these supplements could be easily introduced in the daily diet of patients with
DMD.
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PW25-316
IMIPRAMINE TREATMENT INCREASES THE MUSCLE STRENGTH OF MDX MICE
HUCHET-CADIOU C1, CARRE-PIERRAT M2, LAFOUX A1, TANNIOU G2, GERVIER
P1, FOUGEROUSSE F3, SEGALAT L2
(1) UMR CNRS 6204 Universite de Nantes, NANTES, FRANCE. (2) UMR CNRS
5534 Universite de Lyon, LYON, FRANCE. (3) Laboratoire d’Evaluation
Fonctionnelle, Departement Recherche et Developpement, Genethon, EVRY,
FRANCE.
Several bioactive molecules, including antidepressants, efficiently prevent
To contact the author::
[email protected].
muscle necrosis in dystrophin-deficient C. elegans. We tested the effect of the
antidepressant, Imipramine, on the muscle degeneration of mdx mice. The mdx 5cv
strain was preferred to the classical mdx Scsn strain because it has almost no
revertant fibers, and is easier to breed. Groups of 6-8 mice (sex matched) were fed
from 2 to 6 weeks of age with a diet containing 10 and 40 mg/kg/day of Imipramine,
and tested at 6 weeks. The analysis included both histological and functional assays.
The histology was performed on the extensor digitorum longus (EDL), a fast-twitch
muscle, and the diaphragm. Histological parameters (% centrally nucleated fibers,
fiber size variance, necrosis index) were improved 20-40% in treated versus nontreated animals. Treated animals also performed significantly better in the wire test
behavioral assay. Moreover, the analysis of mechanical properties showed that the
force developed by the whole EDL muscle in situ was 25% higher in treated versus
non-treated animals. Surprisingly, the force developed by the EDLs of Imipraminetreated mdx mice was also larger than that of wild-type mice. Finally, using Triton-
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skinned fibers, diaphragm muscle from treated mice exhibited 30% higher maximal
tension than mdx mice. Thus, Imipramine treatment of young mdx mice reduces the
first wave of muscle necrosis and improves muscle strength of fast-twitch muscles.
PW25-317
EFFECT OF ANDROGEN THERAPY ON SKELETAL MUSCLE IN THE AGED
MALE RAT MODEL (ANDROGEN DEPRIVATION)
HOURDÉ C1, VIGNAUD A1, JAGERSCHMIDT C2, BUTLER-BROWNE G1, FERRY A1
(1) INSERM U787, UPMC, Institut de myologie, paris, FRANCE. (2) galapagos,
romainville, FRANCE.
We have analysed the effect of physiological doses of androgens administrated after
To contact the author::
[email protected].
orchidectomy on the skeletal muscle of male rats, as well as the relationships between
muscle performance, hypertrophy as well as the Akt/mTOR signalling pathway which
is involved in the control of anabolic and catabolic muscle metabolism. We studied the
soleus muscles from intact adult rats (SHAM), orchidectomized rats treated during 3
months with vehicle (ORX), nandrolone (NAN) or dehydrotestosterone (DHT). Our
results indicate that NAN and DHT treatments maintained highly androgen dependent
tissues (levator ani muscle, seminal vesicle, prostate, bone) in a normal state after
orchidectomy. We found that there is a trend for absolute and relative (normalized to
muscle weight) maximal tetanic isometric forces and fatigue resistance to be reduced
by orchidectomy. Whereas absolute and relative maximal tetanic isometric forces
(respectively + 69% and + 49%) and fatigue resistance (+35%) in NAN rats were
increased when compared to ORX rats. In contrast, DHT treatment did not improve
muscle function. Interestingly, muscle weights, cross sectional area of muscle fibres,
relative number of muscle fibres expressing slow myosin heavy chain and citrate
synthase activity were identical in NAN, DHT, ORX and SHAM rats. Furthermore,
phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway,
Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were
also similar in NAN, DHT, ORX and SHAM rats. In conclusion, physiological doses of
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androgens are sufficient to prevent virtually all the effects of orchidectomy in rats.
Interestingly, their beneficial effects on muscle performance are not related to muscle
fibre growth or activation of Akt/mTOR signalling pathway in the context of androgen
deprivation.
PW25-318
RESTORATION OF THE CALCIUM SIGNALING IN MDX MOUSE DUODENUM
MYOCYTES BY GENTAMYCIN SULFATE TREATMENT AND DYSTROPHIN EXON
SKIPPING.
DABERTRAND F1, MACREZ N1, MIRONNEAU J2, HENAFF M1, MOREL JL1
(1) umr5228 CNRS université Bordeaux, talence, FRANCE. (2) retired, umr5017
CNRS université bordeaux, bordeaux, FRANCE.
To contact the author::
[email protected].
RESTORATION OF THE CALCIUM SIGNALING IN MDX MOUSE DUODENUM
MYOCYTES BY GENTAMYCIN SULFATE TREATMENT AND DYSTROPHIN EXON
SKIPPING.
We have previously described that the loss of dystrophin expression in
duodenum myocytes from mdx mouse induces the decrease of ryanodine receptor
subtype 2 (RYR2)-dependent calcium signalling. This effect is due to a lower level of
RYR2 expression in mdx duodenum myocytes [Morel et al., 2004]. Both
aminoglycoside treatment and dystrophin exon skipping are proposed as therapeutic
ways for the Duchenne muscular dystrophy. After 14 days of gentamycin treatment,
the activation of RYR2 by caffeine in mdx duodenum myocytes induced the same
calcium responses than those observed in wild-type mice and an increase of the
expression of RYR2. In addition, mdx mice were treated by antisense oligo-2’ OMethyl (O-Me) nucleotides directed against the dystrophin exon23 (asDYS) to induce
exon skipping and the expression of truncated dystrophin. The Cy5 labelling of asDYS
enables to follow its presence in duodenum myocytes. As observed in gentamycin
treated mdx mice, the treatment with asDYS was able to increase the RYR2
expression and restore the calcium signals encoded by RYR activation. These results
suggest that gentamycin as well as antisense strategy could be used to restore the
Ca2+ signalling in smooth muscle from mdx mice. Both of these therapeutic ways have
benefits and inconvenience but are possibly usable to restore the smooth muscle
function for patients affected by Duchenne muscular dystrophy.
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PW25-319
BENEFICIAL EFFECT OF MOLSIDOMINE ON MDX MICE MUSCLE MEMBRANE
EVIDENCED BY IN SITU MALDI PROFILING
BENABDELLAH F1, TAHALLAH N1, TOUBOUL D1, YU H2, BRUNELLE A1,
LAPRÉVOTE O1, DE LA PORTE S2
(1) Institut de Chimie des Substances Naturelles, Laboratoire de Spectrométrie de
Masse, CNRS - UPR 2301, Gif-sur-Yvette, FRANCE. (2) Institut de Neurobiologie
Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire,
CNRS -UPR 9040, Gif-sur-Yvette, FRANCE.
Duchenne muscular dystrophy (DMD) is the most common genetic disorder
characterized by the lack of dystrophin, a sub-sarcolemmal protein necessary for
To contact the author::
[email protected].
normal muscle functions. Among therapeutic approaches, the upregulation of utrophin
to replace defectious dystrophin is developed. Previous studies demonstrate an
increase of utrophin labelling, a decrease of necrotic surface, a diminution of creatine
kinase release in the serum, and an improvement of isometric strength as
consequences of improved NO production via the injection of L-arginine, the substrat
for NO synthase or of molsidomine, an NO donor commercialized as Corvasal®.
While histology indicates that molsidomine can reverse the degenerating process
induced in mdx mice, the mechanism of action on the muscle structure is not really
known. We examine the part of the phospholipid composition in membrane myofibers
by addressing the phospholipid intensity ratio PC34:2(PC C16:0/C18:2)/PC34:1(PC
C16:0/C18:1). This ratio has been demonstrated to be different in normal and
dystrophic mice models by in situ MALDI-MS profiling. This variation was recently
confirmed by cluster-TOF-SIMS (Time-of-flight Secondary Ion Mass Spectrometry)
imaging on human dystrophic tissue sections. On the other hand, cell cultures were
performed and the same intensity ratio inversion was shown during the differentiation
from myoblasts to myotubes.It was thus suggested that this ratio could be a marker of
the regenerating process of muscle cell lines. After the treatment of mdx mice with
molsidomine, we evidenced by MALDI-TOF MS profiling a restoration of a membrane
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lipid composition equivalent to those of wild-type mice. This restoration was
associated with an increase of the regeneration process in the mice. This indicates
that NO donors restore a normal membrane structure mainly via a regeneration
process although the eventuality of a simple compensation of the membrane structure
by incorporation of specific class of fatty acids cannot be excluded.
PW 26:
Gene therapy
of muscular dystrophy
PW26-320
UPGRADING U7SNRNA TO COMPLETE EFFICIENT RESCUE OF DYSTROPHIN
BY EXON-SKIPPING IN DMD PATIENTS
GOYENVALLE A1, BABBS A1, GARCIA L2, DAVIES KE1
(1) Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford,
UNITED-KINGDOM. (2) INSERM, S UMR 787, Groupe myologie, Paris, FRANCE.
To contact the author::
aurelie.goyenvalle@dpa
g.ox.ac.uk.
Most cases of Duchenne muscular dystrophy (DMD) are caused by
mutations that disrupt the dystrophin mRNA reading frame. In many cases, skipping of
a single exon could supposedly restore the reading frame, giving rise to a shorter but
still functional quasi-dystrophin protein. It has previously been proposed to use small
nuclear RNAs, especially U7snRNA, to shuttle antisense sequences designed to
mask key elements involved in the splicing of targeted exons.
Our present project focuses on the upgrading of U7snRNA to complete
rescue of dystrophin by exon-skipping in DMD patients. We indeed developed
bifunctional U7snRNAs carrying a complementary sequence to the targeted exon and
a free tail harbouring canonical binding sites for the heterogeneous nuclear
ribonucleoproteins A1/A2 (hnRNP) that are powerful splicing repressors. The
presence of this generic strong silencer tail could indeed circumvent the always tricky
and time-consuming specific optimization required for each new exon-target.
We first focused on the exon 51 of the dystrophin gene and therefore
designed new tailed U7snRNA constructs. Each construct has been inserted into
lentiviral vectors for in vitro analysis on myoblasts from DMD patients. After
transduction of these cells with lentiviral vectors encoding the tailed U7-ex51, we
confirmed the skipping of the exon 51 by nested RT-PCR and dystrophin restoration
by Western blot. By Comparison with their controls with a mutated tail, we could show
that the skipping efficiency of these constructs was due to the tail carrying silencer
motifs, therefore confirming its splicing repressor action, and not the annealing
sequence to the exon.
These very encouraging results on exon 51 provide evidence that
bifunctional U7snRNA can achieve efficient exon-skipping in myoblasts from DMD
patients. Since these tailed U7 could be very useful for the development of the exonskipping strategy for the other dystrophin exons, the confirmation of their efficiency
shown in this study offers very promising tools for clinical treatment of DMD.
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PW26-321
THE USE OF IMMORTALISED HUMAN FIBROBLASTS FROM A DMD PATIENT
TO TEST EXON SKIPPING IN VIVO
CHAOUCH S1, GOYENVALLE A1, MAMCHAOUI K1, ; BUTLER-BROWNE G1, DI
SANTO J2, TORRENTE Y3, MOULY V1, GARCIA L1, FURLING D1
(1) UMR S 787 Groupe Myologie INSERM/UPMC-ParisVI, Institut de Myologie, Paris,
FRANCE. (2) Cytokines and Lymphoid Development Unit, Immunology Department,
Institut Pasteur, Paris, FRANCE. (3) Fondazione IRCCS Ospedale Maggiore
Policlinico of Milan, Department of Neurological Sciences, Dino Ferrari Center,
University of Milan, Milan, ITALY.
We are now at a point in time where gene therapy is becoming a reality. However, in
order to validate these strategies, it is essential to have in vitro human cell culture
models. For the study of neuro-muscular diseases, the use of patient myoblasts is not
To contact the author::
always possible due to their drastically decreased proliferative capacity induced by the
[email protected]. repeated cycles of degeneration and regeneration. Therefore it is neccessary to
fr.
envisage new in vitro models. In the pioneering studies of Weintraub et al, it had been
shown that the forced expression of the myogenic transcription factor myoD was able
to convert fibroblasts into myogenic cells. Based on this, we have developed a
universal in vitro model from skin fibroblasts which have been immortalised using
hTERT and then converted into myoblasts by a lentivirus containing an inducible
myoD contruct. We have then used this model to validate a strategy for exon skipping
using fibroblasts isolated from a DMD patient. These fibroblasts were immortalized
and then transduced using an inducible myoD construct. We first confirmed the
expression of myoD in vitro and the potential of these cells to form differentiated
myotubes. These cells were then transfected with an U7 construct that promote exon
skipping in the mutated dystrophin transcript. In order to test if these cells could
reconstitue muscle fibres in vivo expressing human dystrophin, they were injected into
cryodamaged TA muscles of immunodeficient RAG-/- gammaC-/- C5-/- mice. Muscle
were analysed after 27 days of regeneration and fibres expressing human dystrophin
were observed. Therefore this cellular model provides us with an alternative model
system to test different therapeutic strategies for various neuromuscular diseases
when patient myoblasts are not available.
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PW26-322
SYSTEMIC DELIVERY OF ANTISENSE OLIGONUCLEOTIDES RESTORES
DYSTROPHIN EXPRESSION AND FUNCTIONALITY IN THE MDX MOUSE
VAN PUTTEN M1, DE WINTER C1, HEEMSKERK H1, DE KIMPE S2, VAN
DEUTEKOM J2, VAN OMMEN G-J1, AARTSMA-RUS A1
(1) Leiden University Medical Center, Leiden, THE NETHERLANDS. (2) Prosensa
BV, Leiden, THE NETHERLANDS.
To contact the author::
[email protected].
Duchenne Muscular Dystrophy (DMD) is a chromosome X-inherited disorder,
characterised by the absence of the protein dystrophin which plays a role in the
protection of muscle fibers against damage. Patients display progressive degeneration
of skeletal muscles leading to functional impairment and premature death.
One of the most promising therapeutic approaches for DMD involves the
restoration of the disrupted reading frame through exon skipping with antisense
oligonucleotides (AON). In cultured cells from patients and in the mdx mouse model
AON treatment resulted in the generation of dystrophin proteins similar to those seen
in Becker Muscular Dystrophy patients, who generally display a milder phenotype than
Duchenne patients.
Recently, we showed local restoration of dystrophin expression after
intramuscular injection of an exon 51 specific 2’-O-methyl phosphorothioate AON in
four Duchenne patients in a first-in-man clinical trial. For future clinical studies on full
body treatment we are currently optimizing systemic AON delivery. We here
compared different doses (25 mg/kg to 100 mg/kg) and treatment periods (4 to 8
weeks) with murine exon 23 specific AONs in mdx mice applying subcutaneous
administration.
The exon skip levels varied between specific muscle groups from 8% in the
diaphragm to 26% in the extensor muscles of the lower forelimb and up to 2% in the
heart, after twice weekly repeated injections with a 100 mg/kg AON dose during 8
weeks treatment. Dystrophin expression was observed in all samples analysed. This
was accompanied by trends of decreased CK and improved rota-rod running times of
treated mice compared to saline injected control mice. Markers for kidney and liver
function remained unaffected, indicating that repeated AON treatment was well
tolerated even when performed over longer time periods. Our results show that
systemic injections of 2’-O-methyl phosphorothioate AON lead to restoration of
dystrophin and an improved functionality without apparent toxicity.
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PW26-323
IN VIVO DELIVERY OF NAKED AND LIPID-COMPLEXED ANTISENSE OLIGOS IN
MDX MICE: EFFECTS ON SKELETAL AND CARDIAC MUSCLE
PIGOZZO S1, REPELE A1, REGGIANI C2, AUSONI S3, ZAGLIA T3, BARONI MD1,
AMBEGIA E4, MCLACHLAN I4, VITIELLO L1
(1) Department of Biology, University of Padova, Padova, ITALY. (2) Department of
Anatomy and Physiology, University of Padova, Padova, ITALY. (3) Department of
Biomedical Sciences, University of Padova, Padova, ITALY. (4) Protiva
Biotherapeutics Inc., Burnaby, CANADA.
To contact the author::
[email protected].
Antisense-mediated exon skipping holds great potential for the treatment of DMD. In
mdx mice, functional recovery of skeletal muscle has been reported upon systemic
delivery of “naked” oligonucleotides or viral vectors encoding for antisense snRNAs.
However, only one study achieved dystrophin restoration in cardiac muscle (using an
adeno-associated vector). Here we report the in vivo delivery of morpholino oligos in
aged mdx mice, both in skeletal muscle, via intra-arterial injection, and in cardiac
muscle, via intramuscular injection. Intra-arterial delivery yielded levels of dystrophin
restoration comparable to those reported in the literature with the intra-venous
approach, but with smaller amounts of oligonucleotides. Intra-cardiac injections, on
the other hand, showed that the level and duration of the skipping effect found in
cardiac muscle were greatly decreased compared to skeletal muscle. This latter
finding provides the first direct evidence that antisense-mediated dystrophin
restoration in cardiac muscle might suffers from limitations that do not exist in skeletal
muscle.
All data published so far have indicated that systemic delivery via the vasculature
requires large amount of naked oligos to achieve therapeutically significant results.
Here we also report that the use of lipid carriers has the potential to greatly improve
the delivery efficiency; in particular, we found that the use of lipid-encapsulated oligo
RNA allowed to detect dystrophin re-expression with a single dose of ~40 µg of oligos
per adult mdx mouse. Importantly, dystrophin restoration could be seen not only in
skeletal and but also (albeit to a smaller extent) in cardiac muscle.
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PW26-324
NON-VIRAL VECTORS BASED ON AMPHIPHILIC POLYMERS: PARAMETERS
INFLUENCING THEIR IN VIVO EFFICIENCY
ROQUES C1, BOUCHEMAL K2, SALMON A1, FATTAL E2, FROMES Y1
(1) Institut de Myologie - INSERM U582, Paris, FRANCE. (2) UMR CNRS 8612 Université Paris 11, Châtenay-Malabry, FRANCE.
To contact the author::
[email protected].
Gene transfer is an interesting therapeutic approach for inherited muscular
dystrophies with no curative treatment currently available. Nevertheless, DNA does
not freely cross the membranes as it is a hydrophilic, negatively charge
macromolecule, rendering thus formulation of nucleic acids a major concern. Synthetic
vectors appear as a promising approach and particularly polymer based formulations.
Among those, Polyethyleneimine (PEI), a polymer exhibiting a high density of positive
charges and amphiphilic Tetronic 304 and Pluronic L64, displaying few or no charges,
seem of particular interest to transfer DNA. Our work has focused on determining the
influence of several formulation parameters on the organization of polymer/DNA
systems. We have studied the correlation between these modifications and the toxicity
and efficiency of the systems in vivo.
Complexation of PEI with DNA is leading to small nanoparticles displaying relatively
strong interactions. After in vivo administration, PEI/DNA complexes exhibited a high
toxicity towards skeletal muscle. Amphiphilic polymers associated to DNA are
generating more complex systems displaying weaker interactions. In vivo, no lesions
were detected with amphiphilic polymers based formulations. Moreover, these
formulations allowed significant improvement of gene transfer to the skeletal muscle
with reference to naked DNA, even at low DNA doses. Our experiments highlighted
the role of the medium and the temperature to optimize in vivo efficiency of Pluronic
L64 vectors. Moreover, these findings could be correlated to modifications of the
supramolecular organization of these systems depending on the conditions utilized.
Our studies have emphasized the interest of amphiphilic polymers displaying few or
no charges to transfer DNA in the skeletal muscle. The supramolecular organization of
Pluronic L64 based formulations, as well as the interactions between polymer and
DNA, is strongly dependent on the temperature and the medium used. These
modifications have a direct impact on the in vivo efficiency of such vectors.
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PW26-325
CLINICAL DEVELOPMENT OF A GENE THERAPY PRODUCT FOR THE
TREATMENT OF LIMB GIRDLE MUSCULAR DYSTROPHY TYPE 2C
HADDAD H1, RIGOLET A2, CAIZERGUES D1, DOUAR AM1, MILLOT L1, LAMBERT
I1, LIABEUF M1, LAFORÊT P2, LETURCQ F3, VOIT T4, BÉHIN A2, EYMARD B2,
CARLIER P4, BENVENISTE O2, LEMOINE F2, HOGREL JY4, ROSIER-MONTUS M1,
DENÈFLE P1, HERSON S2, MASQUELIER AM1
(1) Généthon, Evry, FRANCE. (2) Hôpital Pitié-Salpêtrière, Paris, FRANCE. (3)
Hôpital Cochin, Paris, FRANCE. (4) Institut de Myologie, Paris, FRANCE.
In late November 2006, GENETHON initiated its first gene therapy clinical trial in
gamma-sarcoglycanopathy (LGMD2C), a rare autosomal recessive muscular disorder
To contact the author::
[email protected].
caused by mutations in the gamma-sarcoglycan gene. Patients commonly present
with proximal and progressive muscular weakness before the age of 10 and loose
ambulance by age 12 on average. Cardiomyopathy and respiratory insufficiency may
develop during the course of the disease, leading to poor prognosis and premature
death.
Earlier during the clinical development, GENETHON had obtained an Orphan Drug
Designation by EMEA. Approval from the French Agencies was obtained in November
2006 to initiate the phase I/IIa trial, which is being held at Pitié–Salpêtrière Hospital in
Paris. The investigational product, a serotype 1 adeno-associated virus (AAV1) vector
harboring the human gamma-sarcoglycan gene, is administered by a single
intramuscular injection into the carpi radialis muscle.
The primary objective of this trial is to evaluate the clinical safety of local intramuscular
injection of the gene therapy product. Secondary objectives are to monitor local and
systemic immune responses, assess histological modifications and gene transfer into
injected muscles. 9 patients, aged above 15, will be enrolled sequentially in the study,
assigned to 3 cohorts with a single dose-escalation and followed-up for 6 months.
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Evaluation will address clinical, histological, biological, immunological and functional
parameters as well as MRI.
As of today, four patients have been treated. The first cohort has been completed and
the first patient of the second cohort has been treated. Encouraging data will be
presented on the status of the product activity, immunologic response and patient
compliance to the treatment.
As the clinical trial is halfway, a next phase IIb clinical trial is being planned, in which
systemic administration will be performed. This implies important preclinical and
clinical points to consider, as well as a series of preclinical studies to achieve.
PW26-326
PARTIAL FUNCTIONALITY OF A MINI-DYSFERLIN MOLECULE IDENTIFIED IN A
PATIENT AFFECTED WITH MODERATELY SEVERE PRIMARY
DYSFERLINOPATHY
KRAHN M1, WEIN N2, LOSTAL W 1, BOURG-ALIBERT N1, NGUYEN K2, COURRIER
S3, VIAL C4, LABELLE V2, DE PETRIS D2, BORGES A2, MATTEI MG2, ROUDAUT C1,
MIYAKE K5, MCNEIL P5, CAU P2, LETURCQ F6, BARTOLI M1, LEVY N2, RICHARD I1
(1) Genethon, CNRS FRE 3087, Evry, FRANCE. (2) AP-HM, Departement de
Genetique Medicale, Hopital d'enfants de la Timone and INSERM U910, Equipe:,
Marseille, FRANCE. (3) Centre d'Enseignement et de Recherche en Genetique
medicale, Faculte de Medecine de Marseille, Marseille, FRANCE. (4) Hopital
neurologique Pierre Wertheimer, Lyon, FRANCE. (5) Medical College of Georgia,
Augusta, USA. (6) Laboratoire de Biochimie Genetique, Hopital Cochin, Paris,
FRANCE.
To contact the author::
[email protected].
Primary dysferlinopathies are a heterogeneous group of autosomal recessive
muscular dystrophies, caused by mutations in the large-sized DYSF gene encoding
dysferlin. Dysfunction of dysferlin causes deficient sarcolemmal repair and leads to
muscle fiber degeneration.
In a patient born from a distantly consanguineous union, presenting with
moderately severe primary dysferlinopathy, we identified a homozygous multi-exonic
internal deletion using genomic, transcriptional and F.I.S.H. analyses. Currently, this
patient presents with proximo-distal weakness, but is still ambulant without a cane at
41 years.
Bioinformatic analysis of putative Open-Reading-Frames within the resulting,
deleted transcript predicts a possible translation into a truncated protein maintaining
two C2 domains and the transmembrane domain. While muscle biopsy samples were
not available, dysferlin protein analyses on monocytes obtained from the patient
evidenced expression of a truncated molecule at the expected size, which localizes to
the plasma membrane. Ectopic expression of the predicted Open-Reading-Frame
using transfection into fibroblasts confirmed targeting to the plasma membrane.
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To further evaluate the functionality of this Mini-dysferlin, we constructed an
AAV-vector containing the predicted Open-Reading-Frame identified in the patient
under a muscle specific promoter. Intra-muscular injection of the Mini-DYSF-AAV
vector in wild-type and dysferlin-deficient mice led to high-level expression of the
truncated protein. Using a membrane repair assay, based on membrane wounding of
transduced isolated muscle fibers with a two-photon laser-scanning microscope, we
demonstrated that the Mini-dysferlin efficiently reseals the damaged sarcolemma.
Therefore, this Mini-dysferlin is at least partially functional. Importantly, this
demonstrates the modularity of dysferlin.
In Duchenne muscular dystrophy, the identification of functional Minidystrophins led to the development of therapeutic strategies such as gene transfer of
Mini-dystrophin and exon-skipping approaches. Our findings constitute a prerequisite
for similar therapeutic strategies for a subset of primary dysferlinopathies. (MK and
NW; NL and IR contributed equally to this work)
PW26-327
CORRECTION OF MUSCLE FUNCTION IN MYOTUBULAR MYOPATHY BY AAVMEDIATED MTM1 REPLACEMENT
BUJ BELLO A1, FOUGEROUSSE F2, JAMET T1, DURAND M2, KRETZ C1, DANOS
O2, DOUAR AM2, MONTUS M2, DENÈFLE P2, MANDEL JL1
(1) IGBMC, INSERM U596, CNRS UMR7104, ULP, Collège de France, Illkirch,
FRANCE. (2) Généthon, Evry, FRANCE.
To contact the author::
[email protected].
Myotubular myopathy (XLMTM) is a severe congenital muscular
disease due to mutations in the myotubularin gene (MTM1) and
characterized by the presence of small non-regenerative
myofibres with frequent occurence of internalized nuclei. No
specific treatment exists to date. Recombinant adeno-associated
virus (rAAV) vectors appear as one of the most promising tools
for gene therapy of muscular disorders. We have constructed
rAAV vectors expressing myotubularin under either a ubiquitous
(CMV) or a muscle-specific (desmin) promoter in order test their
therapeutic potential in a faithful XLMTM mouse model. We
show that a single intramuscular injection of either of these
vectors in symptomatic Mtm1-deficient mice rescues the
pathological phenotype. Myotubularin replacement corrects
nuclei and mitochondria positioning in myofibres and leads to a
strong increase in muscle volume. Importantly, the contractile
force of mutant muscle becomes comparable to that of wild-type
animals 4 weeks after rAAV transduction. This study provides a
proof of principle that viral-mediated Mtm1 gene delivery may be
an effective therapeutic approach for patients with myotubular
myopathy.
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PW26-328
NO NEUROMUSCULAR DISEASE CLINICAL TRIAL WITHOUT GMP
PHARMACEUTICAL CLINICAL LOT PRODUCTION.
NOGUIEZ-HELLIN P1
(1) GENETHON-ETGC, EVRY, FRANCE.
To contact the author::
[email protected].
One way to design drugs for neuromuscular diseases goes through gene therapy
products from Biodrugs list. Regarding the regulatory legislation, gene therapy
products are considered as pharmaceutical products and their manufacturing requires
compliance with Good Manufacturing Practices (GMP).
Manufacturing processes complexity in addition with genetically modified organisms
manipulation entail for biodrugs production a pharmaceutical structure as well as a
know-how that Genethon has implemented more than three years ago.
This structure named ETGC has obtained a GMP certification. It has been inspected
by Afssaps and has received the authorisation to produce gene therapy vectors for
phase I and II clinical trials. We are able to produce AAV, HIV and MLV vectors up to
50 litres scale. Our production capacity is 6 lots per year and per production suite.
From the 2200 m2 facility surface, approximately 800m2 are dedicated to clean rooms
where clinical lots are produced. The other rooms are used for raw materials and
media preparation, storage, quality control laboratory, quality-assurance and
administration areas.
Facility qualification including materials and clean rooms must be done and annually
validated, according to Q10 ICH guidelines.
Production processes development including upstream and downstream process,
technological transfers to pharmaceutical GMP norms, aseptic filling validation, in
process and final products controls are as many necessary steps that must be
validated. .
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Today Genethon has the expertise to produce, control and aseptic fill gene therapy
products including AAV, HIV and MLV vectors.
We have produced different AAV vectors clinical lots which are already, or about to
be, used in neuromuscular disease clinical trials (for example gammasarcoglycanopathy phase 1 trial or polymiositis disease).
Our capacity today is sufficient to meet the local intramuscular phase 1 clinical trial
approach, but as we already have assessed, the systemic treatment for
neuromuscular disease will require huge amounts of GMP products. We already have
started, in collaboration with the bioprocess development department of Genethon,
the industrial vectors production scale, for GMP vector production up to 300 litres.
PW26-329
GENE THERAPY PRODUCTS MANUFACTURING: FRAME AND LIMITATION OF
VIRAL CLEANING AND DECONTAMINATION
PAUTREL I1, CRAPIE C1, JACQUES D1, SEQUESTRA N1, NOGUIEZ-HELLIN P1
(1) GENETHON-ETGC, EVRY, FRANCE.
To contact the author::
[email protected].
Généthon’s ETGC is working in 5 production rooms - dispatched on two sites - among
which 3 are dedicated to viral vectors production for development of therapeutic
products for phase I and II clinical trials. These productions are performed in
compliance with GMP standards.
One of the standards applies to cross contaminations control during manufacturing
and recommends production in separate zones, implementation of air locks and air
extraction device, protective clothing, use of manufacturing closed system, checking
for absence of residues and cleaning as well as decontamination processes known for
their efficacy.
To prove the efficacy of cleaning or decontamination, attachment 15 of GMP
standards recommends validation of disinfectants efficacy on facilities and equipments
used at all different production steps.
At ETGC, cross contamination control concerns viral vectors, like lentivirus and rAAV
vector, produced in GMP zones. Validation of disinfectants virucidal activity must be
validated in this context. According to NF72-180 norm, a virucidal activity is defined as
follows: “a disinfectant virucidal activity must allow a decrease of at least 10 4 times the
number of infectious units”.
Validation is performed on surface disinfection (liquid-liquid validation) and facilities air
disinfection. Validation first step consists in selecting one or several marketed
disinfectants with virucidal activity beyond the 4 logs recommended by norm.
Among all marketed disinfectants, for liquid-liquid validation, 10 products have been
tested and only 2 have a virucidal activity decreasing lentivirus and rAAV vector by 4
logs.
For air disinfection, 10 products have been tested and only 2 may be efficient enough
to be selected further to validation steps.
Disinfection validations have shown a strong resistance of AAV vector to disinfectants
(surface and air) usually used in laboratories. One of the most efficient disinfectants
still is Javel water at 2,6% on a period of 30 minutes
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PW26-330
GMP GENE THERAPY VECTORS MANUFACTURING
FAUCHILLE S1, ADIN P1, BARNAY-TOUTAIN F1, BURIE C1, DUFOUR D1, HALBOUT
C1, LE PROVOST G1, MALEAU G1, SANDER B1, NOGUIEZ-HELLIN P1
(1) GENETHON-ETGC, EVRY, FRANCE.
Genethon has been provided with GMP manufacturing facilities of about 600 m 2 for
the production of viral vectors for human gene therapy trials. All steps are performed
To contact the author::
[email protected].
on the site: plasmid production, cell banking, cell culture, purification and final filling.
Genethon can assume a complete process for AAV, HIV and MLV viral vectors.
Cell culture can be performed in flasks (up to 24 CF 10 cell factories) or bioreactors
(up to 10 liters volume). The viral vectors are purified by ion exchange or affinity
chromatographic steps. The batch size, depending on the process, is from 10 13 to
1014 total physical particles. The process covers 3 to 5 weeks.
The production site is operational since 2005. Genethon is producing clinical batches
for four inner projects but also for outer customers, depending on the available
production slots. Genethon’s facilities are also designed for feasibility studies in pilot
laboratories before GMP production.
The production is performed on two independent sites: one is L2 classified and the
other one L3.
In 2010, new facilities will be built for Genethon and designed for large scale
production and will be equipped with 300 liters bioreactors and 4 independent
production suites. It will be located in Evry.
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PW26-331
STRATEGY OF CONTROLS FOR GENE THERAPY CLINICAL BATCHES
DUGUE C1, AMMOUR M1, ANTONELLI D1, BASTIANI C1, BLIN J1, CUENOT P1,
DUPUIS C1, NOGUIEZ-HELLIN P1
(1) GENETHON-ETGC, EVRY, FRANCE.
To contact the author::
[email protected].
Généthon’s aim is to find medicine for rare and heavy neuromuscular or
immunological diseases. In the ‘90s, Généthon’s adopted the Gene Therapy to find
solutions for these diseases.
In this purpose, the E.T.G.C. from Généthon (Cell and Gene Therapy Company) was
created in 2005 for the production and control of viral vector batches from AAV, HIV
or MLV. These batches are dedicated to Phase 1 for human clinical trials. The
AFSSAPS (French regulatory for Health products authorisation) accredited the ETGC
to produce these batches following restrictive quality rules.
In consequence, each step of the production is controlled:
Raw material involved into the production process was elected in regards to quality
standards (respect to European Pharmacopeia for example), its traceability, and
agreement of each subcontractor to a quality charter written by Généthon. Each
subcontractor is then audited every two years to verify the Charter regulation
engagements. Raw material is also controlled a second time at the E.T.G.C. by
operators. These operators are qualified to work according to the GMPs
recommendations: Identity testing is then performed on chemical raw materials
whereas sterility, endotoxin and viability on culture media.
In process control consisting in Physical and Infectious Particles quantification is done
to calculate the yield of the production.
On the final product, at least 15 tests are performed: Identity testing (sequencing, gel
migration), purity (SDS-Page, residual or total proteins quantification) and functionality
testing are realised in the QC team. Security viral testing (as human adventitious
viruses, viral contaminants, replicative viruses, infectious genomes quantification…)
are however done by qualified subcontractors.
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PW26-332
EFFICIENT IN VIVO DYSFERLIN EXPRESSION BY DUAL AAV VECTORS
RECONSTRUCTION.
LOSTAL W 1, BOURG N1, ROUDAUT C1, MIYAKE K2, MCNEIL P2, BARTOLI M1,
RICHARD I1
(1) GENETHON, EVRY, FRANCE. (2) Medical College of Georgia, Augusta, USA.
To contact the author::
[email protected].
Dysferlinopathies are recessive muscular disorders caused by defects in dysferlin.
Genetic mutations are responsible for two major phenotypes: Limb Girdle Muscular
Dystrophy type 2B and Distal Miyoshi Myopathy. These skeletal muscle diseases are
characterized by progressive loss of muscle integrity and strength. Recently, dysferlin
was demonstrated to be involved in membrane repair process, providing a preliminary
understanding of the pathophysiological mechanism in these diseases.
Currently, no treatment is available. Considering the recessive nature of
dysferlinopathies, a possible therapeutic strategy is gene transfer. To date, the best
vector for gene transfer in muscle is Adeno Associated Virus (AAV). However, the
human dysferlin cDNA size approximates 7 kb, preventing its direct incorporation into
a single AAV vector, since the encapsidation limit is around 4.7 kb. In order to bypass
this limitation, we set up a strategy taking advantage of the concatemerization ability
of AAV vectors. The dysferlin cDNA was separated into two AAV2/1 vectors, one
carrying a muscle specific promoter followed by the 5’ half of dysferlin cDNA and a 5’
splicing signal and another one carrying a 3’ splicing signal, the remaining dysferlin
sequence and a polyadenylation signal. We demonstrated the ability of these vectors
to produce a human full-length dysferlin mRNA in a cellular model. To test this
approach in vivo, intramuscular injection of both vectors was performed into dysferlin
deficient mice. Dysferlin mRNA was correctly spliced, expressed at a level close to the
endogenous one and was stable for an entire year. At the protein level, dysferlin was
detected at 237 kDa, the size of the expected full-length protein. The functionality of
dysferlin was demonstrated by a membrane repair assay based on 2-photon injury on
isolated muscle fibers. Taken together, all these data prove that gene transfer based
on AAV concatemerization allows the expression of a full-length functional dysferlin.
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PW26-333
CHARACTERIZATION OF THE IMMUNE RESPONSES TO AAV2/1 VECTORS
FOLLOWING INTRAVENOUS INJECTION IN MICE.
SUDRES M1, GALY A1
(1) Immunology and Gene Therapy Group - INSERM U790- Genethon, Evry,
FRANCE.
Clinical gene therapy studies have demonstrated the immunogenicity of AAV vectors
since neutralizing antibodies and cytolytic T-cell responses have been elicited against
To contact the author::
[email protected].
the capsid, in some patients. For further clinical development, the immunogenicity of
AAV vectors should be better understood in preclinical models. We are interested in
rAAV2/1 vectors which are promising tools for gene transfer into skeletal muscle. We
evaluated the innate and adaptive immune responses following a bolus intravenous
(IV) administration in mice. Innate immune responses triggered by AAV vectors are
thought to be weak, yet hepatic inflammatory reactions were described in mice
following IV administration (Zaiss et al., J. Virol 2002). In our hands, the IV injection of
1011 vg endotoxin-free AAV2/1 vector did not trigger a detectable cytokine response.
On the contrary, research-grade AAV2/1 preparations induced rapid but transient
increases in TNFa, IL6, CCL5 and CXCL10 mRNAs, presumably an effect of the LPS
contained in these preparations. Endotoxin-free AAV2/1 vectors triggered strong and
neutralizing humoral responses initiated with IgM followed by IgG conversion peaking
respectively at 1 and 2.5 weeks. A dose-dependent increase in IgG2a and IgG3 levels
was observed with increasing amounts (1010, 1011 or 1012 vg) of vector whereas IgG2b
were high at all doses and little IgG1 was detected. We found no evidence of CD4 + or
CD8+ T-cell priming based on proliferation or activation markers.
In conclusion, a bolus IV administration of rAAV2/1 vector in mice induces a strong
humoral immune response against the capsid but little to no inflammation or cellular
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immunity. However, antibody isotype profiles suggest an underlying IFNg or TGFbmediated T cell response. Our current prospects are (i) to further characterize the
cellular immune response to the AAV1 capsid using reporter epitopes (ii) to assess
the pro-inflammatory potential of AAV1 vector through activation of TLRs or others
pathway of inflammasome.
PW 27:
Cell therapies – Myoblasts
PW27-334
To contact the author::
thomas.laumonier@med
ecine.unige.ch.
IN VIVO BIOLUMINESCENCE IMAGING OF TRANSPLANTED HUMAN
MYOBLASTS USING RENILLA-LUCIFERASE TRANSGENE EXPRESSION
LAUMONIER T1, KONIG S2, BADER C2, BERNHEIM L2, HOFFMEYER P1,
MENETREY J1
(1) Department of Orthopaedic Surgery, University Hospital of Geneva, Geneva,
SWITZERLAND. (2) Department of Basic Neurosciences, University Medical Center,
Geneva, SWITZERLAND.
Cellular therapies for Duchenne muscular dystrophy and other muscle diseases are
limited by a massive early cell death following injections. The quantification of cell
survival is an essential step to evaluate the efficiency of myoblast transplantation. In
the present study, we describe an approach for in vivo follow-up of human myoblast
grafts transplanted in mice using non-invasive imaging techniques (bioluminescence).
Human myoblast clones, each derived from single post-natal human satellite cell,
were transduced with a lentivirus containing the renilla-luciferase (Rluc) gene under a
HSV-TK promoter. Reporter gene expression had no adverse effects on myoblast
viability, proliferation, or differentiation in vitro. A robust correlation was found between
Rluc signals and cell numbers by ex vivo imaging analysis (R 2 = 0.98) and by in vitro
enzyme assay (R2 = 0.93). Similar correlations were observed between Rluc signals
and the number of infectious virus particles per cell (multiplicity of infection).
Afterward, up to 1 x 10(6) Rluc or control myoblasts were injected into the tibialis
anterior (TA) muscle of mice. Bioluminescence imaging 6 hours after transplantation
showed a correlation between Rluc signals and the number of cells injected, while
only background level of bioluminescence was observed within control cells. These
results demonstrate that optical imaging of human myoblasts containing renillaluciferase reporter gene can be used for the rapid and accurate evaluation of cell
survival post transplantation. With further development, this approach will help to
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improve myoblast transplantation biology, notably by facilitating the screening for
therapeutic agents acting on grafts.
PW27-335
HEAT SHOCK TREATMENT INCREASES ENGRAFTMENT OF HUMAN
TRANSPLANTED MYOBLASTS INTO IMMUNODEFICIENT MICE
BENCZE M1, RIEDERER I1, NEGRONI E1, BIGOT A1, DI SANTO JP2, BUTLERBROWNE GS1, MOULY V1
(1) UMR S 787 - Groupe Myologie-Inserm / UPMC / Institut de Myologie-105, bld de
l'hôpital., Paris, FRANCE. (2) Unité des Cytokines et Développement Lymphoïde,
Département d'Immunologie, Institut Pasteur., Paris, FRANCE.
One of the phenomena known to limit the success of myoblast transfer therapy (MTT)
To contact the author::
is the massive and early cell death observed during the first few hours after cell
[email protected] injection. A major part of injected cells die by necrosis and apoptosis, thus decreasing
.
the number of muscular progenitors that could be involved in fusion events during
muscle regeneration. In the mouse it has already been shown that a short incubation
of myoblasts at a high temperature (42°C/107°F) increases their resistance to death
in vivo and in vitro.
Our objective was to determine if heat shock (HS) treatment can increase the survival
of human myoblasts leading to a better participation of the injected cells in muscle
regeneration, and to quantify this potential improvement. We show in vitro that HS
treatment of human myoblasts doesn’t change significantly their proliferation, but does
increase their resistance to staurosporin induced apoptosis, as compared to untreated
cells. Control or HS treated human myoblasts were also injected in vivo into Tibialis
anterior muscles of immuno-deficient RAG-/- C-/- C5-/- mice. We have found that, 24
hours post-injection, the number of HS cells in proliferation was the same as that of
control cells, while the number of apoptotic cells was significally lower in muscles
transplanted with heat shocked myoblasts. The contribution of human myoblasts to
host regeneration was assessed using species specific antibodies, and a dramatically
increase in the number of human fibres was observed in muscles injected with HS
treated cells, as compared to untreated cells. These results suggest that heat shock
treatment of human myoblasts promotes a significant protection against apoptotic cell
death and improves muscle regeneration in vivo. This treatment could be applied to
clinical trials involving intra-muscular cell therapy.
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PW27-336
To contact the author::
[email protected].
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OPTIMIZATION OF HUMAN MYOBLAST TRANSPLANTATION USING THE RAG
IMMUNODEFICIENT MOUSE MODEL
RIEDERER I1, NEGRONI E2, BENCZE M2, WOLFF A2, DI SANTO JP4, SAVINO W 3,
BUTLER-BROWNE GS2, SILVA-BARBOSA S3, MOULY V2
(1) UMRS787 – Groupe Myologie; Inserm / UPMC-ParisVI; Institut de Myologie Oswaldo Cruz Foundation, Department of Immunology, Laboratory on Thymus
Research, Paris/Rio de Janeiro, FRANCE. (2) UMRS787 – Groupe Myologie; Inserm
/ UPMC-ParisVI; Institut de Myologie, Paris, FRANCE. (3) Oswaldo Cruz Foundation,
Department of Immunology, Laboratory on Thymus Research, Rio de Janeiro,
BRAZIL. (4) Institut Pasteur – Departement d\' Immunologie, Unité des Cytokines et
Développement Lymphoïde, Paris, FRANCE.
Myoblast transplant therapy (MTT) can be envisioned as a clinical alternative in the
treatment of certain diseases. The problems that still remain to be solved to improve
the efficiency of MTT are the massive early cell death, the limited proliferation and the
inefficient migration of these cells. Recently, we showed that immunodeficient mice
such as RAG-/-gammaC-/-C5-/- and RAG-/-gammaC-/- are both attractive and
efficient systems to support engraftment of human myoblasts. We have used this
model to study early events which occur following human myoblast transplantation.
5x105 myoblasts were injected into Tibialis Anterior (TA) muscles. Mice were
sacrificed after 0h, 1h, 3h, 6h, 24h, 12h, 3 days, 5 days and TA muscles were
analyzed by immunofluorescence. Human cells were detected with a human specific
lamin A/C antibody. We observed a progressive decrease in the percentage of
proliferating human myoblasts from 0h to 5 days pos-transplantation. A peak in cell
death was observed at 12 and 24h whereas by 5 days there was virtually no cell
death. After cell injection we observed cell dispersion, but this was only clearly
detected after 24h. Cell dispersion following transplantation was correlated with a
deposition of laminin within the pocket of human cells that peaked between 12h and
24h, a time at which there is a huge macrophage infiltration in the area of injected
cells. However, despite a significant increase in the area occupied by the injected cells
between 3 and 5 days, this area corresponded to only a portion of the total muscle.
Interestingly, markers of differentiation were expressed by the human myoblasts after
24h, indicating an “early” differentiation of human cells following transplantation. A
better knowledge of the mechanisms involved in survival, proliferation migration and
differentiation of transplanted human myoblasts, will certainly be important to design
new strategies for cell mediated therapy.
PW27-337
IN VITRO ARRESTED MUSCLE CELLS AS MODELS FOR THE IDENTIFICATION
OF GENES INVOLVED IN THE QUIESCENCE OF SKELETAL MUSCLE
SATELLITE CELLS
KANDALLA P1, JACQUEMIN V1, REIDERER I1, BUTLER-BROWNE G1, MOULY V1
(1) UMRS 787- Groupe Myologie; Inserm / UPMC-Paris VI; Institut de Myologie,
Paris, FRANCE.
Satellite cells, the post-natal muscle progenitors, are responsible for the regenerative
capacity of skeletal muscle. They remain quiescent on the edge of muscle fibers, and
To contact the author::
become activated when fibres degenerate, either after trauma or in genetic diseases.
[email protected]. Once the regeneration process is terminated, satellite cells, which have not engaged
fr.
into the myogenic process, return to quiescence on the edge of the newly formed
fibres. Therefore, the asymmetric division that sends progenitors back to quiescence
will define the number of cells available for regeneration, and thus the future
regenerative capacity. Furthermore, the group of Partridge showed that
transplantation of single fibres with their resident satellite cells is by far more efficient
in intra-muscular transplantation than any other type or state of progenitor, thus
raising the possibility that quiescence may confer this increased efficiency. Our aim is
to try to identify genes involved in the state of quiescence in human muscle
progenitors. In order to reach this goal, we use two in vitro human cell culture models the Reserve cells model and the suspension culture model of muscle stem cells. We
are currently characterizing these models in terms of viability, proliferation, cell cycle
profiling, myogenic markers, cell cycle regulators and expression of specific genes.
Our preliminary work on reserve cells model and the suspension cultures of muscle
cells showed that the cells are desmin positive, Pax7 positive, myogenin negative,
MyoD negative. 90 – 92 % of the Reserve cells are in G0/G1 phase of the cell cycle.
In addition a micro-array experiment comparing reserve cells, myoblasts and
myotubes has been carried out and we are screening and validating the genes
specifically upregulated in the reserve cells. These two in vitro models will be used to
further identify, validate and characterize quiescence-induced genes.
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PW27-338
A SEMI-MANUAL DEVICE FOR MULTIPLE REPETITIVE INTRAMUSCULAR CELL
INJECTIONS
RICHARD P-L1, GOSSELIN C1, PARADIS M2, GOULET M2, TREMBLAY JP2, SKUK
D2
(1) Laboratory of Robotics, Laval University, Quebec, CANADA. (2) Human Genetics
Unit, CHUL Research Center, Quebec, CANADA.
To contact the author::
[email protected]
.ca.
Intramuscular transplantation of muscle-precursor cells in nonhuman primates
and humans requires meticulous repetitive injections. Performed with single
syringes operated manually throughout large regions, the procedure takes a lot
of time, becoming tiring and thus imprecise. Trying to solve this difficulty, we
decided to develop a semi-manual cell injector specially conceived for this task.
Diverse mechanical principles and mechatronic components were examined to
choice the optimal mechanical arrangement to create a prototype. Simulations
were done to verify the concept before made-up a functional prototype. First tests
were carried out focusing on control, precision, mechanical behavior and
robustness. Thereafter, we tested the prototype by performing intramuscular
myoblast transplantations in monkeys. The prototype was able to repeatedly
inject a cell suspension simultaneously through six needles or less, delivering
few microliters equally through the intramuscular trajectory of each needle. The
success of the graft was comparable to that previously observed in monkeys by
our standard methods, but the task is facilitated by several facts. (1) Injecting
through several needles at the same time accelerates the procedure. (2) The
operator needs to be concentrated only in positioning the needles for each cycle
of injections, since the distance of needle penetration to reach the muscle and
the distance of cell delivery into the muscle can be preset in the device. (3) The
course of the needles into the muscle is done automatically after pushing a
button. (4) Continuous refilling of the cell suspension for each round of injections
is done automatically from a container in the device. This device could be useful
not only for intramuscular cell transplantations, but also for intramuscular
injections of gene vectors.
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PW27-339
INTRA-ARTERIAL DELIVERY OF MYOBLASTS TO SKELETAL MUSCLES IN
NONHUMAN PRIMATES.
SKUK D1, PARADIS M1, GOULET M1, TREMBLAY J1
(1) Human Genetics Unit, CHUL Research Center, Quebec, CANADA.
To contact the author::
[email protected]
.ca.
The main constraint of the therapeutic strategy of intramuscularly injecting
myogenic cells is that the implanted cells fuse only with the myofibers reached by
the injection trajectories. An intravascular delivery of myogenic cells may be
obviously a better strategy of cell delivery, but so far this strategy seemed to
work only with special cells such as the so-called “mesoangioblasts”. Previous
experiments of intravascular delivery of myoblasts in mice produced limited
results, but this animal model is quite different from human in several aspects of
the myoblast transplantation biology. According to our experience, nonhuman
primates are more appropriate models for human extrapolations in this field:
transplantation biology and myoblast culture are very similar. Thus, we
performed intra-arterial injections of beta-galactosidase-labeled allogeneic
myoblasts in tacrolimus-immunosuppressed cynomolgus monkeys. The myoblast
suspension was injected in one femoral artery. Some muscles were damaged
with a 27G needle 3 days before or at the time of the cell infusion. Several
organs and muscles were biopsied 1 hour, 1 day and 1 month posttransplantation, and the biopsies were analyzed by histology. We observed that
most intra-arterial delivered myoblasts were retained mainly in the capillaries of
the skeletal muscles of the leg ipsilateral to the cell injection. Scarce betagalactosidase-positive cells were observed in the lungs only at 1 hour posttransplantation, and no beta-galactosidase-positive cells were observed in other
organs (liver, brain, spleen, heart, gut) or other muscles. One month posttransplantation, beta-galactosidase-positive myofibers were observed only in the
skeletal muscles of the ipsilateral leg to the injection, when these muscles were
damaged at the time of the cell injection.
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PW27-340
MUSCLE REGENERATION POTENTIAL OF CANINE MUSCLE-DERIVED STEM
CELLS IN GRMD DOG MODEL
ROUGER K1, DUBREIL L1, FORNASARI B1, DELORME B2, JOUVION G1, LEROUXGOUBAULT I1, LEDEVIN M1, DESCHAMPS JY1, CHEREL Y1
(1) INRA, UMR 703, Ecole Nationale Vétérinaire de Nantes, Nantes, FRANCE. (2)
INSERM, ESPRI EA3855, Faculté de Médecine de Tours, Tours, FRANCE.
To contact the author::
[email protected].
The last decade, multi-lineage stem cells encountered in various adult tissues have
been described as exhibiting myogenic potential in experimental conditions. These
stem cells include Side Population cells, mesoangioblasts, CD133 + progenitors and
Muscle-Derived Stem Cells (MDSC). The last ones have been initially selected in mice
as late-adherent cells, using preplating technique.
Here, we focused on the canine MDSC isolation and their in vivo myogenic potential.
Muscle-derived cells (MDC) were extracted from healthy dogs and submitted to
classical preplating technique. MDSC were analyzed in primary and clonal cultures,
and investigated for their cell cycle status, multi-lineage differentiation potential and
phenotype. MDSC and myoblasts were transduced with nls LacZ retrovirus, then intramuscularly injected in 4-month old GRMD dogs, whom muscles show massive fiber
necrosis with endomysial and perimysial connective tissue proliferation. After 5 weeks,
the injected muscles were dissected and histologically analyzed for the presence of gal+ nuclei.
We demonstrated that MDSC could be isolated in large animal model : MDSC
represented 2.9% of all MDC. In addition to their proliferation/fusion behavior in vitro,
MDSC displayed other stem cell features, including differentiation into adipogenic and
osteogenic cells, G0-cell cycle arrest and reduced expression of myogenic regulatory
factors. Many thousand of -gal+ nuclei are observed in MDSC-injected muscles while
anyone is identified in myoblasts-injected ones : 55-64% are observed into muscle
myofibers, 25-35% in satellite cell niche and 10% in interstital tissue. Some of MDSC
in satellite cell localization display Pax7 expression, revealing that they participate to
maintain the satellite cell pool of dystrophic muscle. -gal+ nuclei presence is regularly
associated with dystrophin expression. Also,
-dystroglycan and sarcoglycans
( and
) are present throughout the sarcolemma of many hybrid fibers while
utrophin is down-expressed.
The outcome of our project provides novel insights into the myogenic potential of
MDSC in clinically relevant context.
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PW27-341
MACROPHAGES IMPROVE CELL SURVIVAL AND MIGRATION DURING
MYOBLAST TRANSFER THERAPY
LESAULT PF1, GHERARDI K1, TREMBLAY P2, CHAZAUD B1
(1) INSERM U841, Creteil, FRANCE. (2) CRCHUL, Quebec, CANADA.
Main limitations to efficient cell therapy in skeletal muscle include massive cell
death, limited diffusion and poor fusion of the transplanted cells. Numerous attempts
To contact the author::
of myogenic cell transplantation in skeletal muscle have been performed in both
benedicte.chazaud@ins
animals and humans. Massive death of transplanted cells is observed even in
erm.fr.
autologous grafts or immunosupressed recipients and is distinct from rejection. Acute
deprivation in survival cues likely participates to massive death of myogenic cells that
are mass-injected. They therefore have lost their privileged relationship with their
stromal environment.
We have previously shown that macrophages are important for skeletal muscle
regeneration and may exert beneficial effects on myogenic cell growth through
mitogenic and anti-apoptotic activities. Our aim was to evaluate the potential beneficial
effect of co-injection of macrophages on myogenic precursor cell (mpc) survival and
migration after transplantation into mdx skeletal muscle.
Co-injection of macrophages with mpcs increased in a dose-dependent way the
signal associated with mpcs, indicating a beneficial effect of macrophages on
myogenic cell survival. This has been evaluated by using [ 14C]thymidine loaded mpcs
and mpcs isolated from Tg:CAG-GFP mouse. After injection of GFP-mpcs with
macrophages, the GFP signal even increased with time, suggesting a beneficial effect
of macrophages on proliferation of myogenic cells, as we have previously
demonstrated in vitro.
We have also shown that coinjection of macrophages with mpcs stimulated their
migration within the muscle tissue, in a dose-dependent manner.
These data show that co-injection of macrophages improve myogenic precursor cell
transplantation by several effects: 1) limitation of mpc post-transplantation mortality; 2)
stimulation of mpc proliferation; 3) stimulation of mpc migration within the muscle
tissue. Macrophages may be therefore used as a stromal adjuvant for cell therapies.
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PW27-342
RHOE CONTROLS MYOBLAST ALIGNMENT PRIOR FUSION THROUGH RHOA
AND ROCK
FORTIER M1, COMUNALE F1, KUCHARCZAK J2, BLANGY A1, CHARRASSE S1,
GAUTHIER ROUVIERE C1
(1) Universités Montpellier 2 et 1, CRBM, CNRS, UMR 5237, IFR 122, Montpellier,
FRANCE. (2) IBCP UMR 5086 CNRS Université de Lyon, Lyon, FRANCE.
To contact the author::
[email protected]
rs.fr.
Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step
process orchestrated by several signaling pathways. The Rho small G protein family
plays critical roles both during myogenesis induction and myoblast fusion.
We report here that, in C2C12 myoblasts, expression of RhoE, an atypical member of
this family, increases until the onset of myoblast fusion before resuming its basal level
once fusion has occurred. We show that RhoE accumulates in elongated, aligned
myoblats prior fusion and that its expression is also increased during injury-induced
skeletal muscle regeneration.
Moreover, although RhoE is not required for myogenesis induction, it is essential for
myoblast elongation and alignement before fusion and for M-cadherin expression and
accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present defective
p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE
interacts also with the RhoA effector ROCKI, whose activity must be down-regulated
to allow myoblast fusion. Consistently, we show that pharmacological inactivation of
RhoA or ROCK restores myoblast fusion in RhoE deficient myoblasts. RhoE
physiological up-regulation before myoblast fusion is responsible for the decrease in
RhoA and ROCKI activities, which are required for the fusion process.
Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.
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PW27-343
SATELLITE CELL HETEROGENEITY BOTH BETWEEN, AND WITHIN, MUSCLES
ONO Y1, ZAMMIT P1
(1) King's College London, London, UNITED-KINGDOM.
To contact the author::
[email protected].
Homeostasis, hypertrophy and repair of adult skeletal muscle are carried out by
resident stem cells called satellite cells, located on the surface of the myofibre, below
the ensheathing basal lamina. Normally mitotically quiescent, satellite cells must first
be activated to undergo extensive proliferation to generate myoblasts that eventually
differentiate to repair/replace myofibres. Satellite cell self-renewal is the primary
mechanism responsible for maintaining a viable satellite cell pool. This process can be
modelled in culture where satellite cell progeny adopt divergent fates. Quiescent
satellite cells express Pax7, and when activated, co-express Pax7 with MyoD. After
proliferation, most then down-regulate Pax7 and differentiate. In contrast, other
satellite cell progeny maintain Pax7 but lose MyoD and withdraw from both cell cycle
and immediate myogenic differentiation, returning to a quiescent-like state.
Here we have explored functional heterogeneity both between, and within, the satellite
cell pool of specific muscles; namely the extensor digitorum longus (EDL) and soleus
in the hindlimb, the extensor carpi radialis longus (ECRL) in the forelimb, and the
masseter in the head. Satellite cell numbers and differentiation potential are higher in
the Soleus than the EDL, which in turn is higher than the masseter. Even within a
specific muscle, there is a large range of proliferative potential and self-renewal
capacity. In summary, the work to be presented demonstrates marked functional
heterogeneity amongst satellite cells.
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PW27-344
TRACKING OF A LIVE POPULATION OF IMMORTALIZED MYOBLASTS BY
CONFOCAL MICROSCOPY.
DUGUEZ S1, RAJA B2, PARTRIDGE T1
(1) Center for Genetic Medicine, Children's Research Institute, Children's National
Medical Center,, Washington DC, USA. (2) Flow Cytometry Facility, Children's
Research Institute, Children's National Medical Center, Washington DC, USA.
Cell proliferation is an important parameter in the behavior of many cell types.
Fluorescence activated cell sorting (FACS) in combination with cell tracker dyes
To contact the author::
allows tracking of cell divisions over time but is restricted to large numbers of dead
sduguez@cnmcresearch (fixed) cells. We sought to develop a method by which cell division in a single small
.org.
population of live cells could be followed. Current cell tracker dyes such as
carboxyfluorescein diacetate succinimidyl ester (CFSE) label cell volume uniformly
and are fully retained over time, segregating between daughter cells in proportion to
cytosolic volume. Equal distribution of cytosolic volume between daughter cells
results in halving of fluorescence intensity per cell per generation. Use of FACS to
distinguish cells by size, complexity, and fluorescence intensity enables determination
of generation time, proliferation index, and division index. Confocal microscopy allows
accurate measurement of fluorescence intensity but intensity varies with cell volume.
We used two cell trackers, CFSE and CMRA orange, to measure both dilution of
labeling and final cell volume. This facilitated measurement of number of divisions.
The confocal method was validated by a complementary FACS analysis. We applied
this method to the H2K immortalized myoblast cell line and determined a highly
synchronous generation time of 22.45 ± 1.21h. We were able to follow cell division out
to four days. This method facilitates the tracking of cell division in a small population of
live cells over time.
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PW27-345
EVALUATION OF THE AMPLIFICATION OF MYOBLASTS ON MICROCARRIERS
JENNY C1, ALBERT V1, VOVARD F1, DENEFLE P1, MERTEN OW 1
(1) GENETHON, Evry, FRANCE.
To contact the author::
[email protected].
Myogenic progenitor cells are promising tools for cell therapy to treat neuromuscular
disorders. But a challenging question is their amplification to a satisfying scale for
clinical applications.
Until now, most of myogenic progenitor cells have been cultivated on plastic surfaces
as they are naturally adherent cells. The scaling up of such procedures results in the
multiplication of culture surface (eg. Cell Factories), which will be rapidly limited. As
myogenic progenitor cells are not able to grow in suspension, we have decided to test
an alternative strategy to grow adherent cells in suspension: use of microcarriers.
Human myoblasts (CHQ5B) have been chosen as a study model. Different
microcarriers (Cytodex, Cultisphers, …) have been tested in order to find the surface
best adapted for cell growth. The selected microcarriers (Cultisphers) are entirely
made from gelatine and thus can be degraded by an enzymatic means (trypsine,
collegenase…) and cells are collected by a simple centrifugation step. Preliminary
results indicate that by using a 50ml spinner flask, an amplification of 1 week allows
the production of 15.106 cells (starting with an inoculum of 2.5.10 6 cells). Further
studies will address scale-up issues for getting 108 cells / spinner run. These amplified
cells are characterized for their myogenic potential.
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PW27-346
CELL AUTONOMOUS ROLE FOR ATRX IN MYOBLAST SURVIVAL AND
EXPANSION
HUH M1, GRENIER G2, RUDNICKI M1, PARKS R1, PICKETTS D1
(1) Ottawa Health Research Institute, Ottawa, CANADA. (2) University of
Sherbrooke, Sherbrooke, CANADA.
To contact the author::
[email protected].
Boys born with the ATR-X syndrome have severe skeletal muscle weakness and
hypotonia at birth. The resulting muscle weakness delays developmental milestones
such as sitting, standing, and walking and many never become ambulatory during
their life. To investigate the role of ATRX in muscle development a Cre-loxP approach
was used to inactivate the ATRX gene specifically in skeletal muscle.
ATRXf/y:Myf5Cre+/- mice were born with a reduced muscle mass, smaller fibres, and a
normal number of satellite cells. Primary myoblasts from ATRXf/y:Myf5Cre+/- muscle
failed to proliferate in culture suggesting a defect in satellite cell activation or
proliferation. To further characterize ATRX function, we infected adult derived ATRXf/y
primary myoblasts with Cre recombinase expressing adenovirus (ATRXf/y:Ad-Cre).
ATRXf/y:Ad-Cre myoblasts display a lower proliferative capacity than ATRXf/y:Lac-Z
controls in high mitogen growth media. Despite their inability to expand in culture,
ATRX deficient primary myoblasts were capable of sequentially expressing the early
and terminal markers myogenin and Myosin Heavy Chain. The activation and
proliferative potential of ATRX deficient myoblasts were tested in vivo by cardiotoxin
(CTX) muscle injury experiments. CTX treated ATRXf/y:Myf5Cre+/- mice
demonstrated a poor ability to regenerate, highlighted by fewer numbers of centrally
located nuclei and the presence of improperly regenerated fibrotic tissue. Taken
together, ATRX is required for the expansion of satellite cells that is necessary for the
growth and regeneration of skeletal muscle.
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PW27-347
RGD-COUPLING TO COLLAGEN SCAFFOLD IMPROVES HUMAN MYOBLAST
MIGRATION AND DIFFERENTIATION: NEW POSSIBILITIES FOR HUMAN
SKELETAL MUSCLE ENGINEERING
COIRAULT C1, MAMCHAOUI K2, ALLAMAND V1, MOULY V2, BONNE G1
(1) INSERM U582, Paris, FRANCE. (2) Inserm UMRS 787, Paris, FRANCE.
There is increasing agreement that 3D culture of skeletal muscle satellite cells
and myoblasts holds great promise to investigate pathophysiological mechanisms and
To contact the author::
[email protected].
to test therapeutic strategies for neuromuscular disorders. However, the construction of
functional, tissue-engineered skeletal muscles (TESM) containing more than a few
layers of mature muscle cells remains challenging. Obvious concerns relate to
incomplete myotube differentiation, impaired nutrient diffusion and limited cell viability
in bioartificial constructs. Because the Arg-Gly-Asp (RGD) motif of fibronectin plays a
critical role in cell-extracellular matrix interactions through a specific interaction with
integrins 51, we hypothesized that TESM development could be improved by
coupling RGD peptides to collagen scaffold. The method used for the RGD-collagen
scaffold coupling allows i) high coupling yields and complete washout of excess
reagent and by-products with no need for chromatography; ii) spectroscopic
quantification of RGD coupling; iii) a spacer arm of 36 angstroms, a length reported as
optimal for RGD peptide presentation and favorable for integrin receptor clustering and
subsequent activation. A nonfunctional RGE peptide was used as control. Collagencross-linked RGD or RGE scaffolds were seeded with immortalized human myoblasts
(hMS, clone 9) and incubated in proliferative and then differentiation medium.
Morphological analysis demonstrated that both collagen scaffolds have the ability to
support hMS cell proliferation and differentiation. However, the presence of RGD
improved cell attachment by activating 5 integrin signalling pathway, enhanced
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proliferation and cell migration within the matrix, and enhanced differentiation of
myotubes. In conclusion, we report a novel method of engineering a highly effective
and stable mature tissue-engineered human skeletal muscle based on collagen matrix
cross-linked to RGD peptides. It should provide a useful tool for basic research and for
identifying new therapeutic strategies in human myopathies.
PW 28:
Cell therapies –
Myogenic precursor cells
PW28-348
To contact the author::
[email protected].
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ROLE OF NECDIN IN SKELETAL MUSCLE REGENERATION AND IN THE
DIFFERENTIATION OF MESOANGIOBLAST STEM CELLS
PESSINA P1, FRANÇOIS S1, AZZONI E2, COSSU G2, BRUNELLI S2
(1) DIMS-University Milano Bicocca, Milano, ITALY. (2) DIBIT HSR, Milano, ITALY.
Muscular dystrophies are heterogeneous diseases characterized by
a primary wasting of skeletal muscle. Replacement of diseased
muscles with new healthy and functional muscle fibers has been for
a long time a major therapeutic strategy for muscular dystrophies.
In particular the mesoangioblasts have been show to contribute to
muscle repair in dystrophic mice and dogs when injected intraarterially.
Despite of the identification of mesoangioblasts as potential source
of skeletal muscle, the molecular mechanisms regulating their
growth and differentiation into skeletal muscle remained
unexplored. Detailed studies on the molecular pathways regulating
their response to specific cues are needed to manipulate their fate,
since it would be of great interest to be able to improve their ability
to differentiate and fuse to the fiber, as well as to proliferate and
resist to cell death and as such act as pool of resident stem cells.
Necdin is a member of the MAGE protein family, a large family of
genes initially isolated from melanomas. Ndn-/- animals show a
defect in muscle regeneration. On the other hand Necdin appears
to improve muscle regeneration in gain of function transgenic mice
by promoting satellite cells survival and differentiation: it would
therefore be of great interest if this could be exploited in
mesoangioblasts. Preliminary experiments show that transient
overexpression of Necdin increases the differentiation ability of
mesoangioblasts in vitro.
We are isolating mesoangioblasts from the dorsal aorta of MlcNec2
and Ndn-/- embryos at. In parallel with studies carried on on
satellite cells, we are investigating mesoangioblasts proliferation,
migration, response to apoptotic stimuli and myogenic
differentiation potential in vitro and in vivo in comparison with wt
cell.. We will also evaluate their migration potential.
PW28-349
To contact the author::
[email protected].
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IN VIVO MYOGENIC POTENTIAL OF HUMAN AC133 MUSCLE-DERIVED STEM
CELLS
NEGRONI E1, RIEDERER I1, WOLFF A1, DI SANTO J2, TORRENTE Y3, MOULY V1,
BUTLER-BROWNE GS1
(1) UMRS787 – Groupe Myologie; Inserm / UPMC-ParisVI; Institut de Myologie,
Paris, FRANCE. (2) Institut Pasteur – Departement d' Immunologie, Unité des
Cytokines et Développement Lymphoïde, Paris, FRANCE. (3) Stem Cell Laboratory –
Departement of Neurological Sciences, Fondazione IRCCS Ospedale Maggiore
Policlinico, Centro Dino Ferrari, University of Milan, Milan, ITALY.
After birth, adult skeletal muscle growth and repair are mediated by a population of
cells, normally mitotically quiescent and located under the basal lamina of the
myofibers, called satellite cells. In response to injury, resident satellite cells become
activated and proliferate, differentiate and fuse to form new muscle fibers.
We have investigated the myogenic potential of human muscle-derived cells based on
the expression of two stem cells markers, CD34 and AC133, as compared to bona
fide satellite cells.
The efficiency of these cell populations to participate to muscle regeneration and
contribute to replenish the satellite cell pool is evaluated in an ex vivo model, the
RAG-/- gammaC-/- C5-/- immunodeficient mouse in which degeneration of the Tibialis
Anterior (TA) is induced by cryoinjury. Human cells were then injected into the
regenerating TA, each animal receiving satellite cells in one leg and stem cells in the
other. 1 month post-injection, human nuclei are visualised using an human-specific
antibody directed against lamin A/C, while fibres containing human proteins are
identified with an antibody directed against human spectrin.
Our results demonstrate that human muscle-derived AC133+ cells showed a better
regenerative capacity than human myoblasts derived from satellite cells. The number
of fibres expressing human proteins, the number of human nuclei and the number of
human cells in a satellite cell position are all increased in TA injected with AC133+
cells as compared to those injected with human myoblasts. In addition
AC133+/CD34+ cells exhibited a better longitudinal dispersion in the host muscle
when compared to human myoblasts.
We propose that human muscle-derived AC133+ cells could be a potentially attractive
candidate for cellular therapy, provided that sufficient numbers of cells could be
available either at isolation or more likely after their amplification in vitro.
PW28-350
MYOGENIC AND ADIPOGENIC POTENTIAL OF HUMAN SKELETAL MUSCLEDERIVED CD34-SORTED CELLS
PISANI D.F1, DECHESNE CA1, DESNUELLE C2, BELMONTE N2, DELPLACE S2,
COCHET O1, BAGNIS C3, DI SANTO J4, KURZENNE JY5, DANI C1, SACCONI S2
(1) UMR 6543 CNR/UNSA, Nice, FRANCE. (2) UFR Médecine, Nice, FRANCE. (3)
Etablissement Français du Sang, Marseille, FRANCE. (4) Institut Pasteur, Paris,
FRANCE. (5) Hôpital de l’Archet, Nice, FRANCE.
Myoblast transplantation in clinical trials is based on intramuscular injection of a
population of muscle-derived cells. Up to date, homogeneity of this population
To contact the author::
[email protected].
throughout culture has been evaluated using the CD56 marker. According to our data,
the CD56 population contains stem cells able to give rise to adipocytes in vitro.
Differentiation of stem cells after transplantation into muscles is driven towards the
myogenic lineage. However, adipocyte accumulation is observed in human dystrophic
muscular diseases and likely, stem cells transplanted in a muscle environment
permissive to fat development may be committed towards adipogenesis at the expense
of myogenesis. Therefore, for muscular cell therapy, it is critical to identify a cell
population with a high myogenic and a low adipogenic potential from the mixture of
cells to be transplanted.
The stem cell marker CD34 allowed us to sort two distinct populations from human
pediatric and adult muscle biopsies. In vitro, the CD34+ cells were myogenic and
adipogenic whereas the CD34- cells were only myogenic. Both cell populations have
muscle regeneration potential after transplantation in cryo-injuried muscle of
immunodeficient Rag2-/- c-/- mice.
To our knowledge, there is no convenient mouse model fully mimicking human
muscular dystrophies, i.e. fat infiltration in regenerative muscles. However, we have
observed a higher fat development in cryo-injured tibialis anterior muscle of Rag2/gc-/-
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mice when using clodronate-containing liposomes. Therefore, experiments are carrying
out to determine the fate of CD34 cell populations after their muscle transplantation in
this new mouse model having a micro-environment permissive for differentiation of
transplanted cells into adipocytes. In conclusion, the muscle CD34 negative cell
population could represent a new alternative cell population in cell therapy of muscular
dystrophy.
PW28-351
MYOGENIC POTENTIAL OF GENETICALLY-COMMITTED HUMAN MULTIPOTENT
ADIPOSE-DERIVED STEM CELLS
GOUDENEGE S1, PISANI DF1, DI SANTO JP2, DANI C1, DECHESNE CA1
(1) CNRS UMR 6543, Institute of Signaling Developmental Biology and Cancer, Nice,
FRANCE. (2) Inserm U668, Pasteur Institute, Paris, FRANCE.
We have previously shown that human multipotent adipose-derived stem (hMADS)
cells have a myogenic potential. They contribute to skeletal muscle regeneration after
To contact the author::
[email protected].
transplantation into mdx mouse muscle although only a very small proportion of cells
shows in vitro myogenic commitment under myogenic culture conditions. In addition,
co-culture experiments led us to propose that hMADS cells participate in myotube
formation via cell fusion and that only a sub-population has the capacity to fuse with
myoblasts. The goal of our work is to optimize hMADS cells myogenic potential to
evaluate their interest in cell therapy, since adipose tissue is easily available. One
option is to experimentally commit hMADS cells to myogenic differentiation. Here we
present the effect of a myogenic pre-commitment of hMADS cells that were
genetically modified by MyoD-forced expression. Myogenic potential of
wild-type
hMADS and MyoD-hMADS cells was assessed in vitro and in vivo. In vitro amplified
hMADS cells isolated from a fat pad of the prepubic area of a 4-month old boy were
transduced with a lentivirus vector expressing human MyoD. Transduced hMADS
cells formed numerous characteristic multinucleated myotubes and expressed muscle
markers at RNA and protein levels when maintained for 1-2 weeks in myogenic
differentiation medium. Thus, forced expression of MyoD dramatically increased the in
vitro myogenic potential of hMADS cells. Then, we investigated the capacity of MyoDhMADS cells to participate to muscle regeneration. Cells were injected into cryoinjuried regenerating Tibialis anterior muscles of RAG2 (-/-)
-/-)
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mice and
-sarcoglycan and
-PCR. Contribution of
wild-type hMADS and MyoD-hMADS cells to muscle regeneration was clearly
observed in this mouse model. Efficiency quantitative comparisons between wild-type
hMADS and MyoD-hMADS cells will be presented and discussed.
PW28-352
ARE SATELLITE CELLS AN HOMOGENOUS POPULATION? ADIPOGENIC
POTENTIAL IS LINKED TO THEIR PROLIFERATIVE CAPACITY.
ROSSI CA1, DITADI A1, MALERBA A1, FRANZIN C2, SANNA M2, POZZOBON M1,
VETTOR R2, DE COPPI P1
(1) Pediatric Oncohematology, Stem Cell Transplantation Unit, Department of
Pediatrics, University of Padova, Padova, ITALY. (2) Endocrine-metabolic laboratory,
Department of Medical and Surgical Sciences, University of Padova, Padova, ITALY.
Cell therapy represents a valid tool for tissue replacement, in particular in the contest
of muscle dystrophies or structural defects. Satellite cells (SCs) have been frequently
To contact the author::
used as source of cells for skeletal muscle replacement, because they represent in
carloalberto.rossi@unipd vivo the pool of myogenic precursors. SCs are located between the basal lamina and
.it.
the plasma membrane of skeletal myofibers. They offer the possibility of in vitro
expansion and autologous transplantation. According to recent studies, they seem to
be divided into two subpopulations, one of committed muscle precursors and one of
cells with more stem-like properties. This distinction correlates to both a diversity in
markers expression and differentiation potential.
In this study, for the first time, two subpopulations of SCs obtained from rat flexor
digitorum brevis muscle through single fiber selection and disgregation, were
distinguished based on both different proliferative and differentiative capacities.
Quantitative analyses showed that there is an almost fixed proportion of SCs that
possess a great proliferative potential, and that spontaneously give rise to adipocytes
in culture. Immunofluorescence and PCR analyses showed that while initially SCs are
homogenously positive for early myogenic markers such as Pax7 and Myf5,
pluripotent clones lose in culture myogenic markers and form lipid droplets in
cytoplasm, becoming adipocytes.
These observations could be relevant in muscle regeneration therapies. Selection of
satellite cells by their proliferative ability could have implication in their in vivo
regeneration potential.
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PW28-353
To contact the author::
[email protected].
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RESTORING CELL-BASAL LAMINA INTERACTION TO RESCUE TISSUE
DEGENERATION IN CONGENITAL MUSCULAR DYSTROPHY
PORRELLO E1, CAPOTONDO A2, TRIOLO D1, SAMPAOLESI M3, BRUNELLI S3,
COMI G1, RUEGG M4, COSSU G3, BIFFI A2, QUATTRINI A1, PREVITALI S1
(1) Dept. of Neurology, S. Raffaele Scientific Institute, Milan, ITALY. (2) TIGET, S.
Raffaele Scientific Institute, Milan, ITALY. (3) SCRI, S. Raffaele Scientific Institute,
Milan, ITALY. (4) Biozentrum, University of Basel, Basel, SWITZERLAND.
Congenital Muscular Dystrophy (CMD) is characterized by progressive wasting
muscular dystrophy and dysmyelinating neuropathy with variable involvement of the
central nervous system, which may lead to severe disability in early childhood. The
most frequent form is due to mutations of the LAMA2 gene encoding the laminin
alpha2 chain, which forms merosin the predominant laminin isoform of muscle and
nerve basement membrane. Although much is known about clinical aspects and
genetic causes of CMD, and about the pathological mechanisms that lead to muscle
and nerve degeneration, no useful therapy to arrest neuromuscular degeneration and
to promote tissue repair is available to date. As a proof of principle the overexpression
of laminin2 or mini-agrin, a cross-linker molecule that allows reconnection of the
basement membrane to the resident cells, showed amelioration of CMD in animal
models. However, at present direct viral transduction of exogenous proteins into
human tissues is not feasible. Cell therapy may instead constitute a promising tool to
speed translation into clinical practice. Mesoangioblasts have shown promising results
in terms of amelioration of muscular dystrophy phenotype and reconstitution of
missing proteins in pre-clinical experiments. We infected mesoangioblasts with
lentivirus vectors carrying a mouse mini-agrin gene. Mesoangioblasts can synthesize
and deliver mini-agrin in vitro and in vivo. We injected the engineered
mesoangioblasts in the vein tail of CMD model, dy2J/dy2J mice. Mesoangioblasts
carrying the mini-agrin gene were able to fuse into myotubes of dy2J mice, many of
these myotubes expressed the mini-agrin protein, and these mice displayed
amelioration of muscle histology and clinical phenotype.
PW28-354
RESIDENT CD34+/AC133+ FETAL MUSCLE-DERIVED CELLS : LOCATION AND
MYOGENIC POTENTIAL
AUDA-BOUCHER G1, ROUAUD T1, ROUGER K2, FONTAINE-PÉRUS J1, CHÉREL
Y2, GARDAHAUT MF1
(1) CNRS UMR 6204 Fac Sciences, 44322 Nantes Cedex 3, FRANCE. (2) INRA
UMR 703 Ecole Nat Vét, 44307 Nantes Cedex 3, FRANCE.
We previously showed that CD34+ mouse fetal muscle-derived cells transplanted into
To contact the author::
mdx dystrophic mice efficiently regenerate skeletal muscle and improve its function.
Gwenola.Boucher@univ- Following several reports on the existence of heterogeneity in CD34 + adult musclenantes.fr.
derived cells we attempted to identify the specific subset of fetal CD34+ population
that possessed the highest myogenic potential. CD34+ fetal cells sorted by magnetic
bead selection were characterized on the basis of their expression for several markers
as Sca1, CD31, Ac133, CD45 using FACS analysis. CD31, Ac133 and Sca1 were
expressed at different levels by CD34+ cells. In contrast the CD34+cells were negative
for CD45 indicating that the sorted CD34 + population comprised muscle resident cells.
In fetal muscle sections CD34+/CD31+ cells were found in all vessel endothelium,
whereas CD34+/Ac133+ cells were observed on endothelium of some vessels. Double
staining for CD34+ and Sca1+ recognized cells lodged in connective tissue surrounding
muscle fibers and muscle bundles. We then examined the myogenic potency of these
three cell subsets (isolated from GFP mice) after transplantation into mice EDL
muscle. Our results indicated that CD34 +/ CD31+ cells preferentially differentiated into
endothelial cells while CD34+/Sca1+ cells were shown to have both endothelial and
adipogenic potentials. If CD34+/Ac133+ subpopulation had a high tendency to
differentiate into myogenic cells, nevertheless it displayed less efficient myogenic
activity compared to the original whole CD34 + sorted population. These observations
suggest that the other subpopulations identified into whole CD34 + fetal muscle-derived
cell population facilitate myogenic differentiation of CD34 +/Ac133+ fraction. In vitro and
in vivo experiments are currently in progress to understand and define the conditions
that promote the myogenic capacities of CD34+/Ac133+ fetal muscle-derived cells.
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PW28-355
To contact the author::
[email protected].
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MESENCHYMAL-LIKE CELL POPULATIONS IN HUMAN SKELETAL MUSCLE.
LECOURT S1, MAROLLEAU JP2, FROMIGUÉ O3, VAUCHEZ K4, TERNAUX B5,
LACASSAGNE MN5, ROBERT I5, PRAUD C1, ANDRIAMANALIJAONA R6,
BOUMEDIENE K6, CHEREAU F4, MARIE P3, LARGHÉRO J5, FISZMAN M1, VILQUIN
JT1
(1) Inserm, U582, Institut de Myologie, UPMC Univ Paris 06, UMR S582, IFR14,
Paris, FRANCE. (2) CHU Amiens Hôpital Sud, Service Hématologie Clinique, UPJV,
Amiens, FRANCE. (3) Inserm U606, Université Paris 7, Hôpital Lariboisière, Paris,
FRANCE. (4) Genzyme SA, St-Germain en Laye, FRANCE. (5) Laboratoire de
Thérapie Cellulaire, Hôpital Saint Louis, Paris, FRANCE. (6) Laboratoire de Biochimie
des Tissus Conjonctifs, Faculté de Médecine, Caen, FRANCE.
Purpose: The comprehension of the human skeletal muscle development,
homeostasis and physiopathology, and the set up of new therapeutic tools, mandate
the cellular investigation of skeletal muscle compartments. In situ, we assessed the
phenotype and localization of muscle cells on biopsy sections. In vitro, we analysed
the main cell populations from the onset of cell culture, their potential relationships
and phenotypical evolutions, and their differentiation abilities.
Methods: Immunohistochemistry was performed on human muscle cryostat sections.
Primary cell cultures were established upon enzymatic dissociation.
Immunophenotypic analyses were performed by flow cytometry and completed by
immunocytochemistry. The two main cell populations were separated using
immunomagnetic beads, and expanded. Differentiation studies were performed in vitro
using specific media.
Results: Classical mesenchymal (MSC) markers and additional ones (CD 10, 13, 15,
29, 34, 44, 47, 49, 56, 62, 73, 90, 105, 106, 146) were expressed by resident muscle
cells, which were classified according to their position relatively to muscle fibers,
basement membranes, and intramuscular vessels. CD56+ satellite cells co-expressed
rarely the CD90, CD146 or CD34 markers but no other MSC marker. In culture, two
main populations were identified, separated, and distinguished by expression of
CD56+ or CD15+ antigens, while a third population of CD34+ cells disappeared
rapidly. When expanded, CD56+ and CD15+ cells expressed all MSC markers, similar
to that harbored by human BM-MSC, while keeping heterogeneity. The CD56+ cells
expressed myogenic and chondrogenic abilities and osteogenic markers, while CD15+
presented adipogenic and chondrogenic capacities, and osteogenic markers.
Conclusions: These data underline the diversity of cells participating to human
muscle structures. They suggest that different populations express phenotypical
markers typical of myogenic and/or MSC, and present mutually exclusive lineage
restrictions.
Supported by grants from Genostem and the AFM.
PW28-356
To contact the author::
[email protected].
MUSCLE-DERIVED STEM CELLS ISOLATED AS NON-ADHERENT POPULATION
GIVE RISE TO CARDIAC, SKELETAL MUSCLE AND NEURAL LINEAGES
ARSIC N1, MAMAEVA D1, LAMB N1, FERNANDEZ A1
(1) Institut de Genetique Humaine, CNRS, Montpellier, FRANCE.
Stem cells with the ability to differentiate in specialized cell types can be
extracted from a wide array of adult tissues including skeletal muscle. Here we have
characterized a population of stem cells from skeletal muscle that can be reproducibly
isolated and grown as a non-adherent, floating population. These cells express the
stem cell surface markers Sca-1 and Bcrp-1. Although capable of growing as nonattached spheres for months, when given an appropriate matrix, these cells adhere
and give rise to skeletal muscle, neurons and beating cardiac myocytes. Interestingly
no similar cell population could be isolated from either bone marrow or cardiac tissue
suggesting their specificity to skeletal muscle. When injected into damaged muscle,
these muscle-derived floating stem cells are retrieved expressing Pax7, in a
sublaminar position characterizing satellite cells and participate in forming new
myofibers. These data show that a non adherent-stem cell population can be
specifically isolated and expanded from skeletal muscle and this population
spontaneously differentiates into muscle, cardiac and neuronal lineages in vitro and
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contributes significantly to the repair of injured muscle in vivo. These findings support
the potential use of a similar muscle derived non-adherent cell population from human
muscle in the treatment of neuromuscular disorders.
(Key words: muscle; stem cells; multi-lineage differentiation: tissue regeneration )
PW 29:
Cell therapies –
Muscle embryogenesis
PW29-357
THE COFACTOR VESTIGIAL-LIKE 2 IS ASSOCIATED WITH SKELETAL MUSCLE
DIFFERENTIATION IN CHICK LIMBS
BONNET A1, DAI F2, BRAND-SABERI B2, DUPREZ D1
(1) Université Pierre et Marie Curie (ParisVI), CNRS, UMR7622, Laboratoire de
Biologie du Développement, Paris, FRANCE. (2) Institute of Anatomy and Cell
Biology, Department of Molecular Embryology, University of Freiburg, Freiburg,
GERMANY.
To contact the author::
[email protected]
u.fr.
Skeletal muscle formation is crucially dependent on four basic-helix-loop-helix (bHLH)
transcription factors, Myf5, MyoD, Mrf4 and Myogenin, which are named the myogenic
regulatory factors (MRFs). Beside this recognized master role of the MRFs in
triggering myogenesis in Vertebrates, there is emerging evidence that other
transcription factors are important for muscle formation. Mammalian Vestigial-like 2
(Vgl2), a cofactor of TEF-1 and MEF2 transcription factors, has been shown to
promote skeletal muscle differentiation in vitro.
We analyzed the expression of Vgl2 transcripts during muscle formation in chick
limbs. Vgl2 is expressed specifically in embryonic skeletal muscles. Comparison of
Vgl2 transcript location with that of the known muscle markers such as Pax3, FgfR4,
MyoD at different stages of limb development showed that Vgl2 transcripts are not
expressed in muscle precursors migrating from the dermomyotome to the limb. Vgl2
expression starts at E4, in a domain similar to that of MyoD. At this stage, Vgl2 is
expressed in a subpopulation of MyoD positive cells as shown by double in situ
hybridization. When differentiated fibers appear, Vgl2 transcripts are present both in
myoblasts and muscle fibers as are MyoD transcripts. We have previously shown that
activation of the Notch pathway inhibits muscle differentiation without affecting the
expression of the Pax3 and Myf5 genes. Similarly to the expression of MyoD, that of
Vgl2 is clearly down regulated after Notch activation. The transcriptional relationship
between MyoD and Vgl2 was analyzed in the neural tube and somite contexts by
using electroporation. Experiments in both contexts showed that Vgl2 is a target of
MyoD.
All these results indicate that Vgl2 is involved in muscle differentiation downstream of
MyoD. We are currently performing gain and loss of function experiments in chick
embryos in order to understand Vgl2 function in myogenesis.
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PW29-3 58
FUNCTIONAL CHARACTERIZATION OF SMALP IN SKELETAL MUSCLE
POMIÈS P1, BECKERLE M2
(1) CRBM - CNRS UMR5237, Montpellier, FRANCE. (2) Huntsman Cancer Institute University of Utah, Salt Lake City, USA.
To contact the author::
[email protected]
rs.fr.
The two ALP (-actinin associated LIM protein) isoforms, smALP and skALP,
which exhibit identical N-terminal PDZ domains and C-terminal LIM motifs with a
variable central core, are produced as a result of alternative splicing and are known to
interact with the actin-crosslinking protein, -actinin. skALP is specifically expressed in
adult skeletal muscle while smALP was supposed to be only expressed in smooth and
cardiac muscles. Recently, using an antisense RNA strategy, we have shown that
disruption of ALP isoform expression severely affects muscle differentiation (Pomiès
et al., 2007).
Here, using a novel isoform-specific antibody directed against smALP, we
show that smALP expression is strikingly upregulated upon induction of differentiation
of C2C12 cells and that smALP is also expressed in adult skeletal muscle.
Furthermore, using indirect immunofluorescence microscopy, we show that
upregulation of smALP expression at the early stage of myogenesis is concomitant
with its translocation from the cytoplasm to the nucleus of C2C12 cells. This nuclear
localization is confirmed by cellular fractionation using differentiating C2C12 cell
lysates.
Actually, using transitory transfections of C2C12 cells, we are mapping the
domain of smALP that target the protein to the nucleus of differentiating skeletal
muscle cells. Different smALP sequences such as the PDZ domain, the LIM motif and
the unique central region of smALP coupled to a 6-histidine tag are expressed in
C2C12 cells and are visualized by immunofluorescence microscopy. Furthermore,
knowing that -actinin is the only ALP-interacting protein described to date, we are
actually searching for smALP binding partners. We are therefore using an
immunoprecipitation strategy to identify the binding partner repertoire of smALP from
differentiating C2C12 nuclear extracts. Our hypothesis is that smALP is a key
regulator of skeletal muscle differentiation which is translocated into the nucleus of
myoblasts upon induction of differentiation in order to participate to the transcription
machinery.
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PW29-359
KIR2.1-LINKED HYPERPOLARIZATION SPECIFICALLY CONTROLS MEF-2A
AND MEF-2C EXPRESSION DURING HUMAN PRIMARY MYOBLAST
DIFFERENTIATION
KONIG S1, BADER CR2, BERNHEIM L1
(1) Department of Basic Neurosciences, Geneva, SWITZERLAND. (2) Department of
Clinical Neurosciences and Dermatology, Geneva, SWITZERLAND.
Our work is based on human primary myoblast cultures derived from single satellite
To contact the author::
cells. Human myoblasts are able to proliferate for several weeks in culture, and
stephane.konig@medeci terminal differentiation into myotubes can be induced by serum withdrawal. In human,
ne.unige.ch.
as in other species, it is well known that myoblast differentiation is mainly controlled by
two families of transcription factors, the myogenic bHLH family (including MyoD and
myogenin) and the MEF-2 (A-D) family.
We have previously shown that, during the differentiation process of human
myoblasts, the expression of myogenic transcription factors follows a
hyperpolarization of the membrane myoblast resting potential to -70 mV due to Kir2.1
channel activation. Inhibition of this hyperpolarization strongly decreases myogenic
transcription factor expression and myoblast differentiation, indicating that the
hyperpolarization is a prerequisite for myogenic factor expression. We have shown, in
addition, that the Kir2.1-linked hyperpolarization initiates the differentiation process by
generating a cytoplasmic calcium signal which specifically activates the calcineurin
pathway, although p38-MAPK, PI3K and CaMK pathways are also required for an
optimal myoblast differentiation. When induced to differentiate, human myoblasts first
hyperpolarize and then express myogenin and, slightly later, MEF-2A and MEF-2C.
MEF-2D is already present during proliferation, although its expression increases
during differentiation.
Recently, we observed that preventing the Kir2.1-linked hyperpolarization with 10 mM
cesium inhibited MEF-2A and MEF-2C expression whereas myogenin and MEF-2D
expression were unaffected. These results suggest that the Kir2.1-linked
hyperpolarization and the related calcium signal activating the calcineurin pathway
triggers MEF-2A and MEF-2C, while myogenin and MEF-2D must be controlled by
other pathways. We thus propose that the initiation of the differentiation process via
activation of the calcineurin pathway mainly occurs through the induction of the
expression of two specific members of the MEF-2 transcription factors, MEF-2A and
MEF-2C.
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PW29-360
MOLECULAR MECHANISMS OF KIR2.1 ACTIVATION AT THE ONSET OF HUMAN
MYOBLAST DIFFERENTIATION
HINARD V1, LEROY M1, BADER CR1, BERNHEIM L1
(1) University of Geneva, Geneva, SWITZERLAND.
Myoblasts are mononucleated cells that fuse together to form skeletal muscle fibers.
To contact the author::
We have shown previously that human myoblast differentiation requires a
marina.leroy@medecine. hyperpolarization of the resting membrane potential to take place. This
unige.ch.
hyperpolarization is due to the activity of particular potassium channels, the Kir2.1
channels. Kir2.1 activation is, so far, the earliest detectable event during human
myoblast differentiation. The purpose of this work was to elucidate the molecular
mechanisms controlling Kir2.1 activation at the onset of differentiation.
Last published results showed that Kir2.1 activity, as well as myoblast fusion, can be
modulated by inhibitors of tyrosine kinases and tyrosine phosphatases. We observed
that Kir2.1 channels are phosphorylated on tyrosine 242 in proliferating myoblasts,
and that this phosphorylation decreases significantly during the first hours of
differentiation. We thus proposed that phosphorylation of the tyrosine 242 maintains
Kir2.1 channels in an inactive state during the proliferating phase of human myoblasts.
Other groups, in other cell types, have shown that the Kir2.1 channels can be
inactivated through EGF receptor activation. We therefore looked, in human
myoblasts, whether receptor tyrosine kinases (RTKs) could be involved in Kir2.1
phosphorylation. Our first results suggest that the activation state of EGF/ErbB
receptors and, to a lesser extent, Insulin/IGF receptors vary between proliferation and
differentiation. We also observed that application of a high dose of EGF or
surexpression of EGF-R slow down the differentiation process. Based on these
preliminary data, we plan 1) to analyze the effect of EGF-R modulation on the Kir2.1
current and 2) to determine how the EGF-R activation is modulated during
differentiation (internalization, heterodimerization).
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PW29-361
TISSUE AND MOLECULAR INTERACTIONS REGULATING THE ONSET OF
MYOGENESIS
MIMAULT B1, CHERAUD Y1, FONTAINE-PERUS J1
(1) UMR CNRS 6204 Biorégulation, NANTES, FRANCE.
The tissular and molecular interactions that govern the onset of myogenesis during
To contact the author::
[email protected] embryogenesis are not yet entirely elucidated. The aim of our research is to use the
-nantes.fr.
advantages of the chick embryo to analyze the emergence of myoblasts in early
muscle development. We believe that the in vivo manipulation remains a powerful
technique allowing to discriminate the role played by a tissue in an embryonic
process. In this context, the chick embryo is a model of choice to perform the
manipulations since it is readily accessible to experimentation. We wish to identify the
neural signals that participate in myogenesis by gain and loss function approach using
electroporation and mouse-chick chimera method. Our experiments are specifically
addressed to the transcription factor Pax3. Caudal neural tube of 1,5 day-old chick
embryos are electroporated with constructs inducing over and under expression of the
Pax3 gene. In our mouse-chick chimeras, reciprocal exchanges of precisely defined
regions of neural tubes are performed between chick and mutant (Pax3-/GFP+)
mouse embryos.
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We analyze the myogenic commitment of somites formed at the experimental zones.
The expression level of Wnt11, MyoD, Sim1, Bmp4, Msx1, Pax3, Wnt1, Wnt3a, and
Wnt7a is investigated.
PW29-362
COMPUTATIONAL PREDICTION OF TRANSCRIPTION FACTORS INVOLVED IN
MYOGENIC DIFFERENTIATION AND THEIR BINDING SITES.
HESTAND MS1, VAN GALEN M1, VILLERIUS MP1, VAN OMMEN GJB1, DEN
DUNNEN JT1, 'T HOEN PAC1
(1) Center for Human and Clinical Genetics, Leiden University Medical Center,
Leiden, THE NETHERLANDS.
To contact the author::
[email protected].
The computational identification of transcription factor binding sites is difficult
due to their small size, resulting in large numbers of false positives and negatives in
current approaches. Two computational methods to reduce false positives are to look
for over-representation of transcription factor binding sites in a set of similarly
regulated promoters or look for conservation in orthologous promoter alignments.
We have developed a novel tool titled CORE_TF (Conserved and OverREpresented Transcription Factors) that identifies common transcription factor binding
sites in promoters of co-regulated genes. To improve upon existing binding site
predictions, the tool searches for TransFac R matrices that are over-represented
compared to a random set of promoters and identifies cross-species conservation in
the predicted transcription factor binding sites. The algorithm has been evaluated
using expression array data from several literature and in house studies on myogenic
differentiation.
CORE_TF is accessible as a web interface at www.LGTC.nl/CORE_TF. It
provides a table of over-represented transcription factor binding sites in a user-defined
set of promoters and a graphical view on evolutionary conserved transcription factor
binding sites. In our myogenic test data sets it successfully predicts target
transcription factors and their binding sites. Binding sites for the transcription factors
MAF, NF-1, and Runx2 were significantly over-represented in the upregulated genes
from all microarray studies analyzed. In addition to other known muscle-related
transcription factors, we have predicted the involvement of transcription factors not
previously known to function in myogenesis. We are in the process of verifying results
with high throughput sequencing of chromatin-immunoprecipitated samples. The
combination of in silico and empirical approaches will assist in the identification of
transcription factors with a role in the regulation of myogenic differentiation and
associated with myogenic defects seen in many neuromuscular disorders.
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PW29-363
MYOGENIG DIFFERENTIATION OF HUMAN EMBRYOID BODIES (EBS)
PETERSSON SJ1, FRANDSEN U2, JENSEN CH3, KASSEM M3, SCHROEDER HD1
(1) Department of Clinical Pathology, Institute of Clinical Research, University of
Southern Denmark, Odense, DENMARK. (2) Immunology and Microbiology, Institute
of Medical Biology, University of Southern Denmark, Odense, DENMARK. (3)
Medical Biotechnology Center, Institute of Medical Biology, University of Southern
Denmark, Odense, DENMARK.
To contact the author::
[email protected].
Human EBs were co-cultured with C2C12 mouse myoblasts to investigate if the
myogenic cells could direct the embryonic stem cells into myogenic differentiation. In
vitro EB differentiation is disorganized and frequently varies from one EB to another in
the same culture. C2C12 myoblasts are cultured on Extra Cellular Matrix-coated
coverslips with DMEM containing 10%FCS and 1%P/S. The EBs are then added
directly to an established C2C12 culture. The EBs adhere to the C2C12 cells and
spread out. The co-cultures are kept for one week during which the C2C12 cells are
induced to differentiate by changing to DMEM supplemented with 2%HS.
Few myotubes of human origin were found in the marginal zone of the EB, visualized
by ICC using human specific anti-nuclei antibody (MAB1281). The myotubes produce
Desmin, NCAM, and Myogenin. Double-staining with human specific CD56 (Leu19)
and MAB1281 was used to distinguish myotubes formed by fusion of mouse
myoblasts and human stem cells from myotubes of genuine human origin.
EBs were prior to the co-culture experiments kept in suspension culture for 20 days.
During this period they were induced to differentiate with and without ActivinB. No
difference in myotube formation was detected between induced and control EBs. The
myogenic enrichment seemed to be dependent on a direct contact with live,
proliferating C2C12 mouse myoblasts cells. Culturing EBs alone on ECM-coated
surfaces in conditioned medium from C2C12 myoblasts did not result in myotube
formation, neither did co-culture with fixated C2C12 cells with and without conditioned
medium. Co-culturing of DiI-incorporated EBs and human myoblasts resulted in
formation of very few myotubes.
For embryonic stem cells to achieve their clinical potential as a source for cell-based
technologies, methods for generating large quantities of the desired end products
must be developed. This is not possible solely with the methods demonstrated in this
study.
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PW29-364
DISSECTING THE ROLE OF PITX2C DURING MYOGENESIS
FRANCO D1, VELASCO E1, MARTINEZ S1, LYONS G2, NAVARRO F1, ARANEGA A1
(1) University of Jaen, Jaen, SPAIN. (2) University of Wisconsin, Madison, USA.
Pitx2 is a member of the bicoid family of homeodomain transcription factors that plays
a relevant role in morphogenesis. Pitx2 expression has been detected in many tissues
To contact the author::
[email protected].
during development, including myotomes as well as in migrating myoblasts. Its
expression is also maintained in Pax3 positive cells that have completed migration at
the proximal limb bud. We have previously documented that overexpression Pitx2c –
isoform in undifferentiated myoblasts (Sol8 myogenic cell line) resulted in upregulation
of cell cycle genes (c-myc, cyclinD1 and D2) while it arrests differentiation into mature
myotubes by upregulating Pax3 and downregulating myogenic transcription factors
such as MyoD and myogenin. These observations indicate that c-isoform of Pitx2
plays a pivotal role modulating proliferation vs differentiation during skeletal
myogenesis. We report herein that transient tranfections leading to overexpression
Pitx2c in Sol8 myoblasts demonstrate that the Pitx2c effects in this cell line are dosedependent. Therefore, we have determined at which doses of transfection Pitx2c
began to induce changes in cell phenotype, inhibiting myocyte differentiation and
myoyube formation. Real-time PCR analysis after Pitx2c-transfection reveals that cell
cycle genes (Cyclin D1 and Cyclin D2) as well as Pax3 upregulation coincides with
induced changes in cell phenotype, whereas myogenic regulatory factors (MyoD,
Myogenin) becomes down-regulated at low doses of Pitx2c-transfection before the
onset of changes in the phenotype. These data suggests that regulation of genes
involved in the maintenance of proliferative stages in myoblast and genes involved in
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the onset of differentiation (MyoD and Myogenin) require different Pitx2c doses.
Interestingly, we found Pax7 down-regulation after low doses of Pitx2c-transfection
coinciding with MyoD and Myogenin down-regulation suggesting that Pitx2 could play
a role modulating Pax3/7 function in adult satellite cells.
PW29-365
EXPRESSION PROFILE OF MUSCLE PROGENITOR MATURATION DURING
MOUSE DEVELOPMENT AND ADULTHOOD
ROCHAT A1, ALONSO S1, MORAIS J1, RELAIX F1
(1) Mouse Molecular Genetics group, UMR S 787 - Groupe Myologie, INSERM UPMC-Paris VI – Institut de Myologie, Faculté de Médecine Pitié-Salpétrière, Paris,
FRANCE.
To contact the author::
[email protected]
Growth and repair of adult skeletal muscle is achieved by a population of
m.
progenitor/stem cells, the satellite cells. Satellite cells are derived from a fetal
population of Pax3-expressing progenitor cells providing successive waves of
myogenic cells to sustain muscle growth during development
We are interested in the identification of genes involved in the acquisition of
stem-cell properties by the fetal progenitor when they progress into the post-natal
satellite cell lineage.
We took advantage of the Pax3GFP/+ mice, in which GFP labels the muscle
progenitor cells (Relaix et al., 2005), to perform an expression profiling of muscle
progenitor cells from early embryonic development to adulthood. RNAs of the FACSsorted GFP-positive cells were subjected to a microarray analysis. By comparing
different time points, it revealed the progressive modifications of the progenitor cell
transcriptome over time. Strikingly, more than 3000 transcripts are regulated during
the transition from primary to secondary myogenesis while about 1000 transcripts are
regulated at later time points, suggesting that the adult muscle progenitor cells are
molecularly defined at the fetal stage.
In addition to the analysis and validation of the screen, we will present
preliminary work on new factors which might be involved in satellite cells formation.
Relaix, F., Rocancourt, D., Mansouri, A. and Buckingham, M. (2005). A Pax3/Pax7dependent population of skeletal muscle progenitor cells. Nature, 435, 948-953.
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PW29-366
EXPRESSION OF VOLTAGE ACTIVATED CHLORIDE CHANNEL CLC-1 DURING
SKELETAL MUSCLE SATELLITE CELLS DIFFERENTIATION
ROSAS-SÁNCHEZ F1, MARTÍNEZ-TORRES A2
(1) Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro,
MEXICO. (2) Instituto de Neurobiología, Universidad Nacional Autónoma de México,
Querétaro, MEXICO.
To contact the author::
[email protected]
om.mx.
Muscle satellite cells (SMCs) are quiescent precursors of myoblast with specific
localization and cytology properties (Mauro, 1961; Muir et al., 1965; Gibson et al.,
1982; Schmalbruch y Hellhammer., 1977). SMCs are able to proliferate, differentiate
in myoblast and fusion to create new myofibers in response to damage or muscle
disease (Bischoff, 1994; Zammit et al., 2004).
Some myophaties are characterized by skeletal muscle degeneration as consequence
of genetic mutations, particularly in one chloride channel of the ClC family: ClC-1
(Koch et al., 1992), and little is known about molecular mechanism that induce the
precise temporal gene expression that codified for ClC-1, a specific protein in skeletal
muscle, including the differentiation processes of SMCs.
In another hand, calcium ions play a critical rule in cellular fusion processes that occur
during skeletal muscle differentiation and regeneration (Shainberg et al., 1969),
evidence of that is the spontaneous intracellular calcium transient release during
muscular embryogenesis (Strube at al., 2000) and during SMCs differentiation
processes (Bidaud et al., 2006).
Similar events precede the presence of ClC-1. Previous to muscle cell differentiation
the proper expression of ClC-1 secures a key component for the regulation of the
electrical excitability of the plasma membrane. How is this balance achieved? Thus far
we don’t know if the transcription of ClC-1 in the SMCs relays on the expression and
activity of VDCCs or VDDC currents relays the transcription of ClC-1; furthermore it
may responds to a genetically programmed pattern, and expression of myogenic or
cell-specific transcription factors.
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PW29-367
In the present study we will determine: a) the time expression of ClC-1 and VDCCs, Ttype and L-Type, in mammalian SMCs after muscle injury, b) the correlation between
the expression of ClC-1, VDCCs and myogenic markers during the differentiation
process of SMCs and c) if ClC-1 expression affect VDCCs expression pattern or
viceversa.
EFFECTS OF WNT4 ON THE MYOGENIC DIFFERENTIATION
BERNARDI H1, GAY S1, FEDON Y1, BOLZEC T1, BACOU F1
(1) INRA, UMR 866, Laboratoire de Différenciation Cellulaire et Croissance,
Montpellier, FRANCE.
The molecular signals that regulate satellite cell function remain largely obscure.
However, it was recently demonstrated that Wnts participate in the temporal control of
satellite cell expansion versus differentiation during adult muscle regeneration (Brack
To contact the author::
et al, 2008). Thus, differentiation of myoblasts in vitro and in vivo is correlated with an
[email protected] upregulation of canonical Wnt signaling. Furthermore, ectopic Wnt induces premature
.
muscle differentiation whereas inhibition of Wnt signaling interferes with muscle
differentiation. In this context, the fact that myostatin -a member of the TGF-β
superfamily that specifically regulates muscle mass- was shown to implicate Wnt4
signaling in postnatal skeletal muscle hypertrophy is of the upmost importance
(Steelman et al, 2006). This was corroborated by Takata et al, (2007) showing the
involvement of Wnt4 signaling during myogenic proliferation and dfferentiation of
skeletal muscle. In this context, we first established by SQ-PCR that a limited number
of Wnts was expressed during proliferation and differentiation of C2C12 myoblasts
and satellite cells (SC). Amongst the 19 Wnts examined, we noticed that only the
expression of Wnt4 was lacking during proliferation and was highly induced during
differentiation of both cell types. The aim of this study was thus to characterize the
role of Wnt4 protein in muscle homeostasis. The effects of Wnt4 on myogenic
differentiation were examined by modulating the expression level of this factor. We
showed that over-expression of Wnt4 in proliferative state by transient or stable
transfection was responsible for spontaneous differentiation (C2C12) or an important
myotube hypertrophy (SC). Conversely, inhibition of Wnt4 expression by siRNA
silencing on SC leaded to an atrophy of differentiated myotubes (myotube areas
correspond to 40% of siRNA luciferase controls) and a decrease in fusion index
(24%). Treatment of C2C12 myoblasts by Wnt4 siRNA induced a strong inhibition of
differentiation associated with a decrease in the expression of Myf5.
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PW29-368
WNT NON-CANNONICAL SIGNALING REGULATES STEM CELL SELFRENEWAL WITHIN THE MUSCLE SATELLITE CELL NICHE
LE GRAND F1, JONES A1, SCIMÈ A1, RUDNICKI M1
(1) Ottawa Health Research Institute, Ottawa, CANADA.
To contact the author::
[email protected]
m.
Skeletal muscle growth and regeneration are attributed to satellite cells which are
myogenic cells lying between the myofiber sarcolemma and basal lamina. Recent
findings in our lab indicated that the satellite cell pool can be divided in 2 lineages on
the basis of the activation of either Myf5-LacZ or Myf5-Cre reporter alleles. Myf5+
satellite cells represent a committed progenitor whereas the Myf5- sub-population
possesses repopulating ability. We designed a FACS strategy for the specific
selection of both satellite cell lineages and subsequently performed genetic differential
screening utilizing suppression subtractive analysis (SSH). Adult satellite cells were
FACS-sorted from muscle by 7integrin and CD34 immunoreactivity. Targeted
satellite cell populations were then separated on the basis of the expression of the
Myf5-YFP reporter. Real-Time PCR analysis showed both populations express
numerous satellite cell-specific genes at a similar level. Selected YFP- and YFP+ cells
were able to proliferate in vitro and give rise to functional cycling myoblasts and
differentiated myotubes. RNA was extracted from quiescent cells and their
proliferating progeny and subjected to SSH to identify differentially expressed
transcripts in proliferating myoblasts and YFP+ and YFP- satellite cells. This approach
allowed us to identify numerous genes specifically expressed in quiescent cells, and
showed parallels with embryonic myogenic progenitors. Comparison of YFP- and
YFP+ satellite cells demonstrated that more than 30 genes are differentially expressed
between these two populations. Further analysis of specific cDNAs suggested that the
Notch pathway, as well as Wnt non-canonical signaling, are involved in satellite stem
cells maintenance. We investigated the impact, in vitro and in vivo, of the modulation
of these molecular pathways on the commitment of muscle stem cells and
demonstrated that Wnt7a signaling controls symmetric division and self-renewal in the
satellite niche. Our findings may help design new therapies to alleviate muscular
dystrophy by enhancing muscle regeneration.
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PW29-369
IMPLICATION OF PITX GENES DURING SKELETAL MUSCLE DEVELOPMENT
L'HONORÉ A1, COULON V1, DROUIN J1
(1) Institut de Recherches Cliniques de Montreal, Montreal, CANADA.
The myogenic program of cell differentiation is controlled by different groups of
transcription factors acting during muscle development. We reported the expression
To contact the author::
[email protected].
of Pitx transcription factors throughout muscle development. The earliest member of
this homeobox subfamily to be expressed in myogenesis is Pitx2 that is expressed in
dermomyotome delaminating cells, and in proliferative muscle progenitors located
within myotome and limb muscles masses. Pitx3 expression is then concomitant with
myoblast determination of progenitors. While Pitx2 expression decreases in
differentiated cells, Pitx3 is maintained until birth in all skeletal muscles of the body
and limbs. We first analyzed Pitx3 function by use of a natural mouse mutant of this
gene. Indeed, the aphakia (ak) mouse was shown to have severely deficient Pitx3
expression in midbrain and eyes, and thus constitute a model of loss-of-Pitx3 function.
However, we found unaltered Pitx3 expression in muscles of ak mice. This led us to
identify and characterize in transgenic mice a muscle-specific Pitx3 promoter that is
intact in the ak mice. To achieve Pitx3 loss-of-function in muscle, we generated a
complete Pitx3 knockout mouse. Pitx3 deficiency does not significantly perturb muscle
development but results in complete compensation through maintenance of Pitx2
expression in all skeletal muscles until birth. These experiments indicate that the level
of Pitx mRNAs is maintained through compensatory mechanisms in skeletal muscles
and this suggests that Pitx2 and Pitx3 may redundantly serve critical functions in
muscle development. To assess these functions, we are currently investigating Pitx2
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PW29-370
and Pitx3 complete and muscle-specific double knock-out mice phenotype.
PROTEOME DYNAMICS OF BOVINE MUSCLE DURING FOETAL MYOGENESIS
CHAZE T1, MEUNIER B1, CHAMBON C2, JURIE C1, PICARD B1
(1) INRA, UR 1213, Unité de Recherche sur les Herbivores, Saint GenèsChampanelle, FRANCE. (2) UR 370 Plateforme Protéomique du Centre INRA de
Clermont Ferrand Theix, Saint Genès-Champanelle, FRANCE.
To contact the author::
[email protected]
.
Pre-natal period is crucial for muscle development in large species such as Bos taurus
since the most part of differentiation process is completed at birth. Previous bovine
studies displayed developmental stages representative of particular myogenic events
across the nine months of gestation. Production of bovine foetuses at these specific
stages is really interesting to begin multidisciplinary studies using in vivo biological
material. Beside classical methods (histology, immunodetection), proteomic is a
method of choice that can give access to the entire genome expression during
myogenesis. Two-dimensional electrophoresis and mass spectrometry identification of
proteins were conducted to display the muscular proteome dynamics through bovine
myogenesis. This study allowed the identification of 250 proteins grouped in different
expression profiles using Principal Component Analysis and Hierarchical Clustering
Analysis. Most part of proteins referred to contractile apparatus, energy metabolism,
cytoskeleton component and cell cycle actors. Early myogenic events were strongly
characterized first by a large number of differentially expressed proteins and second
by a large class of proteins involved in the control of the balance proliferation
/apoptosis. On the other hand, the last third of pre-natal life was mainly characterized
by i) few differentially expressed proteins, ii) many isoforms changes of contractile and
metabolic proteins and iii) a more important number of common proteins across the
last developmental stages.
This study allows a wide vision of proteome dynamics across foetal myogenesis and
is in agreement with previous genomic approach conducted with transcriptomic tool on
total mRNA. These results are of fundamental interest to myogenesis in general, and
could be important for human biology since bovine and human share a lot of common
features during skeletal myogenesis from the gestation time to the course of the
proliferation of different myoblasts generations. Studies based on bovine model could
potentially conduct to new insights for human knowledge.
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PW29-371
SIX1 AND SIX4 GENES ARE REQUIRED TO ACTIVATE THE FAST-TYPE
MUSCLE GENE PROGRAM IN THE MOUSE EMBRYO.
NIRO C1, DEMIGNON J1, GRIFONE R2, GIORDANI J1, MAIRE P1
(1) INSTITUT COCHIN INSERM U567 CNRS UMR8104, PARIS, FRANCE. (2)
Developmental Biology Institute of Marseilles UMR 6216, MARSEILLES, FRANCE.
To contact the author::
[email protected].
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The mechanisms that participate to the adult muscle slow fibre phenotype have been
well characterized. These mechanisms emphasize resting calcium concentration as a
key cofactor relaying slow motoneuron firing and controlling the activity of several
signalling pathways (calcineurin, CamK) that activate different transcription factors
(NFAT, MEF2, PGC1). On the contrary, the mechanisms that govern the genesis of
muscle fibre heterogeneity during development are still unresolved in mammals.
Several signalling pathways have been discovered in zebrafish that trigger slow
(SHH/Blipm1) and fast-type (FGF8/Pbx-Myod) muscle lineages. However their
relevance is not established in other species. We have previously shown that the Six1
homeoprotein is specifically enriched in muscle nuclei of fast-type muscle fibres and
that forced expression of Six1 and its Eya1 cofactor in the soleus converts its slow
oxidative phenotype to a fast glycolytic one.
We now show that Six proteins are responsible for the activation of the fast muscle
program during the earliest steps of muscle development in the mouse embryo : fasttype muscle genes expression is not detected by in situ hybridization in six1-/-six4-/embryos at E10.5 whereas slow muscle gene isoforms are still expressed in the
remaining myocytes. While several muscle defects have been already characterized
in six1-/- embryos, but not in six4-/-, we have shown an aggravation of the phenotype
in six1-/-six4-/- embryos, suggesting some compensatory functions between these two
genes. We now show that Six1 null embryos are not impaired in their fast-type muscle
genes activation. Thus activation of the fast muscle program in the primary myotome
can be achieved with Six4 homeoprotein alone, showing that Six1 has no intrinsic
properties as compared with Six4 in regard of this induction. We also provide
evidence that the known Eya cofactors of Six homeoproteins are dispensable to
achieve fast-type muscle genes specific activation in the mouse E10 embryo.
PW 30:
Myogenesis and cell
transplantation
PW30-372
NUCLEAR MOVEMENT DURING MYOFIBER FORMATION
CADOT B1, GACHE V1, GOMES E1
(1) INSERM U787, PARIS, FRANCE.
To contact the author::
[email protected].
Nuclear movement during myofiber formation
The formation of a myofiber requires the fusion of myoblasts to form a myotube which
then differentiate into a mature myofiber. During all these steps, the position of the
nuclei changes: during the formation of the myotube, the nuclei from myoblasts move
from the site of fusion to the center of the myotube. During myofiber formation, the
nuclei move from the center to the periphery of the myotube. Moreover, in the mature
myofiber, some nuclei become anchored close to the clusters of acetylcholine
receptors, precursor of neuromuscular synapse. It has been proposed that the
distribution of nuclei in the mature myofiber forms distinct functional domains and this
is probably important for muscle function since mis-distribution of nuclei is observed
during muscle regeneration and in some muscular disorders. The question of how the
position of nucleus is established in muscle cells and what are the molecular
mechanisms responsible for the position of the nucleus in muscle cells has not been
addressed. To answer this question we are identifying the nuclear position events that
occur during myotube formation, using both primary and immortalized myoblasts. We
are using high content time-lapse microscope to determined the trajectories of nuclei
after cell fusion. Using this approach, we will identify the cytoskeleton elements
involved in nuclear position (actin and microtubules). Furthermore, we will
characterize the pathways that regulate these nuclear movement events.
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PW30-373
BARX2 REGULATES SATELLITE CELLS ACTIVATION AND DIFFERENTIATION
DURING MUSCLE DEVELOPMENT AND REGENERATION.
MEECH R2, PICKLE L1, GONZALEZ K2, MAKARENKOVA H1
(1) The Neurosciences Institute, San Diego, USA. (2) The Scripps Research
Institute, La Jolla, USA.
To contact the author::
[email protected].
Satellite cells are muscle progenitor cells that are involved in normal muscle
growth and provide a reserve capacity to replace damaged muscle fibers following
injury or disease. However, the factors that regulate satellite cell self-renewal,
activation and differentiation are not fully defined. In this study we show that
homeodomain transcription factor Barx2 is a novel regulator of muscle development
and repair. Barx2 is expressed in embryonic myoblasts and adult satellite cells and
interacts with other muscle-expressed transcription factors. Barx2 is strongly
upregulated in Pax7-expressing satellite cells after muscle injury suggesting a role for
Barx2 in satellite cell activation. Consistent with this notion, mice lacking the Barx2
gene show reduced body and muscle mass and defective repair after acute muscle
injury, as well as decreased Pax7 expression. In addition, loss of Barx2 in dystrophic
mdx mice (Barx2/mdx double null) leads to a much more severe muscle phenotype
than either parental strain alone. In satellite cell cultures, Barx2 regulates early events
of differentiation and directly controls the expression of muscle-specific genes in
cooperation with MyoD and SRF. Moreover, satellite cell cultures prepared from
Barx2-/- muscle show a decreased proliferation rate and delayed differentiation
together with downregulation of smooth muscle actin and other differentiation markers
such as myogenin and myosin heavy chain, suggesting that Barx2 could control
differentiation of satellite cells. Moreover, cultured Barx2-/- satellite cells show
decreased substrate attachment and migration abilities. Taken together these data
suggest that Barx2 is an important factor for satellite cell activation and differentiation.
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PW30-374
REGULATION OF RESERVE CELLS BY ANGIOPOIETIN 1/TIE-2 SYSTEM WITHIN
HUMAN MYOGENIC PRECURSOR CELL POPULATION
ABOU-KHALIL R1, GHERARDI K1, CHAZAUD B1
(1) INSERM U841, Creteil, FRANCE.
Satellite cells are now considered as true stem cells as they both participate to
To contact the author::
benedicte.chazaud@ins
erm.fr.
skeletal muscle repair by contributing to the formation of new myofibres and provide
new quiescent satellite cells or reserve cells presenting the same properties. We have
previously shown that in adult normal skeletal muscle, satellite cells are located close
to capillaries. In vitro, endothelial cells and myogenic precursor cells (mpcs) have
privileged interactions and may act in a paracrine way (Christov et al. 2007). One of
the main molecular systems that regulate vascular homeostasis is the angiopoietin
(Ang)/ Tie system. Particularly, Angiopoietin 1 (Ang1) binding to its tyrosine kinase
Tie-2 receptor is required to maintain stabilization of the vessel and endothelial cell
survival. Beside its role in the vascular system, Ang1/Tie-2 interaction has been also
involved in hematopoietic stem cell quiescence by promoting their survival and
maintain in the G0 phase.
We have explored the role of Angs and Tie-2 system during in vitro myogenesis and
in the regulation of myogenic cell population homeostasis. Expression of Ang1
decreased as cells differentiated, whereas Tie-2 expression increased. Both were
upregulated in the reserve cells. Adding Ang1 to mpc cultures led to inhibition of both
mpc growth and differentiation. Ang1 also protected mpcs from apoptosis through
ERK1/2 signalling. These effects were mediated through Tie-2 receptor since a
specific antagonist abolished Ang1 effect on mpcs. In whole mpc cultures, Ang1
treatment increased Pax7 expression and decreased MyoD expression. Inversely,
silencing Tie-2 decreased Pax7 expression and increased MyoD expression, and
increased the number of both proliferating and differentiating cells. In vivo, isolated
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quiescent satellite cells from both human and murine muscle expressed Tie-2 and
Ang1. These results suggest that Ang1 binding to its receptor Tie-2 is involved in the
regulation of the reserve cell pool within human myogenic precursor cell population.
PW30-375
DUAL ROLE OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY
MACROPHAGES DURING SKELETAL MUSCLE REGENERATION AND
MYOGENESIS
YACOUB-YOUSSEF H1, ARNOLD L1, GHERARDI K1, CHAZAUD B1
(1) INSERM U841, Creteil, FRANCE.
To contact the author::
benedicte.chazaud@ins
erm.fr.
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PW30-376
Macrophages are important for skeletal muscle regeneration and may exert
beneficial effects on myogenic cell growth through mitogenic and anti-apoptotic
activities. However, macrophages are highly versatile and may exert various, and
even opposite, functions depending on their activation state. We have characterized
the phenotype of monocyte/macrophages, studied their role during skeletal muscle
regeneration and analyzed their effect on in vitro myogenesis.
After injury, skeletal muscle recruited CX3CR1 lo/Gr-1+ monocytes from blood that
exhibited a non-dividing proinflammatory profile. Then, within muscle, these cells
switched their phenotype to become proliferating anti-inflammatory CX3CR1hi/Gr-1cells that further differentiated into F4/80 hi macrophages. We have shown that in vitro,
phagocytosis of necrotic muscle cells induced a switch of proinflammatory
macrophages toward an anti-inflammatory phenotype releasing TGFbeta1 suggesting
that phenotype transition may occur in vivo upon phagocytosis of muscle debris.
Depletion of circulating monocytes in CD11b-DTR mouse at time of injury totally
prevented muscle regeneration, myofibres remaining in a necrotic state, indicating that
macrophages are necessary for muscle repair. Depletion of the intramuscular F4/80 hi
macrophages at later stages after injury reduced the diameter of regenerating fibres
suggesting that they are involved in myofibre growth.
In vitro, we showed that pro-inflammatory macrophages stimulated human myogenic
precursor cell (mpc) proliferation and prevented their differentiation whereas antiinflammatory macrophages exhibited differentiating activity, assessed by myogenin
expression and fusion into myotubes. We are investigating each step of in vitro
myogenesis: migration, differentiation (expression of the myogenic programme) and
fusion itself. Our first data showed that pro-inflammatory macrophages stimulated mpc
migration and inhibited their differentiation and fusion. Inversely, anti-inflammatory
macrophages slowed down mpc migration and stimulated their fusion into
multinucleated myotubes.
Finally, skeletal muscle regeneration would be sequentially associated with two main
types of macrophages: first, inflammatory macrophages that sustain mpc proliferation,
then anti-inflammatory macrophages that stimulate myogenesis and fiber growth.
STIM1 AND ORAI CHANNELS CONTROL EARLY POST-NATAL HUMAN
MYOBLAST DIFFERENTIATION
DARBELLAY B1, ARNAUDEAU S2, KÖNIG S2, JOUSSET H3, BADER CR1,
DEMAUREX N3, BERNHEIM L2
(1) Dept of Clinical Neurosciences and Dermatology - Geneva University Hospital,
Geneva, SWITZERLAND. (2) Dept of Basic Neurosciences - University of Geneva
Medical Center, Geneva, SWITZERLAND. (3) Dept of Cell Physiology and
Metabolism - University of Geneva Medical Center, Geneva, SWITZERLAND.
To contact the author::
Basile.Darbellay@mede
cine.unige.ch.
Specific intracellular calcium signals are required to induce myoblast differentiation.
To generate these calcium signals, myoblasts can rely on two major sources: releases
from intracellular calcium stores and influxes from extracellular medium. We have
shown previously in cultured human myoblasts that store operated calcium entry
(SOCE) is a possible source of calcium for differentiation to proceed. In the present
study, we show that STIM1 and the Orai family of calcium channels regulate resting
cytosolic calcium, intracellular calcium store level and SOCE in myoblasts. The
inhibition of SOCE using siRNA strategy directed against the proteins STIM1 or Orai 1
and 3 fully prevented the expression of the transcription factor MEF2, a specific
marker of myoblast differentiation. Conversely, MEF2 production was slightly
accelerated by increasing SOCE with STIM1 and Orai1 over-expression. Despite a
marked up-regulation during late differentiation, STIM1 and Orai1 are crucial only
during the very first steps of the differentiation process as differentiation and fusion
were not impeded by a 48h delayed SOCE knockdown. Inhibition of the different
players of SOCE allowed us to show a strong correlation between the amplitude of
SOCE measured at the onset of myoblast differentiation and the MEF2 expression,
confirming that myoblast differentiation critically depend on the overall store operated
calcium entry.
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PW30-377
INVESTIGATING THE FUNCTION OF TSHZ3 DURING SKELETAL MUSCLE
REGENERATION IN MOUSE.
FARALLI H1, CAUBIT X1, CORE N1, FASANO L1
(1) Institut de Biologie du Developpement de Marseille Luminy, Marseille, FRANCE.
Muscle growth and repair depend on Satellite Cells (SCs), myogenic stem cells
located between the plasma membrane and the basal lamina of the myofiber. When
muscles are damaged, SCs become activated, proliferate and differentiate to form
To contact the author::
multinucleated myofibers. Skeletal muscle differentiation is initiated by the
[email protected]. transcription factor MyoD, which binds directly to the regulatory regions of genes
expressed during skeletal muscle differentiation and initiates chromatin remodelling at
specific promoter. The modulation of the MyoD activity affects the balance between
proliferation and differentiation of the activated SCs. The identification of the different
players, which are implicated in this step, appears important.
We found that, following treatment with cardiotoxin the zinc finger protein Tshz3 was
strongly expressed in the SCs of regerating adult skeletal muscle. Our
immunohistochemical analyses indicated that Tshz3 is expressed in quiescent and
activated SCs. In primary myofibers culture, Tshz3 expression was gradually
downregulated when SCs differentiate into mature myofibers. Transfection and forced
expression of Tshz3 in C2C12 myoblast cells resulted in delay of myogenic
differentiation. Tshz3 might have a potential role in activation, proliferation and/or
controlling differentiation of myogenic cells. Moreover, a luciferase reporter assay with
the 184bp promoter of the Myogenin shows that Tshz3 represses MyoD dependent
activation on this element.
These results suggest that Tshz3 plays important roles in the molecular mechanisms
opering in activated SCs when there are poised between proliferation and
differentiation, probably through the regulation of the MyoD-dependent activation.
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PW30-378
THE ROLE OF NECDIN IN THE MUSCLE REGENERATION
FRANÇOIS S. 1, DEPONTI D. 2, PESSINA P. 1, AZZONI E. 1, MAGGIONI M. 1,
CLEMENTI E. 2, MENEVERI R. 1, COSSU G. 2, BRUNELLI S. 1
(1) Università Milano Bicocca- Dipartimento di Medicina Sperimentale, Monza, ITALY.
(2) SCRI-Stem Cell Research Institute-Dibit San Raffaele, Milano, ITALY.
To contact the author::
[email protected]
mib.it.
Necdin is a transcriptional co-factor of the MAGE protein family and deletion of this
gene in human is associated with Prader-Willi syndrome (PWS). Mice lacking Necdin
display a variety of phenotypes mimicking some aspects of the PWS. Previous studies
suggested that necdin might act as growth suppressor in neurons, facilitating cell
cycle exit and differentiation and inhibiting apoptosis. However Necdin role is not
limited to the CNS and it has been demonstrated in our group that Necdin is also
expressed during skeletal myogenesis, in myogenic precursor cells and when
overexpressed leads to hypertrophy in C2C12 myoblasts.
We have produced transgenic mice overexpressing Necdin under the control of
MLC1F skeletal muscle specific promoter and we have also obtained, from our
collaborators, a line of Necdin loss of function mice. We show that mice carrying the
transgene show an accelerated capacity to regenerate damaged muscle fibres, while
KO mice show decreased regeneration ability, accompanied by an increased level of
apoptosis. These data indicates that Necdin plays an important role in muscle
regeneration. Satellite cells are the main player in adult muscle regeneration and the
molecular mechanisms playing a role in this process are similar to those involved in
muscle development. We would like to get insight into the role of Necdin in the
molecular regulation of satellite cells activation and differentiation.
Muscle regeneration is also a key process in the development of cell therapy
strategies for muscle dystrophy. Our actual aims are to identify the molecular partners
of Necdin in the satellite cells using a two hybrid screening and GST-pull down and
study how these interactions modulate Necdin activity during satellite cell activation
and differentiation. We also intend to compare and study the differentiation properties
and expression profiles of satellite cells derived from the Necdin gain and loss of
function mice.
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PW30-379
INVOLVEMENT OF CALPAINS IN MYOBLAST ADHESION AND MIGRATION
ROUMES H1, DAURY L1, COTTIN P1, BRUSTIS JJ1
(1) Université Bordeaux I, Unité Protéolyse, Croissance et Développement
Musculaire, INRA USC-2009, avenue des Facultés, 33405 Talence, FRANCE.
To contact the author::
[email protected].
Ubiquitous calpains (µ- and m-calpain) are proteases of which enzymatic and
structural properties are well characterized. Their implication in the early stages of
myogenesis and more particularly in the fusion and migration of myoblasts seems well
established. In the aim of improving myoblasts transplantation therapy, our study
focuses on the role of the different actors of the proteolytic neutral calcium dependant
system during adhesion and migration of murin (C2C12) and human (LHCN-M2)
myoblasts.
The adhesion of both cell lines is similar and rapid. In presence of calpain inhibitor,
this phenomenon is delayed and reduced by about 55%. The characteristics of the
migration phenomenon (cell morphology, velocity and migration area) have been
analysed using video-microscopy. The morphology of both migrating cell lines as well
as their migration velocity are similar. During this migration process, the global
proteolytic activity of calpains has been measured on living cells using a fluorescent
substrate (t-Boc-LM-CMAC). The calpain activity is higher in C2C12 cells than in
LHCN-M2 myoblasts. The addition of a specific inhibitor decreases dramatically the
velocity of myoblasts migration as well as the migration area of myoblasts.
The impact of this inhibition has been observed on the actin cytoskeleton known to be
the motor of migration. The stress fibres are disorganised, the cells have a rounded
morphology and failed to form membrane protrusion.
In conclusion, calpain activity seems to play a pivotal role for migration process and
dispersion of human myoblasts. At the molecular level, these results suggest the
implication of calpains in the organisation of stress fibres probably by a limited
proteolysis of proteins involved in the organisation of the actin cytoskeleton.
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PW30-380
OVERCOMING IMMUNE REJECTION IN MODELS OF TRANSPLANTATION IN
DYSTROPHIC MUSCLE
GROSS DA1, VIGNAUD L1, DA ROCHA RODRIGUES S1, GJATA B1, CHARLES S1,
GEORGER C1, SCHERMAN D1, ISRAELI D1
(1) Genethon CNRS FRE 3087, Evry, FRANCE.
To contact the author::
[email protected].
Transplantation of muscle precursor cells (MPC) into dystrophic muscle is a major
therapeutic approach in muscular dystrophies. However the clinical benefit of this
approach is seriously compromised by the low survival rate of the transplanted cells.
One principal reason for the low survival of transplanted MPC is their attack by the
host immune response. Immune response in an allogenic transplantation is evoked
principally by donor to host incompatibility of the major histocompatibility complex.
However, mismatch of minor histocompatibility (mH) contributes to transplant rejection
too. At present, the only way to overcome rejection is the continuous administration of
non-specific immunosuppressive drugs, a treatment not without serious risk, which do
not lead to transplantation tolerance.
CD4+CD25+ regulatory T cells (Treg) are a unique subpopulation of CD4 T cells
required for the maintenance of peripheral tolerance and with the ability to suppress
deleterious immune response in many approaches.
Here we demonstrate that immune response to the male mH antigen HY is sufficient
to compromised survival of transplanted MPC. Importantly, infusing the host mouse
with Treg specific for the male antigen HY, we were able to inhibit both the anti-HY
immune response and the rejection process in two models of transplantation in
muscle. Moreover, this induced immune tolerance was spread over to other rejection
antigens presented in the engrafted cells, opening the way for large choice of
therapeutic applications.
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PW30-381
INTRAMUSCULAR MIGRATION OF SUBCUTANEOUSLY IMPLANTED
MYOBLASTS IN NONHUMAN PRIMATES.
SKUK D1, PARADIS M1, GOULET M1, TREMBLAY JP1
(1) Human Genetics Unit, CHUL Research Center, Quebec, CANADA.
To contact the author::
[email protected]
.ca.
The main constraint of the therapeutic strategy of intramuscular myogenic-cell
delivery is that the transplanted cells fuse only with the myofibers reached by the
injection trajectories. This phenomenon is traditionally attributed to a “lack of
migration” of the implanted cells. However, recent observations of the author in
monkeys suggest that intramuscularly injected myoblasts always fuse with
myofibers following an intra-fascicular migration, although this migration seems
to be only towards the myofibers damaged by the injections. To confirm this
migration capacity of the implanted myoblasts, we perform an experiment in
nonhuman primates, the animal model more appropriate for human
extrapolations in the field of myoblast transplantation. Skeletal muscles of
cynomolgus monkeys were damaged with a 27G needle (100 parallel
penetrations per cm2) and 1 hour later 1/2 ml of a cell suspension of allogeneic
beta-galactosidase-labeled myoblasts was injected subcutaneously over the
damaged region and over non-damaged regions. Monkeys were
immunosuppressed with tacrolimus. One month later, the damaged regions were
biopsied and analyzed by histology. In the damaged regions, beta-galactosidasepositive myofibers were observed up to 1 cm distant from the muscle surface (the
depth of each needle penetration). The distribution pattern of the betagalactosidase-positive myofibers followed the pattern of the previous needle
trajectories. This indicates that in nonhuman primates the implanted myoblasts
are capable to migrate for long distances into the recipient’s muscle, although to
reach and to fuse with damaged myofibers. The problem is thus not a “lack of
migration capacity” but rather the incapacity to diffuse throughout a nondamaged muscle to fuse indiscriminately and spontaneously with nonregenerating myofibers.
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PW30-382
MONITORING CELL TRANSPLANTATION PROTOCOLS BY 1H-NMR IMAGING :
WHICH CLASS OF CONTRAST AGENT?
VAUCHEZ K1, BALIGAND C2, VILQUIN JT3, FISZMAN M3, CARLIER P2
(1) Genzyme SA, Saint Germain en Laye, FRANCE. (2) NMR Laboratory,
Association Institute of Myology and CEA, IFR14, Pitié-Salpêtrière University Hospital,
Paris, FRANCE. (3) Inserm, U582, Institut de Myologie, UPMC Univ Paris 06, UMR
S582, IFR14, Paris, FRANCE.
To contact the author::
[email protected].
Purpose: High spatial resolution and non-invasiveness feature NMR imaging, and
may allow longitudinal assessment of cell therapies. However, cells must be preloaded with an appropriate NMR contrast agent (CA). We compared the ability of two
classes of CA, iron oxide nanoparticles (SPIO) and lanthanide complexes (Gd-DTPA),
to monitor the fate of labeled myoblasts transplanted in a xenogenic context.
Methods: Primary human myoblasts were loaded by direct incubation with 25mM of
Gd-DTPA (Magnevist) or 100µg/mL of SPIO (Endorem). 2.106 labelled cells were
injected in the tibialis anterior of C57/Bl6 immunocompetent mice. NMR acquisitions
were performed in a 4T Bruker Biospec NMR spectrometer. Interleaved axial T1weighted spin echo images (in-plane resolution:120x120µm) were acquired
immediately after cell injection (D0) and repeated on D1, D4, D6, D8, D11, D14, D21
and 3 months after transplantation. Size and contrast of the labeled area were
measured. Comparatively, muscle cryosections were labeled with anti-human COX-2
(mitochondrial localization) and lamin A/C (nuclear localization) antibodies
(immunohistofluorescence study). Prussian blue staining detected SPIO in tissue.
Results: T1-weighted images showed hyper-intensity (Gd-DTPA) and hypo-intensity
(SPIO) signals at injection site on D0. The size of SPIO spots halved between D1
and D11 and plateaued thereafter, while the contrast remained stable over time. The
Gd-DTPA label significantly decreased and totally disappeared at D21.
Immunolabelings performed on D8 and D11 confirmed the immunorejection of human
cells as expected. However, Prussian blue staining revealed the presence of SPIO at
D8, D11 and at 3 months, in agreement with the persistence of the NMR signal.
Conclusions: Time-courses of CAs detection were different after intramuscular
injection of loaded cells and the remanence of Gd-DTPA exceeded the known survival
of xenografted loaded cells. Most importantly, SPIO label was still visible after 3
months, confirming its poor relevance for therapeutic cell monitoring in vivo.
Supported by grants from AFM.
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PW30-383
GENE EXPRESSION PROFILING OF PURIFIED ADULT SKELETAL MUSCLE
PROGENITOR CELLS
PALLAFACCHINA G1, MONTARRAS D1, CUMANO A2, BUCKINGHAM M1
(1) Pasteur Institute, Dept. Dev. Biol., Paris, FRANCE. (2) Pasteur Institute, Dept.
Immun., Paris, FRANCE.
Satellite cells are the main progenitor cells for skeletal muscle growth and
regeneration under physiological conditions. The difficulty of purifying these quiescent
To contact the author::
[email protected].
cells in sufficient number has precluded their biochemical characterization. Pax3 and
Pax7 paired-box transcription factors play critical roles in skeletal myogenesis by
directing progenitor cells into the myogenic programme and by ensuring their survival.
In adult muscle, satellite cells are marked by the expression of Pax7 and, in a subset
of muscles, also of Pax3. The generation of a Pax3GFP/+ mouse line permitted the
purification of satellite cells expressing the GFP marker by flow cytometry and the
demonstration of their major role in muscle repair together with their capacity to self
renew as adult muscle progenitor cells in vivo (Montarras et al, 2005). This purification
procedure has opened the way for biochemical characterization of these freshly
isolated Pax3GFP/+ quiescent cells. The gene expression profile of quiescent satellite
cells has been established by comparison with activated cells. Activated cells were
obtained in three ways: in vitro, by culturing freshly isolated cells for 3 days in growth
medium, and in vivo, by isolating them from growing post-natal muscles and from mdx
mice. These mice lack dystrophin, a structural protein that is mutated in Duchenne
muscular dystrophy patients. In mdx mice, fibre degeneration is chronic but constantly
compensated by regeneration, which results from the continuous mobilization and
activation of satellite cells. This approach gives new insights into satellite cell function
and regulation and should lead to the identification of novel specific markers such as
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those of use for isolating satellite cells from other species, including humans.
Montarras et al., Science 2005;309(5743):2064-7.
PW30-384
A NEW ROLE FOR SRF IN SKELETAL MUSCLE AGING
LAHOUTE C1, SOTIROPOULOS A1, FAVIER M1, GUILLET-DENIAU I1, SCHMITT A1,
METZGER D2, TUIL D1, DAEGELEN D1
(1) Institut Cochin, INSERM U567 CNRS UMR8104, PARIS, FRANCE. (2) Institut de
Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) INSERM/CNRS/ULP,
ILLKIRCH, FRANCE.
The Serum Response Factor (SRF) is a crucial transcription factor for muscle-specific
To contact the author::
gene expression. We previously demonstrated that SRF is needed for postnatal
[email protected] skeletal muscle growth.
r.
To further investigate the role of SRF in adult skeletal muscle maintenance and
regeneration, we developed a new model of knock-out mice allowing tamoxifeninducible muscle-specific loss of SRF. We could thus trigger a rapid and efficient loss
of SRF expression in post-mitotic myofibers, leading to the rapid down regulation of
transcripts coding for SRF known target genes such as skeletal and cardiac alphaactin as well as muscle creatine kinase. We also confirm in this model the importance
of SRF expression for IGF-1 gene transcription.
Muscles lacking SRF displayed no obvious phenotype until 5 months after tamoxifen
injection. However SRF-KO muscles later developed an atrophy due to the reduction
of myofiber cross-section area, a sarcomere disorganization and an aggravated
accumulation of tubular aggregates. Moreover, these muscles displayed an altered
lipid metabolism showing inter- and intra-myofiber lipid accumulation, particularly in
oxidative fibers. After cardiotoxin injury, SRF-KO muscles also exhibited an altered
regeneration process, showing smaller regenerated fibers and a persistent fibrosis.
This phenotype is strongly reminiscent of age-related skeletal muscle abnormalities,
suggesting that triggering SRF loss could accelerate the aging process.
Interestingly, in skeletal muscle of control mice, we could observe a progressive and
important decrease in SRF protein expression associated with muscle aging.
Our data suggest that a naturally occurring SRF down-regulation or an induced SRF
loss could contribute to the muscle aging process. The molecular mechanisms
underlying such an accelerated muscle aging in the absence of SRF are under
investigation.
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PW30-385
ENHANCED MUSCLE RECONSTRUCTION AND SELECTION OF STEM-LIKE
CELLS BY MYOGENIC MACROPHAGE-SECRETED FACTORS
MALERBA A1, VITIELLO L1, SEGAT D1, DAZZO E1, FRIGO M1, SCAMBI I1, DE
COPPI P2, BOLDRIN L2, MARTELLI L1, PASUT A1, ROMUALDI C1, BARONI D1
(1) Department of Biology, University of Padova, Padua, ITALY. (2) Department of
Pediatrics, University of Padova, Padua, ITALY.
Skeletal muscle regeneration relies onto a specific population of myogenic precursors,
To contact the author::
[email protected].
named satellite cells. Inflammation also has a determinant role, as upon injuring
macrophages are attracted by the damaged myofibers and the activated satellite cells
and act as key elements of dynamic muscle supportive stroma. Yet, it is not kwown
how macrophages interact with the more profound stem cells of the satellite cell niche.
In this study we show that in the presence of a murine macrophage conditioned
medium (mMCM) a subpopulation of stem-like cells could be selected and expanded
from adult rat muscle with serial platings. These cells were small, round, poorly
adhesive, slow-growing and showed mesenchimal differentiation plasticity. mMCM
also inhibited the mesenchimal potential towards adipogenesis of satellite cells
mechanically isolated from suspensions of single myofibers. mMCM-treated myogenic
cells in mixed primary muscle cultures from neonatal rats showed a growth rate
increase, spindle-like morphology and alignment before forming an impressive array
of contracting myotubes; comparison with cultures from adult muscles suggested that
mMCM-sensitive cells are more abundant in developing muscles. In vivo,
intramuscolar administrations of concentrated mMCM in a model of extensive surgical
ablation of rat tibialis anterior dramatically improved muscle regeneration. Altogether,
these findings suggest that macrophagic factors could be of great help in developing
therapeutic protocols with myogenic stem cells.
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PW 31:
Satellite cells
and muscle homeostasis
PW31-386
EXPRESSION OF DLK1 AND MYOSTATIN IN SKELETAL MUSCLE AND
SATELLITE CELLS
SELLATHURAI J1, JOERGENSEN LH1, PETERSSON SJ1, JENSEN CH2, DHAWAN
J3, SCHROEDER HD4
(1) Institute of Clinical Research, University of Southern Denmark, Odense,
DENMARK. (2) Institute of Medical Biology, University of Southern Denmark,
Odense, DENMARK. (3) Center for Cellular and Molecular Biology, Hyderabad,
INDIA. (4) Institute of Clinical Research, University of Southern Denmark and
Department of Clinical Pathology, Odense University Hospital, Odense, DENMARK.
Muscular dystrophies are a heterogeneous group of muscle degenerating diseases for
To contact the author::
[email protected] which no effective treatment exist. A potential treatment strategy could be developed
gionsyddanmark.dk.
by studying DLK1 and myostatin regulation. DKL1 and myostatin are known to
generate muscle hypertrophy phenotypes and therefore these may be potential
targets for intervention or be used in gene therapeutic strategies of muscular
disorders. DLK1 is involved in embryonic muscle development and it has been shown
to induce muscle hypertrophy in transgenic mice and callipyge sheep. Myostatin
negatively regulates myogenesis, and inactivation leads to heavy muscle growth.
The expression of DLK1 and myostatin was studied in a set of experiments. In
transgenic mice over expressing DLK1 it was found that myostatin mRNA levels were
significantly lower compared to the controls.
The effect on proliferation and differentiation were tested in vitro by transfecting
C2C12 cells with full length mouse Dlk1. DLK1 did not affect the myogenic potential of
the cells; but it appeared to keep the cells in a proliferative state for a longer time
before initiation of differentiation. Like in the regeneration studies, myostatin mRNA
was down regulated in DLK1-C2C12 cells. In addition we found decorin upregulated.
Furthermore, the expression of DLK1 and myostatin was studied in human satellite
cell cultures. A high level of DLK1 was observed in nonproliferating G0 cells but during
proliferation and differentiation the level of DLK1 was highly down regulated. The
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expression of myostatin was down regulated in G0 and proliferating human satellite
cell cultures, but was highly up regulated in differentiating cultures. Thus, the results
again indicate that DLK1 interact with the expression of myostatin.
The studies were made using Taqman® RT-qPCR and immunohistochemistry.
PW31-387
MUSCLE GROWTH STIMULATION AFTER MYOSTATIN BLOCKADE IN ADULT
MUSCLE IS INDEPENDENT OF SATELLITE CELL ACTIVITY
MOUISEL E1, VULIN A1, HOURDE C1, DUMONCEAUX J1, RELAIX F1, GARCIA L1,
AMTHOR H1
(1) UMR S 787-Institut de Myologie, Paris, FRANCE.
Previously, we have shown that null mutation of myostatin resulted in muscle fibre
hypertrophy that is independent of satellite cell activity. Here, we investigated the
To contact the author::
effect of myostatin blockade in mature mouse muscle. Myostatin was blocked after
[email protected] intramuscular injection of recombinant AAV-2/1 coding for the myostatin propeptide
ussieu.fr.
(AAV-prop) into tibialis anterior (TA) muscle of 6 weeks old female C57Bl6 mice.
The local overexpression of myostatin propeptide resulted in a 28% increase of
muscle weight after 28 days of treatment compared to the contralateral side that was
injected with a AAV-Mseap control vector (P<0.001). Morphometric analysis revealed
a 16% increase of mean single fibre area following AAV-prop injection compared to
the controls (P=0.004). The mean fibre number after AAV-prop vector injection was
3328 compared to 3420 after control vector injection (P=0.47). Thus, excessive growth
after myostatin blockade resulted exclusively from fibre hypertrophy.
Next, the number of myonuclei was determined on cross sections of TA muscles.
Muscle fibres contained on average 0.65 myonuclei after injection of AAV-prop
compared to 0.63 myonuclei after control vector injection (P=0.77).
We finally determined the effect of myostatin blockade on satellite cell number. We
injected AAV-prop vector into TA muscle of Myf5nlacZ/+ mice, in which satellite cells
express the transgene lacZ. One month after injection of AAV-prop, muscle fibres
contained on average 32 satellite cells versus 25 satellite cells per 1000 fibres after
control vector injection, a difference that was statistically not significant (P=0.31). As
Myf-5 is expressed in quiescent as well as in activated proliferating satellite cells, we
can conclude that satellite cell activity is not increased after myostatin blockade.
Above results confirm findings from previous analysis of constitutive myostatin
knockout mice. Myostatin blockade in adult muscle resulted in fibre hypertrophy
without concomitant increase in myonuclear number and without any evidence of
increased satellite cell activity.
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PW31-388
CHARACTERIZATION OF THE HUMAN SATELLITE CELLS SECRETOME
DURING THE IN VITRO SENESCENCE
LE BIHAN MC1, ROGOWSKA-WRZESINSKA A2, BIGOT A1, FURLING D1,
COULTON G3, MOULY V1, BUTLER-BROWNE G1
(1) UMRS787 – Groupe Myologie; Inserm / UPMC-ParisVI; Institut de Myologie,
Paris, FRANCE. (2) Department of Biochemistry & Molecular Biology, University of
Southern Denmark, Odense, DENMARK. (3) St. George's University of London,
London, UNITED-KINGDOM.
With age, there is a gradual decline in the effectiveness of the regenerative response
of skeletal muscle to damage which is accompanied by muscle fiber atrophy and a
.
general loss of muscle mass and function. Age-related muscle wasting, like the
decline in the regenerative capacity may be due to the decrease in the number of
muscle precursor cells (satellite cells) as well as to an age-related decline in satellite
cell function. It has been suggested that the decreased level of the circulating trophic
factors that occurs with age could also contribute to the decrease in muscle mass,
force and regenerative capacity described in the elderly. The aim of this study was to
identify secreted muscle proteins essential for the maintenance of the regenerative
capacity of the resident satellite cells and to characterize differences in the global
pattern of protein expression observed in a model of muscle ageing: the replicative
senescence of human satellite cells in vitro. Conditioned medium from differentiating
primary cultures at an early passage and at senescence were analyzed by a
proteomic approach using 3 different expression profiling strategies: 1) 2D gel
electrophoresis/mass spectrometry (2DE/MS); 2) Luminex based assay; 3) mass
spectrometry (MS) based approach. A time course of myoblast secretion at early
passage and late passage in “Differentiation medium” revealed a 3 to 4-fold decrease
in the total amount of protein secreted during the senescence process associated with
considerable qualitative and quantitative changes in the secretome. Preliminary data
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from 2DE/MS & Luminex expression profiling has enabled us to identify 44 molecules
differentially “secreted” during muscle ageing in vitro: senescent myoblasts expressed
a similar panel of inflammatory cytokines as previously reported for senescent
fibroblasts associated with an upregulation of key regulators of matrix remodelling.
Results of the MS based approach will be obtained in the near future.
PW31-389
To contact the author::
[email protected].
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MUSCLE AGING AND CALCIUM-DEPENDENT PROTEOLYTIC SYSTEM.
BRULE C1, DARGELOS E1, COTTIN P1, POUSSARD S1
(1) UPCDM université Bordeaux 1, TALENCE, FRANCE.
The calcium-dependent proteolytic system is composed of cysteine
proteases named calpains. They are ubiquitous or tissue-specific enzymes and the
two best characterised isoforms are the ubiquitously expressed µ- and m-calpains.
Besides its regulation by calcium, calpain activity is tightly regulated by calpastatin
(which is the specific endogenous inhibitor), binding to phospholipids, autoproteolysis
and phosphorylation. Calpains are responsible for limited proteolytic events. Among
the multitude of substrates identified so far are cytoskeletal and membrane proteins,
enzymes and transcription factors. Calpains are involved in a large number of
physiological processes such as muscle growth and differentiation, and pathological
conditions such as muscular dystrophies.
Aging is associated with a progressive and involuntary loss of muscle mass
also known as sarcopenia. This condition represents a major public health concern.
Although sarcopenia is well documented, the molecular mechanisms of this condition
still remains unclear. The aim of this study was to determine if the proteolytic system
could be involved in the phenotype associated with sarcopenia. Calpains and
calpastatin levels, subcellular distributions and activities were compared between
muscles from young (3 months) and old (24 months) rats. While the subcellular
localisation of calpains did not change between young and old rat samples, their
enzymatic activity significantly increase with age. In the meantime, calpastatin
specific activity and protein level decreased in old samples. Altogether, our data
showed an overall increase in calpain activity associated with muscle aging. These
findings suggest that the calcium-dependent proteolytic system is indeed involved in
sarcopenia.
PW31-390
To contact the author::
[email protected].
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ANALYSES OF METABOLIC AND CONTRACTILE PLASTICITY DURING IN VITRO
MYOGENESIS OF NEW MURINE CELL LINES
PELTZER J1, COLMAN L2, CEBRIAN J3, MUSA H2, MARTELLY I1, PECKHAM M2,
KELLER A1
(1) Laboratoire CRRET, UMR CNRS 7149, Créteil, FRANCE. (2) Institute of Mol and
Cell Biol, University of Leeds, Leeds, UNITED-KINGDOM. (3) Génomes et Cancers,
FRE CNRS 2939, Institut Gustave Roussy, Villejuif, FRANCE.
Our general objective is to contribute to the elucidation of factors involved in
coordinated regulations of metabolic and contractile pathways in myofibres. For that
purpose we have established myogenic clones derived from satellite cells of the
transgenic immortomouse capable of expressing adult fast-glycolytic and/or slowoxidative markers in myotubes. We have investigated the expression, by such clones,
of metabolic enzymes and contractile markers, indicative of coordinated modulations,
as a function of time and differentiation stage. The WTt clone, that differentiates into
myofibres of a mixed fast and slow phenotype was chosen for attempts to direct its
phenotype towards a more homogeneous slow-oxidative or fast-glycolytic phenotype.
To reach a slow-oxidative phenotype, we have selected stably infected WTt clones
over-expressing the PPAR delta transcription factor known to be involved in the
balance of oxidative energy metabolism. PPAR delta over expression enhanced the
slow-contractile phenotype of the WTt myogenic clone. To reach a fast-glycolytic
phenotype, we have attempted to stabilize the HIF-1alpha protein of WTt cells by
using cobalt chloride (CoCl2). This pseudo-hypoxic treatment induces HIF-1alpha
stabilisation, which in turn activates target genes, including enzymes of the glycolytic
pathway. Interestingly, a short CoCl2 treatment (24 hours) favoured the accumulation
of fast MHC isoforms. Only longer treatments (96 hours) also induced a decrease in
expression of the slow phenotypic markers, suggesting that HIF-1alpha might regulate
fast-glycolytic and slow-oxidative myofibre phenotypes through independent
pathways. Our data support the idea that modifying oxidative or glycolytic metabolism
will induce coordinated changes in contractile phenotype of muscle cells. It will be
worth investigating whether the sequences we found on MHC genes, carrying putative
-1alpha transcription factors, might be active.
PW31-391
To contact the author::
[email protected]
.
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DYSTROGLYCAN, TKS5 AND SRC MEDIATED ASSEMBLY OF PODOSOMES IN
MYOBLASTS
THOMPSON O1, KLEINO I2, CRIMALDI L3, GIMONA M3, SAKSELA K2, WINDER S1
(1) University of Sheffield, Sheffield, UNITED-KINGDOM. (2) University of Helsinki,
Helsinki, FINLAND. (3) Mario Negri Sud, Santa Maria Imbaro, ITALY.
Dystroglycan is an essential component of the dystrophin glycoprotein
complex of skeletal muscle (DGC), an important mediator of
connections to the actin cytoskeleton and a scaffold for signalling
molecules in a number of other cell types. In addition dystroglycan is
also an important polarity determinant and is dysregulated in the
majority of epithelial-derived tumours. We have examined the role of
dystroglycan in the early stages of myoblast adhesion and spreading
and found that dystroglycan initially associates with other adhesion
proteins in small puncta that precede the formation of true focal
adhesions in these cells. The complement of proteins localised in
these puncta have all the hallmarks of podosomes. Using a phage
display library of all 296 human SH3 domains we identified Tks5, a
key regulator of podosomes, as interacti
-dystroglycan. We
verified the interaction by immunoprecipitation, GST-pulldown and
immunfluorescence localisation. Both proteins localise to small puncta
during early phases of spreading, but importantly following stimulation
by PDBu also localise to structures indistinguishable from podosomes.
-dystroglycan and Tks5 relies of
-dystroglycan by Src. Dystroglycan
depletion by siRNA, not only reduces overall cell adhesion and cell
motility- 135 -but also the formation of podosomes and recruitment of
Tks5 and cortactin to these structures. Interestingly dystroglycan
overexpression also inhibited podosome formation, but mutation of
Tyr892, previously identified as a Src substrate, restored podosome
formation. We therefore propose that Src-dependent phosphorylation
-dystroglycan drives the SH3-mediated association between
dystroglycan and Tks5 which together regulate podosome formation.
PW31-392
CHANGES IN GLYCOSAMINOGLYCANS DURING SKELETAL MUSCLE
REGENERATION AND SATELLITE CELL DIFFERENTIATION IN VITRO.
OUDGHIR M1, BARBOSA I2, ZIMOWSKA M3, JENNISKENS G4, DUCHESNAY A5,
PAPY-GARCIA D5, MARTELLY I5
(1) Faculty of Sciences, Cady Ayyat University, marrakech, MOROCCO. (2) CRRET
laboratory, present address Sanofi-Aventis, Vitry, FRANCE. (3) Department of
Cytology, Faculty of Biololgy, University of Warsaw, Warsaw, POLAND. (4) ModiQuest
Company, Nijmegen, THE NETHERLANDS. (5) CRRET laboratory, University ParisEst, Faculté de Sciences et Technologie, Paris 12, Créteil, FRANCE.
Together with other extracellular matrix and membrane-associated components
that contribute to the cellular environment of muscle cells, proteoglycans (PG) have
To contact the author::
[email protected].
been implicated in numerous physiological and pathological processes including
regulation of enzymes and growth factor bioavailability, cellular growth and
differentiation. The known effects of PG are mainly due to the sulphated
glycosaminoglycan (GAG), namely dermatan sulphate/chondroitin sulphate
(DS/CS) and heparan sulphate (HS). It is generally accepted that unique tissue
specific HS sequences are generated by biosynthetic enzymes that produce this
type of molecules, key regulators of cell signaling. It is therefore of special interest
to analyze the spatio-temporal pattern of GAG expression during skeletal muscle
regeneration.
We have used a crush-induced muscle regeneration model that generates
differences in rat fast EDL and slow Soleus regenerating muscles. Using a simple
sensitive method of GAG concentration measurement developed in our laboratory,
GAGs were quantitated during regeneration of these muscles. The study was
further extended to in vitro differentiation of primary cultures of satellite cells.
Different GAG epitopes were visualized using specific antibodies applied on
regenerating muscle sections and on myoblasts differentiating in vitro.
We have demonstrated changes in the composition of GAG extracted from EDL and
Soleus muscles during regeneration. In both muscle types, total GAG amounts were
collapsed one day after crush, then increased during muscle repair. HS that was less
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abundant than DS/CS during the first week after crush, and became the most
important GAG species starting at the second week of muscle reconstruction. Similar
changes in GAG composition were observed during in vitro satellite cell differentiation.
Time-dependent specificities were revealed that depended on muscle of origin (EDL or
Soleus), in both in vivo and in vitro studies. We propose that changes in GAG
environment of myogenic cells might alter signalling events associated to myogenesis.
PW31-393
To contact the author::
[email protected].
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OPPOSITE ROLES OF CONTROLLED EXPRESSION OF THE INITIATION
FACTOR EIF3-F IN SKELETAL MUSCLES.
CSIBI A1, LAGIRAND-CANTALOUBE J1, OFFNER N1, LEIBOVITCH MP1,
BARBOIRON C2, PICARD B2, LEIBOVITCH S1
(1) Laboratoire de Génomique Fonctionnelle et Myogénèse, UMR 866 DCC, INRA,
Campus INRA/Supagro, Montpellier, FRANCE. (2) URH Theix, INRA, ClermontFerrand, FRANCE.
Skeletal muscle size depends upon a dynamic balance between anabolic and
catabolic processes. The E3 ubiquitin-ligase MAFBx/Atrogin-1 is upregulated during
muscle atrophy caused by a variety of conditions, including cancer, AIDS, stress,
diabetes and starvation. However, its precise function in muscle wasting is not yet
completely elucidated. With the aim to identify new MAFbx targets during atrophy, we
screened a human adult skeletal muscle library yeast two-hybrids and identified eIF3fp47 as an interacting partner. MAFbx also targeted eIF3f for its ubiquitylation and
subsequent proteasome degradation. Conversely, blocking MAFbx expression by
shRNAi prevented eIF3f degradation during muscle atrophy. To address the question
of how functionally relevant is eIF3f in muscle homeostasis, we used a controllable
protein knockout method by using full-length eIF3f-antisense-RNA. Genetic activation
of eIF3f caused hypertrophy and blocked atrophy in myotubes, whereas blocking
eIF3f expression induced atrophy. Finally, overexpression of eIF3f showed an
increase of sarcomeric proteins and hypertrophy in both myotubes and mouse skeletal
muscle, as confirmed by comparative proteomics. Our results indicate that eIF3f is a
key target for MAFbx during atrophy and plays a major role in skeletal muscle
hypertrophy.
PW31-394
To contact the author::
[email protected].
ASB2BETA, A NOVEL ACTOR OF MUSCLE DIFFERENTIATION
BELLO NF1, HEUZÉ ML1, MÉTAIS A1, DUPREZ D2, MOOG-LUTZ C1, LUTZ PG1
(1) Institut de Pharmacologie et de Biologie Structurale, UMR 5089 CNRS, Université
Paul Sabatier, Toulouse, FRANCE. (2) UMR 7622 CNRS, Université Pierre et Marie
Curie, Paris, FRANCE.
The covalent linkage of a polyubiquitin chain to a protein and its subsequent
targeting to the 26S proteasome is one of the major mechanisms for controlled
proteolysis. The specificity of this degradation pathway is due to E3 ubiquitin ligases
involved in the recruitment of specific substrate(s). Ubiquitin-mediated protein
degradation is crucial for muscle development and for maintenance of muscle
homeostasis. The ubiquitin-proteasome pathway also regulates the rapid proteolysis
associated with muscle wasting which is induced by metabolic or catabolic diseases.
However, few E3 ubiquitin ligases involved in these processes and their specific
substrates have been described so far.
The ankyrin repeat-containing protein with a suppressor of cytokine signaling
box 2 (ASB2) gene that we originally identified as induced during differentiation of
myeloid leukaemia cells encodes the specificity subunit of a multimeric E3 ubiquitin
ligase complex. This suggests that ASB2 regulates the stability of specific proteins via
their polyubiquitination and proteosomal degradation. We provide the first evidence
that a novel ASB2 isoform, ASB2
in vitro
muscle differentiation and appeared with the differentiation commitment of C2C12
myoblasts. Its inhibition by shRNAs during induced-differentiation of C2C12 cells
delayed myotube formation and expression of muscle contractile proteins. Moreover,
ASB2 is upregulated during induced-atrophy of C2C12 myotubes suggesting that it
might be also involved in this catabolic state. We also showe
assemble with the Elongin BC complex and a Cullin5/Rbx2 module to reconstitute an
active E3 ubiquitin ligase complex.
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PW31-395
Altogether, our results suggest that ASB2
is involved in muscle
differentiation likely through the targeting of crucial muscle proteins to destruction by
the proteasome.
CYTOKINES ALTER NUMBER AND FUNCTION OF CELL POPULATIONS
RELEVANT TO SKELETAL MUSCLE HOMEOSTASIS
COLETTI D1, BERARDI E1, AULINO P1, MORESI V1, PRISTERÀ A1, SASSOON D2,
MOLINARO M1, ADAMO S1
(1) Sapienza University, Rome, ITALY. (2) Groupe Myologie, Paris, FRANCE.
Chronic exposure to tumor necrosis factor-alpha (TNF) triggers muscle wasting
To contact the author::
reminiscent of cachexia (1), a debilitating syndrome characterized by skeletal muscle
[email protected] wasting (2). In addition to TNF-treated muscle we exploited tumor (C26)-bearing mice
.
as a model of cachexia to study whether stem cell number and function are altered in
muscle wasting. We observed that in the presence of elevated levels of cytokines
several populations of cells with myogenic potential are stable or increased in muscle,
including satellite (Pax7-expressing) cells, hematopoietic stem (Sca1- CD45expressing) cells and muscle interstitial stem (Sca1- CD34-expressing) cells also
characterized by PW1expression (3,8). PW1 is involved in myogenic cell
differentiation, fiber size control and p53-mediated apoptotic pathways (4-7). The
increase in myogenic cells in cachectic muscle suggests an attempt to cope with
wasting by recruitment and/or activation of a myogenic response. Nonetheless, we
observed that the regenerative capacity of skeletal muscle is reduced by cytokines (1,
8). To characterize the molecular mechanisms underlying stem cell impairment in
cachexia, we treated injured muscle with TNF (8). TNF negatively affected the onset
of regenerating fibers, characterized by centrally located nuclei, without exacerbating
fiber death following the initial trauma. Several cells showed caspase activity during
regeneration and the number of caspase activated cells was markedly increased by
TNF, concomitant with an inhibition in regeneration. Caspase activation did not involve
caspase-3 nor led to apoptosis. Inhibition of caspase activity improved muscle
regeneration either in the absence or presence of TNF, suggesting a non apoptotic
role for this pathway in the myogenic program. Cells with caspase activity, were
localized in the interstitial compartment and could be identified by the expression of
Sca1, CD34 and PW1. Perturbation of PW1 activity blocked caspase activation and
improved regeneration indicating a pivotal role for PW1 in controlling stem cell
function and muscle regeneration (8).
REFERENCES
1) Coletti D. et al.Genesis. 2005;43(3):120-8.
2) Coletti D. et al. Basic Appl Myol. 2006; 16(5&6):131-139.
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3) Berardi E. et al., Neurol Res., in press.
4) Coletti D. et al. EMBO J. 2002;21(4):631-42.
5) Schwarzkopf M. et al. Genes Dev. 2006; 20(24):3440-52.
6) Relaix F. et al. Proc Natl Acad Sci U S A. 2000;97(5):2105-10.
7) Relaix F. et al. Dev Biol. 1996;177(2):383-96.
8) Moresi et al. Stem Cells, in press.
PW31-396
MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE
HOMEOSTASIS: EFFECTS OF V1A VASOPRESSIN RECEPTOR OVEREXPRESSION
SCICCHITANO B1, TOSCHI A1, MURFUNI I1, MOLINARO M1, ADAMO S1
(1) Sapienza University, Rome, ITALY.
To contact the author::
bianca.scicchitano@unir
oma1.it.
The maintenance of a working skeletal musculature is conferred by its remarkable
ability to regenerate after mechanical or pathological injury. However muscle atrophies
are characterized by the progressive loss of muscle tissue due to alterations of
skeletal muscle homeostasis. In particular cachexia is a severe syndrome consisting
of marked skeletal muscle atrophy, characterized by a dramatic loss of muscle mass
associated with a compromised regenerative ability. Arg-vasopressin (AVP) is a
potent myogenesis promoting factor and activates both the calcineurin and CaMK
pathways, whose combined activation leads to the formation of transcription factor
complexes in vitro (2, 3). The local over-expression of the V1a AVP receptor (V1aR) in
injured muscle results in enhanced regeneration. V1aR over-expressing muscle
exhibits: early activation of satellite cells and regeneration markers, accelerated
differentiation, increased cell population expressing hematopoietic stem cell markers
and its conversion to the myogenic lineage.Here we investigate the role of V1aR overexpression in animals undergoing cachexia as a result of muscle over-expression of a
specific cytokine (TNF) (1). In these conditions, the local V1aR over-expression
counteracts the negative effects of TNF on muscle, as demonstrated by morphological
and biochemical analysis. In particular, the presence of V1aR results in increased
Pax-7, myogenin and myosin expression levels both in control and in cachectic
muscles. We demonstrate that V1aR over-expressing muscle increases calcineurin
and IL-4 expression levels, and induces the phosphorylation of FOXO trascription
factors, inhibiting the expression of atrophic genes.This study highlights a novel in vivo
role for the AVP-dependent pathways which may represent a potential gene therapy
approach for many diseases affecting muscle homeostasis.
Reference List
1 -Coletti,D., Moresi,V., Adamo,S., Molinaro,M., Sassoon,D. (2005). Tumor necrosis
factor-alpha gene transfer induces cachexia and inhibits muscle regeneration.
Genesis. 43, 120-128.
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2 -Scicchitano,B.M., Spath,L., Musaro,A., Molinaro,M., Adamo,S., Nervi,C. (2002).
AVP induces myogenesis through the transcriptional activation of the myocyte
enhancer factor 2. Mol.Endocrinol. 16, 1407-1416.
3 -Scicchitano,B.M., Spath,L., Musaro,A., Molinaro,M., Rosenthal,N., Nervi,C.,
Adamo,S. (2005). Vasopressin-dependent Myogenic Cell Differentiation Is Mediated
by Both Ca2+/Calmodulin-dependent Kinase and Calcineurin Pathways. Mol.Biol.Cell.
16(8):3632-41.
PW31-397
GENE EXPRESSION CHANGES IN ISOLATED MYOFIBRES
DUDDY W 1, COHEN T1, PARTRIDGE T1
(1) Children's National Medical Center, Washington DC, USA.
To contact the author::
bduddy@cnmcresearch.
org.
Loss of muscle mass through atrophy is associated with aging, cachexia, inactivity
through illness or injury, and many neuromuscular disorders. Regulation of muscle
mass is important for strength and fitness of healthy individuals. Change in muscle
mass involves modification of myofibre size or number. Modification may be effected
during regeneration events involving myoblasts and other cell types, or by the direct
action of growth modulators on the myofibre itself. Signalling between fibres and their
cellular environment may also be important. Aberrant regulation of fibre size is
associated with many neuromuscular disorders, though it is unclear whether greater
fibre size protects against necrosis. Myoblast cell lines have provided much data
concerning the effects of modulators of muscle growth on the proliferation and
differentiation events necessary for regeneration, but they tell us little about effects on
the myofibre itself. We have characterized the isolated myofibre model by microarray
expression profiling one and two days post-isolation. Over 2,000 genes are altered.
Results suggest some atrophic character, with down-regulation of muscle contractile
proteins, and up-regulation of the proteasome and ubiquitinylation. The model will be
further characterized at the protein level and a complementary in vivo denervation and
tenectomy study is underway. A set of genes is selected that change post-isolation
and are involved in pathways related to atrophy. Regulation of these target genes is
being followed by rtPCR of myofibres treated with IGF-1, Follistatin, and Myostatin.
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PW31-398
GROWTH CHARACTERISTICS OF SATELLITE CELLS IN MIXED CULTURES AND
SINGLE CLONES FROM THE SAME INDIVIDUAL.
MAIER A1, COHEN R1, BLOM J1, WESTENDORP R1
(1) Leiden University Medical Center, Department of Gerontology and Geriatrics,
Leiden, THE NETHERLANDS.
To contact the author::
[email protected].
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Sarcopenia is defined as a decrease in skeletal muscle mass that is particularly
caused by satellite cells being unable to proliferate. The number of satellite cells, as
well as the proliferative capacity of isolated satellite cells in vitro decreases with
increasing chronological age. However, even at the end of human lifespan, myoblast
cultures can be established out of muscle biopsies even in the presence of
sarcopenia. As satellite cell characteristics have predominantly been assessed using
mixed cultures and not compared to studies using single clones, the importance of
these in vitro characteristics are uncertain.
We established a mixed myoblast culture and three clonal myoblast cultures
out of the same muscle biopsy obtained from a middle aged man and cultured these
cells for 100 days. We found a significantly lower replicative capacity of the myoblast
clones when compared to the mixed culture. Replicative capacity was inversely
related to the beta-galactosidase activity after exposure to oxidative stress when
cultures were tested at an earlier passage. Remaining replicative capacity was greatly
enhanced in all four cultures when carnosine was supplemented to the medium,
whereas the beta-galactosidase activity at the early replicative stage remained
unchanged.
We conclude that proliferative capacity of satellite cell in mixed cultures in
vitro do not reflect characteristics of single clones. The in vitro characteristics of a
mixed culture is likely to represent few dominant clones. The impact of the huge
variation in replicative potential of each single cell on the in vivo situation remains to
be established.
PW 32:
Functional assessment
of muscle
PW32-399
SCANNING ION CONDUCTANCE MICROSCOPY (SICM): A NEW TECHNIQUE TO
INVESTIGATE THE DEVELOPMENT OF PATHOPHYSIOLOGICAL PHENOTYPE
OF MEMBRANE SURFACE IN DYSTROPHIN-DEFICIENT MUSCLE CELLS?
COGNARD C1, CONSTANTIN B1, DUCLOHIER H1, SEBILLE S1
(1) IPBC 6187 Université de Poitiers/CNRS, Poitiers, FRANCE.
To contact the author::
[email protected].
Since the cytoskeleton provides the cell with structure and shape, and participates in
building specialized domains with specific functions, it will be interesting to compare
the membrane surface topography in control muscle cells with the one of dystrophindeficient cells.
A glass nanopipette is filled with a physiological electrolyte and lowered
through a Petri dish containing the same electrolyte and the living cells while the
conductance between an electrode inside the pipette and a reference electrode in the
bath is monitored. As the tip of the micropipette approaches the cell surface, the ion
conductance decreases because the space through which ions can flow is decreased.
The pipette is then scanned (in X-Y two dimensions) over the membrane while a
feedback device raises and lowers the micropipette to keep the conductance constant.
Thus, the path of the pipette tip follows the topography of the surface. A key feature of
the technique is its scanning 'algorithm' which maintains constant the distance
between the pipette tip and the sample.
In our experiments membrane surfaces of myotubes from minidystrophinexpressing cell line (SolD) and from dystrophin-deficient cell line (SolC) in culture
were scanned and the topographical images built and compared. An unexpected
longitudinally grooved and complex surface was observed especially in dystrophinexpressing myotubes. This constitutes the first observations of cell surface in skeletal
muscle preparations with this technique.
Further experiments will be conducted to check if the membrane topography
differences observed in the present preliminary experiments were directly related to
absence/presence of dystrophin, and to elucidate the possible link with the
normal/pathological function of the membrane embedded channels permeable to
calcium which are reported to be involved in calcium homeostasis perturbation
observed in dystrophin-deficiency related diseases like Duchenne muscular
dystrophy.
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PW 32-400
ASSESSMENT OF MUSCLE CONTRACTION BY DIFFUSION TENSOR IMAGING
DEUX JF1, BASSEZ G2, MALZY P3, PARAGIOS N4, LUCIANI A1, ROUDOTTHORAVAL F5, VIGNAUD A6, RAHMOUNI A1
(1) Service d’Imagerie Médicale, CHU H. Mondor, APHP, Créteil, FRANCE. (2)
INSERM U841-E10 (Institut Mondor de Recherche Biomédicale), Paris 12 University,
Créteil, FRANCE. (3) Service d’Imagerie Médicale, CHU Lariboisière, Paris,
FRANCE. (4) Ecole Centrale de Paris, Chatenay Malabris, FRANCE. (5) Centre
d’Investigation Clinique, CHU H. Mondor, APHP, Créteil, FRANCE. (6) Siemens
Medical division, Paris, FRANCE.
To contact the author::
guillaume.bassez@hmn.
aphp.fr.
The goal of this study was to assess the changes of water diffusion during
contraction and elongation of calf muscles using Diffusion Tensor (DT) MRI in normal
volunteers. Twenty volunteers (mean age, 29 ± 4 years) underwent DT MRI
examination of the right calf. Echo Planar Imaging sequence was performed at rest,
during dorsal flexion and during plantar flexion. The 3 eigenvalues (λ1, λ2, and λ3),
Apparent Diffusion Coefficient (ADC) and Fractional Anisotropy (FA) of the diffusion
tensor were calculated for medial gastrocnemius (mGM) and tibialis anterior (TA). A
fiber tractography was performed on both muscles. Non parametric Wilcoxon and
Mann Whitney tests were used for statistical evaluation. At rest, λ1, λ2 and ADC of
mGM were higher than their counterparts of TA (P<0.01). During dorsal flexion, the 3
eigenvalues and ADC of TA significantly increased (P<005) as their counterparts of
mGM slightly decreased (P=NS). Opposite variations were detected during plantar
flexion of the foot. Visual analysis evidenced a relationship between 3D
representations of MRI fibers and physiological state of muscles. Contraction of calf
muscles produces changes in DT parameters, which are related to the physiological
state of the muscle.
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PW32-401
EFFECT OF MATURATION ON THE RELATIONSHIP BETWEEN MUSCLE SIZE
AND FORCE PRODUCTION
TONSON A1, RATEL S2, LE FUR Y1, COZZONE P1, BENDAHAN D1
(1) Center for Magnetic Resonance in Biology and Medicine (CRMBM), UMR CNRS
6612, MARSEILLE, FRANCE. (2) Laboratory of Exercise Biology (BAPS), EA 3533,
AUBIERE, FRANCE.
To contact the author::
david.bendahan@univm
ed.fr.
The purpose of this investigation was to determine whether maturation affects the
relationship between muscle size and maximal strength and to investigate the reasons
accounting for the discrepancies among previous studies. Methods: Maximal
isometric handgrip force (Fmax) and forearm muscles size were measured in 14 prepubertal boys (11.3 ± 0.8 y.o.), 16 adolescents (13.3 ± 1.4 y.o.) and 16 men (35.4 ±
6.4 y.o.). MRI was used to measure anatomical maximal cross-sectional area (MCSA)
and muscle volume (VM). VM was compared with anthropometric measurements of
muscle volume (VL). Results: Fmax was linearly correlated with VM (r²=0.90), VL
(r²=0.85) and MCSA (r²=0.87). The Fmax/VM ratio did not differ between groups. By
contrast, Fmax/VL and Fmax/MCSA were significantly higher in adults than in children
and adolescents. Additionally, VM was strongly correlated to VL (r²=0.90). This
relationship demonstrated that, when compared to MRI, anthropometric
measurements lead to a systematic overestimation of muscle volume which was
significantly larger in children and adolescents than in adults (43.1 %, 38.5 % and 20.5
% p< 0.05 respectively). Conclusion: Our results showed that the maximal isometric
strength which can be exerted by the forearm human muscles is proportional to its
size whatever the age. During growth VM was the best index of muscle size. We
suggest that the previously reported increased ability to produce maximal strength
from childhood to adulthood could be explained by systematic bias introduced by the
method used in order to characterize muscle size and not by physiological or neural
changes.
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PW32-402
A NEW EXPERIMENTAL SETUP FOR INVESTIGATING SKELETAL MUSCLE
FUNCTION STRICTLY NON-INVASIVELY IN RAT USING NUCLEAR MAGNETIC
RESONANCE SPECTROSCOPY AND IMAGING
GIANNESINI B1, IZQUIERDO M1, LE FUR Y1, COZZONE PJ1, FINGERLE J2,
HIMBER J2, KÜNNECKE B2, VON KIENLIN M2, BENDAHAN D1
(1) Centre de Résonance Magnétique Biologique et Médicale (CRMBM), UMR 6612
CNRS - Université de la Méditerranée, Faculté de Médecine de la Timone, Marseille,
FRANCE. (2) F. Hoffmann-La Roche Ltd, Discovery Biology, Basel, SWITZERLAND.
Introduction. Magnetic resonance (MR) techniques have proven their ability to
investigate skeletal muscle function in situ. However, in animal research, the benefit
To contact the author::
provided by MR techniques in terms of non-invasiveness is lost because of the
benoit.giannesini@univm utilization of invasive procedures for inducing muscular contractions (via nerve
ed.fr.
stimulation) and for measuring force output (via attachment of a transducer to the
muscle tendon). Required surgical preparation thus prohibits repeated investigations
on the same animal. We have developed a new experimental setup allowing strictly
non-invasive MR investigations of muscle function in contracting rat gastrocnemius
muscle. In this study, we have compared its performance with that of a traditional
invasive setup.
Methods. The new setup integrates four non-invasive systems allowing prolonged
anesthesia with control of the animal’s body temperature, transcutaneous electrical
stimulation of the gastrocnemius muscle, force measurement with a dedicated
ergometer, and two MR probes for 31-phosphorus spectroscopy and proton imaging.
Muscle function was investigated in 20 rats through a fatiguing stimulation protocol,
either with this new setup (n = 10 rats) or with a traditional setup (n = 10).
Results. Muscle functional MR imaging demonstrated that transcutaneous stimulation
specifically activated the gastrocnemius muscle. During the stimulation period,
changes in isometric force production and energy metabolism (intramuscular pH and
phosphorylated compounds) were similar for both setups.
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PW32-403
Conclusion. The non-invasive setup is then suitable for investigating skeletal muscle
function in situ, allowing mechanical performance, energy metabolism, anatomy and
physiology to be accessed strictly non-invasively in contracting rat gastrocnemius
muscle. This non-invasive alternative makes surgical preparations obsolete and
represents a major advance for the future investigations of skeletal muscle function in
animal models. Longitudinal studies become readily feasible and each rat can serve
as its own control, thus reducing inter-individual variability, workload and costs, hence
opening up new perspectives in various research areas including pharmaceutical
research.
DEVELOPMENT OF MUSCULAR STRENGTH IN SARCOPENIA : COMPARING
THREE DIFFERENT TRAINING METHODOLOGIES
IODICE P1, DI TANO G1, DORIA C1, SAGGINI A2, SAGGINI R1
(1) Dept. Basic and Applied Medical Science, "G. dAnnunzio" University, Chieti,
ITALY. (2) La Sapienza University, Roma, ITALY.
To contact the author::
pierpaolo_iodice@yahoo
.it.
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PW32-404
Aging is characterised by a gradual decrease in muscle mass and muscle
strength which contributes to a decline in physical functions, increase
disability, frailty, and loss of independence. Age related loss of muscle mass is
referred to as sarcopenia (Argiles JM et al., Adv Gerontol.18:39-54,2006).
Regular training programs are a concrete means to prevent and/or reduce
functional decline due to aging (ACSM, Med Sci Sports Exerc.1998
Jun;30(6):992-1008), although the optimum regime specific for older adults
remains unclear. Twenty-eight subjects (12♂ and 16♀) of 75±10 years old with
a diagnosis of grade 3 Sarcopenia (CDC) were randomly assigned to three
different training programs, 3 day/wk for 12 wk, high intensity local vibrational
program (intensity: 300hz), endurance program (intensity: 60-70% HRmax)
and resistance program (intensity:60-80%Fmax, 10-12 repetitions for 3sets).
Before and after the training programs a muscle samples were collected from
vastus lateralis muscle by biopsy. These were used to analyse the specific
tension development of single fibers.
At the same time, the Isometric lower limb force was measured by
dynamometer. The Myoton-2 equipment was used to describe the viscoelastic
parameters of the skeletal muscles: the frequency of damped mechanical
oscillation of the muscle tissue (Hz), logarithmic decrement of the oscillations
(Θ) and stiffness (Nm-1) of the muscle tissue. As follow-up the subjects was
tested 3 months after protocol end.
Our results indicate that resistance and vibrational training increase muscular
strength(p<0.05). This improvement was maintained at 3 months. This study
shows increase of muscular elasticity and muscular tension. Variation in
muscular stiffness is not significant. The relation between strength, the
viscoelastics parameters and the specific tension of fibers have been
analysed.
In conclusion, different training programs cause specific adaptation in muscles
in the elderly. Resistance and vibrational programs seem to have better results
in counteracting muscles decline due to aging.
CELLULAR AND MOLECULAR MODIFICATIONS INDUCED BY THREE SPECIFIC
TRAINING IN AGED HUMAN SKELETAL MUSCLE
PIETRANGELO T1, PUGLIELLI C1, BOSCO G1, TONIOLO L2, REGGIANI C2,
BELLOMO RG3, DI PANCRAZIO L3, FANÒ G1
(1) Dept. Basic and Applied Medical Science, Interuniversity Institute of
Myology,University G. d’Annunzio, Chieti, ITALY. (2) Dept. of Human Anatomy and
Physiology, Interuniversity Institute of Myology, University of Padova, Padova, ITALY.
(3) Dept. Basic and Applied Medical Science, University G. d’Annunzio, Chieti,
ITALY.
To contact the author::
[email protected].
Sarcopenia is a scientific term indicating the physiological reduction of skeletal muscle
mass and strength in older people. Sarcopenia has a multifactorial origin linked to:
oxidative damage of fibers (Fulle et al, Exp. Gerontol 40:189, 2005), mitochondrial
damage (Brunk UT et al, Eur J Biochem, 269(8):1996-2002, 2002) reduced levels of
GH, IGF-1, steroids and reduced myogenesis (Beccafico et al., ANNALS 1100:345352, 2007). The physical activity is able to slow down and/or revert this condition
(Taaffe DR et al, Clin Physiol 16:381- 391, 1996). We analysed the effects of three
different physical activity programs (endurance training, resistance training and local
vibrational energy) on vastus lateralis biopsy derived by 65-85 years old people,
before and after the training.
From these muscle fragments we analysed: (i) the specific tension development of
single fibers and the expression of myosin heavy chain proteins; (ii) the transcriptional
profile and (iii) the regenerative capacity of satellite cells. The single fiber strength
development do not change with any training protocol. Considering the gene
expression profiles, each physical activity share a stimulation of a specific metabolic
pathway; both endurance and vibrational training increase the aerobic metabolism
while the resistance training stimulates the creatine metabolism. All the training, in a
different manner, stimulate the expression of sarcomeric and cytoskeletal proteins and
in particular the vibrational training stimulates proteins linked to Z-line. Moreover, the
endurance training induces the expression of protein for neuronal chemotaxis. We
studied also the behavior of satellite stem cells after the specific training and their
contribution to regeneration process and fiber trophism. In conclusion, our results
suggest that all our training counteract Sarcopenia progression and each of them are
able to stimulate a specific molecular signaling. The effects are specific because it
exists coherence between exercise typology and stimulated metabolism.
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PW32-405
ASSESSMENT OF GRIP STRENGTH USING PRECISION DYNAMOMETRY
HOGREL JY1, LI K2, DUCHENE J2, HEWSON D2
(1) Institut de Myologie, Paris, FRANCE. (2) Institut Charles Delaunay - Université de
Technologie de Troyes, Troyes, FRANCE.
To contact the author::
[email protected].
Grip dynamometry is an important and easy-gathered method to evaluate hand
function. Reliable and valid evaluation of grip strength depends on the quantified
measurements and standardised testing procedures. A major drawback of all
commercially available grip handles is that they are not adapted to weak patients,
particularly during therapeutic trials.
To this end, the Myogrip was designed for the assessment of grip strength of weak
patients suffering from various neuromuscular disorders such as muscle dystrophies.
The device was built around a precision force sensor (full scale: 89 daN; accuracy:
0.05 daN; sensitivity: 0.01 daN). The Myogrip can complete several functions: real
time display, maximal force recording, wireless communication with a computer,
RS232 or BNC connections. The handle size is finely adjustable.
Several works are undergone using the Myogrip, aiming at:
- testing the effect of elbow positioning and grip handle size on maximal grip
strength
- comparing the Myogrip with the gold standard (represented by the Jamar)
and another available device (Martin Vigorimeter)
- developing a normative database on 450 subjects aged from 5 to 80 years
- assessing the reproducibility and repeatability of the measure on 120
subjects
- proposing a predictive model of grip strength to compute patient deficit
- testing the validity of the Myogrip for the evaluation of weak patients
Prelimlinary results showed no effect of elbow positioning. However when the elbow is
fully extended, patient evaluation was more reproducible, probably due to less
possibility of compensation and better patient maintaining by the evaluator. A
significant effect of grip handle size was observed. Results obtained with Myogrip and
Jamar were highly correlated (0.93). Test-retest reliability of all grip devices was
excellent (ICC higher than 0.96).
The Myogrip is a new device devoted to the measure of weak patients. Its validation is
ongoing and preliminary results show excellent reliability.
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PW32-406
QUALITY ASSURANCE AND METROLOGY STANDARDS IN FUNCTIONAL
EVALUATION OF NEUROMUSCULAR DISORDERS
OLLIVIER G1, TOUITA KABBAJ A1, CANAL A1, ROQUES S1, HOGREL JY1
(1) Institut de Myologie, Paris, FRANCE.
To contact the author::
[email protected].
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PW32-407
Many techniques and methods exist to evaluate the functional capacity of patients
suffering from neuromuscular disorders. Because each evaluator/physiotherapist (PT)
has different skills and because each trial centre has different tools and practices, it is
essential to standardize techniques and methods for the functional assessment of
patients, particularly for multicentric studies, in order to improve the reproducibility and
consistency of results.
At the Institute of Myology, the Neuromuscular Physiology and Evaluation Lab,
composed of 6 persons including 3 Physiotherapists, is developing a quality system
based on the international standard ISO/CEI 17025 "General requirements concerning
the competence of the laboratories of calibrations and tests".
The aim of this process is:
- to guarantee the reproducibility and robustness of the results delivered
- to reinforce our credibility with investigators, industrial partners and drug
companies
The first section concerns the management of the laboratory. Main requirements are:
- to understand study objectives fully and adapt functional evaluations in
consultation with investigators
- to manage all the documents linked to a clinical trial according to formal
procedures
- to improve communication and follow-up with investigators by meetings,
contracts and questionnaires
- to detect all variations from correct procedures in order to improve the
system
The second section concerns measurement and metrology to ensure:
- competence of the personnel by training and checking the reproducibility
within- and between-PTs
- proper control of equipment (functioning and calibration)
- respect of evaluation procedures
- metrological quality of equipment (compliance with national standards)
- development and validation of new methods
ISO 17025 is an industrial standard which needs some adaptations to be transferred
to clinical settings. We plan to be accredited at the end of the year 2008 by the
COFRAC (French Committee of accreditation). For scientific, ethical and financial
reasons, it is fundamental to apply formal and rigorous methodology to ensure the
quality of measurements. Quality assurance and metrology standards help towards
reaching this goal.
ASSESSMENT OF FLEXION AND EXTENSION TORQUES OF WRIST AND
ANKLE USING SPECIFIC DYNAMOMETERS
HOGREL JY1
(1) Institut de Myologie, Paris, FRANCE.
To contact the author::
[email protected].
Evaluation of wrist and ankle flexion and extension has rarely been considered in
neuromuscular disorders, particularly because their assessment is difficult using
classical methods, either by hand-held dynamometry or by quantified muscle testing.
This work aimed to develop two different dynamometers to study these muscle
functions.
The wrist device allows the measurement of the torque generated around the wrist
joint during voluntary isometric contractions in the flexion/extension directions. The
wrist dynamometer is built around a static torque sensor presenting a full scale
(nominal torque) of 25 Nm, an accuracy of 0.05 Nm and a sensitivity of 0.0025 Nm.
The ankle device was designed to measure strength of ankle flexion and extension
using two different settings. The device is made of a foot plate under which two
precision load cells are fixed. From each load cell is attached an extremity of a strap.
This strap is then tighten over the fifth metacarpal joint (for ankle flexion) or over the
knee (for ankle extension). The full scale is 200 daN; the accuracy is 0.1 daN and the
sensitivity is 0.01 daN. Force values (in N) are converted into torque values (Nm) by
measuring the lever arm.
Fatigue assessment during static contractions and timed tests are also possible with
both dynamometers because they can be connected to a computer.
The work was firstly devoted to test the technical validity and the reliability of both
instruments. At the present time maximal voluntary contractions of wrist and ankle
flexion and extension were measured on 250 subjects aged from 5 to 80 years to build
normative data. Repeatability and reproducibility were tested on 60 subjects. Wrist
flexion and extension and ankle flexion presented excellent intraclass correlation
coefficients (ICC higher than 0.9) whereas ankle extension presented lower ICC
(about 0.7). Both devices were also tested in some very weak patients suffering from
various neuromuscular disorders and demonstrated their ability to detect small
contractions and their good reproducibility.
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PW32-408
GAIT ANALYSIS OF NEUROMUSCULAR PATIENTS USING ACCELEROMETRY
HOGREL JY1, CANAL A1, BARREY E2, OLLIVIER G1
(1) Institut de Myologie, Paris, FRANCE. (2) LEPHE-INRA - Genopole, Evry,
FRANCE.
To contact the author::
[email protected].
Quality of gait is a fundamental outcome measurement for the follow-up of
neuromuscular patients either during the natural history of their disorder or during a
therapeutic trial. Precise gait analysis can be performed using kinematic systems in
lab conditions. Unless very useful, the measurement and analysis procedures are long
and complex and not always suitable for clinical routine depending on the patients.
Accelerometry can bring much useful information on gait without many constraints for
the patient or the medical staff. This has been considered in the present work during
six-minute walk tests (6MWT).
The gait analysis system used in this study (Locometrix-2™) includes three
accelerometers in a small (20 x 40 x 80 mm) and light (50 g) box and a data logger.
The apparatus is incorporated into a semi-elastic belt, which is fastened around the
subject's waist, close to the centre of gravity. Signals were recorded with a sampling
frequency of 100 Hz. The recorded signals were transferred to a computer and
analyzed by a specific software. Several gait variables were computed on a sample of
10 s of stationary gait: walking velocity, stride frequency and length, symmetry and
regularity indexes, mechanical power and high frequency shocks.
As a pilot study, the measurements were performed on 12 patients suffering from acid
maltase deficiency during 6MWT. Their results were compared to normative data
available from more than 400 healthy subjects and show deep modifications of their
gait depending on the severity of their disorder.
Accelerometry is a very simple tool to assess gait quality. Measurements will also be
considered at home in further studies.
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PW32-409
MEASUREMENT OF BODY HEIGHT IN PERSONS WITH NEUROMUSCULAR
DISEASES
ZUPAN A1, BALANTIC Z2, CERU B3
(1) Institute for rehabilitation of the Republic of Slovenia, Ljubljana, SLOVENIA. (2)
Faculty of organizational sciences, Kranj, SLOVENIA. (3) Institute for rehabilitation of
the Republic of Slovenia, Ljubljana, SLOVENIA.
To contact the author::
[email protected]
es.si.
Information about a person's body height is needed for calculating numerous formulas
such as for vital capacity. Classical method of measuring body height (client in
standing position) is not usable in wheelchair-bound persons with neuro-muscular
diseases (NMD), particularly not in those with physical deformations. We tried to find a
new method of measuring and predicting body height on the basis of measuring
certain body parameters – segments in upper and lower limbs. We chose 12 body
segments, which were considered to be measured easily in persons with physical
deformities. First we conducted a pilot study on 18 healthy persons, whose body
heights were measured by classical method. Subsequently the study was performed
in 60 persons with NMD, whose body heights were measured with a tailor's tape
measure in lying position and we were trying to consider all physical deformations,
above all scoliosis and flexor contractures in hips, knees and ankles. A good
correlation between the chosen parameters and the body heights was found in healthy
and also in persons with NMD. Statistical analysis proved that by measuring one of
the newly defined parameters we can assess the body height with 0,63 reliability rate.
The study has proven that by measuring the newly defined parameters the body
height can be well assessed. We made a mathematical model to help us to define the
body height, by using at least three chosen body parameters, those three, which
correlate best with the body height and which, considering the specific physical
disability and the present body deformities, can be measured most easily and
objectively.
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PW32-410
REHABILITATION PROGRAMS
DISEASES IN SLOVENIA.
FOR
PERSONS
WITH
NEUROMUSCULAR
ZUPAN A1, PRAZNIKAR A1, SARDOC M2
(1) Institute for rehabilitation of the Republic of Slovenia, Ljubljana, SLOVENIA. (2)
Muscular Dystrophy Association of Slovenia, Ljubljana, SLOVENIA.
To contact the author::
[email protected]
es.si.
Neuromuscular diseases are inherited, chronic, degenerative and progressive. The
main characteristics of neuromuscular diseases are: muscular weakness,
contractures, scoliosis, respiratory insufficiency, cardiac affection, nutrition
disturbances, dependence on the help of others, possible social isolation and
physiological problems. Appropriate rehabilitation programs should influence all
mentioned characteristics. A special unit for rehabilitation of patients with
neuromuscular diseases within the Institute for rehabilitation of the Republic of
Slovenia was established in 1993 at the initiative of the Muscular Dystrophy
Association of Slovenia. The main aim of this establishment was to try to guide the
patient and his family through the course of the disease. The article described the
work of the mentioned unit. Different clinical rehabilitation programs for persons with
neuromuscular diseases are presented and some research results of the unit are
mentioned.
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PW32-411
DYNAMIC, MULTI-PARAMETER NMR IMAGING QUANTIFICATION IN HUMAN
CALF AT REST AND IN CONDITIONS OF REACTIVE HYPEREMIA
LOUREIRO DE SOUSA P1, VIGNAUD A2, CARLIER PG1
(1) Laboratoire de RMN AIM - CEA Institut de Myologie, Paris, FRANCE. (2)
Siemens Medical Solutions, Saint-Denis, FRANCE.
To contact the author::
[email protected].
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PW32-412
NMRI (Nuclear Magnetic Resonance Imaging) investigations have attempted
to exploit the Blood-Oxygen-Level-Dependent (BOLD) contrast in the skeletal muscle
as an estimator of physiological parameters. However, beside changes in hemoglobin
saturation, which induce changes in apparent T2 and T2*, other mechanisms are
involved in the BOLD contrast. For instance, it has been shown that BOLD contrast
depends both on perfusion and on vascular filling during reactive hyperemia (RH) in
human skeletal muscle [1].
To improve understanding of the origins of BOLD signal changes during RH,
interleaved multi-parameter measurements have been used [2]. Such experiments
have confirmed T2* and T2 changes, with no significant alteration in T1 and M0 [2]. In
the present work we investigated the feasibility of simultaneous measurement of NMR
parameters (T1, T2 and M0).
Experimental data were acquired on a 3.0 T whole body scanner (Siemens
TimTrio, Erlangen, Germany). Multiple parameters were obtained in human calf using
the IR TrueFISP [3] sequence. Image resolution = 2.3 x 2.3 mm 2, slice thickness = 8.0
mm.
Dynamic multi-parameter measurements were performed in healthy
volunteers who underwent a three phase protocol: rest (10 min), ischemia of the calf
(5-10 min), reactive hyperemia (15 min). Time resolution for these multiparametric
measurements was ~12 s. At rest, T1 and T2 maps showed excellent agreement with
those obtained from standard methods. In all volunteers parameter time-courses
showed a complex pattern, with concomitant changes in T1, T2 and M0, whose origins
need to be disentangled. The ability to monitor dynamically and simultaneously the
main parameters of the NMR signal might help to define indices that reflect muscle
oxygenation more closely than does standard BOLD contrast.
[1] Duteil et al, MRM 55, 450-4 (2006)
[2] Klarhöfer et al, Proc. ISMRM 3800 (2007)
[3] Schmitt et. al., MRM 51, 661-7 (2004)
INFLUENCE OF WORKLOAD ON HUMAN CALF MUSCLE FOLLOWING AEROBIC
EXERCISE:PERFUSION AND BOLD ASSESSMENT
MONNET A1, WARY C1, WUYAM B2, VERGES S2, DUTEIL S1, CARLIER PG1
(1) Laboratoire de RMN, AIM - CEA, Institut de Myologie, Paris, FRANCE. (2) CHU,
Université Joseph Fourier, Grenoble, FRANCE.
Introduction: NMR is a powerful tool to assess musculo-skeletal perfusion and
metabolism non-invasively and with a high time resolution. Blood oxygen level
To contact the author::
[email protected].
dependent (BOLD) NMR imaging readily provides contrast which depends on
oxygenation but also on perfusion and other intricate factors. Arterial spin labelled
(ASL) NMR imaging maps skeletal muscle perfusion, while 31P NMR spectroscopy
monitors high energy phosphates metabolism. When combined in a single protocol,
multiparametric functional NMR can provide new insight into muscle regulatory
mechanisms, into time course of muscle recovery after dynamic aerobic exercise in
healthy and diseased muscle.
Methods: To explore the time course of muscle recovery from aerobic exercise, and
in particular the "luxury perfusion", we studied exercise and recovery in 5 healthy
volunteers who performed 10 min bouts of single leg plantar flexion at 4 graded
workloads on a custom-built ergometer, while lying in a 4T magnet. Perfusion data
with positive and negative ASL were acquired in the calf muscle during and after
aerobic exercises and BOLD signal was directly measured from the mean of the same
images. In the meantime, phosphorous spectra were acquired to evaluate
phosphocreatine inorganic phosphate and pH changes. Muscle cell oxygenation was
concomitantly explored by deoxymyoglobin proton NMR spectroscopy.
Results: Recovery perfusion profiles were significantly prolonged with increasing
workload (p<0.05), time x perfusion integrals and time of peak perfusion were linearly
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dependent on PCr depletion (R2=0.86). In contrast, BOLD curves were not
significantly different at different loads.
Conclusion: Our results illustrate how mpf-NMR can investigate non-invasively
control
mechanisms
between
energy
demand
and
microvascular
perfusion
adaptations in exercising muscles. They confirm that BOLD contrast is not a suitable
substitute for perfusion assessment. However, it may be taken in combination with
other variables, such as perfusion and intracellular oxygenation, to improve
understanding of the physiology of exercising muscle.
PW 33:
Animal models (Part 1)
PW33-413
ELECTROMYOGRAPHIC EVALUATION OF MUSCLE ACTIVITY IN AN ANIMAL
MODEL OF DELTA-SARCOGLYCAN DEFICIENCY.
GIROUX-METGÈS MA1, HOGREL JY1, FISZMAN MY1, FROMES Y1
(1) Institut de Myologie, Paris, FRANCE.
To contact the author::
[email protected].
CHF147 hamster strain is an animal model of delta-sarcoglycan deficiency. Skeletal
muscular dystrophy features observed in this model are very similar to those
described in humans with more severe disease in proximal than distal muscles.
Muscle activation in this limb girdle muscular dystrophy has not yet been studied. Our
goal was to develop a standardized protocol for electromyography (EMG) study during
either voluntary or electrically elicited contractions.
Prior to surgery hamsters were trained to run at different speeds on a motor-driven
treadmill. Tibialis anterior (TA) and vastus lateralis (VL) were instrumented in CHF147
and control animals during global anesthesia. In each muscle, electrodes were made
up of two teflon insulated multistranded stainless wires (diameter = 110 µm).
Recording surfaces were made by removing 1 mm of insulation and the distance
between bared regions let to 2-2.5 mm centre-to-centre. The first recordings started at
day 6 after surgery. Two kinds of measurements were performed. First, global EMG
activity of TA and VL for each animal was recorded during 10-seconds runs at
increasing treadmill velocities (from 15 to 35 cm.s -1). Secondly, electrical stimulation
on anesthetized animals was performed in order to analyze muscle response during
recruitment and fatigue.
The methodology used in the present work generally yields EMG signals of good
quality either during voluntary or during electrically elicited contractions. Intramuscular
wires do not influence leg movements in hamsters. Various results in both contraction
modes can be depicted. For example, during repeated stimulation at 20 Hz,
compound muscle action potential amplitude decreased while its duration increased
associated to spectral compression. The rate of change of such parameters can be
used to assess muscle fatigability.
This standardized procedure associated to reliable electrophysiological parameters
may be useful for the follow-up of animals during the natural progression of the
disease or during therapeutic trials.
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PW33-414
THE ACETYLCHOLINESTERASE DEFICIENCY SEEN AT THE
NEUROMUSCULAR JUNCTION IN THE MOUSE MODEL OF SCHWARTZ-JAMPEL
SYNDROME IS PROBABLY NECESSARY BUT IS NOT SUFFICIENT TO INDUCE
NEUROMYOTONIA
STUM M1, BANGRATZ M1, GIRARD E2, BERNARD V3, DAVOINE CS1, TABTI N1,
WILLER JC4, ECHANIZ-LAGUNA A5, RENÉ F5, MARCEL C5, FONTAINE B1, MOLGO
J2, KREJCI E3, NICOLE S1
(1) Inserm, U546, Paris, FRANCE. (2) CNRS, UPR9040, Gif sur Yvette, FRANCE.
(3) Inserm, U686, Paris, FRANCE. (4) Assistance Publique-Hopitaux de Paris, Paris,
FRANCE. (5) Inserm, U692, Strasbourg, FRANCE.
To contact the author::
[email protected].
Schwartz-Jampel syndrome (SJS) is a recessive disorder characterized by
neuromyotonia with complex repetitive discharges at electromyography (EMG). SJS
results from hypomorphic mutations of perlecan, a ubiquitous proteoglycan secreted
within basement membranes (BM). The muscle hyperactivity in SJS is proposed to
result from synaptic acetylcholinesterase (AChE) deficiency as perlecan is crucial for
the anchorage of AChE at the neuromuscular junction (NMJ). However, EMG patterns
typical of AChE deficiency at the NMJ is not observed in patients with SJS. To
determine whether the AChE hypothesis is correct, we studied the mutants of our SJS
mouse model (see the abstract of Bangratz et al. for details on the model).
Partial AChE deficiency at the NMJ was seen in all striated muscles tested. Lack of
pretzel-like organization of postsynaptic acetylcholine receptors, poor branching of
nerve terminals, and partially denervated NMJs were also observed. These alterations
probably resulted from the major loss of synaptic perlecan as no disorganization of the
extrasynaptic muscle BM nor muscle degeneration were seen, that suggested a role
of perlecan in the maturation and/or maintenance of the NMJ. However, the AChE
deficiency seen at the NMJ was primary to the synaptic deficiency of perlecan and
was not due to denervation events as checked by transcriptional and co-staining
analyses. EMG performed on the mutants demonstrated the occurrence of
neuromyotonic discharges without decrement during repetitive stimulation in all
muscles tested except the diaphragm. Despite the absence of neuromyotonic
discharges, measures of contractile force and synaptic transmission parameters on
isolated hemidiaphragm preparations showed features of AChE deficiency. These
results demonstrate an electrophysiological effect of the partial AChE deficiency seen
at the NMJ, and suggest that the AChE deficiency is necessary but is not sufficient to
induce the neuromyotonia in SJS.
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PW33-415
INVERSE CORRELATION BETWEEN THE LEVEL OF SECRETED PERLECAN
AND THE SEVERITY OF THE ASSOCIATED PHENOTYPE IN A MOUSE MODEL
OF SCHWARTZ-JAMPEL SYNDROME
STUM M1, BANGRATZ M1, BERNARD V2, DAVOINE CS1, FONTAINE B1, KREJCI
E2, NICOLE S1
(1) Inserm, U546, Paris, FRANCE. (2) Inserm, U686, Paris, FRANCE.
To contact the author::
[email protected].
Schwartz-Jampel syndrome (SJS) is a recessive disorder with neuromyotonia and
chondrodysplasia that appear during childhood and slowly progress until adulthood.
SJS results from hypomorphic mutations in the perlecan gene, a proteoglycan
secreted within basement membranes, and is allelic to DDSH, a recessive
chondrodysplasia lethal at birth. A gene dosage effect is currently proposed to
account for the difference in severity: the residual level of perlecan secreted in SJS
would rescue the lethality due to the complete lack of perlecan in DDSH. To test this
hypothesis and understand the pathophysiology of SJS, we developed one mouse
model by introducing the p.C1532Y missense mutation into the perlecan gene by
homologous recombination. We developed two mouse lines: one (Neo+) still
contained the Neo box in addition to p.C1532Y, and the second (Neo-) contained only
the p.C1532Y mutation.
Homozygous mutant mice from the two lines were viable. A reduced secretion of
perlecan was seen in the Neo+ and Neo- mutants, with a lower level in Neo+
compared to Neo- due to splicing events between the perlecan gene and Neo. In the
two lines, the adult mutants were smaller than controls and develop a neuromuscular
phenotype with delayed opening of the eyelids and flexion of the hind paw when
suspended by the tail. Alterations of neuromuscular junctions with lack of pretzel-like
shape and acetylcholinesterase deficiency, and histological changes of skeletal
muscles similar to those observed in patients with SJS were observed in the two lines,
but they were more severe in Neo+. Altogether, our results argue for an inverse
correlation between the level of secreted perlecan and the severity of the phenotype,
and propose increasing the level of secreted perlecan by acting on the hypomorph
effect of the SJS mutations as a rational therapeutic strategy for SJS.
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PW33-416
THE ROLE OF THE DYSTROPHIN-GENE PRODUCT DP71 IN EXCITATORY
SYNAPSE ORGANIZATION, GLUTAMATERGIC TRANSMISSION, SYNAPTIC
PLASTICITY, AND SELECTIVE BEHAVIORAL FUNCTIONS.
DAOUD F1, CANDELARIO-MARTINEZ A3, BILLARD J4, AVITAL A5, KHELFAOUI M2,
ROZENVALD Y6, GUEGAN M7, MORNET D9, NUDEL U6, CHELLY J1, MARTINEZROJAS D3, LAROCHE S7, YAFFE D6, VAILLEND C8
(1) Institut Cochin, Université Paris-Descartes, CNRS, UMR 8104, PARIS, FRANCE.
(2) 2 INSERM U 567, PARIS, FRANCE. (3) CINVESTAV, Physiology, Biophysics and
Neurosciences, MEXICO City, MEXICO. (4) Université Paris-Descartes, Faculté de
médecine René Descartes, Neurobiologie de la Croissance et de la Sénescence,,
PARIS, FRANCE. (5) Weizmann Institute of Science, Neurobiology, Max Stern
Yezreel Valley College, Behavioral Sciences,, REHOVOT, ISRAEL. (6) Weizmann
Institute of Science, Molecular Cell Biology, REHOVOT, ISRAEL. (7) CNRS,
Neurobiologie de l’Apprentissage, de la Mémoire et de la Communication, UMR 8620,,
ORSAY, FRANCE. (8) Univ Paris-Sud, ORSAY, FRANCE. (9) 10 INSERM, Equipe
ESPRI 25 Muscle et Pathologies, Université de Montpellier I, UFR de Médecine, EA
4202, MONTPELLIER, FRANCE.
To contact the author::
[email protected].
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PW33-417
Duchenne muscular dystrophy (DMD) is caused by deficient
expression of the cytoskeletal protein, dystrophin. One third of
DMD patients also have mental retardation (MR), likely due to
mutations preventing expression of dystrophin and other brain
products of the DMD gene translated from distinct internal
promoters. Loss of Dp71, the major DMD-gene product in brain,
is thought to contribute to the severity of MR; however, the
specific function of Dp71 is poorly understood. Here we used
complementary approaches to explore the role of Dp71 in
neuronal function and identify putative mechanisms by which
Dp71 loss may impair neuronal and cognitive functions. We used
neuronal cell cultures and fractionation studies to assess the
subcellular localization of Dp71 and found expression at both
pre-and postsynaptic compartments of excitatory synapses. We
found that Dp71-associated protein complexes interact with
specialized modular scaffolds that cluster glutamate receptors
and organize signaling in postsynaptic densities. We further
showed that Dp71-null mice display abnormal synapse
organization and maturation in vitro, abnormally enhanced
glutamatergic transmission and altered synaptic plasticity in CA1
hippocampal area. Analysis of the behavioral phenotype of
Dp71-null mice revealed mild behavioral disturbances
characterized by reduced exploratory behavior and delayed
spatial learning. These findings suggest that Dp71 acts as a key
regulator of glutamatergic synapse organization and function.
This link between genetic loss of Dp71 and altered glutamatergic
synapse function provides a mechanism which may underlie the
increased severity of MR when inactivation of Dp71 is associated
with that of other brain products of the DMD gene.
INCLUSION BODY MYOSITIS IN A RHESUS MONKEY.
SKUK D1, PARADIS M1, GOULET M1, TREMBLAY J1
(1) Human Genetics Unit, CHUL Research Center, Quebec, CANADA.
To contact the author::
[email protected]
.ca.
Nonhuman primates are the ideal animal models for preclinical transplantation
studies. In addition, in the case of myoblast transplantation, the behavior of these
cells is similar to humans, in contrast to other animal models. Unfortunately,
there are not colonies of myopathic nonhuman primates available for cell
transplantation experiments. Here, we report the case of a rhesus (Macaca
mulata) monkey with a myopathy exhibiting a histological pattern similar to the
human inclusion body myositis. The monkey was detected among a group of
male rhesus received for studies in our research center. The size and weight of
the monkey were less than a half the values of other rhesus of the same age and
sex. Since the general state of the animal deteriorated rapidly, the veterinary
decided to perform analyses before to proceed to euthanasia. In the biochemical
analyses, the values of creatine kinase and LDH were abnormally high. Clinically,
we observed generalized severe muscle atrophies and musculotendinous
contractures. We performed biopsies in the biceps brachii, quadriceps femoris
and gastrocnemius. The biopsies showed several pathological modifications. The
most prominent was the presence of several myofibers with rimmed vacuoles
and/or eosinophilic hyaline inclusions, together with lymphocyte accumulations
around myofibers, necrotic and regenerating myofibers, and thickening of the
endomysium. Rimmed vacuoles contained lamellar structures in semithin
sections. This veterinary case report point-out the importance to be vigilant in
detecting symptoms of neuromuscular diseases in the colonies of nonhuman
primates, trying to find candidates to use in neuromuscular research.
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PW33-418
AN EXPERIMENTAL MODEL OF FIBROSIS IN TIBIALIS ANTERIOR MUSCLE OF
MDX MOUSE
DESGUERRE I1, ARNOLD L1, GHERARDI K1, FERRY A2, CHAZAUD B1
(1) INSERM U841, Creteil, FRANCE. (2) INSERM U787, Paris, FRANCE.
Duchenne muscular dystrophy (DMD) is a progressive disease, with patients
gradually losing most of their skeletal muscle strength with increasing fibrosis and fatty
To contact the author::
tissue infiltration. Endomysial fibrosis is believed to be in part responsible of muscle
benedicte.chazaud@ins
weakness, since it is correlated to motor deficit.
erm.fr.
The mdx mouse also lacks dystrophin. From 3 weeks of age, time of a massive
myofibre destruction, mdx skeletal muscle presents the same characteristics that
DMD muscle including necrotic and regenerating myofibres, surrounded by
inflammatory cells. However, excepted the diaphragm muscle that presents similar
histology than DMD muscle, limb muscles do not present hallmark of fibrosis,
excepted at very old age (at least 20 months). Moreover, adult mdx mouse does not
present sign of muscle weakness (excepted after excentric exercise) while diaphragm
presents dysfunction and loss of compliance.
Our aim was to establish a fibrotic model of dystrophinopathy in adult - not ederlymdx mouse by repeated mechanic microinjuries in hindlimb muscle. One week after
the end of microinjuries, histological analysis and strength evaluation were performed.
Histological stainings and collagene I immunolabellings showed the presence of
endomysial fibrosis. Number of centrally nucleated myofibres was unchanged while
the diameter of the fibres was decreased in treated muscle. Maximal force was
decreased by more than 30% suggesting the involvement of endomysial fibrosis in
muscle weakness. After one month resting, endomysial collagene I area was strongly
decreased and force raised near control values, suggesting a fibrolytic process.
The experimental model of skeletal muscle endomysial fibrosis we propose is
transient, showing sequentially the establishment of fibrosis in the interstitial
compartment, as it is observed in DMD, then a disappearance of this endomysial
fibrosis. This model will be helpfull to study the cellular and molecular mechanisms
involved in both fibrogenesis and fibrolysis processes in dystrophic skeletal muscle.
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PW33-419
TRIADIN KNOCK-OUT MOUSE : A MODEL FOR CORE MYOPATHIES ?
ODDOUX S1, FAURÉ J1, BROCARD J1, BROCARD J2, FOUREST A1, LUNARDI J1,
MARTY I1
(1) Grenoble Institut des Neurosciences - Inserm U836 - Equipe Muscle et
Pathologies - UJF Site Santé - Bat Edmond J Safra - BP 170 - 38042, Grenoble,
FRANCE. (2) Grenoble Institut des Neurosciences - Inserm U836 - Equipe
Physiopathologie du Cytosquelette - UJF Site Santé - Bat Edmond J Safra - BP 170 38042, Grenoble, FRANCE.
To contact the author::
[email protected].
In skeletal muscle, release of calcium leading to contraction (a mechanism also called
“excitation contraction coupling”) is performed by a macromolecular complex,
composed of two calcium channels (ryanodine receptor, RyR, and dihydropyridine
receptor, DHPR). A number of associated proteins (triadin, calsequestrin, …) are also
part of this complex, they could regulate the calcium channels. Mutations of RYR1
and DHPR resulting in an alteration of the calcium homeostasis have been
associated with various muscle diseases including malignant hyperthermia and core
associated congenital myopathies such as Central Core Disease (CCD) or Multi-mini
Cores Disease (MmD). However in a number of patients, no mutation has been
identified in either of the calcium channels, pointing to potential involvement of other
proteins in the physiopathology of these diseases. Our research project aims at
understanding the function of triadin in muscle and in the calcium release complex,
and its possible involvement in these myopathies. We have previously shown that
overexpression of one triadin isoform, Trisk 95, abolishes excitation-contraction
coupling, and therefore that the precise stoichiometry of triadin compared to RyR
seems to be important for a correct function of the calcium release complex. In order
to go deeper in the identification of the function of triadin, we have developed a triadin
knock-out mouse, in collaboration with ICS-Strasbourg. The first characterization
(Western blot analysis, immunolabelling on muscle section) of this mouse will be
presented, and the possible correlation with a human pathology will be discussed.
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PW33-420
LOSS OF SKELETAL MUSCLE STRENGTH IN JP-45 KNOCK OUT MICE
DELBONO O1, XIA J2, TREVES S2, WANG ZM1, JIMENEZ-MORENO R1, PAINE A1,
MESSI LM1, NISHI M4, BRIGUET A3, SCHAERER F3, TAKESHIMA H4, ZORZATO F2
(1) Wake Forest School of Medicine, Winston-Salem, USA. (2) University Basel
Hospital, Basel, SWITZERLAND. (3) Santhera Parmaceuticals, Liestal,
SWITZERLAND. (4) Kyoto University, Kyoto, JAPAN.
Skeletal muscles from young adult mice containing homozygous
deletion of the gene for the sarcoplasmic reticulum protein JP-45
were studied and compared to age-matched wild-type litter
mates. Analysis of spontaneous motor activity with a running
wheel revealed that dark phase running distance ran by the JP45 KO mice was significantly lower compared to wild type. In
vitro EDL and soleus muscle mechanical property analysis
showed slower twitch and tetanic absolute and specific force in
JP-45 KO mice compared to wild-type, while muscle size, wet
weight and cross sectional area of muscle fibers were not
different. Isolated sarcotubular membranes isolated from 3
month and 6 months old JP-45 KO mouse muscles exhibited
reduced [H3]PN200-110 binding indicating down regulation of
CaV1.1 protein. SR Ca2+ release in voltage-clamped muscle
fibers of JP-45 KO was significantly reduced compared to wildtype.
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PW33-421
Our results show that ablation of JP-45 reduces functional
CaV1.1 protein expression and/or targeting to the t-tubule
membrane, impairing the EC coupling signal between CaV1.1
and RyR1, thereby reducing skeletal muscle force
IMPAIRED MTOR SIGNALING IN MOUSE SKELETAL MUSCLE LEADS TO A
SEVERE MYOPATHY
GANGLOFF YG1, RISSON V1, CORNELOUP C1, MAZELIN L1, SANCHEZ H2,
ROCERRI M3, RICHARD H2, VIGNAUD A4, BAUCHE S5, HANTAI D5, MUELLER M6,
KOZMA S7, THOMAS G7, FERRY A4, PENDE M3, BIGARD X2, KOULMANN N2,
SCHAEFFER L1
(1) CNRS-UMR5239-IFR128, Ecole Normale Supérieure, Lyon, FRANCE. (2)
CRSSA, Grenoble, FRANCE. (3) INSERM U810, Faculté de médecine Necker, Paris,
FRANCE. (4) UMR 787, Université Pierre et Marie Curie-Paris 6, Paris, FRANCE. (5)
INSERM U582, Institut de Myologie, Paris, FRANCE. (6) Novartis Pharma AG, Basel,
SWITZERLAND. (7) Department of Genome Science, Cincinatti, USA.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that
To contact the author::
yann-gael.gangloff@ens- coordinates cellular responses to nutritional and growth factor conditions by
lyon.fr.
controlling transcription, translation, cell size, cytoskeletal organization and
autophagy. mTOR is encoded by an essential gene and its function in vivo has
been mainly inferred by employing the mTOR inhibitor, rapamycin. By this
approach, mTOR signaling has been shown to be necessary for skeletal muscle
growth. However, mTOR is part of two distinct complexes, mTORC1 and
mTORC2, which display differential sensitivity to rapamycin. Therefore, we
generated and characterized skeletal muscle-specific mTOR knock-out mice to
identify the full spectrum of mTOR functions in the post-natal development of
skeletal muscle. We show that these mice develop a severe myopathy
displaying characteristics of dystrophies and metabolic myopathies as they
exhibit muscle fiber regeneration, increased glycogen content, decreased
glycolytic capacity as well as altered oxidative metabolism. We find that mTORdepleted muscles are weaker and that they show slower contractile properties
and reduced fatigue resistance. Moreover, our data show significantly altered
expression of genes critical for muscle structure and energy metabolism in
mutant muscles that could underlie the pathology. These results demonstrate a
critical role for mTOR in maintenance of muscle fiber integrity. Our findings
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suggest that alterations of mTOR-mediated physiological processes could
contribute to the pathogenesis of a broad range of myopathies and emphasizes
the therapeutic interest in developing activators of this pathway.
PW33-422
ASSESSMENT OF GAIT QUALITY IN DYSTROPHIN-DEFICIENT DOGS USING
ACCELEROMETRY
BARTHELEMY I1, BARREY E3, THIBAUD JL1, URIARTE A1, BLOT S1, HOGREL JY2
(1) Laboratoire de Neurobiologie - ENVA, Maisons-Alfort, FRANCE. (2) Institut de
Myologie - GH Pitié-Salpêtrière, Paris, FRANCE. (3) Laboratoire d'Etude de la
Physiologie de l'Exercice - INSERM 902 - Genopole, Evry, FRANCE.
To contact the author::
[email protected].
Dystrophin-deficient dogs (GRMD, LRMD) exhibit similar pathophysiological and
clinical features as Duchenne patients and represent therefore the best animal model
to evaluate a therapeutic benefit. Emerging systemic approaches and the associate
necessity to assess their clinical efficiency have led us to develop a quantitative,
discriminating and non-invasive method to analyse locomotion. Accelerometry, a gait
analysis technique based on 3D recording of accelerations, seems to be easy to
perform in dogs and a source of several quantified parameters.
In order to test this method, 11 dystrophin-deficient and 5 healthy adult dogs were
encouraged to walk or trot along a 20 metres corridor, as spontaneously as possible.
Three axial accelerations were recorded close to the center of gravity, the
accelerometer being placed under the sternum, using a light elastic belt tightened
around the thorax (Equimetrix device ®). Recorded data were then analysed using
specific gait analysis software on ten seconds-samples of steady state locomotion.
The accelerometer device was well tolerated by the dogs. The trot was spontaneously
adopted by healthy, and by less affected dystrophic dogs, whereas gait of severely
affected animals was restricted to walk. The mean regularity of dorso-ventral
accelerations was found to be significantly higher in healthy than in trotting dystrophic
dogs. Total mechanical power of gait (W/kg) was also observed to be significantly
decreased in dystrophic dogs. Interestingly, the medio-lateral component of the power
was significantly increased in dystrophic dogs, demonstrating a swaying component in
the gait of these dogs.
These preliminary results show that accelerometry is a simple and non-invasive
method of functional evaluation of dog’s gait and is efficient to quantitatively
discriminate dystrophic from healthy dogs, using specific signal processing methods.
Longitudinal studies are ongoing in order to describe gait evolutions of healthy versus
dystrophic dogs at various ages, with various phenotypes and after treatment.
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PW33-423
PHYSIOPATHOLOGICAL CONSEQUENCES OF EXCITATION-CONTRACTION
UNCOUPLING
PIÉTRI-ROUXEL F1, GENTIL C1, FERRY A1, VIGNAUD A1, GARCIA L1
(1) UMRS 787 Inserm-UPMC, Institut de Myologie, Paris, FRANCE.
Adipose infiltration is a characteristic common to many muscular dystrophies. In
certain cases, the infiltration is such that all of the muscle fibers are replaced by
To contact the author::
[email protected].
adipocytes, preventing or at least limiting, the rational of gene or cell therapies. Our
previous studies on denervated muscle have demonstrated an unsuspected plasticity
of muscle fibres. Indeed, the muscle syncytium, reserved the capacity to convert into
mononucleated cells able to develop into adipocytes. These results enable us to reexamine the physiopathology of Duchenne myopathy and the progressive emergence
of adipose metaplasia. The primum movens remains the absence of dystrophin, which
is at the origin of the extreme fragility of DMD muscle fibres. This fragility results in
spontaneous microlesions. The number and repetition of necrotic events are such that
certain microlesions probably escape the repair process thus causing the electric
disconnection of distal parts of segmented fibers. These distal segments would then
develop like denervated muscle, resulting in a fatty transformation. The rational
mentioned above encouraged us to explore the consequences of an interruption of the
functional activity of muscle fibers without affecting the nerve-muscle communication.
This can be carried out by destroying the skeletal excitation-contraction coupling
system. The excitation-contraction coupling is guaranteed at the level of the triads,
forming “electric synapses” between membranes of the transverse tubules and the
sarcoplasmic reticulum. It has been known that the alpha1s sub-unit of the slow Ca2+
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channel (DHP-R) ensures the “voltage sensor”. To study the morphogenetic
consequences related to an inactivation of this sensor, we have developed AAV
vectors harboring constructions coding snRNA-U7 directed against exons crucial for
the synthesis of the alpha1s sub-unit. Kinetics of muscle atrophy, tissue modifications
and expression of molecular markers following this type of paralysis were analyzed.
PW33-424
GLIAL CELLS AT NEUROMUSCULAR JUNCTION PARTICIPATE TO THE MOTOR
END-PLATE PHENOTYPE.
MUSARELLA M1, BELLOUZE S1, CAILLOL G1, AUTILLO-TOUATI A1
(1) UMR641, INSERM, Faculté de medecine Nord, Université de la Méditerranée,
Marseille, FRANCE.
To contact the author::
[email protected].
Mouse mutants provide good models for the study of degenerative neuromuscular
diseases in human. Although recent data indicate a pivotal role for Terminal Schwann
Cells (TSC) in maintaining synaptic structure and function, their role in pathologies
affecting the neuromuscular junction (NMJ) has been poorly investigated until now.
The study we conducted help to re-evaluate the importance of TSC both in the normal
junction and in diseased individuals.
In med mice (motor endplate disease), neuromuscular transmission is severely
impaired and death occurs 20 days after birth. The homozygous med mice are voltagegated sodium channel Nav1.6 null mutants.
As published (Musarella et al., 2006) :
1.
In wild-type mice peripheral nervous system, this isoform is not only
expressed at nodes of Ranvier but also in TSC.
2.
The number of TSC per NMJ is dramatically decreased at terminal stage of
med pathology (in the wild-type, 86 % of NMJ had associated TSC whereas
only 36 % in the mutant).
In the present work, we investigate the time course of the loss of TSC, at a preclinical stage (P7), at the onset of the disease (P10) and at the terminal stage (P19).
By confocal microscopy on sterno-mastoid muscle sections (40µm), we quantified the
number of TSC per NMJ identified by S100 immunostaining in vicinity to alphabungarotoxin staining and the number of apoptotic nuclei localised by ” In Situ CeIl
Death Détection Kit, Fluorescein (Roche) ".
Progressive loss of TSC occurs before the onset of the phenotype and is due to an
increase of glial cell apoptosis.
These results suggest that defects in Terminal Schwann Cells contribute to the med
phenotype.
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Grant sponsor : Association Française contre les Myopathies (AFM), n° 11352.
Acknowledgments : The authors thank MP. Blanchard (CMI, plateforme R.I.O.) and C.
Gomez.
PW 34:
Animal models (Part 2)
PW34-425
TRANSGENIC MICE WITH THE FULL-LENGTH HUMAN DMD GENE FACILITATE
STUDY OF TRANSCRIPTION REGULATION AND GENETIC THERAPIES FOR
DUCHENNE MUSCULAR DYSTROPHY
'T HOEN P1, DE MEIJER E1, AARTSMA-RUS A1, VAN OMMEN G1, VAN
DEUTEKOM J1, DEN DUNNEN J1
(1) Leiden University Medical Center, Department of Human Genetics, Leiden, THE
NETHERLANDS.
To contact the author::
[email protected].
We have generated mice with an intact and functional copy of the 2.3 Mb human
dystrophin gene (hDMD), the largest functional stretch of human DNA thus far
integrated into a mouse chromosome. For this, yeast spheroplasts containing an
artificial chromosome with the full-length hDMD gene were fused with mouse
embryonic stem cells, and were subsequently injected in mouse blastocyts to produce
transgenic hDMD mice. Human-specific PCR, Southern blotting and fluorescent in situ
hybridization techniques demonstrated the intactness and stable chromosomal
integration of the hDMD gene on mouse chromosome 5. Expression of the transgene
was confirmed by RT-PCR and Western blotting. The tissue-specific expression
pattern of the different DMD transcripts was maintained. However, the human Dp427p
and Dp427m transcripts were expressed at two-fold higher levels, and human Dp427c
and Dp260 transcripts at two- and four-fold lower levels than their endogenous
counterparts. Ultimate functional proof of the hDMD transgene was obtained by
crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and
utrophin-deficient mdx x Utrn-/- mice. The hDMD transgene rescued the lethal
dystrophic phenotype of the mdx x Utrn-/- mice. All signs of muscular dystrophy
disappeared in the rescued mice, as demonstrated by histological staining of muscle
sections and gene expression profiling experiments. Currently, hDMD mice are
extensively used for pre-clinical efficacy testing of exon
skipping antisense
oligonucleotides, which restore the reading frame in patients with Duchenne muscular
dystrophy. In addition, the hDMD mouse can be used to study the influence of the
genomic context on deletion and recombination frequencies, genome stability, and
gene expression regulation.
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PW34-426
QUANTITATIVE CHARACTERISATION OF DYSTROPHIC MUSCLE IN GRMD
DOGS BY NMR IMAGING.
THIBAUD JL1, BERTOLDI D2, MONNET A2, BARTHÉLÉMY I1, BLOT S1, CARLIER
PG2
(1) Laboratoire de Neurobiologie ENVA, Maisons-Alfort, FRANCE. (2) Laboratoire de
RMN AIM - CEA Institut de myologie, Hopital Pitié-Salpétrière, Paris, FRANCE.
The Golden Retriever Muscular Dystrophy dog lacks dystrophin and shares
To contact the author::
[email protected].
pathological and clinical similarities with the Duchenne patients. The model,
increasingly used in pre-clinical trials, needs to be further characterized. We defined
NMR imaging indices of canine dystrophic muscle. Six two-month old control and 6
GRMD dogs were examined at 4 T. Three control and 5 GRMD dogs were examined
at 3 T at the age of 2, 4 and 6 months. Standard and fat-saturated T1-weighted images
were acquired, followed by T2-weighted images. After Gd-DTPA injection, the timecourse of muscle enhancement was monitored with fat-saturated T1-weighted imaging
during 2 hours. Extensor carpi radialis and flexor carpi ulnaris were studied. Indices
were calculated as follows: T2w/T1w signal ratio (SR= T2w Signal x T1w ref gain)/ T1w
Signal x T2w ref gain), T1w and T2w heterogeneity (H=√(SD2- SD2noise/0,655) ). An
exponential decay was fitted to the signal decrease post Gd-DTPA injection; maximal
relative enhancement (RE) and time-constant of decay were compared. A three-way
analysis of variance was performed. T2w/T1w SR, H2 and RE were found significantly
increased in dystrophic muscles at 4 T. These findings were confirmed at 3 T, at all
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ages. H1 was also found significantly increased in dystrophic muscle at 4 and 6
months at 3 T.
These quantitative indices differentiate dystrophic from normal muscles and might be
proposed as non-invasive evaluation tools of therapeutic trials.
PW34-427
CENTRONUCLEAR MYOPATHY: DECIPHERING PHYSIOPATHOLOGICAL
MECHANISMS IN SKELETAL MUSCLES AND VISCERAL ORGANS THROUGH
THE ANALYSIS OF THE PTPLA GENE FUNCTION IN LABRADOR RETRIEVERS
AFFECTED BY CNM.
MAURER M1, MARY J1, DROUGARD C1, BARTHÉLÉMY I2, BERNEX F1, GUILLAUD
L1, KESSLER JL1, PELÉ M1, PANTHIER JJ1, BLOT S2, TIRET L1
(1) UMR955 INRA-ENVA, ENV Alfort, Maisons-Alfort, FRANCE. (2) Laboratoire de
Neurobiologie, ENV Alfort, Maisons-Alfort, FRANCE.
To contact the author::
[email protected].
The autosomal recessive centronuclear myopathy of the Labrador Retriever (cnm) is a
relevant clinical and histopathological model for the human nosological group of
myotubular/centronuclear myopathies. We previously showed that the diseasecausing gene was PTPLA, encoding the Protein Tyrosine Phosphatase-Like, member
A. In skeletal muscles of affected dogs, we demonstrated that the two copies of the
PTPLAalf mutated allele were prone to several splicing abnormalities, eventually
resulting in the production of only 1% of the normal level of wild-type transcripts. In
Labradors, the cnm mutation is therefore hypomorphic at the transcriptional level.
In man, the genomic coding sequence of the orthologous PTPLA gene has been
assessed in some affected individuals. No significant functional polymorphism could
be found. Thus, although not a true molecular homologue of the human disorder, the
canine cnm model remains highly relevant to analyze and compare the
physiopathological mechanisms leading to similar diseases in both species.
In mammals, the function of the PTPLA protein remains undeciphered. Using
antibodies currently being fully characterized, we have initiated the description of its
cellular and subcellular expression pattern in healthy and affected dogs, as well as in
cell lines and mouse embryos. Two splicing variants of PTPLA (PTPLAfl and
PTPLAd5) were exclusively expressed in different organs. PTPLAfl was expressed
mainly in skeletal muscles and heart. In affected dogs, we found that the muscular Ttubule network and its associated proteins (DHPR, BIN1) was abnormally spred in
some fibers. The excitation-contraction coupling could therefore be implicated in the
physiopathological mechanisms of the disease. PTPLAd5 was present in several
smooth muscle fibers containing organs, and sub-clinical defects could be observed in
some of them in healthy carriers and affected dogs. This highlighted an unexpected
key role played by PTPLA in cellular homeostasis, yet to be investigated in man.
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PW34-428
SYNAPSE AND AXON DEGENERATION IN THY1 YFP MICE WITH
PROGRESSIVE MOTOR NEURONOPATHY
BIELLI S2, SCHÄFER M1, BELLOUZE S1, HAASE G1
(1) Equipe Avenir, INSERM-Université de la Méditerranée, Marseille, FRANCE. (2)
Trophos SA, Marseille, FRANCE.
To contact the author::
[email protected].
Progressive motor neuronopathy (pmn) is a particularly aggressive form of motor
neuron disease characterized by early loss of neuromuscular synapses and axonal
dying back (Schmalbruch et al., 1991). We previously identified the genetic defect of
pmn mice as a missense mutation in TBCE, one of five tubulin chaperones required
for tubulin folding and dimerisation (Martin et al. 2002). We further demonstrated that
TBCE is a Golgi-associated protein that controls axonal tubulin routing and
microtubule maintenance (Schäfer et al. 2007).
To monitor the morphological and molecular correlates of progressive motor
neuronopathy we now crossbred pmn mice with transgenic thy1-YFP-reporter mice
(Feng et al. 2000) and labelled whole mount nerve-muscle preparations from
endstage mice for VAChT (vesicular acetylcholine transporter), synaptic vesicle
proteins and acetylcholine receptors. Axon degeneration was attested by calibre
irregularities of YFP-positive axons, axonal spheroids and loss of axonal continuity.
Degeneration of neuromuscular synapses was evidenced by reduced VaChT levels in
presynaptic terminals and by altered distribution of postsynaptic acetylcholine receptor
clusters. These degenerative changes were most pronounced in the diaphragm and
more moderate in the gluteus and superficial abdominal muscles. Thy1 YFP mice thus
represent a powerful tool to monitor temporal progression and spatial distribution of
axon and synapse degeneration in mouse motor neuron disease.
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PW34-429
MEF2C IS PRECOCIOUSLY INVOLVED IN XENOPUS LAEVIS TENDON
DEVELOPMENT
DELLA GASPERA B1, ARMAND AS1, LECOLLE S1, CHARBONNIER F1, CHANOINE
C1
(1) Université Paris Descartes, CNRS, Paris, FRANCE.
The myocyte enhancer factor 2 (MEF2) family of MADS (MCMI, agamous, deficiens,
serum response factor)-box transcription factors has four members in vertebrates,
To contact the author::
christophe.chanoine@un MEF2A, -B, -C and –D. The four MEF2 genes are expressed in complex and
iv-paris5.fr.
overlapping patterns in embryonic and adult tissues. MEF2 family members have
been shown to play a pivotal role in morphogenesis and myogenesis of skeletal,
cardiac and smooth muscles cells differentiation and also regulates neuronal and
immune cell differentiation. In mice, two unanticipated roles for MEF2C have been
recently identified showing that MEF2C controls both chondrocyte hypertrophy and
bone development and that the expression of MEF2C in the neural crest is required
for craniofacial development.
In this study, we describe the cloning of multiple Xenopus MEF2 splicing isoforms
differentially expressed during development and show that the accumulation of
XMEF2C mRNA was initially specifically located in forming and migrating neural crest.
In the tadpole stages, XMEF2C expression was restricted to intersomitic regions and
to the peripheral edges of epaxial and cranial muscle masses and was coexpressed
with scleraxis, a specific marker for tendons and ligaments.
We show that overexpression of XMEF2C inhibited myogenesis, whereas the use of
an hormone-inducive MEF2C construct is able to induce scleraxis expression.
Furthermore, XMEF2C and scleraxis act cooperatively to induce betaig-h3, a gene
expressed in collagen-rich tissues.
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These findings highlight a previously unappreciated role for XMEF2C in the regulation
of tendon development and identify a novel gene transactivation pathway where
XMEF2C enhance the ability of the bHLH protein, scleraxis, to activate specific gene
expression.
PW34-430
DROSOPHILA NEUROMUSCULAR JUNCTION (NMJ) PRUNING: A POTENTIAL
MODEL TO FIND THERAPIES FOR NMJ DEGENERATION DISEASES
BOULANGER A1, RAMANOUDJAME C1, DURA JM1
(1) Institut de Génétique Humaine CNRS, Montpellier, FRANCE.
To contact the author::
[email protected].
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PW34-431
At the neuromuscular junction (NMJ) individual muscle fibers are first contacted by
many motoneurons, later in development synapse elimination and denervation
followed by branch pruning occurs, so that, only one motoneuron innervates a same
muscle fiber. In motoneuron diseases as ALS, one of the first signs of the diseases is
also the denervation of neuromuscular junctions. Indeed, it is known that
neurodevelopment and pathological neurodegeneration converge.
The aim of this study is to identify the mechanisms involved in motoneurons
pathological degeneration using as a model the axonal dismantling of drosophila NMJ
during development
In this study:
1) We have performed a precise description of the NMJ dismantling during drosophila
development using immunohistochemistry, and shown that muscles degenerate
before motoneurons. Thus, after muscle degeneration signs are observed, cell
adhesion molecules are lost and the motoneuron cytoskeleton pulls-back by a
retraction mechanism.
2) We have evidenced cellular (p35-mediated apoptosis) and genetic (ecdysone)
pathways involved in NMJ dismantling. Accordingly, we have been able to stop NMJ
dismantling by overexpressing or blocking specific ecdysone regulated nuclear
receptors and by expressing apoptosis inhibitor proteins in muscle.
Together these results suggest that muscle degeneration is the trigger of the
dismantling process since :
-muscle degeneration takes place before motoneuron pruning.
-Blocking muscle degeneration stops motoneuron pruning. However, blocking
motoneuron pruning do not stop muscle lost.
We are actually trying to decipher the molecular nature of the message sent from the
muscle to the motoneuron which induce motoneuron pruning. For this we are using a
gene candidate screening approach.
Importantly, we have set up an in vivo developmental assay of NMJ dismantling in
wich denervation can be stopped. The assay is ready to test the potential motoneuron
or muscle degeneration-protective human genes, and can be used as a first test
before gene therapy.
THE UCS FACTOR UNC-45B INTERACTS WITH THE HEAT SHOCK PROTEIN
HSP90A DURING MYOFIBRILLOGENESIS AND SHUTTLES BETWEEN THE ABAND AND THE Z-LINE OF THE MYOFIBRIL
ETARD C1, ROOSTALU U1, BEHRA M2, STRÄHLE U1
(1) Institute for Toxicology and Genetics; Forschungszentrum Karlsruhe, Karlsruhe,
GERMANY. (2) NIH/NHGRI, Bethesda, USA.
To contact the author::
[email protected]
e.
Contraction of muscles is mediated by highly organized arrays of myosin motor
proteins. We report here the characterisation of a mutation of an UCS gene named
steif/unc45b that is required for the formation of ordered myofibrils in both the skeletal
and cardiac muscles of zebrafish. We show that Steif/Unc45b interacts with the
chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal
musculature and knock-down of Hsp90a leads to impaired myofibril formation in the
same manner as lack of Steif/Unc45b activity. Our data indicate a requirement of
Steif/unc45b and Hsp90a for the assembly of the contractile apparatus in the
vertebrate skeletal musculature. I addition we show that Unc45b and Hsp90a colocalize with myosin during myofibrillogenesis and associate with the Z-line when
myofibril assembly is completed. In response to stress or damage to the myofiber,
Unc45b and Hsp90a dissociate from the Z-line and transiently associate with myosin.
We propose that the Z-line serves as a reservoir for chaperones, allowing a rapid
mobilization in response to muscle damage
_0MS0G0OLK
PW34-432
COLLAGEN XXII, A NEW COMPONENT OF THE MYOTENDINOUS JUNCTION: A
STRUCTURE-FUNCTION STUDY USING THE ZEBRAFISH MODEL.
CHARVET B1, BADER H1, SCHULZE J2, KOCH M2, RUGGIERO F1
(1) Institut de biologie et chimie des protéines, UMR 5086, Université Lyon 1, IFR128
Lyon Biosciences, Lyon, FRANCE. (2) Institut for Biochemistry II, Medical Faculty of
the university of Cologne, Cologne, GERMANY.
The myotendinous junction (MTJ) provides a structural link between the muscle
cell cytoskeleton and the extracellular matrix (ECM) of tendons. Lack of one of the
To contact the author::
[email protected].
MTJ components can alter dramatically muscle cell anchoring to the tendon. Still, its
morphogenesis remains elusive and has been neglected by biologists.
Collagen XXII is a novel component of MTJ (Koch et al, 2004). It belongs to the
FACIT (Fibril-associated collagen with interrupted triple helices) subset of the collagen
family which are characterized by their capacity to associate with the fibrillar collagens
and to mediate protein interactions. We aim at analyzing the role of collagen XXII in
MTJ morphogenesis in zebrafish. Visual direct observations of embryos and rapid and
efficient protein knock-down unmatched in other animal models make zebrafish a
particularly valuable model for exploring muscle/tendon development. We identify the
zebrafish ortholog of the human collagen XXII gene (COL22A1) and show that its
transcripts are exclusively expressed in myotomal muscle at 24 hpf. As muscle cells
differentiate, the signal concentrates at the extremities of muscle fibers close to the
MTJ and sandwiches the myosepta, the structure equivalent to mammalian tendons.
Confocal double immunofluorescence with antibodies to dystrophin and to zebrafish
collagen XXII shows that collagen XXII is deposited at the MTJ. Morpholino-based
knock-down of col22a1 causes bending of the tail and important morphofunctional
alterations in muscles/tendon formation: myosepta interruptions and presence of giant
muscle cells spanning two myotomes at the interruption sites, muscle fiber
_0MS0G0OLK
detachment from the MTJ… Several mutations in ECM genes have been associated
to myopathies. The genetic heterogeneity of the disorders suggests that other matrix
proteins could account for unexplained cases. Our results suggest that collagen XXII
plays a crucial role in muscle homeostasis and thereby represents a good candidate
gene for muscular dystrophies.
PW34-433
APPROACHING THE MECHANISMS UNDERLYING THE EARLY LOSS OF LIMB
MUSCLE FUNCTION IN ACUTE QUADRIPLEGIC MYOPATHY USING A PORCINE
ANIMAL MODEL
OCHALA J1, BANDUSEELA V1, LARSSON L1
(1) Uppsala University, Uppsala, SWEDEN.
To contact the author::
julien.ochala@neurofys.
uu.se.
_0MS0G0OLK
Acute quadriplegic myopathy (AQM) is commonly observed in patients suffering from
critical illness, and is notably characterized by an early severe limb muscle weakness.
The basic mechanisms underlying such muscle weakness remain poorly understood.
It may be related to various components, i.e., sepsis, prolonged mechanical
ventilation, postsynaptic block of neuromuscular transmission (NMB), systemic
corticosteroid hormone treatment (CS), and muscle unloading. The present study
aimed at exploring the relative importance of each component on the early
development of limb muscle weakness using a porcine model. 21 pigs were
mechanically ventilated and exposed to various combinations of NMB, CS and/or
endotoxin-induced sepsis, for a period of five days. Biopsy specimens from biceps
femoris on day 1 and 5, allowed the measurements of muscle function (single muscle
fiber preparation), protein (12% SDS-PAGE gels, ELISA assay) and mRNA (Real-time
PCR) expressions. Results showed interesting findings pointing out the major
components that seriously accounts for the early development of AQM. On day 5,
single muscle cell force-generating capacity was dramatically altered, the severity was
related to the exposition to the various components: combination of sepsis, CS and
NMB > sepsis > CS > NMB. This impairment may be directly related to the lower
expression of contractile proteins, related to degradation in the early phase of the
disease.
PW 35:
Cardiac, smooth and
other muscles
PW35-434
HUMAN SERCA2A CONTROLS VASCULAR SMOOTH MUSCLE CELL
PROLIFERATION VIA INHIBITION OF BASAL CA2+ INFLUX AND NFAT.
LIPSKAIA L1, COLOMBE A2, LEHOUX S3, ESPOSITO B3, ATASSI F2, LE PRINCE P4,
BONNET N4, LE FEUVRE C4, HATEM S2, HAJJAR R1, LOMPRE AM2
(1) MSSM, New York, USA. (2) INSERM U621, Paris, FRANCE. (3) INSERM 689,
Paris, FRANCE. (4) Hôpital Pitié-Salpêtrière, Paris, FRANCE.
Coronary restenosis, results mainly from the proliferation of vascular
To contact the author::
[email protected] smooth muscle cells (VSMC). Here, we determine whether in human
arteries, the cardiac isoform of the sarco/endoplasmic reticulum calcium
.
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PW35-435
ATPase, SERCA2a controls VSMC proliferation via calcium-dependent
calcineurin/NFAT signalling pathway. In atherosclerotic coronary arteries
(CA) SERCA2a and the ryanodine receptor (RyR2) were expressed only in
differentiated VSMC from the media but not in the neointima, containing
mainly dedifferentiated cells. Primary CA VSMCs lost SERCA2a and RyR
within 3 days after induction of proliferation. SERCA2a gene transfer
inhibited VSMC proliferation through inhibition of NFAT leading to down
regulation of cell cycle controlling proteins cyclin D1 and Erg1. Singlechannel patch-clamp recording showed that SERCA2a gene transfer
inhibited basal voltage-independent Ca2+ influx. Promoter-reporter assay
showed that in human VSMCs NFAT was activated by store-dependent Ca2+
influx, inhibited by depolarisation-induced Ca2+ influx and its transcriptional
activity strongly correlated with proliferative state, as demonstrated by Ca 2+
channels blockers, expression of VIVIT (NFAT inhibitory peptide) or
constitutively active NFAT. Furthermore, SERCA2a gene transfer prevented
vascular remodelling and reduced neointima formation in an ex-vivo model of
injury: the left internal mammary arteries (MA), injured and kept 2 weeks in
organ culture under constant pressure and flow (intima/media ratio was 0.07
± 0.01 vs 0.40
-infected arteries, p<0.001, n=5). In conclusion,
in human SERCA2a controls proliferation of VSMC and neointima formation
via inhibition of basal voltage-independent Ca2+ influx and transcription factor
NFAT. These findings could have potential implications for treatment of
intervention-induced restenosis.
MOLECULAR CONTROLS OF VISCERAL SMOOTH MUSCLE CELL
DIFFERENTIATION IN VERTEBRATE EMBRYOS
NOTARNICOLA C1, LE GUEN L1, DE SANTA BARBARA P1
(1) INSERM ERI 25, Muscle and Pathologies, Montpellier, FRANCE.
To contact the author::
[email protected]
m.fr.
Despite significant advances in the description of molecular controls of
gut development in different animal models, little works have been done on the
pathways involved during visceral smooth muscle cell differentiation. This
differentiation is often affected in patients with congenital gut malformations. These
malformations account for a significant percentage of all congenital defects, and
the molecular description of gut development would be beneficial in diagnostic and
therapeutic treatment of these common human gastrointestinal disorders.
Our group focuses on the identification and the functional study of the
molecular mechanisms that control the differentiation of the visceral smooth muscle
cells (SMCs). We use the chick embryo as animal model, and techniques from
development (misexpression of cDNAs by avian retroviral infection in the mesodermal
layer). With a microarray approach, we found only 55 genes regulated during the
differentiation of SMCs and let us hypothesize that we identified important molecular
players that could be essential to trigger the differentiation of the mesenchymal cells
into SMCs. The limited number of genes gave us the opportunities to study the
expression patterns of different candidates. We already analyzed the status of the
FGF pathways activation and found that the FGF signaling pathways is activated in
the mesodermal cells, but this pathway is specifically down-regulated during the
differentiation of the visceral SMCs. In addition, when we maintained the activation of
the FGF pathways, we were able to avoid the visceral SMCs differentiation. These
experiments showed that the control and timing of FGF pathway activation is essential
for tigger of SMCs differentiation. New potential smooth muscle markers have been
isolated such as Hermes, a RNA-binding protein containing an RNA-recognition motif.
We observed expression of Hermes in the differentiated SMCs in the digestive tract
close to
expression. Functional studies of these factors are currently
investigated in our laboratory.
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PW35-436
INTERMUSCULAR TENDONS ARE ESSENTIAL FOR THE DEVELOPMENT OF
VERTEBRATE STOMACH
LE GUEN L1, NOTARNICOLA C1, DE SANTA BARBARA P1
(1) INSERM ERI 25, Muscle and Pathologies, Montpellier, FRANCE.
The gastrointestinal tract is a vital organ system present in all multicellular animals
initially derived from a simple tubal structure. The morphology of the gut requires
reciprocal signaling between the mesoderm and endoderm during development. In
To contact the author:
order to identify and functionally study the mechanisms that control the specific
[email protected] development of the stomach, we performed a microarray study to obtain the gene
expression profiles in the stomach, and in the colon. Analyses of these results
confirmed some genes (BapX1, Hoxa5) and gave us new candidate genes
(Scleraxis, Tenomodulin).
Scleraxis, a member of the basic-helix-loop-helix family has been reported as a
marker of tendons. We identified cells expressing Scleraxis in the stomach as tendon
cells adjacent to visceral smooth muscle cells. We found that Scleraxis expression is
present in mouse stomach, with expression limited to small domains, highlest
adaptation of the stomach between species. In order to investigate more directly the
role of Scleraxis during the development of the stomach, we used the avian retroviral
system to specifically misexpress or inactived Scleraxis in the stomach mesoderm.
Using specific Scleraxis siRNA cloned into avian retroviruses, we observed that thin
malformed stomachs are associated with a strong diminution of Scleraxis expression
suggesting that Scleraxis expression is essential for the correct development of the
stomach. Ectopic expression of Scleraxis into the whole stomach mesoderm leads to
drastic morphological change characterized by a thick stomach associated with a
strong downregulation of smooth muscle markers but no induction of ectopic
tendinous structure. These experiments showed that Scleraxis is able to inhibit the
differentiation of the smooth muscle cells and more surprising to inhibit the
differentiation of the tendon.
In summary, we identified the presence of intermuscular tendons in vertebrate
stomach, and showed that the function of Scleraxis could be to pattern the tendon
domains into avian stomach.
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PW35-437
INVESTIGATING THE FUNCTION OF TBX1 IN CRANIOFACIAL MUSCLE
DEVELOPMENT
GRIFONE R1, JARRY T1, KELLY RG1
(1) Developmental Biology Institute of Marseilles - Luminy, Marseilles, FRANCE.
To contact the author::
[email protected].
Vertebrate craniofacial and trunk myogenesis are regulated by distinct genetic
programs. Branchiomeric craniofacial muscles regulate jaw opening and closing, facial
expression and laryngeal and pharyngeal function. These muscles correspond to the
gill musculature of fish and originate not from the somites, source of trunk and limb
muscles, but from pharyngeal mesoderm. Prior to skeletal muscle specification
branchiomeric muscle progenitor cells share the genetic program of a population of
adjacent myocardial progenitor cells termed the second heart field which give rise to
the right ventricle and outflow tract of the heart (Grifone and Kelly, 2007, Trends
Genet 23, 365-9). Tbx1, murine homologue of the major DiGeorge/del22q11.2
syndrome candidate gene TBX1, is required for robust specification of branchiomeric
craniofacial muscles in pharyngeal mesoderm (Kelly et al, 2004, Hum Mol Genet 13,
289-40). Here we examine the properties of branchiomeric and somite-derived muscle
development in Tbx1 mutant embryos. Using immunohistochemistry we demonstrate
that the myogenic regulatory factors Myf5 and MyoD are first activated in Tbx1 and
Isl1-positive cells within core arch mesoderm. Despite altered fiber-type distributions
and pharyngeal muscle weakness in TBX1 haploinsufficient del22q11.2 syndrome
patients, myosin heavy chain type II and type I fiber distributions are indistinguishable
in Tbx1+/+ and Tbx1+/- adult masseter and pharyngeal constrictor muscles.
Furthermore, type I fibers are distributed normally in hypoplastic branchiomeric
muscles which form sporadically in Tbx1-/- embryos. These sporadic muscles contain
Pax7 positive cells and primary myocytes isolated from Tbx1-/- muscles have
indistinguishable proliferative and myogenic differentiation properties from wild-type
muscles in culture. A subset of somite-derived muscles express Tbx1, including
certain limb muscles. These muscles display normal patterning, growth, fiber-type
distribution and maturation in the absence of Tbx1. Together, our results suggest that
the major role of Tbx1 during skeletal muscle development is restricted to myogenic
specification of branchiomeric muscles.
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PW35-438
OCT-4 A PRIMARY EVENT IN SOX17-MEDIATED SPECIFICATION OF
CARDIAC MESODERM
STEFANOVIC S1, ABBOUD N1, DESILETS S1, PUCEAT M1
(1) INSERM/Evry University UMR861, I-STEM/AFM, Evry, FRANCE.
The embryonic stem cell (ESC) specific protein Oct-4, is one of the earliest
transcription factor in the embryo. Together with Sox2 and Nanog, Oct-4
cooperatively maintains the pluripotency of ESC through a tightly regulated
transcriptional loop. However, a new function of this transcription factor has
recently emerged. There are now valuable insights regarding the role of Oct-4 in
the process of cell differentiation. We recently showed that upregulation of Oct-4
drives Mouse ESC toward a cardiogenic fate. We now report that this
phenomenon is conserved in Human ESC. Furthermore, we uncovered the
transcriptional mechanisms underlying this event. We found that the primary
event triggered by Oct-4 upregulation is the induction of Sox17, an endodermal
marker recently reported as a key factor in the formation of cardiac mesoderm.
Using a ChIP assay in both Mouse and Human ESC, we found that an increase in
Oct-4 level of expression displaces its interaction with Sox2 to target Sox17 DNA
elements. This results in a transcriptional activation of Sox17 as monitored by real
time PCR and cell immunofluorescence. Then induced endodermal cells act
through a paracrine effect. These cells secrete cardiogenic factors in the medium
surrounding colonies of ESC.This results in a specification of both Mouse and
Human ESC toward a cardiac lineage. We thus delineated an Oct-4 mediated
cardiogenic transcriptional network, conserved in both Mouse and Human
ESC.These findings should advance our understanding of biological processes
underlying early cardiogenesis and in turn better comprehend origins of genetic
congenital diseases.
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PW35-439
ECTOPIC FETAL CARDIAC GRAFT : A CONVENIENT EXPERIMENTAL MODEL
OF HEART RECONSTRUCTION
DELRÉE P1, COULIC V2, COLLETTE J3, GOTHOT A3
(1) IRSPG, Gosselies, BELGIUM. (2) CHU Brugmann (ULB), Brussels, BELGIUM.
(3) Université de Liège (Ulg), Liège, BELGIUM.
PURPOSE :in order to easily study heart development or reconstruction, we have
developed a model of ectopic (in time and location) foetal heart graft.
To contact the author::
[email protected].
RESULTS : In this model ,rat foetal heart (E15-E20) , is grafted in the pavilion ear of
an adult rat. Location in the pavilion ear protects the graft from automutilation, and
allows easy studies of the development and function of the graft, during the whole
course of his development, using external recording technologies. Histological studies
demonstrate an early stage of extensive ischemic necrosis, followed some days
after,by a reconstruction process , grossly recapitulating heart ontogenesis. This often
leads to a partially functional heart with blood filled cavities, pace-maker and beating
activities and to some extent with blood flow, as assessed by histological, ultrasound,
IRM and electrical activity studies. Immunohistochemistry and microelectronic studies
demonstrate progressive acquisition of adult myocyte phenotype of the graft cells.
Furthermore the fact of grafting induces a elevation of serum IGF1 level.(50%).
DISCUSSION : this experimental model allows study of the cellular and cytokine
environment associated with cardiac reconstruction of a foetal heart .That knowledge
could potentially give insights to optimize adult cardiac repair using cellular therapy.
By example, modifying cytokine network during a cellular therapy of genetic diseased
heart, in a way mimicking the one observed during the foetal heart graft,, could
potentially favour engineered cells to engraft successfully in the diseased heart. In
that context of cellular therapy, we intend to study the potential mobilisation of host
stem cell induced by foetal heart grafting as well as their potential engraftment in the
foetal graft.
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PW35-440
BIOCOMPATIBLE NANOFILMS DRIVE DIFFERENTIATION OF STEM CELLS
TOWARD A CARDIOGENIC FATE
LOUIS-TISSERAND M1, BLIN G1, PICART C2, PUCÉAT M1
(1) INSERM/UEVE UMR 861, I-Stem, AFM, Evry, FRANCE. (2) INSERM CNRSUMR 5235, Montpellier, FRANCE.
Cell therapy has emerged as a promising therapeutic option for heart failure. To such
an aim, optimisation of cell engraftment is mandatory. Both cell survival and secure
differentiation are likely to require an extracellular scaffold. Using nanotechnology, we
To contact the author::
engineered biocompatible polypeptide and polysacharide multilayer films (PEMS).
[email protected]. These included Poly (L-lysine) hydrobromide (PLL) and Hyaluronan (HA). Crosslinking
of films using different amounts of dimethylaminopropyl carbodiimide (EDC) defines
their stiffness.
We found that the most plastic cells (i.e, embryonic stem cells) respond to the force
exerted by their attachment to the film. This force is translated into induction of a
mesodermal cardiogenic genetic program. Indeed, stem cells cultured on PEMS
(PLL/HA) with increasing stiffness turned on expression of mesodermal genes. This
was associated with changes in cell shape. Real time RT-PCR revealed that the most
rigid films (EDC:100), mimicking the infarction scar, induced a 4, 7, 6, 4.5 fold
increase in expression of Brachyury, Myocardin, Tbx6 and Mesp1,2, respectively, in
comparison with the non cross-linked ones. In fact, more the films were cross-linked
and in turn stiff, more the cells adhered, changed their shape and expressed
mesodermal cardiogenic genes. Expression of the protein Brachyury was also
observed in cells grown on rigid films (from EDC 40). Furthermore, the proliferation
rate of cells also increased with the level of stiffness. Our study shows that the
stiffness of films is crucial for the differentiation of stem cells towards mesodermal
cardiac lineage.
By exerting traction forces on a substrate, stem cells sense the stiffness and show
dissimilar morphology and adhesive properties translated in gene transcription.
Biocompatible nanofilms might represent in the future a mean to secure a cell product
in therapy of heart failure.
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PW35-441
ECTOPIC FETAL INTESTINE GRAFT AS A MODEL FOR CELLULAR THERAPY IN
HIRSCHPRUNG DISEASE
COULIC V1, DELRÉE P2, COLLETTE J3, GOTHOT A3
(1) CHU Brugmann (ULB), Brussels, BELGIUM. (2) IRSPG, Gosselies, BELGIUM.
(3) University of Liege (Ulg), Liege, BELGIUM.
To contact the author::
[email protected].
Hirschprung disease may be considered as an intestinal neuropathy with
muscular dysfunction. Some hope for its treatment has appeared with cellular
therapy.
Aim: to test the possibility of intestinal nervous ganglia reconstruction .
Material, methods: To study intestinal reconstruction features, syngenic fetal
intestine implantation was provided in 30 Fischer rats (site: subcutaneous
pouch of the ear pavilion). HE , connexin-43, nestin, vimentin and actin
staining allowed evaluation of graft maturation state. Host cell participation in
graft development, was evaluated by grafing fetal intestine of “wild type” mice
C57Bl (WT) to 30 green fluorescent protein (b-actin-GFP) transgenic mice
(same site). HE , GFP and SOX-10 staining were used to identify host cells in
the graft and to detect neuronal precursors.
Results. After initial graft ischaemic destruction, reconstruction and growth of the
organ were observed. The reconstruction beginning corresponded to an increase of
IGF-1 levels in the recipient serum, and of nestin and connexin-43 expression in the
graft. Actin and vimentin were first absent and reappeared as smooth muscle
differentiation started. After 1 month, muscular layers and myenteric plexuses were
well-developed. In mice, GFP positive cells, coming only from the GFP host indeed,
were identified in the implant, including muscular layers and many neurons.
_0MS0G0OLK
PW35-442
Conclusion. Remaining alive initially grafted cells, as well as host cell, incorporated
in the graft, grew and differentiated into the different layers of intestine, including
neuronal ganglia, according to ontogenic patterns. . Moreover the graft modifies host
metabolism, as increased host serum IGF-1 level testimonies. . These observations
opens potentialities for cellular therapy in Hirschprung disease
RAE-1 IS A NEW MARKER FOR NEURAL STEM CELLS INVOLVED IN CELL
PROLIFERATION
CEDILE O1, POPA N1, POLLET-VILLARD X1, MONTI G2, BAGNIS C3, DURBEC P2,
BOUCRAUT J1
(1) CRN2M Centre de Recherche en Neurobiologie; CNRS UMR6231, Marseille,
FRANCE. (2) IBDML Institut de Biologie du Developpement de Marseille Luminy;
CNRS UMR6216, Marseille, FRANCE. (3) EFS Etablissement Français du Sang
Alpes Méditérannée; laboratoire de thérapie génique et cellulaire, Marseille, FRANCE.
More and more experiments show the role of immune cells and immunological
To contact the author::
molecules in the physiology of the nervous system including neurogenesis. A better
[email protected] knowledge of interactions between immune cells and neural stem cells is important for
r.
improving and controlling the capacity of either endogenous or grafted adult neural
stem cells to repair the nervous system.
We address the question of the role of Major Histocompatibility Complex-I (MHC-I)
molecules in neurogenesis in the mouse model. Our main results concern the
expression and functions of Rae-1, a molecule related to the MHC-I family. Rae-1 was
expressed by neural stem cells and was down regulated after their differentiation.
Rae-1 transcripts were known to be present only in early embryonic stages particularly
in the nervous tissue. Moreover, we showed that Rae-1 was also expressed in the
regions of adult neurogenesis and its expression was induced in the olfactory bulbs
after olfactory axotomy.
Rae-1 is recognized by NKG2D, the main activator receptor of the NK, NKT and T δ
lymphocytes. In the model of olfactory axotomy, we demonstrated a recruitment of
lymphocytes in the olfactory bulbs which correlated with an increase in NKG2D
transcripts. However, no NKG2D expression was detected in the subventricular zone
in physiological or pathological condition. We thus questioned a role of Rae-1 in the
absence of immune cells. Using a transduction approach with lentivirus coding for
shRNAs targeting Rae-1, we demonstrated that Rae-1 is involved in the proliferation
of neural stem cells.
Deciphering the roles in vivo of Rae-1 in neurogenesis is now our main objective.
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PW35-443
EGR1 AND EGR2 CONTROL VERTEBRATE TENDON CELL DIFFERENTIATION
BY REGULATING SCLERAXIS AND COLLAGEN EXPRESSION
BLAIS F1, LÉJARD V1, MARO GS2, GILARDI-HEBENSTREIT P2, ROSSERT J3,
DUPREZ D1
(1) CNRS UMR 7622, Université Pierre et Marie Curie, PARIS, FRANCE. (2)
INSERM U784, Ecole Normale Supérieure, PARIS, FRANCE. (3) Equipe 3 Centre de
recherche des Cordeliers, Université Paris-Descartes, PARIS, FRANCE.
To contact the author::
[email protected]
ssieu.
The molecular mechanisms underlying tendon formation during vertebrate
embryogenesis are still largely unknown. In Drosophila, tendon differentiation relies
upon the transcription factor stripe, an Early growth response (Egr)-like transcription
factor. Based on the requirement for stripe in tendon formation in Drosophila, we
investigated the involvement of the homologous Egr family members, Egr1 and Egr2
in vertebrate tendon formation. We established that Egr1 and Egr2 transcripts are
located in forming and differentiating limb tendons during chick and mouse
embryogenesis. The secreted factor fibroblast growth factor-4 activated the
expression of both Egr genes within 4 hours before the activation of the differentiation
tendon markers Scleraxis, EphA4 and the collagens I, V and XII. Misexpression
experiments using the chick model allowed us to establish that Egr genes are
sufficient for the expression of Scleraxis and tendon-associated collagens. Analysis of
embryonic limbs from mouse mutants for Egr1 or Egr2 did not exhibit major
modifications in tendon formation, based on the expression of Scleraxis and collagen
I, suggesting redundancy between both genes in tendon formation. Finally, we
showed that Egr2 enhanced the activity of the col1a1 tendon promoter, indicating that
Egr2 is part of the DNA-binding protein network involved in the regulation the col1a1
expression in tendons. The endogenous tendon expression of the Egrs, their
regulation by Fgf4, their sufficiency to induce tendon gene expression and their ability
to bind the tendon promoter of col1a1 all indicate that Egr genes control tendon cell
differentiation by regulating Scleraxis and collagen expression. This represents a rare
example of conserved regulatory function between homologous genes in invertebrates
and vertebrates.
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PW35-444
TSHZ3 DEFICIENCY CAUSES FUNCTIONAL RENAL TRACT OBSTRUCTION BY
IMPEDING URETERIC SMOOTH MUSCLE DIFFERENTIATION
MARTIN E1, GANNON C2, CAUBIT X1, CORÉ N1, FILLIPI P1, VOLA C1, GARRATT
AN3, WOOLF A2, FASANO L1
(1) IBDML - Development Biology Institute of Marseille Luminy, Marseille, FRANCE.
(2) Nephro-Urology Unit, UCL Institute of Child Health, London, UNITED-KINGDOM.
(3) Max-Delbrück-Center for Molecular Medicine, Berlin, GERMANY.
To contact the author::
[email protected].
Teashirt (Tshz) genes encode transcription factors conserved between flies and
mammals. We show that mouse ureteric smooth muscle cell (SMC) precursors
express Tshz3, and that Tshz3 null mutant mice have congenital hydronephrosis
without anatomically-impaired urine flow. In vivo, failed ureteric muscle differentiation
antedated urinary tract dilatation while ex vivo, wild-type but not mutant fetal proximal
ureter segments contracted spontaneously. Moreover, the expression of myocardin,
an essential component of the regulatory pathway for SMC differentiation, and of
myocardin-dependent SMC genes was markedly downregulated in Tshz3-null mouse
ureters prior to the onset of hydronephrosis. The data are consistent with a model in
which Tshz3 expression is required for SMC differentiation by pathways involving
myocardin-dependent SMC transcription. These findings provide new insights into
molecular pathways linking visceral SMC differentiation with muscle function, and they
also emphasise the central role of the proximal ureter in the physiological transit of
fetal urine from the kidney to the urinary bladder, with defects leading to congenital
hydronephrosis
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PW35-445
ROLE OF SRF IN THE RESPONSE OF VASCULAR SMOOTH MUSCLE CELLS TO
MECHANICAL STRESS.
GALMICHE G1, MERICSKAY M1, BLANC J1, LI Z1
(1) UPMC/CNRS UMR7079, Physiologie et physiopathologie, Paris, FRANCE.
Vascular smooth muscle cells (VSMCs) anomalies are directly involved in different
forms of myopathies including Duchene myopathy and desminopathies. The alteration
To contact the author::
of cytoskeletal mechanostransduction and transcriptional pathways play an important
guillaume.galmiche@snv
role in the evolution of the vasculopathies. Serum response factor (SRF) is a key
.jussieu.fr.
transcription factor in SMCs. The activity of SRF is modulated by the RhoA-actin
pathway and growth factors signaling pathways. Our aim is to study the role of SRF in
adult VSMCs in vivo and in vitro by conditional invalidation of SRF gene using a
tamoxifen-inducible and smooth muscle-specific Cre recombinase, SM22-CREERT2(ki).
In order to identify the targets of SRF in VSMCs in vivo, RNA was isolated from aorta
8 days after tamoxifen injection in control and mutant mice. Preliminary analysis of
gene expression microarrays shows that more than 100 genes are differentially
expressed in the aorta of the SRF mutants. The validation of these data by Q-PCR is
ongoing. In order to evaluate the role of SRF in the transcriptional response of VSMCs
to experimental cyclic stretch, we isolated VSMCs from the aorta of floxed SRF mice,
and optimized the conditions for maintaining VSMCs differentiation in culture with a
modified serum-free medium. We infected primary VSMCs cultures with a
recombinant adenovirus expressing the Cre recombinase to inactivate SRF in vitro.
More than 95% decrease of SRF mRNA expression was obtained by this approach.
The effect of stretch on SMC-specific genes expression is currently evaluated using
the FlexerCell system in control and SRF-deficient VSMCs. Chemical inhibition or
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dominant negative approach will be used to decipher the pathways linking
biomechanical stretch to SRF activation.
PW 36:
Muscle assessment,
scales and
patients’ registries
PW36-447
SPARSE SHAPE MODELS WITH OPTIMAL IMAGE SUPPORT FOR MRI CALF
MUSCLE SEGMENTATION
ESSAFI S1, LANGS G1, BASSEZ G2, DEUX JF2, VIGNAUD A3, RAHMOUNI A2,
PARAGIOS N1
(1) GALEN Group, Laboratoire MAS, Ecole Centrale Paris/ INRIA Saclay - Île-deFrance, Parc Orsay Université, Chatenay-Malabry, FRANCE. (2) Centre Hospitalier
Universitaire Henri Mondor, Créteil, FRANCE. (3) Siemens Medical Solutions, Saint
Denis, FRANCE.
To contact the author::
[email protected].
The aim of this study is to evaluate a model based method for the automatic
segmentation of human calf muscles in T1-MRI data. Recent advances in biomedical
imaging have made possible the in-vivo non invasive visualization of anatomical and
functional information on the muscle state. These modalities offer new means to
assess the state of the muscle and the performance of different
therapeutic strategies. A crucial pre-requisite is the accurate segmentation of
individual muscles, a challenging task due to the lack of contrast between different
regions.
Sparse shape models (SSM) learn a statistical model of shape and local textures from
an annotated training set and derive a sparse representation and reconstruction
mechanism for individual muscles. SSMs take the heterogeneous distribution of
salient
features
in
the
muscle
MRI
data
that causes standard segmentation methods to fail. During application, SSMs use the
sparse model to automatically and accurately segment the muscle in a T1-MRI
volume. The evaluation of the accuracy of SSM segmentation was performed on 4 T1MRI data sets with a spatial resolution of 0.5859x0.5859x7 mm. For all data sets a
manual expert annotation of the medial gastrocnemius served as standard-ofreference. In addition SSM accuracy is compared to a standard approach that does
not take the heterogeneous data into account. The segmentation error was found to
be sufficient for further processing of the data. The mean segmentation error of SSMs
is 9.53, while standard segmentation achieves 37.78. On all examples, SSM
outperform the standard approach.
Thus, SSMs provide for an accurate segmentation of human calf muscles in T1-MRI
data. Their performance is superior to standard methods. The work aims at a noninvasive analysis of the structure of muscles affected by myopathies.
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PW36-448
NON INVASIVE ASSESSMENT OF MOUSE MUSCLE VOLUME USING 3D µECHOGRAPHY
NEJJARI M1, JANIER M1, GOURDON G2, PUYMIRAT J3, HIBA B1
(1) Université Lyon1, ANIMAGE, Lyon, FRANCE. (2) INSERM UR 383, Necker
Hospital, Paris, FRANCE. (3) Human Genetics Unit, Laval CHU, Québec, CANADA.
Introduction: Mouse models are now widely used for drug discovery and muscle
disorder studies (e.g. myopathy). Therefore a quantitative method to determine
muscle volume in vivo will help us for the follow-up of the muscle disease. In the
To contact the author::
present work, we report a method based on 3D ultrasound (3D-US) imaging to
[email protected]. quantify Tibialis Anterior muscle (TA) volume with high accuracy and precision.
Methods: Mice were anesthetized and the lower leg was scanned along the TA
muscle (over a 20 mm, 0.2 mm steps) using a 3D-US high-resolution probe (spatial
lateral resolution 40µm). A semi-automated segmentation algorithm was used to
delineate the TA muscle in the acquired images and assess its volume.
Validation and reproducibility of the measurement was investigated in 5 mice (C57bl6
of 10 months) examined 4 times and Intra-reader was also investigated.
Results: Mean muscle volume measured by 3D-US was 40.4 ± 1.1 mm3 in control
mice. Statistical analysis showed an intra-reader deviation equal to or less than 2%. In
addition, a high correlation between TA muscle ex vivo weights and the volumes
obtained using 3D-US was found for the investigated mice (R > 0.95).
The proposed method was used to follow-up TA volume evolution in 7 normal mice
from age 2 to age 7 months. We observed a mean muscle volume increase from 17.6
± 1.3 mm3 to 22.5 ± 1.4 mm3, which correspond to an increase of 28%.
Conclusion: 3-D US imaging provided a good precision and accuracy in the
measurements of muscle volume in small animal models (e.g. TA muscle). This
method could be very useful for the quantification of disease progression and for
evaluation of the efficacy of new therapies.
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PW36-449
ASSESSING NEUROMUSCULAR FUNCTION FOR PATIENT FOLLOW-UP USING
VARIOUS METHODOLOGIES: CORRELATIONS AND VARIABILITIES
HOGREL JY1, OLLIVIER G1, CANAL A1, CALPAÏN STUDY GROUP T1
(1) Institut de Myologie, Paris, FRANCE.
To contact the author::
[email protected].
Assessment of the neuromuscular function during the time course of neuromuscular
disorders is much informative for patient follow-up either during the natural history of
his disease or during a therapeutic intervention. Within this last frame, the choice of
the evaluation criteria is crucial for many reasons. Many methods exist to assess the
various aspects of the neuromuscular function. This was the aim of this study to
perform and to analyze a selection of different tests for the follow-up of patients during
the natural history of their disease.
This study is a small part of a larger multicentric study about a multi-parametric
approach to natural history knowledge of calpainopathies for efficacy assessment of
therapies. Twenty one patients suffering from LGMD 2A (calpainopathy) were followed
every six months during two years. Each patient visit included Manuel Muscle Testing
(MMT), Quantified Muscle Testing (QMT), Muscle Function Measure (MFM), isometric
dynamometry (Biodex) and timed functional tests. Elbow and knee flexion and
extension were particularly analyzed. Their reproducibility and variability were
assessed. Correlations between the measurements obtained using different methods
were analysed.
This study revealed that the results obtained with QMT or Biodex were not always
highly correlated and may differ depending on the muscle function studied. For
instance, the evaluation of elbow and knee extensions seems to be much more
reliable than the evaluation of elbow and knee flexions for this patient population.
Moreover, both QMT and Biodex were unsuitable to detect small strengths in very
weak patients. From longitudinal data, it was possible to compute percentage of
variations to be considered significant of true strength changes.
Natural history of disorders may help to identify the muscle function to be studied in
further therapeutic trials and to provide the amount of change significant of a true
evolution.
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PW36-450
THE 6-MINUTE WALK TEST AS A CLINICAL TRIAL OUTCOME MEASURE IN
DUCHENNE MUSCULAR DYSTROPHY: RELIABILITY AND CORRELATION WITH
OTHER MEASURES OF DISEASE SEVERITY
MCDONALD CM1, HENRICSON EK1, HAN JJ1, NICORICI AR1, ABRESCH RT1,
ATKINSON LA2, REHA AL2, ELFRING GL2, MILLER LL2
(1) University of California, Davis Medical Center, Sacramento CA, USA. (2) PTC
Therapeutics, South Plainfield NJ, USA.
To contact the author::
[email protected]
du.
Background: The 6-minute walk test (6MWT) is a commonly used measure of
cardiorespiratory endurance. Researchers have begun to use it as a strength-related
outcome measure in clinical trials in neuromuscular disease. We evaluated the
6MWT differentiation between boys with Duchenne muscular dystrophy (DMD) and
healthy controls, test-retest variability in boys with DMD, and correlation of the 6MWT
with timed functional measures.
Methods: We enrolled ambulatory boys 5-12 years old with DMD (n=15) and without
(n=20). Boys with DMD were tested 7 days apart using a modified American Thoracic
Society 6MWT and standard clinical timed function testing. Healthy controls
underwent testing at a single time point.
Results: Across all ages, distance traveled differed between boys with DMD and
healthy controls. In boys aged 5-6, those with DMD averaged 367 ± 74 m compared
to 574 ± 35 m (p<.01) for the healthy controls. In boys aged 7-9, those with DMD
averaged 354 +/- 31 m compared to 622 ± 50 m (p<.001) for the healthy controls. In
boys aged 10-12, those with DMD averaged 265 ± 146 m compared to 646 ± 49 m
(p<.001) for the healthy controls. As age increased in the DMD group, percent
predicted scores decreased from 82% at age 5 to 56% at age 12. 6MWT test-retest
correlation was high (r=.92). In the DMD group, the 6MWT correlated well with time to
walk 10 m (r=.80), time to walk 25 m (r=.80), time to climb 4 standard stairs (r=.77),
and time to stand (r=.64).
Conclusion: A modified 6MWT in ambulatory boys with DMD is reproducible,
differentiates boys with DMD from healthy controls at all ages, and correlates with
other measures of disease severity.
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PW36-451
QUALITY OF LIFE OF ADOLESCENTS WITH NEUROMUSCULAR DISEASES: HERE’S
WHAT THEY SAY
VUILLEROT C1, HODGKINSON I1, BERARD C1, ECOCHARD R2, D'ANJOU MC3,
COMMARE MC4
(1) Hospices Civils de Lyon, CHU Lyon Sud, L'Escale, Service de Médecine Physique et de
Réadaptation Pédiatrique, Pierre Benite, FRANCE. (2) Hospices Civils de Lyon, Service de
Biostatistique, LYON, FRANCE. (3) CHU St Etienne, Service de MPR pédiatrique, St
Etienne, FRANCE. (4) CHU Grenoble, service MPR Pédiatrique, Grenoble, FRANCE.
Little is known about quality of life of adolescents with neuromuscular diseases or the factors
To contact the author::
[email protected].
that influence it. We searched whether physical impairment, physical disability, and medical
complications were predictors of low quality of life. Motor function, health, orthopaedic status,
and rehabilitation were assessed in 43 adolescents with neuromuscular diseases (age:
13.8±1.7; sex ratio 2.9/1). Quality of life was measured with the VSP-A (Vécu Santé Perçu par
l’Adolescent), a validated health-related quality of life self-perception test. A multiple linear
mixed regression related quality of life to impairment, disability, and respiratory status.
Comparisons were made with data from an age/sex-matched healthy population. On the
average, the VSP-A scores in diseased adolescents were: i) similar to those of the healthy
population as regards vitality, body image, relationships with parents and friends, and physical
and psychological well-being ; ii) higher as regards school performance (68 vs. 52.8%) and
relationships with teachers (67.4 vs. 43.2%); iii) lower as regards leisure activities (43.9 vs.
60.9%). Physical disability and physical impairment were not significantly associated with
seven out of the nine domains but scores for leisure activities and vitality were significantly
associated with physical impairment (p=0,001 and p=0,006 respectively). Adolescents with
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ventilatory support did not express lower scores than non-ventilated ones (67.7% +/- 11 vs.
62.9%+/-15, p=0.39). These surprising results should question our medical, educational, and
rehabilitation practices. Already well-managed disabled adolescents should benefit from a
less compassionate and more daring and dynamic interpersonal contacts.
Conclusions - These surprising results should question our medical, educational, and
rehabilitation practices. Already well-managed disabled adolescents should benefit from a
less compassionate and more daring and dynamic interpersonal contacts .
PW36-452
THE MOTOR FUNCTION MEASURE (MFM) SCALE WAS USED TO EVALUATE
PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY (DMD).
BERARD C1, VUILLEROT C1, PAYAN C2, GIRARDOT F1
(1) Hospices Civils de Lyon, CHU Lyon Sud, L'Escale, Service de Médecine Physique et
de Réadaptation Pédiatrique, Pierre Benite, FRANCE. (2) Institut de myologie, Paris,
FRANCE.
Study participants and setting: Patients without medication were evaluated at 3 months
To contact the author::
[email protected].
for 13 patients (mean age 11y 7 mo; SD 1y 10mo) and at one year for 41 patients (mean
age 14y 1 mo; SD 5y 5mo) in a referral center. Twelve patients treated with steroids
(mean age 10 y 2 mo; SD 2y 2 mo) were evaluated after one year of treatment and
compared with 12 age-matched DMD controls.
Results: In a 3-month period, subscore D1 showed a significant average decrease of
4.7% (p<0,01). In a 1-year period, all scores showed significant decreases: D1 4.9%
(p<0,01), D2 7.7% (p<0,01), D3 4.3% (p<0,03) and Total Score 5.8% (p<0,01). For the
11 walking patients at the beginning of the study, the average value of D1 annual
decrease is 26.1%. For the non-ambulant patients, the annual average decrease was
11.8% for D2 and 6.3% for Total Score. A sensitive threshold value for the loss of the
ability to walk and a predictive value a year before loss of ambulation could be
estimated. In the group of patients treated with steroids, compared to controls, it was
evidenced a stabilization of the total score (-0.59 vs. -5.87, p<0,02 ) and dimension D2
(0.98 vs. -8.50, p<0,01).
Conclusions: Our preliminary results are promising for the use of the MFM in clinical
trials to evidence either deterioration of disease or lack of deterioration induced by
therapeutics.
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PW36-453
THE ONLINE WEB VERSION OF THE NMD GENE TABLE OF NEUROMUSCULAR
DISORDERS
HAMROUN D1, BEROUD C1, CLAUSTRES M1, KAPLAN JC2
(1) INSERM, U827, Montpellier, FRANCE. (2) Université Paris Descartes/CNRS,
Institut Cochin, et Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Cochin,
PARIS, FRANCE.
The first gene table of neuromuscular disorders (Neuromuscular Disorders,
1991, Vol 1, N°1, 75-76) was a list of seven identified genes and sixteen mapped
To contact the author::
loci awaiting identification of the causative gene. In the January 2008 printed
[email protected].
version of the table there are 213 identified genes, 103 mapped but not yet
identified loci, 536 phenotypes and 742 key references (ibid, 2008, Vol 18, N°1,
101-129). This increase has generated a high degree of complexity, because
mutations in a given gene may produce a large number of patho-phenotypes,
such as in laminopathies; and because, conversely, a given patho-phenotype
may be caused by mutations affecting one among several possible genes, such
as in CMT. Thus the printed updated table, now published once a year, has
reached a size making it difficult to maintain and to consult. In order to help find
one's way through this chaos we started in 2005 a computerized online version
of the NMD gene table (see http://194.167.35.195), which is regularly updated
and implemented. The main advantage of the web version is its interactivity
facilitated by : (i) cross-referencing the different items pertaining to each entry
(name of disease, mode of inheritance, locus symbol with OMIM number,
chromosomal localization, approved gene symbol with OMIM number; approved
protein name, key references, other allelic disease phenotypes); (ii) introducing
outward links providing instantaneous connection to the following databases:
Leiden Muscular Dystrophy, OMIM, NCBI, Genatlas. A query tool allows one to
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ask many kinds of questions. In the future other external links will be added, in
particular to Locus Specific Databases
giving access to the mutational
spectrum of each gene. The online NMD gene table should allow clinicians to
obtain molecular diagnosis far more easily.
PW36-454
FRENCH DATABASE FOR MYOTONIC DYSTROPHIES (DM1-DM2): GENERAL
PRINCIPLES AND DESCRIPTION
BASSEZ G1, GUIRAUD-DOGAN C1, HAMROUN D2, BEROUD C2
(1) Reference center for neuromuscular diseases, CHU Henri Mondor, Créteil,
FRANCE. (2) Institut Universitaire de Recherche Clinique, CHU de Montpellier,
Montpellier, FRANCE.
Myotonic dystrophy is the commonest muscular dystrophy in adults and the most
To contact the author::
guillaume.bassez@hmn.
aphp.fr.
variable neuromuscular disorder. This high variability of the multisystemic involvement
creates particular challenges for both management and the design of optimal
therapeutic trial. Therefore, a database specifically dedicated to myotonic dystrophies
may be a valuable tool for promoting clinical research and optimizing the management
of the patients. This database will contain clinical and paramedical data collected
during the medical consultations of DM patients in several French neuromuscular
centres and sent to the Henri Mondor hospital for recording. Basic informations will be
first reported in an inclusion document (identification, expansion size of the mutation,
clinical history, clinical evaluation of neuromuscular and systemic signs, and
professional and social consequences of the disease) and next completed by an
annual follow-up section reporting the recent clinical events and an actualised clinical
evaluation. The database may then allow us: 1) to identify prognostic factors (muscle
weakness, cardiac and respiratory involvement, swallowing disturbances, intellectual
impairment) and to study their potential interrelations; 2) to compare the main features
of both diseases in patients from two different countries. In particular, the study will
compare the DM1 genetically homogenous population of Quebec to the genetically
heterogeneous French patients 3) to study mortality and morbidity; 4) to search for
genotype/phenotype correlations; and 5) to compare DM1 and DM2 features.
Furthermore, recent research has provided more information on the underlying
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molecular pathomechanisms involved in myotonic dystrophies that creates new
opportunities for more specific therapy. The database will be of great interest to select
patients for future therapeutic trials.
PW36-455
UMD-DMD FRANCE: A NATIONAL KNOWLEDGEBASE OF MOLECULAR
DEFECTS IN THE DYSTROPHIN GENE
TUFFERY-GIRAUD S1, BÉROUD C2, LETURCQ F3, BEN YAOU R4, HAMROUN D5,
DESMET FO2, MICHEL-CALEMARD L6, KHAU VAN KIEN P5, HUMBERTCLAUDE
V5, KAPLAN JC3, CHELLY J3, CLAUSTRES M2
(1) Université Montpellier 1, UFR Médecine, Montpellier, F-34000, FRANCE. (2)
Inserm U827, Montpellier, F-34000, FRANCE. (3) CHU Hôpital Cochin, Laboratoire
de Biochimie et Génétique Moléculaire, Paris, FRANCE. (4) INSERM, U582, IFR14,
Institut de Myologie, Paris, FRANCE. (5) CHU Montpellier, Laboratoire de Génétique
Moléculaire, Montpellier, F-34000, FRANCE. (6) French Collaborative Network of
molecular diagnostic laboratories, Lyon, FRANCE.
UMD-DMD France is a national locus-specific database (LSDB) dedicated to
dystrophinopathies. It has been developed through a multi-center academic effort to
To contact the author::
provide an up-to-date resource of curated information covering all identified and fully
[email protected]
validated mutations in patients with dystrophinopathies in France. Whenever
erm.fr.
necessary, mutations have been reevaluated at the light of the currently available
techniques. The database includes 2270 entries corresponding to 2070 independent
mutational events identified in either male patients (2034) or symptomatic female
carriers (36). These mutations consist in 1420 deletions, 261 duplications, and 449
small rearrangements of which 39.1% are nonsense. Experts in the DMD gene and
related diseases are responsible for data quality and accuracy. In addition to gather
mutations, the UMD-DMD France includes available data on dystrophin and RNA
analysis, phenotypic groups, and transmission. The database aims at further including
extensive description of phenotypes associated with the reported mutations to better
delineate the clinical spectrum of dystrophinopathies and to allow genotype/phenotype
correlations. New tools have been specifically developed in the UMD software to
facilitate large-scale mutation analyses of the DMD gene. UMD-DMD will benefit to all
the scientific community interested in dystrophinopathies including geneticists,
clinicians, and researchers involved in the design of therapeutic strategies. Also it will
prove useful to implement forthcoming global registries of patients for clinical trials as
ultimate goal within the European Network of Excellence for treatment of
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neuromuscular disorders (TREAT-NMD). Network of French Laboratories: R.
Bernard (Marseille), E Bieth (Toulouse), M. Blayau (Rennes), P Boisseau (Nantes), L
Calemard (Lyon), M Cossée (Strasbourg), I Creveau (Clermont-Ferrand), B De
Martinville (Lille), A Guiochon (Kremlin Bicêtre), P Khau van Kien (Montpellier). F
Leturcq (Cochin), MP Moizard (Tours), N Monnier (Grenoble), C Philippe (Nancy).
PW36-456
TREAT-NMD UMD PATIENTS’ REGISTRIES: FROM PATIENTS TO THERAPY
HUMBERTCLAUDE V2, TUFFERY-GIRAUD S3, HAMROUN D2, DESMET FO1, LALANDE
M1, COLLOD-BÉROUD G1, LOCHMÜLLER H4, CLAUSTRES M1, BÉROUD C1
(1) INSERM U827 F-34000, Montpellier, FRANCE. (2) CHU Montpellier, Laboratoire de
Génétique Moléculaire, Montpellier, FRANCE. (3) Université Montpellier 1, UFR
Médecine, Montpellier, FRANCE. (4) TREAT-NMD network, Newcastle upon Tyne,
UNITED-KINGDOM.
The TREAT-NMD (Translational Research in Europe for the Assessment and Treatment
of Neuromuscular Diseases) ‘network of excellence’ is funded by the European Union and
To contact the author::
[email protected]
serm.fr.
aimed at improving treatment and finding cures for patients with neuromuscular disorders.
The development of the TREAT-NMD UMD patients’ registries will allow identifying
patients with respect to their genetic defect and clinical status. Duchenne-Becker muscular
dystrophies and spinal muscular atrophy will be specifically targeted. The primary
objective of these European patients’ registries is to facilitate the planning of appropriate
clinical trials and supports the recruitment of patients. Data collection is performed either
from a "patient report" filled out directly by the patient (or his legal representative) or from
a "professional report” filled out by a clinician and/or a geneticist. Three levels of data have
been defined: (1) mandatory items (requested for patients’ inclusion into the database), (2)
highly encouraged items (very important items but not requested for patient’s inclusion)
and finally (3) optional items (useful for secondary purposes of the registries). The
mandatory items contain patient’s personal data (encrypted), mutation name (reported
according to the international nomenclature system) and clinical data (motor function,
scoliosis surgery, steroid therapy (DMD only) and feeding function (SMA only)). In each
national registry, curators validate the data in order to maintain high-quality data and to
insure the respect of both nationals and European legislations. Authorized medical experts
and researchers will have access to encrypted data if their research project fits the goals
of TREAT-NMD and after approval by the TREAT-NMD global registry oversight
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committee.
To our knowledge, this is the first example of such a large international initiative to build
patients’ registries. In the next years, we will need to convert this try to speed up clinical
trials development and a rapid enrollment of patients.
AFTER READING, DELETE THIS MESSAGE
PW 37:
Myotonias and
miscellaneous
PW37-457
To contact the author::
[email protected].
SEROLOGICAL ASSAY FOR THE DIAGNOSIS OF AUTOIMMUNE K+
CHANNELOPATHIES
MARTIN-MOUTOT N1, BERTHOMIEU M1, SEAGAR M1
(1) INSERM U641, Universite de la Mediterranee-Aix Marseille 2, Marseille,
FRANCE.
Autoimmune responses against voltage-gated ion channels are suspected in
several diseases, but evidence is compelling in the case of two: Lambert-Eaton
myasthenic syndrome (voltage-gated Ca2+ channels, Cav) (O’Neill et al. 1988) and
neuromyotonia (voltage-gated K+ channels, Kv) (Benetar, 2000). These disorders
affect the PNS and principally the axonal terminals of motoneurones.
Acquired neuromyotonia (NM) or Isaac’s syndrome is a disease in which
peripheral nerve hyperactivity leads to myokymia (muscle twitching, cramps and
impaired relaxation resulting from spontaneous motoneurone discharge). In some
patients NM is associated with CNS symptoms (hallucinations, mood changes,
insomnia) and this has been designated as Morvan’s syndrome. Patients with NM
respond to plasmapheresis and passive transfer has been demonstrated. Mice
injected with IgG from an NM patient displayed increased quantal content, probably
resulting from an increment in Ca2+ influx owing to increased action potential duration
(Sinha et al. 1991). Thus the effects of NM IgG resemble those of certain K+ channel
blockers, suggesting that NM patients produce anti-Kv autoantibodies.
Based on published studies and our experience in serological assay of Cav
autoantibodies (Martin-Moutot et al. 2006), our laboratory has set-up serological assay
for circulating antibodies against Kv as an aid to diagnosis. Routine assays for
autoantibodies rely on immunoprecipitation of channel / 125I-ligand complexes from
solubilized mammalian brain membranes. Potassium channels in detergent extracts of
rat brain membranes were labeled with a radioligand ( 125I-dendrotoxine) specific for
these channels. Patient autoantibodies that immunoprecipitate the ligand/channel
complex can thus be titrated.
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Benetar M, (2000) Q J Med 93: 787-797.
Martin-Moutot N, de Haro L, Santos RG, Mori Y, Seagar M (2006) Neurobiol Dis. 22,
57-63.
O’Neill J. H., Murray N.M.F., Newsom-Davis J. (1988) Brain 111, 577-596.
Sinha S, Newsom-Davis J, Mills K, Byrne N, Lang B, Vincent A. (1991) Lancet 338,
75-77.
PW37-458
To contact the author::
[email protected].
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SPECIFICITY OF ELECTROMYOGRAPHIC EXERCISE TESTS FOR
DIFFERENTIATION BETWEEN THE INVOLVED ION CHANNELS AND TYPES OF
MUTATIONS IN INHERITED MUSCLE CHANNELOPATHIES AND IN MYOTONIC
DYSTROPHY TYPE 2
LEONARDIS L1, ZIDAR J1
(1) Institute of Clinical Neurophysiology, University Medical Centre Ljubljana,
Ljubljana, SLOVENIA.
The role of electromyographic short and long exercise tests in diagnosing inherited
muscle channelopaties is well established. It has been reported that such tests can
point to the affected genes or specific mutations. Our aim was to re-examine this
hypothesis and, in addition, to test patients with myotonic dystrophy type 2 (DM2).
We examined 24 patients: 11 with potassium aggravated myotonia (PAM) (SCN4A: 9
Val445Leu mutation, 2 Ala699Thr), 6 with myotonia congenita (MC) (different CLCN1
mutations), two with hypokalaemic periodic paralysis (HypoK-PP) (CACNA1S and
KCNJ2 mutations), and 5 with DM2, where aberrantly spliced RNA produces altered
chloride channel protein.
Results of the long and short exercise tests were normal or near to normal in 9 PAM
(all Val445Leu) patients. A decrease of the compound muscle action potential (CMAP)
late after completion of the long exercise and also after short exercise was noted in
one of the two Ala699Thr patients but was normal in the other. Five of the 6 MC
patients had autosomal dominant and one recessive form of the disease. In none of
them, the results of tests differed significantly from normality. The HypoK-PP1 patient,
tested when taking acetazolamide, had no abnormalities. In patient with AndersenTawil’s syndrome, a progressive decline in CMAP amplitudes after long exercise was
noted. In DM2 patients, we found minor deviations from normality with no specific
pattern.
Number of the examined patients is rather small, especially in the subgroups. The
results were largely normal in PAM Val445Leu patients while one Ala699Thr patient a
decline of muscle response after long exercise was noted. All MC patients, those with
dominantly and recessively inherited disease forms, were normal on these tests. The
Hypo-PP patient’s CMAPs declined on long exercise. Specificity of the
electromyographic exercise tests seems to be limited, but they may help in decisions
for genetic analyses.
PW37-459
THE PRESYNAPTIC CALCIUM CHANNEL P/Q TYPE AS A POTENTIAL PARTNER
FOR THE BASEMENT MEMBRANE PROTEIN PERLECAN.
OERTEL J1, WATSCHINGER K2, FONTAINE B1, NICOLE S1
(1) Inserm U546, Paris, FRANCE. (2) Universität Innsbruck, Institut für Pharmazie,
Abteilung für Pharmakologie und Toxikologie, Innsbruck, AUSTRIA.
Hereditary skeletal muscle channelopathies are characterized by abnormal muscle
excitability. They result from mutations in genes coding for skeletal muscle voltageTo contact the author::
[email protected].
gated ion channels involved in the propagation of action potential and excitationcontraction coupling. Related diseases that clinically resemble these channelopathies
include Schwartz-Jampel syndrome (SJS, MIM 255800), which is a rare autosomal
recessive human disorder. It is characterized by muscle hyperactivity clinically related
to myotonia. SJS is due to a lack of perlecan, the major heparan sulfate proteoglycan
of basement membranes (BMs). The synaptic BM is rich in laminin isoforms that play
a crucial role in the organization of neuromuscular junctions (NMJs). The C-terminus
of the laminin beta2 subunit binds directly to the pore forming alpha1 subunit of the
presynaptic P/Q type voltage-gated calcium channel (CaV2.1) by a leucine-arginineglutamate (LRE)-motif. Two LRE-motifs are present in the human perlecan (aa 41494151 and 4299-4301), which is highly enriched at the BM of NMJs. This observation
led us to study a possible interaction between the BM protein perlecan and CaV2.1 in
vivo and in vitro. First, the SJS mouse mutant line was used to analyze the correlation
between the CaV2.1 expression and the absence of perlecan. Immunostaining and
confocal microscopy of dissected longitudinal and transversal muscle slices revealed
an up-regulation of CaV2.1 at the NMJ in the SJS line compared to wildtype mice.
Secondly,
immunocytochemical
experiments
using
the recombinant
HEK293
expression system showed a selective colocalization at the outer membrane of
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endogenously expressed perlecan and the transfected alpha1 subunits of CaV2.1.
These preliminary results suggest an interaction, direct or indirect, between the
presynaptic calcium channel and perlecan. We currently investigate this issue further
by biochemical analyses.
PW37-460
AUTOSOMAL RECESSIVE MYOTONIA CONGENITA : CLINICAL,
ELECTROPHYSIOLOGICAL AND GENETIC STUDY IN 6 FAMILIES
BIROUK N1, BELAÏDI H1, REGRAGUI W 1, KABLY B1, URTIZBEREA A2, OUAZZANI
R1
(1) Service de Neurophysiologie clinique, Hôpital des Spécialités, Rabat, MOROCCO.
(2) Institut de Myologie, Hôpital de la Salpêtrière, Paris, FRANCE.
To contact the author::
[email protected].
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PW37-461
Myotonia congenita (MC) can be Autosomal recessive (Becker)
or Autosomal dominant (Thomsen). These 2 forms are due to
mutations in CLCN1 gene coding for chloride channel of muscle
membrane. This is a study of the clinical and electrophysiological
phenotypic features and genetic data in 6 Moroccan families.
Twelve patients with Becker MC had clinical evaluation for age at
onset, myotonia distribution, muscle hypertrophy and weakness
and electrophysiological examination for the pattern of chloride
channelopathy. Family investigation consisted of clinical and
electrophysiological examination of at risk relatives. Molecular
analysis was performed in one family.
Age at onset ranged from 2 and 10 years. Myotonia was the
predominant symptom that improved by exercise in all cases and
worsened by cold in 5 patients. It was noticed in orbicularis oculi
muscles in 5 cases. Eight patients had moderate weakness in
limb girdle muscles. Muscle hypertrophy was a constant sign. All
patients had myotonic discharges at needle EMG examination.
The effort test and repetitive stimulation showed typical patterns
in 4 cases.
Autosomal recessive inheritance was confirmed by established
consanguinity and normal clinical and electrophysiologic
examination of the parents in all families. An homozygous
mutation in the CLCN1 gene was demonstrated in one patient.
Functional disability was moderate in all cases due mostly to
myotonia which responded partially to symptomatic treatment.
Becker MC has homogeneous phenotype. The clinical signs are
explained by loss of function in the chloride channel responsible
of muscle membrane hyperpolarisation.
INTRANUCLEAR LOCALIZATION OF PHOSPHO-B-DYSTROGLYCAN (PY892) AT
RAT BRAIN
RODRIGUEZ-MUÑOZ R1, MORNET D2, MARTÍNEZ-ROJAS D1
(1) CINVESTAV-IPN, Physiology, Biophysic and Neurosciences Department, México
D. F., MEXICO. (2) Institut National de la Sante´ et de la Recherche Me´dicale,
Equipe ERI 25, Muscle et Pathologies, Universite´ de Montpelier1, Unite´ de
Formation et de Recherche de Me´decine, Montpellier, France, FRANCE.
-
-DG) is a transmembrane protein that links the extracellular matrix
with the cytoskeleton. This protein is a Dystrophin-associated protein (DAP) that has
To contact the author::
an important role in cell signaling, and cytoplasm and nuclear organization ([email protected]
-DG undergoes tyrosine
.mx.
phosphorylation in a cell adhesion-
-
DG has not been detected in nuclei.
In the present work, we studied the sub-
-DG and its
phosphorylated status at nuclei from cerebral cortex of rat. We found that the nuclear
-
-DG band of
47kDa was detected in rat brain extract with anti- -DG antibodies (NCL-43DG, G5
and JAF), but was undetectable with anti-phospho Y892 antibody that recognizes the
-DG C-terminal phosphorylated tyrosine 892 residue, indicating that it is not
-DG band was almost undetected in nuclei, nuclear matrix and
nuclear membrane fractions. However, some protein bands with upward or downward
mobility shifts (250, 190, 115, 65, 44, 41, 38 and 35 kDa) were detected in nuclear
extract and nuclear matrix fraction. These bands were more immunoreactive in
nuclear that in rat brain extracts. We hipothethized, that these nuclear proteins are
post-
-DG. With the anti-phospho Y892 antibody, we
-DG bands expressed in nuclei are phosphorylated. The phospho- -
DG bands of upward mobility shifts may be poly-glycosilated isoforms.
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For the first time, we have demonstrated the enriched expression of phospho- -DG
isoforms at nuclei from cerebral cortex and nuclear matrix fraction. These results give
new insights about the possible functions of phospho- -DGs in the nuclear
architecture and the anchorage of signaling proteins at nuclei.
PW37-462
EFFECTS OF CTGF OVEREXPRESSION ON MYOGENIC CELLS IN VITRO.
AMBROSI I1, NOIREZ P1, MOULY V2, FISZMAN M1, KELLER A3, DUBOIS C4,
ALAMEDDINE H1
(1) Inserm U582, Université Paris 6, Institut de Myologie, Paris, FRANCE. (2) Inserm
U787, Université Paris 6, Institut de Myologie, Paris, FRANCE. (3) CNRS UMR 7149
– CRRET, Université Paris 12 – Val de Marne, Paris, FRANCE. (4) Inserm U515,
Université Paris 6, Paris, FRANCE.
To contact the author::
[email protected].
Fibrosis, characterized by excessive accumulation of extracellular matrix (ECM), is a
hallmark of muscle biopsies in several muscular dystrophies such as Congenital
(CMD) or Duchenne Muscular Dystrophies (DMD). Cell therapy trials have shown that
increased connective tissue hindered the dispersion and incorporation of grafted cells
into regenerating myofibers. Consequently, the success of this therapeutic strategy
depends on the comprehension of molecular mechanisms leading to dysregulation of
the balance between production and/or hydrolysis of ECM components.
The involvement of Transforming Growth Factor beta (TGFβ) in fibrotic processes is
well established. Other factors, among which Connective Tissue Growth Factor
(CTGF), have equally been implicated. This protein, member of the CCN family
(Cyr61/Ctgf/Nov), appears to be involved in the development of fibrosis in various
pathological situations. It was shown to act as a downstream mediator of TGFβ but
could also act through a TGFβ independent mechanism. In dystrophic muscles, the
role played by CTGF in the fibrotic process is emerging and remains to be fully
assessed.
In this study, we have examined the consequences of exposing myogenic cells to
CTGF. At first, we have investigated the production of endogenous CTGF by
myogenic cells and its regulation during myogenesis in vitro. Then, we have
overexpressed human-CTGF in myoblasts to explore its effect on cell proliferation and
differentiation and compared them to control cells. Stably transfected clones were
screened for h-CTGF expression by western blotting and quantitative real time Q-PCR
has confirmed CTGF overexpression. The effect of CTGF on myoblast proliferation
was quantified using the Neutral Red assay. Myogenic differentiation was evaluated
by comparing the fusion capacity of clones overexpressing the protein versus their
control clones. Data on CTGF expression, regulation and the functional consequences
resulting from myoblasts exposure to CTGF will be presented.
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PW37-463
NEUROMUSCULAR DISORDERS IN AVIATION MEDICINE
MARTINEZ PEREA MDC1, LISTE H2, RUGGIERO M3, ANDRADA L4, CANAVERIS
G5
(1) HOSPITAL AERONAUTICO CENTRAL.JEFE SERVICIO NEUROLOGIA
INFANTIL, BUENOS AIRES, ARGENTINA. (2) HOSPITAL AERONAUTICO
CENTRAL.NEUROLOGIA, BUENOS AIRES, ARGENTINA. (3) INSTITUTO DE
MEDICINA AERONAUTICA Y ESPACIAL, BUENOS AIRES, ARGENTINA. (4) JEFE
SERV. CLINICA MEDICA.INSTITUTO DE REHABILITACION PSICOFISICA,
BUENOS AIRES, ARGENTINA. (5) INSTITUTO DE MEDICINA AERONAUTICA Y
ESPACIAL, BUENOS AIRES, ARGENTINA.
To contact the author::
mdeposadas@intramed.
net.
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PW37-464
NEUROMUSCULAR DISORDERS in AVIATION MEDICINE
Dres. *Martínez de Posadas, M. ;*Liste H, *** Ruggiero
,# Andrade L.
Hospital Aeronáutico Central, * Neuropediatric, ** Neurology, *** Instituto de Medicina
Aeronáutica y Espacial, #Instituto de Rehabilitación Psicofísica. Buenos Aires. Rca
ARGENTINA.
INTRODUCTION: The physicians need to be aware of the NMD and physiological
flight conditions. The primary difference between the aircraft and ground is the
barometric pressure reduced. Although most healthy travelers can compensate for
this hypoxemia, this may not be true for Neuromuscular Disorders (NMD).
OBJECTIVES: To present NMD in aviation, special jobs dispositions, and airline
travel passenger’s medical condition.
Methods: This paper reports three cases: 2 with DM (Myotonic Dystrophy), and 1
patient with DMD (Duchenne Muscular Dystrophy). Methodology was used, including
the historical information, neurophysiologic studies, and molecular test.
Case 1: Male Patient, 19 years that enters in specific tasks for Security Force
presenting myotonic stiffness of the hand with generalized myotonia in right arm, while
presses trigger of gun. The molecular study confirms DM (more than 99 of CTG
triplets’ repeats).
Case 2: Male patient 36 years, private aircrew, in a routine annual flight examination
show impaired in muscular relaxation.
Case 3: Male child, 10 years, presents DMD. His molecular test presents deletion
Xp21 exon 48. He needs fly to Hospital for medical controls. His pulmonary function
was normal. At 15 years he becomes wheelchair bound, cardiac muscle and
pulmonary function are affected. He request permission to travel by air.
Results Some forms of NMD inevitably preclude military /security jobs aeromedical
dispositions. Patient 1: had been transferred to other Department. Patient 2: After
wide consultation wasn’t returned to fly. Patient 3, at the first time he could airline
travel; at 15 years, couldn’t travel.
Conclusions: These cases offer some more generally applicable lessons in
occupational, aviation medicine and Neuromuscular Disorders. Recognition of this
disease during the early stages is the primary hurdle for the physician in screening to
selective occupational jobs. The diagnosis has ramification for the patients and their
offspring‘s futures and will necessitate genetic counseling.
PRACTICAL EDUCATION IN BIOLOGY FOR GENETIC DISEASE ASSOCIATIONS
: A FRENCH ACTION UNIQUE IN EUROPE !
MATHIEU M1, KARLIN D1, THIMONIER J1, LANGLET C1, HAMMOND C1
(1) Association Tous Chercheurs, Marseille, FRANCE.
To contact the author::
[email protected].
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Rare diseases associations are increasingly interested and involved in research, from
which they hope to gain better knowledge and prevention of their disease. Because
the time scales of research and patients waiting for treatments intrinsically differ, it is
essential to train members of disease associations with the various aspects of
research, in order to contribute to a closer relationship between the scientific
community and these associations.
Accordingly, since 2004, the association DNA school in Marseilles (renamed Tous
Chercheurs in 2007) has developed practical training in molecular biology and
genetics for rare disease associations. These 3-day sessions, unique in Europe, take
place in a laboratory where the trainees work “as” researchers, together with trainers
who are experienced research fellows. They learn to observe, formulate hypotheses
and carry out experiments. In addition to the practical work, every session includes
discussions with researchers specialized in the pathology under the scope of training.
The trainees thus understand concretely how researchers work and their constraints.
Between 2004 and 2007, Tous Chercheurs formed more than 150 patients during 17
sessions, highlighting that such an approach is meeting a crucial need and desire
from patient associations. At the same time, more and more associations want to
benefit from these training sessions. To achieve this, and to make access easier for
associations from all over France, it was essential to expand our action to a national
scale. This was possible in 2007 thanks to the French-speaking Federation of DNA
Schools, built around 6 members distributed throughout France, to whom we
transferred our skills. Acting as initiator and catalyser of this type of associations
training, we look forward to extend this successful story to a Europe-wide level.
The development of this innovative action was made possible thanks to the support of
the AFM, but also the Inserm and CNRS.
PW 38:
Young investigator
symposium
Posters
PW38-465
To contact the author::
[email protected]
nrs-mrs.fr.
MORPHOMETRIC ANALYSIS REVEALED ABNORMAL COMPLEX DENDRITES IN
POSTNATAL LUMBAR MOTONEURONS OF MOUSE MODEL OF AMYOTROPHIC
LATERAL SCLEROSIS (ALS).
AMENDOLA J1, DURAND J1
(1) CNRS-P3M, Marseille, FRANCE.
The early mechanisms leading to the progressive loss of motoneurons during ALS are
still unknown. Using the in vitro preparation of brainstem/spinal cord isolated from
wild-type and SOD1G85R transgenic mice (ALS model), we have recently reported
alterations of the input resistance and gain of SOD1G85R lumbar motoneurons (Mns)
during the second postnatal week [1]. These results suggested that the morphology of
these cells could have been modified. To address this question, we have stained
developing lumbar Mn with neurobiotin tracer and fully reconstructed them in 3D using
the Neurolucida system. The somata of labelled Mns (SOD1 G85R, n=6; control (WT)
n=8) were located within the same ventro-lateral region and directions of the dendrites
depended on the location of the cell bodies. Dendrites mainly projected in three
directions (dorsal, dorso-lateral and median). In the rostro-caudal plane, the labelled
Mns extended up to 750 µm. This staining procedure revealed a highly complex
morphology of SOD1G85R Mns labelled from postnatal day 8 (P8) to P9 mice. The
number of branching nodes in SOD1G85R Mns was almost twice than that of WT.
Moreover the total length and surface of dendritic trees were higher in SOD1G85R Mns.
The increase in complexity of the SOD1G85R arborizations was due to higher
occurrence of branching points at different distances from the soma.
We had also found correlations between the total size of Mns (cell body, dendrites and
axone) and several electrical properties like the input resistance, rheobase current,
the gain, spike after-hyperpolarization (AHP) duration and AHP decay-time.
In conclusion, the alterations of electrical properties of SOD1G85R lumbar motoneurons
are associated with morphological changes. These motoneuronal alterations took
place during the period of maturation and could have important consequence on the
pathophysiology of ALS.
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[1]- Bories et al, 2007, Eur. J. Neurosci. 25(2):451-9.
PW38-466
A LARGE NUCLEAR RETAINED NON-CODING RNA REGULATES
SYNAPTOGENESIS.
BERNARD D1, PRASANTH KV1, SEDEL F1, TRILLER A1, SPECTOR DL1, BESSIS A1
(1) INSERM U789, Ecole Normale Supérieure, Paris, FRANCE.
During physiological development of spinal cord, half of the motoneurons die few days
after their generation. It has been shown that motoneurons are not committed to death
but rather acquire the competence to die upon a transient microglial TNF signalling
(Sedel et al., 2004). In order to identify motoneuron molecules implicated in this
process, we isolated 24 candidate genes expressed by motoneurons during the
acquisition of the competence to die. One of these candidates is a non-coding RNA
(ncRNA) referred to as metastasis associated lung adenocarcinoma transcript-1
(Malat1) (Ji et al., 2003). Malat1 is a mammal specific 7kb nuclear ncRNA that is
highly expressed in motoneurons during development and in adult. Malat1 localizes to
nuclear speckles (Lamond and Spector, 2003) and knockdown studies revealed that it
is required for the proper recruitment of SF2/ASF splicing factor. In neurons, Malat1 is
detected concomitantly with synaptogenesis. Consistent with this observation,
inhibition and overexpression of this ncRNA regulates positively synaptogenesis in
cultured neurons, which correlates with its effect on the expression of at least two
genes involved in synaptogenesis : neuroligin1 (Nlgn1) and synaptic cell adhesion
molecule (SynCAM) (Chih et al., 2005 ; Biederer et al., 2002). Preliminary results now
show that Malat1 is upregulated in pathological motoneurons such as axotomized and
SOD mutant motoneurons.
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PW38-467
FUNCTIONAL CHARACTERIZATION OF A NOVEL MITOCHONDRIAL PROTEIN
CAMBIER L1, RASSAM P1, AUFFRAY C2, POMIÈS P1
(1) CRBM-CNRS UMR 5237, montpellier, FRANCE. (2) UMR 7091-LGN CNRS,
villejuif, FRANCE.
To contact the author::
[email protected]
s.fr.
By quantitative hybridization of a high-density cDNA array, fourteen novel gene
transcripts, called GENX, preferentially expressed in human muscles have been
identified (Piétu et al., 1996). My research project is to identify and to characterize the
cellular function of a protein, called PROTX-70612, encoded by one of these genes,
localized on a human chromosome 6p21-31.
Northern blot analysis has shown that this gene is specifically expressed in cardiac
and skeletal muscles. Nevertheless, using RT-PCR, we show that it is in fact
ubiquitously expressed in different adult mouse organs and that PROTX-70612 is
ubiquitously expressed in these organs with a higher level in heart and brain.
In the myogenic C2C12 mouse cell line, PROTX-70612 expression increases
progressively during the differentiation process. Furthermore, down-regulation of
PROTX-70612 expression in C2C12 cells using ShRNA and SiRNA techniques
affects the expression of myosin suggesting that a disruption of PROTX-70612
expression inhibits muscle differentiation.
Using indirect immunofluorescence microscopy and electronic transmission
microscopy, we show that PROTX-70612 is localized in mitochondria of muscle cells.
Mitochondria are organelles that play a central role in energy production for the cell
and in the signaling of apoptosis. This localization can explain the high level of
PROTX-70612 expression in organs like heart and brain, which consume a lot of
energy. Furthermore, bioinformatic and biochemical analysis of the PROTX-70612
sequence show the presence of a N-terminal -helix domain, which is necessary and
sufficient to import the protein in mitochondria.
Using submitochondrial fractionation techniques, we are defining the precise PROTX70612 localization in the mitochondria. We are also trying to determine binding
partners for PROTX-70612 that could indicate the signaling pathway in which the
protein is implicated.
Collectively, these results show the identification of a novel mitochondrial protein
playing a role during the differentiation process of skeletal muscle cells.
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PW38-468
EXPRESSION AND ROLES OF THE ORPHAN RECEPTOR ALK IN THE
DEVELOPMENT OF THE NERVOUS SYSTEM
DEGOUTIN J1, BRUNET-DE CARVALHO N1, GOUZI JY2, CIFUENTES-DIAZ C1,
VIGNY M1
(1) Institut du Fer à Moulin UMR_S839 INSERM/UPMC, Paris, FRANCE. (2) BSRC
'Alexander Fleming', Vari, GREECE.
To contact the author::
[email protected].
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PW38-469
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently
expressed in specific regions of the central and peripheral nervous systems,
suggesting a role in its normal development and function. The nature of the cognate
ligands of ALK in vertebrate is still a matter of debate. We produced a panel of
monoclonal antibodies (mAbs) directed against the extracellular domain of the human
receptor. Two mAbs induced the differentiation of PC12 cells transiently transfected
with ALK. In HEK 293 cells stably expressing ALK, we showed that these two Mabs
strongly activated the receptor and subsequently the MAP kinase pathway and a
specific activation of STAT3. Interestingly, others mAbs present all the characteristics
of blocking antibodies. We also studied the primary events occurring at the plasma
membrane triggered by ALK activation and driving MAP-kinase activation and
subsequently PC12 cells differentiation. We focused on two adaptor proteins Shc and
FRS2, and their specific role in the neuron-like differentiation of PC12 cells. We
showed that both adaptors could interact with ALK in an activation dependent manner.
We also characterized the functional role of Shc adaptor in the neuron-like
differentiation of PC12 cells. Recently, we studied the expression of ALK in DRG
(dorsal root ganglia) neurons both in vivo and in culture. Our results on the level of
expression showed a maximum reach at P0 (birth) in the rat DRG.
Immunofluorescence assay on section of DRG showed a specific localization of ALK
in a subtype of neurons. Moreover, our data suggested that ALK could be involved in
the relation between neurons and Schwann cells.
Thus, in absence of clearly established ligand(s) in vertebrates, the availability of
mAbs allowing the activation or the inhibition of the receptor will be essential to better
understand the roles of ALK.
PLASMINOGEN INDUCES AN INTRACELLULAR SIGNALLING ACTIVATION
THROUGH ITS BINDING TO ALPHA-ENOLASE IN MYOGENIC CELLS.
DIAZ-RAMOS A1, LLORENS A1, LOPEZ-ALEMANY R1
(1) Biomedical Research Institute of Bellvitge (IDIBELL), L'Hospitalet de Llobregat
(Barcelona), SPAIN.
To contact the author::
[email protected].
The plasminogen activation (PA) system is a group of serine proteases that
participate in tissue remodeling by degrading most of components of the extracellular
matrix. Different studies have shown that PA system components (plasminogen and
urokinase-plasminogen activator) play a role in myogenesis in vitro and in muscle
regeneration in vivo. Alpha-enolase constitutes a receptor for plasminogen in several
cell types, where it acts focalizing proteolytic activity on the cell surface. We have
previously shown that alpha-enolase/plasminogen binding is required for myogenic
differentiation, fusion, migration and invasion processes, in an in vitro model of
myogenesis. Moreover, the blockage of alpha-enolase/plasminogen binding in an
animal model of muscle regeneration impairs the regeneration process, indicating an
important role of cell surface-associated plasminogen, in an alpha-enolase-dependent
way.
In the last years, evidences have appeared showing that plasmin(ogen)
induces an intracellular response after binding to cell surface in several cell types, but
it has never been evaluated in myogenic cells.
We have analysed the intracellular signalling response of plasminogen
binding in myoblasts C2C12 and in primary cultures of Muscle Precursor Cells
(MPCs). Our results showed that plasmin(ogen) induces PI3K/AKT and MEK/ERK
phosphorylation in C2C12 myoblasts and in MPCs. This activation is alphaenolase/plasmin(ogen) binding-dependent because inhibitors of plasminogen-cell
surface binding (as MAb11G1 and EACA) abrogates this activation. Plasminogen
induced invasion and migration were also inhibited by MAb11G1 and EACA,
suggesting an important role for alpha-enolase/plasminogen binding induced
response in these processes.
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As alpha-enolase lacks a transmembrane or intracellular domain, it is
presumed to act through association with other membrane proteins. Experiments are
being performed to identify such molecular partner that could collaborate with alphaenolase to transduce plasmin(ogen)-induced intracellular signalling.
To our knowledge, the role of alpha-enolase as a mediator of plasminogeninduced intracellular signalling in myogenic cells is demonstrated for the first time.
PW38-470
A NEW MODEL FOR MICROTUBULE DYNAMIC INSTABILITY.
DIMITROV A1, QUESNOIT M2, MOUTEL S3, CANTALOUBE I4, POÜS C4, PEREZ F1
(1) CNRS UMR144 – Institut Curie, Paris, FRANCE. (2) Univ.Paris-Sud IFR141 Faculté de Pharmacie / CNRS UMR144 – Institut Curie, Châtenay-Malabry / Paris,
FRANCE. (3) Translational Research Department – Institut Curie, Paris, FRANCE.
(4) Univ.Paris-Sud IFR141 - Faculté de Pharmacie, Châtenay-Malabry, FRANCE.
To contact the author::
[email protected].
Microtubules are highly dynamic tubulin polymers essential for intracellular
organization and cell division. They display a dynamic instability, alternating phases of
growth and shrinkage separated by catastrophe and rescue transitions. Tubulin
polymerises in a GTP-bound form and hydrolyses GTP in the polymer with a slight
delay. This creates a GTP-cap at the growing end of microtubules that is essential to
prevent microtubule catastrophes. Loss of the GTP-cap would promote catastrophe
events while microtubule rescue would be due to yet uncharacterized stochastic
events.
The existence of a GTP-cap at the extremity of microtubules has however not yet
been documented in vivo. We selected a recombinant antibody (MB11) that
specifically recognizes GTP-bound tubulin conformation in microtubules and we show
here that GTP-tubulin is indeed present at the plus end extremity of more than half of
cellular microtubules. However we also observe an unexpected staining. This
suggests a new model for microtubule dynamic instability. We anticipate that the
conformational antibody MB11 will be important both to understand the regulation of
microtubule dynamic instability and to find new proteins and drugs that modulate the
conformation and the dynamics of the polymer.
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PW38-471
OVERVIEW OF TOR1A-LINKED DYSTONIA IN FRANCE
FRÉDÉRIC M1, CLOT F2, BLANCHARD A1, DHAENENS CM3, LESCA G4, CIF L5,
DÜRR A3, VIDAILHET M2, SABLONNIERE B3, CALENDER A3, BRICE A2,
CLAUSTRES M1, TUFFERY-GIRAUD S1, COLLOD-BEROUD G1
(1) INSERM, U827, Montpellier, FRANCE. (2) INSERM, UMR_S679, Paris, FRANCE.
(3) INSERM, U837, Lille, FRANCE. (4) Hôpital Edouard Herriot, Lyon, FRANCE. (5)
CHU Montpellier, Hôpital guy de Chauliac, Montpellier, FRANCE.
Early-Onset Torsion Dystonia (EOTD) are rare movement disorders developing in
childhood with a neurological origin. They begin in a limb and potentially spread to
To contact the author::
[email protected] other parts of the body to become generalized. Molecular defect is known for only a
nserm.fr.
subgroup, consisting of a unique and recurrent mutation (c.907delGAG) in the TOR1A
gene. Although the molecular cause of this particular form of EOTD is well known,
there is very little epidemiologic data regarding mutation carriers. We first investigated
the incidence at birth of the mutation in a population of 12,000 newborns from SouthEastern France and found only one positive. Our results suggest that the prevalence of
the disease in France is in the lowest estimations, compared to previous
epidemiological surveys (about 1/10,000 to 1/30,000 among non-Jews). We then
undertook the census of French TOR1A-mutation carriers and the assessment of
clinically associated signs to realize an overview of this population. Collaborations
were established between the French laboratories involved in the molecular diagnosis
of TOR1A-linked dystonia (Lille, Lyon, Paris and Montpellier). Family history, clinical
data and DNA samples were gathered for each of the 53 identified index cases and for
their relatives. From these data, we estimate disease frequency to be at least
0.13:100,000 and mutation frequency of 0.17:100,000 in France. Haplotypes linked to
the c.907delGAG TOR1A mutation were constructed based on the analysis of flanking
microsatellites. The previously reported Ashkenazi-Jewish haplotype, known to be
linked with a founder effect, was found in 11 families. Only three of the remaining
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unrelated families shared the same haplotype suggesting that the mutation probably
occurred independently several times in the French population. Finally, it was the first
exhaustive nation-wide study of a genetically ascertained population of TOR1A
carriers. Our results confirm the scarcity of this disease in the French population.
PW38-472
GENETIC HETEROGENEITY OF EMERY-DREIFUSS MUSCULAR DYSTROPHY:
RESPECTIVE CONTRIBUTION OF LMNA, EMD AND OTHER GENES.
GUENEAU L1, BEN YAOU R1, DEMAY L3, LLENSE S2, CHIKHAOUI K1, TRABELSI
M2, BEUGNET C2, DEBURGRAVE N2, LETURCQ F2, CHELLY J2, RICHARD P3,
BONNE G1
(1) Institut National de la Santé et de la Recherche Médicale, U582, IFR14, Institut de
Myologie, PARIS, FRANCE. (2) AP-HP, Groupe hospitalier Cochin, Laboratoire de
Génétique Moléculaire, Pavillon Cassini, PARIS, FRANCE. (3) AP-HP, Groupe
Hospitalier Pitié-Salpêtrière, U.F. Myogénétique et Cardiogénétique, service de
Biochimie Métabolique, PARIS, FRANCE.
To contact the author::
[email protected].
Emery-Dreifuss Muscular Dystrophy (EDMD) is a rare autosomal or X-linked
recessive condition, associating muscular dystrophy, joint contractures and cardiac
disease. Mutations in 2 genes, EMD (emerin) and LMNA (Lamins A/C) encoding
nuclear envelope proteins account for only 40% of EDMD cases, suggesting
additional genetic heterogeneity. Our objective was to estimate the respective
contribution of LMNA and EMD mutations among an EDMD cohort screened in our
labs.
We reviewed medical records of 2200 patients screened for LMNA and/or EMD.
EDMD clinical criteria were the coexistence of muscle involvement in a humeroperoneal, proximal or diffuse distribution; early tendons contractures; conduction
defect and arrhythmias with or without dilated cardiomyopathy. EMD and LMNA genes
were analyzed by dHPLC/sequencing with a preliminary analysis of emerin protein
(western-blot, immuno-histochemistry).
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656 patients matched with our EDMD inclusion criteria. This study revealed the
respective proportion of LMNA mutations (176 patients, 27%), EMD mutations (48
patients, 7%) detected following an abnormal emerin expression. 90% of EMD
mutations were truncating ones whereas 94% of those found in LMNA gene were nontruncating. No mutation of these 2 genes was identified for 432 EDMD patients (66%)
underlining the large number of patients lacking genetic diagnosis. Of these, 28
patients (6.5%) were eventually found to be mutated in other genes, as EDMD
clinically overlaps with several other myopathies (LGMDs, CMDs, collagenopathies…)
thus highlighting the importance of an accurate and careful diagnosis of this disease.
From 96 patients consistent with X-linked transmission and for whom emerin protein
was normal, analysis of EMD gene did not identify any mutation, confirming that
emerin protein analysis remains a powerful diagnostic tool to detect EMD mutations.
We finally characterized a global cohort of 404 patients eligible to search new EDMD
genes. Six informative families are currently being genotyped. Meanwhile, candidate
genes are being sequenced among our global cohort.
CONTROL OF PLANAR DIVISIONS BY THE G-PROTEIN REGULATOR LGN
MAINTAINS PROGENITORS IN THE CHICK NEUROEPITHELIUM
MORIN X1, JAOUEN F1, DURBEC P1
(1) Institute of Developmental Biology of Marseille–Luminy, Centre National de la
Recherche ScientifiqueUMR6216, Marseille, FRANCE.
To contact the author::
[email protected].
The spatio-temporal regulation of symmetrical as opposed to asymmetric cell
divisions directs the fate and location of cells in the developing CNS. In
invertebrates, G-protein regulators control spindle orientation in asymmetric
divisions, which generate progeny with different identities. We investigated
the role of the G-protein regulator LGN (also called Gpsm2) in spindle
orientation and cell-fate determination in the spinal cord neuroepithelium of
the developing chick embryo. We show that LGN is located at the cell cortex
and spindle poles of neural progenitors, and that it regulates spindle
movements and orientation. LGN promotes planar divisions in the early
spinal cord. Interfering with LGN function randomizes the plane of division.
Notably, this does not affect cell fate, but frequently leads one daughter of
proliferative symmetric divisions to exit the neuroepithelium prematurely and
to proliferate aberrantly in the mantle zone. Hence, tight control of planar
spindle orientation maintains neural progenitors in the neuroepithelium, and
regulates the proper development of the nervous system
The first two authors contributed equally to this work.
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PW38-474
HETEROLOGOUS EXPRESSION AND FUNCTIONAL ANALYSIS OF HUMAN
MYOTUBULARIN IN THE YEAST MODEL SYSTEM S. CEREVISIAE
KANEVA G1, LAPORTE J2, WINSOR B1, FRIANT S1
(1) Unité Mixte de Recherche 7156 Centre National de la Recherche Scientifique –
Université Louis Pasteur, Departement de Génétique Moléculaire, Génomique et
Microbiologie, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, FRANCE. (2)
Department de Neurobiologie et Génétique, Institut de Génétique, Biologie
Moléculaire et Cellulaire, Illkirch, FRANCE.
To contact the author::
[email protected].
The human myotubularin gene (hMTM1) that is mutated in a severe congenital
neuromuscular disease, X-linked myotubular myopathy (XLMTM), encodes a
phosphatase specific for phosphatidylinositol 3-phosphate (PtdIns(3)P) and
PtdIns(3,5)P2. In Saccharomyces cerevisiae, Ymr1p is the hMTM1 orthologue and the
unique member of the myotubularin family. In order to study myotubularin intracellular
functions as well as the impact of disease-causing mutations, hMTM1 wild-type and
mutants constructs were introduced in yeast. Heterologous expression of hMTM1 wild
type led to an enlarged vacuole phenotype, while a phosphatase inactive mutant
(C375S) had no effect. The results obtained suggest that dephosphorylation of
PtdIns(3)P and PtdIns(3,5)P2 by human MTM1 in yeast impairs membrane trafficking,
in particular the trafficking of carboxypeptidase Y (CPY). Depending on the mutation
carried there is a difference in the severity of the vacuole phenotype in both wt and
ymr1Δ strains. Mutation N180K responsible for a very mild form of XLMTM provoked a
phenotype similar to that of the wild type myotubularin, Mutation V49F responsible for
severe XLMTM triggered an increase in vacuole size, although less pronounced, and
mutation R421Q (severe XLMTM) did not have an effect when compared to the
C375S phosphatase inactive mutant. Surprisingly, mutation R69C (intermediate form
of XLMTM) caused an enlargement of the vacuole that was more pronounced than the
wild-type construct, suggesting a possible dominant negative effect of the mutation in
this system. Apart from establishing a suitable model for studying the function of
human myotubularin, the present work sheds light on the possible causes of X-linked
myotubular myopathy such as defects in membrane trafficking. Similar studies for
other members of the myotubularin family such as hMTMR2, mutated in CharcotMarie-Tooth peripheral neuropathy, could give information about the pathological
processes related to other neuro-muscular diseases.
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ACCURATE WORK-RATE MEASUREMENTS DURING IN VIVO MRS STUDIES OF HUMAN
QUADRICEPS EXERCISING MUSCLE
LAYEC G1, BRINGARD A2, VILMEN C1, MICALLEF JP3, LEFUR Y1, PERREY S2, COZZONE
P1, BENDAHAN D1
(1) CRMBM CNRS 6612, Marseille, FRANCE. (2) EA 2991, Montpellier, FRANCE. (3)
INSERM ADR 08, Montpellier, FRANCE.
Reliable investigation of exercising muscle within a superconducting magnet requires
To contact the author::
gwenael.layec@univme the construction of dedicated ergometers in order to perform standardised exercise protocols
d.fr.
and to record mechanical variables. A few ergometers designed for quadriceps dynamic
exercise have been described, but the corresponding mechanical data typically rely on the
constancy of imposed work rates which is not satisfactory. Given that we have reached a point
in the field of muscle energetics where absolute measurements are warranted to take the area
forward, we designed an ergometer, including two force and two displacement transducers,
allowing dynamic and isometric knee extension within a Magnetic Resonance (MR) system
and accurate measurements of power output.
On the basis of repeated measurements, the force and displacement transducers
accuracy was 0.5% for values ranging from 0 to 394N and 3 % for values ranging from 0 to 20
cm. In addition, measurements were not affected by magnetic field and we were able to
distinguish mechanical output during eccentric and concentric phases of exercise. MRS
experiments in exercising muscle were conducted in 8 subjects. They performed two
standardized dynamic alternate leg extension exercises (25 and 35 % of MVC) while the
corresponding metabolic changes were measured using
31P-MRS.
The mean power output produced during both exercises were 62 ± 17 and 79 ± 12 W.
The corresponding metabolic changes were significant with a 20 to 40% PCr depletion and an
end of exercise pH ranging from 7.0 to 6.3 pH units.
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Overall, the present ergometer is MR compatible. Dynamic and isometric leg
extensions are possible while power output can be accurately quantified separately during the
concentric and eccentric phases of exercise. Such an ergometer should be useful for future
metabolic studies conducted in control subjects and patients for whom muscle energetics is
impaired. Therapy follow-up is also possible.
PW38-477
SAFETY AND EFFICACY OF REGIONAL INTRAVENOUS (RI) VERSUS
INTRAMUSCULAR (IM) DELIVERY OF RAAV1 AND RAAV8 TO NONHUMAN
PRIMATE SKELETAL MUSCLE
TOROMANOFF A1, CHEREL Y2, GUILBAUD M1, PENAUD-BUDLOO M1, SNYDER
R3, HASKINS M4, DESCHAMPS JY2, GUIGAND L2, PODEVIN G5, ARRUDA V6,
HIGH K6, STEDMAN H7, ROLLING F1, ANEGON I8, MOULLIER P1, LE GUINER C1
(1) INSERM UMR 649, CHU de Nantes, Université de Nantes, Nantes, FRANCE. (2)
INRA UMR 703, Ecole Nationale Vétérinaire, Nantes, FRANCE. (3) Department of
Molecular Genetics and Microbiology department, University of Florida, Gainesville,
USA. (4) School of Veterinary Medecine, University of Pennsylvania, Philadelphia,
USA. (5) CHU Hôtel Dieu, Nantes, FRANCE. (6) Department of Pediatrics, University
of Pennsylvania, The Children's Hospital of Philadelphia, Philadelphia, USA. (7)
Department of Surgery, University of Pennsylvania, Philadelphia, USA. (8) INSERM
UMR 643, CHU de Nantes, Institut de Transplantation et de Recherche en
Transplantation, Université de Nantes, Nantes, FRANCE.
To contact the author::
[email protected].
We developed in the nonhuman primate (NHP) a drug-free regional
intravenous (RI) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and
8 to an entire limb and compared it with the intramuscular (IM) delivery of the same
dose of vector. We show that RI delivery of both serotypes was remarkably well
tolerated with no adverse side effects. After IM, muscle transduction was restricted to
the site of injection with a high number of vector copies per cell for rAAV1, whereas RI
delivery resulted in lower vector copy per cell but detectable in the vast majority of
muscles of the injected limb. The amounts of circulating infectious rAAV were similar
for both serotypes and modes of delivery. At autopsy, up to 34 months post vector
administration, similar biodistribution patterns were found for both vectors and modes
of delivery, with numerous organs positive for vector sequence by PCR and Southernblot. Altogether, we demonstrated that RI is a simple and efficient transduction
protocol in NHP, resulting in higher expression of the transgene with a lower number
of vector genomes per cell. However, regardless of the mode of delivery, concerns
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were raised by vector sequence detected at distant sites.
DROSOPHILA MODEL FOR STUDYING LEG MYOGENESIS: ROLE OF
LADYBIRD, A HOMOLOG OF VERTEBRATE LBX1 GENE AND REGULATORY
INPUTS OF FGF AND WG SIGNALING PATHWAYS
MAQBOOL T1, SOLER C2, JAGLA T1, JAGLA K1
(1) INSERM U384, Faculté de Medecine, Clermont- Ferrand, FRANCE. (2) School of
Biosciences, Cardiff University, Cardiff, UNITED-KINGDOM.
To contact the author::
[email protected].
Leg muscles of Drosophila display a unique vertebrate-like multi-fiber organization.
They form a highly stereotyped pattern of dorsal and ventral multi-fiber muscle units,
which are attached to the internal tendons in the adult leg (soler et al., 2004)
Ladybird, a homeodomain transcription factor, is found to be expressed specifically in
the myoblasts that form leg muscles. It precedes that of founder cell marker
dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties
of developing myotubes affecting their inherent ability to grow and to interact with the
internal tendons and epithelial attachment sites. It also leads to affected sarcomeric
ultrastructure contributing to the reduced leg muscle performance and altered mobility
of surviving flies. Furthermore, the over-expression of ladybird results in abnormal
pattern of dorsally located leg muscles indicating different requirements for ladybird in
dorsal versus ventral muscles. This differential effect is consistent with the higher level
of Ladybird in ventrally located myoblasts and with positive ladybird regulation by
extrinsic Wingless signaling from the ventral epithelium. Moreover, ladybird
expression correlates with that of FGF receptor Heartless and the read-out of FGF
signaling DOF. FGF signals set up the number of leg disc associated myoblasts and
resulting fibers and when over-expressed are able to accelerate myogenic
differentiation by activating ladybird prematurely and leads to ectopic muscle
formation, implying that FGF signaling may also be cooperating with extrinsic Wg
signaling to regulate ladybird in leg myoblasts (Maqbool et al., 2006).
To determine transcriptional gene activity in leg myoblasts and tendon cells, a protocol
was developed for dissection, dissociation of myoblasts and tendon cells. The
investigation of the candidate genes from microarray analysis, and understanding of
how the coordinated development of muscle and tendon is orchestrated at the gene
transcriptional level is expected to shed light on the molecular basis of myo-tendonous
dysfunctions observed in a large spectrum of myopathies.
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PW38-479
REGULATION OF UTROPHIN A IRES-MEDIATED TRANSLATION BY
GLUCOCORTICOID TREATMENT IN SKELETAL MUSCLE CELLS
MIURA P1, CORIATI A1, SARKAR M1, ANDREWS M2, HOLCIK M2, JASMIN B1
(1) Department of Cellular and Molecular Medicine and Centre for Neuromuscular
Disease, Faculty of Medicine, University of Ottawa, Ottawa, CANADA. (2) Apoptosis
Research Centre, Children's Hospital of Eastern Ontario, Ottawa, CANADA.
To contact the author::
[email protected].
Glucocorticoids are currently the only drugs recognized to benefit Duchenne muscular
dystrophy (DMD) patients. The mechanisms that underlie the beneficial effects still
remain incompletely understood. In agreement with previous observations, we show
here that treatment of myotubes with the glucocorticoid 6-alpha-methylprednisolone21 sodium succinate (PDN) results in enhanced expression of utrophin A without
concomitant increases in mRNA levels, thereby suggesting that translational
regulation contributes to the increase. Previously, we established that the utrophin A
5’UTR contains an Internal Ribosome Entry Site (IRES) that is activated in vivo during
muscle regeneration (J Biol Chem. 280:32997-3005, 2005). In the present study,
using monocistronic and bicistronic reporter assays, we demonstrate that activity of
this IRES is enhanced by PDN treatment. Analysis of polysomes from PDN treated
cells show an increase in polysome association of endogenous utrophin A mRNAs
and reporter mRNAs harbouring the utrophin A 5’UTR, while global translation rates
were found to be depressed. Additional experiments identified a distinct region within
the utrophin A 5’UTR that contains the inducible IRES activity and displays enhanced
binding to multiple proteins following PDN treatment. Together, these studies
demonstrate that a translational regulatory mechanism involving increased IRES
activation mediates, at least partially, the enhanced expression of utrophin A in
muscle cells treated with glucocorticoids. As part of this work we have also created
several lines of transgenic mice harbouring the utrophin A IRES reporter in order to
assess the tissue distribution of utrophin A IRES activity and its regulation in response
to both PDN treatment and muscle regeneration in vivo. In addition, we are also
investigating the involvement of the utrophin 3’UTR in regulating utrophin IRES
activity. These studies may contribute to the development of drugs that specifically
target the utrophin UTRs to drive increased expression of utrophin in the muscle fibers
of DMD patients.
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STRUCTURE-FONCTION ANALYSIS OF THE S.CEREVISIAE PROTEIN BCS1P
WHICH HUMAN HOMOLOGUE IS INVOLVED IN SEVERAL MITOCHONDRIAL
PATHOLOGIES
NOUET C1, TRUAN G1, DUJARDIN G1
(1) Centre de Génétique Moléculaire, Gif sur Yvette, FRANCE.
To contact the author::
[email protected].
In man, mutations in the gene BCS1L are responsible for pathologies with varying
clinical presentations: GRACILE syndrome (iron overload in liver), Bjornstad
syndrome (pili torti and sensorineural deafness) and several pathologies characterized
by deficiencies in the bc1 complex. The human protein, Bcs1, is 40% identical to
Bcs1p of S.cerevisiae. In yeast Bcs1p is located in the mitochondrial inner membrane
with a large domain protruding into the matrix. Bcs1p is composed of an N-terminal
domain required for import into mitochondria, a central domain of unknown function
and a C-terminal domain characteristic of AAA proteins (ATPases Associated with
various cellular activities). Bcs1p is required for the assembly of the Rieske/FeS
protein within the complex bc1. However, the molecular mechanism by which Bcs1p
mediates assembly remains unknown. My thesis project focuses on the use of yeast
genetic tools to further our understanding of the function of the different domains of
the protein. I have undertaken a structure-function analysis based on a strategy of
random PCR mutagenesis. I obtained a collection of a hundred of point mutants
displaying respiratory deficiency. The mutations identified by sequencing the BCS1
gene affected all parts of the protein. Three mutants affecting sub-domains that are
only conserved in the Bcs1-like proteins, have been further characterized
biochemically. Moreover an UV mutagenesis led to the isolation of intragenic
compensatory mutations. To explain the effects of the primary and compensatory
yeast mutations and to further understand the organization and the interactions
between the different domains of Bcs1p, we have produced an homology-based
model of the yeast and human Bcs1 proteins.
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PW38-481
COLLAGEN XV, A NOVEL FACTOR IN ZEBRAFISH NOTOCHORD
DIFFERENTIATION AND MUSCLE DEVELOPMENT
PAGNON-MINOT A1, MALBOUYRES M1, HAFTEK-TERREAU Z1, H KIM R2, SASAKI
T3, THISSE C4, THISSE B4, INGHAM PW 2, RUGGIERO F1, LE GUELLEC D1
(1) IBCP,UMR CNRS 5086, Université Lyon 1, IFR 128 Biosciences Gerland, Lyon,
FRANCE. (2) MRC Centre for Developmental and Biomedical Genetics, University of
Sheffield, Sheffield, UNITED-KINGDOM. (3) Max-Planck Institut für Biochemie,
Martinsried, GERMANY. (4) IGBMC, CNRS/INSERM/Université Louis Pasteur,
Illkirch, FRANCE.
To contact the author::
[email protected].
Muscle cells are surrounded by extracellular matrix, the components of which play an
important role in signalling mechanisms involved in their development. In mice, loss of
collagen XV, a component of basement membranes expressed primarily in skeletal
muscles, results in a mild skeletal myopathy. We have determined the complete
zebrafish collagen XV primary sequence and analysed its expression and function in
embryogenesis. During the segmentation period, expression of the Col15a1 gene is
mainly found in the notochord and its protein product is deposited exclusively in the
peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1
causes defects in notochord differentiation and in fast and slow muscle formation as
shown by persistence of axial mesodermal marker gene expression, disorganization
of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome.
In addition, the number of medial fast-twitch muscle fibers was substantially
increased, suggesting that the signalling by notochord derived Hh proteins is
enhanced by loss of collagen XV. Consistent with this, there is a concomitant
expansion of patched-1 expression in the myotome of morphant embryos. Together,
these results indicate that collagen XV is required for notochord differentiation and
muscle development in the zebrafish embryo and that it interplays with Shh signalling.
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PW38-482
IN VITRO AND IN CELLULO EVIDENCES FOR ASSOCIATION OF THE SURVIVAL
OF MOTOR NEURON COMPLEX WITH THE FRAGILE X MENTAL RETARDATION
PROTEIN
PIAZZON N1, RAGE F2, SCHLOTTER F1, MOINE H3, BRANLANT C1, MASSENET S1
(1) Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567
CNRS-UHP Nancy I, VANDOEUVRE, FRANCE. (2) Institut de Génétique Moléculaire
de Montpellier, UMR 5535, MONTPELLIER, FRANCE. (3) Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, STRASBOURG, FRANCE.
The common neurodegenerative disease spinal muscular atrophy (SMA) is caused by
reduced levels of the survival of motor neuron (SMN) protein. SMN associates with
To contact the author::
several proteins to form a large complex that is essential for the assembly and
nathalie.piazzon@maem metabolism of spliceosomal U snRNPs. It is still not understood why reduced levels of
.uhp-nancy.fr.
the ubiquitously expressed SMN protein specifically cause motor neuron
degeneration. Recently, several lines of evidence support additional neuron-specific
functions of SMN in mRNA transport and translation regulation in neuronal processes.
FMRP (Fragile X Mental Retardation Portein), the defective protein in Fragile X mental
retardation syndrome, is thought to play a role in the transport of mRNPs from the
nucleus to the cytoplasm and may be crucial in neurons to repress translation of
specific mRNAs during their transport as silent mRNPs from the cell body to growth
cones and synapses. Therefore, we examined possible relationships of SMN with
FMRP. We observed granules containing both transiently expressed RFP-tagged
SMN and GFP-tagged FMRP in cell bodies and processes of rat primary neurons of
hypothalamus in culture. By immunoprecipitation experiment, we detected an
association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells
and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we
demonstrated that the SMN protein is essential for this association. We showed that
the C-terminal region of FMRP, as well as the conserved YG box and the region
encoded by exon7 of SMN, are required for the interaction. Our findings suggest a link
between the SMN complex and FMRP in neuronal cells.
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PW38-483
ROLE OF SIX HOMEOPROTEINS IN THE ACQUISITION OF THE FAST
GLYCOLYTIC PHENOTYPE DURING MOUSE EMBRYOGENESIS
RICHARD A1, FAVIER M2, GUILLET-DENIAU I2, MAIRE P2
(1) Université pierre et marie curie, Paris, FRANCE. (2) inserm U567, paris,
FRANCE.
Previous studies have shown the importance of Six homeoproteins in different steps
of myogenesis. Six homeoproteins control Pax3, myogenin and MRF4 genes in the
embryo as well as aldolase A gene which is expressed specifically in fast glycolytic
To contact the author::
fibres. Moreover Six1 accumulates preferentially in the nucleus of adult fast-glycolytic
[email protected]. fibres and forced expression of Six1 and its Eya1 cofactor in adult soleus fibres was
fr.
able to switch their phenotype toward a fast glycolytic one. To define the role of Six
homeoproteins during the genesis of muscle fibre type diversity, we generated a
double knock-out model. six1-/- six4-/- mice die at birth and show severe muscle
hypoplasia. At the truncal level, musculature is drastically reduced and residual
myofibres show a disorganized ultrastructure. To characterize the slow/fast phenotype
of fibres formed in absence of Six homeoproteins, we have collected embryos from 14
days of development until birth, and we analyzed the expression of different myosin
heavy chain isoforms using immunohistochemistry. In six1-/- six4-/- fetuses, the
number of myosin slow positive fibres was greatly increased, showing an alteration of
muscle patterning. Furthermore microarrays experiments showed that in absence of
Six homeoproteins, the expression of a great number of genes involved in
intramuscular calcium metabolism ( e.g. calsequestrin 1, ……) as well as in the
contractile apparatus of the fast-type fibres was down-regulated, whereas the
expression of slow-type isoforms was not modified. In parallel, we performed primary
cultures of satellite cells isolated from residual muscles. Myoblasts were able to
proliferate quite normally, but fusion was dramatically impaired. As it was the case in
residual muscles fibres, satellite cells showed a 20 to 100-fold decrease in genes
expressed in fast-type fibres (MyhIIX, MyhIIB…). Altogether our results demonstrate
that Six1 and Six4 genes are necessary to switch on fast-type specific genes in
myogenic cells during embryogenesis.
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PW38-484
REPROGRAMMING RNA MESSENGER BY SPLICEOSOME MEDIATED RNA
TRANS-SPLICING:
EVALUATION IN THE CANINE MUCOPOLYSACCHARIDOSIS VII MODEL
SACHOT S1, ROLLING F1, MOULLIER P1, LE GUINER C1
(1) Laboratoire de Thérapie Génique INSERM U649, Nantes, FRANCE.
To contact the author::
[email protected].
Background:
An approach to restore protein function involves the recombination of two
RNA molecules in trans by a mechanism named Spliceosome Mediated RNA Transsplicing (SMaRTTM), in which splicing occurs between two independently transcribed
pre-mRNA molecules. SMaRTTM requires an RNA molecule, the Pre Trans-Splicing
Molecule (PTM), introduced artificially in cells.
An advantage of trans-splicing method is the small length of the PTM to
transfer, allowing the use of viral vectors such as rAAV. This strategy would also
preserve the natural regulation of genes and limit immune responses often seen after
gene transfer in large animal models, in which high numbers of transgene copies are
transferred per cell.
Our current hypothesis is to determine if the reprogramming of defective
genes can be done, firstly in vitro, by the delivery of 5’, 3’ and double PTM, which
permit the replacement of an internal exon, in canine mucopolysaccharidosis VII
(MPS VII) cells.
Method:
5’, 3’ and double PTM have been designed, associated with various binding
domain (BD) from 25bp to 220bp, and tested for their ability to trans-splice with a
target transcribed from an artificial GusB Minigene and with the physiologically
expressed GusB pre-messenger RNA, in MPS VII cells.
Results:
Our data demonstrated that reprogramming of artificially and physiologically
expressed GusB pre-messengers is feasible in vitro in the MPS VII canine model
using 5’ and 3’ PTM, as seen by RT-PCR and Western Blot. We also show
illegitimate reprogrammations in the GusB minigene context.
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PW38-485
Conclusion:
We have identified several functional 5’ and 3’ PTM. There ability to
reprogram the physiologically expressed GusB messenger will allow us to generate
double PTM, and test them for such a reprogrammation. We are also planning to
develop strategies to improve the trans-splicing reaction efficiency. Finally, the more
active molecule will be tested in MPS VII dog after a scAAV8 intravenous injection.
COUPLING GLUTAMATE RELEASE TO VASODILATION IN VIVO
TIRET P1, LECOQ J1, CHAIGNEAU E1, DUCROS M1, KNOPFEL T2, CHARPAK S1
(1) U603 Laboratory of Neurophysiology and New Microscopies, Université Paris
Descartes, Paris, FRANCE. (2) Laboratory for Neural Circuit Dynamics, Riken Brain
Science Institute, Saitama, JAPAN.
To contact the author::
[email protected].
Several techniques of human functional brain imaging measure changes in blood flow
parameters to localize activated cerebral regions. At present, the study of the spatiotemporal coupling between neuronal activity and associated vascular parameters is
becoming a hot topic. Analysis of this coupling requires to measure the dynamics of
red blood cell (RBC) flow in individual capillaries that precisely irrigate activated
neurons. In a recent study, Charpak's team has used two-photon laser scanning
microscopy, in vivo, to measure blood flow parameters in control condition and during
odor stimulation in the dorsal olfactory bulb of anaesthetized rat (Chaigneau et al.,
PNAS, 2003). They found that capillary vascular responses are odorant- and
glomerulus-specific. My PhD. project has consisted in using two-photon microscopy,
in vivo, to determine the involvement of astrocytes and of smooth muscle cells in
triggering vascular response. Such a project has initially required to assess the validity
of a recent hypothesis that proposes that astrocytes activation is sufficient to trigger
vasodilation of arterioles. Using two experimental models, the rat and the G-CaMP2
transgenic mice, I have demonstrated that this hypothesis was wrong and that
neuronal postsynaptic activation is a necessary step for neurovascular coupling
(Chaigneau et al., Journal of Neuroscience, 2007). More recently, I have been
investigating the cellular and molecular pathway(s) that couples : neuronal
postsynaptic activation, astrocytes activation and vasodilation. Precisely, I am
examining the intermediate role of NO in these pathways. This in situ analysis of the
regulation of the vascular tone should bring new insights in the understanding of fMRI
signals detected during neuronal activation in the normal and pathological brain.
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PW38-486
DELAYED GDNF GENE DELIVERY IN THE STRIATUM OF 6-OHDA-TREATED
RATS PROMOTES A REVERSIBLE INCREASE OF TYROSINE HYDROXYLASE
BUT NOT DOPAMINE LEVELS.
YANG X1, MERTENS B2, LEHTONEN E1, VERCAMMEN L3, BOCKSTAEL O1,
CHTARTO A1, LEVIVIER M4, BROTCHI J4, SARRE S2, TENENBAUM L1
(1) Laboratory of Experimental Neurosurgery/IRIBHM ULB, Brussels, BELGIUM. (2)
Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information VUB,
Brussels, BELGIUM. (3) Laboratory for Neurobiology and Gene Therapy, Katholieke
Universiteit, Leuven, BELGIUM. (4) Laboratory of Experimental Neurosurgery ULB,
Brussels, BELGIUM.
To contact the author::
[email protected].
A tetracycline (tet)-inducible adeno-associated viral vector expressing human GDNF
cDNA (AAV-tetON-GDNF) was administered in the striatum of rats 5 weeks after
lesioning by intrastriatal 6-hydroxydopamine injection. A significant tet-dependent
improvement of motor symptoms was evidenced 4 weeks post-vector injection. At
later time points, a partial behavioural recovery was observed in all groups but no
further improvement could be evidenced in GDNF-treated animals. Fourteen weeks
post-vector injection, tyrosine hydroxylase (TH) levels were significantly higher and
striatal TH-positive reinnervation significantly increased in GDNF-treated rats but the
striatal dopamine content remained unchanged. We show that the lack of functionality
of the additional TH-positive fibers in GDNF-treated animals could be due to a defect
in TH phosphorylation. In addition, the TH increase was not permanent since
withdrawal of tet after 4 weeks, resulted in TH levels comparable to the control
groups. These data suggest that delayed GDNF gene delivery stimulates some
components of dopamine metabolism but does not rescue dopaminergic neurons.
_0MS0G0OLK
PW38-487
IN SILICO IDENTIFICATION OF ABD-A TARGET GENES IN THE DROSOPHILA
HEART FROM GENE EXPRESSION DATA
ZEITOUNI B1, TÉVY MF2, AERTS S3, HERRMANN C1, POTIER D1, SÉMÉRIVA M1,
CAPOVILLA M2, PERRIN L1
(1) Developmental Biology Institute of Marseille-Luminy (IBDML) CNRS UMR 6216,
Marseille, FRANCE. (2) Dulbecco Telethon Institute, Dept. of Biology and Evolution,
University of Ferrara, Ferrara, ITALY. (3) Laboratory of Neurogenetics, Dept. of
Molecular and Developmental Genetics, University of Leuven, Leuven, BELGIUM.
To contact the author::
[email protected].
_0MS0G0OLK
A number of inherited cardiomyopathies affect cardiac muscle organogenesis
emphasizing the need to improve our knowledge of heart formation. Recent advances
have shed light on patterning informations that lead to cardiac muscle formation.
However, how the involved molecular mechanisms interact to regulate heart
organogenesis remains to be elucidated.
We have demonstrated that the homeotic (Hox) genes, encoding homeobox
transcription factors are required for cardiac tube formation in Drosophila. The
remarkable conservation, between vertebrates and invertebrates, of the mechanisms
responsible for heart formation, warrants the choice of this model organism. To gain
insight into the downstream genetic control responsible for cardiac cell differentiation,
we have applied a whole-genome expression analysis of the cardiac tube, where the
Hox gene abdominal-A (abd-A), differentially expressed according to the
anterioposterior axis, is known to be required for working cardiac cell differentiation.
By this procedure, we pointed out a large set of genes up-regulated in the functional
part of the heart, and 15 genes were further validated by hybridation in situ to share
the same heart expression pattern as that of Abd-A. This group of co-expressed
genes is likely to be regulated by Abd-A and common transcription factors (TFs), our
current approach aims at identifying putative direct AbdA cis-regulatory sequences
from these heart target genes. The conserved non-coding sequences of these genes
(from DNA alignments between the D.melanogaster and D.pseudoobscura genomes)
were ranked according to a bioinformatic motif search pipeline based on conserved
clusters of multiple Abd-A and known cardiac TF binding sites. By attaching the
genomic sequence to a GFP reporter and expressing the construct in transgenic
embryos, we show that 3 of 18 tested enhancers are new heart-specific enhancers.
We are now determining whether they are direct Abd-A targets, demonstrating the role
of a Hox gene in the realization of heart identity.
PW 39:
Young investigator
Symposium
Lectures
(Tuesday May, 27 – Afternoon)
PW39-488
MUSCLE PERFUSION AND BIOENERGETICS MEASURED IN VIVO IN THE
MOUSE BY FUNCTIONAL 1H-NMR IMAGING AND 31P-NMR SPECTROSCOPY.
BALIGAND C1, WARY C1, MÉNARD J1, BERTOLDI D1, GIACOMINI E1, CARLIER
PG1
(1) Laboratoire de RMN AIM - CEA, Institut de Myologie, Paris, FRANCE.
1H-NMR
To contact the author::
[email protected]
ups.jussieu.fr.
_0MS0G0OLK
imaging combined with arterial spin labeling (ASL) provides dynamic,
quantitative and non invasive measurements of muscle perfusion as well as blood
oxygenation level dependent (BOLD) contrast. In Parallel, 31P-NMRS has long been
used to investigate muscle exercise metabolism but remains challenging in mouse
because of the small size of the animal. The cross analysis of these three parameters
is rarely explored, however valuable for an accurate characterization of muscular
diseases. In this work, a complete experimental set-up has been developed to
simultaneously and non-invasively assess perfusion, BOLD and phosphorus
metabolites in mouse, in response to electrical stimulation.
Experiments were performed inside a Bruker Biospec 4T NMR system using custombuilt coils. Muscle contractions were induced with an electrostimulator through
subcutaneous electrodes. In order to compensate for minimal 31P signal, we explored
the possibility to sum the results of 10 consecutive exercises. C57/bl6 mice were
repeatedly subjected to a “rest(30s)- exercise(30s)- recovery(9min)” protocol.
Perfusion/BOLD and phosphocreatine content (PCr) were monitored with a time
resolution of 9s and 2s, respectively. Time courses of rephosphorylation of PCr were
fitted with a single exponential function.
Exercise systematically resulted in a total consumption of PCr. Owing to high time
resolution, we could measure the subsequent initial rate of PCr recovery, a direct
estimate of mitochondrial oxidative capacity, to have a time constant PCr= 77 +/16s. Perfusion was 6.6 +/- 3.4 ml.min-1.100g-1 at rest and rose to 141 +/- 13 ml.min1.100g-1 immediately after the end of the exercise. We also observed a transient
increase in BOLD signal.
This is the first report of a totally non-invasive assessment of mouse skeletal muscle
function in vivo by combined NMR imaging and spectroscopy. This tool offers new
perspectives to study muscle pathology in mice models and monitor longitudinal effect
of therapeutic protocols.
PW39-489
To contact the author::
[email protected].
EXPRESSION OF SELENOPROTEIN N IN MICE DURING DEVELOPMENT
CASTETS P1, MAUGENRE S1, GARTIOUX C1, REDERSTORFF M2, LESCURE A2,
KROL A2, TAJBAKHSH S3, ALLAMAND V1, GUICHENEY P1
(1) INSERM UMR582, UPMC Paris 6, Institut de Myologie, IFR14, Paris, FRANCE.
(2) UPR 9002, CNRS, IBMC, Strasbourg, FRANCE. (3) CNRS, URA 2578, Institut
Pasteur, Paris, FRANCE.
SEPN1-related myopathy is a rare disorder characterized by axial muscle weakness,
scoliosis and respiratory failure, and caused by mutations in the SEPN1 gene,
encoding selenoprotein N (SelN). This entity gathers four autosomal recessive
muscular pathologies with molecular, clinical and morphological overlap: Rigid Spine
Muscular Dystrophy, the classical form of Multi-minicore Disease, rare cases of
Desmin-Related Myopathy with Mallory Body–like Inclusions and Congenital Fibre
Type Disproportion. SelN is a glycoprotein of as yet unknown function, localized in the
membrane of the endoplasmic reticulum. As all selenoproteins, SelN is characterized
by a specific selenocysteine residue in its peptidic sequence.
The clinical features observed in patients and the defects in muscle organization seen
in zebrafish mutants, led us to hypothesize that SelN may play a role during muscle
development and/or maintenance. To precise Sepn1 expression during murine
development and due to a lack of robust antibodies against SelN, we used RNAbased approaches: quantitative RT-PCR (qRT-PCR), performed on cDNA obtained
from isolated adult and embryonic tissues, and whole mount in situ hybridization.
We demonstrated that Sepn1 is expressed early during embryogenesis, with a strong
expression already detected at E9. By qRT-PCR, we showed that this expression
increases until E12 and then markedly decreases. Between E15 and E18, its decline
is also detected in most isolated tissues. After birth, a strong reduction is observed
with age in most tissues, leading to barely detectable levels at 6 weeks. In some rare
tissues, such as liver, Sepn1 expression was not detectable, neither in embryos nor
after birth.
_0MS0G0OLK
These results indicate that SelN is developmentally regulated in mice and correlate
with data obtained in human that showed it is strongly down regulated during
myoblasts differentiation. We are currently investigating muscle embryonic
development in SelN deficient mice by characterizing the expression of myogenic
factors in Sepn1-/- embryos.
PW39-490
TRANSDUCTION OF LOWER MOTOR NEURONS USING PERIPHERAL
INJECTION OF AAV VECTORS
DUQUE S1, JOUSSEMET B2, RIVIÈRE C3, MARAIS T1, DUBREIL L4, DOUAR AM3,
MOULLIER P2, COLLE MA4, BARKATS M1
(1) CNRS FRE 3018, Evry, FRANCE. (2) INSERM UMR 649, Nantes, FRANCE. (3)
GENETHON, Evry, FRANCE. (4) INRA U703, Nantes, FRANCE.
Gene therapy for Central Nervous System (CNS) disorders is impeded by the
Blood Brain Barrier (BBB), a physical barrier preventing the entry of viral vectors. For
motor neuron diseases (e.g. Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral
Sclerosis (ALS)), this problem has been previously circumvented by injecting viral
vectors directly into the spinal cord. This approach resulted only in the transduction of
cells close to the injection site. The development of strategies less invasive and
allowing a larger dispersion of viral vectors and/or therapeutic proteins in the CNS is
therefore required for future clinical application.
Here, we describe a new gene transfer method that allows efficient
transduction of lower motor neurons after peripheral injection of recombinant AAV
vectors. We first injected single strand (ss) and self-complementary (sc) AAV vectors
of serotype 1 and 9 intraperitoneally, intramuscularly or intravenously in neonatal and
adult C57Bl6 mice and analyzed transgene expression in the CNS. We found that
both recombinant ss- and scAAV9 vectors targeted epithelial cells of both the choroids
plexus and the ependyma, as well as neural cells including motor neurons. In mice,
the most impressive transduction was observed after intravenous administration of
scAAV9 vectors. Futhermore, the ability of scAAV9 to efficiently cross the BBB and
transduce motor neurons was confirmed in a feline model of SMA. In these animals,
_0MS0G0OLK
intravenous vector injections led to a transduction of 38% of motor neurons.
In conclusion, this study describes the first efficient and non invasive
procedure that allows a global transgene delivery to the spinal cord via a single
intravenous injection. This new gene therapy tool offers new hope for the treatment of
motor neuron diseases.
PW39-491
USE OF NONSENSE-MEDIATED MRNA DECAY (NMD) INHIBITORS TO
CORRECT GENETIC PATHOLOGIES
DURAND S1, TAZI J1, LEJEUNE F2
(1) IGMM-CNRS-UMR5535, Montpellier, FRANCE. (2) Institut Pasteur de Lille, Lille,
FRANCE.
Nonsense-Mediated mRNA Decay (NMD) is an mRNA quality control process that
degrades mRNA containing premature termination codons (PTC) in order to avoid the
To contact the author::
production of truncated proteins with potential deleterious effects for cells. One third of
sebastien.durand@igmm inherited and acquired diseases are due to nonsense mutations that elicit NMD. In
.cnrs.fr.
some cases such as Duchenne Muscular Dystrophies (DMD), NMD could be
detrimental to the production of truncated proteins that has kept the activity of wildtype proteins. Consequently, an interesting challenge would be to inhibit NMD in order
to restore truncated protein whose expression could lead to clinical rescue. We have
screened a small chemical library to find inhibitors of NMD. We have identified the first
chemical inhibitor that blocks efficiently and specifically NMD (J. Cell Biol 2007 sept
24; 178(7):1145-1160). We have shown that this NMD inhibitor called NMDI 1
(Nonsense-mediated mRNA Decay Inhibitor 1) disrupts the interaction between
hSMG5 and the NMD factor hUPF1 thereby leading to the accumulation of
hyperphosphorylated isoforms of hUPF1 in cytoplasmic granules so called
processing-bodies (P-Bodies). NMDI 1 stabilizes PTC-containing mRNAs in P-Bodies
and could give rise to truncated proteins. To address the question of the therapeutic
potential of NMDI 1, we have used cell lines from DMD patients but also different
mouse models of NMD: the mdx mice that contain a PTC in the exon 23 of the
dystrophin gene and the KIM mice that carry a PTC in the exon 3 of the gene coding
the µ opioïd receptor. Preliminary results have shown that NMDI 1 is able to stabilize
dystrophin mRNA in patient cell line and lead to truncated protein expression rescue
when NMDI 1 is directly injected in mice.
_0MS0G0OLK
PW39-492
MS2PH-DB : TOWARD THE INTEGRATION OF PROTEIN STRUCTURAL
BEHAVIOUR TO STUDY PHENOTYPIC IMPACTS OF MUTATIONS
FRIEDRICH A1, GARNIER N2, BLANDIN G3, BETTLER E2, RICHARD I3, MOULINIER
L1, POCH O1
(1) IGBMC, Illkirch, FRANCE. (2) IBCP, Lyon, FRANCE. (3) Généthon, Evry,
FRANCE.
To contact the author::
[email protected].
_0MS0G0OLK
PW39-493
The MS2PH project (from Structural Mutation to Human Pathologies Phenotypes)
aims at investigating how mutations impact protein structures and to which extent this
change could affect pathological phenotypes.
This project is part of the Decrypthon program (www.decrypthon.fr) where we have
implemented the MS2PH-db relational database (http://ms2phdb-pbil.ibcp.fr/). This
database focuses on 1915 proteins implicated in human monogenic diseases,
including 70 proteins directly implicated in various muscular dystrophies, and offers
access to mined, predicted and pre-computed data.
MS2PH-db provides instant access to three types of data for each entry protein:
- an evolutionary overview through pre-computed hierarchized multiple alignments of
complete sequences,
- a structural view through an automatically generated three-dimensional model of the
protein,
- an additional layer of information composed of structural and functional annotations,
as well as mutations and phenotypic data related to the pathology.
These three views are interconnected through a user-friendly graphical interface
which allows an interactive approach to study the combination of structural, functional
and clinical data within the framework of the evolutionary relevance of the sequence
information.
Using this tool, we have studied the distribution of all mapped mutations within the
1915 MS2PH proteins with respect to known functional domains. We observed that
roughly 78% of the mutated residues associated with a phenotypical impact are
located in close or medium proximity of a conserved residue.
In the frame of the newly developed Decrypthon Data Center, we are currently
integrating the structural and evolutionary information with interactomic data, using
protein interactions data provided by complementary sources: experimental interaction
data generated in the Muscular Interactome project, functional and physical
interactions mined from the STRING database and interacting interface predictions.
The preliminary results obtained with this integrative approach are showing the way to
the establishment of a system dedicated to the prediction of the link between
mutations and clinical phenotypes.
TROPISM CHARACTERIZATION OF DIFFERENT VIRAL VECTORS IN THE
PERIPHERAL NERVOUS SYSTEM
HOMS J1, ARIZA L1, RODRÍGUEZ E1, CHILLÓN M1, CHILLÓN M2, BOSCH A1
(1) Centre of Animal Biotechnology and Gene Therapy (CBATEG) and Department of
Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Barcelona,
SPAIN. (2) Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona,
SPAIN.
To contact the author::
[email protected].
_0MS0G0OLK
PW39-494
Central nervous system transduction has been extensively studied with a wide variety
of viral vectors. However, peripheral nervous system transduction has not brought that
much attention to researchers so far, despite the importance of acquired and inherited
neuropathies, neuromuscular diseases or pain treatment, among other pathologies.
Thus, we have characterized the tropism and transduction efficiency of different AAV
pseudotypes in primary and established Schwann cell lines as well as in vivo, through
sciatic nerve injection in the mouse. Among the AAV pseudotypes tested, AAV2/8
transduced Schwann cells more efficiently, both in vitro and in vivo. On the other
hand, AAV2/2 infected preferentially sensory neurons and AAV2/1 transduced both
Schwann cells and neurons. Expression of marker genes coded by the different
vectors was still present 10 weeks after administration, the overall duration of the
experiment. We also analyzed the generation of neutralizing antibodies against AAVs
in the infected mice. Antibody titers were higher against AAV1 than against AAV2 or
AAV8. Indeed, animals injected with AAV8 showed the lower titers of neutralizing
antibodies against this serotype, correlating with higher expression overtime. These
results suggest that AAV8 may be a suitable tool for gene therapy treatment of
diseases affecting Schwann cells.
To improve transduction of peripheral neurons, we infected organotypic cultures of
murine dorsal root ganglia with different serotypes of AAVs and human and canine
adenoviruses. These vectors were also tested in vivo by intrathecal administration to
the lumbar region of mice. We observed high levels of expression in sensory neurons
with AAV2/1 and with human Ad5. We are currently evaluating efficiency of canine
adenovirus and human Ad40 serotype in targeting sensory and motoneurons. In the
near future, helper-dependent adenovirus will be assayed with therapeutic genes in a
mouse model of disease.
Financed by AFM (#12277; AFM2007/12763AE), ISCIII (PI051705) and Generalitat de
Catalunya (2006FI00762; 2004FI00970).
SKELETAL MUSCLE CELL FATE DECISIONS IN THE EMBRYO AND THE ROLE
OF NUMB
JORY A1, TAJBAKHSH S1
(1) Stem cells and development, Pasteur Institute, Paris, FRANCE.
To contact the author::
[email protected].
Although much is known about skeletal muscle biology, we have only
recently begun to appreciate how stem and progenitor cells establish this tissue
through the action of key regulatory genes. In the mouse embryo, the dermomyotome
(DM) harbors stem cells for multiple cell lignages including muscle. The DM is the
epithelium of the somite which assures a continuous source of muscle progenitor
cells (MPCs) from the stem cells expressing Pax3 and Pax7. Muscle progenitors
generate myoblasts which differentiate to form the first skeletal mass in the somite,
the myotome.
To study self-renewal and differentiation of MPCs, we are investigating selfrenewal via asymmetric divisions. The cell fate determinant Numb has been
implicated in asymmetric division and involved in binary cell fate choices via Notch
inhibition in Drosophila. However, the mechanisms and the role of Numb involved in
generating cell diversity in vertebrates remains unclear. To further assess the role of
Numb in driving cell fate in the mouse dermomyotome and in establishing the
myotome, several transgenics were made to study the effects of Numb-GFP overexpression in the somite using the epaxial enhancer of Myf5.
First we show that these transgenics do drive NumbGFP over-expression in
most of the stem cells of the dorsal somite. Unexpectedly this strategy revealed that
the overexpression of Numb-GFP in the dorsal dermomyotome only very mildly affects
cell fate decisions and does not seem to have an effect on Notch signalling in this
context. We do not observe an overt increase of muscle differentiation in the dorsal
somite. Thus, we believe that Numb and Notch act in particular cell states in lineage
progression. Interestingly, this strategy revealed a novel role for Numb in somite
patterning, in the epithelial organisation of the dermomyotome, as well as in myofiber
organisation in the underlying myotome.
_0MS0G0OLK
PW39-495
ANCHORING OF ACETYLCHOLINESTERASE IN MEMBRANES BY PRIMA
NOUREDDINE H1, CARVALHO S1, SCHMITT C1, MASSOULIE J1, BON S1
(1) Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supérieure,
Paris, FRANCE.
The nervous tissue and muscles of mammals express the T splice variant of
acetylcholinesterase (ACHET), characterized by its 40-residues C-terminal peptide (t
To contact the author::
peptide), which allows its association with anchoring proteins, the collagen ColQ and
Hiba.noureddine@biolog the transmembrane protein PRiMA, producing respectively collagen-tailed forms and
ie.ens.fr.
membrane-bound tetramers. These interactions are important since they condition the
functional anchoring of AChE in cholinergic tissues. The collagen-tailed forms are
inserted in the basal lamina at neuromuscular junction, while the membrane-bound
tetramers are anchored at the cell surface through the transmembrane domain of
PRiMA. The membrane-bound tetramers represent the major enzyme species in the
brain ; they are also expressed in muscles, where their level is regulated by exercise.
The association of AChET subunits with ColQ has been extensively studied : it is based
on an tight interaction between four t peptides and a proline-rich motif, called PRAD
(Proline-Rich Attachment Domain), located in the N-terminal non collagenous region
of ColQ. The structure of a complexe between synthetic t and PRAD peptides has
been determined by cristallography. The association of AChE T subunits with PRiMA
appears similar because this transmembrane protein also contains a proline-rich motif,
but there are significant differences in the number of prolines (8 in ColQ, 14 in PRiMA)
and in the number and positions of cysteines that might form intercatenary disulfide
bonds with the cysteine located near the C-terminus of the t peptides. Therefore, we
have undertaken an analysis of the association of AChET with PRiMA. Using deletions
and point mutations in PRiMA, we defined a minimal motif in PRiMA, which could
associate with AChET subunits.
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PW39-496
CTCF AND A-TYPE LAMINS COUPLE INSULATION AND PERINUCLEAR
LOCALIZATION OF THE D4Z4 SUBTELOMERIC ELEMENT IN FSHD.
OTTAVIANI A1, RIVAL-GERVIER S2, BOUSSOUAR A1, FÖRSTER A3, RONDIER D1,
BAUWENS S1, GILSON E1, MAGDINIER F1
(1) Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure de
Lyon, Lyon, FRANCE. (2) UMR 1198. Biologie du développement et de la
Reproduction, INRA-CNRS-ENVA, Domaine de Vilvert, Jouy-en-Josas, FRANCE. (3)
Gregor Mendel-Institute of Molecular Plant Biology, Vienna, AUSTRIA.
Both genetic and epigenetic alterations contribute to the Facio-Scapulo-Humeral
Dystrophy (FSHD) linked to the reduction of a number of D4Z4 repeated elements at
To contact the author::
the 4q35 locus. The consequence of this rearrangement remains enigmatic but
alexandre.ottaviani@ens deletion of these repeats to a threshold of 11 copies might epigenetically dysregulate
-lyon.fr.
the FSHD gene(s) in patients through position effect variegation (PEV) and our goal
was to test the function of D4Z4 on the regulation of gene silencing.
We generated several constructs where different parts or different numbers of D4Z4
were cloned downstream of a reporter gene and followed the expression of the gene
over an extended time in culture. We showed that D4Z4 is a bona fide insulator
element protecting from PEV and able to block enhancer-promoter communication.
The multivalent CTCF protein and A-type Lamins bind to D4Z4 and participate in this
insulation activity.
Unlike other human telomeres, the 4q35 locus is localized at the periphery of the
nucleus and might interact with components of the lamina. We showed that D4Z4
displaces a telomere toward the nuclear periphery in the presence of CTCF and
Lamins A/C suggesting that both factors coordinate the long-range chromatin
organization of D4Z4 and the tethering of a locus to a specialized nuclear
compartment.
We further demonstrate that insulation and perinuclear activities of D4Z4 are lost upon
multimerization of the repeat, suggesting that the D4Z4 array at the 4q35 locus acts
as a CTCF-dependent insulator in FSHD patients but not in normal individuals and
might impact on the expression of the genes causing FSHD through the alteration of
the 4q35 locus microenvironment.
_0MS0G0OLK
PW39-497
UROCORTINS IMPROVE SKELETAL MUSCLE STRUCTURE AND FUNCTION OF
DYSTROPHIC MDX MOUSE VIA A CYCLIC AMP/PKA DEPENDENT PATHWAY
REUTENAUER J1, DORCHIES O1, BOITTIN FX2, PATTHEY-VUADENS O1, RUEGG
U1
(1) Laboratory of Pharmacology, Geneva Lausanne School of Pharmaceutical
Sciences, University of Geneva, Geneva, SWITZERLAND. (2) Laboratory of Vascular
Cell Physiology, Department of Zoology & Animal Biology, University of Geneva,
Geneva, SWITZERLAND.
Urocortins are structurally related peptides of the corticotrophin-releasing factor
(CRF), which has been widely implicated in responses to physical, emotional and
To contact the author::
environmental stress. Recently, it has been reported that a CRF-receptor-2 (CRFR2)
julie.reutenauer@pharm. agonist increased muscle mass and slowed disease progression in the mdx mouse
unige.ch.
model. Here, we report on our investigations on urocortins in protecting mdx muscle
from necrosis and on the mechanism involved.
Three week-old mdx5Cv mice were treated daily by sub-cutaneous injection for
2 weeks with either urocortin 1 at 300 µg/kg, urocortin 2 (30, 100 or 300 µg/kg), or
vehicle. Isometric force recordings of the triceps surae (comprising soleus, plantaris
and gastrocnemius muscles) showed that contraction and relaxation kinetics were
shorter than in untreated mice. Moreover, the force-frequency curve was shifted to the
right, suggesting a change in the calcium homeostasis or in the distribution of fibres
toward a faster phenotype. Interestingly, hindlimb muscles from mice receiving
urocortins displayed a higher resistance to mechanical stress and muscle mass was
significantly increased. Histology demonstrated that urocortins remarkably protected
diaphragm, EDL and soleus muscles from necrosis. Finally, urocortins administration
lowered plasma creatine kinase levels up to 49% compared with vehicle.
In addition, basal calcium influx was measured in whole dystrophic muscle (EDL and
soleus) and in diaphragm strips. Urocortin 2 concentration-dependently decreased the
permeability of the dystrophic muscle to calcium by 20-40%. This effect was
completely abolished by the selective CRFR2 antagonist astressin 2-B or an inhibitor of
protein kinase A (PKA) while addition of forskolin, an activator of adenylate cyclase,
mimicked the effect of urocortin 2.
We conclude that urocortins improve both the structure and the function of dystrophic
skeletal muscle via the stimulation of a cAMP/PKA-dependent signal transduction
pathway. Thus, urocortins should be considered as potential candidates to counteract
the impairment of calcium homeostasis observed in Duchenne Muscular Dystrophy.
_0MS0G0OLK
INDEX OF AUTHORS
Last name and Initial
Abstract Reference
AARTSMA-RUS A
ABADI N
ABBACI K
ABBOUD N
ABOU-KHALIL R
ABRESCH RT
ABRIAT A
ACHARJEE S
ACHIRON A
ACQUAIRE J
ADAMO S
ADAMOPOULOS S
ADAMY C
ADIN P
AEBISCHER J
AERTS S
AGUENNOUZ M
ÅHLBERG G
AHMED R
ALABANESE P
ALAMEDDINE H
ALBERT V
ALBERTS M
PW2-024, PW26-322, PW34-425
PW7-077, PW11-137, PW20-242
PW9-106
PW35-438
PW30-374
PW36-450
PW15-192
PW2-027
PW7-085
PW12-148
PW31-395, PW31-396
PW22-271
PW13-162
PW26-330
PW6-074
PW38-487
PW25-315
PW16-204
PW17-213
PW25-307
PW37-462
PW27-345
PW21-268
ALEXANDER C
ALEXANDRI N
ALLAMAND V
AUTILLO-TOUATI A
AVILA D
AVITAL A
AVRIL-DELPLANQUE A
AZPITARTE M
AZULAY JP
AZZEDDINE H
AZZONI E
PW10-126
PW18-214
PW13-166, PW17-209, PW17-209, PW17-211,
PW27-347, PW39-489
PW4-043, PW9-105, PW14-175
PW10-119
PW21-267
PW17-209
PW9-110
PW29-365
PW14-175
PW23-289
PW7-083
PW26-323
PW37-462
PW38-465
PW23-290, PW23-290
PW26-331
PW21-263
PW16-201
PW31-387
PW8-100
PW37-463
PW24-302
PW38-479
PW28-355
PW38-477
PW11-129
PW24-294
PW20-245
PW10-115
PW26-331
PW21-265
PW7-078, PW7-080
PW29-364
PW14-174
PW13-161, PW13-162
PW39-493
PW34-429
PW4-046
PW30-376
PW21-264
PW6-066
PW30-375, PW33-418
PW21-269, PW21-270
PW18-221
PW16-194, PW24-296
PW38-477
PW28-356
PW1-006
PW22-271, PW22-279
PW7-079, PW23-287
PW7-085
PW13-168
PW35-434
PW36-450
PW21-264
PW7-086, PW21-262, PW23-282
PW15-190
PW12-149
PW24-302
PW28-354
PW38-467
PW31-395
PW21-261
PW26-323
PW11-139, PW21-253, PW21-254, PW21-258, PW21-259,
PW21-265, PW21-269
PW33-424
PW17-207
PW33-416
PW2-021
PW8-092
PW19-228, PW21-262
PW13-163, PW23-285
PW25-305, PW28-348, PW30-378
B HUOT N
BABBS A
BACHINSKI L
BACHOUD-LEVI AC
BACOU F
BADER C
BADER H
PW5-057
PW26-320
PW11-141
PW21-259
PW18-220, PW29-367
PW27-334, PW29-359, PW29-360, PW30-376
PW34-432
ALLARD B
ALLEGRA M
ALLENBACH Y
ALLOUCHE S
ALLYSON J
ALONSO S
AL-QUSAIRI L
ALTERIO J
AMAROF K
AMBEGIA E
AMBROSI I
AMENDOLA J
AMER EL KHEDDOUD W
AMMOUR M
AMOURA Z
AMSILI S
AMTHOR H
ANDERSON L
ANDRADA L
ANDRESEN B.S
ANDREWS M
ANDRIAMANALIJAONA R
ANEGON I
ANGEARD N
ANGELINI C
ANNANE D
ANSSEAU E
ANTONELLI D
AOUBA A
APOSTOL P
ARANEGA A
ARBOGAST S
ARIMURA T
ARIZA L
ARMAND AS
ARNAUD MC
ARNAUDEAU S
ARNE-BES MC
ARNOLD AS
ARNOLD L
ARNOULT C
ARRIGHI N
ARRIGO AP
ARRUDA V
ARSIC N
ARTHI C
ARVANITIS DA
ASSAMI S
ASTORD S
ASTUDILLO A
ATASSI F
ATKINSON LA
ATTAL M
ATTARIAN S
AUBERT P
AUBERT S
AUBEY F
AUDA-BOUCHER G
AUFFRAY C
AULINO P
AURANGABADKAR K
AUSONI S
AUTHIER FJ
BADSI D
BAGHDOYAN S
BAGNIS C
BAGUET JP
BAIAZITOV R
BALANTIC Z
BALCH F
BALGHI H
BALIGAND C
BALL S
BALOTTIN U
BANDUSEELA V
BANGRATZ M
BARAD M
BARAKAT-WALTER I
BARANOV VS
BARBARA G
BARBET P
BARBIER-CHASSEFIÈRE V
BARBOIRON C
BARBOSA I
BARBOT JC
BARBRY P
BARDONI B
BARHANIN J
BARKATS M
BARNAY-TOUTAIN F
BARONCHELLI C
BARONI D
BARONI MD
BARRAULT L
BARREY E
BARRO M
BARTHELEMY I
BARTHÉLÉMY I
BARTOLI M
BARZAGHI P
BASHIR R
BASSE N
BASSEM H
BASSEZ G
BASTIANI C
BASTIN J
BASTUJY-GARIN S
BATISTA L
BAUCHE S
BAUCHÉ S
BAUDOT C
BAUTISTA-LORITE J
BAUWENS S
BAYLE n
BEAUFRÈRE AM
BEAULIEU D
BECANE H
BECHARA E
BECK B
BECKER-COHEN M
BECKERLE M
BEGGS AH
BEHARI M
BEHIN A
BÉHIN A
BEHRA M
BEILLEVAIRE T
BELAÏDI H
BELANGER G
BELAYEW A
BELICCHI M
BELLANCE R
BELLO NF
BELLOMO RG
BELLOUZE S
BELMONTE N
BEN AMMAR A
BEN YAOU R
BENABDALLAH BF
BENABDELLAH F
BENARD R
BENCZE M
BENDAHAN D
BENDAHHOU S
PW11-132
PW12-149
PW28-350, PW35-442
PW20-247
PW2-027
PW32-409
PW3-030
PW5-063
PW30-382, PW39-488
PW20-250
PW1-007
PW34-433
PW33-414, PW33-415
PW6-071
PW12-143
PW7-084
PW23-291
PW4-051, PW4-052
PW22-278
PW31-393
PW31-392
PW9-103
PW22-276
PW10-120
PW18-221
PW7-085, PW39-490
PW26-330
PW9-107
PW30-385
PW26-323
PW9-109, PW24-295
PW32-408, PW33-422
PW10-115, PW10-118
PW33-422
PW2-022, PW34-426, PW34-427
PW8-098, PW9-109, PW9-109, PW9-112, PW24-295, PW 26-326,
PW26- 332
PW4-044, PW17-212, PW24-300, PW24-300
PW8-100
PW9-106, PW24-294
PW11-138
PW8-097, PW11-128, PW11-130, PW11-139, PW21-253,
PW21-254, PW21-258, PW32-400, PW36-447, PW36-454
PW26-331
PW24-302
PW11-139
PW20-246
PW33-421
PW18-214, PW18-217, PW25-309
PW23-280, PW23-283
PW19-233, PW19-234
PW39-496
PW11-136
PW15-191
PW12-154
PW1-001, PW11-128, PW16-200
PW10-120
PW3-030
PW16-201, PW17-208
PW29-358
PW14-175
PW21-256, PW21-261
PW16-199, PW16-200, PW18-215
PW26-325, PW26-325
PW34-431
PW18-215
PW13-163, PW23-285, PW37-460
PW12-144
PW10-115
PW2-018, PW2-019, PW4-047, PW5-056, PW8-098
PW7-083
PW31-394
PW32-404
PW33-424, PW34-428
PW28-350
PW18-214, PW18-217
PW13-159, PW13-164, PW36-455, PW38-472
PW5-062
PW25-319
PW4-054
PW27-335, PW27-336
PW9-102, PW14-173, PW15-192, PW32-401, PW32-402,
PW38-476
PW18-221
BENEDETTI S
BENHASSINE T
BENLATRECHE C
BENNDORF R
BENOMAR A
BENVENISTE O
BENYAOU R
BERARD C
BERARDI E
BERARDINELLI A
BERBEY C
BERGE J
BERNARD A
BERNARD D
BERNARD N
BERNARD R
BERNARD V
BERNARDI H
BERNARDI P
BERNEX F
BERNHEIM L
BEROUD C
BÉROUD C
BERRIH-AKNIN S
BERTHIER C
BERTHOMIEU M
BERTINI E
BERTOLDI D
BERTRAND A
BERTRAND J
BESNIER-PENISSON I
BESSE S
BESSIS A
BESTANDJI K
BETTLER E
BETTO R
BEUGNET C
BEVILACQUA J
BHATIA R
BIANCALANA V
BICHRAOUI H
BIELLI S
BIFFI A
BIGARD X
BIGOT A
BILLARD J
BIONDI O
BIROUK N
BISMUTH J
BITOUN M
BITTO A
BLAINEAU S
BLAIS F
BLANC J
BLANCHARD A
BLANCHARD C
BLANCHARD S
BLANDIN G
BLANGY A
BLAZQUEZ L
BLEDSOE C
BLIN G
BLIN J
BLOM J
BLOT S
BLUMEN N
BLUMEN SC
BOBAN BLAGAIC A
BOCCACCIO I
BOCKSTAEL O
BOËRIO D
BOESPFLUG-TANGUY O
BOHL D
BÖHM J
BOISSE MF
BOITTIN FX
BOLDRIN L
BOLLINI G
BOLZEC T
BOMMELAER C
BOMONT P
BON S
BONALDO P
BONANNI E
PW13-156
PW7-079, PW23-290
PW7-077, PW11-137, PW20-242
PW16-195, PW23-281, PW23-281
PW13-163, PW23-285
PW21-263, PW21-263, PW21-267, PW26-325
PW13-166
PW1-012, PW36-451, PW36-452
PW31-395
PW1-007
PW4-043, PW14-175
PW3-029
PW19-241
PW38-466
PW6-075
PW8-090, PW8-091, PW8-097, PW23-282, PW23-284, PW23-290
PW33-414, PW33-415
PW29-367
PW17-210
PW34-427
PW27-334, PW29-359, PW29-360, PW30-376
PW36-453, PW36-454
PW1-002, PW8-091, PW8-097, PW36-455, PW36-456
PW18-223, PW18-224, PW18-225
PW9-105, PW9-106, PW9-106
PW37-457
PW7-082, PW17-210, PW17-210
PW34-426, PW39-488
PW13-161
PW25-312
PW13-167
PW22-278
PW38-466
PW7-077
PW39-492
PW24-294
PW9-103, PW13-164, PW38-472
PW8-096, PW14-177, PW14-179, PW14-181
PW1-010
PW14-177
PW9-105
PW34-428
PW28-353
PW25-310, PW33-421
PW12-150, PW27-335, PW31-388
PW33-416
PW6-076
PW13-163, PW23-285, PW37-460
PW18-223
PW14-177, PW14-179, PW14-180, PW14-181
PW25-315
PW9-106
PW35-443
PW15-190, PW35-445
PW38-471
PW20-244, PW21-255
PW6-071
PW9-109, PW39-492
PW27-342
PW8-092
PW3-032
PW35-440
PW26-331
PW31-398
PW2-022, PW33-422, PW34-426, PW34-427
PW7-085
PW7-085
PW25-314
PW23-283
PW38-486
PW11-130
PW15-191
PW6-071
PW14-178
PW21-259
PW39-497
PW30-385
PW23-286, PW23-286
PW29-367
PW15-191
PW23-288
PW39-495
PW17-210
PW11-131
BONDOIN L
BONDON A
BONELLO-PALOT N
BONET-KERRACHE A
BONGIOANNI P
BONNE G
BONNET A
BONNET N
BONNIEU A
BOREL P
BORGES A
BORRUAT FX
BOSCH A
BOSCO G
BOTEZ S
BOUAZZA B
BOUCHARD JP
BOUCHE P
BOUCHEMAL K
BOUCHENTOUF M
BOUCHET C
BOUCHETEIL J
BOUCRAUT J
BOUÉ P
BOUHOUCHE A
BOUHOUR F
BOULAAJAJ F
BOULANGER A
BOULAY C
BOULEFKHAD A
BOUMEDIENE K
BOUREZGUI M
BOURG N
BOURG – ALIBERT
BOURGEOIS F
BOURMEYSTER N
BOURRET A
BOUSQUET N
BOUSSOUAR A
BOUTILIER JK
BOUVIER-DURAND M
BOVOLENTA M
BOWERMAN M
BOYER F
BOYER O
BRAIS B
BRAND-SABERI B
BRANLANT C
BRAVERMAN I
BRCIC I
BRCIC L
BREGESTOVSKI P
BRESOLIN N
BRICE A
BRIGITTE M
BRIGUET A
BRINAS L
BRIÑAS L
BRINGARD A
BROCARD J
BROCHIER G
BROGLIO L
BRONICKI L
BROSSET C
BROTCHI J
BRUGIERES P
BRULE C
BRUNELLE A
BRUNELLI S
BRUNET-DE CARVALHO N
BRUNO C
BRUSTIS JJ
BUCKINGHAM M
BUÉE L
BUHLER E
BUJ BELLO A
BULDAKOVA S
BURIE C
BURLOIU C
BUTLER-BROWNE G
BUTOIANU N
PW21-255
PW1-003, PW4-049
PW23-282
PW2-028
PW12-142
PW13-155, PW13-157, PW13-159, PW13-160, PW13-161,
PW13-162, PW13-164, PW13-166, PW17-209, PW22-275
PW29-357
PW35-434
PW24-297
PW8-093
PW26-326
PW18-222
PW39-493
PW32-404
PW18-222
PW10-124, PW10-125
PW9-110
PW16-200, PW19-231
PW26-324
PW5-059, PW5-062
PW17-206, PW17-207
PW9-109
PW21-262, PW35-442
PW8-094
PW13-163, PW23-285
PW11-134
PW19-230
PW34-430
PW23-286
PW7-077, PW20-242
PW28-355
PW19-230
PW8-098, PW26-332
PW 26-326
PW18-219
PW5-064
PW10-122
PW8-093, PW8-094
PW39-496
PW2-023
PW9-106
PW2-025, PW17-210
PW6-073
PW11-136
PW21-269, PW21-270
PW9-110, PW10-126
PW29-357
PW38-482
PW7-085, PW7-085
PW25-314
PW25-314
PW23-292
PW2-018, PW2-019, PW4-047
PW38-471, PW38-471
PW21-268
PW4-044, PW33-420
PW17-209
PW17-211
PW38-476
PW14-170, PW33-419, PW33-419
PW14-177, PW16-199
PW9-107
PW6-068
PW15-192
PW38-486
PW21-259
PW31-389
PW25-319
PW25-305, PW28-348, PW28-353, PW30-378
PW38-468
PW1-009, PW1-015, PW14-176
PW30-379
PW30-383
PW12-146
PW6-071
PW14-175, PW26-327
PW23-292
PW26-330
PW7-078, PW7-080
PW26-321, PW4-045, PW10-124, PW10-125, PW12-152,
PW12-153, PW18-220, PW21-266, PW21-267, PW25-317,
PW27-335, PW27-336, PW27-337, PW28-349, PW31-388
PW7-078, PW7-080
BUYSE G
BYRNE B
PW24-300, PW24-301
PW20-249, PW20-252
CABELLO A
CABIANCA D
CACHEUX M
CACOUB P
CADOT B
CAILLAUD C
CAILLET-BOUDIN ML
CAILLOL G
CAIZERGUES D
CALBO S
CALENDER A
CALPAIN STUDY GROUP _
CALPAÏN STUDY GROUP T
CALSOLARO V
CAMBIER L
CAMOZZI D
CANAL A
CANAVERIS G
CANDELARIO-MARTINEZ A
CANDELARIO-MARTÍNEZ A
CANKI-KLAIN N
CANTALOUBE I
CAPOTONDO A
CAPOVILLA M
CAPRI Y
CAPRON F
CARAMELLE P
CARASSO RL
CARLIER P
PW13-168
PW10-116
PW14-170
PW21-263
PW30-372
PW20-246
PW12-146
PW33-424
PW26-325
PW21-269, PW21-270
PW38-471
PW8-093, PW8-094
PW36-449
PW9-114, PW11-131
PW38-467
PW13-157
PW32-406, PW32-408, PW36-449
PW37-463
PW33-416
PW4-053
PW9-113
PW38-470
PW28-353
PW38-487
PW15-191
PW21-263
PW13-162
PW7-085
PW20-245, PW26-325, PW30-382, PW32-411, PW32-412,
PW34-426, PW39-488
CARLIER R
CARLSON CG
PW20-245
PW3-029, PW3-030, PW3-030, PW3-031, PW3-032, PW3-032,
PW3-033, PW3-034
PW10-118, PW24-297
PW23-281
PW25-316
PW22-273
PW18-218
PW18-218
PW39-495
PW20-252
PW5-061
PW16-197
PW39-489
PW22-275
PW24-293
PW2-019
PW8-099, PW 26-326
PW30-377, PW35-444
PW31-390
PW21-266
PW9-113
PW35-442
PW32-409
PW21-259
PW24-301
PW20-247
PW9-108, PW23-286
PW17-213
PW38-485
PW29-370
PW12-149
PW6-076, PW34-429
PW7-079, PW23-283, PW23-290
PW17-211, PW26-321
PW12-154
PW21-260
PW8-091, PW17-209, PW18-221
PW11-139
PW6-076, PW34-429
PW30-380
PW12-146
PW24-304
PW38-485
PW27-342
PW10-127
PW8-095
PW34-432
PW18-220
PW21-268, PW27-341, PW30-374, PW30-375, PW33-418
CARNAC G
CARRA S
CARRE-PIERRAT M
CARRIER L
CARTAUD A
CARTAUD J
CARVALHO S
CASE L
CASSAR-MALEK I
CASTERAS-SIMON M
CASTETS P
CATELAIN C
CATOIRE H
CATTANEO A
CAU P
CAUBIT X
CEBRIAN J
CECCALDI PE
CECIO MR
CEDILE O
CERU B
CESARO P
CEULEMANS B
CHABRE O
CHABROL B
CHAHNEZ T
CHAIGNEAU E
CHAMBON C
CHAMPON B
CHANOINE C
CHAOUCH M
CHAOUCH S
CHAPEDELAINE P
CHAPELON-ABRIC C
CHAPON F
CHAPOY E
CHARBONNIER F
CHARLES S
CHARLET N
CHARLIN C
CHARPAK S
CHARRASSE S
CHARTIER A
CHARTON K
CHARVET B
CHATONNET A
CHAZAUD B
CHAZE T
CHELBI M
CHELH I
CHELLY J
CHERAUD Y
CHEREAU F
CHEREL Y
CHÉREL Y
CHEVALIER C
CHEVALIER X
CHEVESSIER F
CHIAMULERA C
CHIKHAOUI K
CHILLÓN M
CHISARI C
CHOI K
CHOPARD A
CHOUERY E
CHRETIEN F
CHRISTEN Y
CHRISTOV C
CHTARTO A
CIAFALONI E
CIF L
CIFUENTES-DIAZ C
CIMPONERIU D
CINTAS P
CIZERON-CLAIRAC G
CLAEYS K
CLARKE N
CLAUSTRES M
CLEMENTI E
CLEVELAND D
CLOT F
CLOW C
COBO AM
COCHET O
COGNARD C
COHEN R
COHEN T
COIRAULT C
COISY-QUIVY M
COLASSE S
COLETTI D
COLLE MA
COLLETTE J
COLLOD-BÉROUD G
COLLURA V
COLMAN L
COLOMBE A
COLOMER J
COLUMBARO M
COMI G
COMMARE MC
COMMERE V
COMUNALE F
COMYN S
CONSTANTIN B
CONTE G
COOPER S
COPPÉE F
CORAL R
CORBEL L
CORE N
CORÉ N
CORIATI A
CORNELOUP C
COROMINAS JM
COSSÉE M
COSSU G
COTÉ C
CÔTÉ J
COTTIN P
COUBES C
COUCHOUX H
COUETTE B
COUETTE M
COULIC V
COULON V
COULTON G
COURDIER-FRUH I
COURRIER S
PW29-370
PW17-207
PW5-061
PW1-001, PW13-164, PW2-021, PW9-103, PW33-416,
PW36-455, PW36-455, PW38-472
PW29-361
PW28-355, PW28-355
PW27-340, PW38-477
PW28-354
PW5-061
PW21-259
PW18-217
PW19-236
PW38-472
PW39-493, PW39-493
PW12-142
PW18-227
PW6-068
PW23-283
PW21-268
PW25-309
PW1-001, PW4-051, PW4-052, PW21-253
PW38-486
PW1-008
PW38-471
PW38-468
PW7-078, PW7-080
PW21-264
PW18-223
PW14-177, PW15-188, PW16-200, PW18-215
PW13-155, PW14-171, PW17-206
PW1-002, PW2-025, PW36-453, PW36-455, PW36-456,
PW38-471
PW25-305, PW30-378
PW23-288
PW38-471
PW3-037
PW13-158
PW28-350
PW5-063, PW5-064, PW32-399
PW31-398
PW31-397
PW22-273, PW27-347
PW10-122
PW6-070
PW31-395
PW39-490
PW35-439, PW35-441
PW36-456, PW38-471
PW9-109
PW31-390
PW35-434
PW17-210
PW13-157
PW13-156, PW28-353
PW36-451
PW9-103
PW27-342
PW25-306, PW25-313
PW5-063, PW5-064, PW32-399
PW14-176
PW14-171, PW15-185
PW10-115
PW1-016
PW10-119
PW30-377
PW35-444
PW38-479
PW33-421
PW11-140
PW1-002
PW2-018, PW25-305, PW28-348, PW28-353, PW30-378
PW11-134, PW11-138
PW6-065, PW12-144
PW8-089, PW30-379, PW31-389
PW1-002
PW9-105, PW9-106
PW24-298
PW21-259
PW35-439, PW35-441
PW29-369
PW31-388
PW4-044, PW24-300
PW8-099, PW 26-326
COURTY J
COZZONE P
CRAIU D
CRAPIE C
CRASSOUS B
CREED KE
CRIMALDI L
CSIBI A
CUENOT P
CUISSET JM
CUMANO A
CUSTAUD M
PW25-307
PW9-102, PW14-173, PW15-192, PW32-402, PW32-401,
PW38-476
PW7-078
PW26-329
PW25-310
PW20-248
PW31-391
PW31-393
PW26-331
PW2-025, PW17-206
PW30-383
PW23-286
DA ROCHA RODRIGUES S
DABERTRAND F
DAEGELEN D
DAI F
DANDONNEAU M
D'ANGELO MG
DANI C
DANIELE N
DANIÈLE N
DANIELI-BETTO D
D'ANJOU MC
DANOS O
DAOUD F
D'APICE MR
DARBELLAY B
DARGELOS E
DASHI F
DAURY L
DAVIDOVIC L
DAVIES KE
DAVOINE CS
DAZZO E
DE CASTRO D
DE COPPI P
DE GROOT I
DE GUZMAN C
DE KIMPE S
DE LA FUENTE-CORTEZ B
DE LA PORTE S
DE MEIJER E
DE PASQUALE MG
DE PETRIS D
DE SANDRE-GIOVANNOLI A
DE SANTA BARBARA P
DE WINTER C
DEBURGRAVE N
DECAUX JF
DECHESNE CA
DECONINCK N
DECOSTRE V
DEFORGES S
DEGOUTIN J
DEGROUARD J
DELAGRÈVERIE H
DELAGUE V
DELBONO O
DELDICQUE L
DELLA GASPERA B
DELMONT E
DELORME B
DELPLACE S
DELRÉE P
DELRIEU J
DEMARQUOY J
DEMAUREX N
DEMAY L
DEMAY-THOMAS E
DEMIGNON J
DEN DUNNEN J
DENEFLE P
DENÈFLE P
DENGLER R
DENIS J
DEPONTI D.
DEROIDE N
DESBIEZ M
DESCHAMPS JY
DESCHAMPS R
DESDOUITS M
DESGROSEILLERS L
PW30-380
PW3-041, PW25-318
PW22-274, PW30-384
PW29-357
PW22-277
PW2-018
PW28-350, PW28-351
PW8-095
PW9-109, PW9-112
PW10-117
PW36-451
PW26-327
PW9-103, PW2-021, PW33-416
PW14-176
PW30-376
PW31-389
PW21-260
PW30-379
PW10-120
PW26-320
PW33-414, PW33-415
PW30-385
PW20-243
PW28-352, PW30-385
PW24-301
PW23-281
PW2-024, PW26-322
PW19-237
PW3-042, PW25-309, PW25-319
PW34-425
PW25-315
PW 26-326
PW23-290
PW35-435, PW35-436
PW2-024, PW26-322
PW9-103, PW13-164, PW38-472
PW22-274
PW28-350, PW28-351
PW13-166
PW13-160, PW13-161, PW13-162, PW22-275
PW6-076
PW38-468
PW3-039
PW21-269, PW21-270
PW23-280, PW23-283, PW23-290
PW33-420
PW25-310
PW34-429
PW21-262
PW27-340
PW10-119, PW10-120, PW28-350
PW35-439, PW35-441
PW21-264
PW19-241
PW30-376
PW13-155, PW13-159, PW17-209, PW38-472
PW18-217
PW29-371
PW29-362, PW34-425
PW27-345
PW26-325, PW26-327
PW7-088
PW12-149
PW30-378
PW7-088
PW15-191
PW27-340, PW38-477
PW7-083
PW21-266
PW12-144
DESGUERRE I
DESILETS S
DESMET FO
DESNUELLE C
DUBREIL L
DUCHENE J
DUCHESNAY A
DUCLOHIER H
DUCROS M
DUDDY W
DUFOUR D
DUGUE C
DUGUEZ S
DUJARDIN G
DUMONCEAUX J
DUNAND M
DUPAS B
DUPLAN L
DUPREZ D
DUPUIS C
DUQUE S
DURA JM
DURAND J
DURAND M
DURAND S
DURBEC P
DURIEUX AC
DÜRR A
DUTEIL S
DUTRAIT M
PW1-001, PW4-051, PW4-052, PW33-418
PW35-438
PW36-455, PW36-456
PW10-119, PW10-119, PW10-120, PW10-121, PW18-221,
PW28-350
PW32-400, PW36-447
PW12-146, PW38-471
PW3-036
PW31-386
PW24-300
PW19-236
PW9-113
PW32-404
PW26-321, PW27-335, PW27-336, PW28-349, PW28-350,
PW28-351
PW32-403
PW16-194
PW3-040, PW38-469
PW10-126
PW1-011, PW11-135
PW21-253, PW21-258
PW38-470
PW16-194
PW13-166
PW28-352
PW24-302
PW10-119
PW1-009, PW1-015
PW15-185
PW5-059
PW22-279
PW15-186
PW8-094, PW20-243, PW24-303
PW25-306, PW25-313, PW39-497
PW32-403
PW22-271
PW26-325, PW26-327, PW39-490
PW20-246
PW2-021, PW2-022
PW25-314
PW34-427
PW14-172, PW15-182
PW29-369
PW8-091
PW1-008
PW4-044
PW11-128, PW16-200, PW16-200
PW37-462
PW13-163, PW16-199, PW16-200, PW16-202, PW21-260,
PW21-267, PW23-285
PW4-046, PW27-340, PW39-490
PW32-405
PW31-392
PW32-399
PW38-485
PW31-397
PW26-330
PW26-331
PW27-344
PW38-480
PW31-387
PW18-222
PW4-054
PW6-075
PW29-357, PW31-394, PW35-443
PW26-331
PW39-490
PW34-430
PW38-465
PW9-102, PW26-327
PW2-028, PW39-491
PW35-442, PW38-473
PW14-180, PW14-181
PW38-471
PW32-412
PW18-218
EBIHARA M
ECHANIZ-LAGUNA A
ECOCHARD R
EDSTRÖM L
EISENBERG I
EL MOUTAWAKIL B
ELFRING GL
PW10-126
PW7-088, PW33-414
PW36-451
PW16-204
PW4-048
PW19-230
PW36-450
DEUX JF
DHAENENS C
DHAMANE E
DHAWAN J
D'HOOGE J
DI CHIO M
DI GREGORIO MG
DI PANCRAZIO L
DI SANTO J
DI TANO G
DIAZ-LATOUD C
DIAZ-RAMOS A
DICAIRE MJ
DILEK N
DIMITRI D
DIMITROV A
DIMITROVA V
DION E
DITADI A
DJOUADI F
DODILLE-PLAISANT M
DOGLIO L
DOMAZETOVSKA A
DOMINIQUE JC
DONG M
DONNER K
DOPPLER V
DORCHIES O
DORIA C
DORN II GW
DOUAR AM
DOUILLARD-GUILLOUX G
DREYFUS P
DRMIC D
DROUGARD C
DROUHIN S
DROUIN J
DROUIN-GARRAUD V
DRUSCHEL C
DUBACH-POWELL J
DUBOC D
DUBOIS C
DUBOURG O
EMDIN M
ENOND C
ERAN A
ERB M
ESCOBAR MG
ESCOBAR RE
ESCOUBET B
ESPIGAT-GEORGER A
ESPOSITO A
ESPOSITO B
ESQUITIN N
ESSAFI S
ESTOURNET B
ETARD C
EVANS C
EYMARD B
EYMARD-PIERRE E
FABBRI G
FABBRIZIO E
FABRIS M
FAIZA F
FALORNI M
FALZARANO S
FANIN M
FANÒ G
FAOUZI B
FARALLI H
FARAUT B
FARDEAU M
FARINI A
FASANO L
FATTAL E
FAUCHILLE S
FAULKNER G
FAURÉ J
FAVIER M
FAYET G
FEDON Y
FEIL R
FÉRÉOL S
FERLINI A
FERNANDEZ A
FERNÁNDEZ JM
FERON M
FERRARI A
FERRARI M
FERREIRO A
FERREIRO V
FERRER I
FERRER X
FERRY A
FIGARELLA D
FIGARELLA-BRANGER D
FILAUT S
FILLIPI P
FILOSTO M
FINGERLE J
FINI S
FINKEL R
FISZMAN M
FLAVIER S
FLETCHER S
FLORENCE M
FOIRY L
FONTAINE B
FONTAINE JM
FONTAINE-PERUS J
FONTAINE-PÉRUS J
FONTE C
FORMSTECHER E
FORNASARI B
FÖRSTER A
FORSTER V
FORT P
FORT PE
FORTIER M
FOUGEROUSSE F
FOUREST A
PW11-131
PW13-162
PW4-048
PW4-044, PW17-212, PW24-300
PW1-016
PW1-016
PW22-274
PW13-165
PW10-117
PW35-434
PW1-016
PW36-447
PW17-206, PW17-207, PW17-211
PW34-431
PW4-050
PW11-128, PW11-129, PW11-139, PW13-166, PW13-167, PW14177, PW14-181, PW15-188, PW16-199, PW16-200,
PW15-191
PW10-121
PW2-028
PW2-025, PW17-210
PW17-213
PW9-114, PW11-131
PW2-025
PW24-294
PW32-404
PW17-213
PW30-377
PW18-217
PW14-177, PW14-179, PW14-181, PW15-188, PW16-199,
PW17-206, PW18-214
PW2-018, PW2-019, PW4-047, PW5-056, PW8-098
PW30-377, PW35-444
PW26-324
PW26-330
PW16-199
PW14-170, PW33-419
PW30-384, PW38-483
PW19-240
PW29-367
PW15-190
PW23-289
PW2-025, PW17-210
PW28-356
PW13-168
PW4-046
PW1-013
PW13-156
PW13-166, PW14-174, PW14-177, PW14-177, PW16-200,
PW17-209
PW1-017
PW13-158
PW13-167
PW4-045, PW12-153, PW18-220, PW25-317, PW33-418,
PW33-421, PW33-423
PW9-108
PW8-097, PW9-111, PW15-192, PW19-228
PW19-239
PW35-444
PW9-107, PW19-236
PW32-402
PW17-210
PW7-082
PW9-101, PW22-272, PW22-275, PW28-355, PW30-382,
PW33-413, PW37-462
PW10-118
PW2-026, PW6-067
PW19-235
PW12-148
PW18-221, PW33-414, PW33-414, PW33-415, PW37-459
PW16-195, PW23-281
PW29-361
PW28-354
PW24-299
PW9-109
PW27-340
PW39-496
PW4-055
PW10-122
PW4-055
PW27-342
PW8-093, PW8-094, PW9-102, PW18-220, PW25-316, PW26-327
PW33-419
FOUREST-LIEUVIN A
FOURNIER E
FOUTRIER-MORELLO C
FOX D
FRANCANNET C
FRANCAUX M
FRANCIPANE L
FRANCO D
FRANÇOIS S
FRANDSEN U
FRANZIN C
FRANZOSO S
FRAYSSE B
FRECHTEL G
FRÉDÉRIC M
FRIANT S
FRICKER B
FRIEDRICH A
FRIESEN W
FRIGO M
FRITZ N
FROMES Y
FROMIGUÉ O
FUJII I
FUKAO T
FURLING D
PW14-170
PW16-200, PW18-214
PW14-173
PW1-008
PW15-191
PW25-310
PW1-017
PW29-364
PW28-348, PW30-378
PW29-363
PW28-352
PW24-294
PW9-101
PW1-017
PW38-471
PW38-474
PW7-088
PW39-492
PW2-027
PW30-385
PW3-041
PW9-101, PW22-272, PW26-324, PW33-413,
PW28-355
PW5-057
PW24-302
PW12-149, PW12-150, PW12-152, PW12-153, PW26-321,
PW31-388
GABELLINI D
GACHE V
GAILLY P
GALAN L
GALLAIS B
GALLIEN CL
GALLO C
GALMICHE G
GALY A
GANGLOFF YG
GANNON C
GANTELET E
GARCIA L
PW10-116
PW30-372
PW5-058
PW7-087
PW24-303
PW6-076
PW3-036
PW35-445
PW26-333
PW33-421
PW35-444
PW12-143
PW2-021, PW2-022, PW26-320, PW26-321, PW31-387,
PW33-423
PW8-090
PW13-158
PW22-278
PW28-354
PW19-237
PW11-129, PW24-303
PW39-492
PW35-444
PW17-209, PW17-211, PW39-489
PW12-150
PW24-294
PW3-029, PW3-032
PW18-214, PW18-215
PW1-012
PW27-342
PW7-078, PW7-080
PW29-367
PW3-042
PW8-096
PW6-069
PW9-106
PW23-290
PW33-423
PW30-380
PW10-117
PW25-312, PW25-316
PW21-266
PW12-146
PW1-001, PW4-051, PW4-052, PW11-139, PW21-253,
PW21-254, PW21-258, PW21-259, PW21-268, PW27-341,
PW30-374, PW30-375, PW33-418
PW39-488
PW9-102, PW32-402
PW16-194
PW9-109, PW24-295
PW12-149
PW35-443
PW1-017
PW39-496
PW31-391
PW29-371
PW33-414
GARCIA-BRAGADO F
GARCÍA-BRAGADO F
GARCIA-FILIPE S
GARDAHAUT MF
GARESSE R
GARGIULO M
GARNIER N
GARRATT AN
GARTIOUX C
GASNIER E
GASTALDELLO S
GATTI F
GAUDON K
GAUTHERON V
GAUTHIER ROUVIERE C
GAVRILA L
GAY S
GAYRAUD G
GEJMAN R
GENDRON MC
GENIN E
GÉNIN E
GENTIL C
GEORGER C
GERMINARIO E
GERVIER P
GESSAIN A
GHANEM D
GHERARDI R
GIACOMINI E
GIANNESINI B
GIBERT B
GICQUEL E
GIDE J
GILARDI-HEBENSTREIT P
GILIBERTO F
GILSON E
GIMONA M
GIORDANI J
GIRARD E
GIRARDOT F
GIRAUD-TRIBOULT K
GIROUX-METGÈS MA
GITIAUX C
GJATA B
GLARD Y
GOBIN-LIMBALLE S
GOEMANS N
GOICOECHEA M
GOMES E
GOMES-PEREIRA M
GONNET F
GONZALEZ DE AGUILAR JL
GONZALEZ K
GONZALEZ S
GONZÁLEZ-PÉREZ P
GORNI K
GOSSELIN C
GOTHOT A
GOTO J
GOUD B
GOUDEAU B
GOUDENEGE S
GOULET M
GOULVESTRE C
GOURDON G
GOUZI JY
GOYAL V
GOYENVALLE A
GRAHAM K
GRÉGOIRE S
GRENIER G
GRENIER J
GRID D
GRIFONE R
GRIGOLI L
GROSS DA
GRUMATI P
GUALANDI F
GUEGAN M
GUELLICH A
GUENEAU L
GUERCHET N
GUERRERO A
GUEVEL L
GUICHENEY P
GUIDY C
GUIGAND L
GUIGUET M
GUILBAUD M
GUILLAUD L
GUILLERY O
GUILLET-DENIAU I
GUILLEVIN L
GUIRAUD-DOGAN C
GULATI G
GUREVICH M
GUTIÉRREZ E
H KIM R
HAAPASALO H
HAASE G
HACKMAN P
HADDAD H
HAFTEK-TERREAU Z
HAJJAR R
HALBERT C
HALBOUT C
HALTER B
HAMADOUCHE T
HAMAÏ S
HAMMOND C
HAMMOUDA H
HAMRI A
HAMROUN D
HAN JJ
HANDSCHIN C
HANTAI D
HANTAÏ D
HARDY S
HASKINS M
HATEM S
PW36-452
PW12-149
PW33-413
PW21-253
PW30-380
PW23-286
PW24-302
PW24-301
PW8-090, PW8-092
PW30-372
PW12-148
PW10-125
PW7-088
PW30-373
PW8-096
PW19-233, PW19-234
PW1-007
PW27-338
PW35-439, PW35-441
PW3-038
PW19-238
PW16-197, PW16-198, PW16-200
PW28-351
PW1-014, PW27-338, PW27-339, PW30-381, PW33-417
PW21-265
PW12-143, PW12-148, PW12-153, PW36-448
PW38-468
PW21-256, PW21-261
PW26-320, PW26-321
PW3-029
PW21-267
PW27-346
PW24-299
PW23-283, PW23-287, PW23-290
PW29-371, PW35-437
PW19-236
PW30-380
PW17-210
PW2-025, PW17-210
PW33-416
PW22-273
PW13-161, PW13-164, PW38-472
PW9-102
PW7-087
PW4-046
PW13-155, PW14-177, PW14-179, PW14-180, PW14-181,
PW14-181, PW17-206, PW17-207, PW17-211, PW39-489
PW16-199
PW38-477
PW21-263
PW38-477
PW34-427
PW19-235, PW19-238
PW30-384, PW38-483
PW21-258, PW21-265
PW36-454
PW1-010
PW7-085
PW13-168
PW38-481
PW11-141, PW13-169
PW6-071, PW23-283, PW34-428
PW16-203, PW16-204
PW26-325
PW38-481
PW35-434
PW9-108
PW26-330
PW7-088
PW7-079, PW23-280, PW23-283, PW23-284, PW23-287,
PW23-290,
PW6-069
PW37-464
PW8-097, PW13-167
PW7-077, PW11-137, PW20-242
PW36-453, PW36-454, PW36-455, PW36-456
PW36-450
PW6-066
PW18-215, PW33-421
PW18-214, PW18-216, PW18-217, PW25-309
PW4-049
PW38-477
PW35-434
HAUW JJ
HEBERT L
HÉBERT L
HEEMSKERK H
HENAFF M
HENLEY S
HENNEBOEHLE M
HENRICSON EK
HERIJGERS P
HERLICOVIEZ D
HÉRON D
HERRMANN C
HERSON A
HERSON S
HERZNER H
HESTAND MS
HEUVELMANS N
HEUZÉ ML
HEWSON D
HEZODE M
HIBA B
HIBAOUI Y
HIGH K
HILLOCK S
HIMBER J
HINARD V
HINDERLICH S
HIRASAKA K
HIRN C
HNIA K
HOCQUETTE JF
HODGKINSON I
HOFF H
HOFFMAN E
HOFFMEYER P
HOGREL JY
HOLCIK M
HOLLO S
HOLMBERG M
HOMS J
HOPPE A
HORVÁTH G
HOURDE C
HOURDÉ C
HOVERS V
HOWELL JMC
HUBERT JF
HUCHET-CADIOU C
HUERRE M
HUGON G
HUGUET A
HUH M
HUMBERTCLAUDE V
HUOVINEN S
HUYNH M
HWU WL
PW19-231
PW11-138
PW11-134
PW2-024, PW26-322
PW3-041, PW25-318
PW3-030
PW4-044
PW36-450
PW24-300
PW17-209
PW11-129
PW38-487
PW24-303
PW21-263, PW21-267, PW24-303, PW26-325
PW4-044
PW29-362
PW2-024
PW31-394
PW32-405
PW18-215
PW11-134, PW36-448
PW25-308
PW38-477, PW38-477
PW6-068
PW32-402
PW29-360
PW16-201
PW3-038
PW3-035
PW3-042
PW5-061
PW36-451
PW3-030, PW3-032
PW13-160
PW27-334
PW8-094, PW11-130, PW11-133, PW20-243, PW26-325,
PW32-405, PW32-406, PW32-407, PW32-408, PW33-413,
PW33-422, PW36-449
PW38-479
PW16-204
PW20-251
PW39-493
PW16-195, PW23-281
PW21-257
PW31-387
PW4-045, PW18-220, PW25-317
PW17-211
PW20-248
PW1-003, PW4-049
PW25-312, PW25-316
PW21-266
PW3-042, PW24-297
PW12-148, PW12-153
PW27-346
PW1-012, PW36-455, PW36-456
PW13-169
PW22-278
PW20-249
IACCHETTI E
ILIC S
ILKOVSKI B
INAMO J
INDAKOETXEA B
INGHAM PW
IODICE P
ISRAELI D
IVERSEN PL
IZMIRYAN A
IZQUIERDO M
PW2-019
PW25-314
PW15-185
PW7-083
PW13-158
PW38-481
PW32-403
PW7-085, PW30-380
PW2-023
PW16-196
PW32-402
JABBOUR R
JACOBS MS
JACOPIN S
JACQUEMIER M
JACQUEMIN V
JACQUEMOND V
JACQUEMONT S
JACQUES D
JACQUETTE A
JACQUIER A
JAGANNATHAN N
JAGERSCHMIDT C
JAGLA K
PW23-283
PW25-307
PW23-286
PW23-286
PW27-337
PW9-105, PW14-175
PW8-090
PW26-329
PW11-129
PW6-071, PW23-283
PW21-256
PW25-317
PW15-191, PW38-478
JAGLA T
JAILLARD D
JAMET T
JANIER M
JAOUEN F
JARA A
JARDEL C
JARRY T
JASMIN B
JEANNET PY
JENISKENS G
JENNISKENS G
JENNY C
JENSEN CH
JIMENEZ-MORENO R
JOERGENSEN LH
JONES A
JORY A
JOUK PS
JOURNOT L
JOUSSEMET B
JOUSSET H
JOUVE JL
JOUVION G
JUNGHANS R
JURIE C
PW38-478
PW3-039
PW26-327
PW11-134, PW11-134, PW11-138, PW36-448
PW38-473
PW24-300
PW19-230, PW19-231, PW19-232, PW19-239
PW35-437
PW3-037, PW6-068, PW12-144, PW38-479
PW8-090, PW15-189, PW17-211, PW17-211
PW22-278
PW31-392
PW27-345
PW29-363, PW31-386
PW33-420
PW31-386
PW29-368
PW39-494
PW1-002, PW15-182
PW24-298
PW39-490
PW30-376
PW23-286
PW27-340
PW12-147
PW29-370
KABLY B
KALRA K
KANDALLA P
KANEVA G
KANG PB
KAPLAN JC
KARLIN D
KASSAR-DUCHOSSOY L
KASSEM M
KASSOURI N
KAVIAN N
KAZAKOV V
KELLER A
KELLINSALMI M
KELLY R
KERE J
KERLERO DE ROSBO N
KESARI A
KESSLER JL
KHATTAR P
KHAU VAN KIEN P
KHELFAOUI M
KHOOBARRY K
KHOUZAMI L
KIM JJ
KISELEV AV
KISHNANI P
KLATZMANN D
KLEIN AF
KLEINO I
KLEMEN A
KNOPFEL T
KOCH M
KOECHLIN C
KOEING M
KOENIG H
KOENIG J
PW37-460
PW1-010
PW27-337
PW38-474
PW4-048
PW2-021, PW9-103, PW36-453, PW36-455
PW37-464
PW12-149
PW29-363
PW23-283, PW23-290
PW9-103
PW10-123
PW31-390, PW37-462
PW15-184
PW22-277, PW35-437
PW16-204
PW18-224, PW18-225
PW13-160
PW34-427
PW22-275
PW1-002, PW1-002, PW36-455
PW33-416
PW6-070
PW13-162
PW24-302
PW7-084
PW20-249, PW20-252
PW21-267
PW10-126
PW31-391
PW7-081
PW38-485
PW34-432
PW3-042, PW24-297
PW11-137
PW25-309
PW18-214, PW18-216, PW18-217, PW18-217, PW25-309,
PW25-309
PW4-048
PW5-060
PW22-271
PW10-123
PW7-084
PW27-334, PW29-359
PW30-376
PW22-271
PW9-104
PW6-073
PW25-310, PW33-421
PW14-173, PW15-192
PW33-421
PW12-143
PW11-141
PW8-090, PW8-091, PW8-096, PW8-097, PW8-098, PW8-099,
PW9-108, PW9-111, PW 26-326
PW7-081
PW22-271, PW22-271, PW22-279
KOHANE IS
KOLLIAS H
KOLOKATHIS F
KOLYNIN V
KOMANTZEV V
KONIG S
KÖNIG S
KONTROGIANNI-KONSTANTOPOULOS A
KÖSTER D
KOTHARY R
KOULMANN N
KOZAK G
KOZMA S
KRAFTSIK R
KRAHE R
KRAHN M
KRAJNIK J
KRANIAS EG
KRATASSIOUK G
KREJCI E
KREMASTINOS DTh
KRESS W
KRETZ C
KROL A
KUCHARCZAK J
KUNKEL LM
KÜNNECKE B
KUNTZER T
KURZ J
KURZENNE JY
PW10-124
PW33-414, PW33-415
PW22-271
PW11-141
PW14-175, PW14-178, PW26-327
PW39-489
PW27-342
PW4-048
PW32-402
PW12-143, PW18-222
PW3-030
PW28-350
LABARRE VILA A
LABARRE-VILA A
LABELLE V
LACAMPAGNE A
LACASSAGNE MN
LACÈNE E
LACHEY J
LAFORET P
PW20-247
PW14-172, PW15-182
PW8-097, PW8-099, PW 26-326
PW3-042
PW28-355
PW13-161, PW14-177, PW19-240
PW25-311
PW8-091, PW14-177, PW11-128, PW13-167, PW15-188,
PW16-200, PW18-215, PW19-231, PW19-232, PW20-243,
PW20-245, PW24-303, PW26-325
PW25-312, PW25-316
PW5-059
PW31-393
PW30-384
PW15-183
PW1-004, PW1-006
PW36-456
PW20-243
PW14-172
PW9-104
PW28-356
PW18-219
PW26-325
PW5-064
PW22-274
PW23-281
PW37-464
PW36-447
PW10-115, PW10-118
PW5-057
PW14-175, PW14-175, PW14-178, PW38-474
PW25-319
PW28-355
PW3-039, PW33-416
PW18-221
PW15-187, PW34-433
PW18-215, PW21-260
PW23-282
PW13-157
PW27-334
PW23-286
PW16-198
PW9-112
PW8-093
PW12-145
PW12-149
PW3-032
PW38-476
PW17-206, PW20-250
PW31-388
PW4-049
PW19-241
PW24-298
PW35-434
PW32-401, PW32-402
PW29-368
PW38-481
PW35-435, PW35-436
PW13-163, PW23-285
PW38-477, PW38-484
PW18-223
PW35-434
PW26-330
PW9-106
PW1-003, PW4-049
PW20-244, PW21-255
PW22-273
PW16-193
PW34-429
PW6-076
PW38-485
LAFOUX A
LAFRENIÈRE JF
LAGIRAND-CANTALOUBE J
LAHOUTE C
LAING NG
LAKSHMI R
LALANDE M
LALOUI K
LAMARIA M
LAMAZE C
LAMB N
LAMBERGEON M
LAMBERT I
LAMICHE C
LAMOTTE L
LANDRY J
LANGLET C
LANGS G
LAOUDJ-CHENIVESSE D
LAPOINTE E
LAPORTE J
LAPRÉVOTE O
LARGHÉRO J
LAROCHE S
LARROQUE MM
LARSSON L
LARUE S
LATOUR P
LATTANZI G
LAUMONIER T
LAUNAY F
LAUNAY N
LAURE L
LAURENT E
LAURENT FX
LAUSTRIAT D
LAVIN J
LAYEC G
LAZARO L
LE BIHAN MC
LE BIHAN O
LE BORGNE F
LE DIGARCHER A
LE FEUVRE C
LE FUR Y
LE GRAND F
LE GUELLEC D
LE GUEN L
LE GUERN E
LE GUINER C
LE PANSE R
LE PRINCE P
LE PROVOST G
LE RAVALEC V
LE RUMEUR E
LEBLANC A
LECARPENTIER Y
LECHAIRE JP
LECOLLE S
LÉCOLLE S
LECOQ J
LECOURT S
LECUNFF M
LECUYER C
LEDEUIL C
LEDEVIN M
LEE S
LEFAUCHEUR JP
LEFEBVRE S
LEFUR Y
LEGARDINIER S
LEGAY C
LEGER J
LEGRAND B
LEGRAND C
LEHOUX S
LEHTOKARI VL
LEHTONEN E
LEIBOVITCH MP
LEIBOVITCH S
LÉJARD V
LEJEUNE F
LEMERCIER C
LEMOINE F
LEONARDIS L
LEROUX-GOUBAULT I
LEROY M
LESAULT PF
LESCA G
LESCOP C
LESCURE A
LESLIE N
LESSARD J
LETURCQ F
LEVIVIER M
LEVY N
L'HONORÉ A
LI K
LI Z
LIABEUF M
LIDOV HG
LIDOVE O
LILIENBAUM A
LIND L
LIPECKA J
LIPSKAIA L
LISSANDRON V
LISTE H
LITIM K
LIU H
LIU JM
LLENSE S
LLORENS A
LOBRINUS JA
LOCHMÜLLER H
LOEFFLER JP
LOFASO F
LOISEL L
LOMBÈS A
LOMBES A.
LOMPRE AM
LOPA R
LOPES P
LÓPEZ DE MUNAIN A
LOPEZ-ALEMANY R
LORY P
LOSTAL W
LOUBAT A
LOUIS M
LOUIS-TISSERAND M
LOUREIRO DE SOUSA P
LOVATO L
LUCIANI A
LUNARDI J
LUNDE J
LUNKKA-HYTÖNEN M
LUQUE H
LUSTREMANT C
PW28-355
PW10-121
PW12-151
PW17-209, PW17-211
PW27-340
PW2-027
PW11-130
PW6-069, PW6-070, PW6-072
PW38-476
PW1-003, PW4-049
PW18-219
PW10-121, PW18-219
PW1-003
PW4-043, PW9-105
PW35-434
PW15-184
PW38-486
PW31-393
PW31-393
PW35-443
PW2-028, PW39-491
PW12-152
PW26-325
PW37-458
PW27-340
PW29-360
PW27-341
PW38-471
PW4-044
PW39-489
PW20-249
PW22-274
PW1-001, PW1-002, PW2-021, PW8-099, PW8-091, PW9-103,
PW13-164, PW17-209, PW 26-326, PW26-325, PW36-455,
PW38-472
PW38-486
PW8-096, PW8-098, PW8-099, PW9-111, PW23-280, PW23-284
PW8-090, PW8-091, PW8-097, PW23-282, PW23-283,
PW23-290,
PW 26- 326
PW29-369
PW32-405
PW5-060, PW15-190, PW16-196, PW22-274, PW35-445
PW26-325
PW4-048
PW21-260
PW16-198
PW20-251
PW18-217
PW35-434
PW18-227
PW19-229, PW37-463
PW11-132
PW6-073
PW22-272
PW9-103, PW38-472
PW3-040, PW38-469
PW8-090, PW15-189
PW36-456
PW7-088
PW20-245
PW9-110
PW19-231, PW19-232, PW19-235, PW19-238, PW19-239,
PW19-240
PW19-230
PW35-434, PW35-434
PW2-018
PW6-076
PW8-090, PW8-092, PW8-094, PW13-167
PW3-040, PW38-469
PW23-291
PW 26-326, PW26-332
PW10-119
PW5-058
PW35-440
PW32-411
PW19-236
PW32-400
PW14-170, PW14-171, PW14-172, PW14-173, PW15-182,
PW15-192, PW20-247, PW33-419
PW6-068
PW15-184
PW16-204
PW2-020
LUTZ PG
LYNN M
LYONS G
PW31-394
PW4-050
PW29-364
MACARTHUR DG
MACIOTTA S
MACREZ N
MADELEINE M
MAGAUD C
MAGDINIER F
MAGGIONI M.
MAGUREANU S
MAGY L
MAGYAR J
MAHEMUTI L
MAIER A
MAIOLI M
MAIRE P
MAISON P
MAISONOBE T
PW16-201
PW4-047, PW5-056
PW3-041, PW25-318
PW17-213
PW5-064
PW39-496
PW30-378
PW7-078, PW7-080
PW23-287
PW4-044, PW4-044
PW10-115
PW31-398
PW17-210
PW29-371, PW38-483
PW21-259
PW13-163, PW16-199, PW16-200, PW16-202, PW19-231, PW21260, PW23-285
PW30-373
PW5-057
PW24-299
PW7-079, PW13-155, PW15-183, PW17-211
PW38-481
PW26-330
PW28-352, PW30-385
PW13-161
PW3-029
PW7-084
PW32-400
PW28-356
PW26-321, PW27-347
PW20-249
PW14-175, PW14-178, PW26-327
PW16-199, PW16-199
PW14-177
PW38-478
PW39-490
PW33-414
PW1-017
PW9-109
PW2-018
PW17-207, PW17-207, PW20-250, PW21-255
PW10-115
PW12-145
PW28-355
PW6-075
PW19-236
PW8-100
PW35-443
PW28-355
PW17-210
PW30-385
PW22-278, PW25-307, PW31-390, PW31-392
PW1-011, PW11-135
PW12-151
PW35-444
PW16-195, PW18-227, PW23-281
PW12-149, PW12-149, PW12-151
PW19-229, PW37-463
PW29-364
PW4-053, PW33-416, PW37-461
PW29-366
PW13-158
PW23-282
PW37-457
PW2-025, PW17-210
PW11-140
PW15-186
PW14-170, PW14-172, PW15-182, PW33-419
PW34-427
PW26-325
PW24-299
PW38-482
PW39-495
PW3-042
PW37-464
PW7-087
PW7-081
PW 26-326
PW14-176
PW13-155, PW14-177, PW17-206, PW17-207, PW39-489
MAKARENKOVA H
MAKOTO M
MAKOUKJI J
MAKRI S
MALBOUYRES M
MALEAU G
MALERBA A
MALISSEN M
MALNASSY G
MALYSHEVA OV
MALZY P
MAMAEVA D
MAMCHAOUI K
MANDEL H
MANDEL JL
MANERE L
MANÉRÉ L
MAQBOOL T
MARAIS T
MARCEL C
MARCESE D
MARCHAND S
MARCHESI C
MARCORELLES P
MARCOWYCZ A
MARIE J
MARIE P
MARIN P
MARINI M
MARLOW G
MARO GS
MAROLLEAU JP
MARROSU G
MARTELLI L
MARTELLY I
MARTENS W
MARTEYN A
MARTIN E
MARTIN J
MARTINAT C
MARTINEZ PEREA MDC
MARTINEZ S
MARTÍNEZ-ROJAS D
MARTÍNEZ-TORRES A
MARTÍN-GARCÍA A
MARTINI N
MARTIN-MOUTOT N
MARTONI E
MARTORELL L
MARTTILA M
MARTY I
MARY J
MASQUELIER AM
MASSAAD C
MASSENET S
MASSOULIE J
MATECKI S
MATHIEU M
MATIAS-GUIU J
MATJACIC Z
MATTEI MG
MATTIOLI G
MAUGENRE S
MAURAGE CA
MAURER M
MAXIM D
MAYENÇON M
MAYER M
MAZELIN L
MAZUE F
MAZZEO A
MCDONALD CM
MCLACHLAN I
MCNEIL P
MÉCHIN D
MECHREF Y
MEDINA I
MEDJA F
MEECH R
MÉGARBANÉ A
MEGENEY L
MEIER T
MEIL A
MEINEN S
MEJAT A
MENARD D
MÉNARD J
MENEGUZZI A
MENETREY J
MENEVERI R.
MERCIER J
MERCIER S
MERCURI E
MERDES A
MEREGALLI M
MERICSKAY M
MERLINI L
MERTEN OW
MERTENS B
MERTENS L
MESSADDEQ N
MESSI LM
MESSINA S
MÉTAIS A
METZ G
METZGER D
MEUNE C
MEUNIER B
MEZGHRANI A
MEZIN P
MICALLEF JP
MICHEL-CALEMARD L
MICHIEL M
MICHON CC
MIGLIORATO A
MIGNARD C
MIGNARD D
MIHOVIL I
MILANI P
MILES M
MILLER LL
MILLMAN G
MILLOT L
MILLS P
MILMAN I
MIMAULT B
MINCIU I
MINETTI C
MINUZ P
MIQUEROL L
MIRANDA A
MIRANDA R
MIRONNEAU C
MIRONNEAU J
MISEREY-LENKEI S
MISTELI T
MITRANI-ROSENBAUM S
MITRPANT C
MIURA P
MIYAKE K
MOINE H
MOLANO J
MOLGO J
MOLINARO M
MONDIN L
MONGUE-DIN H
MONNET A
PW14-181
PW34-427
PW1-005
PW23-282
PW1-001
PW33-421
PW19-241
PW25-315
PW36-450
PW26-323
PW 26-326, PW26-332
PW1-002
PW19-236
PW6-071
PW19-232
PW30-373
PW17-206, PW23-282, PW23-283, PW23-283
PW4-046
PW4-044, PW17-212, PW24-300, PW24-301
PW9-109
PW17-212
PW13-160
PW20-250
PW39-488
PW19-236
PW27-334
PW30-378
PW10-118, PW10-122
PW2-021
PW17-210
PW13-165
PW2-018, PW2-019, PW4-047, PW5-056, PW8-098
PW15-190, PW22-274, PW35-445
PW17-206, PW17-206, PW17-210, PW17-211, PW20-252
PW27-345
PW38-486
PW24-300, PW24-301
PW14-175
PW33-420
PW17-210, PW25-315
PW31-394
PW4-044
PW30-384
PW11-128, PW13-162
PW5-061, PW29-370
PW23-291
PW15-182, PW20-247
PW38-476
PW36-455
PW16-193
PW24-303
PW25-315
PW8-094, PW24-304
PW24-304
PW25-314
PW2-019
PW3-030, PW3-031
PW36-450
PW3-029, PW3-034
PW26-325
PW5-059
PW16-205
PW29-361
PW7-080
PW1-009, PW1-015, PW14-176
PW19-236
PW22-277
PW1-016
PW3-039
PW3-041
PW3-041, PW25-318
PW19-238
PW13-160
PW16-201, PW16-205, PW17-208
PW2-026, PW6-067
PW38-479
PW 26-326, PW26-332
PW38-482
PW13-158
PW25-309, PW33-414
PW31-395, PW31-396
PW5-063
PW22-272
PW20-245, PW32-412, PW34-426
MONNIER N
MOUMEN A
MOUSAVI K
MOUTEL S
MOUTHON L
MOXLEY R
MUELLER M
MUKHTAROV M
MUÑIZ GARCIA N
MUNNICH A
MUNTONI F
MURFUNI I
MUSA H
MUSARELLA M
MUSSET L
PW14-170, PW14-171, PW14-172, PW14-173, PW14-179,
PW15-182, PW15-192, PW20-247
PW25-305
PW6-069
PW30-383
PW23-291
PW35-442
PW26-327
PW2-020
PW11-131
PW31-394
PW2-027
PW24-298
PW29-365
PW13-156
PW3-041, PW25-318
PW31-395
PW15-190
PW22-278, PW25-307
PW38-473
PW4-053, PW4-055, PW24-297, PW3-042, PW33-416, PW37-461
PW1-007
PW4-045, PW18-220, PW31-387
PW39-492
PW38-477, PW38-484, PW39-490
PW2-023
PW10-125, PW12-150, PW21-266, PW26-321, PW27-335,
PW27-336, PW27-337, PW27-347, PW28-349, PW31-388
PW6-074
PW3-037
PW38-470
PW21-258
PW1-008, PW1-011, PW11-135
PW33-421, PW33-421
PW23-292
PW1-017
PW12-148
PW14-174
PW31-396
PW31-390
PW33-424
PW21-263, PW21-267
NACIM L
NADARAJAN T
NAGI S
NAKAE Y
NANCY P
NARASSIMPRAKASH H
NARGEOT J
NASSOY P
NAVARRO C
NAVARRO F
NAVAS LLORET P
NAZZAL D
NEGRONI E
NEJJARI M
NELSON I
NERI C
NERI M
NEUNLIST M
NEVO Y
NGUYEN K
NICOLE A
NICOLE S
NICOLINO M
NICORICI AR
NICOT AS
NIGRO V
NIKAWA T
NIRO C
NISHI M
NIVET AL
NOGUIEZ-HELLIN P
NOIREZ P
NORTH K
NOTARNICOLA C
NOTHIAS F
NOUET C
NOUIOUA S
NOULET F
NOUREDDINE H
NOVELLI G
NOVOTNY M
PW17-213
PW2-027
PW9-101
PW3-038
PW18-224
PW22-278
PW23-291, PW24-298
PW9-104
PW8-098, PW13-168
PW29-364
PW19-233, PW19-234
PW18-225
PW27-335, PW27-336, PW28-349
PW11-134, PW36-448
PW19-232, PW19-239, PW19-240
PW24-293
PW2-025
PW15-190
PW17-208
PW8-097, PW 26-326
PW12-148
PW33-414, PW33-415, PW37-459
PW20-249
PW36-450
PW14-178
PW9-113,
PW3-038
PW29-371
PW33-420
PW2-020
PW26-328, PW26-329, PW26-330, PW26-331
PW37-462
PW14-171, PW15-185, PW16-201
PW35-435, PW35-436
PW23-289
PW38-480
PW23-284, PW23-287, PW23-290
PW9-109
PW39-495
PW14-176
PW19-236
MONOPOLI A
MONTAGNE JJ
MONTARRAS D
MONTEIL A
MONTI G
MONTUS M
MONVILLE C
MONZANI F
MOOG-LUTZ C
MOON YC
MOORE-MORRIS T
MORAIS J
MORANDI L
MOREL JL
MORESI V
MORIEZ R
MORIN C
MORIN X
MORNET D
MOTTA C
MOUISEL E
MOULINIER L
MOULLIER P
MOULTON HM
MOULY V
NUDEL U
NURY D
NUSBAUM P
NUUTINEN E
PW4-054, PW4-055, PW33-416
PW22-276
PW2-021
PW15-186
OCHALA J
OCHOA-MORALES H
ODDOUX S
OERTEL J
OFFNER N
OLDFORS A
OLENŠEK O
OLIVÉ M
OLIVERO P
OLLILA S
OLLIVIER G
OLPIN S
OLSSON A
ONGINI E
ONO Y
ORCESI S
ORLIKOWSKI D
OTAEGUI D
OTELEA D
OTTAVIANI A
OTTONELLO G
OUALI A
OUAZZANI R
OUBAICHE M
OUDGHIR M
OUDRER N
OZDEN S
PW15-187, PW34-433
PW19-237
PW14-170, PW33-419
PW37-459
PW31-393
PW14-177, PW14-178, PW14-181
PW7-081
PW13-158
PW22-273
PW15-186
PW20-243, PW32-406, PW32-408, PW36-449
PW24-302
PW20-251
PW25-305
PW27-343
PW1-007
PW17-207, PW18-214, PW20-245
PW8-092
PW1-005
PW39-496
PW1-009, PW14-176
PW9-112
PW13-163, PW13-163, PW23-285, PW37-460
PW11-132
PW31-392
PW11-132
PW21-266
P. TREMBLAY J
PADOVANI A
PAGNI S
PAGNON-MINOT A
PAINE A
PÁL E
PALLAFACCHINA G
PALMIERI B
PANAITE PA
PANDYA S
PANTHIER JJ
PAPALOUKA V
PAPO T
PAPPONE C
PAPY-GARCIA D
PAQUETTE B
PAQUIS V
PARADIS M
PARAGIOS N
PARASKEVAIDIS IA
PARISET C
PARKER A
PARKS R
PAROLINI D
PARTRIDGE T
PASCO M
PASCUAL J
PASDELOUP M
PASSAMANO L
PASUT A
PATO C
PATTHEY-VUADENS O
PATURNEAU-JOUAS M
PAULIN D
PAUTREL I
PAYAN C
PEARSALL RS
PÉCHEUX C
PECKER F
PECKHAM M
PEDEMONTE M
PEDRAZA L
PEGORARO E
PELÉ M
PELIN K
PELLEGRINI N
PELLISSIER JF
PELTZER J
PENA-SEGURA JL
PENAUD-BUDLOO M
PENDE M
PW5-057
PW9-107
PW23-286
PW38-481
PW33-420
PW21-257
PW30-383
PW1-013, PW3-032
PW12-143
PW1-008, PW1-011, PW11-135
PW34-427
PW22-279
PW21-260
PW13-156
PW22-278, PW25-307, PW31-392
PW6-065
PW19-228
PW1-014, PW27-338, PW27-339, PW30-381, PW33-417
PW32-400, PW36-447
PW22-271
PW6-076
PW24-293
PW27-346
PW2-018, PW4-047, PW5-056, PW8-098
PW27-344, PW31-397
PW24-293
PW11-140
PW25-310
PW9-113
PW30-385
PW18-218
PW39-497
PW18-214
PW16-196
PW26-329
PW8-094, PW20-243, PW24-303, PW36-452
PW25-311
PW8-090, PW8-091, PW23-282
PW13-162, PW13-162
PW31-390
PW1-009, PW1-015, PW14-176
PW8-096
PW13-156
PW34-427
PW15-184, PW15-186
PW18-214, PW20-245
PW9-111, PW11-139, PW15-192
PW31-390
PW8-090
PW38-477
PW33-421
PENISSON-BESNIER I
PEREZ F
PERIE S
PERIER M
PERNIGOTTI I
PERON S
PERREY S
PERRIN L
PESCHANSKI M
PESSINA P
PETERLIN B
PETERMANN O
PETERSEN Y
PETERSSON SJ
PETIOT P
PETIT F
PETRI S
PETTMANN B
PEUDENIER S
PFUND Z
PHILIPS A
PIAZZON N
PICARD B
PICART C
PICKETTS D
PICKLE L
PICOZZI P
PIETRANGELO T
PIÉTRI-ROUXEL F
PIETROBELLI A
PIETTE JC
PIETU G
PIGNOL B
PIGOZZO S
PILUSO G
PISANI D.F
PLANCHERON A
PLATENBURG G
PLONQUET A
POCH O
PODEVIN G
POITELON Y
POLISIAKEIWICZ L
POLITANO L
POLLET-VILLARD X
POMERO V
POMIÈS P
PONZIO G
POPA N
PORON F
PORRELLO E
PORRETTI L
PORTE F
POSADAS MARTINEZ MM
POTIER D
POU A
POUGET J
POÜS C
POUSSARD S
POZA J
POZZOBON M
PRAS E
PRASANTH KV
PRAUD C
PRAZNIKAR A
PRAžNIKAR A
PREVITALI S
PRIGENT H
PRISTERÀ A
PROST J
PRUDHON B
PUCEAT M
PUCÉAT M
PUCELLE M
PUGLIELLI C
PULLEN A
PUYMIRAT J
PW8-091
PW38-470
PW10-124, PW10-125
PW13-162
PW1-015
PW10-117
PW38-476
PW38-487
PW2-020, PW12-149, PW12-151
PW28-348, PW30-378
PW13-159
PW3-036
PW18-226
PW29-363, PW31-386
PW18-214
PW18-214
PW7-088
PW6-074, PW6-075
PW17-207
PW21-257
PW10-122
PW38-482
PW5-061, PW29-370, PW31-393
PW35-440
PW27-346
PW30-373
PW10-116
PW32-404
PW33-423
PW1-013
PW21-263
PW12-149, PW12-151
PW25-309
PW26-323
PW9-113
PW28-350, PW28-351
PW2-020
PW2-024
PW21-254, PW21-268
PW39-492
PW38-477
PW23-280, PW23-283
PW3-034
PW9-113
PW35-442
PW23-286
PW2-028, PW29-358, PW38-467
PW10-119
PW35-442
PW4-051
PW28-353
PW2-018, PW2-019
PW7-085
PW19-229
PW38-487
PW11-140
PW7-086, PW8-097, PW9-111, PW19-228, PW21-262, PW23-282
PW38-470
PW31-389
PW8-094, PW13-158
PW28-352
PW17-208
PW38-466
PW28-355
PW32-410
PW7-081
PW13-156, PW17-210, PW28-353, PW28-353
PW20-245
PW31-395
PW19-241
PW14-177, PW14-180, PW14-181
PW35-438
PW22-276, PW35-440
PW19-235
PW32-404
PW25-311
PW11-134, PW11-138, PW12-154, PW36-448
QUATTRINI A
QUESADA JF
QUESNOIT M
QUIJANO-ROY S
QUINN C
PW28-353
PW13-158
PW38-470
PW17-206, PW17-207, PW17-211
PW11-135
RABEN N
RACHMIEL M
RADIC B
RADVANYI H
RADVANYI-HOFFMAN H
RAFAI MA
RAGE F
RAGUÉNÈS-NICOL C
RAHEEM O
RAHMOUNI A
RAJA B
RAMACHANDRAN U
RAMANOUDJAME C
RAOUL C
RASSAM P
RATEL S
RATHORE C
RAUSCENT H
RAUTENSTRAUSS B
RAVEL-CHAPUIS A
RAYMOND G
RAZINI P
REBOUD-RAVAUX M
RECOUVREUR M
REDERSTORFF M
REECY J
REGGIANI C
REGRAGUI W
REHA AL
REIDERER I
RELAIX F
REMEC JF
RENDON A
RENE F
RENÉ F
RENOU L
RENVOISÉ B
REPELE A
RESNIK-WOLF H
REUTENAUER J
RHODES J
RIBAS T
RICHARD A
RICHARD E
RICHARD H
RICHARD I
RICHARD N
RICHARD P
RICHARD P-L
RICHARD-BULTEAU H
RICHELME C
RIEDERER I
RIGAL O
RIGAULT C
RIGOLET A
RIMESSI P
RISSON V
RIVAL-GERVIER S
RIVAS-INFANTE E
RIVET J
RIVIER F
RIVIÈRE C
RIVIERE JP
ROBERT I
ROBIN V
ROBITAILLE Y
ROCERRI M
ROCHAT A
ROCHCONGAR P
ROCHER C
ROCHON-BEAUCOURT C
RODER IV
RODRÍGUEZ E
RODRIGUEZ M
RODRIGUEZ-MUÑOZ R
ROELENS F
ROELENS P
ROGOWSKA-WRZESINSKA A
ROJO M
ROLLING F
ROMERO N
PW20-246
PW17-208
PW25-314
PW11-129
PW11-139
PW19-230
PW38-482
PW1-003, PW4-049
PW11-141, PW13-169
PW32-400, PW36-447
PW27-344
PW8-100
PW34-430
PW6-074, PW6-075
PW38-467
PW32-401
PW21-256
PW1-012
PW23-287
PW3-037, PW12-144
PW5-063
PW2-019, PW5-056, PW8-098
PW9-106, PW24-294
PW18-218
PW39-489
PW5-061
PW26-323, PW32-404
PW37-460
PW36-450
PW27-337
PW29-365, PW31-387,
PW11-134
PW4-054, PW4-055
PW7-088
PW33-414
PW13-159, PW13-160, PW13-161
PW6-069, PW6-070, PW6-072
PW26-323
PW17-208
PW25-313, PW39-497
PW3-030
PW13-168
PW38-483
PW20-246
PW33-421
PW8-094, PW8-094, PW8-095, PW8-098, PW8-098, PW9-102,
PW9-109, PW16-203, PW24-295, PW 26-326, PW26-332,
PW39-492,
PW11-134
PW13-155, PW13-159, PW13-166, PW13-167, PW15-189,
PW16-199, PW16-200, PW17-209, PW17-211, PW18-214,
PW27-338
PW25-310
PW1-012
PW27-335, PW27-336, PW28-349
PW18-215
PW19-241
PW21-263, PW26-325
PW2-025
PW33-421
PW39-496
PW19-233, PW19-234
PW5-064
PW1-002, PW3-042
PW39-490
PW21-266
PW28-355
PW7-085
PW9-110
PW33-421
PW29-365
PW20-250
PW4-049
PW12-149
PW18-226, PW18-227
PW39-493
PW1-016
PW4-053, PW37-461
PW17-211
PW24-304
PW31-388
PW19-235, PW19-238
PW38-477, PW38-484
PW8-090, PW8-090, PW8-091, PW14-177, PW14-179,
ROMERO-DIAZ VJ
ROMUALDI C
RONDIER D
RONJAT M
ROOSTALU U
ROQUE M
ROQUES C
ROQUES S
ROSAS-SÁNCHEZ F
ROSENTHAL N
ROSIER-MONTUS M
ROSSERT J
ROSSI B
ROSSI CA
ROSSI M
ROSSIER J
ROUAUD T
ROUCHE A
ROUDAUT C
ROUDOT-THORAVAL F
ROUGER K
ROULEAU GA
ROULET E
ROUMES H
ROUSSEAU J
ROUX MJ
ROUX-LOMBARD P
ROZENVALD Y
RUDENKO D
RUDNICKI M
RUDOLF R
RÜEGG M
RUEGG U
RUGGIERO F
RUGGIERO M
RUHLMANN N
RUTTER J
SABATELLI P
SABLONNIERE B
SABOURIN J
SACCO F
SACCONE V
SACCONI S
SACHOT S
SAENZ A
SÁENZ A
SAGGINI A
SAGGINI R
SAHEL JA
SAKANYAN V
SAKSELA K
SAKTHIVEL M
SALAMA I
SALEKI K
SALIM C
SALMON A
SALOMON B
SALORT-CAMPANA E
SALVIATI L
SALVO E
SAMADI A
SAMPAOLESI M
SAN MILLÁN B
SANCHEZ H
SÁNCHEZ-ALCÁZAR JA
SANDER B
SANDONÀ D
SANNA M
SANOUDOU D
SANTOS G
SARDOC M
SARKAR C
SARKAR M
SARPARANTA J
SARRAZIN E
SARRE S
SASAKI T
SASSOON D
SAULNIER J
SAVINO W
SBLENDORIO V
PW14-181, PW17-206, PW17-207
PW19-237
PW30-385, PW30-385
PW39-496
PW9-105
PW34-431
PW1-017
PW26-324
PW32-406
PW29-366
PW22-274
PW26-325
PW35-443
PW10-119, PW10-119, PW12-142
PW28-352
PW1-007
PW18-217
PW28-354
PW18-214, PW25-309
PW9-112, PW 26-326, PW26-332
PW32-400
PW4-046, PW27-340, PW28-354
PW10-126
PW3-035, PW3-036, PW25-308
PW30-379
PW5-062
PW4-054
PW18-222
PW33-416
PW10-123
PW27-346, PW29-368
PW18-226, PW18-227
PW17-212, PW28-353
PW3-036, PW25-306, PW25-308, PW25-313, PW39-497
PW34-432, PW38-481
PW37-463
PW18-223
PW3-032
PW2-025, PW17-210,
PW12-146, PW38-471
PW5-064
PW13-156
PW9-113
PW10-119, PW10-119, PW10-120, PW10-121, PW18-221,
PW28-350, PW28-350
PW38-484
PW8-090
PW8-092
PW32-403
PW32-403
PW4-054, PW4-055
PW4-046
PW31-391
PW1-004, PW1-006
PW16-205
PW8-100
PW23-289
PW9-101, PW13-162, PW22-272, PW26-324
PW21-267
PW9-111, PW19-228
PW10-121
PW8-097
PW3-033, PW3-034
PW28-353
PW13-168
PW33-421
PW19-233, PW19-234
PW26-330
PW24-294
PW28-352
PW14-175, PW22-271, PW22-279
PW4-044
PW32-410
PW1-010, PW21-261
PW38-479
PW16-203
PW7-083
PW38-486
PW38-481
PW31-395
PW11-134
PW27-336
PW1-013
SCAMBI I
SCAPOLAN S
SCARPELLI M
SCHAEFFER L
SCHAERER F
SCHÄFER M
SCHARA U
SCHENA E
SCHERMAN D
SCHLOTTER F
SCHMIED A
SCHMITT A
SCHMITT C
SCHRAEN-MASCHKE S
SCHRÖDER R
SCHROEDER HD
SCHULZE J
SCHUMACHER M
SCHWARTZ K
SCICCHITANO B
SCIMÈ A
SCIORATI C
SCUTIFERO M
SEAGAR M
SEBILLE S
SEBRIÉ C
SEDEL F
SEEHRA J
SEGALAT L
SEGAT D
SEIWERTH S
SELLATHURAI J
SÉMÉRIVA M
SENE A
SENS P
SEOW Y
SEQUESTRA N
SERGEANT N
SERMON K
SERRURIER B
SERVAN J
SETA N
SHAFEY D
SHAPOVALOV G
SHARMA M
SHARMA MC
SHIN G
SHONO M
SHUKLA G
SIALA O
SICILIANO G
SIEGEL A
SIENDT H
SIFI K
SIFI Y
SIGOILLOT S
SIKIRIC P
SILVA-BARBOSA S
SIMON S
SIMONELIG M
SINGH R
SINGH S
SINHA B
SINKOVEC M
SKOURI-PANET F
SKUK D
SLASSI I
SMADJA D
SMITH R
SNYDER R
SOHEILI T
SOLER C
SOLLY S
SOTIROPOULOS A
SPECTOR DL
SPEHRS-CIAFFI V
SPITALI P
SQUADRITO F
SQUARZONI S
SROUR M
ST GUILY JL
STANCIU R
STARKE J
PW30-385
PW1-009, PW1-015, PW14-176
PW9-107, PW19-236, PW19-236
PW33-421
PW33-420
PW34-428
PW24-301
PW13-157
PW30-380
PW38-482
PW7-086
PW30-384
PW39-495
PW12-146
PW16-199
PW29-363, PW31-386
PW34-432
PW24-299
PW22-275
PW31-396
PW29-368
PW25-305
PW9-113
PW37-457
PW5-063, PW5-064, PW32-399
PW3-039
PW38-466
PW25-311
PW25-316
PW30-385
PW25-314
PW31-386
PW38-487
PW4-054, PW4-055
PW9-104
PW2-023
PW26-329
PW12-146
PW12-149, PW12-151
PW25-310
PW21-260
PW17-206, PW17-207
PW6-073
PW3-035
PW1-010
PW21-261
PW3-030
PW3-038
PW21-256
PW17-213
PW9-114, PW11-131
PW3-030
PW4-044
PW7-077, PW11-137, PW20-242
PW7-077, PW11-137, PW20-242
PW18-219
PW25-314
PW27-336
PW16-193, PW16-194, PW16-195, PW16-197, PW23-281,
PW24-296
PW10-127
PW3-029, PW3-032, PW3-033, PW3-034
PW1-010, PW21-256, PW21-261
PW9-104
PW13-159
PW16-193
PW1-014, PW27-338, PW27-339, PW30-381, PW33-417
PW13-163, PW19-230
PW7-083
PW14-171
PW38-477
PW24-295
PW38-478
PW21-267
PW30-384
PW38-466
PW15-189
PW2-025
PW25-315
PW13-157
PW9-110
PW10-124, PW10-125
PW21-263
PW3-032
STAVARACHI M
STEDMAN H
STEFANOVIC S
STEINBRECHER A
STERNBERG D
STETZKOWSKI-MARDEN F
STEWART C L
STOCKHOLM D
STOJKOVIC T
STOLTENBURG-DIDINGER G
STORA S
STOWARD PJ
STRÄHLE U
STREICHENBERGER N
STROBER J
STUELSATZ P
STUM M
SUDRES M
SUEL-PETAT L
SUMSKI C
SUN X
SUOMINEN T
SÜTÖ G
SVINARTCHOUK F
SWEENEY L
SZIJAN I
PW7-078, PW7-080
PW38-477
PW35-438
PW17-207
PW18-215, PW18-221
PW18-218
PW13-160
PW8-093, PW8-094
PW13-166, PW13-167, PW14-181, PW16-199, PW16-200,
PW18-214
PW14-177, PW16-199, PW16-200, PW18-214, PW18-215,
PW18-215
PW15-188
PW13-159
PW3-038
PW34-431
PW8-090
PW8-090
PW8-089
PW33-414, PW33-415
PW26-333
PW8-095, PW9-112
PW3-034
PW16-195, PW23-281
PW11-141, PW13-169, PW16-199, PW16-202
PW21-257
PW10-124, PW10-125
PW2-027
PW1-017
T HOEN P
TABTI N
TACCHINO C
TADAYONI R
TAHALLAH N
TAJBAKHSH S
TAKESHIMA H
TANGUY A
TANNIOU G
TANVEER N
TARATUTO A
TARDIEU A
TASCON C
TASSIN A
TASSISTRO V
TAXONERA C
TAZI J
TAZIR M
TCHIKVILADZÉ M
TEDESCO V
TEIJEIRA S
TEISSIER N
TENENBAUM L
TENTORIO M
TERKI N
TERNAUX B
TERRIER B
TESTARD H
TETREAULT M
TEUSAN R
TÉVY MF
THEODORAKIS G
THÉRIER P
THÉVENIAU-RUISSY M
THIBAUD JL
THIFFAULT I
THIJS D
THILOTHAMMAL N
THIMONIER J
THISSE B
THISSE C
THOMAS G
THOMPSON O
THOREL D
THORNELL LE
THORNTON C
TIRET L
TIRET P
TOKOLA M
TOMA M
TOMELLERI G
TOMIZAWA Y
TOMŠIC I
TONIN P
PW29-362, PW34-425
PW33-414
PW1-015
PW4-054
PW25-319
PW21-268, PW39-489, PW39-494
PW33-420, PW33-420
PW15-191
PW25-316
PW1-010
PW14-179
PW16-193
PW4-049
PW10-115
PW24-297
PW7-087
PW39-491
PW7-079, PW23-284, PW23-287, PW23-290
PW19-231, PW19-232
PW19-236
PW13-168
PW14-172
PW38-486
PW9-107
PW13-155, PW15-183
PW28-355
PW21-265
PW8-090
PW9-110
PW10-121
PW38-487
PW22-271
PW14-172
PW22-277
PW33-422, PW34-426
PW9-110
PW24-301
PW1-004
PW37-464
PW38-481
PW38-481
PW33-421
PW31-391
PW1-002
PW20-251
PW12-144
PW34-427
PW38-485
PW16-204
PW7-078, PW7-080
PW9-107, PW19-236
PW2-027
PW7-081
PW9-107, PW19-236
STOLTENBURG G
TONIOLO L
TONSON A
TOPALOGLU H
TOROMANOFF A
TORRENTE Y
TOSCH V
TOSCHI A
TOUBOUL D
TOUITA KABBAJ A
TOUITOU D
TOURETTE C
TOUSSAINT A
TOUSSAINT B
TOUVRON M
TRABANELLI C
TRABELSI M
TRAN H
TREMBLAY JP
TREVES S
TREVISAN C
TRILLER A
TRIOLO D
TRITSCH E
TRON F
TROUSSON A
TRUAN G
TRUFFAULT F
TUFFERY-GIRAUD S
TUIL D
TUPLER R
TURIN E
UDD B
PW32-404
PW32-401
PW17-211
PW38-477
PW2-018, PW2-018, PW2-019, PW4-047, PW5-056, PW8-098,
PW26-321, PW28-349
PW14-178
PW31-396
PW25-319
PW32-406
PW21-263
PW24-293
PW14-178
PW20-247
PW22-274
PW17-210
PW38-472, PW9-103
PW12-146
PW1-014, PW5-059, PW5-062, PW27-338, PW27-339,
PW27-341, PW30-381, PW33-417
PW33-420
PW13-156
PW38-466
PW28-353
PW15-190
PW21-269
PW24-299
PW38-480
PW18-223, PW18-225
PW1-002, PW2-025, PW36-455, PW36-456, PW38-471
PW22-274, PW30-384
PW10-117, PW10-121
PW3-032, PW3-034
URIARTE A
URO-COSTE E
URTIZBEREA JA
UZENOT D
PW8-095, PW11-141, PW13-169, PW16-199, PW16-200,
PW16-202, PW16-203, PW16-204
PW33-422
PW21-264
PW8-090, PW8-091, PW8-094, PW8-097, PW37-460
PW19-228, PW21-262
VAFIADAKI E
VAILLEND C
VAKHARLOVSKY VG
VALA C
VALLAT JM
VAN BOKHOVEN H
VAN BRUSSELS E
VAN DEN BERGH A
VAN DEN BERGH P
VAN DEN HAUWE M
VAN DER MAAREL SM
VAN DER MIEREN G
VAN DER PLOEG A
VAN DEUTEKOM J
VAN GALEN M
VAN KUIK-ROMEIJN P
VAN OMMEN G-J
VAN PUTTEN M
VAN REEUWIJK J
VAN ZOGGEL J
VANASSE M
VANDEBROUCK A
VANDERPLANCK C
VARNOUS S
VARRAULT A
VASSETZKY Y
VASSON A
VATTEMI G
VAUCHEZ K
VEBER H
VELA A
VELASCO E
VELASQUEZ AC
VENTRIGLIA VM
VENTUROLI A
VERBEKEN E
VERCAMMEN L
VERGES S
VERGNAUD S
VERNUS B
VERRIPS T
VERSCHUEREN A
VESCHAMBRE P
PW22-271
PW3-039, PW33-416
PW7-084
PW9-106
PW23-284, PW23-287, PW23-290
PW17-206
PW12-146
PW24-300
PW17-206, PW17-207
PW24-301
PW10-127
PW24-300
PW20-252
PW2-024, PW26-322, PW34-425
PW29-362
PW2-024
PW2-024, PW26-322, PW29-362, PW34-425
PW26-322
PW17-206
PW25-307
PW9-110
PW15-185
PW10-115
PW13-162
PW24-298
PW10-118
PW9-103
PW9-107, PW19-236
PW28-355, PW30-382
PW21-260
PW7-087
PW29-364
PW1-016
PW9-113
PW17-210
PW24-300
PW38-486
PW32-412
PW20-247
PW24-297
PW10-127
PW21-262
PW8-089
VETTOR R
VIAL C
VICART P
VIGNAUD L
VIGNIER N
VIGNY M
VIHOLA A
VILCHEZ J
VILLERIUS MP
VILMA-LOTTA L
VILMEN C
VILQUIN JT
VINH J
VIOLLET L
VISWANATHAN V
VITA G
VITIELLO L
VOIT T
VOLA C
VOLPI L
VON BOXBERG Y
VON KIENLIN M
VOVARD F
VUADENS O
VUILLAUMIER-BARROT S
VUILLEROT C
VUKOJA I
VULIN A
PW28-352
PW11-134, PW 26-326
PW16-193, PW16-195, PW16-197, PW16-198, PW16-199,
PW16-200, PW23-281
PW18-221
PW38-471
PW24-294, PW9-106
PW23-286
PW13-168
PW4-045, PW12-153, PW18-220, PW25-317, PW32-400,
PW32-411, PW33-421, PW33-423, PW36-447
PW7-085, PW30-380
PW22-273
PW38-468
PW16-203
PW8-092
PW29-362
PW15-183
PW38-476
PW10-121, PW22-275, PW28-355, PW30-382
PW18-217
PW7-085, PW17-206, PW18-214
PW1-004, PW1-006
PW25-315
PW26-323, PW30-385
PW15-188, PW16-199, PW26-325
PW35-444
PW9-114, PW11-131
PW23-289
PW32-402
PW27-345
PW25-313
PW17-206, PW17-207
PW36-451, PW36-452
PW25-314
PW2-022, PW31-387
WADDELL L
WAGNER K
WAHBI K
WALLGREN-PETTERSSON C
WANDERS R.J
WANG HS
WANG ZM
WARY C
WATSCHINGER K
WDZIECZAK-BAKALA J
WEHNERT M
WEIN N
WEISS N
WELCH EM
WELSH M
WESTENDORP R
WEYERMANN P
WILLER JC
WILTON S
WINDER S
WINDERS T
WINSOR B
WITZEMANN V
WOLFF A
WONG V
WOOD MJA
WOOLF A
WRAITH J
WUYAM B
PW14-171
PW5-060
PW11-128, PW16-200
PW15-183, PW15-184, PW15-186
PW24-302
PW22-279
PW33-420
PW32-412, PW39-488
PW37-459
PW22-272
PW13-157
PW8-098, PW8-099, PW 26-326
PW4-043, PW9-105, PW14-175
PW2-027
PW16-195, PW23-281
PW31-398
PW4-044
PW33-414
PW2-026, PW6-067
PW31-391
PW3-032
PW38-474
PW18-217
PW27-336, PW28-349
PW25-311
PW2-023
PW35-444
PW20-249
PW20-247, PW32-412
XIA J
XUE Z
PW33-420
PW16-196
YACOUB-YOUSSEF H
YAFFE D
YAHYAOUI M
YAMANAKA K
YANAGISAWA A
YANG X
YARON Y
YIN H
YOSHIDA M
YU H
PW30-375
PW4-054, PW4-055, PW33-416
PW13-163, PW23-285
PW23-288
PW17-206
PW38-486
PW17-208
PW2-023
PW3-038
PW25-319
ZACCOLO M
ZAGLIA T
ZAGNOLI F
PW18-227
PW26-323
PW18-214, PW18-214, PW20-244, PW21-255
VICART S
VIDAILHET M
VIDAL J
VIEHWEGER E
VIÉITEZ I
VIGNAUD A
ZAMMIT P
ZANOU N
ZEITOUNI B
ZELLER R
ZENTOUT H
ZER H
ZERBINI G
ZHAO W
ZIDAR J
ZIMMERMAN A
ZIMOWSKA M
ZORZATO F
ZUPAN A
PW27-343
PW5-058
PW38-487
PW1-001
PW11-132
PW16-201
PW13-156
PW22-279
PW37-458
PW3-030
PW31-392
PW33-420
PW7-081, PW32-409, PW32-410
*********************