Disrupted-in-Schizophrenia-1 expression is regulated
by β-site amyloid precursor protein cleaving
Saurav Seshadria, Atsushi Kamiyaa,1, Yukako Yokotab,1, Ingrid Prikulisc, Shin-ichi Kanoa, Akiko Hayashi-Takagia,
Amelia Stancob, Tae-Yeon Eomb, Sarada Raob, Koko Ishizukaa, Philip Wongd,e, Carsten Korthc, E. S. Antonb,2,
and Akira Sawaa,d,2
Departments ofaPsychiatry,dNeuroscience, andePathology, Johns Hopkins University School of Medicine, Baltimore, MD 21287;bUniversity of North Carolina
Neuroscience Center and Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
cDepartment of Neuropathology, Heinrich Heine University of Düsseldorf, 40225 Düsseldorf, Germany
Edited* by Solomon Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved February 2, 2010 (received for review August
Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are
promising susceptibility factors for schizophrenia. Both are multi-
functional proteins with roles in a variety of neurodevelopmental
processes, including progenitor cell proliferation, migration, and
differentiation. Here, we provide evidence linking these factors
together in a single pathway, which is mediated by ErbB receptors
and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not
NRG3, increase expression of an isoform of DISC1 in vitro. Recep-
tors ErbB2 and ErbB3, but not ErbB4, are responsible for transduc-
ing this effect, and PI3K/Akt signaling is also required. In NRG1
knockout mice, this DISC1 isoform is selectively reduced during
neurodevelopment. Furthermore, a similar decrease in DISC1 expres-
sion is seen in β-site amyloid precursor protein cleaving enzyme–1
(BACE1) knockout mice, in which NRG1/Akt signaling is reportedly
impaired. In contrast to neuronal DISC1 that was reported and char-
acterized, expression of DISC1 in other types of cells in the brain has
not been addressed. Here we demonstrate that DISC1, like NRG and
ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes,
microglia, and radial progenitors. These findings may connect NRG1,
ErbBs, Akt, and DISC1 in a common pathway, which may regulate
neurodevelopmentand contribute tosusceptibility toschizophrenia.
ErbB proteins are associated with schizophrenia (1). The NRG
family of proteins activates the ErbB family of receptor tyrosine
kinases, which play numerous roles in neurodevelopment, in
processes such as progenitor cell proliferation, radial and tan-
gential migration, neurite outgrowth, dendritic arborization, and
myelination (2, 3). NRG1 is cleaved by β-site amyloid precursor
protein cleaving enzyme–1 (BACE1), which was originally found
to cleave amyloid-β precursor protein (APP) and thereby con-
tribute to the pathophysiology of Alzheimer’s disease (4). This
cleavage of NRG1 results in the release of the extracellular EGF-
like domain, which in turn activates ErbB receptors and down-
stream intracellular signaling pathways, including the PI3K/Akt
and JAK/STAT cascades, in a context-dependent manner (5).
Consequently, decreased levels of cleaved NRG1 and diminished
activation of Akt have been reported in BACE1 knockout
function model for NRG1 signaling (6).
Disrupted-in-Schizophrenia–1 (DISC1) is another promising
genetic susceptibility factor for a wide range of major mental dis-
orders, including schizophrenia (1, 7). DISC1 is expressed in the
neocortex and other brain regions, especially during neuro-
development (8). Like NRG family proteins, DISC1 is multifunc-
tional, and has been shown to have a strong influence on
neurodevelopmental processes including progenitor cell pro-
liferation, radial migration, dendritic arborization, and synapse
formation, with its effects often mediated by protein–protein
inkage and association studies in diverse populations have
suggested that the genes encoding for neuregulin (NRG) and
interactions (3, 9–13). There is evidence linking DISC1 to cAMP
signaling through its ability to bind PDE4 enzymes (14, 15), which
can influence brain functioning (16). In addition, PDE4 (17) and
cAMP (18) can influence Akt signaling.
Various genetically engineered mice have been generated for
NRG proteins, ErbB proteins, DISC1, and BACE1 (19–21).
These mice display overlapping abnormal phenotypes. For
example, whereas homozygous NRG1 knockout (NRG1-KO)
mice lacking the EGF-like domain do not survive past the
embryonic stage of development (22), heterozygous NRG1-KO
mice show impairments in prepulse inhibition and working
memory (23–25). Several types of DISC1 genetically engineered
mice have been generated (20, 21); in addition to common
deficits of prepulse inhibition, some of these mice display deficits
in working memory. Very interestingly, BACE1 knockout mice,
originally generated for studies on Alzheimer’s disease, also
show deficits in prepulse inhibition and working memory, pos-
sibly associated with disturbed NRG1 signaling (26). In addition
to these results from murine models, a recent report that
examined a zebrafish model indicated that knockdown of NRG1
and DISC1 could result in similar phenotypes in development of
oligodendrocytes and neurons from olig2-expressing precursor
cells (27). Although these data are highly suggestive, a direct link
between NRG1 signaling and DISC1 has not yet been elucidated
at the molecular level.
Here we present evidence that NRG1 regulates expression of
DISC1, mediated via ErbB2/3 and PI3K/Akt signaling. The
influence of NRG1 on expression on an isoform of DISC1 is
observed in both in vitro neuron cultures and in vivo. Fur-
thermore, we also provide evidence that DISC1 is expressed in
glial cells, including astrocytes, oligodendrocytes, and microglia.
NRG Signaling Increases Expression of an Isoform of DISC1 in Primary
Neuron Cultures. To address a possible link between NRG sig-
naling and DISC1 at the molecular level, we investigated whether
NRG ligands might influence the expression of DISC1. We used
recombinant NRG1-β-GST (GST), NRG2-β-GST, or NRG3-GST
Author contributions: S.S., A.K., C.K., E.S.A., and A. Sawa designed research; S.S., Y.Y., I.P.,
A. Stanco, T.E., S.R., and C.K. performed research; S.K., A.H.-T., P.W., C.K., and E.S.A.
contributed new reagents/analytic tools; S.S., A.K., K.I., and P.W. analyzed data; and
S.S. and A. Sawa wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1A.K. and Y.Y. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or anton@med.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| March 23, 2010
| vol. 107
| no. 12www.pnas.org/cgi/doi/10.1073/pnas.0909284107
proteins (NRG1, NRG2, and NRG3, respectively, in this study),
consisting of the GST-tagged external EGF-like domain of the
NRG1 β isoform, NRG2 β isoform, or NRG3, which in all cases is
reportedly necessary and sufficient for activation of the ErbB
family of receptors and downstream intracellular signaling (28).
Equal levels of activation of the downstream signaling were con-
firmed by assessing ErbB4 phosphorylation in HEK293 cells fol-
lowing exposure to each recombinant NRG protein (Fig. S1A).
Western blotting with DISC1 antibody D27 revealed that treat-
ment with NRG1 and NRG2, but not NRG3, increased the levels
of DISC1 immunoreactivity at 130 kDa in immature primary
neuron cultures compared with treatment with GST alone (Untr)
(Fig. 1A). Immature neurons were chosen as the main subject of
this study because the NRG and DISC1 cascades play important
roles during neurodevelopment (3); however, a similar induction
of DISC1 by NRG1 was also observed in mature neuron cultures
(Fig. S1B). Strong induction of 130 kDa DISC1 was observed with
NRG1 at 3 nM concentration (Fig. S1C), 2 h after treatment,
persisting up to at least 24 h (Fig. S1D). DISC1 has multiple
isoforms, including an isoform at ∼100 kDa that is thought to
represent the full-length protein; therefore, to confirm that the
130 kDa signal reflects an isoform of DISC1, we have used two
independent antibodies (D27 and mExon3) generated and pub-
lished by different research groups (8, 29). Induction of this 130
kDa isoform of DISC1 by NRGs was consistently observed with
these two antibodies (Fig. S2A).
Immunoprecipitation of neuronal lysates using mExon3, fol-
previously characterized RNAi to DISC1 (9); and this RNAi also
prevented induction of the 130-kDa band by treatment with
C and D). In the present study, we mainly used D27 for Western
of minimized background signal with each antibody when opti-
mized for the respective experimental paradigm. Of interest, the
influence of NRG1 and NRG2 was observed only on 130 kDa
DISC1, but not on previously reported isoforms at 100 and 105
kDa (Fig. S2E). These results suggest that NRG1 and NRG2 may
influence DISC1 expression via a signaling pathway not activated
before and performed immunofluorescent cell staining. Although
DISC1 expression in the nucleus and cell body was relatively
increased in treated cultures (Fig. 1B).
ErbB2/3 and PI3K/Akt Signaling Mediate the Effect of NRG1 on DISC1
Expression. To determine which receptor and secondary signaling
pathway activated by NRG1 is responsible for transducing its
effect on expression of DISC1 130 kDa isoform, we first used
lentivirus-mediated RNAi to knock down expression of each
receptor in primary cortical neurons, and treated cultures with
recombinant NRG1 as before. RNAi sequences directed against
ErbB2, ErbB3, and ErbB4 were confirmed to be specific for each
receptor (Fig. S3A). Western blotting with D27 and mExon3
revealed an increase in 130 kDa DISC1 expression in control
RNAi- and ErbB4 RNAi-infected neurons following treatment
with NRG1 (compared with untreated control RNAi-infected
neurons), suggesting that ErbB4 is not necessary for transducing
this effect, whereas NRG1-treated ErbB3 RNAi– and ErbB2
these receptors are required (Fig. 2A). To determine the secon-
dary signaling pathway involved, NRG1 treatment was performed
in conjunction with blockade of PI3K/Akt or JAK2/STAT3 sig-
naling by using pharmacological agents (10 μM LY294002 and 5
by Western blotting for phospho-Akt (for LY294002) and phos-
pho-STAT3 (for AG490) (Fig. S3 B and C). Increases in 130 kDa
with vehicle (DMSO) or AG490 compared with untreated cells
with vehicle, but this effect was abolished by cotreatment with
LY294002,indicating thatPI3K/Aktsignalingmediates thiseffect
did not affect induction of 130 kDa DISC1 (Fig. S3D). However,
NRG1-induced increase in 130 kDa DISC1 expression was
blocked by cotreatment with transcription inhibitor Actinomycin
D (Fig. S3E), suggesting that this isoform may arise from a tran-
scriptionally regulated mechanism. These results indicate that
NRG1 signaling via ErbB2, ErbB3, and PI3K/Akt signaling is
required for the induction of 130 kDa DISC1.
DISC1 Expression Is Decreased in NRG1 Knockout Mice. Todetermine
whether NRG1 signaling influences DISC1 expression during
neurodevelopment in vivo, we studied DISC1 expression in
NRG1-KO mice. In these mice, the EGF-like domain common to
all isoforms of NRG1 is replaced with the neomycin resistance
gene, thereby eliminating NRG1 signaling (22). Homozygous
NRG1-KO mice do not survive beyond embryonic day 11 (E11),
whereas heterozygous knockout is not embryonically lethal. We
performed Western blotting for DISC1 and NRG1 in cortices
reduction in cleaved NRG1 (Fig. 3A). In these animals, 130 kDa
DISC1 expression was decreased while DISC1 100 and 105 kDa
isoforms were unaffected (Fig. 3A and Fig. S4A). This selective
influenceofNRG1on the130kDaisoformofDISC1 isconsistent
with observations in primary neuron cultures (Fig. 1A). Immu-
NRG1 NRG2 NRG3 Untr
Neurite DISC1 expression (A.U.)
130 kDa DISC1 expression (A.U.)
manner. (A) Western blotting for DISC1 in NRG-treated neurons. Western
blotting with D27 shows increase in isoform of DISC1 at 130 kDa (arrow)
following treatment with NRG1 (mean ± SD, 1.77 ± 0.23-fold increase, P =
0.007, n = 5) and NRG2 (1.53 ± 0.13-fold increase, P = 0.008, n = 4), whereas
bands at 100 and 105 kDa were unaffected (arrowheads). GAPDH was used
as a loading control. (B) Immunofluorescent cell staining and quantification
of DISC1 in NRG1-treated neurons. Cell staining for DISC1 with antibody
mExon3 was quantified and normalized to neurite length indicated by
β-tubulin staining in primary neurons treated with NRG1. DISC1 in neurites
increased after NRG1 treatment (1.72 ± 0.33-fold increase, P = 0.01, n = 3).
Magnification, 20×. (Scale bar, 50 μm.)
NRG treatment affects DISC1 expression in an isoform-specific
Seshadri et al.PNAS
| March 23, 2010
| vol. 107
| no. 12
nohistochemistry for DISC1 showed widespread reduction in
DISC1 expression in the telencephalon of homozygous NRG1
knockout mice at E10 (Fig. 3B). Costaining with cell type–specific
expression of DISC1 in these cell types in wild-type embryos, and
reduction in DISC1 expression in knockout mice, verified by
quantification of fluorescent intensity in each cell type (Fig. 3B).
These results indicate that NRG1 signaling is important in main-
taining expression of 130 kDa DISC1 in the developing mouse
brain in vivo.
130 kDa DISC1 expression (A.U.)
130 kDa DISC1 expression (A.U.)
knockdown by RNAi. Primary cortical neurons infected with lentiviral shRNA against ErbB4, ErbB3, and ErbB2 (Fig. S3A) and treated with NRG1. Western
blotting was performed for 130 kDa DISC1 with DISC1 antibodies D27 and mExon3 and quantified (*P < 0.01, n = 3). (B) Western blotting for DISC1 after NRG1
treatment with pharmacological blockade of secondary signaling pathways. Primary cortical neurons were treated with LY294002 and AG490, then treated
with NRG1. Western blotting was done for 130 kDa DISC1 with DISC1 antibodies D27 and mExon3 and quantified (*P < 0.01, n = 3). Magnification 200× and
400×. (Scale bar, 50 μm and 5 μm.)
NRG1 signaling affects DISC1 expression via ErbB3/ErbB2 and Akt activation. (A) Western blotting for DISC1 after NRG1 treatment with receptor
BACE1 -/- WT
130 kDa DISC1
BACE1 -/- WT
130 kDa DISC1
Percentage of cells with
DISC1 in neurites
DISC1 expression (A.U.)
NRG1 -/- WT
DISC1 expression (A.U.)
shows reduced DISC1 expression (arrow) in cortex of heterozygous P0 NRG1-KO mice compared with wild-type littermates (23.6% decrease). A C-terminal–
NRG1 knockout mice. Neurons (Tuj1) and radial glia (RC2) in E10.5 telencephalon of WT and homozygous NRG1-KO mice were colabeled with DISC1. Both
(arbitrary units) verifies decreased DISC1 in neurons (NRG1−/−: mean ± SD, 9.62 ± 1.24, WT: 34.91 ± 1.17), and radial glia (NRG1−/−: 8.04 ± 0.9, WT: 15.87 ± 1.2).
Magnification,630×.(Scalebar,10μm.)(C)Western blotting for DISC1inBACE1knockoutmice. ImmunoblottingwithDISC1 antibodyD27showsreducedDISC1
expression (arrow) in cortex of homozygous P0 BACE1-KO mice compared with wild-type littermates (44.1% decrease, n = 3). A C-terminal–directed NRG1
antibody was used to verify a reduction in cleaved NRG1 in these mice. GAPDH used as loading control. (D) Immunohistochemistry for DISC1 in BACE1 knockout
mice. Staining with DISC1 antibody mExon3 shows reduced DISC1 in the cortex of homozygous P0 BACE1-KO mice compared with wild-type littermates, with a
reduction in the percent of neurons with visibly stained neurites (76.4% reduction, n = 3). Magnification 630×. (Scale bar, 50 μm and 10 μm.)
| www.pnas.org/cgi/doi/10.1073/pnas.0909284107Seshadri et al.
DISC1 Expression Is Decreased in BACE1 Knockout Mice. As NRG1
signaling via Akt activation appears to modulate DISC1 expres-
altered in BACE1-KO mice during neurodevelopment. Con-
sistent with previous publications (6, 26), reduction in cleaved
NRG1 and accumulation of uncleaved NRG1 was verified in
reduction appeared to be specific to the 130 kDa isoform, as
expression of the 100- and 105-kDa DISC1 isoforms remained
relatively unchanged. Immunohistochemistry using DISC1 anti-
body mExon3 showed similar DISC1 expression in nuclei and cell
bodies but decreased expression in cellular processes in the cortex
of homozygous BACE1-KO mice compared with wild-type mice
(Fig. 3D and Fig. S4D). Marked reduction in the percentage of
DISC1-stained neurons with visible neurites was observed in
neurites, consistent with in vitro results showing an increase of
DISC1 in neurites following NRG1 treatment (Fig. 1B). Finally,
subcellular fractionation revealed that 130 kDa DISC1 was
with their wild-type littermates (Fig. S4F).
DISC1 Is Expressed in Neurons, Astrocytes, Oligodendrocytes, and
Microglia. NRG1 signaling plays important roles in both neuronal
and glial cells (3, 5). Here we have demonstrated a direct
molecular link between NRG1 signaling and DISC1; however,
despite some indications of DISC1 expression in glial cells (27),
most studies thus far have focused on neuronal DISC1 (3).
Therefore, to address whether DISC1 is truly expressed in glial
cells, we first used a well-characterized and published affinity-
purified monoclonal antibody 3D4 against human DISC1 (30) to
investigate DISC1 expression with autopsied human brains. We
confirmed high levels of DISC1 expression in pyramidal neurons
costained with a neuronal marker MAP2 (Fig. 4A). We also
observed equivalent levels of DISC1 expression in astrocytes and
microglia, costained with markers glial fibrillary acidic protein
(GFAP) and KP-1, respectively (Fig. 4A). Weak staining of
DISC1 colocalized with oligodendrocyte marker nogoA was also
observed (Fig. 4A). Next, we investigated expression of DISC1 in
rat primary cortical neuron, astrocyte, oligodendrocyte, or
microglial cultures, with established DISC1 antibody mExon3
(29). In neurons costained with neuronal marker MAP2, DISC1
was seen in the nucleus, cell body, and neurites (Fig. 4B). In
astrocytes and microglia costained with markers GFAP and
Cd11b, respectively, DISC1 was strongly expressed in the nucleus
and cell body, and was seen to a lesser extent in cellular pro-
cesses, whereas in oligodendrocytes costained with marker
A2B5, DISC1 expression was restricted to the nucleus and cell
body (Fig. 4B). Colocalization of DISC1 and GFAP were also
observed in adult mouse brain, confirming the expression of
DISC1 in astrocytes (Fig. S5).
This study reports two major findings. First, NRG1 signaling
increases the expression of an isoform of DISC1 at 130 kDa and
maintains expression of this isoform in the developing mouse
signaling-impaired murine models, NRG1-KO and BACE1-KO
mice, is consistent with in vitro results and suggests the possibility
that DISC1 deficiency may contribute to their phenotypic abnor-
among these knockout mice and DISC1 genetically engineered
models (3, 20, 21). For example, as DISC1 appears to play an
DISC1 may underlie the alterations in spine morphology and
of DISC1 in these mice could help to determine the functional
significance of the NRG1-PI3K/Akt-DISC1 pathway. Second,
DISC1 is expressed in both neuronal and glial lineages, including
astrocytes, oligodendrocytes, and microglia, in human brains and
rat cortical cultures. We believe that our systematic approach,
including costaining with cell type–specific markers to verify
neuronal and glial lineage, establishes that DISC1, like NRG and
ErbB family proteins, is expressed in several distinct cell types in
the human and rodent cortex. DISC1 has been shown to have
several functions in neurons, involving multiple subcellular pools
capable of interaction with synaptic proteins, centrosomal pro-
teins, and transcription factors (3); however, the role of DISC1 in
glia remains to be elucidated. Roles for NRG1 and DISC1 sig-
naling cross-talk across neuronal–glial interactions may well
become an important question.
Although we have verified the 130-kDa isoform of DISC1 by
Western blotting and immunoprecipitation using well-established
antibodies generated from different groups (Fig. S2B) and by
verifying its knockdown by DISC1-specific shRNA (Fig. S2 C and
D), the true nature of this isoform remains uncertain. These two
DISC1 antibodies used have been described previously (29) and
were raised against amino acids 360–374 (mExon3) and 734–753
(D27) of mouse DISC1, corresponding to portions of exons 3 and
10, respectively. Full-length DISC1 is 854 amino acids in length
and is thought to be detected at ∼100 kDa; however, alternative
DISC1 andcell type–specificmarkersinhumancorticalsections. Costainingfor
human DISC1 (brown) with purified monoclonal antibody 3D4 and the fol-
lowing markers (green) was performed: MAP2 (neurons), GFAP (astrocytes),
nogoA (oligodendrocytes), and KP-1 (microglia). Magnification 100×. (Scale
bar, 50 μm.) (B) Immunofluorescent cell staining for DISC1 and cell type–spe-
cific markers in rat primary cortical cultures. Costaining for DISC1 with anti-
body mExon3 (red) and the following markers (green) was performed: MAP2
Magnification 200×. (Scale bar, 10 μm.)
Seshadri et al. PNAS
| March 23, 2010
| vol. 107
| no. 12
splicing or posttranslational modification could give rise to the
larger DISC1 isoform. The similarity in the magnitude of increase
of 130 kDa DISC1 and DISC1 in neurites after NRG1 treatment
(Fig. 1 A and B), suggests that this isoform may be neurite-specific
decreased 130-kDa DISC1 and corresponding loss of DISC1 in
Thus, to validate 130 kDa DISC1 signal, we used two antibodies
against DISC1 in Western blotting after immunoprecipitation. At
least at present, the observation that cotreatment with transcrip-
tional inhibitor actinomycin D prevents induction of 130 kDa
DISC1 by NRG1 suggests involvement of a transcriptional
mechanism in this induction.
We demonstrate here that NRG1 and NRG2, but not NRG3,
can increase expression of 130 kDa DISC1 (Fig. 1A). Interest-
ingly, whereas NRGs can activate receptor ErbB4, only NRG1
and NRG2 can bind ErbB3 (27); furthermore, ErbB3 lacks the
receptor tyrosine kinase domain necessary for activation, and
ErbB2 lacks an extracellular ligand binding domain, implying
that these receptors must heterodimerize to transduce NRG
signaling (5). Our results are fully consistent with these obser-
vations and directly implicate receptors ErbB2 and ErbB3 as
mediators of the effect of NRG1 on DISC1 expression (Fig. 2A).
Both receptors are expressed in neurons and radial glia of the
developing cortex (33): we also show DISC1 expression in these
cell types in vivo and diminished expression of DISC1 in
homozygous NRG1-KO mice before embryonic lethality (Fig.
3B). These results suggest that NRG1 maintains DISC1 expres-
sion in vivo by signaling via ErbB2/3 heterodimers, in a mecha-
nism that also appears to involve PI3K/Akt signaling (Fig. 2B).
As there is evidence associating impaired Akt signaling with
schizophrenia (29), this pathway may tie together three prom-
inent schizophrenia susceptibility genes (genes for NRG1, Akt,
and DISC1) previously thought to be independent. Although two
loss-of-function murine models of NRG1 signaling have been
used in this study, previous studies have shown increases in
NRG1 mRNA expression and increased NRG1/ErbB4 signaling,
but unchanged NRG1 protein levels, in postmortem brains from
patients with schizophrenia (34–36); this implies that study of
gain-of-function model for NRG1 signaling as well as further
examination of NRG1/DISC1 cross-talk at different devel-
opmental (including adult) stages could better characterize roles
of these genetic susceptibility factors for schizophrenia.
Materials and Methods
Reagents. Rabbit polyclonal DISC1 antibody D27 (used at 1:50 dilution) was a
(1:200) was raised against AA 360–374 of mouse DISC1 and exhibits similar
immunoreactivity to D27 (29). Affinity-purified monoclonal primary antibody
3D4 against human DISC1 was reported previously (30). Antibodies against
NRG1 C-term (sc-348, 1:300), ErbB4 (sc-283, 1:200), ErbB3 (sc-285, 1:100), ErbB2
Cruz Biotechnology. Other antibodies used were mouse anti-A2B5 (R&D Sys-
tems.1:400), mouse anti-GFAP (Millipore, 1:400), rat ant-GFAP (Ambion, 1:400),
mouse anti-MAP2 (1:500), mouse anti-Cd11b (1:200), mouse anti–β-tubulin
(1:1,000), and mouse anti-GAPDH (1:10,000) (all AbD Serotec), GFAP (DAKO
Z0334; 1:2,000), nogoA (Santa Cruz sc-25660; 1:500), anti-CD68, clone KP1
(DAKO M0814; 1:500). Secondary antibodies used were horse anti-mouse bio-
tinylated IgG (Vector BA-2000, 1:1,000), goat anti-rabbit IgG-POD conjugated
(Pierce #31460; 1:2,000), goat anti-mouse IgG-POD conjugate (Pierce 31444;
1:2,000), and Alexa Fluor 488 and 568 (Invitrogen, 1:400). Other reagents used
were DAB solution (Vector SK-410), streptavidin-peroxidase conjugate (Roche
1089153, 1:2,000), Histogreen (Linaris E109, Wertheim, Germany), and target
retrieval solution pH 6.1 (DAKO S1699).
Cell Culture and Transfection. HEK-293FT cells were maintained in High-
Glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS at 37°C
with a 5% CO2atmosphere in a humidified incubator. 293FT cells were
transfected using PolyFect Transfection Reagent (Qiagen) following the
manufacturer’s protocol, with 5 μg DNA. Cortical primary neuron cultures
were prepared from E17-18 Sprague-Dawley rats, and maintained in neu-
ronal medium (Invitrogen neurobasal medium, 2% B27 supplement, 0.5 mM
glutamine). Neurons at 5 days in vitro (DIV) were considered immature, and
at 21 DIV were considered mature. Glial cell cultures were prepared as fol-
lows [adapted from (37) and (38)]: cortical cells from postnatal day 0 (P0)
Sprague-Dawley rats were seeded in Poly-D-Lysine (PDL)–coated T-75 flasks
in NM-15 medium (MEM with L-glutamine, 15% FBS, 6 mg/mL D-glucose, 100
U/mL penicillin, and 100 μg/mL streptomycin). Homogeneous glial cultures
were isolated as follows: after 9 days, medium was replaced and cultures
were shaken at 190 rpm for 18 h to separate oligodendrocyte precursor cells
(OPCs) and microglia, leaving astrocytes. The medium was removed and
added to uncoated 10-cm dishes for 1 h to separate adherent microglia. The
remaining supernatant fraction containing OPCs was seeded on PDL-coated
dishes, and medium was replaced with differentiation medium the follow-
ing morning to obtain oligodendrocytes.
Treatment of Primary Neurons. Immature primary cortical neurons at 3–7 DIV
were treated with various compounds (including recombinant proteins,
lentivirus, and pharmacological agents) by replacing half of the culture
medium with new medium containing the compound. Cells were treated
with recombinant NRG proteins at 3 nM concentration for 2 h. Lentiviral
infection was carried out by replacing half of the cell culture medium with
medium containing lentivirus, incubating cells at 37 °C for 8 h, followed by
addition of complete medium and incubation for 5 days. LY294002 in DMSO
(Invitrogen) was added to medium at 10 μM concentration concurrently with
NRG1. AG490 was reconstituted in DMSO and added to medium at 5 μM
concentration 30 min before NRG1 treatment. Rolipram was reconstituted in
DMSO and added to medium at 5 μM concentration 1 h before NRG1
treatment. Actinomycin D was reconstituted in DMSO and added to medium
1 h before NRG1 treatment. All pharmacological treatment was conducted
based on previously reported protocols (39–42).
Western Blotting and Quantification. Cells were harvested by scraping into PBS
and lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0%
Triton X-100, 1% deoxycholate, and 5 mM EDTA). Protein concentrations
were determined by BSA Protein Assay (Thermo Scientific), and Western
blotting was carried out using 8% Tris-Glycine gels, as previously described
(29). Exposed films were scanned and densitometric analysis was performed
using ImageJ software. Raw intensity was normalized to background and
loading control levels.
Immunofluorescent Cell Staining and Quantification. All cell staining (including
neurons and glia) was performed as previously described (13). Briefly, cells
cultured on PDL-coated coverslips were fixed (4% formaldehyde, 2% sucrose
in PBS) and blocked (2% normal goat serum, 0.1% Triton X-100 in PBS) at
room temperature and incubated in primary antibody diluted in blocking
reagent overnight at 4 °C. The following day, cells were washed and incu-
bated in secondary antibody and stained with DAPI by incubation for 10 min
in 300 ng/mL DAPI (Invitrogen) in PBS. Coverslips were mounted in DABCO
anti-fade reagent and observed after drying overnight. To quantify DISC1
expression in neurites, a confocal microscope was used to obtain z-stack
images of neurites. MetaMorph software was used to measure integrated
signal intensity in the whole cell and intensity in the cell body was sub-
tracted to obtain neurite intensity. Total neurite length was measured by
tracing β-tubulin–stained neurites in Metamorph and used to normalize
DISC1 signal intensity.
Immunohistochemistry in Human Brains. Diagnostic human brain samples and
of the Heinrich Heine University of Düsseldorf Medical School. Forty-eight
hours after incubation in 5% buffered formalin, brain was embedded in
paraffin and cut into 5-mm-thick sections. Sections were deparaffinized and
endogenous peroxidase was inactivated by incubation in methanol/0.3%
H2O2for 30 min. Sections were then rehydrated in an ethanol gradient
and incubated in anti-DISC1 antibody 3D4 overnight at 4 °C, washed, and
incubated in secondary antibody, washed and incubated with streptavidin-
peroxidase conjugate, and finally developed with DAB solution. For double
immunolabeling, before incubation with second antibody, residual perox-
idase activity was blocked with PBS/0.3% H2O2 for 30 min. For primary
antibodies, incubation was done overnight at 4 °C, followed by secondary
antibody and signal development by using Histogreen (Linaris, Wertheim,
Germany). For KP-1, pretreatment with target retrieval solution at pH 6.1
(DAKO S1699) was performed before application of primary antibody.
| www.pnas.org/cgi/doi/10.1073/pnas.0909284107 Seshadri et al.
ACKNOWLEDGMENTS. We thank Yukiko Lema and Dr. P. Talalay for manu- Download full-text
script preparation. We appreciate Dr. Gabrial Corfas for participating in scien-
tificdiscussions.WethankDr. Nicholas Brandon andDr. Cary Laifor providing
us with reagents, and Rocky Cheung for technical assistance. This work was
supported by Silvo O. Conte Center MH-084018 (to A.S.), MH-069853 (to A.S.),
MH-51134 (to E.S.A.), as well as by grants from Stanley (to A.S.), Cure Hunting-
ton’s Disease Initiative (to A.S.), HighQ (to A.S.), S-R (to A.S.), National Alliance
Award fellowship. F31MH081475 from the National Institute of Mental Health.
All work is solely the responsibility of the authors.
1. Harrison PJ, Weinberger DR (2005) Schizophrenia genes, gene expression, and
neuropathology: On the matter of their convergence. Mol Psychiatry 10:40–68.
2. Corfas G, Roy K, Buxbaum JD (2004) Neuregulin 1-erbB signaling and the molecular/
cellular basis of schizophrenia. Nat Neurosci 7:575–580.
3. Jaaro-Peled H, et al. (2009) Neurodevelopmental mechanisms of schizophrenia:
Understanding disturbed postnatal brain maturation through neuregulin-1-ErbB4
and DISC1. Trends Neurosci 32:485–495.
4. Willem M, et al. (2006) Control of peripheral nerve myelination by the beta-secretase
BACE1. Science 314:664–666.
5. Mei L, Xiong WC (2008) Neuregulin 1 in neural development, synaptic plasticity and
schizophrenia. Nat Rev Neurosci 9:437–452.
6. Hu X, et al. (2006) Bace1 modulates myelination in the central and peripheral nervous
system. Nat Neurosci 9:1520–1525.
7. Hennah W, Thomson P, Peltonen L, Porteous D (2006) Genes and schizophrenia:
Beyond schizophrenia: The role of DISC1 in major mental illness. Schizophr Bull 32:
8. Schurov IL, Handford EJ, Brandon NJ, Whiting PJ (2004) Expression of disrupted in
schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its
role in neurodevelopment. Mol Psychiatry 9:1100–1110.
9. Kamiya A, et al. (2005) A schizophrenia-associated mutation of DISC1 perturbs
cerebral cortex development. Nat Cell Biol 7:1167–1178.
10. Duan X, et al. (2007) Disrupted-In-Schizophrenia 1 regulates integration of newly
generated neurons in the adult brain. Cell 130:1146–1158.
11. Camargo LM, et al. (2007) Disrupted in Schizophrenia 1 Interactome: Evidence for the
close connectivity of risk genes and a potential synaptic basis for schizophrenia. Mol
12. Mao Y, et al. (2009) Disrupted in schizophrenia 1 regulates neuronal progenitor
proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136:1017–1031.
13. Kamiya A, et al. (2006) DISC1-NDEL1/NUDEL protein interaction, an essential
component for neurite outgrowth, is modulated by genetic variations of DISC1. Hum
Mol Genet 15:3313–3323.
14. Murdoch H, et al. (2007) Isoform-selective susceptibility of DISC1/phosphodiesterase-4
complexes to dissociation by elevated intracellular cAMP levels. J Neurosci 27:
15. Millar JK, et al. (2005) DISC1 and PDE4B are interacting genetic factors in
schizophrenia that regulate cAMP signaling. Science 310:1187–1191.
16. Zhang HT, Crissman AM, Dorairaj NR, Chandler LJ, O’Donnell JM (2000) Inhibition of
cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA
receptor antagonism. Neuropsychopharmacology 23:198–204.
17. Huston E, et al. (2008) EPAC and PKA allow cAMP dual control over DNA-PK nuclear
translocation. Proc Natl Acad Sci USA 105:12791–12796.
18. Monje PV, Bartlett Bunge M, Wood PM (2006) Cyclic AMP synergistically enhances
neuregulin-dependent ERK and Akt activation and cell cycle progression in Schwann
cells. Glia 53:649–659.
19. Dominguez D, et al. (2005) Phenotypic and biochemical analyses of BACE1- and
BACE2-deficient mice. J Biol Chem 280:30797–30806.
20. Kellendonk C, Simpson EH, Kandel ER (2009) Modeling cognitive endophenotypes of
schizophrenia in mice. Trends Neurosci 32:347–358.
21. Wang Q, Jaaro-Peled H, Sawa A, Brandon NJ (2008) How has DISC1 enabled drug
discovery? Mol Cell Neurosci 37:187–195.
22. Meyer D, Birchmeier C (1995) Multiple essential functions of neuregulin in
development. Nature 378:386–390.
23. Gerlai R, Pisacane P, Erickson S (2000) Heregulin, but not ErbB2 or ErbB3,
heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks. Behav
Brain Res 109:219–227.
24. Stefansson H, et al. (2002) Neuregulin 1 and susceptibility to schizophrenia. Am J Hum
25. Moy SM, et al. (2009) Deficient NRG1-ERBB signaling alters social approach: Relevance
to genetic mouse models of schizophrenia. J Neurodev Disord 4:302–312.
26. Savonenko AV, et al. (2008) Alteration of BACE1-dependent NRG1/ErbB4 signaling
and schizophrenia-like phenotypes in BACE1-null mice. Proc Natl Acad Sci USA 105:
27. Wood JD, Bonath F, Kumar S, Ross CA, Cunliffe VT (2009) Disrupted-in-schizophrenia
1 and neuregulin 1 are required for the specification of oligodendrocytes and
neurones in the zebrafish brain. Hum Mol Genet 18:391–404.
28. Buonanno A, Fischbach GD (2001) Neuregulin and ErbB receptor signaling pathways
in the nervous system. Curr Opin Neurobiol 11:287–296.
29. Ishizuka K, et al. (2007) Evidence that many of the DISC1 isoforms in C57BL/6J mice are
also expressed in 129S6/SvEv mice. Mol Psychiatry 12:897–899.
30. Leliveld SR, et al. (2008) Insolubility of disrupted-in-schizophrenia 1 disrupts oligomer-
dependent interactions with nuclear distribution element 1 and is associated with
sporadic mental disease. J Neurosci 28:3839–3845.
31. Kvajo M, et al. (2008) A mutation in mouse Disc1 that models a schizophrenia risk
allele leads to specific alterations in neuronal architecture and cognition. Proc Natl
Acad Sci USA 105:7076–7081.
32. Sawamura N, Sawamura-Yamamoto T, Ozeki Y, Ross CA, Sawa A (2005) A form of
DISC1 enriched in nucleus: Altered subcellular distribution in orbitofrontal cortex in
psychosis and substance/alcohol abuse. Proc Natl Acad Sci USA 102:1187–1192.
33. Anton ES, Marchionni MA, Lee KF, Rakic P (1997) Role of GGF/neuregulin signaling in
interactions between migrating neurons and radial glia in the developing cerebral
cortex. Development 124:3501–3510.
34. Hashimoto R, et al. (2004) Expression analysis of neuregulin-1 in the dorsolateral
prefrontal cortex in schizophrenia. Mol Psychiatry 9:299–307.
35. Hahn CG, et al. (2006) Altered neuregulin 1-erbB4 signaling contributes to NMDA
receptor hypofunction in schizophrenia. Nat Med 12:824–828.
36. Law AJ, et al. (2006) Neuregulin 1 transcripts are differentially expressed in
schizophrenia and regulated by 5′ SNPs associated with the disease. Proc Natl Acad Sci
37. Barres BA, et al. (1992) Cell death and control of cell survival in the oligodendrocyte
lineage. Cell 70:31–46.
38. Tang DG, Tokumoto YM, Raff MC (2000) Long-term culture of purified postnatal
oligodendrocyte precursor cells. Evidence for an intrinsic maturation program that
plays out over months. J Cell Biol 148:971–984.
39. Yoshii A, Constantine-Paton M (2007) BDNF induces transport of PSD-95 to dendrites
through PI3K-AKT signaling after NMDA receptor activation. Nat Neurosci 10:
40. Barnabé-Heider F, et al. (2005) Evidence that embryonic neurons regulate the onset
of cortical gliogenesis via cardiotrophin-1. Neuron 48:253–265.
41. Yamashita N, Yamauchi M, Baba J, Sawa A (1997) Phosphodiesterase type 4 that
regulates cAMP level in cortical neurons shows high sensitivity to rolipram. Eur J
42. Joo CH, et al. (2002) Coxsackievirus B4-induced neuronal apoptosis in rat cortical
cultures. Neurosci Lett 326:175–178.
Seshadri et al.PNAS
| March 23, 2010
| vol. 107
| no. 12