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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).


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).


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 (+/+).


[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.


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.



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


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.


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