ChengLong Huang

ChengLong Rao

Dong Wu

HaiYang Shi

Jian Zhou

JianYong Tang

Jin Chen

Ke Cheng

Li Liao

Peng Xie

YiRen Song

YongTao Yang

You Wu

PubMed

Springer - Publisher Connector

LETTER
Comparative proteomic analysis of plasma
from bipolar depression and depressive
disorder: identiﬁcation of proteins associated
with immune regulatory
Dear Editor,
Bipolar disorder (BD) is a debilitating psychiatric mood dis-
order affecting approximately 1%–3% of the population
worldwide (Merikangas, 2007). Bipolar disorder is character-
ized by recurrent episodes of depression, hypomania, mania,
or mixed states, and it has a poor outcome, with high rates of
relapse, lingering residual symptoms, cognitive impairment,
and functional impairment (Moreno et al., 2007) Although
various etiopathological hypotheses concerning the disease
have been reported, the pathophysiology underlying BD
remains poorly understood (Gawryluk and Young, 2011).
According to the DSM-5 or ICD-10 criteria, the diagnosis
of BD is still largely reliant on behavioral observations, and
given the similarities in the clinical presentations between
BD and unipolar major depressive disorder (MDD), espe-
cially bipolar II, approximately 40% of BD patients are initially
misdiagnosed with MDD (Forte et al., 2015), and this may
misguide the medication of BD patients. A better under-
standing of the pathophysiology underlying BD is thus
essential to improve the diagnosis rate and pharmacother-
apies for this disorder.
Proteomics, the quantitative analysis of protein expres-
sion in biosamples, is considered a powerful tool by which
novel molecules and biomarkers of psychiatric disease can
be identiﬁed (Roepstorff et al., 2012). Previous proteomic
analyses of BD used mainly postmortem brain tissue, pro-
viding essential insight into BD (Behan et al., 2009); how-
ever, such analyses are inevitably affected by postmortem
sample collection times. Antecedently, we performed com-
parative proteomics to distinguish protein expression differ-
ences between varying states of BD and healthy control
(HC) subjects, and between MDD and HC groups (Song
et al., 2015; Xu et al., 2012). To our knowledge, proteomic
expression across BD and MDD has not been studied
before; thus, there is an urgent need for this aspect to be
investigated. The available evidence seems to indicate that
brain imbalances can be reﬂected in the peripheral
circulation (Herberth et al., 2010); using peripheral blood is
therefore a practical means to achieving an improved
understanding of the pathophysiological mechanisms
underlying BD, with minimal collection risk and cost.
Here, a proteomic analysis based on two-dimensional
electrophoresis (2-DE) coupled with matrix-assisted
laser desorption/ionization-time-of-ﬂight/time-of-ﬂight tan-
dem mass spectrometry (MALDI-TOF/TOF MS) was carried
out on plasma samples from drug-naïve bipolar II and MDD
patients, In order to overcome the limitations of a narrow
linear range and large variations in 2-DE, 15 samples were
mixed within each study group, and for each group, 2-DE
was repeated in triplicate to limit run-to-run variations to
about 20%, the criterion of 2-fold change was used as a
cut off to ensure the reliability of apparent differences.
PDQuest analysis revealed 2-fold differences for 25 distinct
protein spots (P < 0.05). These 25 protein spots were
successfully identiﬁed by MALDI-TOF MS/MS analysis
(Fig. S1, Table S2). The expression levels of eight proteins
were found to be signiﬁcantly elevated and those of the
other 17 proteins were signiﬁcantly lower in bipolar II sub-
jects compared with MDD subjects.
The DAVID Bioinformatics Resource v6.7 (http://david.
abcc.ncifcrf.gov/home.js p) (Dennis et al., 2003) was used to
obtain gene ontology (GO) terms and to carry out enrichment
analysis to determine the most relevant GO and Kyoto
encyclopedia of genes and genomes (KEGG) terms asso-
ciated with the identiﬁed proteins. The cellular component
(Fig. 1A) attributed to the differentially expressed proteins
includes extracellular region, extracellular space, extracel-
lular region part, high-density lipoprotein particle, plasma
lipoprotein particle, and protein-lipid complex; and all the
enriched biological processes (Fig. 1B) of the differentially
expressed proteins were immune regulatory, including
defense response, acute inﬂammatory response, response
to wounding, and inﬂammatory response. All differently
expressed proteins were mapped onto the KEGG database
to determine the altered biological pathways associated with
© The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn
Protein Cell 2015, 6(12):908–911
DOI 10.1007/s13238-015-0218-5 Protein&Cell
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these plasma proteins. Notably, the complement and coag-
ulation cascade pathways were enriched (P < 0.01) and ﬁve
proteins were found to be involved in the pathway: C3, CFI,
C4BPα,kininogen-1, and antithrombin. Among these, C3,
CFI, and C4BPα were included in the pathway of comple-
ment cascades (P < 0.05).
Next, we choose the three signiﬁcant differentially
expressed complement proteins (C3, CFI, and C4BPα) for
ELISA validation by using individual samples from the bipolar
II (n = 20), MDD (n = 30), and HC groups (n = 30), plasma
samples were diluted 1:10,000 for C3 analysis, 1:2000 for
C4BPα analysis, and 1:1000 for CFI analysis. The quantita-
tive results trends in expression patters were consistent with
the MALDI -TOF/TOF MS ﬁndings, although the changes
were smaller than those determined by 2-DE, the differences
were statistically signiﬁcant, and the expression levels of
these three proteins being signiﬁcantly dysregulated with the
following proﬁles: C3, MDD > bipolar II > HC subjects; CFI
and C4BPα, HC > MDD > bipolar II subjects (Fig. 2A–C).
The ﬁndings reported here provide evidence demon-
strating that autoimmune dysregulation is involved in the
pathophysiological mechanisms either bipolar II or MDD.
Consistent with our ﬁndings, different lines of research have
conﬁrmed dysfunction in inﬂammation being closely related
to the pathophysiology of BD and MDD (Leboyer et al.,
2012). Our previous comparative proteomic analysis also
showed immune dysregulation plays a role in the patho-
physiology of BD and MDD (Song et al., 2015; Xu et al.,
2012). C3 was considered as a signiﬁcant feature of com-
plement pathway activation because of its key role in all
A
B
5%
5%
5%
24%
24%
37%
19%
23%
16%
16%
26%
Extracellular region
Extracellular region part
High-density lipoprotein particle
Plasma lipoprotein particle
Protein-lipid complex
Extracellular space
Defense response
Acute inflammatory response
Innate immune response
Response to wounding
Inflammatory response
Figure 1. Gene ontology. The most signiﬁcantly enriched (A) cellular component and (B) biological process according to DAVID
Bioinformatics Resources based on the 25 differentially expressed proteins identiﬁed in bipolar II subjects relative to MDD subjects.
Immune dysregulation in bipolar II depression LETTER
© The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn 909
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complement activation pathways (Jones et al., 2013). C4BP
and CFI are important regulators of the complement system,
which controls the activation of the complement cascades
(Nilsson et al., 2011). The imbalance among them may bring
autoimmune dysregulation.
Furthermore, as C3, C4BPα, and CFI display unique tiered
expression proﬁles, the form and extent of the immune dys-
regulation underlying the two mental diseases may differ. The
C3, CFI, and C4BPα proteins may represent a plasma-based
diagnostic biomarker panel, which could aid BD diagnosis or
be used tomonitor BD progression. Current research appears
to support the view that plasma levels of C3 are elevated in
MDDpatients, while changes inC3 in BD is closely associated
with the state of disease (Santos Sória et al., 2012), and there
is also mounting evidence of the C4BPα and CFI levels being
disturbed in several psychiatric disorders, elevated peripheral
C4BP levels have been reported in Alzheimer’s disease
(Trouw et al., 2008), and the mean level of CFI activity in
autistic subjects suggesting that highCFI activitymay have an
impact on the development of autism (Momeni et al., 2012).
These ﬁndings seem to suggest that the autoimmune system
may provide promising biomarkers for psychiatry. Further
investigation into these proteins and the processes they are
involved in is critical to gaining a better understanding of the
underlying pathophysiology of BD.
FOOTNOTES
We are grateful to the patients and control subjects that voluntarily
participated in the study. Thanks to Professors Huaqing Meng and
Delan Yang (Department of Psychiatry, First Afﬁliated Hospital,
Chongqing Medical University) for their assistance in subject
recruitment. This study was supported by the National Basic
Research Program (973 Program) (Nos. 2009CB918300 and
2012CB910602).
Jin Chen, ChengLong Huang, YiRen Song, HaiYang Shi, Dong
Wu, YongTao Yang, ChengLong Rao, Li Liao, You Wu, JianYong
Tang, Ke Cheng, Jian Zhou, and Peng Xie declare that they have no
conﬂict of interest.
All procedures followed were in accordance with the ethical
standards of the responsible committee on human experimentation
(institutional and national) and with the Helsinki Declaration of 1975,
as revised in 2000 (5). Informed consent was obtained from all
patients for being included in the study.
Jin Chen1,2,3, ChengLong Huang2,3,5, YiRen Song1,2,3,
HaiYang Shi1,2,3, Dong Wu2,3,5, YongTao Yang2,3,4,
ChengLong Rao2,3,7, Li Liao2,3,5, You Wu2,3,6,
JianYong Tang2,3,5, Ke Cheng1,2,3, Jian Zhou2,3,
Peng Xie1,2,3&
1 Department of Neurology, The First Afﬁliated Hospital of
Chongqing Medical University, Chongqing 400016, China
2 Chongqing Key Laboratory of Neurobiology, Chongqing 400016,
China
3 Institute of Neuroscience and the Collaborative Innovation Center
for Brain Science, Chongqing Medical University, Chongqing
400016, China
4 Department of Neurology, The Fifth People’s Hospital of
Chongqing, Chongqing 400016, China
5 Key Laboratory of Laboratory Medical Diagnostics of Education
Ministry, Department of Laboratory Medicine, Chongqing Medical
University, Chongqing 400016, China
6 Department of Neurology, Yongchuan Hospital of Chongqing
Medical University, Chongqing 400016, China
A B C140
139
138
137
136
135
402
400
398
396
394
392
50
45
40
35
30
25
C
3 
le
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m
g/
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)
C
4B
P
α 
le
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μg
/m
L)
C
FI
 le
ve
l (
μg
/m
L)
HC Bipolar II MDD HC Bipolar II MDD HC Bipolar II MDD
***
**
**
***
***
**
***
***
*
Figure 2. Complement cascade-associated proteins analyzed by ELISA. Signiﬁcant differences between the bipolar II (n = 20),
MDD (n = 30), and HC groups (n = 30) were observed. The expression levels of plasma C3 (A) in both the disease groups are
signiﬁcantly higher than the level in the HC group, and C3 expression was furthermore signiﬁcantly upregulated in the MDD group
compared with the bipolar II group. In contrast to C3, C4BPα (B) and CFI (C) expression levels were signiﬁcantly downregulated in
both disease groups compared to the HC group, the expression levels of these two proteins were signiﬁcantly lower in bipolar II
subjects compared to MDD subjects. *P < 0.05, **P < 0.001, and ***P = 0.000. Yellow lines highlight the differences between bipolar II
and MDD subjects.
LETTER Jin Chen et al.
910 © The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn
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7 Department of Cell Biology, Chongqing Key Laboratory of Neuro-
biology, Chongqing Medical University, Chongqing 400016, China
& Correspondence: xiepeng@cqmu.edu.cn (P. Xie)
OPEN ACCESS
This article is distributed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to
the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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Jin Chen, ChengLong Huang, YiRen Song, and HaiYang Shi have
contributed equally to this study.
Electronic supplementary material The online version of this
article (doi:10.1007/s13238-015-0218-5) contains supplementary
material, which is available to authorized users.
Immune dysregulation in bipolar II depression LETTER
© The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn 911
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Comparative proteomic analysis of plasma from bipolar depression and depressive disorder: identification of proteins associated with immune regulatory

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Comparative proteomic analysis of plasma from bipolar depression and depressive disorder: identification of proteins associated with immune regulatory

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