Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.

Abstract

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are collectively the most frequent autosomal-recessive neurodegenerative disease of childhood, but the underlying cellular and molecular mechanisms remain unclear. Several lines of evidence have highlighted the important role that non-somatic compartments of neurons (axons and synapses) play in the instigation and progression of NCL pathogenesis. Here, we report a progressive breakdown of axons and synapses in the brains of two different mouse models of NCL: Ppt1(-/-) model of infantile NCL and Cln6(nclf) model of variant late-infantile NCL. Synaptic pathology was evident in the thalamus and cortex of these mice, but occurred much earlier within the thalamus. Quantitative comparisons of expression levels for a subset of proteins previously implicated in regulation of axonal and synaptic vulnerability revealed changes in proteins involved with synaptic function/stability and cell-cycle regulation in both strains of NCL mice. Protein expression changes were present at pre/early-symptomatic stages, occurring in advance of morphologically detectable synaptic or axonal pathology and again displayed regional selectivity, occurring first within the thalamus and only later in the cortex. Although significant differences in individual protein expression profiles existed between the two NCL models studied, 2 of the 15 proteins examined (VDAC1 and Pttg1) displayed robust and significant changes at pre/early-symptomatic time-points in both models. Our study demonstrates that synapses and axons are important early pathological targets in the NCLs and has identified two proteins, VDAC1 and Pttg1, with the potential for use as in vivo biomarkers of pre/early-symptomatic axonal and synaptic vulnerability in the NCLs.

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Molecular correlates of axonal and synaptic
pathology in mouse models of Batten disease
Catherine Kielar1,{, Thomas M. Wishart2,{, Alice Palmer2, Sybille Dihanich1, Andrew M. Wong1,
Shannon L. Macauley3,4, Chun-Hung Chan5, Mark S. Sands3,4, David A. Pearce5,
Jonathan D. Cooper1,{and Thomas H. Gillingwater2,?,{
1Department of Neuroscience, Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King’s College
London, London SE5 9NU, UK,2Centre for Integrative Physiology, University of Edinburgh Medical School, Edinburgh
EH8 9XD, UK,3Department of Internal Medicine and4Department of Genetics, Washington University School of
Medicine, St Louis, MO, USA and5Centre for Neural Development and Disease, University of Rochester School of
Medicine and Dentistry, Rochester, New York 14642, USA
Received June 25, 2009; Revised and Accepted July 26, 2009
Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are collectively the most frequent autosomal-reces-
sive neurodegenerative disease of childhood, but the underlying cellular and molecular mechanisms
remain unclear. Several lines of evidence have highlighted the important role that non-somatic compartments
of neurons (axons and synapses) play in the instigation and progression of NCL pathogenesis. Here, we
report a progressive breakdown of axons and synapses in the brains of two different mouse models of
NCL: Ppt12/2model of infantile NCL and Cln6nclfmodel of variant late-infantile NCL. Synaptic pathology
was evident in the thalamus and cortex of these mice, but occurred much earlier within the thalamus.
Quantitative comparisons of expression levels for a subset of proteins previously implicated in regulation
of axonal and synaptic vulnerability revealed changes in proteins involved with synaptic function/stability
and cell-cycle regulation in both strains of NCL mice. Protein expression changes were present at pre/
early-symptomatic stages, occurring in advance of morphologically detectable synaptic or axonal pathology
and again displayed regional selectivity, occurring first within the thalamus and only later in the cortex.
Although significant differences in individual protein expression profiles existed between the two NCL
models studied, 2 of the 15 proteins examined (VDAC1 and Pttg1) displayed robust and significant changes
at pre/early-symptomatic time-points in both models. Our study demonstrates that synapses and axons are
important early pathological targets in the NCLs and has identified two proteins, VDAC1 and Pttg1, with the
potential for use as in vivo biomarkers of pre/early-symptomatic axonal and synaptic vulnerability in the
NCLs.
INTRODUCTION
Batten disease, or neuronal ceroid lipofuscinosis (NCL), is the
most frequent autosomal-recessive neurodegenerative disease
of childhood (1). At least ten different forms of NCL exist,
each occurring due to mutations in a different gene, but
characterized by the accumulation of autofluorescent storage
material (2,3). The different forms of this lysosomal storage
disorder are classified by the age of onset of the symptoms,
with an increasing number of variant forms being identified
(2,3). However, the pathophysiological mechanisms under-
lying these devastating disorders remain unclear.
Infantile NCL (INCL) is the most prevalent form of NCL in
Finland with an incidence of 1:20 000 (4). INCL is caused by
†The authors wish it to be known that, in their opinion, these authors contributed equally to this study.
?To whom correspondence should be addressed. Tel: þ44 1316503724; Fax: þ44 1316504193; Email: t.gillingwater@ed.ac.uk
# 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Human Molecular Genetics, 2009, Vol. 18, No. 21
doi:10.1093/hmg/ddp355
Advance Access published on July 29, 2009
4066–4080

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mutations in the CLN1 gene encoding palmitoyl protein
thioesterase 1 (PPT1) a soluble lysosomal enzyme (5),
whose function is to remove long-chain fatty acids from modi-
fied cysteine residues (6). Patients with INCL undergo normal
development until around 12 months, with subsequent neur-
onal degeneration in the CNS leading to retinal degeneration
and blindness, cognitive and motor deficits, seizures and a
flat electroencephalogram by 3 years (7,8). These children
may remain in a vegetative state for several years and with
no effective treatment available will invariably die (9).
Mutations in the CLN6 gene, located on chromosome 15q23
(10,11), cause a variant late infantile form of NCL (vLINCL).
The CLN6 gene codes for a highly conserved protein which
localizes to the endoplasmic reticulum (12,13). However, as
for many NCL proteins, the function of the CLN6 gene
product is currently unknown. As in INCL, affected children
develop normally but subsequently develop epilepsy and neur-
onal degeneration that lead to loss of vision and progressive
mental and motor deterioration resulting in the patient existing
in a vegetative state for several years before dying in their
early teens (3).
Data regarding the important role that distal, non-somatic
neuronal compartments (axons and synapses) play in the insti-
gation and progression of NCL pathology is gradually emer-
ging. For example, several NCL proteins are expressed in
the synaptic compartments of neurons (14,15). Furthermore,
early signs of synaptic dysfunction and degeneration have
been demonstrated in Ppt1 deficient neurons in vitro (16)
and synaptic and axonal pathology occur early on in disease
progression in the congenital form of NCL (17) and synaptic
involvement in a mouse model of INCL was also recently
demonstrated (18). However, the mechanisms underlying
axonal and synaptic vulnerability in the NCLs remain poorly
understood and no molecular biomarkers currently exist
capable of providing a readout of the vulnerability status of
axonal and synaptic compartments. A better understanding
of the cellular and molecular characteristics of axonal and
synaptic vulnerability will therefore be important for our
understanding of disease pathogenesis and the effective devel-
opment of therapeutic compounds.
We have now undertaken a correlated cellular/molecular
investigation into synaptic and axonal vulnerability in mouse
models of INCL (Ppt1 deficient mice) and vLINCL (Cln6
deficient mice). The individual proteins chosen for examin-
ation in our molecular experiments were based upon proteins
previously identified in mice carrying a spontaneous genetic
mutation that confers protection on axons and synapses (but
not neuronal soma) in both the central and peripheral
nervous systems: the Wallerian degeneration slow (Wlds)
mutant mouse (19–22). The remarkable Wldsphenotype
delays the degeneration of axons and synapses in response
to a wide range of traumatic, toxic and disease-inducing
stimuli (23–26) and can be transferred across species includ-
ing rodents and Drosophila (27,28). Although the precise
mechanism of action of Wldsremains controversial, several
in vivo and in vitro genomic and proteomic studies have ident-
ified downstream modifications in the expression of genes and
proteins (focused around cell-cycle status, cell stress and mito-
chondrial stability) that are robust indicators of modified
axonal and synaptic vulnerability (29–31). We chose to
study examples of these indicator proteins in mouse models
of Batten disease so as to undertake ‘hypothesis-driven’
research specifically focused on proteins known to be robustly
altered when axons and synapses change their vulnerability
status (29–31). This approach allowed us to obtain sensitive
measurements of expression levels for these candidate proteins
and was deemed preferable to a more ‘discovery-driven’
approach using whole genome or proteome screens where
specific details regarding synaptic and/or axonal genes and
proteins may be missed.
We show that synaptic and axonal pathology can be ident-
ified early on in the disease time-course in both Ppt1 deficient
and Cln6 deficient mice, and that the severity of this pathology
increases as the disease progresses. Thalamic regions were
more severely affected at earlier time-points than cortical
regions in both mouse models. Expression levels of protein
markers of axonal and synaptic vulnerability were also signifi-
cantly modified at pre/early- symptomatic stages of disease
progression in both mouse models of NCL. Once again, pro-
nounced differences were observed between the thalamus
and cortex. These data provide insights into the molecular
pathways underpinning axonal and synaptic vulnerability in
the NCLs and also identify potential biomarkers of early neur-
onal vulnerability in NCL pathology.
RESULTS
Progressive synaptic and axonal pathology in Ppt1 deficient
mice is evident from early-symptomatic time-points
To investigate synaptic changes within the thalamocortical
system of Ppt1 deficient mice, we stained sections from 1
(pre/early-symptomatic), 3, 5 and 7 (late-symptomatic)-
month-old control and Ppt12/2mice for a range of pre-
synaptic markers. We used antibodies against the synaptic
vesicle protein synaptobrevin, the pre-synaptic membrane
protein synaptophysin and the SNARE complex protein
SNAP25. Immunohistochemical staining for these proteins
revealed a complex series of changes over time in Ppt1
deficient mice. SNAP25 immunoreactivity was observed at
similar levels in the VPM/VPL and LGNd nuclei of the thala-
mus of Ppt1 deficient mice and control mice at 1 month of age
(Fig. 1A). However, levels of SNAP25 were markedly reduced
in the thalamus of Ppt1 deficient mice from 3 months of age
onwards, suggesting that synaptic pathology was present at
early-symptomatic time-points (Fig. 1A). SNAP25 immunor-
eactivity continued to decrease at 5 and 7 months of age in
Ppt12/2mice (Fig. 1A). While loss of SNAP25 immunoreac-
tivity was most pronounced in the VPM/VPL and LGNd
nuclei of the thalamus, decreased immunoreactivity was not
confined to these regions, with other thalamic nuclei and
brain regions (e.g. cortex; see following section) showing
changes, especially at later-symptomatic ages. Similar data
were obtained from the VPM/VPL and LGNd nuclei of the
thalamus for synaptophysin (data not shown). The overall
intensity of staining for synaptobrevin was not markedly
changed in VPM/VPL and LGNd of Ppt1 deficient mice but
large globular aggregates of synaptobrevin were evident
around surviving neurons in the LGNd and VPM/VPL as
early as 3 months of age (data not shown).
Human Molecular Genetics, 2009, Vol. 18, No. 214067

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The overall intensity of immunoreactivity for all three
synaptic markers decreased in the cortex (motor and sensory
areas) of Ppt12/2mice, but not to the same extent as that
observed in the thalamus (Fig. 1B). Both SNAP25 and synap-
tophysin immunoreactivity were noticeably decreased by
3 months of age in Ppt12/2mice, but levels of both proteins
subsequently increased over time (Fig. 1B and data not
shown). Interestingly, we also noted an apparent reduction
in SNAP25 expression in wild-type S1BF between 1 and
3 months (Fig. 1B), which may represent ongoing develop-
mental remodelling of synapses in this region. In addition,
increased synaptophysin staining was observed in lamina V
compared with age-matched controls (data not shown). Synap-
tobrevin staining intensity was decreased from 3 months of
age and decreased until 5 months, before subsequently
increasing again at 7 months of age. As in the thalamus,
large globular aggregates of synaptobrevin were evident in
laminae IV and VI in both S1BF and V1 regions of cortex
(data not shown).
To investigate if synaptic changes were accompanied by
axonal degeneration, we carried out a stereological survey of
internal capsule volume in Nissl-stained sections from
control and Ppt1 deficient mice at different stages of disease
progression. The internal capsule was chosen for examination
because it represents a large, easily identifiable white matter
tract containing connections between the cortex and the thala-
mus. The volume of the internal capsule was significantly
reduced in Ppt12/2mice versus controls from 3 months of
age onwards (Fig. 1C). This reduction in internal capsule
volume increased with time to reach a 40% difference
between Ppt1 deficient mice and age-matched controls at
7 months of age (Fig. 1C).
Protein changes correlating with axonal and synaptic
pathology in Ppt1 deficient mice
We used quantitative immunofluorescent western blotting to
directly determine relative protein expression levels between
control and Ppt12/2tissue at different stages of disease pro-
gression. A panel of 15 proteins was selected for investigation
based upon their known responsiveness to changes in axonal
and synaptic vulnerability (see Introduction; Table 1) (29–
31). Seven of the proteins examined are involved in the regu-
lation of cell cycle (H2Ax, H2B, BRCA2, cABL, Pttg1, Ccl3,
acetyl H3), seven proteins are involved in synaptic function
and stability (bSNAP, Sti1, GDI2, CRMP2P, VDAC1,
VDAC2, CRMP2total) and one protein has been implicated
in both cell-cycle regulation and synaptic form/function
(Ube1) (29–31).
First, we examined protein expression levels in the thalamus
of Ppt12/2and control littermate mice (Table 2; Figs 2 and 3).
Figure 1. Effects upon SNAP25 expression in the visual and somatosensory
system of Ppt1 deficient mice. (A) Changes in the distribution and intensity
of staining for SNAP25 in the ventral posteromedial and ventral posterolateral
(VPM/VPL) and dorsal lateral geniculate (LGNd) thalamic nuclei of 1, 3, 5
and 7-month-old Ppt1 deficient mice (Ppt1) and controls (þ/þ). Immunohisto-
chemicalstainingforSNAP25withinindividualthalamicnucleirevealssimilar
levels of immunoreactivity in the VPM/VPL and LGNd of Ppt1 deficient mice
and controlmice at 1 month of age. However,levels of SNAP25were markedly
reduced in the thalamus of Ppt1 deficient mice from 3 months of age and con-
tinued to decrease with increased age in these mutant mice, as revealed in more
detail at higher magnification. The boundaries of thalamic nuclei are indicated
by white dashed lines. (B) SNAP25 immunoreactivity in the cortex was notice-
ably decreased by 3 months of age, but subsequently increased in intensity over
time compared with age-matched control mice (þ/þ). Laminar boundaries are
indicatedbyromannumeralsonaNissl-stained sectionthroughthesameregion
of cortex. (C) Stereological survey of internal capsule volume in control and
Ppt12/2tissue at different stages of disease progression. The volume of the
internal capsule was significantly reduced in Ppt12/2mice versus controls
from 3 months of age onwards. This reduction increased with time to reach a
40% difference between Ppt1 deficient mice and age matched controls at 7
months of age. (mean+SEM;
post-hoc Bonferroni analysis). (A and B) show representative images from
experiments on .3 mice per genotype. Scale bar ¼ 200 mm in (A and B);
30 mm in higher magnification views.
??P , 0.01;
???P , 0.001; ANOVA with
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Although neither synaptic nor axonal pathology were readily
identifiable in the thalamus of Ppt12/2mice at 1 month of
age, 66% of the proteins examined (10 out of 15) had
expression levels modified by .10% in Ppt12/2mice
versus controls at 1 month of age (Fig. 3A; +10% was
selected as the minimum change required to be considered
above background noise, based on low-level changes in
control proteins such as tubulin). Five of these changes
(33% of the 15 proteins examined) were found to be statisti-
cally significant (Table 2). The variability detected between
samples in these experiments is likely to reflect the high sen-
sitivity of some individual proteins (as is evident when observ-
ing dynamic temporal expression profiles in Fig. 3) to subtle
differences in the rate of disease progression observed
between animals.
Changes in protein expression were found in both
cell-cycle-related and synaptic proteins (Fig. 3B and C).
Expression levels for the majority of proteins were found to
change dynamically with disease progression, with temporal
expression profiles conforming to one of four ‘templates’.
Two proteins (H2AX and histone H2B) were found to have
increasing expression levels over time (Fig. 3D). Five proteins
(beta-SNAP, Sti1, GDI2, Ccl3 and acetyl histone H3) showed
decreasing expression levels over time, albeit with Ccl3 and
Sti1 starting from elevated expression levels at 1 month
(Fig. 3E). The latter two proteins are both stress response pro-
teins (32,33), so early expression changes may be reflecting a
pre-symptomatic stress response. Four proteins (cABL, Ube1,
Pttg1 and total CRMP-2) showed an ‘up-down’ expression
profile (Fig. 3F). Interestingly, the peak in expression of these
proteins at 3 months corresponded precisely with the onset of
loss of synaptic markers in the thalamus (Fig. 1A). The remain-
ing four proteins (VDAC1, VDAC2, BRCA2 and phosphory-
lated CRMP-2) showed relatively consistent changes in
expression profiles throughout disease progression (Fig. 3G).
Next, we examined protein expression levels in the cortex
of Ppt12/2and control littermate mice (Table 3; Figs 4 and
Table 2. Protein expression levels in the thalamus of Ppt12/2mice compared
with wild-type littermates ascertained using quantitative fluorescent western
blotting(P-valuescalculatedusing unpairedt-testsonraw arbitaryfluorescence
values from Ppt12/2mice compared with wild-type littermate controls)
ProteinAge
(months)
% change versus
wild-type
S.E.
P-value
Acetyl Histone
H3
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
218.3
224.3
256.4
223
3.8
8.6
6.6
12.6
4.3
1.2
3.4
3.1
20.6
10.6
3.9
36.7
16.2
32.9
7.6
4.7
66.5
1.9
16.6
50.9
13.8
5.2
6.6
3
4.1
28.7
10.2
5.1
6
6.5
2.9
20.9
9.3
7.6
7.4
20.9
4.5
14.8
25.7
60.5
4.9
33.8
9.5
9.9
8.7
27.6
7.5
6.4
11.5
64.3
6.2
7.9
8.6
18.8
7.7
19.2
6.9
5.1
4.6
5.8
???
?
???
ns
ns
???
???
???
ns
ns
??
ns
ns
ns
ns
???
ns
???
ns
ns
ns
ns
??
???
ns
???
ns
???
ns
?
???
???
ns
ns
ns
ns
??
ns
ns
ns
???
?
??
?
???
ns
ns
???
ns
ns
?
ns
???
ns
???
ns
ns
?
ns
?
Beta2SNAP 0.6
213.8
224.6
220.9
31.5
11.3
14.9
26.2
18.4
32.9
BRCA2
cABL
8.9
225.7
115.2
221
22.7
106.3
215.9
21.6
222
219.1
24.5
196.8
213.9
221.6
Ccl3
CRMP2P
CRMP2total
GDI2 5.2
215.1
212.5
225.5
23.6 H2AX
7
8.5
18.8
Histone H2B
215.9
13.2
18.5
81.6
43.8
87.3
29.6
227.8
66.5
38.5
10.8
227.1
20.9
96.7
13.9
212.4
42.9
21.3
34.2
Pttg1
Sti1
Ube1
VDAC1
0.8
7.6VDAC2
213.6
23.5
213.9
?P , 0.05;??P , 0.01;???P , 0.001.
Table 1. Proteins selected for examination in the current study (all are known
to have modified expression levels in mice expressing the Wldsgene that pro-
tects axons and synapses from degeneration)
Protein Direction of
change (Wlds)
Antibody sourceReference
Acetyl Histone H3
Beta-SNAP
BRCA2
cABL
Ccl3
CRMP2P
CRMP2total
GDI2
H2AX
Histone H2B
Pttg1
Sti1
Ube1
VDAC1
VDAC2
"
"
"
"
#
"
"
#
"
"
"
#
"
#
"
Lake Placid Biologicals
Biomol International
Abcam
Abcam
Abcam
Dr Calum Sutherland
Dr Calum Sutherland
Protein Tech Group
Upstate
Lake Placid Biologicals
LabVision Corp.
BD Biosciences
Abcam
GenTex
Abcam
31
30
31
31
31
30
30
30
31
31
29/31
30/31
30/31
30
30
Human Molecular Genetics, 2009, Vol. 18, No. 21 4069

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5). As in the thalamus, 66% of the proteins examined (10 out
of 15) had expression levels modified by .10% in Ppt12/2
mice versus controls at 1 month of age (Fig. 5A). Nine of
these changes (60% of the 15 proteins examined) were
found to be statistically significant (Table 3). Once again,
changes in protein expression were observed in both
cell-cycle-related and synaptic proteins (Fig. 5B and C).
Expression levels for the majority of proteins were found to
change dynamically with disease progression and the temporal
expression profiles conformed to one of four templates.
Examples of proteins conforming to the increasing, decreasing
and no-change temporal profiles previously described in the
thalamus were readily observed in the cortex (Fig. 5D, E
and G). However, proteins conforming to an ‘up-down’
profile identified in the thalamus were not observed. In con-
trast, several of the proteins with ‘up-down’ expression pro-
files in the thalamus showed a mirror-image ‘down-up’
expression profile in the cortex (Ube1 and Pttg1; Fig. 5F).
Taken together, these experiments reveal robust early-onset
protein expression changes correlating with synaptic and
axonal pathology in the thalamus and cortex of Ppt12/2
mice in vivo. These data show that modified axonal and synap-
tic vulnerability in Ppt12/2mice is accompanied by molecu-
lar changes in pathways including those regulating cell-cycle
status and synaptic form/function.
Synaptic and axonal pathology in Cln6 deficient mice
mirrors events occurring in Ppt1 deficient mice
In order to ask whether the axonal and synaptic changes
observed within the thalamocortical system of Ppt1 deficient
mice were specific to INCL, or could be considered more ubi-
quitous pathological events occurring in other forms of NCL,
we repeated our assessment of synaptic markers and internal
capsule volume in Cln6nclfmice modelling vLINCL.
Immunohistochemical staining for synaptic markers in 4
(pre/early-symptomatic) and 10 (late-symptomatic)-month-old
Cln6nclfmice revealed similar changes to those observed in
Ppt1 deficient mice. SNAP25 immunoreactivity was markedly
reduced in the thalamus of Cln6nclfmice at 4 months of age
onwards, suggesting that synaptic pathology was present at
early-symptomatic time-points (Fig. 6A). SNAP25 immunor-
eactivity continued to decrease at 10 months of age in
Cln6nclfmice (Fig. 6A). Similar data were obtained from the
thalamus for synaptophysin (data not shown). The overall
intensity of staining for synaptobrevin was not markedly
changed in VPM/VPL and LGNd of Cln6nclfmice, but large
globular aggregates of synaptobrevin were evident around
surviving neurons in the LGNd and VPM/VPL at 4 months
of age already (data not shown).
In contrast to the thalamus, the overall intensity of immu-
noreactivity for all three synaptic markers increased in the
cortex (both motor and sensory areas) of Cln6nclfmice
(Fig. 6B). Both SNAP25 and synaptophysin immunoreactivity
were already elevated compared with controls at 4 months of
age, and levels of both proteins subsequently increased further
over time (Fig. 6B and data not shown). Increased synaptophy-
sin staining was observed in laminae V and VI compared with
age-matched controls (data not shown). Synaptobrevin stain-
ing intensity was slightly decreased at 4 months of age and
subsequently increased at 10 months of age in laminae V
and VI. As in the thalamus, large globular aggregates of
synaptobrevin were evident in laminae V and VI in both
S1BF and V1 regions of cortex (data not shown).
A stereological survey of axonal integrity (internal capsule
volume) in Nissl-stained sections in control and Cln6nclfmice
at different stages of disease progression revealed a significant
decrease in internal capsule volume with increasing disease
progression in nclf mice compared with age-matched controls
(Fig. 6C). A similar magnitude of reduction in internal capsule
volume was noted for late-symptomatic Cln6nclfmice (?30%
difference; Fig. 6C) as was evident in Ppt1 deficient mice
(Fig. 1C).
Protein changes correlating with initiation of axonal
and synaptic pathology in Cln6 deficient mice
We next asked whether any of the early protein markers of
axonal and synaptic vulnerability observed in the thalamus
of pre/early-symptomatic (1 month old) Ppt1 deficient mice
were also modified in pre/early-symptomatic (3 month old)
Cln6nclfmice. This analysis was designed to reveal any signifi-
cant convergence or divergence in the molecular pathways
underpinning early changes in synaptic and axonal vulner-
ability in the NCLs. Any proteins identified as having
similar modifications in expression profiles across both
models of NCL would therefore have a strong case for being
considered as potential ‘biomarkers’ of pre-symptomatic
modifications in axonal and synaptic vulnerability in vivo.
We compared changes in expression levels within the thala-
mus for all 15 candidate proteins in 1 month old Ppt12/2mice
versus 3-month-old Cln6nclfmice (Fig. 7). As with Ppt12/2
mice, the majority (14 out of 15) of proteins examined
showed modifications in expression levels, with a magnitude
.10% (Fig. 7A). All 14 changes were statistically significant
(Fig. 7A). Two proteins showed consistent directional
expression changes across both mouse models at these early-
symptomatic time-points (Fig. 7B): voltage-dependent anion
channel 1 (VDAC1) and pituitary tumor transforming gene 1
(Pttg1). However, the vast majority of proteins examined did
not show similar expression profiles when comparing Ppt1
deficient mice and Cln6nclfmice (e.g. Ccl3 and Ube1;
Fig. 7B). These data suggest that VDAC1 and Pttg1 might
be useful as early biomarkers of modified axonal and synaptic
vulnerability in the NCLs. They also imply that the molecular
pathways regulating synaptic and axonal degeneration in
INCL and vLINCL impact on similar cellular pathways
Figure 2. Representative fluorescent western blots of protein expression levels
of Pttg1, VDAC1 and tubulin loading control in the thalamus of Ppt12/2mice
(2/2) and littermate controls (þ/þ) at 1, 3, 5 and 7 months.
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Figure 3. Temporal progression of protein expression changes in the thalamus of Ppt12/2mice. (A) Temporal progression of expression profiles for all 15
proteins examined. This graph is included to illustrate the overall complexity of expression changes observed. Data for individual proteins is shown more
clearly on the subsequent graphs. (B) Graph of cell cycle proteins only [data extracted from (A)]. (C) Graph of synaptic proteins only [data extracted from
(A)]. (D–G) Graphs showing expression levels for proteins conforming to a ‘increasing’ profile (D), ‘decreasing’ profile (E), ‘up-down’ profile (F) or ‘no
overall trend’ profile (G). Profiles were determined by the patterns of change in expression following initial measurements in 1-month-old mice, not by the
initial direction of change. Note how proteins conforming to the ‘up-down’ profile reach their peak in 3-month-old mice. All data normalized to control
tubulin levels.
Human Molecular Genetics, 2009, Vol. 18, No. 21 4071

Page 7

(e.g. cell cycle and synaptic stability), but with divergent
responses in the levels of other individual proteins.
Finally, in order to confirm that VDAC1 and Pttg1 may be
useful as clinically relevant biomarkers reporting on neuronal
vulnerability status in vivo, we assessed protein expression
levels in peripherally accessible tissue (muscle) from pre/
early-symptomatic (1 month old) Ppt12/2and wild-type lit-
termate control mice (n ¼ 4 mice per genotype; Fig. 8A).
Both VDAC1 and Pttg1 showed significantly modified
expression levels in hind-limb muscle preparations (including
quadriceps, hamstrings, gastrocnemius and soleus) from
Ppt12/2mice compared with wild-type littermate controls
(Fig. 8B and C). Interestingly, both proteins showed a
similar magnitude of expression change to those observed in
the thalamus. However, the direction of expression change
in pathologically unaffected muscle tissue (T Gillingwater
and J Cooper, unpublished observations) occurred in the oppo-
site direction to those observed in the thalamus for both pro-
teins. This may directly reflect the differing pathological
status of the two tissues examined. Nevertheless, these data
show that both VDAC1 and Pttg1 levels can be robustly deter-
mined in muscle and suggest that altered expression of these
proteins in muscle may be useful as a biomarker for accompa-
nying underlying changes in neuronal vulnerability.
DISCUSSION
The current study has generated three major conclusions. First,
we have demonstrated that axonal and synaptic compartments
of neurons are early pathological targets in two different
mouse models of NCL, with the thalamus being particularly
vulnerable at early stages of disease progression. Second, we
have shown that a range of cell cycle and synaptic proteins
known to be robust markers of axonal and synaptic vulner-
ability have regionally specific modified expression levels in
NCL. Third, we have shown that two proteins (namely
VDAC1 and Pttg1) have the potential to be used as biomarkers
of pre/early-symptomatic changes in axonal and synaptic vul-
nerability in INCL and vLINCL in vivo.
In both mouse models of NCL examined, the onset of
synaptic and axonal pathology was detectable at pre/early-
symptomatic time-points. For example, we have demonstrated
a reduced expression of key synaptic proteins (e.g. SNAP25)
in the thalamus at pre/early-symptomatic ages as well as a
reduction in the size of the internal capsule over time
corresponding with disease progression. These data therefore
support the hypothesis that synapses and axons play a key
Figure 4. Representative fluorescent western blots of protein expression levels
of Pttg1, VDAC1 and tubulin loading control in the cortex of Ppt12/2mice
(2/2) and littermate controls (þ/þ) at 1, 3, 5 and 7 months.
Table 3. Protein expression levels in the cortex of Ppt12/2mice compared
with wild-type littermates ascertained using quantitative fluorescent western
blotting(P-valuescalculatedusingunpairedt-tests onraw arbitaryfluorescence
values from Ppt12/2mice compared with wild-type littermate controls)
Protein Age
(months)
% change versus
wild-type
S.E.
P-value
Acetyl Histone
H3
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
1
3
5
7
218.1
217.6
12.2
4.9
8.9
17.9
6.5
0.3
3.4
3.5
15.2
11
10.6
6.2
2.7
13.7
9
18.6
42.5
7.8
5.4
9.5
3.7
2.4
4.2
5.2
9
7.4
11.4
11.3
7.3
2.1
6.7
5.1
10.5
6.8
24.1
32.3
4
3.6
6.7
12.1
14.6
8.9
20.8
34.2
17.1
12
40.6
35.4
15.5
4.4
7.9
21.4
18.2
12.2
30.1
38.7
9.1
3.3
2.4
4.9
ns
??
ns
ns
ns
???
???
???
??
ns
ns
???
ns
???
ns
ns
?
ns
ns
ns
?
???
?
??
ns
ns
ns
ns
???
???
???
ns
?
??
ns
??
???
???
??
ns
??
?
ns
?
??
ns
??
??
?
???
ns
??
ns
ns
ns
?
ns
??
ns
???
0.8
13.9
2Beta-SNAP
237.3
214.7
218.7
57.2
217.6
10.5
66.4
BRCA2
cABL 7.7
19.5
20.1
12.8
115.6
24.8
20.2
24.9
28.9
226.4
Ccl3
CRMP2P
9.8
218.6
27
214.7
12
212.7
229.7
240.4
228.5
28.7
12
222.3
24.8
61.3
225.5
228.7
222.8
212
56.2
28.1
23.8
79.9
63.3
19.3
144.3
122.7
43.7
258
28.7
67
CRMP2total
GDI2
H2AX
Histone H2B
Pttg1
Sti1
Ube1
VDAC1 4.7
211.3
26.6
96.4
11.7
212.7
24.5
224
VDAC2
?P , 0.05;??P , 0.01;???P , 0.001.
4072Human Molecular Genetics, 2009, Vol. 18, No. 21

Page 8

Figure 5. Temporal progression of protein expression changes in the cortex of Ppt12/2mice. (A) Temporal progression of expression profiles for all 15 proteins
examined. This graph is included to illustrate the overall complexity of expression changes observed. Data for individual proteins is shown more clearly on the
subsequent graphs. (B) Graph of cell cycle proteins only [data extracted from (A)]. (C) Graph of synaptic proteins only [data extracted from (A)]. (D–G) Graphs
showing expression levels for proteins conforming to a ‘increasing’ profile (D), ‘decreasing’ profile (E), ‘down-up’ profile (F) or ‘no overall trend’ profile (G).
Profiles were determined by the patterns of change in expression following initial measurements in 1-month-old mice, not by the initial direction of change. Note
how proteins conforming to the ‘down-up’ profile reach their peak in 3-month-old mice. All data normalized to control tubulin levels.
Human Molecular Genetics, 2009, Vol. 18, No. 214073

Page 9

role in the onset and progression of neurodegeneration in the
NCLs, supporting the findings of several other recent studies
(16–18). As such, the NCLs may need to be considered,
along with many other diseases of the CNS and PNS (includ-
ing Alzheimer’s disease and motor neuron diseases such as
amyotrophic lateral sclerosis and spinal muscular atrophy)
(34–36), as ‘synaptopathies’ and/or ‘distal axonopathies’
(reviewed in 37). Treatments that can directly target axonal
and synaptic compartments of neurons may therefore slow
or halt the onset and/or progression of NCL neuropathology.
Our finding that several proteins whose expression profiles
are known to reflect the vulnerability status of axons and
synapses have modified expression levels in the brains of
two NCL mouse models provides further evidence in
support of synaptic and axonal vulnerability in these disorders.
The data showing that several of these synaptic proteins have
modified expression levels pre/early-symptomatically in NCL
mice suggests that synaptic changes at the molecular level are
instigated in advance of any overt neuropathological changes.
Interestingly, several of the early expression changes observed
in NCL mouse models (e.g. VDAC1) occurred in the opposite
direction to those found in Wlds-expressing cells. However, it
is difficult to directly compare expression changes of individ-
ual proteins in NCL mouse models with those found in
Wlds-expressing cells. This is due to the fact that the Wldsphe-
notype remains constant (in the tissues used to measure protein
expression) while disease progression in NCL is very
dynamic, with corresponding fluxes in expression levels
(Figs 3 and 5). It would, however, be of interest to establish
whether crossing Wldsmice with mouse models of NCL has
any therapeutic benefit. Such crosses would also be useful
for further investigations into the roles of individual proteins
identified in the current study.
Interestingly, several of the protein changes we have ident-
ified in the current study correspond to data presented in
reports on previous microarray experiments carried out on
either whole brain or cortical tissue from mouse models of
Batten disease (38,39). These changes included Pttg1,
histone H2B, CCL3. The modest magnitude of changes in
these genes (and those in other corresponding pathways) is
Figure 6. Synaptic and axonal pathology in early-symptomatic Cln6nclfmice (A) Changes in the distribution and intensity of staining for SNAP25 in the ventral
posteromedial and ventral posterolateral (VPM/VPL) and dorsal lateral geniculate (LGN) thalamic nuclei of 4 and 10-month-old Cln6nclfdeficient mice (nclf)
and controls (þ/þ). SNAP25 immunoreactivity was markedly reduced in the thalamus of Cln6nclfmice at 4 months of age and was decreased further at 10
months of age in Cln6nclfmice, as revealed in more detail at higher magnification. The boundaries of thalamic nuclei are indicated by white dashed lines.
(B) SNAP25 immunoreactivity was already elevated compared with controls (þ/þ) at 4 months of age, and was subsequently more intense in Cln6nclfmice
at 10 months of age. Laminar boundaries are indicated by roman numerals on a Nissl-stained section through the same region of cortex. (C) Stereological
survey of internal capsule volume in control and Cln6nclftissue at different stages of disease progression. The volume of the internal capsule was significantly
reduced in Cln6nclfmice versus controls from 4 months of age onwards. This reduction increased with time to reach a 40% difference between Cln6nclfmice and
age-matched controls at 10 months of age. (mean+SEM;??P , 0.01;???P , 0.001; ANOVA with post-hoc Bonferroni analysis). (A and B) show representa-
tive images from experiments on .3 mice per genotype. Scale bar ¼ 200 mm in (A and B); 30 mm in higher magnification views.
4074Human Molecular Genetics, 2009, Vol. 18, No. 21

Page 10

likely to reflect the heterogeneous nature of the tissue samples
used for the microarray experiments, which did not focus on
the thalamus, the core location of early pathological changes
in these forms of NCL. This suggests that repeating microar-
ray experiments on isolated thalamic tissue might provide
additional important insights into pathways and proteins
beyond the scope of the current study.
The finding that several cell-cycle-related proteins have
modified expression levels provides evidence that the cell-
cycle status of terminally differentiated neurons and/or their
supporting cells is altered in the NCLs. This finding is in
keeping with a growing body of papers showing that altered
cell-cycle status can contribute significantly to neurodegenera-
tive disease. For example, numerous examples of cell-cycle
regulation gone awry have been reported in neurodegenerative
conditions such as motor neuron disease, Alzheimer’s disease,
Parkinson’s disease and stroke (40,41,63,64). Furthermore,
pharmacological manipulation of cell-cycle progression has
been used to confer neuroprotection in animal models of trau-
matic brain injury and stroke (42,43). Our data support the
hypothesis that the influence of cell-cycle status on neuronal
vulnerability extends beyond neurodegenerative mechanisms
resident in cell soma to incorporate independent degenerative
pathways in axonal and synaptic compartments (31). Cell-
cycle pathways may therefore constitute viable therapeutic
targets for the treatment of NCL pathology.
Oneparticularlynovelfindingofthecurrentstudywasthedis-
tinct temporal profiles of protein expression occurring in the
thalamusandcortexofPpt12/2mice.Thefindingthatsomepro-
teins had increased expression levels with disease progression
rules out the possibility that the protein changes observed were
simply occurring due to a loss of total neuronal protein content.
Moreover, the identification of ‘up-down’ profiles in the thala-
mus and ‘down-up’ profiles in the cortex, with peaks occurring
in 3-month-old mice, suggests that the expression levels of
several proteins were intimately associated with the instigation
of synaptic and axonal pathology (as evidenced by our morpho-
logical studies; Fig. 1). We have previously shown that patho-
logical events in Ppt12/2mice are progressive, with acute
astrocytosisfirstappearingat3monthsofageinspecificthalamic
nuclei, followed by loss of neurons relaying different sensory
modalities (44). This thalamic neuron loss was followed by
loss of corresponding cortical target granule neurons that were
only significantly affected in the later stages of disease pro-
gression (44). The age of 3 months in Ppt12/2mice therefore
appearstobeamajorturningpointinthepathogenesisofthisdis-
order,asthisisthepointindiseaseexpressionwhensynapseloss
(andassociatedproteinexpressionchanges),axonlossandastro-
cytosis are all triggered.
The finding that protein expression changes in the cortex did
not precisely mirror those identified in the thalamus is perhaps
not surprising given our understanding of the different pro-
gression of disease-associated pathology in these regions
(44). Moreover, it has previously been suggested that synaptic
reorganization or plasticity may occur in the cortex as a com-
pensatory mechanism against ongoing neuron loss within the
thalamus (17) (J Cooper and T Gillingwater, unpublished
observations). Our morphological data showing more adaptive
changes in synaptic protein levels in the cortex of Ppt12/2
mice supports this hypothesis.
Despite the obvious differences in expression profiles for
proteins in the thalamus and cortex, one protein in Ppt12/2
mice (Ccl3) displayed a similar expression profile in both
regions, with a very high expression at 1 month of age fol-
Figure 7. Protein expression changes in the thalamus of early-symptomatic (3 month old) Cln6nclfmice compared with early-symptomatic (1 month old)
Ppt12/2mice reveal VDAC1 and Pttg1 as potential biomarkers of axonal and synaptic vulnerability. (A) Bar chart showing percentage changes in expression
levels for all 15 proteins examined in Cln6nclfmice (as well as tubulin control levels), compared with wild-type littermate controls. Statistical values were cal-
culated using unpaired t-tests on raw arbitrary fluorescence values from Cln6nclfmice compared with wild-type littermate controls. Note that all but one of the
experimental proteins (VDAC2) had significant changes in expression levels in the Cln6nclfmice. (B) Bar chart showing expression levels (normalized to tubulin)
for all 15 proteins examined. Black bars show data from Cln6nclfmice and white bars show data from Ppt12/2mice (mean+SEM; n ¼ 3 mice per genotype/
protein). Note the distinct expression profiles between the two groups of mice: only 5 out of the 15 proteins examined showed expression changes consistently
changing in the same direction. Two of the proteins examined, however, showed robust increases in expression levels in both mouse models (VDAC1 and Pttg1).
Expression data for CRMP2 total and CRMP2P in Cln6nclfmice are the same as those previously published in (62).
Human Molecular Genetics, 2009, Vol. 18, No. 21 4075

Page 11

lowed by a dramatic decrease from 3 months onwards. This
finding is of potential interest because Ccl3 protein, also
known as macrophage inflammatory protein 1a (MIP-1a), is
a chemokine involved in inflammation (45,46). CNS inflam-
mation is evident in all forms of NCL (44,47–49), and also
in other neurodegenerative disorders (50–52). For example,
elevated levels of Ccl3/MIP-1a were found in a mouse
model of Sandhoff disease (Hexb2/2), another lysosomal
storage disorder, and loss of MIP-1a improves disease
phenotype (53). Our finding therefore supports that hypothesis
that inflammation may contribute to neurodegeneration in
Ppt12/2mice. However, a lack of similar initially increased
expression levels of Ccl3 in Cln6nclfmice suggests that the
inflammatory response is not necessarily conserved between
different forms of the NCLs. It may therefore be informative
to determine whether upregulation of Ccl3/MIP-1a occurs in
other forms of NCL.
Perhaps the most exciting finding of the present study is that
modified expression levels of two distinct proteins (VDAC1
and Pttg1) robustly occur during the pre/early-symptomatic
stages of disease progression in both mouse models of NCL
examined. The role of these proteins in axonal and synaptic
pathology remains unclear, although previous studies have
suggested that Pttg1 may modify ubiquitination pathways in
synapses and axons (29,31) and VDAC1 is known to be a
core regulator of mitochondrial form and function (30).
However, our findings suggest that VDAC1 and Pttg1 proteins
are likely to be useful biomarkers of early changes in axonal
and synaptic vulnerability in INCL and vLINCL, reporting
on molecular changes occurring in advance of morphological
events. These proteins have potential advantages over pre-
viously proposed biomarkers—lysosomal acid phosphotase
(54) and transcriptional changes in extracellular matrix pro-
teins (55)—because they specifically report upon changes
occurring in biologically relevant neuronal compartments
(axons and synapses), can be detected in peripherally accessi-
ble muscle samples and appear to be conserved across multiple
different forms of NCL. It will now be of particular importance
to examine whether similar changes in these proteins occur
across other animal models of the NCLs as well as in human
patients. As the current study was restricted to a relatively
small number of previously identified proteins, experiments
increasing the numbers and diversity of proteins examined
may lead to the identification of additional potential bio-
markers for altered axonal and synaptic vulnerability in NCL.
MATERIALS AND METHODS
Mouse models
The Ppt1-deficient mice (Ppt12/2) used in this study were
originally created through a targeted disruption strategy
which eliminates the last exon in the coding sequence of
Ppt1 (56). These mice were subsequently backcrossed for 10
generations with C57BL/6 control mice, which is generally
considered sufficient to be congenic on this strain background.
C57BL/6 congenic Ppt12/2mice and age-matched C57BL/6
control mice were bred and housed in a barrier facility at
Washington University School of Medicine (St Louis, MO).
C57BL/6 congenic Cln6nclfmice and C57BL/6 control mice
were originally obtained from The Jackson Laboratory (Bar
Harbour, ME) and were bred and housed in the vivarium at
University of Rochester School of Medicine and Dentistry
(Rochester, NY). All diseased and control mice used in this
study were littermates. All animal procedures were carried
out in accordance with NIH guidelines and the Institutional
Animal Care and Use Committee regulations of Washington
University and the University of Rochester.
Figure 8. VDAC1 and Pttg1 protein expression changes were also detectable
in peripherally accessible muscle tissue from 1-month-old Ppt12/2mice. (A)
Bands from quantitative fluorescent western blots showing significantly
reduced expression levels of both VDAC1 and Pttg1 in 1-month-old
Ppt12/2mice (example blots from three separate animals are shown to illus-
trate variability between samples). Actin is shown as a loading control. (B) Bar
chart showing a significant reduction in VDAC1 levels in muscle tissue from
1-month-old Ppt12/2mice (???P , 0.001, Mann–Whitney test, n ¼ 4 mice
per genotype). (C) Bar chart showing a significant reduction in Pttg1 levels
in muscle tissue from 1-month-old Ppt12/2mice (???P , 0.001, Mann–
Whitney test, n ¼ 4 mice per genotype).
4076 Human Molecular Genetics, 2009, Vol. 18, No. 21

Page 12

Histological analysis
Ppt12/2mice present with an INCL-related phenotype and
usually die around 8–8.5 months of age (56). In contrast,
Cln6nclfmice model a vLINCL phenotype and live until
12 months of age (57). To analyse the progression of synaptic
and axonal pathological changes in the Ppt12/2deficient
CNS, the brains of Ppt12/2mice and C57BL/6 controls
were harvested at 1, 3, 5 and 7 months of age (n ¼ 3
Ppt12/2and C57BL/6 control mice at each age). A similar
analysis of the progression of synaptic and axonal pathological
changes in the Cln6 mice was performed on brains harvested
at 4 and 10 months. Brains were immersion fixed for at least
1 week in 4% paraformaldehyde in 0.1 M phosphate-buffered
saline, cryoprotected in a solution of 30% sucrose in Tris buf-
fered saline (TBS: 50 mM Tris, pH 7.6, 150 mM NaCl) and
40 mm frozen coronal sections cut through the rostrocaudal
extent of the cortical mantle (48,58,59). Sections were stored
in a cryoprotectant solution (TBS/30% ethylene glycol/15%
sucrose/0.05% sodium azide) at 2408C prior to histological
processing.
Measurement of internal capsule volume
To provide direct visualization of the internal capsule (the
white matter tract that connects the thalamus and cortex),
every sixth section was slide mounted and Nissl stained as
described previously (48). Unbiased Cavalieri estimates of
the volume of the internal capsule were made from each
animal with no prior knowledge of genotype. A sampling
grid with appropriate spacing (150 mm) was superimposed
over Nissl-stained sections and the number of points covering
the relevant areas counted using ?2.5 objective. Regional
volumes were expressed in mm3and the mean volume of
each region calculated for control and Ppt12/2or nclf mice.
All volume analyses were carried out using StereoInvestigator
software (Microbrightfield Inc., Williston, VT) on a Zeiss
Axioskop2 MOT microscope (Carl Zeiss Ltd., Welwyn
Garden City, UK) linked to a DAGE-MTI CCD-100 camera
(DAGE-MTI Inc., Michigan City, IN, USA).
Immunohistochemical staining
To survey the expression of different synaptic markers, a one
in six series of sections was immunohistochemically stained
for presynaptic markers synaptophysin (Syp), synaptobrevin
(VAMP2) and SNAP 25. These reactions used the following
polyclonal primary antisera (monoclonal mouse anti-Syp,
Upstate, 1:100; chicken anti-VAMP2, Chemicon, 1:500;
monoclonalmouseanti-SNAP25,
1:1000). Sections were then rinsed in TBS with subsequent
incubation in secondary anti-serum (goat anti-mouse [Syp
and SNAP25] and goat anti-chicken [VAMP2], Vector Lab-
oratories, 1:1000) followed by avidin-biotin-peroxidase
complex (Vectastain Elite ABC kit, Vector Laboratories).
Immunoreactivity was visualized by a standard DAB reaction
and sections were mounted onto slides, air-dried, cleared in
xylene and coverslipped with DPX (VWR, Dorset, UK).
BDTransduction,
Protein expression analysis by quantitative
immunofluorescence western blot
We used a quantitative immunofluorescence western blot
approach because of its highly sensitive, reliable and reprodu-
cible nature (30,31,60). Ppt12/2and Cln6nclfbrains, as well as
brains from control littermates, were rapidly dissected
immediately after sacrifice at different ages (n ¼ 3 brains
per genotype per time-point), bisected and flash frozen on
dry ice. The left hemisphere of each brain was then dissected
in order to produce separate samples containing thalamic (T)
and cortical (C) tissue. Protein was extracted and assayed as
previously described (30,31,60). Briefly, tissue was homogen-
ized in 200 ml of ice-cold radioimmunoprecipitation (RIPA)
buffer and 2 ml of protease inhibitors (Pierce Biosciences)
before being gently passed through a 23G needle and a 25G
needle to manually homogenize each sample. The homogenate
was then centrifuged at 14 000 rpm for 20 min at 48C. Finally,
the supernatant was collected into a fresh tube and kept at
2808C until use. Protein concentration was determined
through the use of a MicroBCA assay kit (Pierce Biosciences)
as per manufacturer’s instructions.
In order to separate the proteins according to their molecu-
lar weight, each sample was run on a 4–12% sodium dodecyl
sulphate polyacrylamide pre-cast gel (SDS–PAGE) using a
XCell SureLock Mini-Cell electrophoresis cell (Invitrogen).
Fifteen micrograms of protein sample in a volume of 10 ml
loading buffer (Pierce Biosciences) were prepared. Each
sample was then boiled at 988C for 2 min, spun down and
loaded on the gel. The gel was subsequently run at 120 V
for 45 min. The proteins were then transferred from the gel
onto a PVDF membrane (Invitrogen). The transfer was run
over night.
To visualize the changes in protein expression levels, PVDF
membranes were probed using primary antibodies at 1:5000
dilution (Table 1) after incubation in Odyssey blocking
buffer (Li-COR) for 45 min at room temperature. Membranes
were rinsed 3?5 min with PBS-Tween 0.1% and incubated
with appropriate fluorescent secondary antibodies secondary
antibodies at 1:5000 dilution (Odyssey; Goat anti rabbit
IRDye 680, Goat anti-mouse IRDye 800-Li-COR Biosciences;
Donkey anti-sheep IRDye 800 -Rockland) for 1.5 h at room
temperature in the dark. Beta-tubulin (Abcam) antibodies
were used for loading controls wherever required. The validity
of all antibodies used in current experiments has been con-
firmed in previous studies run in conjunction with proteomic
and SuperArray screens (30,31). In addition, all antibodies
used generated bands at the predicted molecular weight.
Immunoreactive bands were visualized using an Odyssey
Infrared Imaging System (Li-COR Biosciences). Scan resol-
ution of the instrument ranges from 21 to 339 mm and in
this study blots were imaged at 169 mm. Quantification was
performed on single channels with the analysis software pro-
vided. Blot scans were analysed using the Odyssey software
to manually define bands. Odyssey software assigned arbitrary
fluorescence values to these bands, to give relative fluor-
escence intensity between bands on each membrane. Three
mice were used for each brain region and each was scanned
three times at different laser intensities in order to minimize
user error. This gave n ¼ 9 scans, and n ¼ 3 mice/membranes
Human Molecular Genetics, 2009, Vol. 18, No. 214077

Page 13

per brain region for each protein (30,31). Representative blots
were prepared for publication by exporting images from the
Licor Odyssey software as TIFFs before converting to grey-
scale images using Adobe Photoshop.
Muscle protein expression analysis
Hind-limb musculature from 1-month-old Ppt12/2and wild-
type littermate mice were rapidly dissected following sacrifice
and flash frozen. Frozen muscle tissue was then homogenized
using a pestle and mortar. Powdered muscle was solubilized,
protein was extracted and samples processed as described
earlier. Quantitative western blotting was performed as
described earlier using antibodies against VDAC1, Pttg1 and
actin (Abcam; used as a loading control).
Statistical analysis
The statistical significance of differences between genotypes
of internal capsule volume measurements was assessed using
a one-way ANOVA (SPSS 11.5 software, SPSS Inc.,
Chicago, IL), with statistical significance considered at P ?
0.05. The mean co-efficient of error (CE) for all individual
optical fractionator and Cavalieri estimates was calculated
according to the method of Gundersen and Jensen (61) and
was ,0.08 in all these analyses. Data from the quantitative
western blots was entered into Microsoft Excel and GraphPad
Prism v.5 for further analysis. Statistical comparisons of
protein expression levels between diseased mice and wild-type
littermates were undertaken using an unpaired t-test, with stat-
istical significance considered at P ? 0.05 (ns ¼ not signifi-
cant;?P , 0.05;??P , 0.01;???P , 0.001).
ACKNOWLEDGEMENTS
The authors would like to thank Derek Thomson and members
of the Gillingwater, Parson and PSDL laboratories for helpful
advice and assistance with this study and Dr Calum Sutherland
(University of Dundee) for the gift of antibodies.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by grants from the Wellcome Trust
(grant WT084151AIA to T.H.G.; grant GR079491MA to
J.D.C.); Biotechnology and Biological Sciences Research
Council (grant BB/D001722/1 to T.H.G.); National Institutes
of Health (grant NS41930 to J.D.C.; NS40580 and NS44310
to D.A.P.; NS043105 to M.S.S.); European Commission 6th
Framework (grant LSHM-CT-2003-503051
Batten Disease Support and Research Association (to
T.H.G./J.D.C./C.K. and D.A.P.); Natalie Fund (to J.D.C.);
Batten Disease Family Association (to J.D.C./C.K.); and
Remy Fund (to J.D.C.). Funding to pay the Open Access
publication charges for this article was provided by the
Wellcome Trust.
toJ.D.C.);
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