Download Distinct Neuropathologic Phenotypes After Disrupting the

J Neuropathol Exp Neurol
Copyright Ó 2010 by the American Association of Neuropathologists, Inc.
Vol. 69, No. 12
December 2010
pp. 1228Y1246
ORIGINAL ARTICLE
Distinct Neuropathologic Phenotypes After Disrupting the
Chloride Transport Proteins ClC-6 or ClC-7/Ostm1
Sarah N.R. Pressey, MSc, Kieran J. O’Donnell, MSc, Tobias Stauber, PhD, Jens C. Fuhrmann, PhD,
Jaana Tyynelä, PhD, Thomas J. Jentsch, PhD, and Jonathan D. Cooper, PhD
INTRODUCTION
Abstract
The proteins ClC-6 and ClC-7 are expressed in the endosomallysosomal system. Because Clcn6-deficient mice display some features
of neuronal ceroid lipofuscinosis (NCL), CLCN6 may be a candidate
gene for novel forms of NCL. Using landmarks of disease progression
from NCL mouse models as a guide, we examined neuropathologic
alterations in the central nervous system of Clcn6j/j, Clcn7j/j,
and gl mice. gl mice bear a mutation in Ostm1, the A-subunit critical
for Clcn7 function. Severely affected Clcn7j/j and gl mice have
remarkably similar neuropathologic phenotypes, with pronounced
reactive changes and neuron loss in the thalamocortical system, similar
to findings in early-onset forms of NCL. In contrast, Clcn6j/j mice
display slowly progressive, milder neuropathologic features with very
little thalamic involvement or microglial activation. These findings
detail for the first time the markedly different neuropathologic consequences of mutations in these two CLC genes. Clcn7j/j and gl mice
bear a close resemblance to the progressive neuropathologic phenotypes of early onset forms of NCL, whereas the distinct phenotype of
Clcn6-deficient mice suggests that this gene could be a candidate for a
later-onset form of mild neurologic dysfunction with some NCL-like
features.
Key Words: Chloride channel, Chloride/proton exchanger, Grey
lethal, Lysosomal storage disorder, Neuronal ceroid lipofuscinosis/
Batten disease.
From the Pediatric Storage Disorders Laboratory, Department of Neuroscience (SNRP, KJO, JDC), Centre for the Cellular Basis of Behavior,
Institute of Psychiatry, King’s College London, UK; Leibniz Institute for
Molecular Pharmacology [FMP] and Max Delbrück Centre [MDC] for
Molecular Medicine (TS, JCF, TJJ), Berlin, Germany; and Institute of
Biomedicine/ Biochemistry (JT), University of Helsinki, Finland.
Send correspondence and reprint requests to: Jonathan D. Cooper, PhD,
Pediatric Storage Disorders Laboratory, Department of Neuroscience and
Centre for the Cellular Basis of Behavior, MRC Centre for Neurodegeneration Research, James Black Centre, Institute of Psychiatry,
King’s College London, 125 Coldharbour Ln, London, SE5 9NU, UK;
E-mail: [email protected]
Jens C. Fuhrmann is now at Metanomics GmbH, Berlin, Germany.
These studies were supported by National Institutes of Health grant NS41930
(J.D.C.), European Commission 6th Framework Research Grant LSHMCT-2003-503051 (J.D.C.), and a UK Medical Research Council Studentship (S.N.R.P. and J.D.C.), the Deutsche Forschungsgemeinschaft to TJJ
(Je164/7 and SFB 740), and Academy of Finland 1122404 (J.T.). The following nonprofit agencies also contributed financially to this work: The
Batten Disease Support and Research Association (J.D.C.), The Batten
Disease Family Association (J.D.C.), and The Natalie Fund (J.D.C.).
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Members of the CLC family of chloride channels and
transporters have diverse functions in regulating electrical
excitability, performing transepithelial transport, maintaining
electroneutrality during acidification of organelles, and control of ionic homeostasis (1). Much of this information has
come from the disruption of CLC genes in mouse models
(1, 2). Of the 9 mammalian CLCs, 4 are associated with
inherited disorders, with mutations in the genes encoding
ClC-Kb, ClC-1, ClC-5, and ClC-7 that cause Bartter syndrome, myotonia congenita, Dent disease, and osteopetrosis,
respectively (3Y6). ClC-6 and ClC-7 form a distinct branch of
the CLC gene family, sharing 45% sequence homology (7).
ClC-7 requires a A-subunit, Ostm1 (8); mutations in Ostm1
are responsible for the phenotype observed in grey-lethal (gl )
spontaneous mutant mice (9). The gl mice phenotype was
ascribed to a severe loss of ClC-7, which is unstable without
its A-subunit (8). The phenotypes of Clcn6j/j, Clcn7j/j, and
gl mice have unexpectedly suggested links to a group of rare
fatal pediatric storage disorders, the neuronal ceroid lipofuscinoses (NCLs or Batten disease) (10, 11).
Individuals with NCL present with seizures and visual,
intellectual and motor deterioration (12). Because there are no
effective therapies for these autosomal recessive disorders,
their relentless disease course invariably ends in premature
death (12, 13). Disease-causing mutations have been identified
in at least 8 genes (13, 14), but there are still variant and adultonset NCL cases with no characterized molecular basis, suggesting that new disease-causing genes remain to be identified.
The NCLs are characterized by autofluorescent intralysosomal storage material accumulation consisting of a mixture of lipids and proteins, the most abundant being subunit c
of mitochondrial ATPase and saposins (13, 15). This storage
material accumulates widely in central and peripheral tissues,
but there is no evidence that it has a direct role in pathogenesis.
The ultrastructural appearance of the storage material is specific
to different forms of NCL, consisting of granular osmiophilic
deposits, fingerprint, and curvilinear profiles (13, 15). The storage material that accumulates in the lysosomes in the central
nervous system (CNS) of Clcn6j/j, Clcn7j/j, and gl mice
resembles that seen in the NCLs; it is electron-dense, granular
osmiophilic deposit-like, autofluorescent, and stains positively
for subunit c of mitochondrial ATPase (8, 10, 11).
Beyond these common features, the neuronal phenotypes of Clcn6j/j and Clcn7j/j mice differ in many respects.
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J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
Clcn6j/j mice have a normal life span, with mild behavioral
abnormalities and decreased pain sensitivity (11). Upregulation of inflammatory genes in the hippocampi of Clcn6deficient mice suggests that there is an immune response in
these mice (11), similar to that in the NCLs, but this has not
been characterized in detail. gl and Clcn7-deficient mice display disease phenotypes that are very similar to each other and
death occurs by 7 weeks (4, 8). Both of these mice share
neuropathologic phenotypes with the NCLs including severe
retinal and CNS degeneration (4, 8). Clcn7j/j mice show
optic nerve degeneration and associated localized glial activation (4, 10, 16), but this has not yet been investigated in
gl mice. Clcn7j/j and gl mice also display severe osteopetrosis and growth retardation (4, 8). Both CLCN7 and OSTM1
deficiency lead to osteopetrosis in a subset of patients with
juvenile osteopetrosis (4, 9, 17, 18). These patients also have
neurologic signs and brain atrophy as a result of deficiency in
CLCN7 or OSTM1 (19Y21).
Detailed characterization of NCL mouse models has
revealed a series of common themes with respect to the brain
regions and cell types that are affected, although the precise
timing and nature of these events differs among NCL forms
(22Y25). Regional cortical atrophy, localized early gliosis, and
loss of both interneurons and thalamic relay neurons are
strong indicators of disease progression. In light of the
similarities in CNS pathology between the NCLs and Clcn6-,
Clcn7-, and Ostm1-deficient mice, we examined the progression of neuropathologic phenotypes in these mice to determine their status as potential models for new forms of NCL.
These models provide insight into the mechanisms of neuronal storage, the functions of CLC genes in the CNS and the
distinct neuropathologic consequences of deficiencies of these
2 CLC genes.
MATERIALS AND METHODS
Clcn6- and Clcn7-Deficient Mice
Clcn6- and Clcn7-deficient mice (Clcn6j/j and
Clcn7 ) were both generated by targeted disruption strategies, as previously described (4, 11), Clcn6j/j mice were
backcrossed with C57BL/6 control mice and Clcn7j/j mice
were on a mixed C57BL/6-129SV background. Grey-lethal
mice (gl), which have a spontaneous mutation in Ostm1 (8, 9),
were obtained from the Jackson Laboratory (Bar Harbor, ME)
and crossed onto the C57BL/6 background. Clcn6j/j,
Clcn7j/j, and gl mutant mice and age-matched littermate
controls were maintained at the Max Delbrück Centre, Berlin.
All animal procedures performed in accordance with national
and institutional animal welfare directives.
j/j
Histologic Analysis
The brains of deficient mice and age-matched controls
were examined at 3 time points during their life span, when
they were presymptomatic, moderately and more severely
affected. Clcn6j/j mice have a normal life span and were
examined at 55, 92, and 107 weeks. Because Clcn7j/j mice
have an earlier onset disease phenotype and die between 6 and
7 weeks, their brains were examined at 1.5, 3, and 4 weeks.
gl mice were also examined at 4 weeks.
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Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
Brains were removed and immersion fixed in 4% paraformaldehyde (Sigma-Aldrich, Poole, UK) in 0.1 mol/L phosphate buffer, pH 7.4 for 3 days and then cryoprotected in 30%
sucrose, 0.5% sodium azide in 50 mmol/L Tris-buffered saline
(TBS), pH 7.6. Frozen coronal sections 40 Km thick through
the rostrocaudal extent of the cortical mantle were collected
at 1 per well in 96-well plates containing a cryoprotective
solution (30% ethylene glycol [Sigma-Aldrich], 15% sucrose,
0.05% sodium azide in TBS; Sigma-Aldrich) (26). All subsequent histologic analyses were performed blind to genotype.
Histochemical Staining
Every sixth section through each brain was slide-mounted
and incubated in 0.05% cresyl fast violet (Merck, Darmstadt,
Germany), 0.05% acetic acid in water for 30 minutes at 60-C,
rinsed in deionized water, and then differentiated through an
ascending series of alcohols before clearing in xylene and
coverslipped with DPX (VWR, Poole, UK) (23, 26).
Periodic acid Schiff (PAS) staining was used to determine
the presence of accumulated carbohydrate storage material (27).
Sections were slide-mounted and left to dry overnight and then
incubated in periodic acid (Sigma-Aldrich) for 2 minutes and then
rinsed with several changes of dH2O. Sections were next incubated in the Schiff reagent for 2 minutes, followed by washing
for 2 minutes in running tap water and dehydrated through an
alcohol series, cleared in xylene, and coverslipped with DPX.
Immunohistochemistry
A standard immunoperoxidase protocol was used to
detect glial fibrillary acid protein (GFAP), microglia (F4/80),
lysosomes (Cathepsin D [CtsD]), and GABAergic interneurons (somatostatin [SOM], parvalbumin [PV], calretinin [CR],
and calbindin [CB]) (10, 11, 26, 28).
Endogenous peroxidase activity was quenched in 1%
H2O2 (VWR) in TBS for 15 minutes. Sections were then rinsed
in TBS and blocked in 15% normal serum (Vector, Northampton, UK) in TBS with 0.3% Triton-X (TBS-T; SigmaAldrich) before incubating in the appropriate primary antibody;
polyclonal rabbit anti-GFAP (1:4000; DakoCytomation, Ely,
UK), rat anti-mouse F4/80 (1:100; Serotec, Oxford, UK), rabbit
anti-CtsD (1:1000; Millipore-Upstate, Watford, UK), rabbit antiSOM (1:1000; Bachem, Bubendorf, Switzerland), rabbit antiPV (1:5000; Swant, Bellinzona, Switzerland), rabbit anti-CR
(1:10,000; Swant), or rabbit anti-CB (1:20,000; Swant) in 10%
normal serum in TBS-T overnight at 4-C. Sections were rinsed
in TBS and incubated with the appropriate biotinylated secondary antibodies; swine anti-rabbit (1:1000; DakoCytomation),
rabbit anti-rat (1:200; Vector), and goat anti-rabbit (1:1000;
Vector) for 2 hours at room temperature. Subsequently, sections
were rinsed in TBS, followed by incubation in avidin-biotinperoxidase complex (1:1000, Vectastain Elite ABC kit; Vector)
in TBS for 2 hours and then rinsed in TBS. To visualize immunoreactivity (IR), sections were incubated in 0.05% 3,3¶diaminobenzidine tetrahydrochloride (Sigma-Aldrich) containing 0.001% H2O2 in TBS for up to 25 minutes, depending
on antigen, and then rinsed in ice-cold TBS. Finally, sections
were mounted on gelatin-chrome-coated Superfrost microscope
slides (VWR), air-dried overnight, cleared in xylene, and coverslipped with DPX.
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Pressey et al
J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
A standard immunofluorescence protocol was used to
reveal the abundance and distribution of lysosomes (CtsD)
within neurons (NeuN) or microglia (CD68). Sections were
blocked in 15% normal serum in TBS-T, then probed with
the following primary antibodies: rabbit anti-CtsD (1:1000;
Millipore-Upstate), mouse anti-NeuN (1:1000; Millipore),
and rat anti-CD68 (1:2000; Serotec) in 10% normal serum in
TBS-T overnight at 4-C. After rinsing in TBS and incubating
with the appropriate fluorescent secondary Alexa Fluor antibodies (Molecular Probes, Invitrogen, Carlsbad, CA) for 2 hours
at room temperature, sections were rinsed in TBS and mounted
on gelatine-chrome-coated microscope slides and coverslipped
with fluoromount-G (Southern Biotech, Birmingham, AL).
Regional Volume, Cortical Thickness, and
Cell Number Estimation
Stereological estimates were obtained using StereoInvestigator software (Microbrightfield, Inc, Williston, VT),
with regions of interest (ROIs) defined by referring to the
neuroanatomic landmarks described by Paxinos and Franklin
(29). Cavalieri estimates of regional volume (23, 28) were
obtained using the Nissl-stained sections described. A sampling grid was superimposed over the ROI and the number
of points covering the relevant area was assessed. The volume
in cubic micrometers was estimated from a series of sections
through each ROI. Sampling grids and series used were as
follows: for brain subdivisions for Clcn7j/j and gl mice, a
150-Km2 grid was used for all regions; for cortex, 1:12 series;
and all other regions, 1:6 series. For Clcn6j/j mice: cortex,
1:12, 250 Km2; striatum, 1:12, 150 Km2; thalamus, 1:6,
150 Km2; and hippocampus, 1:6, 200 Km2. Volumes of representative cortical subfields that serve different functions
were also obtained from the same sections. These included the
primary somatosensory (S1BF), primary motor (M1), lateral
entorhinal (LEnt), and primary visual (V1) cortices, using a
150-Km2 grid on a 1:6 series for all mice. For the estimation
of the volume of white matter tracts, a 100-Km2 grid was used
for all mice on a 1:12 series for the corpus callosum and a 1:6
series for the internal capsule and cerebral peduncle. Analyses
were carried out on an Olympus BX50 microscope (Olympus,
Southend-on-Sea, UK) linked to a DAGE-MTI CCD-100
camera (Dage-MTI, Michigan City, IN).
The optical fractionator probe was used to estimate
cell numbers (30) in Nissl-stained sections of thalamic relay
nuclei and lamina IV-VI of the S1BF and in sections through
S1BF stained for markers of interneuron populations known to
be vulnerable in this brain region (23, 25, 26). Nissl-stained
cells were only counted if they had a neuronal morphology and
a clearly identifiable nucleus; astrocytes and microglia with
small soma and cells with indistinct morphology were not
counted. A line was traced around the boundary of the ROI, a
grid was superimposed, and cells were counted within a series
of dissector frames placed according to the sampling grid size.
Nissl+ neurons and SOM+, PV+, CR+, or CB+ interneurons
were counted using the 100 and 40 objectives, respectively.
Different grid and dissector sizes were determined according to
each brain region using a coefficient of error value of less than
0.1 to indicate sampling efficiency. Because of the different
brain sizes and ages investigated, these sampling parameters
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were different for each mouse model, with the following
sampling schemes used: For Nissl-stained sections in Clcn7j/j
and gl mice: lateral geniculate nucleus (LGN) grid 125 125 Km, frame 74 42 Km; ventral posterior thalamic nucleus
(VPM/VPL), grid 175 175 Km, frame 74 43 Km; medial
geniculate nucleus (MGN) grid 200 200 Km, frame 50 35 Km; S1BF lamina IV, grid 150 150 Km, frame 41 26
Km; S1BF lamina V, grid 200 200 Km, frame 60 40 Km;
S1BF lamina VI, grid 200 200 Km, frame 60 40 Km. For
Nissl-stained sections in Clcn6j/j mice: VPM/VPL grid
225 225 Km, frame 74 42 Km; S1BF lamina IV, grid
150 150 Km, frame 41 26 Km; S1BF lamina V,
grid 175 175 Km, frame 60 40 Km; and S1BF lamina VI,
grid 200 200 Km, frame 60 40 Km. A 1:6 series was used
for these analyses. For all sections stained for PV: grid 250 200 Km, frame 150 90 Km, 1:12 series. For all sections
stained for SOM, CR and CB grid 250 180 Km, frame
180 100 Km, 1:12 series.
Because of the comparatively low abundance of interneurons in the hippocampus versus the neocortex, stereological methods prove inefficient for estimating hippocampal
interneuron numbers without sampling the entire tissue.
Therefore, to determine the number of interneurons present in
different subfields of the hippocampus, counts of interneurons
were made manually in each defined hippocampal subfield, as
previously described (26, 31). Data were plotted as the mean
number of interneurons per section positive for each marker in
each of these hippocampal subfields.
Quantitative Analysis of Glial Phenotypes
The optical density of GFAP-IR was assessed using
semiautomated thresholding image analysis, as previously
described (28, 32). Using a 40 objective, 40 nonoverlapping
images representing the entire ROI were taken from 4 consecutive sections starting from a defined anatomic landmark.
Image Pro Plus image analysis software (Media Cybernetics,
Chicago, IL) was used to determine the area of IR for each
antigen in each region by applying a threshold that discriminated staining from background in each image. Data
were plotted as the mean percentage area of IR per field T
SEM for each region.
Statistical Analysis
Microsoft Excel (Redmond, WA) was used for data
collection and SPSS (SPSS, Inc, Chicago, IL) for statistical
analysis. To test for significance between genotypes, Student
t test or analysis of variance with post hoc Bonferroni analysis
were used as appropriate. The mean coefficient of error for all
individual optical fractionator and Cavalieri estimates was
calculated according to the method of Gundersen and Jensen
(33) and was less than 0.1 in all these analyses.
RESULTS
Clcn7j/j, gl, and Clcn6j/j mice have a lysosomal
storage disease that resembles the NCLs (8, 10, 11). Detailed
landmarks of disease progression such as selective atrophy, localized glial activation, the loss of interneurons, and
thalamocortical neurons are available for many of the forms of
murine NCL (22). To assess whether similar events occur in
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Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
FIGURE 1. Distinct cortical and white matter atrophy in Clcn7-and Clcn6<deficient mice. (A, B) Unbiased Cavalieri estimates of
regional volume revealed cortex-specific atrophy in severely affected 4-week-old Clcn7j/j mice versus age-matched littermate controls (+/+) that was not evident in 3-week-old mice (A). Hipp = hippocampus. There were no differences in regional volume of
severely affected 92-week-old Clcn6j/j mice (B). (C, D) Cavalieri estimates of the primary motor (M1), lateral entorhinal (LEnt),
primary visual (V1), and somatosensory barrel field (S1BF) cortex revealed selective atrophy of S1BF in 4-week-old Clcn7j/j mice (C)
and of the LEnt in 92-week-old Clcn6j/j mice (D). Both these events occurred late as there was no change in S1BF volume in 3-weekold Clcn7j/j mice (C) or LEnt volume in 55-week-old Clcn6j/j mice (D). (EYG) Cavalieri estimates of white matter tract volumes
revealed atrophy of the corpus callosum in Clcn7j/j mice that became evident at 4 weeks (E), and atrophy of the internal capsule
that became significant at 4 weeks (F). Clcn6j/j mice showed atrophy of the corpus callosum from 55 weeks (G). *, p G 0.05; **,
p G 0.01; ***, p G 0.001; using Student t test. n = 3Y5; error bars = SEM.
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FIGURE 2. Localized astrocytosis in thalamic relay nuclei and basal ganglia in Clcn7-, but not Clcn6-deficient mice. (A) Immunohistochemical staining for glial fibrillary acidic protein (GFAP) revealed pronounced astrocytosis in the basal ganglia and thalamic
nuclei of Clcn7j/j mice at 4 weeks. Thalamic nuclei boundaries are marked with white dashed lines. LGN indicates lateral geniculate nucleus; MGN, medial geniculate nucleus; Rt, reticular thalamic nucleus; VPM/VPL, ventral posterior thalamic nucleus. Basal
ganglia: GP indicates globus pallidus; SN, substantia nigra. (B) GFAP immunoreactivity was first evident in Clcn7j/j mice in the
VPM/VPL at 1.5 weeks and became progressively more widespread with increased age, extending to the MGN in 3-week-old mice.
(C) GFAP+ astrocytes are scattered throughout the thalamus of Clcn6j/j mice with no particular focus. Scale bar = 500 Km.
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Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
FIGURE 3. Distinct patterns of astrocytosis in the cortex of Clcn7j/j and Clcn6j/j mice. Immunohistochemical staining for glial
fibrillary acidic protein (GFAP) revealed progressive activation of astrocytes in the cortex of Clcn7j/j and Clcn6j/j mice. Comparison to adjacent Nissl-stained sections enabled the definition of cortical laminar boundaries. (A) At 1.5 weeks, more prominent
GFAP+ astrocytes were present in Clcn7j/j compared with age-matched control mice (+/+); they are progressively more widespread and intensely stained at 3 and 4 weeks. Dense bands of astrocytosis were evident in lamina IV and the boundary between
lam V and VI in the somatosensory barrel field cortex (S1BF) from 3 weeks. (BYE) In Clcn6j/j mice, there was a more generalized
pattern of astrocytosis with GFAP+ astrocytes scattered throughout the cortex in sensory (B, S1BF) and ventral regions (D, piriform
cortex) occurring most frequently in deep laminae. Quantitative thresholding image analysis in the S1BF (C) and piriform cortex
(E) of Clcn6j/j mice demonstrated consistent upregulation of GFAP immunoreactivity versus age-matched controls (+/+).
*, p G 0.05; ***, p G 0.001; analysis of variance, n = 3Y5; error bars show SEM). Scale bar = 250 Km.
Clcn7j/j and Clcn6j/j mice, the CNS of mutant and control
mice were examined at presymptomatic, moderately and
severely affected stages and were compared with severely
affected gl mice.
Regional Atrophy in Clcn7j/j and Clcn6j/j Mice
The Cavalieri method was used to obtain estimates of
regional volumes (23). This analysis revealed a late-onset
atrophy of the cortex in Clcn7j/j mice (Fig. 1A), but in no
other brain region in severely affected mice at 4 weeks. This
cortical atrophy displayed subfield specificity and was only
significant in the S1BF at this age; there was no significant
difference at earlier ages (Fig. 1C). A similar late-onset atrophy of the LEnt was evident in Clcn6j/j mice (Fig. 1D),
which displayed no other regional atrophy, even at 92 weeks
(Fig. 1B). These data indicate regionally specific effects on
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the sensory cortex of Clcn7j/j mice and on the archicortex
of Clcn6j/j mice toward the end of their disease progression.
There was a significant late-onset atrophy of the corpus
callosum, the internal capsule, and the cerebral peduncles in
severely affected Clcn7j/j mice at 4 weeks (Figs. 1E, F). In
Clcn6j/j mice, only the corpus callosum displayed significant
atrophy that was evident from 55 weeks (Fig. 1G). Thus, there
was widespread atrophy of white matter in Clcn7j/j mice
versus more selective effects on the corpus callosum in
Clcn6j/j mice.
Clcn7j/j Mice But Not Clcn6j/j Mice Display
Pronounced Localized Astrocytosis
Astrocytosis can be a sensitive marker of ongoing
neuronal dysfunction or injury (22, 34, 35). In control mice,
GFAP-IR was confined to fibrous astrocytes within the white
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matter. Only occasional faintly stained protoplasmic astrocytes were scattered in the thalamus of control mice at all ages
(Figs. 2A, C). The GFAP-IR in thalamic nuclei of Clcn7j/j
mice appeared at different stages of disease progression
according to the sensory modality these nuclei relay (Figs. 2A,
B). It started in the VPM/VPL (somatosensory/pain information) and subsequently extended to the MGN (auditory information) (Fig. 2B), LGN (visual information), and reticular
thalamic nucleus (Rt) (provides inhibitory input to the whole
thalamus) with increased age. Another distinctive feature of
Clcn7j/j mice was the presence of intense GFAP-IR within
basal ganglia and midbrain nuclei. This was first evident at
3 weeks in the globus pallidus and substantia nigra (not shown)
and progressed in density and intensity by 4 weeks (Fig. 2A).
This early-onset and progressive astrocytosis in the
thalamus of Clcn7j/j mice was accompanied by similar
laminar-specific events in the corresponding cortical regions
of these mice, which became more widespread with increased
age. At 1.5 weeks, numerous faintly stained astrocytes were
already evident in the S1BF of Clcn7j/j mice (Fig. 3A).
There was an intensely stained band of hypertrophic astrocytes in lamina IV and at the boundary of laminae V and VI
by 3 weeks (Fig. 3A), which further increased in intensity by
4 weeks (Fig. 3A). This phenotype was mirrored in the primary visual and auditory cortex of Clcn7j/j mice without the
intensely stained band of GFAP-IR in lamina IV and at the
border between laminae V and VI (not shown).
In marked contrast, Clcn6j/j mice displayed more
generalized astrocytosis. In the thalamus of these mice,
GFAP+ astrocytes were particularly evident at the level of the
VPM/VPL and LGN (Fig. 2C) but were scattered widely with
no specific focus. Similarly, numerous GFAP+ astrocytes
were also evident throughout the cortical mantle of Clcn6j/j
mice and were most densely packed in deeper laminae of
sensory cortical regions (Fig. 3B) and in ventral areas of the
cortex such as the piriform cortex (Fig. 3D). This phenotype
was evident at 55 weeks and subsequently became slightly
more pronounced with increased age. Thresholding image
analysis revealed that this increase in GFAP-IR in the cortex
of Clcn6 -deficient mice was significant at 55 and 92 weeks in
the S1BF (Fig. 3C) and piriform cortex (Fig. 3E). A brain
from an older mouse killed at 107 weeks displayed no further
IR increase (Figs. 3B, D).
Collectively, these data reveal markedly different degrees
of astrocytosis in Clcn6- and Clcn7-deficient mice, with scattered and diffuse astrocytosis in the thalamus of Clcn6j/j mice
and a more prominent cortical astrocytosis evident part way
through disease progression, but that does not become more
Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
widespread. In contrast, Clcn7j/j mice display more pronounced and rapidly progressing astrocytosis that is initially
localized to individual thalamic nuclei and cortical laminae,
spreading to become widespread with disease progression.
Localized Microglial Activation in Clcn7j/j Mice
Microglial activation is a consistent feature of many
neurodegenerative disorders (34), but its timing and relationship to neuron loss and astrocytosis varies markedly. F4/80 is
commonly used to reveal the progressive morphologic transformation of resting microglia that have numerous thin ramified processes to a brain macrophage-like morphology with an
enlarged cell soma (23, 26, 34).
In Clcn6j/j mice, a faintly stained network of F4/80+
processes was evident in all brain regions (Figs. 4A, C), even
in the oldest mice, with very little difference from the
appearance of microglia in control mice (Figs. 4A, C). In
marked contrast, intense F4/80-IR was evident in the CNS of
4-week-old Clcn7j/j mice in the same brain regions that
exhibited GFAP-IR (Fig. 4). Darkly stained F4/80+ microglia
were present in VPM/VPL, LGN, MGN, and Rt nuclei of
the thalamus and in the globus pallidus and substantia nigra
(Fig. 4A). In the cortical mantle there was an intense band of
F4/80+ microglia in lamina IV of S1BF, the same location as
the most intense GFAP-IR (Figs. 4C, D). Similar to astrocytosis, microglial activation was also progressive and became
more pronounced with increased age (Fig. 4B) but remained
more localized than astrocytosis within each region. The
microglia mostly had many long ramified processes and a cell
soma that was scarcely visible indicating that they had not
become fully activated (Fig. 4A, inserts).
These data provide further new evidence for marked
differences in nature and timing of the neuroimmune responses in Clcn6- and Clcn7-deficient mice. Compared with the
pronounced and localized reactive changes in Clcn7j/j mice,
these responses are far less prominent in Clcn6j/j mice.
Storage Material in Clcn7j/j and Clcn6j/j Mice
The ultrastructural appearance of storage material that
accumulates in lysosomes of neurons in Clcn7j/j and
Clcn6j/j mice resembles that seen in the NCLs (10, 11). In
NCLs, this material accumulates throughout the brain, whereas
neuron loss and gliosis occur in specific brain regions and cell
types (22). The ultrastructural appearance of the storage material has been well characterized in Clcn7j/j and Clcn6j/j
mice, and we examined the distribution of this material.
Periodic acid Schiff staining revealed progressive accumulation of carbohydrates throughout the cortex of Clcn7j/j
FIGURE 4. Microglial activation in Clcn7- but not in Clcn6-deficient mice. (A) Immunohistochemical staining for the microglial
marker F4/80 revealed localized upregulation of microglia in basal ganglia and thalamic nuclei of 4-week-old Clcn7j/j mice but not
in severely affected 92-week-old Clcn6j/j mice. Microglia in Clcn7j/j mice were highly ramified but were not fully transformed
into amoeboid microglia. Thalamic nuclei boundaries are marked with white dashed lines. GP indicates globus pallidus; LGN, lateral
geniculate nucleus; MGN, medial geniculate nucleus; Rt, reticular thalamic nucleus; SN, substantia nigra; VPM/VPL, ventral posterior thalamic nucleus. (B) Microglial activation was progressive in Clcn7j/j mice with a low level of F4/80 immunoreactivity
evident at 1.5 weeks, followed by intense F4/80 immunoreactivity in thalamic nuclei at 3 weeks. (C) There were activated microglia
in lamina IV of the somatosensory barrel field cortex (S1BF) of 4-week-old Clcn7j/j mice, but no F4/80+ microglia were apparent in
the cortex of 92-week-old Clcn6j/j mice. (D) Corresponding fields of F4/80 and GFAP immunoreactivity in the S1BF of Clcn7j/j
mice (white lines mark laminar boundaries). Scale bar = (AYC) 500 Km; higher-power views in A = 20 Km; (D) 250 Km.
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Pressey et al
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J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
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J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
mice, in accordance with Kasper et al (10) (Fig. 5A). Small
cytoplasmic aggregates were initially present throughout all
cortical laminae and subregions but were largest in pyramidal
neurons of lamina V (Fig. 5A, inserts); they became more
pronounced with increased age. The accumulation of carbohydrates in lamina V did not correlate with the most intense
band of astrocytosis and microglial activation in lamina IV,
which contains granule neurons that receive thalamic input
(Fig. 5A, arrowheads).
Compared with the cortex, accumulation of PAS+
material was less prominent and relatively delayed in the
thalamus of Clcn7j/j mice (Fig. 5B). Cellular aggregates
were spread diffusely not affecting every neuron. This generalized distribution of storage in the thalamus was in contrast
to the localized activation of glia within specific thalamic
nuclei of these mice (Figs. 2 and 4).
We also used the lysosomal marker CtsD as a second
marker of the intracellular distribution of storage material. In
the cortex of Clcn7j/j mice, large round clumps of CtsD-IR
were evident in lamina IV, where glial activation was most
intense (Fig. 5A, arrowheads). Costaining with the microglial
marker CD68 demonstrated that these large accumulations of
storage material were present in microglia (Fig. 6, yellow
arrows), a feature seen in many forms of NCL (22, 36). In
contrast in lamina V, CtsD staining revealed long, thin accumulations of lysosomes in the proximal axon segment
(Fig. 5A, insert). Here, costaining with the neuronal marker
NeuN confirmed that the accumulated storage material was
present in neurons (Fig. 6, red arrows). Prominent accumulations of CtsD-IR were also apparent within the thalamus of
Clcn7j/j mice with large round clumps present in the VPM
(Fig. 5B), which was revealed to be storage material in
microglia in this nucleus (Fig. 4A). In contrast, in the adjacent
posterior nucleus, where very few activated microglia were
present, the storage material was visible within neurons as
long, thin clumps from 3 weeks (Fig. 5B).
In Clcn6j/j mice, CtsD-IR lysosomal aggregates
were apparent within neurons throughout the cortex and the
thalamus (Fig. 5C). CtsD-IR was evident in the initial axon
segment of neurons and was clearly seen in lamina II/III and
V of the cortex, in accordance with Poet et al (11). This CtsDIR was confirmed to be in neurons by staining for NeuN
(Fig. 6, red arrows). Although microglial activation is not
pronounced in these Clcn6j/j mice, where microglia were
present, they also showed CtsD-IR (Fig. 6). This widespread
distribution of neuronal storage in Clcn6j/j mice resembles
Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
the generalized and widespread upregulation of GFAP in
these mice.
Neuron Loss in the Thalamocortical System of
Clcn7j/j and Clcn6j/j Mice
Reports of neuron loss have been confined to the hippocampus and cerebellum of Clcn7j/j mice (10). Neurodegeneration has not yet been reported in Clcn6j/j mice (11).
To investigate relationships between astrocytosis, microglial
activation, and neuron loss, we focused on the thalamic nuclei
(VPM/VPL, MGN, and LGN) and cortical laminae (IV, V,
and VI) in S1BF that displayed prominent astrocyte and
microglial activation. We also investigated cortical interneuron populations that are known to degenerate in NCL
models (22, 26).
In 4-week-old Clcn7j/j mice, optical fractionator estimates of neuron number in the S1BF revealed a significant
loss of neurons in laminae IV, V, and VI (Fig. 7A). This
occurred first in laminae that receive projections from the
thalamus (lamina IV) or that project back to the thalamus
(lamina VI) and only subsequently in lamina V (Figs. 7,
AYC). There was also a loss of SOM+, PV+, and CB+ interneurons in the S1BF of Clcn7j/j mice, but CR+ interneurons
in this region were spared (Fig. 7D). In severely affected
Clcn6j/j mice, significant S1BF neuron loss was confined to
lamina VI thalamic feedback neurons, with no loss either of
lamina IV and V neurons (Fig. 7F) or of SOM+ and PV+
interneurons (Fig. 7G). In the thalamus of Clcn7j/j mice,
there was a delayed and significant loss of somatosensory
relay neurons in VPM/VPL (Fig. 7E) but no loss of auditory
or visual relay neurons in MGN or LGN, respectively
(Fig. 7E) or of any subpopulation of thalamic relay neurons
that were severely affected in Clcn6j/j mice (Fig. 7H). Thus,
the extent and pattern of thalamocortical neuron loss differ
markedly in Clcn7j/j and Clcn6j/j mice.
Distinct Hippocampal Pathology in Clcn7j/j
and Clcn6j/j Mice
Hippocampal pathology has been briefly described in
Clcn7j/j mice (10) and is a key feature of murine NCL, with
loss of selected inhibitory interneuron populations (22).
Therefore, we next compared pathologic changes in hippocampi of Clcn7j/j and Clcn6j/j mice. There was a progressive increase in staining for both GFAP and F4/80 in the
hippocampus of Clcn7j/j mice (Fig. 8A) with increased age.
Astrocytosis and microglial activation were most intense in
FIGURE 5. Storage material is evident throughout Clcn7j/j and Clcn6j/j mice. Histochemical staining and immunohistochemistry
(IHC) reveal storage material in the cortex and thalamus of Clcn7j/j and Clcn6j/j mice. (A) Periodic acid Schiff stain reveals
storage material in the cortex of Clcn7j/j mice, from 1.5 weeks; staining becomes more intense at 3 and 4 weeks. Storage material
is most prominent in lamina V (arrows, inserts). Likewise, immunohistochemical staining with Cathepsin D (CtsD) revealed storage
material in the proximal axon segments of neurons in lamina V. This did not correlate with the most intense glial activation which
occurred in lamina IV, where CtsD reactivity revealed storage material accumulation in microglia (arrow heads). (B) Cathepsin D
(CtsD)+ storage material in microglia (arrowheads) that was evident in the thalamus of Clcn7j/j mice from 3 weeks. CtsD+ storage
material is shown in microglia in the ventral posterior medial thalamic nucleus (VPM) and the adjacent posterior thalamic nucleus
(Po) where microglia were not evident. Neuronal storage material is visible in long, thin clumps. (C) Storage material in the cortex
of Clcn6j/j mice in lamina II/III and V; inserts show lamina V and throughout the thalamus; VPM is shown. Scale bar = 100 Km in
cortex strips, 20 Km in cortex inserts, and 10 Km in thalamus images.
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FIGURE 6. Neuronal lysosomal aggregates in Clcn7j/j and Clcn6j/j mice. (A) Costaining with the markers NeuN for neurons,
CD68 for microglia, and cathepsin D (CtsD) for lysosomes shows a punctate distribution of lysosomes (green) in neurons (adjacent
to blue nuclear NeuN) in control mice (+/+). (BYD) In Clcn7j/j and Clcn6j/j mice, CtsD reactivity is clustered together in clumps
(green). In lamina IV in Clcn7j/j mice, CD68+ microglia are full of CtsD (yellow arrows) (B), whereas clumps of CtsD reactivity
adjacent to neuronal nuclei were evident in lamina V (C, red arrows). In Clcn6j/j mice, CtsD reactivity was also clumped in neurons
(D, red arrows) and microglia (yellow arrows). Scale bar = 10 Km.
CA3 of the pyramidal layer (Fig. 8A, asterisk), and there was
an additional region of intense F4/80-IR in the dentate gyrus
(Fig. 8A, 2 asterisks). These reactive changes were accompanied by a progressive increase in storage material, with the
distribution of the most prominent clumps of PAS reactivity
correlating with the intense band of glial upregulation in CA3
(Fig. 8A). Smaller PAS+ aggregates were also present
throughout CA1/2 and the dentate gyrus of Clcn7j/j mice.
CtsD staining showed both storage material in microglia in
CA3 (Fig. 8A, insert) and smaller clumps of neuronal storage
throughout this pyramidal cell layer.
As with the thalamocortical system, the hippocampal
pathology in Clcn6j/j mice was much less pronounced than
that of Clcn7j/j mice. GFAP-IR and F4/80-IR were com-
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parable to those in control mice (not shown). However, there
were clumps of CtsD-IR storage material adjacent to neurons
of the pyramidal cell layer in all hippocampal subfields
(Fig. 8B), which were already evident in 55-week-old mice.
There was a very restricted pattern of hippocampal
neuron loss in both Clcn7j/j and Clcn6j/j mice that was
only evident at the end stages of disease (Fig. 8C). Despite the
loss of CA3 pyramidal neurons evident in Clcn7j/j mice
(10), there was no significant loss of PV+, CR+, or CB+
interneurons in any region of the hippocampus of these mice
(Figs. 8, EYG). Loss of SOM+ interneurons was confined
to the stratum radiatum of aged Clcn7j/j mice (Fig. 8D).
Similar results were obtained in aged Clcn6j/j mice, with no
significant loss of SOM+ or PV+ interneurons in any subfield
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Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
FIGURE 7. Loss of distinct populations of thalamocortical neurons in Clcn7j/j and Clcn6j/j mice. (AYC) Optical fractionator
estimates of Nissl+ neurons in the S1BF of 4-week-old Clcn7j/j mice revealed neuron loss in all lamina examined (IV, V, and VI) (A).
Onset of neuron loss was first evident in lamina IV and VI at 3 weeks (B) but was not seen at 1.5 weeks (C). Neuron loss was
relatively delayed in lamina V and was only evident at 4 weeks (A, B). (D) Assessment of survival of interneurons in the S1BF of
Clcn7j/j mice revealed loss of somatostatin (SOM)+, parvalbumin (PV)+, and calbindin (CB)+ neurons but not of calretinin (CR)+
neurons. (E) Estimates of numbers of Nissl+ thalamic relay neurons revealed the selective loss of neurons from the ventral posterior
thalamic nucleus (VPM/VPL) of 4-week-old Clcn7j/j mice. There was no difference in cell survival between Clcn7j/j mice and agematched controls at 3 weeks. (FYH) In Clcn6j/j mice, there was a selective loss of S1BF Nissl+ lamina VI neurons (F), but no effect
on cell survival of SOM+ or PV+ interneurons in the S1BF (G) or of Nissl+ neurons in the VPM/VPL (H). *, p G 0.05; ***, p G 0.001,
Student t test, n = 4Y5; error bars = SEM.
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FIGURE 8. Distinct hippocampal pathology in Clcn7j/j and Clcn6j/j mice. (A) Immunohistochemical staining for astrocytes (glial
fibrillary acidic protein [GFAP]), microglia (F4/80), and lysosomal storage material (CtsD) and histochemical staining for storage
material (PAS; carbohydrates) identify CA3 pyramidal layer (*, inserts) and the dentate gyrus (**) as sites of pathology in Clcn7j/j
mice. (B) In Clcn6j/j mice, CtsD immunoreactivity reveals accumulation of storage material in the pyramidal layer in long thin
clumps (insert from CA3). (C) Schematic diagram illustrating the hippocampal subfields analyzed for hippocampal survival. (DYG)
Counts of interneurons in discrete hippocampal subfields in Clcn7j/j mice reveal no loss of parvalbumin (PV)+, calretinin (CR)+, or
calbindin (CB)+ interneurons and very restricted loss of somatostatin (SOM)+ interneurons in the radiatum (D) but not in any other
subfield. (H, I) In Clcn6j/j mice, there was no evidence for loss of either SOM+ or PV+ interneurons. Scale bar = 200 Km in lowpower images, 20 Km in inserts (*, p G 0.05, Student t test, n = 3Y4, error bars = SEM).
(Figs. 8H, I). Thus, although glial activation and storage
material accumulation are prominent in the hippocampus of
Clcn7j/j mice, effects on hippocampal interneurons are
much less pronounced than in the cortex where there is significant loss of different types of interneurons.
Similar Pathologic Events in Clcn7j/j and gl Mice
Immunohistochemical staining with markers of glial
activation in gl mice showed localized patches of intense
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GFAP-IR and F4/80-IR subcortically in the VPM/VPL, LGN,
MGN, and Rt thalamic nuclei and in the globus pallidus and
substantia nigra (Fig. 9A), similar to that seen in Clcn7j/j
mice. In the cortex of gl mice, there was widespread astrocytosis with a dark band of intensely stained and densely
packed astrocytes in lamina IV and the boundary of lamina
V/VI of the S1BF (Fig. 9B). The distribution of F4/80-IR in
the cortex of gl mice mirrored that of GFAP-IR, with an
additional intense band of microglial activation in lamina IV
of the S1BF (Fig. 9B). The distribution of storage material
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Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
FIGURE 9. Glial activation and storage material in grey-lethal mice resemble that of Clcn7j/j mice. (A, B) Immunohistochemical
staining for glial fibrillary acidic protein (GFAP), F4/80, cathepsin D (CtsD), and PAS stain revealed a similar pattern of glial activation
and storage material in the thalamus (A) and the cortex (B) of gl and Clcn7j/j mice. Higher magnification images show that
microglia were rarely fully transformed in either mouse model (A, inserts). Thalamic nuclei boundaries are marked with white dashed
lines. GP indicates globus pallidus; LGN, lateral geniculate nucleus; MGN, medial geniculate nucleus; Rt, reticular thalamic nucleus;
SN, substantia nigra; VPM/VPL, ventral posterior thalamic nucleus. Scale bar = (A) 500 Km; insert 20 Km; (B) 250 Km; inserts 20 Km.
also closely resembled that of Clcn7j/j mice, with microglia
full of CtsD+ lysosomes in lamina IV (Fig. 9B, arrows) and
prominent storage material in neurons of lamina V (Fig. 9B,
arrowheads).
gl mice showed overall atrophy of the cortex (Fig. 10A)
that involved atrophy of S1BF, LEnt and V1 but not M1
(Fig. 10B). Consistent with previous qualitative reports (21),
white matter structures were also atrophied in gl mice, with
significantly reduced volume of the corpus callosum, internal
capsule (Fig. 10C) and cerebral peduncle (not shown). Similar
to Clcn7j/j mice, optical fractionator estimates of neuron
number in gl mice showed a significant loss of cortical neurons in laminae IV, V and VI of the S1BF and of SOM+ and
PV+ interneurons in S1BF (Figs. 10D, E) and thalamic relay
neurons in VPM/VPL (Fig. 10F). As in Clcn7j/j mice, hippocampal interneurons were spared (Figs. 10G, H).
Taken together, these data reveal that the neuropathologic phenotypes of gl and Clcn7j/j mice are remarkably similar. The patterns of glial activation and vulnerability
of thalamocortical neuron populations and sparing of hippocampal interneurons suggest that the same regions are pathologic targets of the disease process in these 2 models. There
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was a mild but significant atrophy of the thalamus, hippocampus, LEnt, and V1 in gl mice but not in Clcn7j/j mice.
DISCUSSION
We provide the first detailed comparative neuropathologic characterization of Clcn6j/j, Clcn7j/j, and gl
mice and compare these novel findings to established landmarks of disease progression in the NCLs (Table). Our study
has revealed significant new data of the detailed neurodegenerative and reactive phenotypes of CLC-deficient mice.
The analysis revealed that the underlying neuropathology in
Clcn7j/j and gl mice is remarkably similar, with comparable
patterns of selective cell loss and distribution of reactive
astrocytes, microglia, and storage material. These selective
effects on sensory regions of cortex and the thalamus seen in
Clcn7j/j and gl mice closely mirror those seen in certain
forms of NCL (Table). We also found a much milder and
more slowly progressing pathologic phenotype in Clcn6j/j
mice with astrocytosis confined to the deeper layers of the
cortex that also displayed neuron loss. Thus, the neuropathologic features of Clcn6j/j mice display less resemblance to those of the NCLs (Table). These data reinforce that
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Pressey et al
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FIGURE 10. Similar patterns of atrophy and neuron loss in grey-lethal and Clcn7j/j mice. (A) Cavalieri estimates of regional volume
reveal significant atrophy of cortex, thalamus, and hippocampus of gl mice versus age-matched controls. (B) Cavalieri estimates of
cortical subfields reveal selective atrophy of the somatosensory barrel field cortex (S1BF), primary visual (V1), lateral entorhinal
(LEnt), but not the primary motor cortex (M1) of gl mice. (C) Assessment of white matter tract volumes reveals atrophy of both the
corpus callosum and internal capsule. (DYF) Optical fractionator estimates of neuronal number reveal significant loss of Nissl+
neurons in lamina IVYVI of the S1BF, of somatostatin (SOM)+ and parvalbumin (PV)+ neurons in the S1BF, and of Nissl+ neurons in
the VPM/VPL in gl mice. (G, H) Manual counts of SOM+ and PV+ interneurons in hippocampal subfields did not reveal any
evidence for neuron loss. *, p G 0.05; **, p G 0.01, Student t test, n = 4; error bars = SEM.
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J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
Neuropathology of ClC-6 & ClC-7/Ostm1-Deficient Mice
TABLE. Comparison of Neuropathologic Phenotypes of Clcn7j/j, gl, and Clcn6j/j Mice to a Typical Neuronal Ceroid
Lipofuscinosis Mouse Model
Pathologic Feature
Late-stage cortical atrophy
Selective regional cortical atrophy
Atrophy of white matter tracts
Intense localized astrocytosis in thalamus and cortex
Intense localized microglial activation
Neuronal storage material accumulation
Storage material spread indiscriminately throughout the brain
No direct correlation between storage material distribution and neuron loss
Thalamic relay neuron loss
Cortical neuron loss
Cortical interneuron loss
Hippocampal interneuron loss
NCL
Clcn7j/j / gl
Clcn6j/j
(
(
(
(
(
(
(
(
(
(
(
(
(
( S1BF/V1 and LEnt*
( widespread
(
(
(
,
(
( VPM/VPL only
(
(
,
,
( LEnt only
( corpus callosum only
,
,
(
,
(
,
( Lamina VI only
,
,
Features shared with typical neuronal ceroid lipofuscinosis (NCL) mouse models are indicated with ( ; features that are not typical are indicated as ,.
Clcn7j/j and the naturally occurring gl mutant mice share greater similarities with typical NCL models than do Clcn6j/j mice.
*Atrophy of S1BF, V1 and LEnt where evident in gl mice; selective atrophy of S1BF was evident in Clcn7j/j mice.
LEnt, lateral entorhinal cortex; S1BF, somatosensory barrel field cortex; V1, primary visual cortex; VPM/VPL, ventral posterior thalamic nucleus.
mutations in these two CLC genes have distinctly different
pathologic consequences in the CNS. This study not only
furthers our understanding of the role of these genes within
the brain, but it is also important in their consideration as
candidate genes for variant forms of NCL.
Mice Deficient for Late Endosomal/Lysosomal
CLC Proteins Display Features of NCL
Initial characterization of Clcn7j/j, Clcn6j/j, and
mice revealed some phenotypic similarities to
Clcn3
mouse models of NCL (10, 11, 15, 37, 38). Despite their
severe neurodegeneration, Clcn3j/j mice are not thought to
be an accurate model of NCL because they lack NCL-like
storage material (10). Therefore, we focused on Clcn7j/j and
Clcn6j/j mice. In addition, gl mice bear many similarities to
Clcn7j/j mice and the Ostm1 gene that is mutated in these
mice encodes a A-subunit for ClC-7 (8). Our data provide the
first evidence that the underlying neuropathology of gl mice
also closely resembles that of Clcn7j/j mice. Furthermore,
we found that many of the typical NCL phenotypes are also
apparent in Clcn7j/j mice (Table).
The early onset and rapid progression of the neurodegenerative phenotype of Clcn7j/j mice most closely
resembles that of CtsD -deficient mice (24), which have been
identified as modeling congenital NCL (39). Both mice die
before 2 months and display particularly pronounced pathologic effects within the somatosensory thalamocortical system, including selective loss of somatosensory relay neurons
in VPM/VPL and their cortical targets in lamina IV of S1BF
(Figs. 2Y4 and 7) (24). Clcn7j/j mice also display the
localized astrocytosis and microglial activation within individual thalamic nuclei and loss of cortical interneuron populations that are also evident in multiple forms of NCL
(Table) (23Y26). In contrast to multiple forms of NCL, cortical pathology starts earlier and is more severe than in the
thalamus in Clcn7j/j mice. Neuron loss occurs first and most
profoundly within the cortex; subsequently, more restricted
and late-stage neuron loss was evident in the thalamus of
j/j
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Clcn7j/j mice (Fig. 7). Such predominance of cortical pathology was also recently reported in the Cln5-deficient mouse
model of Finnish variant late infantile NCL (25) and highlights the distinct timing and staging of pathologic events
between models of NCL. Furthermore, despite a loss of pyramidal neurons in CA3 of Clcn7j/j mice (10), hippocampal
inhibitory interneurons were spared in these mice (Fig. 8;
Table). This is surprising considering that loss of hippocampal
interneurons occurs consistently in murine models of infantile, juvenile and variant NCL (26, 28, 31); however, this has
not been characterized in CtsD-deficient mice.
This study also revealed a close correlation between
regions of glial activation and neuron loss in Clcn7j/j mice.
In the thalamus, astrocytosis was first seen in the VPM/VPL
(Fig. 2), the only thalamic nuclei that later displays neuron
loss (Fig. 7). In the cortex, astrocytosis and microglial activation were most pronounced in lamina IV and the boundary
of lamina V and VI (Figs. 3 and 4), and neuron loss was
evident first in lamina IV and VI (Fig. 7). The premature death
of Clcn7j/j mice precludes any investigation of whether
neuron loss and glial activation would have become more
profound in older mice. Inactivation of Clcn7 only in forebrain neurons, however, produces mice that have a normal life
span (16), providing a useful tool to investigate the downstream effects of Clcn7 deficiency in neurons. Clcn7lox/lox;
EMX1cre mice display glial activation solely in the brain
regions in which Clcn7 was deleted and go on to display a
near complete loss of neurons (16). Our data support the
hypothesis that both astrocytes and microglia are activated by
localized factors released by dysfunctional Clcn7-deficient
neurons before they die.
Our analysis also revealed that Clcn6j/j mice display
few similarities to the well-defined neuropathologic phenotypes of NCL mouse models with no evidence of localized
glial responses within the thalamus, loss of thalamic relay
neurons or of GABAergic interneuron populations in the
cortex or hippocampus (Table) (22). However, these mildly
affected mice do show cortical and white matter atrophy,
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Pressey et al
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generalized astrocytosis, and neurodegeneration in lamina VI
of the S1BF where astrocytosis was most pronounced. Thus,
like Clcn7j/j mice, and distinct from most forms of NCL, the
predominant pathologic alterations occur in the cortex of
Clcn6j/j mice.
Finally, our analysis of gl mice revealed that they share
many phenotypes with Clcn7j/j mice, with reactive astrocytes and microglia in the same thalamic nuclei, cortical
laminae, and midbrain nuclei (Figs. 9 and 10). Moreover,
neuron loss in the S1BF and VPM/VPL occurred to a similar
extent in gl mice and Clcn7j/j mice. The only pathologic
discrepancy between these mice was in the small, but significant, atrophy of the hippocampus, thalamus, LEnt, and V1
in gl but not in Clcn7j/j mice. Thus, loss of either Ostm1 or
ClC-7 has remarkably similar consequences on both neuron
populations and glia.
Complex Relationship of Storage Material
With Pathology
The NCLs are characterized by uniform intralysosomal
accumulation of autofluorescent storage material with no
particular preference for any type of neuron or brain region
(13, 15). Indeed, there is no evidence that this storage material
has a direct role in NCL pathogenesis. The present results
suggest that the relationships between accumulation of storage material, distribution of neuron loss, and glial activation
in Clcn7j/j and Clcn6j/j mice are likely to be complex and
may differ between and within brain regions.
In the cortex and thalamus of Clcn7j/j mice, there was
no correlation between storage and neuron loss or glial activation. For example, the most intense band of astrocytosis and
microglial activation occurred in lamina IV and neuron loss
was an early event at 3 weeks; however, there was very little
storage material in this lamina. The opposite was true for
lamina V, in which the most marked storage material was
observed. Very little microglial activation and neuron loss
occurred late, at 4 weeks. In the thalamus of Clcn7j/j mice,
despite the pronounced localized glial activation within individual thalamic nuclei, neuronal storage material was present
in a generalized pattern throughout the thalamus. The clearest
correlation between these pathologic events was, however,
apparent in the hippocampus where astrocytosis, microglial
activation, storage material, and neuron loss were all most
prominent in CA3.
The generalized and less pronounced pathology of
Clcn6j/j mice makes for a more simple comparison; in the
thalamus and hippocampus, the pattern of astrocytosis correlated with the widespread indiscriminate distribution of storage material accumulation with no accompanying neuron
loss. However, in the cortex of these mice, there was a similar
disconnect between events as in Clcn7j/j mice, with neuron
loss restricted to lamina VI and the most prominent storage
material in lamina V.
Candidate Genes for the NCLs
The NCLs are a heterogeneous group of diseases with
mutations in a different gene responsible for each disease
subtype (12). However, there are variant and adult-onset cases
1244
of human NCL with no identified disease-causing mutation
and concerted efforts are being made to identify the underlying genes (http://www.ucl.ac.uk/ncl/RNGC.shtml). Much
valuable information on NCL-like disease phenotypes can be
obtained from engineered or spontaneous mutant mice (22).
For example, Cln8mnd and Cln6nclf were initially identified as
candidate NCL mouse models and were subsequently confirmed as bearing mutations in genes linked to 2 variant forms
of NCL (40Y43). Similarly, the CtsDj/j mouse was reported
to show several NCL-like phenotypes (44, 45), long before
mutations in CTSD were associated with congenital and lateronset forms of NCL (39, 46).
Reports of mice deficient in chloride transporter genes
have raised the possibility of finding another class of genes
that might be responsible for novel forms of NCLs (10, 11).
Our current data support previous observations that Clcn7j/j
mice bear many similarities to NCL models, showing lysosomal storage, retinal and optic nerve degeneration, and
selective loss of hippocampal and cerebellar neurons (10).
Importantly, mice with ClC-7Yrescued osteoclasts live only a
few weeks longer and die of their CNS phenotype (10).
Clinically, patients with mutations in CLCN7 or OSTM1 have
CNS pathology and associated neurologic manifestations, in
addition to osteopetrosis (19, 21). Here, we show for the first
time that mice deficient in Clcn7 or Ostm1 bear a close
resemblance to the progressive neuropathologic phenotype of
early onset forms of NCL (24), with similar populations of
neurons showing vulnerability and a comparable distribution
of glial activation (Table). Although Clcn7 deficiency does
not specifically result in a form of NCL, these data underline
that loss of murine Clcn7 results in CNS outcomes that
resemble NCL with distinct, yet possibly connected, underlying molecular mechanisms. Indeed, the data provide evidence for syndromic forms of NCL in which other symptoms
or disease features are evident in addition to the conventional
presentation of NCL. The use of Bsyndromic NCL[ may be
novel for these disorders but is analogous to the nomenclature used to describe other inherited diseases that have
forms in which additional symptoms are evident, for example,
the osteopetrosis evident in CLCN7 deficiency (4, 17, 18)
or the blindness in Usher syndrome, a form of syndromic
deafness (47Y49).
Clcn7j/j mice and Clcn6j/j mice have distinct phenotypes (Table). Indeed, beyond accumulation of autofluorescent storage material, Clcn6j/j mice display very few
of the common neuropathologic features of NCL. The cortex
is the main site of pathology in Clcn6j/j mice, with no typical early localized thalamic glial activation or cell loss.
Clcn6j/j mice display a distinctive accumulation of storage
material in the proximal segment of the axon hillock (11), a
feature that is not typical of murine NCLs but has been
reported in adult-onset human NCL (50, 51). This feature was
also seen in Clcn7j/j mice in cortical neurons (lamina V; Fig.
5A, inserts) and the thalamus (Fig. 5B, arrows). As such, this
particular phenotype may be specific to these CLC-deficient
mice rather than simply reflecting a delayed disease onset.
Screening a large panel of adult-onset NCL cases did not
reveal any homozygous or compound heterozygous diseasecausing mutations in CLCN6 (11). However, there is currently
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J Neuropathol Exp Neurol Volume 69, Number 12, December 2010
no model for variant late-onset forms of NCL and few histologic data are available from human cases. Indeed, abnormal
pain sensitivity, storage material accumulation (11), and our
data suggest that Clcn6 may prove to be a candidate gene for
late-onset forms of NCL.
Our data also reveal the detailed effects of mutations
in these genes on the CNS. Like many of the proteins
involved in NCL pathogenesis (52), ClC-6, ClC-7, and Ostm1
are expressed within the endosomal/lysosomal system (1)
and colocalize with late endosomal/lysosomal markers (4, 8,
10, 11). Lysosomal Clj conductance was thought to play an
important role in vesicle acidification by neutralizing the H+
conductance created by the vacuolar H+ ATPase (2). The lack
of this Clj current and impaired development of the acidsecreting ruffled border prevents the acidification of the
resorption lacuna in Clcn7j/j osteoclasts, resulting in defective bone resorption and osteopetrosis (4). Nevertheless, the
pH of lysosomes in cultured neurons is indistinguishable
between control and mutant mice (8, 10). The production of
neuropathologic phenotypes shown here and the accumulation of storage material in Clcn6j/j, Clcn7j/j, and gl mice
(8, 10, 11) suggest that the function of these proteins is significant for late endosomal/lysosomal function in neurons.
Indeed, recent evidence from Clcn7j/j kidney cells suggests
that this gene is important for lysosomal protein degradation
(16). Although the mechanisms that underlie chloride transporter deficiency remain poorly understood, it is apparent that
the consequences of mutations in different members of a gene
family can differ markedly, even within the same series of
interconnected pathways. Our current data from Clcn6j/j and
Clcn7j/j mice provide new evidence in support of this
hypothesis with widely divergent and dissimilar neuropathologic phenotypes in these mutant mice
In conclusion, our data clearly highlight that loss of
either Clcn7 or Ostm1 result in similar neuropathologic phenotypes. The broad range of neurologic and neuropathologic
similarities between Clcn7j/j, gl mice, and early-onset forms
of NCL hints at the involvement of convergent functional
pathways in the pathogenesis of these disorders and suggests
that mutations in CLCN7 or OSTM1 may underlie syndromic
NCL. Our data also highlight that loss of Clcn6 has distinct
effects on the CNS and suggest that mutations in CLCN6
could be responsible for NCL-like adult-onset neurologic
dysfunction. Evidence for the physiological function of Clcn6
and Clcn7 as Clj/H+ exchangers is now emerging (2, 53, 54),
and understanding these processes in neurons will provide
crucial insights into the possible mechanisms of cell death and
why materials accumulate within lysosomes.
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