www.elsevier.com/locate/brainres
Brain Research 1023
Research report
Late onset neurodegeneration in the Cln3�/� mouse model of juvenile
neuronal ceroid lipofuscinosis is preceded by low level glial activation
Charlie C. Pontikisa,b,c, Claire V. Cellaa,b, Nisha Parihara,b, Ming J. Lima,c,
Shubhodeep Chakrabartia,b, Hannah M. Mitchisond, William C. Mobleye,
Payam Rezaieb,f, David A. Pearceg,h,i, Jonathan D. Coopera,b,c,e,*
aPediatric 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, UKbDepartment of Neuropathology, Box P040, Institute of Psychiatry, De Crespigny Park, King’s College London, London, SE5 8AF, UK
cDepartment of Neuroscience, Box P040, MRC Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, De Crespigny Park,
King’s College London, London, SE5 8AF, UKdDepartment of Paediatrics and Child Health, Royal Free and University College Medical School, 4th Floor, Rayne Building, 5 University Street,
London, WC1E 6JJ, UKeDepartment of Neurology and Neurological Sciences, Stanford University Medical School, 1201 Welch Road, Palo Alto, CA 94305, USA
fDepartment of Biological Sciences, Faculty of Science, The Open University, Milton Keynes, MK7 6AA, UKgCenter for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USAhDepartment of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
iDepartment of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
Accepted 12 July 2004
Available online 21 August 2004
Abstract
Mouse models of neuronal ceroid lipofuscinosis (NCL) exhibit many features of the human disorder, with widespread regional atrophy
and significant loss of GABAergic interneurons in the hippocampus and neocortex. Reactive gliosis is a characteristic of all forms of NCL,
but it is unclear whether glial activation precedes or is triggered by neuronal loss. To explore this issue we undertook detailed morphological
characterization of the Cln3 null mutant (Cln3�/�) mouse model of juvenile NCL (JNCL) that revealed a delayed onset neurodegenerative
phenotype with no significant regional atrophy, but with widespread loss of hippocampal interneurons that was first evident at 14 months of
age. Quantitative image analysis demonstrated upregulation of markers of astrocytic and microglial activation in presymptomatic Cln3�/�
mice at 5 months of age, many months before significant neuronal loss occurs. These data provide evidence for subtle glial responses early in
JNCL pathogenesis.
D 2004 Elsevier B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Degenerative disease: other
Keywords: Astrocytosis; Microglial activation; GABAergic interneuron; CLN3; JNCL
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.07.030
* Corresponding author. 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. Tel.: +44 20 7848 0286; fax: +44 20 7848 0273.
E-mail address: [email protected] (J.D. Cooper).
1. Introduction
The neuronal ceroid lipofuscinoses (NCLs) are a hetero-
geneous group of at least eight progressive neurodegener-
ative storage disorders, with onset ranging from infancy to
adulthood [9,21]. These autosomal recessive disorders result
from mutations in one of the six different dCLNT genes
(2004) 231–242
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242232
cloned to date [14,21]. However, the precise mechanisms by
which these mutations result in the devastating effects of
these disorders are poorly understood and the therapeutic
outlook for affected individuals is uniformly bleak. Juvenile
NCL (JNCL) is the result of mutations in the Cln3 gene that
codes for a transmembrane protein whose precise function
remains unknown [35]. JNCL typically presents first with
visual failure between 5 and 7 years of age, followed by
progressively more frequent seizures, loss of motor skills,
profound cognitive impairment and an early death [14,21].
Very little detailed quantitative information exists about
which parts of the brain are affected in JNCL and until now,
these studies have been largely restricted to autopsy material
[3–5,37]. However, the recent development of Cln3 null
mutant mice (Cln3�/�) provides an opportunity to inves-
tigate the progressive pathogenesis of the disease [24,27].
Preliminary analysis of these mice on a mixed strain
background revealed characteristic accumulation of auto-
fluorescent storage material and selective pathological
changes in populations of GABAergic interneurons [26], a
phenotype consistent with mice that model other forms of
NCL [2,9,10,25,27].
In addition to widespread neuronal loss, pronounced
gliosis has been described in human NCL autopsy material
[3,4,18–20,36,37]. Similar reactive changes are evident in
mouse models of NCL [2,9,10,17,25,27], but very little is
known about the relative timing of these events. In contrast,
there is an emerging picture of early glial and inflammatory
responses that precede acute neurodegeneration in mouse
models of other storage disorders [22,28,39], prompting us
to investigate whether similar events occur in JNCL.
To begin exploring these issues we have undertaken a
detailed morphological characterization of Cln3�/� mice
and examined the timing and progression of glial activation
compared with the onset of neuronal loss. In this study, we
report that presymptomatic Cln3�/� mice exhibit significant
upregulation of astrocytic and microglial markers at 5
months of age, that is well in advance of the widespread loss
of hippocampal interneurons.
2. Materials and methods
2.1. Animals
Cln3�/� mice inbred on a 129S6/SvEv background and
control (+/+) littermates resulting from heterozygous crosses
were used in this study. Appropriately aged animals were
perfused as described below and fixed brains shipped to the
Pediatric Storage Disorders Laboratory (PSDL), Institute of
Psychiatry for histological analysis. All perfusion proce-
dures were carried out in accordance with the NIH Guide for
the Care and Use of Laboratory Animals (NIH Publications
No. 80-23) and the animal care committee regulations at the
University of Rochester School of Medicine and Dentistry,
and Stanford University Medical School with adequate
measures taken to minimize pain or discomfort. Mice of
both sexes were used for this analysis since previous studies
have revealed no significant difference in NCL-like pheno-
type between male and female mice [26]. Callosal dys-
genesis in mice on the 129S6/SvEv background is well
documented [40], and occurred with equal frequency
between Cln3�/� and control mice used in this study. In
all these analyses we ensured that equivalent numbers of
acallosal mice of either genotype were compared at each age.
2.2. Histological processing
For histological analysis of regional volume and glial
activation, 129S6/SvEv inbred Cln3�/� mice and age-
matched controls (n=3) were perfused at 5 months of age
(asymptomatic), and at 14 months of age, which represent
moderately affected animals (Mitchison, Pearce and Cooper,
unpublished observations). On this strain background
Cln3�/� mice normally survive for 19–20 months of age,
but do not display an obvious seizure phenotype [27].
Analysis of interneuron number was conducted in a further
series of 5 and 14-month-old animals (n=6). All mice were
deeply anesthetized 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 0.1M sodium
phosphate buffer, pH 7.4. Brains were subsequently
removed and post-fixed overnight at 4 8C, cryoprotectedin a solution of 30% sucrose in Tris buffered saline (TBS:
50 mM Tris, pH 7.6) containing 0.05% NaN3 prior to
shipping. 40Am serial coronal sections were cut through the
rostrocaudal extent of each brain (Leitz 1321 freezing
microtome, Leica Microsystems (UK), Milton Keynes, UK),
collected in cryoprotectant solution and stored at �40 8Cprior to histological processing as described previously [2].
2.3. Nissl staining
To provide direct visualization of neuronal morphology a
one-in-six series of sections was mounted onto gelatin-
chrome alum coated Superfrost microscope slides (VWR-
International, UK), air dried overnight and incubated for 45
min at 60 8C in a solution of 0.05% Cresyl Fast Violet
(Sigma, UK) and 0.05% acetic acid (VWR), rinsed in
distilled water and differentiated through a graded series of
alcohols before clearing in xylene (VWR) and coverslipping
with DPX (VWR).
2.4. Immunohistochemistry for interneuron and glial
markers
To survey the survival of hippocampal and cortical
interneurons at 5 and 14 months of age, adjacent one-in-six
series of free-floating frozen sections were stained immu-
nohistochemically to reveal the distribution of neurons
expressing the calcium binding proteins parvalbumin (PV)
Fig. 1. Absence of significant regional atrophy in Cln3�/�. (A, B)
Histograms of unbiased Cavalieri estimates of regional volume do not
reveal significant regional atrophy of any CNS region in Cln3�/� vs. age
matched controls (+/+) at 5 months (A) and 14 months (B) of age. Regions
examined include neocortical mantle (ctx); cerebellum (cereb); hippo-
campus (hp); striatum; thalamus (thal); hypothalamus (hypo).
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242 233
and calbindin (Cb), and the neuropeptide somatostatin
(SOM) [2,10]. These are phenotypic markers that are
normally expressed in subpopulations of cortical and
hippocampal interneurons [13]. The concentrations and
staining conditions were exactly as described previously
[2,10,26], and stained sections were subsequently mounted,
air-dried, cleared in xylene and coverslipped with DPX
(VWR-International, UK).
To examine the extent of glial activation at 5 and 14
months of age, adjacent series of free-floating sections were
immunohistochemically stained using a standard immuno-
peroxidase protocol to detect astrocytes (glial fibrillary
acidic protein, polyclonal rabbit anti-cow GFAP, DAKO,
UK, 1:5000) and microglia (F4/80, monoclonal rat anti-
mouse F4/80, Serotec, Oxford, UK, 1:100) [2]. To provide a
further analysis of microglial phenotype, selected sections
from animals were also stained with CD68, 1:100 or
CD11b, 1:50 (both from Serotec).
Subsequent incubation in the appropriate biotinylated
secondary anti-serum (mouse preadsorbed rabbit anti-rat
IgG, 1:200, F4/80; swine anti-rabbit, 1:400, GFAP; rabbit
anti-rat, 1:250, CD68; rabbit anti-rat, 1:500, CD11b) was
followed by visualization of immunoreactivity according to
standard protocols [2,10]. Immunohistochemistry was pre-
viously optimized and performed on entire batches of
sections to limit interassay variability for subsequent
quantitative image analysis, with staining repeated and
analysis subsequently conducted by two independent
investigators (CCP, NP) who were blind to genotype.
2.5. Measurements of regional volume
We used StereoInvestigator software (Microbrightfield,
Williston, VT) to obtain unbiased Cavalieri estimates of
the volume of the neocortex, hippocampus, striatum,
thalamus and cerebellum in Nissl stained sections from
Cln3�/� and age-matched controls (+/+) at 5 and 14 months
of age, with no prior knowledge of genotype [2]. An
appropriately spaced sampling grid was superimposed over
sections and the number of points covering the relevant areas
assessed using a 2.5� objective. Regional volumes were
expressed in Am3 for each animal and the mean volume of
each region obtained for control and Cln3�/� mice at each
age. All analyses were carried out on a Zeiss, Axioskop 2
MOT microscope (Carl Zeiss, Welwyn Garden City, UK)
linked to a DAGE-MTI CCD-100 camera (DAGE-MTI,
Michigan City, IN).
2.6. Measurements of interneuron number and cross-sec-
tional area
2.6.1. Hippocampus
Due to the comparatively low abundance of interneurons
present in the hippocampus vs. the neocortex, stereological
methods prove inefficient at estimating hippocampal inter-
neuron numbers without sampling the entire tissue [2].
Instead, counts of the number of interneurons expressing,
PV, Cb, and SOM were made exactly as described
previously [2,10,26]. Counts were carried out under a
20� objective and only positively staining cells with clear
neuronal morphology were counted. The number of
interneurons was expressed as the mean number of neurons
per section in each subregion per section and corrected for
oversampling [1].
2.6.2. Entorhinal cortex
The number of GABAergic interneurons expressing PV
in the entorhinal cortex was determined using the design-
based optical fractionator method [42]. This representative
population of cortical interneurons is significantly lost in
another mouse model of NCL [10]. A random starting
section was chosen followed by every sixth section there-
after. Cells were sampled using a series of counting frames
distributed over a grid superimposed onto the section using
Lucivid apparatus and StereoInvestigator software (Micro-
brightfield) [2]. The boundaries of the entorhinal cortex
were defined by comparison with an adjacent series of Nissl
stained sections and anatomical reference points [31]. Only
clearly identifiable PV-positive neurons which fell within
the dissector frame were counted, using a 40� oil objective
(NA 1.30). The following sampling scheme was applied to
Fig. 2. Loss of hippocampal interneurons in aged Cln3�/�. (A–H) Representative photomicrographs of coronal sections through the hippocampus of 14 month
Cln3�/� and age matched controls (+/+) immunohistochemically stained for the interneuron markers somatostatin (SOM in A, B, E, F), and parvalbumin (PV
in C, D, G, H). Aged Cln3�/� had significantly fewer SOM-positive interneurons in the hilus (B) and a less marked reduction in the number of PV-positive
interneurons in the dentate gyrus (D). A similar pattern of neuronal loss was evident in CA1 and the adjacent stratum oriens with significantly fewer SOM-
positive interneurons (F) in aged Cln3�/�, but less pronounced loss of PV-positive interneurons (H).
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242234
the regions of interest. Grid area 32,616 Am2, frame area
20,191 Am2.
2.6.3. Cross-sectional area
As described previously, measurements of the cross-
sectional area of immunoreactive interneurons were made at
maximal equatorial diameter under a 100� objective using
Image-Pro software (Media Cybernetics, Silver Spring,
MD) on digital images captured via a color video camera
(JVC, 3CCD, KY-F55B) [2]. These measurements were
made for at least 50 interneurons positive for each antigen
within each subfield of the hippocampus and for at least 100
PV-positive interneurons in the entorhinal cortex. These
results were presented as cell-size distribution histograms
for corresponding genotypes, per antigen in each subregion,
using a bin size of 20 Am as described previously [2,10,26].
2.7. Quantitative analysis of glial phenotype
The expression of glial markers GFAP and F4/80 was
measured by quantitative thresholding image analysis as
previously described [2,38], with each marker analyzed
blind with respect to genotype. These antigens were
assessed in the striatum, cortical regions M1 and S1BF,
Hilus, CA1, CA2 and CA3 subfields of the hippocampus,
regions which exhibit reactive changes in a mouse models
of infantile NCL [2], using previously defined anatomical
landmarks [2,30]. Briefly, non-overlapping RGB images
were captured across three consecutive sections for each
antigen, providing a thorough and systematic survey
throughout each region. Images were captured via a live
video camera (JVC, 3CCD, KY-F55B), mounted onto a
Zeiss Axioplan microscope using a 40� objective with all
parameters including lamp intensity, video camera setup and
calibration held constant. Subsequently the optimal seg-
mentation of immunoreactive profiles was determined with
the Optimas image analysis system (Media Cybernetics)
using a previously described semi-automated thresholding
method based on the optical density of the immunoreactive
product [2,38]. Foreground immunostaining was accurately
defined according to averaging of the highest and lowest
immunoreactivities within the sample population for each
antigen and this threshold setting was then applied as a
constant to all subsequent images analyzed for this antigen.
Immunoreactive profiles were discriminated in this manner
to determine the specific immunoreactive area (the mean
grey value obtained by subtracting the total mean grey value
from non-immunoreacted value per defined field). Each
field measured 120 Am wide, with a height of 90 Am, the
total area assessed corresponding to 1.8�1.35 mm for each
region. Macros were recorded to transfer the data to a
spreadsheet for subsequent statistical analysis. Data were
separately plotted graphically as the mean percentage area
of immunoreactivity per fieldFS.E.M. for each region.
Fig. 3. Loss of hippocampal interneurons in aged Cln3�/�. (A, B, C)
Histograms of Abercrombie corrected counts of interneuron number in
subfields of the hippocampus of 14-month-old Cln3�/� and age matched
controls (+/+) immunoreactive for parvalbumin (A), somatostatin (B), and
calbindin (C). Significant loss of somatostatin-positive interneurons was
seen in the majority of subfields (B), but with no significant loss of
parvalbumin-positive interneurons (A).
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242 235
2.8. Statistical analysis
The statistical significance of results for GFAP and F4/80
immunoreactivity were assessed via a Mann–Whitney U
and exact probability test. To compare differences between
genotype and age, and also between brain regions, a
repeated measures analysis of variance using multivariate
tests for within variables was conducted. For all other data
statistical significance was assessed by a one-way ANOVA
with post-hoc Bonferroni analysis, P set at b0.05. All
statistical analyses were performed using SPSS software
(SPSS, Chicago, IL). For optical fractionator estimates the
mean co-efficient of error (CE) of individual estimates was
calculated [16] and was less than 0.08 in all these analyses.
3. Results
3.1. Late onset regional atrophy in 129S6/SvEv inbred
Cln3�/�
To screen for progressive neurodegenerative changes in
129S6/SvEv inbred Cln3�/� we carried out a stereological
survey of regional volume at 5 and 14 months of age.
Cln3�/� mice already show significant intracellular accu-
mulation of storage material by 5 months of age [26], a
progressive phenotype that is apparent as early as 21 days of
age (Mitchison, personal communication). The Cavalieri
method [16] was used to obtain unbiased estimates of the
volume of the cortical mantle, striatum, thalamus, hippo-
campus and cerebellum in Nissl stained sections (Fig. 1). No
significant difference in the volume of any CNS region was
present between Cln3�/� and controls at 5 months of age
(Fig. 1A). The volume of cerebral neocortex and hippo-
campus and cerebellum were also not significantly smaller
in Cln3�/� at 14 months of age (Fig. 1B).
3.2. Late onset effects on interneuron survival and cell size
3.2.1. Hippocampal interneuron survival and size
129S6/SvEv inbred Cln3�/� animals displayed a com-
plex pattern of loss of PV, Cb and SOM-positive
subpopulations of hippocampal interneurons that only
became evident with increased age (Fig. 2). To survey
these effects, counts of detectable neuronal number were
made in each of the hippocampal sub regions containing
neurons positive for these antigens at 5 and 14 months of
age. No significant difference in the number of interneurons
positive for any antigen was evident at 5 months of age,
with the exception of SOM-positive neurons in CA1/CA2/
CA3 (control 10.23F0.42 neurons per section; Cln3�/�
7.55F0.66 neurons per section, P=0.014). In contrast, at 14
months of age there was a consistent trend towards reduced
number of interneurons positive for each marker in Cln3�/�
mice (Fig. 3). Due to variation in both controls and
Cln3�/� mice, reductions in the number of PV-positive
interneurons did not reach statistical significance in any
hippocampal subfield of 14-month-old Cln3�/� vs. controls
(Fig. 3A). In contrast, significantly fewer Cb-positive
interneurons were present in the stratum oriens of 14-
month-old Cln3�/� mice vs. controls (Fig. 3C) and
significantly fewer SOM-positive interneurons were present
in all sub-fields, with the exception of the stratum radiatum
(Fig. 3B). The stratum radiatum also exhibited no significant
difference in the number of Cb-positive interneurons in 14-
month-old animals of either genotype (Fig. 3C). Measure-
ments of mean cross-sectional area at 14 months of age
revealed no significant hypertrophy in Cln3�/� mice
compared to controls in any hippocampal interneuron
population examined (Fig. 4).
In other mouse models of NCL we have previously
described the selective loss of hippocampal interneurons
Fig. 4. Lack of hypertrophy of hippocampal neurons in aged Cln3�/�. (A–D) Histograms of cell size distribution reveal the absence of significant hypertrophy
of persisting somatostatin-positive interneurons in the hilus (A), pyramidal cell layers (CA1/CA2/CA3) (B), stratum oriens (C) and stratum radiatum (D) of 14-
month-old Cln3�/� compared with age matched controls (+/+).
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242236
[2,10,25,27]. Our data reveal that 129S6/SvEv inbred
Cln3�/� mice display a similar phenotype; however, this
is only pronounced at 14 months of age and does not
include significant hypertrophy of persisting hippocampal
interneurons.
3.2.2. Cortical interneuron survival and cell size
Morphological examination of the entorhinal cortex of
129S6/SvEv inbred Cln3�/� animals revealed no obvious
change in the number of PV-positive interneurons in layers
II and IV. Optical fractionator estimates revealed no
significant reduction in the number of these neurons
between animals of either genotype at 14 months-of-age
(Fig. 5A). Comparison of cross-sectional area revealed an
increase in the size of these neurons in 14-month-old
Cln3�/� vs. age-matched controls, but this increase did not
reach statistical significance (Fig. 5B). This phenotype of
cortical interneurons in Cln3�/� mice is again less
pronounced than we have described in mouse models of
other NCLs [2,10,25,27].
3.3. Glial responses in Cln3�/� mice
To examine glial responses in Cln3�/� mice we used
immunohistochemical staining of GFAP and F4/80, as
markers of astrocytes and microglia, respectively. Staining
for both markers were more prominent in Cln3�/� mice at 5
months of age compared to age-matched controls, but there
was no obvious astrocytic hypertrophy or brain macro-
phages present, even at 14 months of age (Fig. 6). A careful
survey of complete one-in-six series of F4/80- or GFAP-
stained sections (representing at least 30 sections through
the entire rostrocaudal extent of the CNS in each of 16
animals) found no evidence for the presence of either brain
macrophages (F4/80) or enlarged astrocytes (GFAP) in
Cln3�/� mice at either age.
To document the more subtle effects on glial cell
populations in Cln3�/� mice we used thresholding image
analysis to quantify the expression of GFAP and F4/80 in
the striatum, cerebellum, and different cortical and hippo-
campal subfields. This analysis revealed a widespread and
Fig. 5. Persistence of cortical interneurons in aged Cln3�/�. (A) Histogram
of optical fractionator counts of parvalbumin-positive interneuron number
in the entorhinal cortex reveal no significant loss of these neurons in 14-
month-old Cln3�/� compared with age matched controls (+/+). (B)
Histogram of cell size measurements reveals that although there was a
trend to increased size of persisting parvalbumin positive neurons in 14-
month-old Cln3�/�, this did not reach statistical significance.
Fig. 6. Morphology of astrocytes and microglia in Cln3�/�. Immunohistochemi
astrocytes (GFAP, C) in 5-month-old Cln3�/�. (A) At this age, F4/80-positive mic
Cln3�/� are more intensely stained than in age-matched controls (+/+). (B) This
positive astrocytes are more prevalent in these regions at 5 months in Cln3�/�
pronounced in Cln3�/� at 14 months of age.
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242 237
significant up regulation of these markers in Cln3�/� mice
at 5 months of age (Fig. 7), many months before
significant interneuron loss was pronounced in the hippo-
campus. This data is evidence for the upregulation of glial
markers, in the absence of detectable changes in the size
of astrocytes or the presence of macrophages, and that is
independent of detectable changes in the size or number
of neurons.
3.4. Upregulation of microglial markers in 129S6/SvEv
inbred Cln3�/�
3.4.1. Microglial morphology
F4/80, CD68 and CD11b immunostaining detected
microglia in both Cln3�/� and control tissue at both 5 and
14 months of age, in all CNS regions examined. Compared
to controls F4/80 immunoreactive microglia were more
prominent in Cln3�/� at 5 months of age with ramified cell
processes appearing more pronounced than in control tissue
(Fig. 6A). At 14 months of age F4/80 immunoreactivity in
Cln3�/� remained prominent and was morphologically
similar to that observed at 5 months of age, but was less
markedly different from controls (Fig. 6B). Significantly,
none of these markers revealed microglia with macrophage-
like morphology at any age, irrespective of genotype.
cal staining for glial markers reveals prominent microglia (F4/80, A) and
roglia in the stratum oriens/CA1 and cerebellar granule layer (cereb GL) of
difference is less pronounced in Cln3�/� at 14 months of age. (C) GFAP
compared with age matched controls (+/+). (D) This difference is less
Fig. 7. Upregulation of microglial and astrocytic markers in Cln3�/�. (A–D) Quantitative thresholding image analysis reveals the widespread and significantly
increased expression of both F4/80 (A) and GFAP (C) in Cln3�/� compared with age-matched controls (+/+) at 5 months of age. In contrast, at 14 months of
age the expression of F4/80 (B) and GFAP (D) were significantly reduced in the stratum oriens and CA1, but unchanged in the majority of regions for both
antigens.
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242238
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242 239
3.4.2. Quantitative analysis of F4/80 expression
The mean percentage of F4/80 immunoreactivity was
considerably higher in 5-month-old Cln3�/� vs. controls
(Fig. 7A), with 2–8 fold increased expression in the visual
cortex, stratum oriens/CA1, dentate and striatum all of
which exhibited significant differences between Cln3�/�
and controls (Pb0.001). Less marked increases of up to 2-
fold were evident in the motor cortex, somatosensory cortex
and cerebellar molecular layer areas of 5-month-old
Cln3�/� vs. controls (PV0.05) and no significant difference
was present in the cerebellar granular layer or white matter.
At 14 months of age F4/80 expression was maintained, but
with less pronounced differences in Cln3�/� vs. controls,
apparently due to age-related upregulation of this marker in
these control mice (Fig. 7B).
3.5. Upregulation of GFAP in 129S6/SvEv inbred
Cln3�/�
3.5.1. Astrocyte morphology
GFAP immunoreactive protoplasmic astrocytes were
abundant in the grey matter of both genotypes and ages,
and were most prevalent in the hippocampus. These
GFAP positive cell bodies and cell processes appeared
more prominent in Cln3�/� vs. control tissue at 5
months of age (Fig. 6C), especially in the stratum
oriens/CA1 region, the dentate gyrus and the cerebellum.
In control and Cln3�/� at both ages, GFAP immunor-
eactive fibrous astrocytes (found predominantly within
the white matter) exhibited less pronounced differences
between genotypes.
3.5.2. Quantitative analysis of GFAP expression
The mean percentage of GFAP immunoreactivity varied
between 5 and 14 months of age, with GFAP immunor-
eactivity considerably higher in the 5-month-old Cln3�/�
vs. controls (Fig. 7C), and some regions exhibiting as
much as a 2-fold increase in GFAP expression. Expression
of GFAP was significantly higher in the stratum oriens/
CA1, dentate, striatum, cerebellar molecular layer, cer-
ebellar granular layer and cerebellar white matter in 5-
month-old Cln3�/� vs. controls (Pb0.001), to a lesser
extent in the visual cortex (Pb0.05), but was not
significantly different in primary motor or somatosensory
cortex (Fig. 7C). In marked contrast, at 14 months GFAP
immunoreactivity was comparable between genotypes,
with the exception of the stratum oriens/CA1, which
showed significant reduction in GFAP immunoreactivity at
this age (Fig. 7D).
4. Discussion
This study represents the first detailed neuropatholog-
ical characterization of 129S6/SvEv inbred Cln3�/� mice.
Our data revealed a neurodegenerative phenotype that
resembles models of other forms of NCL, including the
selective loss of interneuron populations. However, this
neurodegenerative phenotype of Cln3�/� had a relatively
late onset and was preceded by subtle changes in glial cell
populations that are distinct from those seen in other
lysosomal storage disorders. These findings raise the
possibility of an early glial-mediated component in JNCL
pathogenesis.
4.1. Late-onset neuropathological phenotype of 129S6/SvEv
inbred Cln3�/�
By systematically analyzing mouse models of NCL at
different stages in disease progression, we are continuing
to build a detailed series of quantitative neuropathological
landmarks in each form of NCL [9]. It is now evident that
many of these mice share several degenerative features
including regional atrophy and the loss of subpopulations
of cortical and hippocampal interneurons [2,9,10,25,26].
Although different strain backgrounds make direct com-
parisons difficult, the timing of neurodegenerative events
differs markedly in mice that model different forms of
NCL [9,27], with PPT1 null mutant mice exhibiting the
earliest and most aggressive phenotype [2,17]. Among
mice on a C57BL/6J strain background, a second Cln3
knockout model exhibits an NCL-like phenotype [23],
much later than models of late infantile variant CLN6 and
CLN8 [10,25]. As such, the delayed neurodegenerative
phenotype of 129S6/SvEv inbred Cln3�/� is not unex-
pected given the more protracted clinical course of human
JNCL [14,21].
4.2. Loss of GABAergic interneurons in Cln3�/�
Although aged 129S6/SvEv inbred Cln3�/� do not
exhibit significant regional atrophy, we present data for
significant loss of subpopulations of hippocampal inter-
neurons in these mice. Significantly, this loss of inter-
neurons in Cln3�/� mice occurs many months after the
accumulation of autofluorescent storage material, a patho-
logical hallmark of this disease [21], emphasizing that the
links between storage material accumulation and neuro-
degeneration remain unclear. It is now apparent that the
specific loss of interneurons is a consistent feature of
murine NCL [2,9,10,25,27], and ovine NCL [29]. More
significantly, these interneuron populations are also
affected at autopsy in different forms of human NCL
[37]. It is still unclear why these GABAergic cell
populations are selectively affected in the NCLs, although
metabolic vulnerability [41], the relative buffering ability
of individual calcium binding proteins [12], and the
presence of GAD65 autoantibodies in both murine and
human JNCL [7,8] may each be contributory factors.
However, neuronal populations in the amygdala that are
immunoreactive for the same calcium binding proteins and
neuropeptide antigens are not significantly affected even
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242240
in aged Cln3�/� (Chakrabarti, Pearce and Cooper,
unpublished observations). As such, site-specific cues
seem likely to influence neuronal survival, rather than
depending solely upon phenotypic identity.
4.3. Glial responses in Cln3�/� mice
Reactive astrocytes and microglia often accompany
neuronal loss and may also serve as early sensitive
indicators of damage within affected areas [15,24,34].
However, in this study we found no evidence of significant
astrocytic hypertrophy or morphological transformation of
microglia in Cln3�/� mice even when significant inter-
neuron loss was evident at 14 months of age. Instead, we
discovered a more subtle glial phenotype of Cln3�/� mice
with significant up regulation of GFAP and F4/80 in
widespread brain regions at 5 months of age. These findings
in Cln3�/� mice may reflect a more gradual and slowly
developing sequence of events in pathogenesis. These may
include prolonged neuronal dysfunction as a result of
accumulated lysosomal storage material [11,26], or chronic
exposure to elevated levels of glutamate [7], which are
already established in early postnatal life.
Microarray studies in Cln3�/� mice have demonstrated
significant upregulation of several genes involved in
immune responses and inflammation [6,7] and autoanti-
bodies to GAD65 are present in Cln3�/� serum as early
as 1 week of age [7]. It is now recognized that the
activation of microglia is a progressive and graded
phenomenon that takes places in different ’stages’ from
extensively ramified cells to full blown brain macro-
phages [31–33]. Nevertheless, it appears that the specific
cues received by microglia and astrocytes are insufficient
to promote their full morphological transformation in
Cln3�/� mice.
4.4. Glial responses in other storage disorders
The lack of prominent glial activation in Cln3�/� mice is
in direct contrast to mouse models of Sandhoff disease [39],
GM1 and GM2 gangliosidosis [22], and mucopolysacchar-
idoses I and IIIB [28], that each display prominent micro-
glial activation or evidence for CNS inflammation early in
pathogenesis. These reactive events may occur in response
to mediators released by neurons whose function is
compromised by excessive storage material [39], or it is
possible that microglia are themselves targeted by disease
[28]. Our data reveal that Cln3�/� mice display more subtle
effects upon astrocyte and microglial cell populations, at a
time when significant intralysosomal accumulation of
storage material is already present [21,26]. As such, these
glial changes in JNCL may lie downstream of storage
material accumulation and it will be important to determine
the precise sequence of events during the earliest stages of
pathogenesis. Further evidence for distinctive glial
responses between individual storage disorders and in other
forms of NCL may provide significant clues to under-
standing disease progression.
The extent of inflammatory response in other storage
disorders appears to correspond to the rate and severity of
the subsequent neurodegenerative decline [22,28,39]. As
such, the delayed neuronal loss and lack of significant
regional atrophy exhibited by Cln3�/� may reflect the
absence of an early inflammatory response in these mice. In
this context, it will be important to reexamine both
neurodegenerative and inflammatory phenotypes in the
Cln3Dex7/8 knock-in mouse that bears the 1.02 kb mutation
present in over 85% of patients [11,35]. Significantly, these
Cln3Dex7/8 knock-in mice exhibit a more aggressive JNCL
phenotype than Cln3�/� and display prominent astrocytosis
during the latter stages of disease progression [11].
4.5. Influence strain background on the phenotype of
Cln3�/�
Our current data from 129S6/SvEv inbred Cln3�/� mice
reveal several differences compared with the phenotype of
these mice bearing the same mutation on a mixed C57BL/
6J/129S6/SvEv strain background [25], and further empha-
size the major influence of strain background upon disease
phenotype [43]. The well-documented callosal dysgenesis
or acallosal phenotype of 129/Sv mouse strains [40], which
we observed with equal frequency in Cln3�/� and 129S6/
SvEv controls, further complicates detailed neuroanatomical
studies in 129S6/SvEv mice. Taken together, these data
emphasize that to make valid comparisons between different
models of NCL it will be essential that these mice are
generated on the same appropriate strain background and
housed under standardized conditions.
Acknowledgements
We would like to thank Dr. Robert Nussbaum for his
continued support, Stephen Shemilt and Noreen Alexander
for their expert advice and the other members of the
PSDL for their valuable contributions; Timothy Curran,
Andrew Serour, Alfredo Ramirez and David Bernard for
skilled technical assistance and Dr. Alison Barnwell for
constructive comments on the manuscript. These studies
were supported by National Institutes of Health (NIH)
grants NS41930 (JDC), NS40580 (DAP), NS44310 (DAP,
JDC), UK Royal College of Paediatrics and Child Health
and WellChild Research Training Fellowship (MJL, JDC),
European Commission 6th Framework Research Grant
LSHM-CT-2003-503051 (JDC, HMM), The Natalie Fund
(JDC, WCM), the NIH Division of Intramural Research,
Medical Research Council, UK (HMM), EJLB Foundation
(DAP), and grants to JDC from the Batten Disease
Support and Research Association, Batten Disease Family
Association, Batten Disease Support and Research Trust
and the Remy Fund.
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242 241
References
[1] M. Abercrombie, Estimation of nuclear populations from microtome
sections, Anat. Rec. 94 (1946) 239–247.
[2] E. Bible, P. Gupta, S.L. Hofmann, J.D. Cooper, Regional and cellular
neuropathology in the palmitoyl protein thioesterase-1 (PPT1) null
mutant mouse model of infantile neuronal ceroid lipofuscinosis,
Neurobiol. Dis. 16 (2004) 346–359.
[3] H. Braak, H.H. Goebel, Loss of pigment-laden stellate cells: a severe
alteration of the isocortex in juvenile neuronal ceroid-lipofuscinosis,
Acta Neuropathol. (Berl) 42 (1978) 53–57.
[4] H. Braak, H.H. Goebel, Pigmentoarchitectonic pathology of the
isocortex in juvenile neuronal ceroid-lipofuscinosis: axonal enlarge-
ments in layer IIIab and cell loss in layer V, Acta Neuropathol. (Berl)
46 (1979) 79–83.
[5] H. Braak, E. Braak, F. Gullotta, H.H. Goebel, Pigment-filled
appendages of the small spiny neurons: a severe pathological change
of the striatum in neuronal ceroid lipofuscinosis, Neuropathol. Appl.
Neurobiol. 5 (1979) 389–394.
[6] A.I. Brooks, S. Chattopadhyay, H.M. Mitchison, R.L. Nussbaum,
D.A. Pearce, Functional categorization of gene expression changes in
the cerebellum of a Cln3-knockout mouse model for Batten disease,
Mol. Genet. Metab. 78 (2003) 17–30.
[7] S. Chattopadhyay, M. Ito, J.D. Cooper, A.I. Brooks, T.M. Curran, J.M.
Powers, D.A. Pearce DA, An autoantibody inhibitory to glutamic acid
decarboxylase in the neurodegenerative disorder Batten disease, Hum.
Mol. Genet. 11 (2002) 1421–1431.
[8] S. Chattopadhyay, E. Kriscenski-Perry, D.A. Wenger, D.A. Pearce, An
autoantibody to GAD65 in sera of patients with juvenile neuronal
ceroid lipofuscinoses, Neurology 59 (2002) 1816–1817.
[9] J.D. Cooper, Progress towards understanding the neurobiology of
Batten disease or neuronal ceroid lipofuscinosis, Curr. Opin. Neurol.
16 (2003) 121–128.
[10] J.D. Cooper, A. Messer, A.K. Feng, J. Chua-Couzens, W.C. Mobley,
Apparent loss and hypertrophy of interneurons in a mouse model
of neuronal ceroid lipofuscinosis: evidence for partial response
to insulin-like growth factor-1 treatment, J. Neurosci. 19 (1999)
2556–2567.
[11] S.L. Cotman, V. Vrbanac, L.A. Lebel, R.L. Lee, K.A. Johnson, L.R.
Donahue, A.M. Teed, K. Antonellis, R.T. Bronson, T.J. Lerner, M.E.
MacDonald, Cln3Dex7/8 knock-in mice with the common JNCL
mutation exhibit progressive neurologic disease that begins before
birth, Hum. Mol. Genet. 11 (2002) 2709–2721.
[12] C. D’Orlando, M.R. Celio, B. Schwaller, Calretinin and calbindin D-
28k, but not parvalbumin protect against glutamate-induced delayed
excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid
cells, Brain Res. 94 (2002) 181–190.
[13] T.F. Freund, G. Buzsaki, Interneurons of the hippocampus, Hippo-
campus 6 (1996) 347–470.
[14] R.M. Gardiner, Clinical features and molecular genetic basis of the
neuronal ceroid lipofuscinoses, Adv. Neurol. 89 (2002) 211–215.
[15] J. Gehrmann, Y. Matsumoto, G.W. Kreutzberg, Microglia: intrinsic
immuneffector cell of the brain, Brain Res. Brain Res. Rev. 20 (1995)
269–287.
[16] H.J. Gundersen, E.B. Jensen, The efficiency of systematic sampling in
stereology and its prediction, J. Microsc. 147 (1987) 229–263.
[17] P. Gupta, A.A. Soyombo, A. Atashband, K.E. Wisniewski, J.M.
Shelton, J.A. Richardson, R.E. Hammer, S.L. Hofmann, Disruption of
PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout
mice, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13566–13571.
[18] M. Haltia, J. Rapola, P. Santavuori, Infantile type of so-called
neuronal ceroid-lipofuscinosis. Histological and electron microscopic
studies, Acta Neuropathol. (Berl) 26 (1973) 157–170.
[19] M. Haltia, J. Rapola, P. Santavuori, A. Keranen, Infantile type of so-
called neuronal ceroid-lipofuscinosis: 2. Morphological and biochem-
ical studies, J. Neurol. Sci. 18 (1973) 269–285.
[20] R. Herva, J. Tyynel7, A. Hirvasniemi, M. Syrjakallio-Ylitalo, M.
Haltia, Northern epilepsy: a novel form of neuronal ceroid-lipo
fuscinosis, Brain Pathol. 10 (2000) 215–222.
[21] S.L. Hofmann, L. Peltonen, The neuronal ceroid lipofuscinosis, in:
C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), 8th ed. The
Metabolic and Molecular Basis of Inherited Disease, vol III, McGraw-
Hill Companies, Montreal, 2001, pp. 3877–3894.
[22] M. Jeyakumar, R. Thomas, E. Elliot-Smith, D.A. Smith, A.C. van der
Spoel, A. d’Azzo, V.H. Perry, T.D. Butters, R.A. Dwek, F.M. Platt,
Central nervous system inflammation is a hallmark of pathogenesis in
mouse models of GM1 and GM2 gangliosidosis, Brain 126 (2003)
974–987.
[23] M.L. Katz, H. Shibuya, P.C. Liu, S. Kaur, C.L. Gao, G.S. Johnson, A
mouse gene knockout model for juvenile ceroid-lipofuscinosis (Batten
disease), J. Neurosci. Res. 57 (1999) 551–556.
[24] G.W. Kreutzberg, Microglia: a sensor for pathological events in the
CNS, Trends Neurosci. 19 (1996) 312–318.
[25] H.H.D. Lam, H.M. Mitchison, N.D.E. Greene, R.L. Nussbaum, W.C.
Mobley, J.D. Cooper, Pathologic involvement of interneurons in
mouse models of neuronal ceroid lipofuscinosis, Abstr.-Soc. Neurosci.
25 (1999) 1593.
[26] H.M. Mitchison, D.J. Bernard, N.D.E. Greene, J.D. Cooper, M.A.
Junaid, R.K. Pullarkat, N. Vos, M.H. Breuning, J.W. Owens, W.C.
Mobley, R.M. Gardiner, B.D. Lake, P.E.M. Taschner, R.L. Nussbaum,
Targeted Disruption of the Cln3 gene provides a mouse model for
Batten Disease. The Batten Mouse Model Consortium, Neurobiol.
Dis. 6 (1999) 321–334.
[27] H.M. Mitchison, M.J. Lim, J.D. Cooper, Selectivity and types of cell
death in the neuronal ceroid lipofuscinoses (NCLs), Brain Pathol. 14
(2004) 86–96.
[28] K. Ohmi, D.S. Greenberg, K.S. Rajavel, S. Ryazantsev, H.H. Li, E.F.
Neufeld, Activated microglia in cortex of mouse models of
mucopolysaccharidoses I and IIIB, Proc. Natl. Acad. Sci. U. S. A.
100 (2003) 1902–1907.
[29] M.J. Oswald, G.W. Kay, D.N. Palmer, Changes in GABAergic
neuron distribution in situ and in neuron cultures in ovine
(OCL6) Batten disease, Eur. J. Paediatr. Neurol. 5 (Suppl. A)
(2001) 135–142.
[30] G. Paxinos, K.B.J. Franklin, The Mouse Brain in Stereotaxic
Coordinates, 2nd edition, 2001, Academic Press, San Diego.
[31] G. Raivich, M. Bohatschek, C.U. Kloss, A. Werner, L.L. Jones,
G.W. Kreutzberg, Neuroglial activation repertoire in the injured
brain: graded response, molecular mechanisms and cues to
physiological function, Brain Res. Brain Res. Rev. 30 (1999)
77–105.
[32] W.J. Streit, The role of microglia in regeneration, Eur. Arch.
Otorhinolaryngol. (1994) S69–S70.
[33] W.J. Streit, Microglial response to brain injury: a brief synopsis,
Toxicol. Pathol. 28 (2000) 28–30.
[34] W.J. Streit, Microglia as neuroprotective, immunocompetent cells of
the CNS, Glia 40 (2002) 133–139.
[35] The International Batten Disease Consortium, Isolation of a novel
gene underlying Batten disease, CLN3, Cell 82 (1995) 949–957.
[36] J. Tyynel7, J. Suopanki, P. Santavuori, M. Baumann, M. Haltia,
Variant late infantile neuronal ceroid-lipofuscinosis: pathology and
biochemistry, J. Neuropathol. Exp. Neurol. 56 (1997) 369–375.
[37] J. Tyynel7, J.D. Cooper, M.N. Khan, S.J.A. Shemilt, M. Haltia,
Specific patterns of storage deposition, neurodegeneration, and glial
activation in the hippocampus of patients with neuronal ceroid-
lipofuscinoses, Brain Pathol. 14 (2004) in press.
[38] U. von Eitzen, R. Egensperger, S. Kosel, E.M. Grasbon-Frodl, Y.
Imai, K. Bise, S. Kohsaka, P. Mehraein, M.B. Graeber, Microglia and
the development of spongiform change in Creutzfeldt–Jakob disease,
J. Neuropathol. Exp. Neurol. 57 (1998) 246–256.
[39] R. Wada, C.J. Tifft, R.L. Proia, Microglial activation precedes acute
neurodegeneration in Sandhoff disease and is suppressed by bone
C.C. Pontikis et al. / Brain Research 1023 (2004) 231–242242
marrow transplantation, Proc. Natl. Acad. Sci. U. S. A. 97 (2000)
10954–10959.
[40] D. Wahlsten, Deficiency of corpus callosum varies with strain and
supplier of the mice, Brain Res. 239 (1982) 329–347.
[41] S.U. Walkley, P.A. March, C.E. Schroeder, S. Wurzelmann, R.D.
Jolly, Pathogenesis of brain dysfunction in Batten disease, Am. J.
Med. Genet. 57 (1995) 196–203.
[42] M.J. West, L. Slomianka, H.J. Gundersen, Unbiased stereological
estimation of the total number of neurons in the subdivisions of the rat
hippocampus using the optical fractionator, Anat. Rec. 231 (1991)
482–497.
[43] D.P. Wolfer, W.E. Crusio, H.P. Lipp, Knockout mice: simple solutions
to the problems of genetic background and flanking genes, Trends
Neurosci. 25 (2002) 336–340.
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