Structural and molecular myelination deficits occur prior to neuronal loss in the YAC128 and BACHD models of Huntington disease
Human Molecular Genetics
Structural and molecular myelination deficits occur prior to neuronal loss in the YAC128 and BACHD models of Huntington disease
Roy Tang Yi Teo 2
Xin Hong 1
Libo Yu-Taeger 0 6
Yihui Huang 2
Liang Juin Tan 2
Yuanyun Xie 5
Xuan Vinh To 1
Ling Guo 1
Reshmi Rajendran 1
Arianna Novati 0 6
Carsten Calaminus 4
Olaf Riess 0 6
Michael R. Hayden 2 3 5
Huu P. Nguyen 0
Kai-Hsiang Chuang 1
Mahmoud A. Pouladi 2 3 7
0 Institute of Medical Genetics and Applied Genomics, University of Tuebingen , 72076 Tuebingen , Germany
1 Singapore Bioimaging Consortium, Agency for Science, Technology and Research , Singapore 138648 , Singapore
2 Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research , Singapore (A
3 Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore , Singapore 117597 , Singapore
4 Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tuebingen , 72076 Tuebingen , Germany
5 Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia , Vancouver, BC V5Z 4H4 , Canada
6 Centre for Rare Diseases, University of Tuebingen , 72076 Tuebingen , Germany
7 STAR) , Singapore 138648 , Singapore
White matter (WM) atrophy is a significant feature of Huntington disease (HD), although its aetiology and early pathological manifestations remain poorly defined. In this study, we aimed to characterize WM-related features in the transgenic YAC128 and BACHD models of HD. Using diffusion tensor magnetic resonance imaging (DT-MRI), we demonstrate that microstructural WM abnormalities occur from an early age in YAC128 mice. Similarly, electron microscopy analysis of myelinated fibres of the corpus callosum indicated that myelin sheaths are thinner in YAC128 mice as early as 1.5 months of age, well before any neuronal loss can be detected. Transcript levels of myelin-related genes in striatal and cortical tissues were significantly lower in YAC128 mice from 2 weeks of age, and these findings were replicated in differentiated primary oligodendrocytes from YAC128 mice, suggesting a possible mechanistic explanation for the observed structural deficits. Concordant with these observations, we demonstrate reduced expression of myelin-related genes at 3 months of age and WM microstructural abnormalities using DT-MRI at 12 months of age in the BACHD rats. These findings indicate that WM
deficits in HD are an early phenotype associated with cell-intrinsic effects of mutant huntingtin on myelin-related transcripts
in oligodendrocytes, and raise the possibility that WM abnormalities may be an early contributing factor to the pathogenesis
Huntington disease (HD) is an autosomal dominant hereditary
disorder characterized by loss of motor control, cognitive
deficits and psychiatric disturbances (1). Although preferential
degeneration of medium spiny neurons in the caudate and
putamen regions of the basal ganglia has long been considered
the major neuropathological hallmark of HD, myelin breakdown
and white matter (WM) atrophy also appear to be universal
features of the disease (2–5). Indeed, in post-mortem brain tissue of
HD patients, there is marked loss of myelin and WM volume, as
well as significant changes in the numbers and turnover rate of
oligodendrocytes, the myelinating cells of the central nervous
system (6–8). Transcriptional analyses have further shown that
the levels of a large number of myelin-related transcripts are
altered in HD brain tissues (9). These findings clearly implicate
WM atrophy in the pathology of HD.
A long-held assumption regarding the aetiology of WM
atrophy in HD is that it is simply a secondary outcome of the
progressive neuronal loss that manifests with advancing disease.
However, imaging-based measures that allow the non-invasive
assessment of brain structure and function during the early
stages of disease have demonstrated that WM abnormalities
are in fact an early event. Indeed, structural magnetic
resonance imaging (MRI) studies of pre-manifest HD gene carriers
reveal that progressive loss of WM volume can be observed
many years before disease onset (3,10,11). In addition to
structural MRI, magnetic resonance diffusion tensor imaging
(MRDTI), which has been widely used to examine the integrity of
neuronal tract connectivity, suggests the presence of
microstructural abnormalities in myelinated tracts not only in
patients with HD but also in pre-symptomatic gene carriers (12–
14). While these studies support the occurrence of WM
abnormalities as an early event in HD, they do not preclude the
possibility that these changes are secondary to neuronal loss.
Furthermore, the nature of these WM abnormalities at a
molecular and microstructural level remains poorly defined.
YAC128 HD mice carry a transgene that expresses the full
length human huntingtin (HTT) protein with 128 polyglutamine
repeats, and develop a number of progressive motor (15,16),
cognitive (17) and affective phenotypes (18,19) that mimic the
symptoms observed in patients with HD. YAC128 HD mice also
develop neuropathological and molecular phenotypes that mimic
key aspects of brain pathology in human HD, including
transcriptional dysregulation as early as 3 months of age (20–22) and
preferential striatal neuronal loss starting at 12 months of age (15).
To better characterize the nature of WM changes in HD and
determine their importance in HD pathogenesis, we analyse changes
in WM structure and myelin-related gene expression in the
YAC128 mouse model. We examine the microstructure of the
WMrich regions of the brain using longitudinal MR-DTI, and the
thickness of the myelin sheaths of YAC128 mice by electron microscopy
(EM), from 1.5 months of age prior to the detection of any neuronal
loss. We also analyse the levels of myelin-related transcripts in
striatal and cortical tissues in YAC128 HD mice from 2 weeks of age
to determine the effects of mutant HTT on early myelination, and
examine whether these changes are intrinsic to oligodendrocytes
using differentiated primary oligodendrocyte cells. Finally, to
corroborate our findings, we evaluate the MR-DTI phenotypes and
the expression of myelin-related genes in the transgenic BACHD
rat model of HD (23). Our results are important for understanding
the contribution of WM abnormalities to the pathogenesis of HD.
MR-DTI reveals early microstructural WM abnormalities
in YAC128 HD mice
DTI is a non-invasive approach widely used to assess the
integrity of WM microstructure. The principal metric derived from
MR-DTI is fractional anisotropy (FA), a measure of the
directionality of water diffusion. In fibre bundles with coherent
orientation, higher FA values indicate that diffusion occurs primarily
along the fibre orientation, correlating with higher overall
integrity of the WM microstructure and organization. Voxel-wise
analysis indicated that FA values were significantly lower in the
WM-rich brain regions of YAC128 mice compared with
wildtype (WT) mice, including in the anterior commissure (AC),
corpus callosum (CC), internal capsule and external capsule (EC)
from 1.5 months of age, and in the cingulum (CG) and cerebral
peduncle from 3 months of age (Fig. 1A and B; Supplementary
Material, Table S1). Quantification of the FA values in the
different brain regions indicated significant reductions over the first
year of life in YAC128 mice compared with WT littermates (Fig
1C). These MR-DTI results indicate that WM microstructure
abnormalities are present in YAC128 HD mice early on, before the
manifestation of behavioural deficits and neuronal atrophy.
Ultrastructural quantitative analysis of myelination in
YAC128 HD mice
To examine whether the observed FA deficits are associated
with altered myelination, we used EM on an independent cohort
of mice to visualize myelinated fibres in the CC at 1.5 and 3
months of age (Fig. 2A). G-ratios, a measure of myelin sheath
thickness calculated as the ratio of axon diameter to myelinated
fibre diameter, were determined for YAC128 HD mice and WT
littermates. The mean g-ratio of myelinated axons from
YAC128 HD mice was significantly higher for YAC128 HD mice
compared with WT controls at both 1.5 and 3 months of age,
indicating that the myelin sheaths were thinner in YAC128 HD
mice (Fig. 2B and E). Plotting g-ratios against axonal diameters
demonstrated that g-ratios of larger calibre axons were higher
in YAC128 HD mice compared with WT littermates (Fig. 2C and
F). In line with these findings, frequency distributions of g-ratios
demonstrated a shift to the right in the population, suggesting a
moderate thinning of myelin sheaths in YAC128 HD mice (Fig.
2D and G). These changes are observed before the manifestation
of behavioural disease phenotypes in 1.5-month-old mice, and
persist following onset of phenotypes in 3-month-old mice.
RNA transcripts of myelin-related genes are altered in
pre-manifest YAC128 HD mice
To investigate whether changes in myelin-related gene
expression are present early in the course of disease prior to neuronal
loss, we measured the levels of the pan-oligodendrocyte
marker 20,30-cyclic-nucleotide 30-phosphodiesterase (CNP),
and the mature myelinating oligodendrocyte markers myelin
basic protein (MBP), and myelin oligodendrocyte glycoprotein
(MOG) in the striatal and cortical tissues of YAC128 mice and
littermate controls at 2, 4 and 12 weeks of age. Compared with
WT mice, YAC128 mice had significantly lower levels of CNP
(56–72%), MBP (50–57%) and MOG (61–69%) at 2 and 4 weeks
of age, and reduced levels of MOG were still apparent at 12
weeks (Fig. 3A). In the cortex, YAC128 mice also had
significantly lower levels of CNP (55–77%) and MOG (63–65%) at
all time points and significantly lower levels of MBP (67–70%)
at 2 and 4 weeks of age (Fig. 3B). This suggests that the
thinner myelin sheaths observed in YAC128 HD mice may
partly be a consequence of deficits in myelin protein
Expression of myelin-related genes in primary
To determine whether changes in myelin-related gene
expression reflect a cell-intrinsic effect of mutant HTT in
oligodendrocytes, we isolated oligodendrocyte precursor cells (OPCs) from
P7 pups using magnetic-activated cell sorting, and cultured
them in oligodendrocyte differentiation media over a 7-day
period. Expression levels of MOG, MBP and CNP were similar in
OPCs from YAC128 and WT mice directly ex vivo (Fig. 3C), and a
significant induction in transcript levels was observed in both
YAC128 and WT mice following the 7-day differentiation period
(Fig. 3C). However, the expression levels of MOG, MBP and CNP
in differentiated oligodendrocytes were significantly lower in
YAC128 compared with WT mice (Fig. 3C). These findings are
consistent with the reduced expression of myelin-related gene
transcripts in the striatal and cortical tissues of YAC128 mice
(Fig. 3A and B), and suggest that the observed deficits reflect
cell-intrinsic effects of mutant HTT in oligodendroglial cells.
Reduced expression of myelin-related genes and abnormal
WM microstructure in BACHD rats
To further validate the WM deficits we observed in the YAC128
mice, we evaluated select WM measures in the BACHD rats, a
transgenic model expressing full-length human mutant HTT
with 97 polyglutamine repeats (23). BACHD rats display a
number of progressive behavioural and neuropathological
phenotypes that mimic clinical and pathological features seen in
patients with HD (23–25). Consistent with the observations in
the YAC128 mice, BACHD rats exhibited significantly lower
DTMRI FA values in the anterior CC, the CG and the EC at 12
months of age compared with WT littermates, suggesting the
presence of WM microstructural abnormalities (Fig. 4A).
Similarly, the BACHD rats showed deficits in the levels of
myelin-related genes at 12 weeks of age, with significantly reduced
levels of MBP transcripts in the cortex (31%) and striatum (38%),
and cortical levels of MOG (17%) transcripts (Fig. 4B). The
transcriptional changes in MBP were paralleled on the protein level
as shown by reduced cortical MBP protein at 12 weeks of age
(Fig. 4C). In addition, the ultrastructural properties of the
BACHD rats were consistent with the YAC128 mice, with
increased average G-ratio and higher frequency distribution of
larger G-ratios pointing to overall thinner myelin sheaths
(Fig. 4D). Taken together, these findings indicate that
WM-related microstructural and molecular changes are not unique to
the YAC128 mice, but are a common early disease feature that
reflects mutant HTT-induced pathology.
WM abnormalities are a well-established pathological feature of
HD, although their molecular and structural characteristics and
aetiology have been largely unexplored. In this study, we
demonstrate that abnormalities in WM microstructure manifest in
YAC128 HD mice well before any neuronal loss. We show
that WM abnormalities appear progressively in different
WMrich regions, starting with the anterior CC and AC at 1.5 months,
the CG at 3 months, the posterior CC at 6 months and the EC at
9 months of age. We further show that the abnormalities in
WM microstructure are paralleled by the presence of
thinner myelin sheaths and lower levels of myelin-related gene
transcripts in these mice. We also show that the WM
microstructural abnormalities and lower myelin-related gene
transcripts are present in a different rodent model of HD, the
BACHD rats. These findings (summarized in Fig. 5) suggest that
WM pathology in HD is an early event and may be, at least in
the nascent stages of the disease process, independent of
Our findings support previous evidence of WM abnormalities
in animal models of HD. Assessment of brain structural changes
revealed progressive atrophy of the myelin-rich CC region in
brains of YAC128 HD mice (26). Furthermore, the volume of the
CC was the most discriminatory structure for discerning
YAC128 from WT mice, indicating the occurrence of high levels
of atrophy specifically in this region of the brain in HD (26).
Diffusion MR imaging studies have also shown abnormal WM
microstructure in the R6/2 and HdhQ250 mouse models, and in
a transgenic rat model of HD carrying a truncated HTT fragment
with 51 CAG repeats (27–29). Thinner myelin sheaths, reflected
by higher g-ratios, have been reported for the BACHD and
HdhQ250 mouse models (27,29). Similar to our findings, these
alterations in myelin sheaths were paralleled by reduced
expression of myelin-related genes, such as MBP and MOG, in R6/2
and HdhQ250 HD mice (27,29).
We show that the deficits in myelin-related gene transcripts
can be seen in differentiated primary oligodendrocytes isolated
from YAC128 HD mice, suggesting that the deficits are a
consequence of cell-intrinsic effects of mutant HTT in
oligodendrocytes. A number of potential pathogenic mechanisms
consistent with a cell-intrinsic origin of abnormal myelination
in HD have been raised previously. The
peroxisome-proliferator-activated receptor gamma coactivator 1 alpha (PGC1alpha),
a transcriptional regulator of energy metabolism, has been
suggested to play a role (27,30). PGC1alpha activity is compromised
in a number of models of HD (31–34). Impaired PGC1alpha
activity, as a result of PGC1alpha knockdown or overexpression of
mutant HTT, has been shown to reduce the levels of MBP and a
number of genes involved in the biosynthesis of cholesterol, a
major constituent of myelin, in oligodendrocytes (27). This is in
agreement with previous studies demonstrating defects in
cholesterol biosynthesis in a number of animal models of HD,
including the YAC128 HD mice (35,36). The myelin regulatory
factor (MYRF), a master transcriptional regulator of
myelin-related gene expression in mature oligodendrocytes, has also
been implicated in HD, and reduced expression levels of MYRF
were observed in the HdhQ250 mouse model (29). Furthermore,
mutant HTT was found to interact with MYRF and affect its
transcriptional activity, leading to reduced expression of
myelin-related genes (37). It should be noted that while these
observations support direct effects of mutant HTT on
oligodendrocytes, we cannot exclude the contribution of
additional non-cell intrinsic effects in vivo.
In addition to myelinating axons to insulate them
electrically and facilitate saltatory conduction along their tracts,
oligodendrocytes play important roles in maintaining axonal
integrity and function (38–40). Oligodendrocytes secrete growth
factors, including BDNF, IGF-1 and GDNF, that have
neuroprotective effects on neurons (38,40). Oligodendrocytes also provide
axonal metabolic support through mechanisms that include
export of lactate through monocarboxylate transporters (38,41,42).
The importance of the latter was demonstrated in a recent
study showing that compromised function of the
oligodendroglia-enriched monocarboxylate transporter 1 results in axonal
damage and neuronal degeneration (43). Thus, it is interesting
to speculate whether, in addition to the deficits in myelination
we describe, mutant HTT interferes with other oligodendroglial
functions which are important to axonal integrity, and whether
such impairments contribute to axonal degeneration and
neuronal dysfunction in HD (44).
An important consideration is whether oligodendrocyte
dysfunction and associated abnormalities in myelination and WM
structure contribute to the clinical manifestations of HD.
Indeed, proper myelination is integral for rapid propagation of
action potentials, and loss of myelination is associated with
severe disabilities in a number of brain disorders (45). In HD, a
number of studies have found significant correlations between
indices of WM structural integrity such as MR-DTI FA and
neuropsychiatric symptoms, including depression, irritability and
apathy (46–48). Furthermore, distinct patterns of abnormalities
in discrete WM tracts are correlated with motor and cognitive
deficits in HD (49). In mice, expression of mutant HTT
specifically in mature oligodendrocytes under the proteolipoprotein
promoter was sufficient to induce a number of pathological and
behavioural abnormalities, including progressive body weight
loss, impaired motor performance and reduced survival (37).
These findings indicate that WM abnormalities are associated
with clinical outcomes and functional decline, and may indeed
contribute to the pathological and behavioural manifestation of
HD. This raises the possibility that interventions aimed at
restoring oligodendrocyte function and proper myelination may
confer therapeutic benefits in HD.
There have been no major efforts aimed specifically at
ameliorating oligodendrocyte dysfunction and maintaining WM
integrity in HD, but some evidence of potential therapeutic value
exists. Improved callosal WM microstructure as a result of
rhythm exercise was associated with improved executive
function in HD (50). Immunotherapy-mediated inhibition of
semaphorin 4D, a negative regulator of oligodendrocyte precursor cell
migration and differentiation, prevented atrophy of the CC and
improved behavioural phenotypes in YAC128 HD mice (51). These
studies suggest that WM-targeted therapeutic approaches may
be beneficial in HD, though this is yet to be directly tested.
In conclusion, we demonstrate that WM abnormalities occur
early, prior to any neuronal loss, and are paralleled by
myelination deficits and reduced myelin-related gene expression by
oligodendrocytes in YAC128 HD mice and BACHD rats. Our
findings support the need for future studies to address the
contribution of WM abnormalities to the clinical manifestations of
HD and to evaluate their potential as targets for therapy in HD.
Materials and Methods
For the YAC128 experiments, male and female mice (line 53)
expressing a full-length human HTT transgene with 128 CAG
repeats maintained on the FVB/N strain (8) were housed with
littermates of mixed genotype in groups of 2–5 on a 12 h light/
dark cycle with free access to food and water. All experiments
were performed with the approval of the Institutional Animal
Care and Use Committee at Biological Resource Centre (BRC),
A*STAR. For the BACHD rats, experiments were approved by the
local ethics committee at Regierungspraesidium Tuebingen,
and carried out in accordance with the German Animal Welfare
Act and the guidelines of the Federation of European Laboratory
Animal Science Associations, based on European Union
legislation (Directive 2010/63/EU).
Real-time quantitative PCR
YAC128 mice: Microdissected striatal and cortical tissues of
YAC128 HD and WT littermate controls (9 per genotype, all males)
were snap frozen, and total RNA was extracted using the RNeasy
Mini Kit (Qiagen). For primary culture experiments, total RNA
from OPCs was extracted from a minimum of two pooled pup
cortices per independent biological replicate (seven pooled
biological replicates per genotype, mixed sex) using the PureLink
RNA Micro Kit (Invitrogen). First-strand cDNA synthesis was
performed using the SuperScript First Strand Synthesis System
(Invitrogen). Following preliminary runs with selected primers
from Primerbank (http://pga.mgh.harvard.edu/primerbank/, last
accessed April 28, 2016) on WT cortical cDNA, the mouse-specific
primers listed in Table 1 were subsequently used on all samples.
All probes were run in triplicate with Sybr Select Universal Master
Mix (Invitrogen) on a StepOnePlus (ABI) to obtain Ct values.
BACHD rats: Rat brains were rapidly dissected on ice and
stored at 80 C. Total mRNA was extracted from frozen tissues
of the striatum and cortex using the RNeasy Lipid Tissue
middle/Large Kit and cDNA was synthesized using a QuantiTect
Reverse Transcription Kit following the manufacturer’s
instructions (Qiagen, Germany). Real-time PCR using QuantiTect SYBR
Green PCR Kits (Qiagen, Germany) was performed for the
analyses of the expressions of MOG, MBP and CNP at cDNA level in
both striatum and cortex. Absolute quantification of cDNA was
performed using the Light Cycler 480 instrument with the aid of
built-in Light Cycler software. ATP5B and Eif4a were chosen as
reference genes for analyzing the cDNA levels in the striatum
and ATP5B and CanX3 for the analyses in the cortex. Forward
and reverse primers (Table 1) of a gene were designed to be at
least one intron apart and to cover all transcription variants of
the target gene. Data were compared between male BACHD TG5
rats and WT littermates at 3 months of age (n ¼ 6). One
transgenic rat and one WT rat were excluded for the analysis of MBP
expression due to the extremely low CT values of both
housekeeping genes detected by real-time PCR.
Animals (3 per genotype, all males) were transcardially perfused
with 2.5% glutaraldehyde and 2.5% PFA in 0.1 M sodium
cacodylate buffer before post-fixing the brains overnight at 4 C in the
same buffer, and then subsequently washing in phosphate
buffered saline. Brain samples were sent to the Harvard Medical
School EM unit for further processing. Briefly, coronal slices at the
level of Bregma -1 mm were made from the central part of the CC
before post-fixation in 1% osmium tetroxide/1.5% potassium
ferrocyanide solution for 1 h. After washing with 1% uranyl acetate
Forward primer (50–30)
Reverse primer (50–30)
in maleate buffer, the samples were dehydrated, infiltrated with
epon, embedded and polymerized at 60 C for 2 days. Ultra-thin
slices (100 nm) were cut before imaging on a transmission
electron microscope. For image analyses, Stereoinvestigator v11 was
employed to allow for systematic random selection of axons,
with a minimum of 500 axons analysed per brain.
MRI acquisition and analysis
YAC128 HD mice: 16 mice (8 WT; and 8 YAC128; 50% male) were
scanned at 1.5, 3, 6, 9 and 12 months old. The mice were
anaesthetized using 1–3% isoflurane mixed with oxygen and air (1:1
ratio) through a nose cone during preparation and MRI
scanning. Respiration rate was maintained at 80 6 10 bpm and
temperature at 36–37 C with an MRI-compatible heater (SAII, NY).
MRI was conducted on a 7T scanner (ClinScan, Bruker BioSpin,
Germany) with four-channel mouse brain array coils. DTI was
acquired using a spin-echo EPI sequence with eight averages of
30 diffusion sensitizing directions, b ¼ 1500 s/mm2, repetition
time (TR) ¼ 10000 ms, echo time (TE) ¼ 40 ms and
0.2 0.2 0.5 mm3 voxel resolution. The acquisition time was
45.5 min. High-resolution structural MRI was acquired with fast
spin-echo with TR ¼ 2760 ms, TE ¼ 43 ms and voxel
size ¼ 0.1 0.1 0.3 mm3. After eddy current distortion and
motion correction, tensor fitting was carried out using the weighted
least squares method with FDT (FMRIB’s Diffusion Toolbox, FSL,
Oxford; http://fsl.fmrib.ox.ac.uk/fsl/, last accessed April 28, 2016)
to obtain FA. For each time point, one animal with the highest
signal-to-noise ratio was chosen to be the initial target, and the
remaining animals’ FA images were non-linearly registered to it
using FNIRT (FMRIB’s nonlinear image registration tool). The
registered images were then averaged to create time
point-specific FA templates. Another round of non-linear registration was
carried out to align all FA images to the corresponding FA
template. After in-plane Gaussian smoothing of 0.3 mm full-width
at half maximum, voxel-wise group analysis was conducted on
the FA map using two-sample t-tests in SPM8 (http://www.fil.
ion.ucl.ac.uk/spm/, last accessed April 28, 2016). The statistical
analysis was restricted to WM regions with FA > 0.175 on the
template. Family wise correction was performed with a cluster
threshold of P < 0.05 determined by a Monte-Carlo simulation
using 3DClustSim in AFNI (NIH; http://afni.nimh.nih.gov/, last
accessed April 28, 2016). Based on the results of the voxel-wise
analysis, regions of interest were manually drawn in the
anterior CC, posterior CC, AC, EC and CG on the templates (Fig. 1B).
The average FA values in those regions were calculated.
BACHD rats: Diffusion tensor images were acquired using an
echo planar imaging with 256 directions (b ¼ 0,1000s/mm2,
54 21 mm field of view, 128 52 mm matrix, twenty-six 1 mm
slices, 60/5500ms TE/TR). FA images were generated using
Inveon Acquisition Workplace. Inveon Research Workplace
software was used for the image analysis, where a volume of
interest approach was implemented on anterior and posterior CC, the
CG and the EC. FA values were compared between male BACHD
rats and WT controls at 12 months of age (TG5:WT ¼ 14:13 for EC,
TG5:WT ¼ 11:10 for the remaining areas).
Primary culture and differentiation of OPCs
Cortices from P6-P7 pups (seven pooled biological replicates per
genotype, mixed sex) were dissected and subsequently
dissociated with the Neural Tissue Dissociation Kit from Miltenyi.
Anti-O4 magnetic microbeads (Miltenyi) were then used to
isolate a pure population of O4þ OPCs. For culture and
differentiation, serum- and antibiotic-free medium comprising 2% B27
(Gibco) and 30 ng/ml thyroid hormone (Sigma) in Neural Basal
Media (Gibco) was used. Medium was changed daily and cells
were harvested after 7 days.
To access protein expression level of MBP in BACHD rats,
western blot was applied using striatal brain lysis of male BACHD
TG5 rats and WT controls at 3 months of age (n ¼ 3). The striata
were stored at 80 C after dissection and were homogenized
with a tissue homogenizer at a speed of 30 000 rpm for 30 s in 10
volumes (w/v) modified RIPA buffer (50 mM Tris pH 7.5, 150 mM
NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS) with Complete
Protease Inhibitor Cocktail tablets (Roche, Germany). After a
further 5-min sonication with bath sonicator for shearing genomic
DNA, the lysates were centrifuged at 4 C for 15 min at 16 200g,
and the supernatant was used for western blot analysis (52).
The blot was probed with polyclonal rat antibody recognizing
rat MBP (1: 5000, ab40390, abcam, USA) followed by incubation
in the HRP-conjugated secondary antibody (1:10 000, ab191866,
abcam, Cambridge, MA, USA). Finally, blots were developed
with ECL Western Blotting Detection Reagent (RPN2134,
Amersham Biosciences, Germany) and followed by visualization
on X-ray film (AGFA, Germany).
Graphpad Prism v6 was used for statistical analyses and data
are expressed as means 6 SEM. Two-way ANOVA was
performed to assess the effects of genotype and age on FA. Unless
otherwise stated, pair-wise comparisons between genotypes at
individual time points were assessed with a Student’s t-test.
Differences were considered statistically significant when
P < 0.05.
Supplementary Material is available at HMG online.
We thank Maria Ericsson and Elizabeth Benecchi (Electron
Microscopy Facility, Harvard Medical School) for electron
microscopy imaging services. We thank Kerry McLaughlin for
Conflict of Interest Statement. None declared.
The work was supported by a Joint Council Grant (1331AFG078)
and a Strategic Positioning Fund for Genetic Orphan Diseases
(SPF2012/005) from the Agency for Science Technology and
Research and by the National University of Singapore to M.A.P.
The BACHD rat work was supported by the European Union
seventh Framework Program (FP7/2012), Project ‘SWITCH-HD’, under
grant agreement No. 324495 to H.P.N. Funding to pay the Open
Access publication charges for this article was provided by the
Agency for Science, Technology and Research (A*STAR),
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