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TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Global signaling effects of a schizophrenia-associated missense
mutation in neuregulin 1: an exploratory study using whole
genome and novel kinome approaches
Ketan K. Marballi •Robert E. McCullumsmith •
Stefani Yates •Michael A. Escamilla •Robin J. Leach •
Henriette Raventos •Consuelo Walss-Bass
Received: 13 August 2013 / Accepted: 12 December 2013
ÓSpringer-Verlag Wien 2013
Abstract Aberrant neuregulin 1-ErbB4 signaling has
been implicated in schizophrenia. We previously identified
a novel schizophrenia-associated missense mutation (valine
to leucine) in the NRG1 transmembrane domain. This
variant inhibits formation of the NRG1 intracellular
domain (ICD) and causes decreases in dendrite formation.
To assess the global effects of this mutation, we used
lymphoblastoid cell lines from unaffected heterozygous
carriers (Val/Leu) and non-carriers (Val/Val). Transcrip-
tome data showed 367 genes differentially expressed
between the two groups (Val/Val N=6, Val/Leu N=5,
Ttest, FDR (1 %), a=0.05, -log
10
pvalue [1.5). Inge-
nuity pathway (IPA) analyses showed inflammation and
NRG1 signaling as the top pathways altered. Within NRG1
signaling, protein kinase C (PKC)–eta (PRKCH) and non-
receptor tyrosine kinase (SRC) were down-regulated in
heterozygous carriers. Novel kinome profiling (serine/
threonine) was performed after stimulating cells (V/V
N=6, V/L N=6) with ErbB4, to induce release of the
NRG1 ICD, and revealed significant effects of treatment on
the phosphorylation of 35 peptides. IPA showed neurite
outgrowth (six peptides) as the top annotated function.
Phosphorylation of these peptides was significantly
decreased in ErbB4-treated Val/Val but not in Val/Leu
cells. These results show that perturbing NRG1 ICD for-
mation has major effects on cell signaling, including
inflammatory and neurite formation pathways, and may
contribute significantly to schizophrenia pathophysiology.
Electronic supplementary material The online version of this
article (doi:10.1007/s00702-013-1142-6) contains supplementary
material, which is available to authorized users.
K. K. Marballi R. J. Leach
Department of Cellular and Structural Biology, University of
Texas Health Science Center at San Antonio,
7703 Floyd Curl Dr., San Antonio, TX 78229, USA
e-mail: marballi@livemail.uthscsa.edu
R. J. Leach
e-mail: leach@uthscsa.edu
K. K. Marballi
Department of Psychiatry, Neuroscience Program, South Texas
Research Facility, 8403 Floyd Curl Dr., San Antonio, TX 78229,
USA
R. E. McCullumsmith S. Yates
Department of Psychiatry, School of Medicine, University of
Alabama at Birmingham, 1719 6th Avenue South, CIRC 576A,
Birmingham, AL 35294, USA
e-mail: smithrob@uab.edu
S. Yates
e-mail: syates@uab.edu
M. A. Escamilla
Paul L. Foster School of Medicine, Texas Tech University
Health Sciences Center, 4800 Alberta Ave, El Paso, TX 79905,
USA
e-mail: m.escamilla@ttuhsc.edu
H. Raventos
School of Biology, University of Costa Rica, San Jose 2060,
Costa Rica
e-mail: hravento@racsa.co.cr
C. Walss-Bass (&)
Department of Psychiatry, University of Texas Health Science
Center at San Antonio, 7703 Floyd Curl Dr., San Antonio,
TX 78229, USA
e-mail: walss@uthscsa.edu
123
J Neural Transm
DOI 10.1007/s00702-013-1142-6
Keywords Neuregulin-1 Transcriptome Kinome
Schizophrenia Lymphoblastoid cells
Introduction
NRG1 is a well-established schizophrenia candidate gene
(Harrison and Law 2006; Tosato et al. 2005; Greenwood
et al. 2012). The NRG1 protein regulates many important
functions in the nervous system by interacting with cognate
receptors belonging to the ErbB family, of which the
NRG1–ErbB4 interaction has been shown to be particularly
relevant to nervous system function (Shamir et al. 2012;
Mei and Xiong 2008). The downstream targets of this
pathway include ERK, AKT, and PKC. Altered phos-
phorylation of these targets, particularly AKT (Keri et al.
2009) and ERK (Funk et al. 2012; Kyosseva et al. 1999)
has been reported in schizophrenia. Of the numerous single
nucleotide polymorphisms (SNPs) identified within the
NRG1 gene to be associated with schizophrenia worldwide,
only one is known to have a direct role in regulating NRG1
function (Talmage 2008; Weickert et al. 2012). This vari-
ant, which causes a change from valine (GTG) to leucine
(TTG) (V [L) in the transmembrane domain of the NRG1
protein, was first identified by our laboratory, and is
associated with schizophrenia in the population of the
Central Valley of Costa Rica (CVCR) (Walss-Bass et al.
2006). We have further found that this variant is associated
with immune dysregulation, indicated by increased levels
of proinflammatory cytokines and autoantibodies in carri-
ers of the variant (Marballi et al. 2010). This is of immense
importance, given the large body of studies showing dys-
regulation of the immune system (Potvin et al. 2008; Strous
and Shoenfeld 2006), including elevated levels of proin-
flammatory cytokines and autoantibodies in schizophrenia.
Other groups subsequently showed that the V [L change
impedes formation of the NRG1 intracellular domain
(ICD) by blocking gamma secretase-mediated intracellular
cleavage of membrane-bound isoforms of NRG1, such as
NRG1 type III (Dejaegere et al. 2008), leading to decreased
dendrite formation in cortical neurons (Chen et al. 2010).
Interestingly, high levels of proinflammatory cytokines
decrease dendrite formation in vitro (Gilmore et al. 2004).
The NRG1 ICD, generated by gamma secretase intra-
cellular cleavage, migrates to the nucleus and regulates
expression of Bcl-X
L
,Bak,Rip and Oct-3genes (Bao et al.
2003). To further explore the impact of the V [L change
on gene expression and cell signaling, specifically NRG1–
ErbB4 signaling, we utilized lymphoblastoid cell lines
(LCLs) from unaffected individuals from the CVCR that
were either heterozygous carriers (Val/Leu) or homozy-
gous non-carriers (Val/Val) to perform whole genome
expression (V/L N=5, V/V N=6) and whole kinome
profiling (V/L N=6, V/V N=6) studies. LCLs are ideal
for the study of the effects of genetic variants on cell
function, as they avoid confounding environmental effects
such as psychotropic drugs used by patients, and allow for
focus solely on mechanistic aspects of genetic perturbation.
We hypothesized that the V [L change that perturbs
formation of the ICD would impact gene expression and
signaling in pathways important for schizophrenia
development.
Materials and methods
Ethics statement
Peripheral leukocytes were isolated from blood of subjects
from the CVCR, at the time of recruitment, as previously
described (Walss-Bass et al. 2006) in accordance with the
principles of the Declaration of Helsinki with approval
from the Institutional Review Boards of the University of
Costa Rica and the University of Texas Health Science
Center at San Antonio. All participants provided written
informed consent.
Lymphoblastoid cell lines: generation and maintenance
Lymphoblastoid cell lines (LCLs) were generated from
leucocytes using LeucoPREP brand cell separation tubes
(Becton–Dickinson Labware, Franklin Lakes, NJ, USA)
and transformed using Epstein–Barr virus (EBV). Cells
were grown in RPMI 1640 medium with 2 mM L-gluta-
mine and 15 % bovine growth serum, 1 % penicillin
streptomycin at 37 °C in a humidified 5 % CO
2
incubator.
As previously described (Marballi et al. 2010), given that
the goal of this study was to determine the association of
the NRG1 V [L mutation with alterations in gene
expression and cell signaling, independent of psychiatric
diagnosis, cell lines used in this study were from unaf-
fected, unrelated individuals who had one first degree rel-
ative with psychosis: homozygous wild type (GG, Val/Val,
N=6; three males, three females, average age
51.4 ±20.54 years) and heterozygous Tallele carriers
(GT, Val/Leu, N=5; two males, three females, average
age 58.5 ±19.15 years). While conducting the experi-
ments, the investigators were blinded to the experimental
groups. Cell viability was determined by neutral red assay
(Sigma, St. Louis, MO, USA). Only viable cells are
capable of incorporating neutral red dye by active trans-
port. In brief, after 24 h of incubation of equal number of
cells, cells were rinsed with PBS and incubated in media
containing 0.033 % neutral red for 2 h. Cells were then
washed several times with PBS and the incorporated neu-
tral red dye was solubilized by gentle rocking for 10 min
K. K. Marballi et al.
123
with a solution of 1 % acetic acid and 50 % ethanol. After
10 min, the solution was collected and the amount of
incorporated neutral red dye was determined spectropho-
tometrically by measuring the absorbance of the solution at
540 nm.
Whole genome expression studies
RNA was extracted from cells using the Trizol method.
RNA samples were cleaned up using RNEasy plus micro
kit and run on an Agilent 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA, USA) to ensure RNA
integrity for whole genome studies. cRNA was prepared,
hybridized to Cy3, loaded as singlicates on to Human WG-
6 v3 beadchips (Illumina, Inc, San Diego, CA, USA), and
scanned using an Illumina iScan reader. All data were
quantile normalized, exported into text files using Bead
studio software and data analyses were performed using
box plots in JMP genomics (version 3.0, SAS Institute Inc.,
Cary, NC, USA). Student’s Ttest was used in JMP to
analyze values, with a Benjamini Hochberg false discovery
rate (FDR) correction of 1 %, a=0.05, and a cutoff of
-log
10
(pvalue) [1.5. The list of significant genes
obtained was imported into Ingenuity pathway analyses
(IPA, Ingenuity
Ò
Systems, Redwood City, CA, USA) for
identification of altered pathways/disease.
Validation of microarray data using real-time PCR
(qPCR)
Two lg of RNA was converted to cDNA using the High
Capacity cDNA Reverse Transcription kit (Life Technol-
ogies, Carlsbad, CA, USA). One hundred ng of cDNA was
used along with Taqman assays (Life Technologies,
Carlsbad, CA, USA) for PRKCH (protein kinase C-eta
isoform-Hs00178933_m1) and SRC (00178494_m1). The
human large ribosomal subunit gene (RPLPO) was used as
the housekeeping gene. Real-time PCR was run using
standard settings on a 7900HT sequence detection system
(Life Technologies, Carlsbad, CA, USA). Each cell line
was run in technical duplicates. Delta Ct (DCt) values were
calculated by subtracting housekeeping gene Ct from the
gene of interest Ct values. 2
-DCt
values were used for
plotting data.
Cell line ErbB4 treatment and preparation for kinome
assay
For these studies, we added an additional sample to the
Val/Leu group to bring the total to N=6 (three males,
three females, average age 62.8 ±19.17 years). For each
cell line, approximately 1 910
7
cells from actively
growing cultures were pelleted, washed once with PBS and
seeded on 6 well plates with 2 ml of serum- free media.
Cells were serum-starved overnight, and then treated with
10 lg/ml ErbB4 (R&D systems, Minneapolis, MN, USA)
or PBS (vehicle) in complete media for 4 h. ErbB4 treat-
ment has been previously shown to stimulate the NRG1
back signaling pathway, i.e., ICD formation (Hancock et al.
2011). Lysates were prepared using the M-PER Mamma-
lian extraction buffer (Thermo Fisher Scientific Inc.,
Rockford, IL, USA) with added protease and phosphatase
inhibitors. In brief, cells were pelleted at 4 °C, washed
twice with ice cold PBS, followed by lysis in the extraction
buffer for 15 min on ice and centrifugation at 10,0009gfor
15 min at 4 °C. Supernatants were collected, aliquots were
flash frozen in liquid nitrogen and stored at -80 °C. Pro-
tein quantitation was performed using nanodrop (Thermo
Fisher Scientific Inc., Rockford, IL, USA) and BCA assay
(Thermo Fisher Scientific Inc., Rockford, IL, USA).
PamStation kinomic analysis
Kinomic profiling was performed using a PamStation
Ò
96
microarray (PamGene International Cambridge, MA, USA)
with STK PamChip
Ò
containing 140 consensus and 4
control phosphopeptide sequences in each well represent-
ing the Serine/Threonine kinome. Each well of the Pam-
Chip
Ò
was first blocked in 2 % bovine serum albumin
(BSA). Following protein concentration determination (by
BCA assay), 1.5 lg of protein was loaded per well of the
PamChip
Ò
along with standard kinase buffer (PamGene),
400 lM ATP, and FITC-labeled anti-phospho Ser and Thr
antibodies (PamGene). Lysate from each cell line was run
as a singlicate (total N=24). The assay mix, containing
the active kinases in the sample lysates, was pumped
through the PamChip
Ò
wells to facilitate interaction with
the specific peptide substrates immobilized in the chip. The
degree of phosphorylation was measured in real time via
the kinetic phosphoserine/threonine antibodies binding to
each phosphorylated peptide substrate every 6 s for the
length of the program (60 min). Following 60 min incu-
bation, the post-wash signals (multiple exposure times)
were integrated for each spot and a log transformation of
the data was carried out. The signal intensities for each
peptide were analyzed using BioNavigator Software
(PamGene) as previously described (Jarboe et al. 2012).
Statistical analyses
Data analyses were carried out using IBM SPSS version
2.0 (IBM Corp, Armonk, NY, USA) using log-transformed
intensity values. Student’s Ttest was used for all experi-
ments comparing Val/Val to Val/Leu individuals (whole
genome array, qPCR). For IPA analysis, the software
identifies gene networks by mapping the connectivity
Global signaling effects of a schizophrenia-associated missense mutation
123
between genes, and computes significant biological/cellular
functions that are overrepresented based on input data
using a right-tailed Fisher’s exact test to calculate pvalues
of identified pathways, based on the number of significant
molecules in the pathway.
IPA also provides canonical pathways significant to the
data, based on known metabolic and signaling pathways
from the literature. For the kinome data, multivariate
analyses (two-way ANOVA) were carried out to assess
changes in phosphorylation of 140 different peptides and
assess effects of genotype and treatment on phosphoryla-
tion. Since we only had two groups per condition [treat-
ment (vehicle or ErbB4); genotype (Val/Val or Val/Leu)],
post hoc testing was not possible in this two-way model.
Based on our previous studies, we had an a priori
hypothesis that we would observe changes between Val/
Val and Val/Leu groups, therefore paired Ttests were used
for comparisons of vehicle and ErbB4-treated Val/Val and
Val/Leu cells. In all cases, statistical significance was
applied at p\0.05. Putative upstream kinases for signifi-
cant peptides were ascertained using the kinexus database
(http://www.phosphonet.ca/).
Results
Differential gene expression between Val/Val and
Val/Leu groups
Whole genome expression analyses showed 367 genes to
be significantly different between groups using Student’s
Ttest and 1 % FDR with a=0.05 and a cutoff of
-log
10
[1.5. 204 genes were down-regulated, while 163
genes were up-regulated in V [L carriers. Ingenuity
Pathway analysis (IPA) showed these genes to be involved
in 18 different networks based on connections with other
genes, the top 6 of which are shown in Table 1. Of the
biological functions within the 18 networks, inflammatory
response and cell cycle were the most overrepresented
(Fig. 1). No significant differences in cell proliferation or
viability were observed when comparing cells from Val/
Val individuals to cells from Val/Leu heterozygous sub-
jects (data not shown). IPA identified the canonical path-
way of NRG1 signaling as having the greatest significance,
with 5 genes being involved in this pathway, including a
disintegrin and metalloproteinase 17 (ADAM 17), neureg-
ulin 2 (NRG2), megakaryocyte associated tyrosine kinase
(MATK), protein kinase C eta (PRKCH) and the non-
receptor tyrosine kinase SRC. PKC and Src are important
secondary messengers downstream of NRG1–ErbB4 sig-
naling, and have both been implicated in schizophrenia.
PRKCH and SRC mRNA levels were 6.6- and 1.6-fold
lower, respectively, in Leu carriers compared to wild type
(Ttest, p\0.05), and this down-regulation was validated
by qPCR (Fig. 2). Of genes within the NRG family, only
NRG2 was differentially expressed between Val/Val and
Val/Leu groups, with increased expression in Leu carriers.
No differences in expression levels of ErbB2, ErbB3 or
ErbB4 were observed between groups.
Val [Leu variant affects phosphorylation of targets
regulating neurite outgrowth
To test the effects of the Val [Leu variant on phosphor-
ylation of targets within NRG1–ErbB4 pathways, we
treated the LCLs with recombinant ErbB4. This treatment
stimulates the intracellular cleavage of NRG1 and forma-
tion of the ICD (Bao et al. 2003; Hancock et al. 2011)
which is perturbed in the presence of the Val [Leu variant
(Dejaegere et al. 2008). Comparisons across the four
groups (Val/Val vehicle, Val/Leu vehicle, Val/Val ErbB4,
Val/Leu ErbB4) using multivariate analyses (two-way
ANOVA) revealed effects of ErbB4 treatment on 35 pep-
tides, effect of genotype on 2 peptides, and effect of
genotype and treatment on 1 peptide (Supplementary Table
S1). IPA analyses revealed 10 of the 35 peptides affected
by ErbB4 treatment to be involved in cell morphology and
nervous system development and function. In both these
functions, ‘‘outgrowth of neurites’’ was the top common
pathway altered, with six molecules being involved: beta 2
adrenergic receptor (ADRB2), cyclic AMP response ele-
ment-binding protein (CREB1), Nuclear factor NF-kappa-
B p105 subunit (NFkB1), C-Rel proto-oncogene protein
(C-REL), ETS domain-containing protein (ELK1) and
Stathmin 2 (STMN2) (Fig. 3). In most cases (4 out of 6),
treatment with ErbB4 caused a significant decrease in
phosphorylation in the Val/Val group (Ttest, p\0.05),
while no significant differences were seen in the Val/Leu
group after treatment (Fig. 3). ADRB2, CREB1, and
NFkB1 share PKA-alpha as an upstream kinase. ADRB2
and CREB1 also share AKT1 as an upstream kinase
(information from kinexus database, http://www.phos
phonet.ca/). ELK1 is phosphorylated at numerous serine/
threonine residues by ERK1/2 (Cruzalegui et al. 1999), two
of which are represented in the array (T363, T368)
(Table 2). CREB1 is phosphorylated by ERK1/2 at Ser 133
(Davis et al. 2000), also represented in the array (Table 2).
Both ERK1/2 and AKT1 are secondary messengers
downstream of NRG1/ErbB4 signaling (Hahn et al. 2006)
and are regulated by PKC (Puente et al. 2006; Uht et al.
2007) and Src (Hu et al. 2009; Lodeiro et al. 2009) (Fig. 4).
ELK1 is also a substrate for Srcasm (Src-activating and
signaling molecule), which is a downstream substrate of
Src (Li et al. 2005). Integrated pathway analysis of mole-
cules identified by the transcriptome and kinome data show
that these molecules share a common pathway (Fig. 4).
K. K. Marballi et al.
123
Discussion
NRG1 is one of the most biologically plausible schizo-
phrenia candidate susceptibility genes (Kanakry et al.
2007; Talmage 2008). Extensive studies in NRG1 function
have shown that it impacts different aspects of the nervous
system such as myelination (Taveggia et al. 2008; Velanac
et al. 2012), radial glia migration (Poluch and Juliano
2007) and neurotransmitter receptor expression (Stefansson
et al. 2002; Corfas et al. 2004). Many of these functions are
mediated through external interaction of NRG1 with the
receptor ErbB4. However, research in the last few years
has shown that internal cleavage of NRG1, on the cyto-
plasmic side of the cell membrane, is equally important
(Hancock et al. 2008,2011). This cleavage, regulated by
gamma secretase, leads to formation of the ICD, which
regulates expression of several different genes (Bao et al.
2003). Our laboratory found a functional transmembrane
mutation (V [L) (Walss-Bass et al. 2006) that has been
shown by other groups to block the NRG1 intracellular
cleavage event (Dejaegere et al. 2008) and caused dendrite
formation deficits (Chen et al. 2010), a process implicated
in schizophrenia development (Glausier and Lewis 2012).
The V [L variant was associated with schizophrenia in
the CVCR population (Walss-Bass et al. 2006) and, more
recently, also associated with elevated levels of autoanti-
bodies and proinflammatory cytokines in plasma and
LCL’s from heterozygous carriers from the CVCR
(Marballi et al. 2010). Elevated levels of inflammatory
cytokines and autoantibodies have been consistently
reported in schizophrenia patients (Potvin et al. 2008).
These studies led us to hypothesize that the Val [Leu
amino acid change may cause dysregulation of gene
expression and cell signaling events in carriers of this
variant, and this may contribute to development of
schizophrenia. Whole genome expression analyses showed
Table 1 Ingenuity pathway analyses of gene expression data arranged by relevance score
ID Molecules in network Score Top functions
1AACS:,ADAM17:,AP2A2;, calcineurin protein(s), calpain, CCR4, CD97;,
E2f, ERK1/2, HIST1H2BJ :(includes EG: 8970), Hsp90, IFN beta, IL12
(complex), immunoglobulin, interferon alpha, LDL, LPP, MARK2;, MATR3,
NFKB1;, NRG2:, P38 MAPK, Pkc(s), PPAP2B, PPP3R2, PRKCH;, RLN3,
RPL23A, S100A9, SNAP23;,SREBF2:, STX11;, Tgf beta, TNNC2, Vegf
40 Reproductive system development and
function, cell cycle, hematological disease
2 Ap1, CD27:, CD180, DOK2, ERK, fibrinogen, HAX1:, IKK (complex), IL1F9,
integrin, LCP2;, LY96, MATK, Mlc, MUC2, NCK, Nfat (family), NFkB
(complex), NFKB1;,NPHS2, PDGF BB, PLC gamma, Rac, Ras, Ras homolog,
RHOB, RIN3, ROCK1;, SH3BP2, SLC3A1, SRC;, SRCIN1, SSH1;, TCR,
VAV, WISP2
29 Inflammatory response, cell death, cellular
assembly and organization
3 ALDH1A1, ALDH8A1, BAI1, BAIAP3 (includes EG: 8938), C2ORF44:,
C9ORF64;, CBR3, DHRS4;,DLG4, DNAJA2, DNAJA3, DNAJB4,
DNAJB9, DNAJC14, DNAJC17:, DTWD1, FEZF2, FYCO1:, HNF4A, HPS3,
HPS6, HPS5 (includes EG: 11234), IL15, MRPL33:, ONECUT1, RUVBL2,
SARS2:,SLC25A32, SREBF1, SUCLG1, TGFB1, TP53, ZNF317:
23 Genetic disorder, drug metabolism, lipid
metabolism
4 ABI3BP, ACMSD:, AGFG1 (includes EG: 3267), AR, BCL7C;, c-Myc/N-Myc,
CDH1, CLEC2A, CSNK1G2,;Erk1/2 dimer, ESR1, FBXW8:, GTF3C4,
KCTD6, MAGEA11, MIR101, MIR214 (includes EG: 406996), MPP5, MYB
(includes EG: 293405), MYC, MYCN, MYO9A, NADSYN1;, PHF5A,
PLSCR4:, retinoic acid, REXO4:, RN5S, RPL41, RPL13A:, RUVBL2,
SCPEP1, SLC25A19, SMARCA4, SNX20;
21 Cell cycle, cancer, dermatological diseases and
conditions
5 26 s proteasome, ADA;, ADRBK2, Akt, BCAS3, caspase, CDKN2A:, DYRK3,
ELP4, ELP6, ELP3 (includes EG: 55140), ERMAP, FABP3;, FOXG1,
FOXO6, FSH, histone h3, histone h4, insulin, JMJD6, Jnk, MAD2L2;,MAF,
Mapk, OPN1LW (includes EG:5956), OPN1SW, PARP10, PAX7:, PHC2,
PI3K, PPP1R1B, PQBP1, RNA polymerase II, SEC14L2, TFF1
20 Neurological disease, genetic disorder,
ophthalmic disease
6 ANXA8, ART2A, BAP1, beta-estradiol, BRCA1, C11ORF82, C13ORF27,
CCL23:,CIC;, CREB1, DEFB1, GABPB2, GBP6, GBP8, GBP4 (includes
EG: 17472), GIP2, GVIN1, HERC2, IFNB1, IFNG, IRGM, KIR2DS1, LY6,
MNS1:,NLRC5;(includes EG:84166), PCBP4:, PRB2, PTPRV, RPL27,
RPL36AL:, RTP4, SNCA, TMEM164;, TP53, TRIM22
19 Cell cycle, cell death, connective tissue
development and function
Table shows top 6 of 18 networks altered between Val/Val and Val/Leu individuals. Genes with increased (:) or decreased (;) expression in Val//
Leu compared to Val/Val groups and present in networks are bolded, related molecules in network are not bolded. Underlined in the top functions
column, are the functions of cell cycle, inflammatory response and cell death, which are overrepresented amongst the top six networks. These
functions are shown in Fig. 1
Global signaling effects of a schizophrenia-associated missense mutation
123
367 genes to be differentially expressed in heterozygous
carriers compared to wild type. Inflammatory response was
a top function altered, which validates our previous results
where we found high levels of proinflammatory cytokines
in the presence of the Val [Leu variant (Marballi et al.
2010). Cell cycle and cell death were also top functions
found to be altered. Examination of cell proliferation and
viability did not show significant differences between Val/
Val and Val/Leu groups at baseline. However, it is possible
that differences would be found under conditions of stress,
and this remains to be investigated. To this respect, it is
known that many of the symptoms shown in schizophrenia
are manifested and exacerbated when patients are under
conditions of stress (Corcoran et al. 2003).
We also found the NRG1 signaling pathway to be the
top canonical pathway altered in Val/Leu carriers. The fact
that expression levels of NRG1 and ErbB2,3,4 were not
found to be significantly different between genotype groups
supports the hypothesis that the observed differences in
global gene expression are due to alterations in NRG1
protein cleavage.
Overall, slightly more genes were down-regulated than
up-regulated in Val/Leu carriers. However, focusing on the
top 6 pathways outlined by IPA analysis (Table 1), there is
no generalized trend for up- or down-regulation. While
changes in expression levels in some cases may be directly
due to inhibited NRG1 ICD formation, other changes may
be due to compensatory mechanisms in attempts to coun-
teract the effects of altered NRG1 cleavage. Therefore, a
detailed analysis of each of the genes in each pathway is
required to specifically assess the effects of alteration in
expression on specific cell functions. For this study, how-
ever, we focused on genes directly relevant to the NRG1–
ErbB4 signaling pathway, PRKCH and SRC.PRKCH was
the top overall gene in terms of fold change (6.6-fold
decreased expression in Leu carriers). This isoform of PKC
regulates keratinocyte differentiation, T cell antigen pre-
sentation, AKT signaling (Shahaf et al. 2012), and interacts
with the ERK pathway to regulate the nose-touch response
in C. elegans (Hyde et al. 2011). The non-receptor tyrosine
kinase (SRC) had the next highest fold change in Val/Leu
compared to Val/Val groups. Importantly, PKC and Src are
not only important effectors of the NRG1–ErbB4 pathway,
but also play crucial roles in regulating NMDA receptor
(NMDAR) function. PKC regulates NMDAR trafficking
through a SNARE-dependent exocytosis mechanism on the
surface of dendrites (Lan et al. 2001). Src has been reported
to play a role in activation of the NMDAR by regulating its
phosphorylation (Kalia et al. 2006; Xu et al. 2012). NRG1–
ErbB4 signaling inhibits Src tyrosine kinase activity
(Pitcher et al. 2011), which may lead to NMDAR hypo-
function. Dysfunction of the NMDAR has been
Fig. 1 Functional analyses of
whole genome data by
Ingenuity pathway analysis
(IPA). Cells from Val/Val
(N=6) and Val Leu (N=5)
groups were subjected to whole
genome analyses using Illumina
Human WG-6 v3 beadchips.
Data were analyzed by JMP
genomics software using
Student’s Ttest with a
Benjamini Hochberg false
discovery rate (FDR) correction
of 1 %, a=0.05, and a cutoff
of -log
10
(pvalue) [1.5.;
output generated was imported
into IPA. Panels show cellular
functions (top) and canonical
pathways (bottom) associated
with differentially expressed
genes and networks (Table 1).
Right-tailed Fisher’s exact test
was used to calculate pvalues
determining the significance of
each biological function and/or
pathway. Threshold =-log
(pvalue) [1.5
K. K. Marballi et al.
123
hypothesized to be relevant in schizophrenia (Weickert
et al. 2012; Hamm et al. 2012), and contributes to cognitive
dysfunction in schizophrenia patients (Pitcher et al. 2011).
Enhanced NRG1-mediated ErbB4 signaling and sub-
sequent decreased activation of NMDARs was found in
postmortem human brains (Hahn et al. 2006). We have
previously found that the NRG1 ICD is decreased in
postmortem brains from schizophrenia patients (Marballi
et al. 2012). Our current results strengthen the ‘‘Src link in
schizophrenia’’ (Hahn 2011) and show that altering NRG1
intracellular cleavage may regulate SRC expression, con-
tributing to NMDAR dysfunction. Given that NRG1 sig-
naling was the top canonical pathway altered between Val/
Val and Val/Leu groups, we were further interested in
finding whether other kinases downstream of SRC and PKC
were affected in V [L carriers. For this, we performed
whole kinome profiling in cells treated with ErbB4, to
stimulate release of the NRG1 ICD (Hancock et al. 2011).
Kinome profiling revealed that phosphorylation of 35
substrates was altered by ErbB4 treatment, with six of these
involved in regulation of neurite formation: ADRB2,
CREB1, ELK1, STMN2, REL, and NFkB1. Interestingly, 5
of the 6 peptides share a common Basic–Basic-X-Ser motif
(Table 2), consistent with possible PKA or PKC involve-
ment. Overall, within the neurite formation molecules,
treatment with ErbB4 caused a significant decrease in
phosphorylation in the Val/Val cell lines, but no differ-
ences in the Val/Leu group (Fig. 3). This was significant in
4 out of the 6 peptides. This suggests that the ICD may be
involved in regulating these phosphorylation events, which
are blocked in the presence of the V [L variant. Decreases
in dendritic spine density have been observed in schizo-
phrenia patients (Jaaro-Peled et al. 2010) and in NRG1 type
III-knockout mice (Chen et al. 2008). Importantly, the
NRG1 V [L variant is reported to cause decreased den-
drite formation in a mouse model (Chen et al. 2010) and
our present results in humans validate these findings. Also,
high levels of proinflammatory cytokines have been shown
to decrease dendrite formation in vitro (Gilmore et al.
2004). Our current results suggest that these deficits in
dendrite formation may be mediated by deficits in NRG1
intracellular cleavage, through inflammatory pathways and
differential phosphorylation of neurite formation media-
tors. It is important to note that NFkB1, a key regulator of
expression of proinflammatory cytokines (Tak and Fire-
stein 2001), is one of the substrates altered in both
expression and phosphorylation (Table 1; Fig. 3), sup-
porting the genome-wide expression results of altered
inflammatory pathways, and our previous findings of
immune system dysregulation in carriers of the V [L
variant (Marballi et al. 2010). Also of interest is that the
phosphorylation of Elk1 is controlled by a downstream
substrate of Srcasm, which in turn is a target of Src (Li
et al. 2005). Given that SRC mRNA levels are decreased in
Val/Leu carriers, this suggests a direct link between the
V[L variant, SRC, and its downstream kinase activity.
Using the kinexus database, we identified PKA, ERK
and AKT as the putative upstream kinases that were altered
in activity. PKA has been implicated in neurite growth and
has been shown to interact with the MAPK pathway in
regulating neuronal differentiation (Vogt Weisenhorn et al.
2001; Yao et al. 1998). ERK is a crucial regulator of
neurite growth (Sarina et al. 2013; Auer et al. 2012) and is
involved in maintaining balance between gliogenesis and
neurogenesis during development (Chang et al. 2011).
Protein Kinase C mediates activation of ERK-regulated
dendritic spine density (Goldin and Segal 2003). The AKT
pathway regulates dendritic size and complexity (Kumar
et al. 2005). Importantly, PKC and SRC are regulators of
both ERK and AKT (Puente et al. 2006; Uht et al. 2007;Hu
et al. 2009; Lodeiro et al. 2009) and they also regulate
neurite formation (Garcia et al. 2013; Liao et al. 2012;
Zhao et al. 2009; Kotani et al. 2007). Taken together, our
preliminary results suggest that the gene expression chan-
ges (PRKCH, SRC) we found in the presence of the V [L
Fig. 2 Quantitative PCR validation of whole genome data using
single gene real-time PCR assays. RNA was extracted from the Val/
Val (N=6) and Val/Leu (N=5) cells, converted to cDNA, and
subjected to qPCR. Relative expression levels are shown with 2
-DCt
values (asterisk represents statistical significance Ttest p\0.05),
SRC (upper panel) and PRKCH (lower panel), with the human large
ribosomal protein (RPLPO) as an endogenous control
Global signaling effects of a schizophrenia-associated missense mutation
123
variant may lead to alterations in activity of PKA, ERK and
AKT kinases, leading to deficits in dendrite formation via
altered phosphorylation of neurite formation mediators.
Integration of the transcriptome and kinome findings by
IPA show that the main molecules altered by gene
expression and phosphorylation are indeed part of a com-
mon pathway (Fig. 4). The NRG1 ICD is released via
ErbB4 treatment and migrates to the nucleus where it both
represses and upregulates expression in a gene-specific
context in neurons (Bao et al. 2003; Hancock et al. 2011).
Therefore, blocking ICD formation could potentially cause
widespread effects on multiple pathways. Based on our
results, we hypothesize that the NRG1 ICD may be
responsible for regulating levels of kinases involved in
phosphorylation of neurite formation mediators, and this
may contribute to constitutive/fine control of dendrite for-
mation. The NRG1 V [L variant has been shown to
inhibit ICD formation (Dejaegere et al. 2008) and to cause
dendrite formation deficits (Chen et al. 2010). Given that
the Val/Leu individuals in our study possess one mutant
allele, the levels of ICD are likely decreased in these
individuals and they do not respond properly to ErbB4
Fig. 3 Differential phosphorylation of molecules involved in neurite
formation. Cells from Val/Val (N=6) and Val/Leu (N=6) groups
were serum-starved and subsequently treated with vehicle or 10
lg/ml recombinant ErbB4 for 4 h; lysates extracted were used for the
kinome array. Average log-transformed intensity values for each
respective peptide for each cell line were used to calculate average
intensity values for peptide phosphorylation in each group. Asterisk
significant Ttest (p\0.05) between Val/Val Veh (vehicle-treated)
and Val/Val ErbB4-treated cell lines
Table 2 Kinome array peptides regulating neurite formation and altered by ErbB4 treatment
ID Description Sequence Ser
a
Thr
a
ADRB2_338_350 Beta-2 adrenergic receptor
Beta-2 adrenoceptor) (Beta-2 adrenoreceptor)
ELLCLRRSSLKAY (345, 346)
CREB1_126_138 cAMP response element-binding protein (CREB) EILSRRPSYRKIL (129, 133)
ELK1_356_368 ETS domain-containing protein Elk-1 LLPTHTLTPVLLT (359, 361, 363, 368)
NFKB1_330_342 Nuclear factor NF-kappa-B p105 subunit
(DNA-binding factor KBF1)
FVQLRRKSDLETS (337, 342) (341)
REL_260_272 C-Rel proto-oncogene protein (C-Rel protein) KMQLRRPSDQEVS (267, 272)
STMN2_90_102 Stathmin-2 (Protein SCG10) (superior cervical
ganglion-10 protein)
AAGERRKSQEAQV (97)
a
Positions of serine (Ser) and threonine (Thr) residues; blank boxes indicate no Ser/Thr residues
K. K. Marballi et al.
123
treatment. This could cause changes in kinase expression,
such as PKC and SRC, leading to ultimately blocking
changes in phosphorylation of neurite formation mediators.
Our study was limited in sample size based on the use of
cells from individuals without a psychiatric diagnosis, and
matched for age and gender. Therefore, we emphasize that
the present results, while important, are preliminary and
must be interpreted with caution. These results must be
replicated using additional and larger cohorts. Furthermore,
it is of immense importance to validate the putative targets
by functional biochemical and/or molecular assays to
understand specifically how the activity of the identified
kinases is modified via NRG1 intracellular signaling.
Whether the trend towards lower kinase activity in V/L vs.
V/V cells is indeed due to disrupted NRG1 intracellular
signaling, or due to inhibition of extracellular autocrine
signaling is an important question that remains to be
answered. However, this work is beyond the scope of the
present study, which was meant to be exploratory and
aimed to identify putative pathways regulated by NRG1
intracellular signaling. We encourage further studies
focusing on the pathways identified here. Another limita-
tion of the study is the use of lymphoblastoid cell lines. As
is the case with immortalized cells, these cell lines have
certain limitations such as differences in baseline growth
rates, EBV copy numbers and ATP levels (Choy et al.
2008). However, the procedure for immortalization was
carried out in an identical manner for both wild-type and
mutation carriers and we did not observe significant dif-
ferences in baseline growth rates or cell viability between
groups. Due to the difficulty of obtaining neuronal cells
from patients, LCLs have been used as a functional model
system to study various mental disorders such as schizo-
phrenia (Cheng et al. 2012) and autism (Granese et al.
2013). This reiterates their utility as an appropriate surro-
gate for brain tissue. A future line of study could involve
use of iPS-derived neuronal cells from Val/Val and Val/
Leu subjects to validate the findings obtained with LCLs.
In conclusion, we have performed an exploratory study
to identify novel cell signaling targets of the NRG1 ICD
using lymphoblastoid cell lines. While the small sample
sizes we used is a limitation, this study is novel in that we
Fig. 4 NRG1 signaling and neurite formation mediators. Highlighted
in grey are molecules within the NRG1–ERBB4 signaling pathway
found to be altered in expression (PRKCH and SRC) or phosphor-
ylation (NFkB1, REL, CREB1, ELK1, STMN2 and ADRB2), or
kinases directly phosphorylating these molecules (AKT, ERK,
MAPK). Other molecules not highlighted in grey are also part of
the pathway/network but not found to be altered in our study.
Triangles represent kinases; rectangles represent ligand-gated nuclear
receptor; concentric circles represent enzyme families; ellipse repre-
sents transcription regulators. Figure was generated using IPA
pathway analysis software. This software uses a right-tailed Fisher’s
exact test to calculate pvalues of identified pathways, based on the
number of significant molecules in the pathway and the fold change of
each molecule
Global signaling effects of a schizophrenia-associated missense mutation
123
have used two different global approaches, which led us to
the same pathways altered, suggesting convergent validity
for our findings. Genome-wide expression studies showed
alterations in expression of PRKCH and SRC (involved in
NRG1 signaling) in the presence of the V [L variant. A
novel kinomics approach identified altered phosphorylation
of neurite formation mediators, downstream of PKC and
Src kinases (Fig. 4). We have for the first time demon-
strated the use of kinomics in peripheral blood cells and
integrated two high throughput techniques to examine the
biological effects of the V [L mutation. These findings
shed light on the role of the NRG1 ICD in cell signaling
and ultimately its contribution to the development of
schizophrenia.
Acknowledgments This work was supported in part by
K01MH077777 and NARSAD: Brain and Behavior Research Foun-
dation grants awarded to CWB; UT system grant: translational sci-
ence training across disciplines awarded to KKM. The authors thank
Dr. Teresa Johnson-Pais, Dr. Carolina Livi, Yasmin Ench and Mandy
Rolando (Genomics core- University of Texas Health Science Center
at San Antonio, UTHSCSA) and Dr. Christopher Willey and Dr.
Joshua Anderson (Kinome core-University of Alabama, Birmingham)
for their services. We thank the families from the CVCR; this
research would not be possible without them. The authors declare no
conflict of interest.
Conflict of interest The authors declare they have no conflict of
interest.
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