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 1998 Oxford University Press
Human Molecular Genetics, 1998, Vol. 7, No. 12 1927–1933
The distribution of SMN protein complex in human fetal
tissues and its alteration in spinal muscular atrophy
P. Burlet, C. Huber, S. Bertrandy, M. A. Ludosky1, I. Zwaenepoel, O. Clermont,
J. Roume2, A. L. Delezoide, J. Cartaud1, A. Munnich and S. Lefebvre*
Unité de Recherches sur les Handicaps Génétiques de l’Enfant, INSERM Unité 393, IFREM, Institut Necker,
Hôpital des Enfants Malades, 149 rue de Sèvres, 75743 Paris cedex 15, France, 1Département de Biologie
Supramoléculaire et Cellulaire, Institut Jacques Monod, CNRS, Université Paris 7, Paris, France and 2Unité de
Foetopathologie, Hôpital Saint-Antoine, Paris, France
Received June 23, 1998; Revised and Accepted August 7, 1998
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized
by degeneration of motor neurons of the spinal cord
and muscular atrophy. SMA is caused by alterations to
the survival of motor neuron (SMN) gene, the function
of which has hitherto been unclear. Here, we present
immunoblot analyses showing that normal SMN protein expression undergoes a marked decay in the postnatal period compared with fetal development. Morphological and immunohistochemical analyses of the
SMN protein in human fetal tissues showed a general
distribution in the cytoplasm, except in muscle cells,
where SMN protein was immunolocalized to large cytoplasmic dot-like structures and was tightly associated
with membrane-free heavy sedimenting complexes.
These cytoplasmic structures were similar in size to
gem. The SMN protein was markedly deficient in tissues derived from type I SMA fetuses, including skeletal muscles and, as previously shown, spinal cord.
While our data do not help decide whether SMA results
from impaired SMN expression in spinal cord, skeletal
muscle or both, they suggest a requirement for SMN
protein during embryo–fetal development.
INTRODUCTION
The survival of motor neuron (SMN) gene has been identified as
the disease-causing gene in spinal muscular atrophy (SMA), a
frequent autosomal recessive neuromuscular disorder (1/6000
live births) characterized by degeneration of motor neurons of the
spinal cord and progressive proximal muscular atrophy (1–5).
The childhood SMAs have been divided into three forms, based
on age of onset and clinical severity: type I (acute form,
Werdnig–Hoffmann disease), type II (intermediate) and type III
SMA (Kugelberg–Welander disease) (6). In >90% of cases, the
disease results from absence of the SMNt gene and SMA patients
retaining the SMNt gene carry intragenic mutations (1,2,7–10).
Further analyses revealed that the absence of SMNt was
associated with a reduction in gene dosage in type I SMA but not
in type III SMA, indicating that the SMNt gene was replaced by
another copy of the SMNc gene (1,11,12). However, severe
forms have also been associated with an increased number of
SMNc copies, suggesting that other factors may modulate clinical
severity (13). Thus, loss of the SMNt gene in SMA patients could
be due either to gene deletion or gene conversion. Yet, rare cases
with no detectable SMNt gene (<1%) have been reported in
asymptomatic relatives of haploidentical SMA type II and III
patients (14,15).
The SMNt gene belongs to a duplicated element (500 kb)
located in a region of chromosome 5q13 prone to large scale
rearrangements (11) and a highly homologous copy gene (SMNc)
maps proximal to the disease gene (1). A tight correlation
between clinical severity and SMNc protein level was demonstrated in tissues and cell lines derived from SMA patients (16,17)
and strong SMN protein expression was shown in normal fetal
lower motor neurons (16). The SMN gene encodes a protein of
hitherto unclear function, detected in the cytoplasm and in nuclear
bodies called gem and found to interact with RNA-binding
proteins (18). More recently, SMN protein has been shown to
form a complex with spliceosomal small nuclear ribonucleoproteins (snRNP) (19) and to be involved in biogenesis of the
spliceosomal snRNPs (20).
Here, we have studied SMN protein expression in various
human tissues during normal fetal and postnatal development and
showed a reduction in SMN protein level in the postnatal period.
Interestingly, SMN protein in fetal muscle cells was concentrated
in large particles distributed within the cytoplasm and similar in
size to gem. A great reduction in SMN protein level was observed
in all tissues of type I SMA fetuses, including skeletal muscles.
RESULTS
Western blot analyses
Immunoblotting experiments on normal human fetal tissues
using the monoclonal anti-SMN antibody 4B3 revealed that the
SMN protein had the same apparent molecular size (38 kDa) in
*To whom correspondence should be addressed. Tel: +33 1 44 49 51 63; Fax: +33 1 47 34 85 14; Email: [email protected]
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Figure 1. SMN protein expression in control and SMA tissues. (A) The monoclonal anti-SMN antibody 4B3 revealed a 38 kDa protein in the various human fetal
tissues tested. (B) Immunoblot studies of total protein preparations from fetal (F) (25 µg) and postnatal control tissue samples (P) (75 µg). SMN protein expression
was reduced in postnatal compared with fetal tissues. (C) Western blot analyses of muscle tissues from control and type I SMA fetuses. (D) Immunoblot analyses of
muscle tissues from control, type II and type III SMA fetuses. Total protein preparations from SMA fetuses showed a marked reduction in SMN protein level as
compared with three control fetuses (C1, C2 and C3). Incubations with monoclonal anti-actin and anti-β-tubulin antibodies were performed as internal controls for
both loading conditions and tissue expression.
lymphoblastoid cell lines (16), skeletal muscle, heart, kidney,
thymus, brain, pancreas and lung (Fig. 1A). The relative amount
of SMN protein was higher in kidney and brain and similar in
skeletal muscle, heart and thymus of 16-week-old fetuses. The
amount of SMN protein in skeletal muscle, heart, kidney and
brain was relatively higher during fetal life than in the postnatal
period, as compared with actin and β-tubulin protein levels (Fig.
1B). Interestingly, the drop in protein level during the postnatal
period was less pronounced in kidney compared with other
tissues. The SMN protein level was markedly deficient in fetal
muscle tissues from all three forms of SMA, being more reduced
in severe than milder forms, as compared with age-matched
controls under our experimental conditions (Fig. 1C and D).
Immunolocalization of SMN protein in human fetal
tissues
Immunofluorescence detection using monoclonal anti-SMN
antibody 4B3 in human fetal tissues (thymus, kidney, lung and
brain) revealed that SMN was present as large gems in the nucleus
and was uniformly distributed throughout the cytoplasm, with no
preferential compartmentation (Fig. 2). No detectable SMN
immunostaining was noted in tissues of type I SMA fetuses (Figs
2 and 3F). Morphological and immunohistochemical analyses of
normal fetal limb showed large dot-like SMN immunolabelling
in the cytoplasm of myotubes and myofibres, and SMN was
occasionally concentrated in small gems in the nucleus (Fig.
3A–E). The size of these large cytoplasmic structures was
estimated to be similar to that of gems in the nucleus, i.e. 0.1–1.0
µm (18). A strong SMN immunostaining was also detected
around blood vessels in endothelial cells (Fig. 3B). Neither
nuclear nor cytoplasmic SMN immunostaining could be detected
in muscle cells from a SMA type I fetus (Fig. 3F). The availability
of the Sol 8 mouse muscle cell line (21) allowed in vitro studies
of SMN protein immunolocalization in the myoblast stage and
during its terminal differentiation into multinucleated myotubes
(Fig. 3G and H). Strong SMN immunostaining was detected
throughout the cytoplasm during the myoblast proliferative state
(Fig. 3G). In contrast, a dramatic reduction in SMN immunodetection was observed in a cell culture differentiated into
multinucleated myotubes (Fig. 3H).
Subcellular localization of SMN protein in the
particulate cellular fraction
The subcellular localization of the SMN protein in skeletal
muscle from human fetal limb was further investigated using the
velocity sedimentation procedure (Fig. 4A; see Materials and
Methods). Immunoscreening of the different subcellular fractions
revealed a specific SMN enrichment in the pellet of the 142 000
g centrifugation (P142 fraction, Fig. 4B). In order to further
characterize SMN protein interactions, the P142 fraction was
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Figure 2. Immunofluorescence staining of SMN in control and SMA fetal tissues. Haematoxylin staining of serial tissue sections showed the morphological aspect
of various tissues (A). Monoclonal anti-SMN antibody 4B3 stained both the cytoplasm and the nuclear gems (arrows) in control thymus, kidney, lung and brain (B).
No detectable immunostaining of the SMN protein was observed in tissues from type I SMA fetuses (C). Bar 10 µm.
either treated with the non-ionic detergent Nonidet P-40 (Fig. 4C)
or analysed by discontinuous sucrose gradient centrifugation
(Fig. 4D). The SMN protein was recovered in the pellet (P),
indicating that neither treatment could dissociate SMN from the
heavy sedimenting particulate fraction. Our results further
support the view that SMN is strongly associated with a
multimeric protein complex with no interaction with membrane
components. These data complement the observations made
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Figure 3. Immunohistological and immunofluorescence analyses of SMN protein expression in muscle cells. Combined haematoxylin staining and
immunohistochemical experiments using the anti-SMN monoclonal antibody 4B3 were performed on human skeletal muscle sections from control fetuses at 16 (A)
and 24 weeks gestation (B and C). Immunofluorescence analyses of human skeletal muscle sections with the same antibody also revealed a dot-like immunolocalization
in the cytoplasm and, occasionally, small gems (arrows) in the nucleus of fetal muscle cells at 8 (D) and 16 weeks gestation (E, DAPI staining of the nucleus). No
detectable SMN immunostaining was shown in a SMA type I fetus at 16 weeks gestation (F). Mouse myogenic cell line Sol 8 in the myoblast proliferative state (G),
and differentiated into multinucleated myotubes (H), was immunostained with anti-SMN monoclonal antibody. The immunodetection of SMN protein was performed
using anti-mouse IgG–FITC (A–D) or anti-mouse IgG–Cy3 (G–H). Bar 10 µm.
using fractionation of HeLa cell cultures (19). This study
hopefully represents a step towards characterization of tissuespecific partners of SMN and unravelling of its function(s).
DISCUSSION
SMN protein function has hitherto been unclear and the
pathogenesis of SMA is still unknown. Yet the disease is caused
by alterations to the telomeric copy of the SMN gene (1–10) and
its severity is tightly correlated with the amount of protein
encoded by its highly homologous gene copy, SMNc (16,17).
Recent studies identified an interaction of SMN with RNAbinding proteins (18,19) and its involvement in the biogenesis of
spliceosomal snRNPs (20).
The present study has shown that the SMN protein level
undergoes a marked drop in the postnatal period in the human
tissues tested, such as skeletal muscle, heart and brain. Also,
analysis of the Sol 8 mouse muscle cell line showed a marked
decay in SMN immunodetection upon in vitro cell differentiation.
These observations suggest that SMN expression undergoes
hitherto unknown developmental and/or hormonal regulation
(22). We have also shown that in fetal muscle cells SMN protein
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Figure 4. Subcellular fractionation and immunoblot studies of control fetal
skeletal muscle. (A) Flow chart for subcellular fractionation by centrifugation.
(B) Western blot analyses with anti-SMN, anti-emerin and anti-SERCA1
antibodies were carried out on skeletal muscle protein homogenate (total) and
centrifugation fractions (P14, P30, P142 and S142) were prepared as described
in (A). The SMN protein was highly enriched in the P142 fraction. (C)
Immunoblot analyses with anti-SMN and anti-SERCA1 antibodies of the pellet
(P) and the supernatant (S) prepared from P142 in an extraction buffer
containing NP-40 (0.5%). SERCA1 is released in the supernatant, while SMN
remains associated with the pellet. (D) Immunoblot analyses using anti-SMN
and anti-αSARC antibodies were performed on the interface of the two sucrose
layers (I) and on the pellet (P). αSARC was detected at the interface while SMN
protein was still found in the pellet.
is concentrated in large cytoplasmic dot-like structures similar in
size to gem (0.1–1.0 µm; 18). To our knowledge, cytoplasmic
structures as large as gem have never been described before. This
immunolocalization of SMN in skeletal muscle contrasts with
results obtained in other normal tissues such as thymus, kidney,
lung and brain or liver and spinal cord (16) or even in cell cultures
(17,18), where a general staining throughout the cytoplasm and
large gems in the nucleus were detected. This distribution might
account for a shorter nuclear half-life of SMN protein. SMN
protein was shown to associate directly with SIP1 (SMN-interacting protein 1) and to form a complex with spliceosomal snRNP
proteins in HeLa cells (19). Further experiments will be necessary
to determine whether accumulation of SMN protein in large
dot-like structures in the cytoplasm of myotubes and myofibres
from fetal limb could be due to post-transcriptional modification
of SMN and/or to tissue-specific partners of SMN.
Immunohistochemical analyses of fetal liver, fetal spinal motor
neurons (16) and of cultured fibroblasts (17) revealed a lack of
and significant reduction in the number of gems in SMA types I
and III, respectively. Here we have extended the range of tissues
tested and shown that SMN protein is greatly reduced in all tissues
derived from type I SMA fetuses. It has recently been reported
that SMN protein levels are markedly reduced in spinal cord of
types I and III SMA fetuses (16) and of type I SMA patients (17).
Immunoblot analyses showed that in postnatal skeletal muscle
derived from type I SMA patients, the amount of SMN protein
was also markedly reduced, but not in a type III patient (17).
Interestingly, the observation of a significant reduction in SMN
in muscle from type III SMA fetuses suggests that expression
and/or the stability of SMNc protein in muscle tissue may differ
between the pre- and postnatal periods. The lack of or the marked
reduction in SMN in skeletal muscle of types I or II and III SMA
fetuses might be related to the pathological defects of muscle
fibres in SMA patients (23,24). These experiments lead to the
observation that in SMA the SMN protein is markedly reduced
prior to onset of the disease, while SMN protein is strongly
expressed in normal tissues. Our present data do not help in
deciding whether SMA results from impaired SMN expression in
spinal cord, skeletal muscle or both, but they raise the hypothesis
that SMA is an embryo–fetal rather than a postnatal disease.
Strong SMN expression in human fetal life along with its
conservation among species (9) and its absolute requirement in
early mouse embryos (25) give strong support to the view that
SMN plays a major role in cell survival.
Ubiquitous SMN expression and its role in RNA metabolism
(26) contrast with the very specific clinical expression of the
disease. One can hypothesize that SMN protein deficiency alters
a subset of tissue-specific RNAs or another function(s) in a
developmental manner. Characterization of SMN protein complexes should contribute to a better understanding of SMN
function(s) and the pathogenesis of SMA.
MATERIALS AND METHODS
Patients
A total of six unrelated control fetuses were selected for
immunohistochemical and immunoblotting analyses. Three fetuses predicted to be affected with type I, three with type II and
two with type III SMA at 13–18 weeks gestation were included
in this study. The probands were diagnosed as SMA according to
the clinical criteria of the International SMA Consortium (6). All
SMA fetuses were homozygous for the absence of SMN exon 7
and retained the SMNc gene (1). Embryos were obtained from
legally terminated pregnancies according to the French ethical
committee recommendations.
Production of monoclonal antibodies against SMN
protein
Anti-SMN antibodies 4B3 and 0B5 were produced by immunization of Balb/c mice with a bacterial fusion protein starting at
amino acid position 85 of the SMN protein expressed in vector
pET30 (Novagen). Hybridoma and ascite fluid preparations were
performed according to standard procedures (Coval Ab, France).
Hybridomas were selected based on the immunoreactivity of the
secreted antibodies in western blotting and immunohistochemical
analyses of control and SMA samples.
Morphological and immunofluorescence analyses
Immunostaining of cryosections (4 µm) from non-muscle fetal
tissues (thymus, kidney, lung and brain) fixed in 3% formaldehyde-buffered solution was carried out as previously described
(16). Fetal muscle sections were prepared from paraffinembedded specimens for immunofluorescence experiments
1932 Human Molecular Genetics, 1998, Vol. 7, No. 12
using the NEN TSA System and immunohistochemical studies
using the DAKO CSA system. Sections were stained with
haematoxylin solution for morphological analyses or stained with
1 µg/ml 4,6-diamidino-2-phenylindole (DAPI) (Sigma) to visualize the nucleus. The human tissue sections were incubated with
monoclonal anti-SMN antibody 4B3 ascite fluid at a
1:1000–4000 dilution (0.25–1 µg/ml) and detected using 1:100
anti-mouse IgG–fluorescein isothiocyanate (FITC) (Biosys). The
Sol 8 mouse muscle cell line was cultured and in vitro
differentiated according to previously published procedures (21).
The cells were fixed with 3% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4), permeabilized with 0.5% Triton and
pre-incubated in blocking solution (5% sheep serum and 1%
BSA). Immunodetection was performed with 1:1000 monoclonal
anti-SMN antibody (Transduction Laboratories) using 1:400
sheep anti-mouse IgG–indocarbocyanide (Cy3) (Sigma). The
immunofluorescence was observed using a Leica DM microscope equipped with a ×10/0.30 or ×100/1.30 objective and dual
bandpass filter for DAPI/FITC or DAPI/rhodamine fluorescence.
organelles and cell debris. The supernatant was centrifuged in a
cold SW41Ti rotor (Beckman Instruments) at 30 000 g for 30 min.
The pellet P30 contained heavy microsomes from the endoplasmic and sarcoplasmic reticulum. The P30 supernatant was
further centrifuged at 142 000 g for 35 min. The resulting pellet
(P142) was resuspended in sucrose buffer and layered on a
discontinuous gradient of 0.303 M sucrose on top of 1.0 M
sucrose in buffer A. The membrane fraction at the interface of the
two sucrose layers and the particulate cellular fraction (pellet, P)
were collected after centrifugation at 150 000 g in a SW41Ti rotor
for 16 h at 4C. Immunodetection with anti-emerin monoclonal
antibody indicated its specific nuclear enrichment. The weak
signal in other fractions reflected the strong homogenization
conditions. The anti-SERCA1 monoclonal antibody revealed
labelling of the P30 (heavy microsomes) and P142 (light
microsomes and particulate cellular) fractions. The membranous
component of the sarcoplasmic reticulum (heavy microsomes)
was probably detected in the P142 fraction, possibly because of
its abundance and its poor fractionation under our homogenization conditions.
Immunoblot analyses
The various human fetal tissues and postnatal skeletal muscles
were homogenized in ice-cold extracting buffer containing 20
mM pyrophosphate, 20 mM phosphate monohydrate, 1 mM
MgCl2, 0.303 M sucrose, 0.5 mM EDTA (pH 7.4) in the presence
of protease inhibitors [2 mM phenylmethylsulfonyl fluoride
(PMSF), 3 µg/ml pepstatin, 3 µg/ml anti-papain, 15 µg/ml
benzaminidine and 40 µg/ml leupeptin]. Following a Bradford
protein assay (Bio-Rad Laboratories) using BSA as standard, the
protein homogenates were diluted in Laemmli sample buffer,
separated by SDS–PAGE (27) and electrotransferred onto
Immobilon-P membranes (28). Whole tissue protein preparations
of normal adult heart, kidney and brain were obtained from
Clontech as electrophoresis-ready solutions. Triplicate immunoblots were incubated with monoclonal antibodies directed against
α-sarcoglycan (α-SARC) (1:100 dilution), emerin (1:250 dilution), sarcoplasmic reticulum Ca2+-dependent ATPase 1
(SERCA1) (1:250 dilution), actin (1:4000 dilution) and β-tubulin
(1:4000 dilution) as recommended by the suppliers (Novocastra
Laboratories and Amersham). The monoclonal anti-SMN antibody 4B3 was used at a 1:1000 dilution. The immunoblots were
incubated with horseradish peroxidase-conjugated secondary
antibody (1:5000–10 000; Amersham) or with the biotin–streptavidin horseradish peroxidase system (1:10 000 dilution; Vector
Laboratories) and detected using chemiluminescent reagents
(ECL; Amersham). The autoradiographs were scanned using a
computerized densitometer as previously described (16).
Subcellular fractionation by centrifugation
Flash frozen fetal muscle samples (500–750 mg) were homogenized with a tight fitting glass pestle in 10 vol ice-cold 0.303 M
sucrose in 20 mM pyrophosphate, 20 mM phosphate monohydrate, 1 mM MgCl2, 0.5 mM EDTA, pH 7.4, buffer (buffer A) in
the presence of protease inhibitors (2 mM PMSF, 3 µg/ml each
pepstatin and anti-papain, 15 µg/ml benzaminidine and 40 µg/ml
leupeptin). Fractionation was performed by velocity sedimentation as previously described (29). Briefly, the homogenate was
centrifuged at 14 000 g for 15 min in a refrigerated bench
centrifuge. The corresponding pellet (P14) contained nuclei,
ACKNOWLEDGEMENTS
We thank the patients, families and doctors who have contributed
to this project and without whom this study would not have been
possible. We thank J.Melki and C.Fallet for tissues derived from
SMA fetuses, D.Montarrat and C.Pinset for providing the Sol 8
mouse muscle cell line, M.Fardeau and F.Tomé for helpful
discussions, M.C.Gubler and E.Viegas-Pequignot for equipment
facilities and M.Recouvreur, V.Raclin and Y.Deris for expert
technical assistance. We thank the members of the International
SMA Consortium for stimulating discussions. S.B. is the
recipient of a MRT Predoctoral Fellowship. I.Z. was a pre-doctoral summer student. This work was supported by Association
Française contre les Myopathies (AFM), Action Concertée des
Sciences du Vivant (ACC-SV2) and Programme Hospitalier de
Recherche Clinique. Work in the J.Cartaud laboratory was
supported by a grant from AFM and Sciences de la Vie, CNRS.
ABBREVIATIONS
Cy3, indocarbocyanide; DAPI, 4,6-diamidino-2-phenylindole;
FITC, fluorescein isothiocyanate; α-SARC, α-sarcoglycan;
SERCA1, sarcoplasmic reticulum Ca2+-ATPase 1; SMA, spinal
muscular atrophy; SMN, survival motor neuron; snRNP, small
nuclear ribonucleoprotein.
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