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Neurobiology of Disease 20 (2005) 823 – 836
Thalamocortical neuron loss and localized astrocytosis in the
Cln3 Dex7/8 knock-in mouse model of Batten disease
Charlie C. Pontikis,a,b Susan L. Cotman,c Marcy E. MacDonald,c and Jonathan D. Cooper a,b,*
a
Pediatric Storage Disorders Laboratory, Box P040, MRC Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry,
De Crespigny Park, King’s College London, London SE5 8AF, UK
b
Department of Neuroscience, Box P040, MRC Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry,
De Crespigny Park, King’s College London, London SE5 8AF, UK
c
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Charlestown, MA 02129, USA
Received 3 April 2005; revised 13 May 2005; accepted 18 May 2005
Available online 11 July 2005
Juvenile neuronal ceroid lipofuscinosis (JNCL) is the result of
mutations in the Cln3 gene. The Cln3 knock-in mouse (Cln3 Dex7/8 )
reproduces the most common Cln3 mutation and we have now
characterized the CNS of these mice at 12 months of age. With the
exception of the thalamus, Cln3 Dex7/8 homozygotes displayed no
significant regional atrophy, but a range of changes in individual
laminar thickness that resulted in variable cortical thinning across
subfields. Stereological analysis revealed a pronounced loss of neurons
within individual laminae of somatosensory cortex of affected mice and
the novel finding of a loss of sensory relay thalamic neurons. These
affected mice also exhibited profound astrocytic reactions that were
most pronounced in the neocortex and thalamus, but diminished in
other brain regions. These data provide the first direct evidence for
neurodegenerative and reactive changes in the thalamocortical system
in JNCL and emphasize the localized nature of these events.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Batten disease; Juvenile neuronal ceroid lipofuscinosis; JNCL;
CLN3; Thalamocortical degeneration; Astrocytosis; Lysosomal storage
disorder
Introduction
The neuronal ceroid lipofuscinoses (NCLs) are the most
common group of inherited neurodegenerative disorders of childhood with a collective incidence of up to 1 in 12,500 live births
(Cooper, 2003; Hofmann and Peltonen, 2001). These fatal
lysosomal disorders are characterized by blindness, marked
* Corresponding author. Pediatric Storage Disorders Laboratory, Department of Neuroscience, Box P040, MRC Social Genetic and Developmental
Psychiatry Centre, Institute of Psychiatry, King’s College London, De
Crespigny Park, London SE5 8AF, UK. Fax: +44 20 7848 0273.
E-mail address: [email protected] (J.D. Cooper).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2005.05.018
psychomotor deterioration and uncontrollable seizures (Hofmann
and Peltonen, 2001; Gardiner, 2002). Juvenile NCL (JNCL) is the
result of a mutation in the CLN3 gene, located in the p12.1 region
of chromosome 16 (International Batten Disease Consortium,
1995). This most frequently occurring form of NCL has an age of
onset between 4 and 10 years and typically results in premature
death before the age of 30 (Gardiner, 2002).
The Cln3 protein is predicted to be a highly hydrophobic 438
amino acid protein, containing multiple transmembrane domains
(Mao et al., 2003). Although very little is known about the normal
function of Cln3, colocalization studies have shown this protein to
be expressed in the lysosome, endosome and synaptosomes
(Haskell et al.; 2000, Jarvela et al., 1999). The most significant
advances in understanding Cln3 function come from yeast that bear
null mutations in Btn1p, the yeast ortholog of Cln3 (Pearce et al.,
1999). The deletion of BTN1 results in a reduced vacuolar pH
during early growth (Pearce et al., 1999), and influences the
transport of basic amino acids into the vacuole (Kim et al., 2003),
suggesting that Btn1p may play a role in pH homeostasis.
Detailed quantitative information about neuropathological
changes in JNCL is limited with qualitative studies restricted to
autopsy material (Braak and Goebel, 1978, 1979). The loss of
cortical neuron populations in JNCL has been well documented
(Braak and Goebel, 1978, 1979; Haltia, 2003), together with
selective neuronal loss and glial activation within the hippocampal
formation (Haltia et al., 2001; Tyynelä et al., 2004). Since human
autopsy material is not widely available, Cln3 null mutant mice
(Cln3 / ) have been generated to enable the study of JNCL
pathogenesis (Katz et al., 1999; Mitchison et al., 1999). These
animals exhibit a JNCL-like phenotype and display widespread
intracellular accumulation of autofluorescent pigment and a
selective loss of GABAergic interneurons (Mitchison et al.,
1999; Katz et al., 1999; Pontikis et al., 2004), together with an
altered threshold for seizure generation (Kriscenski-Perry et al.,
2002). A distinctive feature of Cln3 / mice is the early
occurrence of reactive changes in both astrocytic and microglial
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cell populations, events which occur many months before widespread neuronal loss is evident (Pontikis et al., 2004). Cln3 /
mice and individuals with JNCL also raise autoantibodies to
glutamic acid decarboxylase (GAD65) that inhibit the activity of
this enzyme, resulting in elevated levels of glutamate (Chattopadhyay et al., 2002).
More recently, Cln3 knock-in mice (Cln3 Dex7/8 ) have been
generated which accurately reproduce the 1.02-kb deletion in the
Cln3 gene that is seen in over 85% of JNCL alleles (International
Batten Disease Consortium, 1995), and also exhibit an NCL-like
phenotype (Cotman et al., 2002). This phenotype may be more
aggressive than that seen in Cln3 / mice, with a progressive
neurologic disease and premature death of homozygous Cln3 Dex7/8
mice from 7 months onwards (Cotman et al., 2002).
To further characterize the CNS of homozygous Cln3 Dex7/8
mice, we examined neurodegenerative and reactive phenotypes
that are present in other mouse models of NCL, including Cln3 /
mice (Mitchison et al., 1999; Pontikis et al., 2004). These models
display a range of effects upon cortical thinning and neuronal loss
between sensory and motor cortex (Bible et al., 2004; Cooper et al.,
1999; Mitchison et al., 1999; Pontikis et al., 2004). Our analysis of
these regions in homozygous Cln3 Dex7/8 mice also revealed
different degrees of cortical thinning and selective effects upon
cortical neuron number, together with significant loss of afferent
thalamic neurons. These neuronal changes were accompanied by
graded microglial activation and pronounced hypertrophy, but not
hyperplasia, of astrocytes. These reactive changes also exhibited
marked regional specificity, being most profound in the neocortex
and within restricted nuclei of the thalamus. Taken together with
recent findings from human JNCL (Tyynelä et al., 2004), these data
further highlight the localized nature of reactive and neurodegenerative phenotypes in this disorder.
Brains were subsequently removed and weighed before overnight
post-fixation and cryoprotection at 4-C in a solution containing
30% sucrose in Tris-buffered saline (TBS: 50 mM Tris, pH 7.6)
with 0.05% NaN3. Brains were subsequently processed and
sectioned as described previously (Bible et al., 2004; Pontikis et
al., 2004), with serial 40 Am frozen coronal sections stored at
40-C in cryoprotectant solution (TBS/30% ethylene glycol/15%
sucrose/0.05% sodium azide) prior to histological processing.
Nissl staining
Materials and methods
To provide direct visualization of neuronal morphology every
sixth section through the CNS was stained with the Nissl dye
cresyl violet as previously described (Bible et al., 2004; Pontikis
et al., 2004). Briefly, sections were mounted onto gelatin-chrome
alum-coated Superfrost microscope slides (VWR, Dorset, UK), air
dried overnight and incubated for 20 min at 55-C in 0.05% cresyl
fast violet and 0.05% acetic acid (VWR, Dorset, UK), rinsed in
distilled water and differentiated through a graded series of
alcohol before clearing in xylene (VWR, Dorset, UK) and
coverslipping with DPX (VWR, Dorset, UK). These Nissl-stained
sections were subsequently used for stereological analysis of
regional volume, cortical thinning, cortical and thalamic neuron
number, as described previously (Bible et al., 2004; Pontikis et al.,
2004).
To visualize autofluorescent storage material, selected sections
from each animal were mounted upon Superfrost microscope slides
(VWR, Dorset, UK) and immediately coverslipped with Vectashield aqueous mounting medium (Vector Laboratories, Peterborough, UK) (Bible et al., 2004; Griffey et al., 2003). Sections
were viewed under conventional epifluorescence illumination on a
Zeiss Axioskop2 MOT microscope (Carl Zeiss Ltd., Welwyn
Garden City, UK) and images recorded at multiple wavelengths
using a Zeiss Axiocam and Axiovision software (Carl Zeiss Ltd.,
Welwyn Garden City, UK).
Animals
Immunohistochemistry for interneuron markers
Homozygous Cln3 Dex7/8 knock-in mice and control (+/+) mice
were generated on a mixed 129Sv/Ev/CD1 strain background at the
Molecular Neurogenetics Unit, Massachusetts General Hospital,
Charlestown, MA, as described previously (Cotman et al., 2002).
All mice used in this study were analyzed at 12 months of age and
were littermates produced by crossing heterozygous Cln3 Dex7/8
knock-in mice that were from an F4 CD1 backcross generation.
Mice of both sexes were used for this analysis since we have
previously observed no significant difference in JNCL-like
phenotype between male and female Cln3 Dex7/8 mice (Cotman et
al., 2002). All perfusion procedures were carried out in accordance
with NIH guidelines and under the animal care committee
regulations at Massachusetts General Hospital.
To survey representative interneuron populations in homozygous Cln3 Dex7/8 mice, adjacent one-in-six series of sections were
immunohistochemically stained for the neuropeptide somatostatin
(SOM) or the calcium binding protein parvalbumin (PV), as
previously described (Bible et al., 2004; Pontikis et al., 2004).
Sections were incubated for 15 min in 1% H2O2 in TBS, rinsed in
TBS and blocked for 40 min in TBS/0.3% Triton X-100/15%
normal goat serum (NGS) before overnight incubation at 4-C in the
following polyclonal primary antisera (rabbit anti-SOM, Peninsula,
CA, USA, 1:2000; rabbit anti-PV, Swant, Bellinzona, Switzerland,
1:5000) diluted in TBS with 10% normal goat serum and 0.3%
Triton X-100. Sections were then rinsed in TBS and incubated for
2 h with secondary antiserum (biotinylated goat anti-rabbit IgG,
Vector Laboratories, UK, 1:1000) in TBS/0.3% Triton X-100/10%
NGS. Following rinsing in TBS, sections were incubated for 2 h in
an avidin – biotin – peroxidase complex in TBS (Vectastain Elite
ABC kit, Vector Laboratories, Peterborough, UK). Sections were
next rinsed in TBS and immunoreactivity was visualized by
incubation in 0.05% DAB (Sigma, Dorset, UK) and 0.001% H202
in TBS for 10 min (a time which represents saturation for this
reaction). Sections were then transferred to excess ice-cold TBS
and were rinsed again, mounted, air dried, cleared in xylene and
coverslipped with DPX (VWR, Dorset, UK).
Histological processing
For histological analysis, 12-month-old homozygous Cln3 Dex7/8
knock-in mice and age-matched control littermates (n = 5) were
fixed by transcardial perfusion. Mice were first deeply anaesthetized with sodium pentobarbitone (100 mg/kg) and transcardially
perfused with vascular rinse (0.8% NaCl in 100 mM NaHPO4)
followed by a freshly made and filtered solution of 4%
paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4.
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
825
Immunohistochemistry for glial markers
Measurements of total neuronal number and volume
To assess the extent of glial activation in 12-month-old
homozygous Cln3 Dex7/8 knock-in mice, adjacent one-in-six series
of free-floating frozen sections were immunohistochemically
stained using the standard immunoperoxidase protocol described
above for detection of astrocytic (GFAP, S100h) and microglial
(F4/80) markers using the following polyclonal primary antisera
(polyclonal rabbit anti-cow GFAP, DAKO, Cambridge, UK,
1:1000; polyclonal goat anti-rabbit S100h, DAKO, Cambridge,
UK, 1:1000; monoclonal rat anti-mouse F4/80, Serotec, Oxford,
UK, 1:100). Sections were then rinsed in TBS with subsequent
incubation in secondary anti-serum (swine anti-rabbit [GFAP and
S100h], Vector Laboratories, Peterborough, UK, 1:400; and
mouse adsorbed rabbit anti-rat [F4/80], Vector Laboratories,
Peterborough, UK, 1:1000) followed by avidin – biotin – peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories,
Peterborough, UK). Immunoreactivity was visualized by a standard DAB reaction and sections were mounted onto slides, air
dried, cleared in xylene and coverslipped with DPX (VWR,
Dorset, UK).
To examine neuronal number and volume within individual
cortical laminae, we used StereoInvestigator software to obtain
unbiased optical fractionator estimates of neuronal number and
nucleator estimates of neuronal volume from Nissl-stained
sections. These estimates were obtained for neurons in lamina V
of S1BF and M1 and of neurons in laminae II, III and IV in S1BF,
together with afferent thalamic neurons in the ventral posterior
nucleus (VPM/VPL). These measures were performed as described
previously (Bible et al., 2004), with a random starting section
chosen, followed by every sixth Nissl-stained section thereafter.
The boundaries of M1 and S1BF regions and each individual
lamina were defined by reference to landmarks in Paxinos and
Franklin (2001). Only neurons with a clearly identifiable nucleus
were sampled and all counts were carried out using a 100 oil
objective (NA 1.4). The following sampling scheme was applied to
the regions of interest. Grid area for lamina V M1 and S1BF
122,500 Am2, frame area 2141.3 Am2; grid area for S1BF laminae
II and III of 122,500 Am2, frame area 2141.3 Am2; grid area for
S1BF lamina IV of 40,000 Am2, frame area 1095.82 Am2; grid area
for VPM/VPL of 122,500 Am2, frame area 3382.55 Am2.
Measurements of volume, cortical and laminar thickness
Measurements of interneuron number
Unbiased Cavalieri estimates of the volume of the cortex,
hippocampus, striatum, thalamus, hypothalamus and cerebellum
were made from each animal, as described previously (Bible et al.,
2004; Pontikis et al., 2004), with no prior knowledge of genotype.
A sampling grid with appropriate spacing (500 Am cortex and
cerebellum; 200 Am hippocampus; 300 Am striatum; 200 Am
thalamus and hypothalamus; 400 Am cerebellum) was superimposed over Nissl-stained sections and the number of points
covering the relevant areas counted using 2.5 objective.
Regional volumes were expressed in Am3 and the mean volume
of each region calculated for control and homozygous Cln3 Dex7/8
mice.
Cortical thickness measurements were made on the same onein-six series of Nissl-stained sections for primary motor (M1),
primary somatosensory (S1BF), limbic entorhinal (Lent) and
primary visual cortex (V1) as defined by Paxinos and Franklin
(2001). The length of perpendicular lines extending from the
white matter to the pial surface was measured by placing 10
evenly spaced lines on each of three consecutive sections
spanning each cortical region. These measurements were obtained
blind to genotype using a 5 objective, exactly as described
previously (Bible et al., 2004; Pontikis et al., 2004). Results were
expressed as the mean cortical thickness in Am per region for
control and homozygous Cln3 Dex7/8 animals.
Individual laminar thickness measurements were also performed in M1, S1BF, Lent and V1 using the same Nissl-stained
sections. As with cortical thickness measures, the thickness of
each individual lamina was measured via a series of 10
perpendicular lines drawn in each of three consecutive sections
using a 10 objective. Results were expressed as the mean
laminar thickness in Am per region for control and homozygous
Cln3 Dex7/8 animals. All volume and thickness analyses were
carried out using StereoInvestigator software (Microbrightfield
Inc., Williston, VT) on a Zeiss Axioskop2 MOT microscope
(Carl Zeiss Ltd., Welwyn Garden City, UK) linked to a DAGEMTI CCD-100 camera (DAGE-MTI Inc., Michigan City, IN,
USA).
The number of GABAergic interneurons expressing SOM and
PV in M1 and S1BF was determined using the design-based optical
fractionator method (West et al., 1991). Immunoreactive neurons
were sampled using a series of counting frames distributed over a
grid and superimposed onto the section using StereoInvestigator
software. The rostral and caudal limits of M1 and S1BF regions
were defined according to Paxinos and Franklin (2001). The lateral
to medial extent of M1 and S1BF were identified by comparison
with an adjacent series of Nissl-stained sections and anatomical
reference points. A 40 oil objective (NA 1.30) was then used to
count clearly identifiable immunoreactive neurons, which fell
within the dissector frame. The following sampling scheme was
applied to the regions of interest; Grid area for SOM and PV in M1
and S1BF 38,275 Am2, frame area 20,191 Am2.
Due to the comparatively low abundance of interneurons
present in the hippocampus versus the neocortex, stereological
methods prove inefficient at estimating hippocampal interneuron
numbers without sampling the entire tissue (Bible et al., 2004).
Instead, counts of the number of interneurons expressing SOM or
PV were made, as described previously (Bible et al., 2004; Cooper
et al., 1999; Pontikis et al., 2004) in defined hippocampal
subfields. Counts were carried out under a 20 objective and
only positively stained cells with clear neuronal morphology were
counted. The number of interneurons in each hippocampal subfield
was expressed as the mean number of neurons per section.
Quantitative analysis of glial phenotype
The optical density of GFAP and F4/80 immunoreactivity was
assessed using a semi-automated thresholding image analysis
system (Optimas, Media Cybernetics, Silver Springs, MD). This
analysis was performed blind to genotype, as previously described
(Bible et al., 2004; Pontikis et al., 2004). Forty non-overlapping
images were captured, on three consecutive sections, through the
cortical regions M1, S1BF and V1, striatum, hippocampal dentate
gyrus and a combined measurement for the stratum oriens and
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C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
CA1. All RGB images were captured via a live video camera (JVC,
3CCD, KY-F55B), mounted onto a Zeiss Axioplan microscope
using a 40 objective and saved as JPEGs. All parameters
including lamp intensity, video camera setup and calibration were
maintained constant throughout image capturing.
Images were subsequently analyzed using OPTIMAS 6.2 image
analysis software (Media Cybernetics, Silver Springs, MD), using
an appropriate threshold that selected the foreground immunoreactivity above background. This threshold was then applied as a
constant to all subsequent images analyzed per batch of animals
and reagent used to determine the specific area of immunoreactivity for each antigen in each region. Each field measured 120 Am
wide, with a height of 90 Am. Therefore, the total area compiled
from 40 fields in each region corresponded to 432,000 Am2.
Macros were recorded to transfer the data to a Microsoft Excel
spreadsheet and were subsequently analyzed statistically. Data
were plotted graphically as the mean percentage area of immunoreactivity per field T SEM for each region.
Measurements of astrocytic number
To quantify changes in astrocyte number we obtained designbased optical fractionator estimates of S100h-positive soma,
together with nucleator estimates of astrocyte volume. Immunoreactive astrocytes were sampled in S1BF and CA1/stratum oriens
using a series of counting frames distributed over a grid and
superimposed onto the section using StereoInvestigator software,
exactly as described previously (Bible et al., 2004). The rostral and
caudal limits of S1BF and CA1/stratum oriens regions were
defined as described above. A 40 oil objective (NA 1.30) was
then used to count clearly identifiable immunoreactive astrocytes,
which fell within the dissector frame. The following sampling
scheme was applied to the regions of interest; Grid area for S100h
in S1BF and CA1/oriens 20,191 Am2, frame area 122,500 Am2.
Statistical analysis
The statistical significance of differences between genotypes of
all quantitative data was assessed using a one-way ANOVA (SPSS
11.5 software, SPSS Inc, Chicago, IL), with statistical significance
considered at P < 0.05. The mean coefficient of error (CE) for all
individual optical fractionator and nucleator estimates was calculated according to the method of Gundersen and Jensen (1987) and
was less than 0.08 in all these analyses.
Results
Homozygous Cln3 Dex7/8 mice exhibit widespread accumulation of
autofluorescent storage material
Macroscopic examination of the brains of 12-month-old
Cln3 Dex7/8 homozygotes did not reveal an obvious degenerative
phenotype, with the cortex and cerebellum of affected mice
appearing indistinguishable from controls (data not shown). Consistent with these observations, the brains of homozygous Cln3 Dex7/8
mice were lighter than age-matched controls, but this reduction did
not reach statistical significance (control 494 T 43 mg; Cln3 Dex7/8
456 T 39 mg, P = 0.128, n = 5). Microscopically, homozygous
Cln3 Dex7/8 mice exhibited widespread intracellular accumulation of
autofluorescent storage material within neuronal soma throughout
the CNS (Fig. 1). In mutant mice, this storage material was present as
punctate material that fluoresced at multiple wavelengths, as viewed
by conventional epifluorescence microscopy (Figs. 1B – D, F – G). In
Fig. 1. Accumulation of autofluorescent storage material in homozygous Cln3 Dex7/8 mice. (A – H) Representative images of unstained coronal sections through
the primary motor cortex (A – D) and cerebellum (E – H) viewed by epifluorescence using rhodamine (Rho) or FITC filter sets. Sections from 12-month-old
homozygous Cln3 Dex7/8 mice display widespread intracellular accumulation of storage material (B – D, F – H) that fluoresces with both filter sets and appears
yellow in merged images (D, H). In contrast, sections from littermate controls display a low level of background tissue fluorescence and many fewer scattered
deposits of storage material within the cortex (A), and a low level of autofluorescence within Purkinje neurons of the cerebellum (E).
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
contrast, control littermates displayed a low level of background
autofluorescence and an age-related and sparsely scattered accumulation of autofluorescent material (Fig. 1A) that was more
pronounced in Purkinje neurons of the cerebellum (Fig. 1E).
Lack of significant regional atrophy in homozygous Cln3 Dex7/8
knock-in mice
Since homozygous Cln3 Dex7/8 brains did not display an overt
phenotype macroscopically, we carried out a stereological survey
of regional volume and cortical thickness to look for more subtle
neurodegenerative changes in Nissl-stained sections. Cavalieri
estimates of regional volume revealed that although many CNS
regions including the cortex, hippocampus and cerebellum were
reduced in size in Cln3 Dex7/8 mice, the thalamus was the only
structure that displayed significantly reduced volume in homozygous Cln3 Dex7/8 mice (Fig. 2).
Regional effects upon cortical thinning and lamination in
homozygous Cln3 Dex7/8 mice
Because measurements of overall cortical volume cannot
discriminate between events in different cortical subfields, we
carried out a series of cortical thickness measurements of primary
motor (M1), somatosensory barrel field (S1BF), primary visual
(V1) and lateral entorhinal (Lent) regions (Fig. 3). Homozygous
Cln3 Dex7/8 mice showed variable effects upon cortical thickness in
different regions. There was significant thinning of S1BF and Lent
cortex, but a significant increase in the thickness of M1 (Fig. 3A).
In contrast, V1 exhibited less pronounced thinning in homozygous
Cln3 Dex7/8 mice that was not significant versus littermate controls
(Fig. 3A).
To determine whether these changes were due to laminarspecific events, we made measurements of individual lamina
thickness (Figs. 3B – E). Although significant reductions in the
thickness of lamina I were consistently seen in S1BF, V1 and Lent
and the thickness of laminae II + III in M1, S1BF and Lent (Figs.
3B – D), the remaining laminae in these cortical regions displayed a
827
complex series of contrasting changes in thickness (Table 1). The
most extreme example was in Lent which displayed significant
reductions in the thickness of laminae I and II, but significantly
increased thickness of laminae III and IV (Fig. 3D). The increase in
the thickness of M1 in homozygous Cln3 Dex7/8 mice appeared to be
largely due to a significant increase in the thickness of lamina V
(Fig. 3E), whereas all other laminae were either unchanged (I and
VI) or displayed a small, but significant thinning (II + III).
Regional and laminar effects upon neuronal number in
homozygous Cln3 Dex7/8 mice
To investigate whether these changes in individual laminar
thickness in homozygous Cln3 Dex7/8 mice were the result of effects
upon alterations in neuronal number and/or neuronal size, we
obtained optical fractionator estimates of neuronal number and
nucleator estimates of neuronal volume in Nissl-stained sections.
These data were collected for lamina V pyramidal neurons in S1BF
and M1, two cortical regions which displayed contrasting changes
in the thickness of this lamina, in addition to lamina IV granule
neurons of S1BF and a combined measure of laminae II and III
neurons in S1BF (Fig. 4).
Although there was a trend to reduced neuronal number, lamina
V pyramidal neurons were not significantly lost in either M1 or
S1BF of homozygous Cln3 Dex7/8 mice (Fig. 4A). In contrast, there
was a significant loss of neurons in laminae II and III of S1BF of
mutant mice, together with a significant loss of granule neurons in
lamina IV of this cortical region (Fig. 4A). Nucleator estimates of
neuronal volume revealed only minor changes in cell size in
individual laminae (Fig. 4B), none of which reached statistical
significance.
To determine the effect of this loss of granule neurons in S1BF
upon afferent thalamic neurons, we obtained unbiased stereological
estimates of neuronal number and volume in the ventral posterior
thalamic nucleus (VPM/VPL). These analyses revealed a significant loss of neurons within VPM/VPL of homozygous Cln3 Dex7/8
mice (Fig. 4A), although these neurons were not significantly
hypertrophied (Fig. 4B).
Fig. 2. Absence of significant regional atrophy in homozygous Cln3 Dex7/8 mice. Unbiased Cavalieri estimates of regional volume reveal no significant regional
atrophy in Cln3 Dex7/8 homozygotes versus littermate controls (+/+) at 12 months of age, with the exception of the thalamus (Thal). Regions examined include
cortical mantle (Cortex); hippocampus (Hipp); hypothalamus (Hypoth); striatum (CPu); whole cerebellum (Cb total); cerebellar vermis (Cb vermis); and lateral
hemisphere (Cb lat). *P < 0.05, one-way ANOVA.
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C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
Fig. 3. Subregion- and laminar-specific changes in cortical thickness in homozygous Cln3 Dex7/8 mice. (A) Cortical thickness measurements. Compared with
littermate controls (+/+), thinning of the cortical mantle in Cln3 Dex7/8 homozygotes was more pronounced in primary somatosensory barrel field (S1BF) and
entorhinal (Lent) cortex than primary visual (V1), or primary motor (M1) cortex which exhibited a small, but significant increase in overall thickness. (B – E)
Laminar thickness measurements in these cortical regions reveal a complex series of changes in thickness of individual cortical laminae in 12-month-old
homozygous Cln3 Dex7/8 mice compared with littermate controls (+/+). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. These data are also
summarized in Table 1.
Survival of GABAergic interneurons in homozygous Cln3 Dex7/8
Loss of GABAergic interneurons is a common feature of other
murine models of NCL (Cooper, 2003; Mitchison et al., 2004). To
determine whether these neuronal populations were also affected
in homozygous Cln3 Dex7/8 mice, the expression of parvalbumin
(PV) and somatostatin (SOM) were surveyed in the cortex (Fig.
5A) and hippocampus (Figs. 5B and C). These markers are
usually expressed in representative interneuron populations
(Freund and Buzsáki, 1996), which are consistently affected in
mouse models of infantile NCL (Bible et al., 2004; Jalanko et al.,
2005), juvenile NCL (Mitchison et al., 1999; Pontikis et al., 2004)
and variant late infantile NCL (Cooper et al., 1999; Kopra et al.,
2004).
Primary motor and somatosensory cortex
Optical fractionator estimates revealed a general trend to a
reduced number of interneurons immunoreactive for PV and SOM
in M1 and S1BF, but this loss of interneurons did not reach
significance for interneurons stained for either antigen in these
cortical regions (Fig. 5A).
Hippocampus
We next examined interneuron populations that are immunoreactive for these markers in the hippocampal formation of
homozygous Cln3 Dex7/8 mice. There was an overall trend to
reduced number of SOM-positive interneurons in homozygous
Cln3 Dex7/8 mice, although this did not reach significance in any
hippocampal subfield (Fig. 5C). Effects upon the number of PV-
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
829
Table 1
Tabular depiction of the significant changes in individual laminar thickness
in the primary motor (M1), somatosensory barrel field (S1BF), primary
visual (V1) and entorhinal cortex (Lent) of 12-month-old Cln3 Dex7/8
compared with age-matched controls (+/+)
M1
S1BF
V1
Lent
I
II/III
III
IV
V
VI
Total thickness
NS
,
,
,
,
,
NS
,
na
na
na
j
na
j
,
j
j
,
NS
NS
NS
,
j
NS
j
,
NS
,
Significantly j = thicker in Cln3 Dex7/8 ; , = significantly thinner in Cln3 Dex7/8 ;
NS = not significant; na = not applicable, M1 has no lamina IV. A combined
measurement of laminae II and III was made in all regions except Lent where
these laminae were measured separately.
positive interneurons were more variable between hippocampal
subfields in homozygous Cln3 Dex7/8 mice, but none of these
changes reached statistical significance (Fig. 5B).
Fig. 4. Thalamocortical neuron loss in homozygous Cln3 Dex7/8 mice. (A)
Optical fractionator estimates of neuronal number revealed the significant
loss of neurons in laminae II and III and lamina IV granule neurons, but no
significant loss of lamina V pyramidal neurons in primary somatosensory
barrelfield cortex (S1BF) or primary motor cortex (M1) of homozygous
Cln3 Dex7/8 mice compared with littermate controls (+/+). There was also a
significant reduction in the number of neurons in the ventral posterior
thalamic nucleus (VPM/VPL) of mutant mice. (B) Nucleator estimates of
neuronal volume revealed no significant atrophy or hypertrophy in any
laminae of Cln3 Dex7/8 homozygotes. *P < 0.05, ***P < 0.001, one-way
ANOVA.
Fig. 5. Interneuron populations are not significantly affected in homozygous Cln3 Dex7/8 mice. (A) Persistence of cortical interneurons in aged
Cln3 Dex7/8 homozygotes. Optical fractionator estimates of parvalbumin
(PV) and somatostatin (SOM)-positive interneuron number in the primary
motor (M1) and somatosensory barrelfield (S1BF) cortex reveal no
significant loss of these neurons in 12-month-old Cln3 Dex7/8 homozygotes
compared with littermate controls (+/+). (B – C) Survival of hippocampal
interneurons in aged homozygous Cln3 Dex7/8 mice. Counts of hippocampal
interneuron number revealed no significant loss of interneurons immunoreactive for either parvalbumin (B) or somatostatin (C) in any subfield of 12month-old Cln3 Dex7/8 and age-matched controls (+/+).
Regionally restricted astrocytic and microglial activation in
homozygous Cln3 Dex7/8 mice
To examine glial responses in homozygous Cln3 Dex7/8 mice,
we first surveyed the distribution of cells immunoreactive for the
astrocytic markers GFAP and S100h, in addition to the microglial
marker F4/80. Subsequently, we used image analysis to quantify
the expression of these markers in the striatum, cortical subregions
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C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
M1 and S1BF; and hippocampal subfields hilus, CA1, CA2 and
CA3 using standard methods (Bible et al., 2004; Pontikis et al.,
2004). Reactive astrocytosis may represent hypertrophy of
astrocytes (thickening of processes and changes in cell body
volume) which may be independent of cell proliferation. Therefore, we also obtained optical fractionator estimates of the
number of astrocytes immunoreactive for S100h, a calcium
binding protein that is expressed predominantly in astrocytes
(Boyes et al., 1986).
GFAP- and S100b-positive astrocytes
In the neocortical grey matter of control mice, only few GFAPpositive astrocytes were present, predominantly in lamina VI and in
lamina I adjacent to the pial surface (Fig. 6A). In marked contrast,
homozygous Cln3 Dex7/8 mice displayed a profound astrocytosis
across the cortical mantle with intensely GFAP-immunoreactive
astrocytes present in both superficial and deeper cortical laminae
(Fig. 6B), with positive astrocytes extending in clusters across all
laminae. At higher power, these GFAP-positive astrocytes displayed marked hypertrophy with numerous thickened and branched
processes and enlarged soma in homozygous Cln3 Dex7/8 mice
compared to control littermates (Figs. 6A and B). In marked
contrast to GFAP, S100h immunoreactivity within the neocortex
was present within numerous intensely stained cell somas that were
distributed evenly across laminae and did not differ obviously
between animals of either genotype (Figs. 6C and D).
Subcortically, this astrocytic response was far less pronounced
with reduced staining for GFAP present within the hippocampus
and cerebellum (data not shown). In contrast, the thalamus of
homozygous Cln3 Dex7/8 mice exhibited pronounced GFAP immunoreactivity compared to controls (Figs. 6E and F). However, this
staining for GFAP was not uniformly distributed but was highest in
individual thalamic nuclei, most notably within the ventral
posterior (VPM/VPL), lateral regions of the laterodorsal (LD)
Fig. 6. Prominent astrocytosis in homozygous Cln3 Dex7/8 mice. (A, B) Immunohistochemical staining for GFAP reveals profound astrocytosis in the cortex of
12-month-old Cln3 Dex7/8 homozygotes (B) compared with age-matched controls (+/+, A). Within the primary motor cortex (M1) many hypertrophic GFAPpositive astrocytes bearing numerous thickened processes were present in both deep and superficial laminae of mutant mice (B). In contrast, there were no
obvious differences in the distribution or number of S100h-positive astrocytes in M1 of animals of either genotype (C, D). Compared to controls (E), the
thalamus of Cln3 Dex7/8 homozygotes exhibited pronounced astrocytosis that was confined to individual thalamic nuclei (F), with GFAP-positive astrocytes
prominent in the ventral posterior (VPM/VPL), mediodorsal (MD) and lateral regions of the laterodorsal (LD) thalamic nuclei, but virtually absent in the
adjacent ventromedial (VM), posterior (Po) and centrolateral (CL) thalamic nuclei.
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
and the mediodorsal (MD) thalamic nuclei, but virtually absent in
the adjacent ventromedial (VM), posterior (Po) or intralaminar
thalamic nuclei (Fig. 6F).
Quantitative analysis of astrocytosis
Consistent with these morphologic observations, quantitative
image analysis revealed a significant and widespread increase in
831
GFAP expression in both superficial and deeper cortical layers (Fig.
7A). This upregulation was not confined to any cortical region with
similar increases in GFAP staining in M1, S1BF, V1 and Lent (Fig.
7A). In contrast to these events in the neocortex, quantitative analysis
revealed a range of different astrocyte responses in the hippocampus
(Fig. 7B), cerebellum (Fig. 7C) and subcortical structures of
Cln3 Dex7/8 homozygotes. Unexpectedly, the hippocampal dentate
Fig. 7. Regionally variable astrocytosis in homozygous Cln3 Dex7/8 mice. (A) Quantitative image analysis reveals the widespread and significantly increased
expression of GFAP in the superficial and deep cortical laminae of Cln3 Dex7/8 homozygotes compared with littermate controls (+/+) at 12 months of age. (B –
C) In contrast, hippocampal (B) and cerebellar grey matter (C) display significantly reduced levels of GFAP in mutant mice compared with controls. Areas
surveyed include striatum (CPu), hippocampal dentate gyrus (DG), hippocampal stratum oriens and CA1 (Oriens CA1), cerebellar white matter (WM) and
molecular and granule cell layers (Mol + Gr) in the lateral hemispheres (Lat) and vermis (Verm). (D) Optical fractionator estimates revealed no significant
change in the number of S100h-positive astrocytes in either the primary somatosensory barrel field cortex (S1BF) or hippocampal CA1 and stratum oriens of
homozygous Cln3 Dex7/8 mice, compared with littermate controls (+/+). ***P < 0.001, one-way ANOVA.
832
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
gyrus, stratum oriens and CA1 of homozygous Cln3 Dex7/8 mice
exhibited significantly reduced GFAP expression compared to
control littermates (Fig. 7B). GFAP expression in homozygous
Cln3 Dex7/8 mice was similarly reduced in the grey matter of
cerebellar lateral hemisphere and vermis, but was unchanged in
the white matter within these regions of the cerebellum (Fig. 7C).
To determine whether these changes in GFAP expression
reflected changes in the number of astrocytes, we next obtained
unbiased stereological estimates of the number of S100h-positive
astrocytes. These optical fractionator data were collected within
one representative region that displayed increased GFAP expression (S1BF) and one that displayed decreased GFAP expression
(stratum oriens and CA1 of the hippocampus) but did not reveal
any significant change in astrocyte number within either region
of homozygous Cln3 Dex7/8 mice compared with control littermates (Fig. 7D).
F4/80-positive microglia
Compared to control littermates, F4/80 immunoreactive microglia were consistently more prominent in homozygous Cln3 Dex7/8
mice, with these differences again more prominent in the
neocortex than other CNS regions (Fig. 8). In the neocortex of
control littermates, the widespread distribution of microglia was
revealed by pale F4/80 immunoreactivity within cell soma and
thin processes extending into the neuropil (Figs. 8A and C). In
contrast, throughout the cortical mantle of Cln3 Dex7/8 homozygotes, there were intensely F4/80 immunoreactive microglia with
numerous ramified processes, often with more prominent cell
soma (Figs. 8B and D). In homozygous Cln3 Dex7/8 mice, these F4/
80 immunoreactive microglia were present with no particular
focus and across all laminae, although F4/80-positive cells
frequently displayed more morphological signs of activation in
deeper laminae with fewer thickened processes.
Fig. 8. Activation of microglia in homozygous Cln3 Dex7/8 mice. Immunohistochemical staining for F4/80 revealed graded activation of microglia in 12-monthold homozygous Cln3 Dex7/8 mice compared with littermate controls (+/+). At this age, F4/80-positive microglia with numerous ramified processes in the
primary motor cortex (M1) were more darkly stained in Cln3 Dex7/8 homozygotes (B, D) than littermate controls (A, C). This difference between genotypes was
less pronounced in the striatum (CPu), although numerous partially activated microglia with enlarged soma and short thickened processes were evident in
homozygous Cln3 Dex7/8 mice (F) compared to the microglia with thin ramified processes that were present in littermate controls (E).
C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
Differences in the relative intensity of F4/80 staining between
genotypes were less marked in subcortical structures (Figs. 8E and
F), although homozygous Cln3 Dex7/8 mice again displayed more
morphological evidence of microglial activation in these regions.
Although complete morphological transformation to brain macrophage-like morphology in homozygous Cln3 Dex7/8 mice was
seldom seen, these mice frequently displayed F4/80 immunoreactive microglia with enlarged soma and numerous short
thickened processes (Fig. 8F), compared to the threadlike and
ramified processes in control mice (Fig. 8E).
Discussion
This study represents the first detailed description of the CNS
of homozygous Cln3 Dex7/8 mice, a model that recapitulates the
major deletion present in the vast majority of JNCL alleles
(Cotman et al., 2002). Our data revealed no interneuron loss or
significant regional atrophy in Cln3 Dex7/8 homozygotes by 12
months of age. The marked exception was the thalamus, which
displayed regional atrophy and a significant loss of somatosensory
relay neurons in mutant mice, together with loss of their target
neurons within the somatosensory cortex. These findings provide
significant new data of neuronal loss within the thalamocortical
system in JNCL, which was accompanied by a pronounced astrocytic response within individual thalamic nuclei of Cln3 Dex7/8
homozygous mice. Astrocytic activation was prominent cortically,
yet was diminished within the hippocampus and cerebellum of
mutant mice. These data provide further evidence that both
neuronal and glial responses in JNCL are localized and regionally
specific, as typified by our novel findings within the thalamocortical system of homozygous Cln3 Dex7/8 mice.
Homozygous Cln3 Dex7/8 mice as a model for JNCL
Bearing the major deletion that is present in over 85% of CLN3
alleles in JNCL (International Batten Disease Consortium, 1995),
Cln3 Dex7/8 knock-in mice are an accurate model of this disorder
(Cotman et al., 2002). Certainly, the homozygous Cln3 Dex7/8 mice
used in this study exhibit an early onset of neurologic deficits
(Cotman et al., 2002) that are more pronounced than the relatively
mild retinal and neurological phenotypes of Cln3 / (Katz et al.,
1999; Mitchison et al., 1999; Seigel et al., 2002). Nevertheless, any
comparisons between these mice are complicated by the different
strain backgrounds these models have been raised on (Mitchison et
al., 1999; Cotman et al., 2002). As such, it will be essential to reevaluate their phenotypes once these mouse models of JNCL are
all available upon a common strain background.
Selective loss of cortical neurons in homozygous Cln3 Dex7/8 mice
Although cortical atrophy is a consistent feature of various
mouse models of NCL, including Cln3 / (Mitchison et al., 1999;
Pontikis et al., 2004), PPT1 / (Bible et al., 2004) and TPP1deficient mice (Sleat et al., 2004), atrophy of the neocortex of
homozygous Cln3 Dex7/8 mice was relatively mild and may only
become more pronounced with increased age. Indeed, since
approximately 20% of these mice do not reach 12 months of age
(Cotman et al., 2002), it is possible that by performing our analysis
at this age, we have selected mice that may present with a less
pronounced or delayed neurodegenerative phenotype.
833
Our data from 12-month-old homozygous Cln3 Dex7/8 knock-in
mice did reveal a range of selective effects upon cortical thickness
that varied between different subfields (Fig. 3). These effects
extended to individual laminae and, as highlighted by data from the
somatosensory cortex of homozygous Cln3 Dex7/8 mice, to neuronal
number within these laminae (Fig. 4). The most pronounced loss
within this cortical region was of laminae II and III neurons, which
supply commissural and association projections to other areas of
cortex. The loss of these neurons may potentially have significant
consequences for coordinating neuronal activity between hemispheres and cortical regions. In this respect, it will be important to
determine the functional correlates of these pathological changes in
the cortex of homozygous Cln3 Dex7/8 mice.
Although GABAergic interneuron subpopulations were
depleted to different extents in various cortical and hippocampal
subfields, unlike human JNCL (Tyynelä et al., 2004), the Cln3 /
model of JNCL (Mitchison et al., 1999; Pontikis et al., 2004) or
other mouse models of NCL (Cooper et al., 1999; Bible et al.,
2004; Kopra et al., 2004; Jalanko et al., 2005), these cell
populations were not significantly affected in homozygous
Cln3 Dex7/8 mice. The lack of significant loss of these interneurons
in Cln3 Dex7/8 homozygotes may be characteristic of these mice, or
alternatively, this phenotype may take longer to develop.
Loss of thalamic neurons in homozygous Cln3 Dex7/8 mice
Our analysis of regional volume was informative revealing
thalamic atrophy in homozygous Cln3 Dex7/8 mice, a phenotype that
is also apparent in PPT1-deficient mice (Bible et al., 2004).
Although the thalamus typically displays a reduced MRI signal
intensity in symptomatic INCL and JNCL patients (Autti et al.,
1997; Vanhanen et al., 2004), the cellular basis of this hypointensity has remained unknown. Indeed, in contrast to the wealth of
information available for the cortex and hippocampus (Braak and
Goebel, 1978, 1979; Haltia, 2003; Haltia et al., 2001; Tyynelä et
al., 2004), neuropathological data of neuronal loss within
subcortical structures in JNCL are extremely limited (Braak and
Braak, 1987; Braak et al., 1979). As such, our data from
homozygous Cln3 Dex7/8 mice are significant in providing the first
direct evidence for the loss of thalamic relay neurons in this
disorder. It is unclear whether thalamic neurons are themselves
inherently vulnerable in JNCL, or degenerate in response to events
within the cortex. Determining the precise sequence of events in
the thalamocortical system during pathogenesis will be crucial to
distinguish between these distinct mechanistic possibilities.
The clinical consequences of these events within the thalamocortical system remains unclear, although progressive changes in
somatosensory evoked potentials are consistently reported in
multiple forms of NCL (Tackmann and Kuhlendahl, 1979;
Vercruyssen et al., 1982; Vanhanen et al., 1997; Lauronen et al.,
1997), including JNCL (Lauronen et al., 1997, 1999). Indeed, it has
been suggested that progressive thalamic dysfunction may also
contribute to the sleep disturbances that are characteristic of the
NCLs (Vanhanen et al., 1997). It will be highly informative to
determine whether the loss of thalamic neurons and/or their cortical
targets contributes to similar phenotypes in homozygous Cln3 Dex7/8
knock-in mice.
We have already reported motor coordination problems by 12
months of age in homozygous Cln3 Dex7/8 knock-in mice (Cotman
et al., 2002), but this current study demonstrated no overt effects
within the cerebellum of these mice. Although subtle effects on
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C.C. Pontikis et al. / Neurobiology of Disease 20 (2005) 823 – 836
other neuronal populations may also be present, our data suggest
that neurologic problems in homozygous Cln3 Dex7/8 knock-in mice
may involve altered sensory feedback through the thalamus. In this
context, it will be important to correlate sensory behaviors and
other forms of neurological impairment with pathological changes
in the thalamocortical system during disease progression.
Prominent astrocytosis and graded microglial responses in
Cln3 Dex7/8 homozygotes
Pronounced gliosis is a consistent feature of autopsy material
from individuals with JNCL (Braak and Goebel, 1978, 1979;
Tyynelä et al., 2004) and other forms of NCL (Haltia et al.,
1973a,b; Herva et al., 2000; Tyynelä et al., 1997, 2004). Similar
reactive events are evident in mouse models of CLN8 (Cooper et
al., 1999), and infantile NCL (Bible et al., 2004; Jalanko et al.,
2005), but a considerably more subtle glial phenotype is evident in
Cln3 / (Pontikis et al., 2004). Markers of astrocytic and
microglial activation are significantly elevated by 5 months of
age in Cln3 / , but this activation remains at a low level with no
morphological transformation to brain macrophages or obvious
astrocytic hypertrophy (Pontikis et al., 2004).
Our data from Cln3 Dex7/8 homozygotes provide a distinctly
different picture of reactive changes in these mice, with widespread
astrocyte hypertrophy across the cortical mantle and variable
responses subcortically (Fig. 6). Curiously, there was little
evidence of astrocytic proliferation or hyperplasia in homozygous
Cln3 Dex7/8 mice, with no difference in the number of S100hpositive astrocytes between animals of either genotype. These
S100h data also suggest that reduced GFAP immunoreactivity
evident in the cerebellum and hippocampus of homozygous
Cln3 Dex7/8 mice appears to represent downregulation of this
marker rather than the targeting of astrocytes that has been
proposed in Cln3 / (Pontikis et al., 2004).
Although astrocytes classically mediate responses to neuronal
injury (Raivich et al., 1999), emerging evidence suggests these cell
types play dynamic roles in regulating fluid homeostasis (Simard
and Nedergaard, 2004) and controlling local concentrations of
glutamate, influencing neuronal activity and synaptic efficacy
(Oliet et al., 2001; Piet et al., 2002). Although the localized events
that trigger astrocytosis in homozygous Cln3 Dex7/8 mice remain
unclear, these may involve neuronal damage due to accumulated
storage material. However, in contrast to the widespread accumulation of storage material (Fig. 1), astrocytic activation within the
thalamus was restricted to individual nuclei (Fig. 6), with
particularly prominent GFAP staining within the VPM/VPL which
also exhibited neuronal loss (Fig. 4). These reactive changes may
represent a localized protective response to the effects of the CLN3
mutation, as has been suggested in human JNCL (Tyynelä et al.,
2004). In this respect, investigating the precise spatiotemporal
relationship between astrocytosis and neuronal loss at different
stages of disease progression is likely to be highly informative.
In contrast to these prominent astrocytic responses in Cln3 Dex7/8
homozygotes, the microglial responses in these mice were
markedly less pronounced. The activation of microglia is a
progressive and graded phenomenon that takes place in different
Fstages_ from extensively ramified cells to full blown brain
macrophages (Raivich et al., 1999), and has been considered a
sensitive marker of local neuronal damage (Streit, 2000, 2002). In
Cln3 Dex7/8 homozygous mice this microglial activation rarely
reached its full extent to reveal brain macrophage morphology,
even in areas where a profound astrocytic response was evident.
These data suggest that whatever underlying molecular cues trigger
astrocytosis in Cln3 Dex7/8 knock-in mice, these are not sufficient to
promote full morphological transformation of microglia. Alternatively, microglia may themselves be targeted by the effects of
Cln3 mutation as has been suggested in Cln3 / mice (Pontikis et
al., 2004).
Taken together with data from other mouse models of NCL
(Cooper et al., 1999; Lam et al., 1999; Mitchison et al., 1999; Bible
et al., 2004), sheep with NCL (Oswald et al., 2001, in press) and
human NCL (Tyynelä et al., 2004), our data suggest that highly
regionalized interactions occur between neurons and glia at
different stages of disease progression. Whether these events
contribute directly to subsequent neurodegeneration has not been
demonstrated, but the suggestion that neuron – glial interactions
differ markedly between CNS regions may provide important clues
to the pathogenesis of these disorders.
Acknowledgments
We would like to thank the other members of the PSDL for their
valuable contributions; Noreen Alexander and Stephen Shemilt for
their expert advice; Dr. Payam Rezaie for his continuing
collaboration; and Drs. David Pearce, Alison Barnwell and Jill
Weimer for constructive comments on the manuscript. These
studies were supported by National Institutes of Health grants
NS41930 (JDC), NS33648 (MEM), European Commission 6th
Framework Research Grant LSHM-CT-2003-503051 (JDC), a
post-doctoral fellowship to SLC from the Batten Disease Support
and Research Association, and grants to JDC from The Natalie
Fund, Batten Disease Family Association, Batten Disease Research
Trust and the Remy Fund.
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