DISC1 (Disrupted-in-Schizophrenia-1) Regulates Differentiation of Oligodendrocytes
et al. (2014) DISC1 (Disrupted-in-Schizophrenia-1) Regulates Differentiation of
Oligodendrocytes. PLoS ONE 9(2): e88506. doi:10.1371/journal.pone.0088506
DISC1 (Disrupted-in-Schizophrenia-1) Regulates Differentiation of Oligodendrocytes
Tsuyoshi Hattori 0
Shoko Shimizu 0
Yoshihisa Koyama 0
Hisayo Emoto 0
Yuji Matsumoto 0
Natsuko Kumamoto 0
Kohei Yamada 0
Hironori Takamura 0
Shinsuke Matsuzaki 0
Taiichi Katayama 0
Masaya Tohyama 0
Akira Ito 0
Alexandre Hiroaki Kihara, Universidade Federal do ABC, Brazil
0 1 Department of Molecular Neuropsychiatry, Graduate School of Medicine, Osaka University , Suita, Osaka , Japan , 2 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University , Suita, Osaka , Japan , 3 Department of Child Development & Molecular Brain Science, United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine , Suita, Osaka , Japan , 4 Division of Molecular Brain Science, Research Institute of Traditional Asian Medicine, Kinki University , Sayama, Osaka, Japan, 5 Pharmacology Research Laboratories , Dainippon Sumitomo Pharma Co, Ltd , Suita, Osaka , Japan , 6 Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University , Nagoya, Aichi , Japan
Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a translocation, t(1;11) (q42.1;q14.3), that segregates with major psychiatric disorders, including schizophrenia, recurrent major depression and bipolar affective disorder, in a Scottish family. Here we report that mammalian DISC1 endogenously expressed in oligodendroglial lineage cells negatively regulates differentiation of oligodendrocyte precursor cells into oligodendrocytes. DISC1 expression was detected in oligodendrocytes of the mouse corpus callosum at P14 and P70. DISC1 mRNA was expressed in primary cultured rat cortical oligodendrocyte precursor cells and decreased when oligodendrocyte precursor cells were induced to differentiate by PDGF deprivation. Immunocytochemical analysis showed that overexpressed DISC1 was localized in the cell bodies and processes of oligodendrocyte precursor cells and oligodendrocytes. We show that expression of the myelin related markers, CNPase and MBP, as well as the number of cells with a matured oligodendrocyte morphology, were decreased following full length DISC1 overexpression. Conversely, both expression of CNPase and the number of oligodendrocytes with a mature morphology were increased following knockdown of endogenous DISC1 by RNA interference. Overexpression of a truncated form of DISC1 also resulted in an increase in expression of myelin related proteins and the number of mature oligodendrocytes, potentially acting via a dominant negative mechanism. We also identified involvement of Sox10 and Nkx2.2 in the DISC1 regulatory pathway of oligodendrocyte differentiation, both well-known transcription factors involved in the regulation of myelin genes.
Funding: This work was partially supported by Grant-in-Aid for Scientific Research on Innovative Areas Neural Diversity and Neocortical Organization, from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 23123512 and No. 23700415), and by a grant from Dainippon Sumitomo
Pharma Co., Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Although two authors (HE and YM) have affiliation to Dinippon Sumitomo Pharma Co, Ltd., authors have no competing interest. And this
does not alter the authors adherence to the PLOS ONE policies on sharing data and materials. The authors have declared that no competing interests exist.
DISC1 gene is specifically disrupted by a t(1;11) (q42.1;q14.3)
balanced translocation, in a large Scottish pedigree, which leads to
several major mental illnesses, such as schizophrenia (SZ), bipolar
affective disorder and recurrent major depression . Many
subsequent genetic studies indicated that DISC1 is not only
implicated in schizophrenia and mood disorders, but also in
autism spectrum disorders, Asperger syndrome, attention deficit
and hyperactivity disorder (ADHD) and agenesis of the corpus
callosum . Biological studies have shown that DISC1 plays a
role in multiple process of brain development such as neuronal
proliferation, migration, differentiation, and modulation of DISC1
gene in rodents causes behavioral changes .
Multiple lines of evidence, obtained by brain imaging, studies in
postmortem brains and genetic association studies, have
implicated oligodendrocytes and myelin dysfunction in SZ, major
depressive disorder (MDD), autism and ADHD .
Specifically, compromised white matter (WM)/myelin integrity, a
reduced number and/or altered morphology of oligodendrocytes,
and the aberrant expression and genetic association of
oligodendrocytes/myelin-related genes have been identified by a number
of studies . It can also be inferred from the higher than
chance co-occurrence of WM-diseases, such as multiple sclerosis
(MS), leukodystrophies and velocardiofacial syndrome, with
SZlike psychoses , that oligodendrocytes and myelin
dysfunction may play a key role in the pathophysiology of mental illness.
Despite substantial evidence indicating the role of
oligodendrocyte abnormalities in pathophysiology of psychosis,
neurobiological studies have predominantly focused on neurons. Accordingly,
a large number of studies have shown the key role of DISC1 in
neurons , while only a handful of studies have addressed a
possible role of DISC1 in oligodendrocytes . DISC1
expression in human brain and primary cultured rat cortical
oligodendrocytes was shown by Seshadri et al.  and a critical
requirement for DISC1 in oligodendroglial development, by
promoting specification of olig2-positive cells in the hindbrain
and other brain regions of zebrafish, was reported by Wood et al
. Nevertheless, no study to date, has directly addressed the
functional role of DISC1 expressed in a mammalian cell of glial
By examining the effect of RNA interference (RNAi) on
endogenous DISC1, and also overexpression of either truncated
DISC1 or full length DISC1, we here show for the first time that
endogenous DISC1 expressed in glial cells negatively regulates
mammalian oligodendrocyte development in vitro, acting upstream
of Sox10 and/or Nkx2.2.
Materials and Methods
The study protocol was approved by the Institutional Animal
Care and Use Committee of Osaka University (No. 20-138-006).
Antibodies used in this study are as follows: anti-CNPase
(Sigma, St. Louis, MO, USA); anti-APC (Millipore, Billerica, MA,
USA); anti-MBP (Millipore); anti-b-tubulin (Sigma); anti-GFP
(Abcam, Cambridge, MA, USA); anti-GAPDH (Santa Cruz
Biotechnology, Santa Cruz, CA, USA); HRP-linked anti-IgG
(Cell Signaling, Danvers, MA, USA); Alexa-Flour568-anti-IgG
and Alexa-Flour488-anti-IgG (Molecular Probes). DAPI was
purchased from Invitrogen.
Plasmids and Adenovirus
Complementary DNAs coding full length human DISC1 and
truncated human DISC1 (1598) were cloned into pEGFP-C1. A
recombinant adenovirus expressing either GFP, human DISC1
with GFP fused to the C-terminus, or truncated human DISC1 (1
598) with GFP fused to the C-terminus (GFP-Adv,
DISC1-GFPAdv and trDISC1-GFP-Adv, respectively) was generated using the
ViraPower Adenoviral Expression System (Invitrogen), according
to the manufacturers instructions.
In situ Hybridization-immunohistochemistry
cDNA fragments of mouse DISC1 were amplified by
reversetranscribed-PCR using the sense/antisense primer set of
-39/59TCAGGCCTCGGTTTCCTGAG-39, and used as templates for
probe synthesis. Probe was hydrolyzed and in situ hybridization of
coronal mouse brain sections with DIG-labeled RNA probes was
performed as described previously . The slides were washed
thoroughly in PBS following a colorimetric reaction. Next, the
slides were incubated overnight at 4uC with the primary antibody
(monoclonal mouse anti-APC antibody) at 1:50 in PBS. After
washing in PBS, the slides were incubated for 30 min at RT with
the secondary antibody (biotinylated anti-mouse IgG antibody
from Vector Laboratories). After amplification with the
avidinbiotin complex using ABC kit (Vector Laboratories), reaction
products were visualized with 50 mM Tris-HCl buffer (pH 7.6)
containing 0.02% diaminobenzidine tetrahydrochloride (Sigma)
and 0.01% hydrogen peroxide. After dehydration, the sections
were sealed using Entellan.
siRNA Design and RNA Interference
The targeted sequences of rat DISC1 were:
59-GGCTACATGAGAAGCACAG-39 (DISC1-siRNA-1) and
59CTGGCTGATGCGAGAGAAA-39 (DISC1-siRNA-2). The
DISC1-siRNA-1 was previously shown to knockdown mouse
and rat DISC1 [15,40,41]. The DISC1-siRNA-2 was designed
using online software tool siDirect. We validated knockdown of
endogenous rat DISC1 transcripts by transfecting
DISC1-siRNA1 or DISC1-siRNA-2 in oligodendrocyte precursor cells. The
targeted sequences of rat Sox10 and Nkx2.2 siRNAs were:
59CTGTGTCACTGTCCTAAA-39 (Sox10-siRNA) and
59GTTTGTGTGAGTAGCGATA-39 (Nkx2.2-siRNA). A
scrambled sequence (59-GCGCGCTTTGTAGGATTCGT-39) was
used as a control. Transfection of these siRNAs was carried out
using RNAiMAX (Invitrogen), according to the manufacturers
protocol. To assess the transfection efficiency of siRNA, cells were
transfected with Block-iT Alexa Fluor Red Fluorescent Oligo
(Invitrogen) and examined under fluorescence microscope 24
In vitro Oligodendrocyte Differentiation and Transfection
Primary cultures of rat oligodendrocyte precursor cells were
established and induced to differentiate into oligodendrocytes
according to the method of Chen et al., with some modification
. Single cell suspensions of P1 rat cortex was prepared in
MEM supplemented with 0.292 g/l L-glutamine, 4 g/l D-glucose,
3.2 g/l NaHCO3 and 10% FBS, and plated on poly-L-lysine
(PLL) coated culture flasks (Nunc). These mixed brain cell cultures
were cultured in humidified CO2 incubators for 10 to 14 days with
the medium changed every 3 days. Twelve hours after the last
medium change, the flask was rotated at 200 rpm for 20 hours to
dislodge glial lineage cells. Dislodged cells were plated on
noncoated dishes and incubated for 1 hour to allow astrocytes and
microglia to adhere to the dish. Oligodendrocyte precursor cells
were collected as non-adherent cells, re-suspended in proliferation
medium (PM: Neurobasal medium (Invitrogen) supplemented with
5 ng/ml insulin (Sigma), 5 ng/ml NT3 (Pepro Tech Inc., Rocky
Hill, NJ, USA), 10 ng/ml PDGF (Wako, Osaka, Japan), 2 mM
Lglutamine (Sigma) and B27 (Invitrogen)). Oligodendrocyte
precursor cells plated on PLL coated flasks were maintained for 3 days
with half-medium-changes with PM every second day.
Differentiation of oligodendrocyte precursor cells to oligodendrocytes was
induced by replacing the whole medium with PM deprived of
PDGF (0 hours). After the induction of differentiation, cells were
maintained with half-medium-changes with PDGF deprived PM.
Cells were infected with adenovirus expressing GFP, DISC1-GFP
or trDISC1-GFP, 12 hours before PDGF deprivation at 0 hours.
We confirmed that proportion of cells positive for oligodendrocyte
precursor cells marker was 91.862.4% of the whole cell
population at 0 hours by immunostaining with anti-NG2 antibody.
Transfection of siRNAs was performed at 0 hours and the whole
medium was changed with PM 4 hours after transfection. In
rescue experiments, cells were infected with adenovirus expressing
GFP or DISC1-GFP 24 hours after the siRNA transfection and
the whole medium was changed with PM 12 hours after the
Quantitative Real-time PCR (qRT-PCR)
Total RNA was reverse-transcribed using High-Capacity cDNA
Reverse Transcription Kits (Applied Biosystems, Warrington,
UK), and analyzed by RT-PCR to determine expression levels of
DISC1, CNPase, MBP, Sox10, Nkx2.2, b-actin and GAPDH. For
DISC1 gene, two sets of forward/reverse primers were used: DISC1
CTCATGCCTATGGCTTCGC-39 (DISC1 primer-1) or
(DISC1 primer-2). DISC1 primer-1 and DISC1 primer-2 target
to exon 11 to 12 and exon 5 to 6 of rat DISC1 gene, respectively.
The following sets of forward/reverse primers were used for other
Figure 1. DISC1 mRNA is expressed in oligodendrocytes in the corpus callosum of mouse brain. A Double In situ
hybridizationimmunohistochemistry analysis of hippocampal sections from P70 mice with the antisense RNA probe to DISC1 and antibody against APC. Scale bar,
200 mm. H: hippocampus AS: antisense B As controls, adjacent sections were hybridized with DIG-labeled sense RNA probe. S: sense C, D, Double In
situ hybridization-immunohistochemistry analysis of brain sections from P14 and P70 mice demonstrates the expression of DISC1 mRNA in APC
expressing oligodendrocytes in the corpus callosum of mouse at P14 (C) and P70 (D). High magnified images of the squared region in the left panels
are shown in the adjacent right panels. Arrowheads indicate DISC1+/APC+ cells. Scale bars, 50 mm.
59-AGCCCAGGTGAAGACAGAGA-39/59CCCCTCTAAGGTCGGGATAG-39 and Nkx2.2
59CGGGCTGAGAAAGGTATGGA-39/59TGTGCTGTCGGGTACTGGG-39. To standardize the
experiments, we designed primer sets
(59-CCTGTATGCCTCTGGTCGTA-39/59CCATCTCTTGCTCGAAGTCT-39) to amplify a portion of
the rat GAPDH and b-actin gene respectively. RT-PCR was set up
using Power SYBR Green PCR Master Mix (Applied Biosystems).
Comparison of specific ratios (gene of interest/GAPDH or b-actin)
was used to assess differences in expression levels between groups.
Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4,
containing 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1%
SDS, 0.5% sodium deoxycholate and protease inhibitor mixture).
Western blotting was performed as described previously .
Immunocytochemistry was performed as described . Mouse
anti-b-tubulin, rabbit anti-GFP and mouse anti-CNPase
antibodies were all used at dilutions of 1:200. Confocal microscopy was
performed using an LSM-510 laser scanning microscope (Carl
Zeiss, Oberkochen, Germany). For the analysis of morphological
differentiation, cells were classified to one of the following
morphological categories: simple, bipolar or stellate cells having
short primary branches; intermediate, cells having very long
primary branches and/or secondary branches; or complex
morphology, cells with tertiary branches .
Figure 2. DISC1 expression decreases in the course of oligodendrocyte differentiation. A, Primary cultured cells were harvested at
indicated times after PDGF deprivation and mRNA was quantified by qRT-PCR. Data are expressed as the mean6s.e.m. of at least three independent
experiments. DISC1 mRNA level at 0 hr is higher than later time-points with p,0.01 by one-way ANOVA followed by Tukeys test. B, Intracellular
localization of overexpressed DISC1-GFP in cultured oligodendrocyte precursor cells and oligodendrocytes. Cells were immunostained with anti-GFP
antibody. Scale bar = 50 mm.
Unless otherwise mentioned, all statistical comparisons were
determined by Students t-test, with significant differences
indicated by p,0.05.
DISC1 is Expressed in Oligodendrocytes in the Mouse
Although a previous study reported DISC1 expression in
oligodendrocytes in human brain, DISC1 expression in
oligodendroglial lineage cells in mouse brain had not been investigated
. To investigate whether DISC1 transcripts are expressed in
oligodendrocytes of mouse brain, we performed in situ
hybridization-immunohistochemistry using DIG-labeled RNA probe for
mouse DISC1 and anti-APC antibody. Consistent with previous
studies [41,46], DISC1 mRNA was highly expressed in the
hippocampus at P70 (Fig. 1 A). No signal was detected in sections
hybridized with the sense probe confirming the specificity of our in
situ hybridization probe (Fig. 1 B). DISC1 mRNA was found in
cells expressing APC, an oligodendrocyte marker, both at P14
(Fig. 1 C) and P70 (Fig. 1 D). Furthermore, higher expression of
DISC1 mRNA in oligodendrocytes at P14 than P70 was
suggested. These results show that DISC1 is expressed in
oligodendrocytes of mouse brain.
DISC1 Expression Decreases in the Course of in vitro
As a developmental decrease of DISC1 mRNA in the mouse
corpus callosum was suggested, we investigated DISC1 expression
during in vitro differentiation of oligodendrocyte precursor cells to
oligodendrocytes. Primary cultured rat oligodendrocyte precursor
cells were induced to differentiate to oligodendrocytes by depriving
PDGF from the culture medium. Quantitative PCR analysis using
two sets of primers for DISC1 showed that DISC1 mRNA
expression was reduced after PDGF deprivation (Fig. 2 A). The
decrease of DISC1 expression was confirmed using DISC1
primer-1 and another reference gene (b-actin) (100% for 0 h;
48.9611.3% for 48 h; 26.064.8% for 96 h; 36.4613.5% for
120 h; 22.663.8% for 144 h). These results suggest that DISC1 is
involved in differentiation of oligodendrocyte lineage cells. Next,
we examined the subcellular localization of overexpressed DISC1
in primary cultured oligodendrocyte precursor cells and
oligodendrocytes by immunocytochemistry. Overexpressed DISC1 was
preferentially expressed in the cell soma and cytoplasmic processes
of oligodendrocytes, with marginal nuclear expression. In
oligodendrocyte precursor cells, subcellular localization of DISC1 was
similar to oligodendrocytes (Fig. 2 B). These results demonstrate
that DISC1 expression is more abundant in oligodendrocyte
precursor cells and DISC1 protein is expressed in cell soma and
processes in oligodendrocyte precursor cells and oligodendrocytes.
DISC1 Overexpression Retards Oligodendrocyte
To explore the functional role of decreased expression of DISC1
in the course of oligodendrocytes differentiation (Fig. 2A), DISC1
was overexpressed in oligodendrocyte precursor cells using a
DISC1 expressing adenovirus (DISC1-GFP-Adv). In cells induced
to differentiate 12 hours after DISC1-GFP-Adv infection,
expression of the myelin genes, CNPase (Fig. 3 A, C, D) and MBP (Fig. 3
B, C, E), were decreased at both the mRNA and protein level,
compared with control (GFP-Adv) infected cells. To confirm the
reduced CNPase expression in DISC1 overexpressing cells, we
determined the ratio of GFP expressing cells with CNPase
expression in GFP-Adv or DISC1-GFP-Adv infected cells by
immunostaining with anti-CNPase antibody. The percentage of
CNPase immuno-positive cells decreased 96 hours after PDGF
deprivation, following infection of DISC1-GFP-Adv (70.163.3%)
compared with GFP-Adv (90.662.5%) (Fig. 3 H, I).
Morphological transformation of cells infected by GFP-Adv or
DISC1-GFP-Adv was examined 96 hours after PDGF deprivation.
Infected cells were identified by GFP expression and their
morphology was assessed by immunostaining with anti-b-tubulin.
The morphology of infected cells was classified as simple,
intermediate or complex, as described previously . Of 238
GFP-Adv infected cells, 24.963.4% showed a complex
morphology, characterized by the presence of several interlaced fine
branches indicative of advanced differentiation. Undifferentiated
cells, showing a simple morphology defined by primary branches
and the absence of secondary and tertiary processes, comprised
23.662.2% of the total. In contrast, of 191 DISC1-overexpressing
cells, only 2.560.8% displayed a complex morphology and
53.363.9% showed a simple morphology (Fig. 3 F, G). Thus,
DISC1 overexpression results in a reduction of cells with a
complex morphology, and an increase in cells with a simple
morphology, suggesting that decrease in endogenous DISC1
expression upon PDGF deprivation in oligodendrocyte precursor
DISC1 Knockdown Promotes Oligodendrocyte
To further investigate the role of endogenous DISC1 in
oligodendrocyte differentiation, we treated oligodendrocyte
precursor cells with DISC1 specific siRNA (DISC1-siRNA) and
examined mRNA or protein expression levels of CNPase and
MBP 48 or 72 hours after siRNA transfection. To isolate the effect
of DISC1 knockdown induced by DISC1-siRNA, cells were
maintained in medium with PDGF during the course of the
experiment. The proportion of siRNA-transfected oligodendrocyte
precursor cells determined using Block-iT Alexa Fluor Red
Fluorescent Oligo was 93.661.2% of total cell population. Two
DISC1-siRNAs (DISC1-siRNA-1 and DISC1-siRNA-2) targeting
exon2 and exon5 of the DISC1 gene respectively have already
been shown to effectively suppress rat DISC1 protein expression
[15,40,41]. Suppression of DISC1 expression by these siRNAs was
confirmed by qRT-PCR with two different primer sets for rat
DISC1 (DISC1 primer-1 (100% for control siRNA; 33.764.8%
for DISC1-siRNA-1; 28.562.1% for DISC1-siRNA-2); DISC1
primer-2 (100% for control siRNA; 47.963.6% for DISC1
siRNA-1; 39.068.8% for DISC1 siRNA-2)) (Fig. 4 A). Effective
knockdown of DISC1 expression was also confirmed using another
reference gene (b-actin) (data not shown). Transfection of either of
two siRNAs for DISC1 resulted in an increase of CNPase, at both
the mRNA and protein level, compared with control-siRNA
treated cells (Fig. 4 B, D, E). Although not statistically significant,
we also observed a trend towards increased expression of MBP
(Fig. 4 C, D). Immunostaining of in vitro oligodendroglial lineage
cells with anti-CNPase antibody revealed that DISC1-siRNA
treatment increased the percentage of CNPase positive cells
(DISC1-siRNA-1: 24.660.7%; DISC1-siRNA-2: 22.361.9%),
compared with control-siRNA treated cells (12.262.3%) (Fig. 4
I, J). To confirm the effect of DISC1 knockdown on expression of
CNPase, we performed rescue experiment by co-expression of
DISC1 siRNAs and human DISC1-GFP. Human DISC1 rescued
the increased CNPase expression by DISC1 siRNA (Fig. 4 F).
Morphological transformation of cells transfected with
controlsiRNA or DISC1-siRNAs was examined by immunostaining for
btubulin 72 hours after siRNA transfection. Of 225 control-siRNA
transfected cells, 57.460.9% showed a simple morphology and
only 3.560.7% of cells displayed a complex morphology. In
contrast, DISC1 knockdown decreased the cells with a simple
morphology (DISC1-siRNA-1: 27.964.6%; DISC1-siRNA-2:
33.561.3%), and increased cells with a complex morphology
(DISC1-siRNA-1: 6.262.7%; DISC1-siRNA-2: 14.761.7%)
(Fig. 4 G, H). Our results suggest that endogenous DISC1 in an
oligodendrocyte lineage cell negatively regulates oligodendrocyte
Overexpressed Truncated-DISC1 Promotes
To further confirm the negative regulatory role of DISC1 in
oligodendrocyte differentiation, we overexpressed truncated
DISC1, which has been suggested to be generated when the
DISC1 gene is disrupted by the balanced translocation of
chromosome 1. Truncated DISC1 is predicted to function in a
dominant negative fashion, possibly by competing with the
fulllength form for interacting proteins . When oligodendrocyte
Figure 5. Overexpressed truncated DISC1 promotes oligodendrocyte differentiation. AE Effect of truncated DISC1 overexpression on
CNPase and MBP expression. Oligodendrocyte precursor cells were infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours and induced to
differentiate by PDGF deprivation for 36 hours. mRNA levels of CNPase (A) and MBP (B) were quantified by qRT-PCR. Data are expressed as mean 6
s.e.m. of at least three independent experiments. *p,0.05 vs. GFP-Adv. C, Cells infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours were lysed 60
hours after PDGF deprivation and subjected to western blot analysis. D, E, Quantitation of relative band densities for CNPase (D) and MBP (E) was
performed by scanning densitometry. Data are expressed as mean 6 s.e.m. of at least three independent experiments. *p,0.05 vs. GFP-Adv. FI
Oligodendrocyte precursor cells were infected with GFP-Adv or trDISC1-GFP-Adv for 12 hours and induced to differentiate by depriving PDGF for 60
hours then fixed for immunostaining. Cells were immunostained for GFP and b-tubulin (F, G) or for GFP and CNPase (H, I) and analyzed as described
in figure legend 3. *p,0.05 vs. GFP-Adv. Scale bar = 50 mm.
precursor cells infected with a truncated DISC1 expressing
adenovirus (trDISC1-GFP-Adv) for 12 hours, were deprived of
PDGF, levels of CNPase and MBP, both mRNA and protein
levels, were significantly increased compared with control
(GFPAdv) adenovirus infected cells (Fig. 5 AE). Furthermore,
immunostaining analysis with anti-CNPase antibody showed a
higher proportion of CNPase positive cells in trDISC1-GFP-Adv,
compared with control adenovirus, infected cells (GFP-Adv:
36.061.2%; tr-DISC1-GFP-Adv: 46.963.1%) (Fig. 5 H, I).
Immunostaining of trDISC1-GFP-Adv infected cells with
antib-tubulin antibody showed that upon differentiation, there was a
higher ratio of cells with a complex morphology compared with
control adenovirus infected cells (GFP-Adv: 11.060.8%;
trDISC1GFP-Adv: 32.064.9%) (Fig. 5 F, G). Conversely, the ratio of cells
displaying a simple morphology in trDISC1-GFP-Adv, compared
to GFP-Adv, infected cultures was significantly lower (GFP-Adv:
43.462.1%; trDISC1-GFP-Adv: 23.964.2%). These results
suggest that truncated DISC1 expression promotes differentiation of
oligodendroglial lineage cells, and provides additional supporting
evidence for negative regulation of oligodendrocyte differentiation
by endogenous DISC1.
Involvement of Sox10 and/or Nkx2.2 in the Regulatory
Pathway of Oligodendrocyte Differentiation by DISC1
Transcription factors involved in oligodendrocyte specification
and differentiation include, but are not limited to, the basic
helixloop-helix (bHLH) family members Olig1 and Olig2, the inhibitor
of DNA binding (Id) family of proteins Id2 and Id4, SRY box
containing (Sox) family members Sox10, and the homeobox
containing (Hox) transcription factor Nkx2.2 [47,48].
To examine the potential involvement of transcription factors in
the regulatory pathway of oligodendroglial lineage cells by DISC1,
we investigated the effects of manipulating DISC1 expression on
the mRNA expression of transcription factors. Of the transcription
factors examined (sox10, nkx2.2, mash1, olig1, olig2, Id2 and Id4),
mRNA expression of Sox10 and Nkx2.2 were significantly
decreased in DISC1 overexpressing cells (Fig. 6 A, B) while
expression level of other transcription factors were not significantly
changed compared with control cells (mash1; 10460.3%, olig1;
10760.2%, olig2; 99.561.6%, Id2; 111.866.0%; Id4;
112.068.2%). In contrast, knockdown of endogenous DISC1
resulted in enhanced expression of Sox10 and Nkx2.2 (Fig. 6 C,
D). Furthermore, truncated form of DISC1 also increased Sox10
and Nkx2.2 expression (Fig. 6 E, F).
We examined CNPase expression levels in cells co-transfected
with DISC1-siRNA-1 and siRNAs targeting either Sox10 or
Nkx2.2 (sox-siRNA or nkx-siRNA) to elucidate if DISC1 is acting
upstream or downstream of these transcription factors. When
either Sox10 or Nkx2.2 was simultaneously knocked-down with
DISC1-siRNA-1 treatment, promotion of oligodendrocyte
differentiation by DISC1 knockdown was inhibited (Fig. 6 G). The
effect of DISC1-siRNA-1 on its target mRNA was not altered
when either sox-siRNA or nkx-siRNA was co-transfected (Fig. 6
H). Similarly, DISC1-siRNA-1 co-transfection did not alter the
effect of sox-siRNA or nkx-siRNA (Fig. 6 I, J). Therefore, these
results suggest DISC1 negatively regulates oligodendrocyte
differentiation by acting upstream of Sox10 and/or Nkx2.2 to suppress
The major findings of this study are as follows. First, we show
that DISC1 is expressed in oligodendrocytes in the corpus
callosum of postnatal mouse brain (Fig. 1). Second, DISC1
expression is decreased during in vitro differentiation of
oligodendrocyte precursor cells to oligodendrocytes (Fig. 2 A). Third,
DISC1 endogenously expressed in cells of an oligodendroglial
lineage, negatively regulates differentiation of oligodendrocyte
precursor cells to oligodendrocytes, as shown by promotion of
oligodendrocyte differentiation by either DISC1 knockdown or
overexpression of truncated DISC1 (Fig. 4, 5), while
overexpression of full length DISC1 inhibits oligodendrocyte differentiation
(Fig. 3). Finally, we have implicated Sox10 and/or Nkx2.2 in the
DISC1 regulatory pathway of oligodendrocyte differentiation,
with knockdown of endogenous DISC1 increasing, and DISC1
overexpression decreasing, expression of Sox10 and Nkx2.2.
Additionally, promotion of oligodendrocyte differentiation by
DISC1 knockdown was prevented by simultaneous knockdown
of either Sox10 or Nkx2.2 (Fig. 6).
DISC1 Expression in Oligodendrocytes
Our in situ hybridization analysis shows that DISC1 mRNA is
expressed not only in oligodendrocytes of mouse brain but also in
primary cultured rat oligodendrocytes and oligodendrocyte
precursor cells (Fig. 1, 2). These results are consistent with
previous report by Seshadri et al. showing colocalization of DISC1
with the oligodendrocyte marker in human brain and primary
cultured rat cortical oligodendrocytes . Furthermore, DISC1
expression in oligodendrocytes in the corpus callosum was higher
in developing stage than in adulthood, similar to the
developmental expression pattern of DISC1 in neurons [20,46]. These
results suggest that DISC1 has a developmental role in
oligodendrocyte lineage cells as well as in neurons. Overexpressed DISC1
localized preferentially in cell bodies and processes of
oligodendrocyte precursor cells and oligodendrocytes in vitro, with marginal
nuclear localization (Fig. 2 B). The subcellular localization of
DISC1 in oligodendrocyte precursor cells and oligodendrocytes is
similar to that in neurons, suggesting the possibility that DISC1
has common functional roles between neurons and
oligodendrocyte lineage cells.
DISC1 Function in Oligodendrocytes
DISC1 has been shown to play an important role in immature
neurons, regulating their differentiation, migration and
proliferation [12,17,49]. Thus our findings, namely, decrease of DISC1
expression during the course of oligodendrocyte differentiation
(Fig. 2 A), and higher DISC1 expression in oligodendrocytes in the
mouse corpus callosum at P14 (Fig. 1 C), suggest DISC1 may also
have a developmental role in immature oligodendroglial lineage
cells as well. Supporting evidence is discussed below.
Overexpressed DISC1 disrupts not only induction of CNPase
and MBP expression, but also transformation of oligodendrocytes
to a complex morphology (Fig. 3), indicating a negative regulatory
role of DISC1 in differentiation of oligodendroglial lineage cells
in vitro. Conversely, both expression of CNPase and the number of
matured oligodendrocytes, were increased when endogenously
expressed DISC1 was knocked-down by siRNA, even if the cells
were maintained in medium containing PDGF (Fig. 4). Although
MBP mRNA levels were increased by DISC1 knockdown, the
result did not reach statistical significance. This is likely due to
both the transient nature of DISC1 knockdown by siRNA,
compared to the more stable adenovirus overexpression system,
and also that MBP expression increases at a later stage of
differentiation than CNPase . More robust increases of MBP
expression may be observed at later time-points, with stronger and
more continuous inhibition of DISC1. Truncated DISC1 is
predicted to function in a dominant negative fashion, potentially
by competing with full length DISC1 for interacting proteins.
Thus promotion of oligodendrocyte differentiation by truncated
DISC1 overexpression suggests a negative regulatory role for
DISC1 in oligodendrocyte differentiation (Fig. 5). Further studies
are needed to determine if DISC1 interacts with other proteins in
oligodendrocyte lineage cells, as in neurons [51,52]. Nevertheless,
our results do indicate that decreased level of endogenous DISC1
promotes differentiation of oligodendrocyte precursor cells to
To date, a functional role for endogenous DISC1 expressed in
mammalian oligodendrocyte lineage cells has not been reported. A
critical requirement for DISC1 in oligodendroglial development,
by promoting specification of olig2-positive cells in the hindbrain
and other brain regions of zebrafish, was reported by Wood et al
. Although this report also shows regulation of
oligodendroglial development by DISC1, the low homology between zebrafish
DISC1 and mammalian DISC1 (homologies between zebrafish
and rat, mouse or human are 31, 32, 36% respectively), highlights
the necessity of our study. Furthermore, it is not clear if neuronal
or glial expressed DISC1 is responsible for oligodendroglial
development. Katsel et al., showed that oligodendrocyte-associated
gene/protein expression was changed in the forebrain of
transgenic mice with forebrain restricted expression of mutant
human DISC1 (DhDISC1) at embryonic, neonatal and adulthood
stages . The transgenic mice show neuron-specific
overexpression of DhDISC1, therefore the observed alterations in
oligodendrocyte-associated gene/protein expression are caused
by mutant DISC1 expressed in neurons. However, the results of
our study show that glial expressed DISC1 regulates
oligodendrocyte differentiation. To further determine the role of glial
expressed DISC1 on oligodendroglial development in vivo, studies
using transgenic mice with glia-specific expression of mutant
DISC1 are needed.
Sox10, Nkx2.2 Regulation by DISC1
Each step of oligodendrocyte differentiation is under tight
transcriptional control. Among many transcription factors acting
at different stages, Sox10 and Nkx2.2 play a major role in the
transition from oligodendrocyte precursor cells to pre-myelinating
oligodendrocytes [48,53]. Sox10 is a high-mobility group
transcriptional regulator restricted in the central nervous system to
myelin-forming oligodendrocytes. In Sox10-deficient mice,
oligodendrocyte precursor cells develop but terminal differentiation is
disrupted . A critical role for the homeodomain-containing
protein Nkx2.2 in the development of oligodendroglial lineage
cells has also been reported. The number of MBP-positive and
proteolipid protein (PLPDM-20)-positive oligodendrocytes are
dramatically reduced in the brain of Nkx2.2 null mice .
Positive regulatory roles for Sox10 and Nkx2.2 in oligodendrocyte
differentiation are consistent with our result that DISC1 regulates
oligodendrocyte differentiation via upstream regulation of Sox10
and/or Nkx2.2 (Fig. 6). Negative regulation of Sox10 expression
by DISC1 has previously been reported by Drerup et al., although
they examined cranio-neural crest cells, which become glial
precursors at later stages of development . How DISC1
regulates these transcription factors is not yet known, but
intracellular signaling pathways involving molecules such as Akt,
cAMP, CREB and MAPK are likely candidates, as neuronal
DISC1 regulates these pathways, and moreover, these signaling
pathways have functional roles in differentiation of
oligodendroglial lineage cells .
The pathophysiological role of Sox10 in SZ has been suggested
by a report showing a correlative relationship between the DNA
methylation status of the Sox10 gene and oligodendrocyte
dysfunction in SZ . In addition, a significant association in
the genotype and allelic frequency of a single-nucleotide
polymorphism of the Sox10 gene, between schizophrenic patients
and controls has been reported . Nkx2.2 is known to form a
transcriptional network with Pet1, a molecule involved in
differentiation of serotonergic neurons . It is well known that
serotonergic neurons are both a relevant pathophysiological factor
and therapeutic target in several psychiatric diseases, including SZ,
bipolar disorder, major depression and autism. Therefore our
finding that DISC1, a key psychiatric disease susceptibility gene,
controls Sox10 and/or Nkx2.2 expression is intriguing.
Pathophysiological Role of DISC1 Regulated
Overall, our findings suggest that DISC1 dysfunction may cause
impaired differentiation of oligodendrocytes by affecting Sox10
and/or Nkx2.2 expression, and consequently contribute to the
pathophysiology of psychiatric disorders. Improper myelination of
neuronal axons, resulting from impaired oligodendrocyte
differentiation, may lead to defective neuronal communication, a likely
component in the mechanistic background of structural
disconnectivity, suggested in the pathophysiology of psychiatric
disorders [24,66]. Therefore, it would be of interest to investigate if
WM abnormalities are a feature of the Scottish DISC1 pedigree
that harbors the disrupted DISC1 gene. Furthermore, given that
SZ-like psychosis co-occurs frequently in demyelinating diseases
, it is also warranted to investigate the role of DISC1 in
pathophysiology of demyelinating diseases such as MS,
leukodystrophies and velocardiofacial syndrome.
Conceived and designed the experiments: TH. Performed the experiments:
TH YK HE. Analyzed the data: TH YM NK KY HT SM. Contributed
reagents/materials/analysis tools: TH SS TK. Wrote the paper: TH MT
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