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Neuroscience 230 (2013) 1–12
HIPPOCAMPAL SYNAPTIC DYSREGULATION OF EXO/ENDOCYTOSIS-ASSOCIATED PROTEINS INDUCED IN A CHRONICMILD-STRESSED RAT MODEL
Y. HU, a,b,c� J. ZHOU, b� L. FANG, b� H. LIU, a,b Q. ZHAN, d
D. LUO, a,b C. ZHOU, a,b J. CHEN, b Q. LI a AND P. XIE a,b*
aDepartment of Neurology, The First Affiliated Hospital,
Chongqing Medical University, PR China
bChongqing Key Laboratory of Neurobiology, The Institute
of Neuroscience, Chongqing Medical University, PR ChinacDepartment of Neurology, The Third People’s Hospital of Chengdu,
Sichuan Province, PR China
dDepartment of Neurology, The Fifth People’s Hospital of
Chongqing, PR China
Abstract—Although major depressive disorder (MDD) is a
serious neuropsychiatric illness, it’s pathogenesis remains
unclear. Current evidence suggests that the abnormal trans-
mission and plasticity of hippocampal synapses play an
important role in the pathogenesis of MDD. In this study, a
two-dimensional gel-based proteomic approach to profile
alterations of synaptosome protein expression was applied
in the hippocampus of rats subjected to chronic mild
stress. Through mass spectrometry and database search-
ing, 19 differentially expressed proteins were identified, of
which 5 were up-regulated and 14 were down-regulated in
the chronic mild-stressed group as compared with the con-
trol group. Subsequently, several proteins of interest were
further validated by Western blotting. A detailed analysis
of protein functions and disease relevance revealed that
synaptic exo/endocytosis-associated proteins were dysreg-
ulated in the chronic mild-stressed rats. The present study
is the first reported synaptoproteomic analysis of the
chronic mild-stressed rat hippocampus. The synaptic exo/
endocytosis-associated proteins may participate in a central
0306-4522/12 $36.00 � 2012 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2012.08.026
*Correspondence to: P. Xie, Department of Neurology, The FirstAffiliated Hospital, Chongqing Medical University, 1# Yixue Road,Yuzhong District, 400016 Chongqing, PR China. Tel: +86-23-68485490; fax: +86-23-68485111.
E-mail address: [email protected] (P. Xie).� Y. Hu, J. Zhou and L. Fang contributed equally to this work.
Abbreviations: 2DE, two-dimensional gel electrophoresis; AMPARs,a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors;CHAPS, 3-[(3-cholamidopropyl)dim-ethylammonio]-1-propanesulfonate;CMS, chronic mild stress; GluR2, glutamate receptor subunit 2; HG, handgrinding; IPI, InternationalProtein Index; LNG, liquid nitrogengrinding; LTD,long-term depression; LTP, long-term potentiation; MALDI-TOF/TOF,Matrix-Assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight;MANOVAs, multivariate analysis of variance; MDD, major depressivedisorder; MG,mechanical grinding; NA, noradrenaline; OD, optical density;SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis;SNARE, soluble Nsf attachment protein receptor; SP, sucrose preference;SV, synaptic vesicle; TBST, Tris–buffer saline with 0.05%Tween-20; TEM,transmission electron microscope; V-ATPase, vacuolar-type H+-ATPase.
1
mechanism that underlies the abnormal transmission and
plasticity of hippocampal synapses found in the chronic
mild-stressed rats, and provides guidance to advance our
understanding of the pathogenesis of MDD.
� 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: major depressive disorder, chronic mild stress,
hippocampus, proteomics, synaptosome, two-dimensional
gel electrophoresis.
INTRODUCTION
Major depressive disorder (MDD) is one of the most
widespread and serious mental illnesses, affecting more
than 120 million people annually worldwide and
contributing to increased rates of disability and suicide
(WHO, 2001). Despite its severity, the pathogenesis of
this affective disorder is poorly understood and remains
a challenging scientific problem.
Mounting evidence suggests that the hippocampus, a
cerebral cortical structure associated with behavioral
inhibition, memory, and spatial coding, plays an
important role in MDD pathogenesis. The hippocampus
is anatomically connected to several structures in the
limbic system which regulates emotional behavior,
namely the septum, hypothalamic mammillary body,
and thalamic anterior nuclear complex (Campbell and
Macqueen, 2004). Ineffective synaptic transmission and
disordered synaptic plasticity are widely considered to
be the two key underlying factors in the hippocampal
dysfunction found in MDD. Clinical studies on MDD
patients have reported lower GABA, 5-HT and
noradrenaline (NA) levels in the plasma, urine and
cerebrospinal fluid (CSF), which supports the deficiency
in synaptic transmission found in the brain (Brambilla
et al., 2003; Belmaker and Agam, 2008). These
abnormal synaptic transmissions underly the behavioral
and cognitive dysfunction in MDD (Sarter et al., 2007).
On the other hand, a fundamental property of the
synapse is its ability to self-modify the efficacy of its
own transmission, which is referred to as synaptic
plasticity. Concretely, synaptic transmission was
enhanced by synaptic long-term potentiation (LTP) and
weakened by synaptic long-term depression (LTD)
(Linden, 1999). Recent preclinical and clinical
investigations have indicated that stress and aversive
experiences can decrease the amount of LTP and
enhance LTD in the hippocampus (Popoli et al., 2002;
d.
2 Y. Hu et al. / Neuroscience 230 (2013) 1–12
Holderbach et al., 2007). Moreover, alterations of
synaptic plasticity may impair function of brain circuits
involved in the pathophysiology of MDD (Holderbach
et al., 2007).
From the foregoing studies, it can be concluded that the
hippocampal synapses play an important role in the
molecular mechanism underlying MDD. Therefore,
profiling the protein expression patterns of the
hippocampal synapses should help to decipher the
pathways and chemical species involved in MDD
pathogenesis. In previous proteomic studies on depre-
ssed animal models, analysis has mainly focused on
protein expression in the whole hippocampus. Thus, due
to the complexity of the samples, little information specific
to synaptic proteins was discovered (Carboni et al., 2006;
Mu et al., 2007; Marais et al., 2009; Kedracka-Krok et al.,
2010).
Fortunately, synaptoproteomic technology provides a
powerful tool to reduce this proteome complexity. More
significant information can be revealed by profiling the
differential expression of synaptic proteins under specific
pathophysiologic conditions. This technology has been
widely employed in mental disorder research (Moron
et al., 2007; Zhou et al., 2010). However, to date, few
studies focusing on the synaptic proteins in animal
models of depression have been conducted (Mallei
et al., 2008, 2011).
In the presentwork, differential analysis of hippocampal
synaptic proteins using the synaptoproteomic approach
was applied in rats subjected to chronic mild stress
(CMS), a well-validated model of MDD. After
hippocampal synapses were purified down to so-called
‘‘synaptosomes’’ by differential and density-gradient
centrifugation, the constituent proteins were extracted
and separated by two-dimensional gel electrophoresis
(2DE). Simultaneously, the differential spots obtained by
PDQuest software were subjected to in-gel digestion and
identified by Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight/Time-of-Flight (MALDI-TOF/TOF) mass
spectrometry. This study should provide a valuable
resource for deciphering the molecular mechanism
underlying the abnormal synaptic transmission and
plasticity in MDD.
EXPERIMENTAL PROCEDURES
Comparison of three classic grindings inhippocampal synaptosome preparation
After 30 healthy adult male Sprague–Dawley rats (Chongqing
Medical University, Chongqing, China) were sacrificed by
decapitation, hippocampal tissue was excised. The fresh tissue
samples were collected and divided into three groups of similar
weight. For comparison, each group was ground using a different
method: liquid nitrogen grinding (LNG), hand grinding (HG) and
mechanical grinding (MG). In the LNG method, tissue was
ground in a mortar and pestle under liquid nitrogen, and the
resulting powder was suspended in solution A (0.32 M sucrose,
1 mM NaHCO3, 1 mM MgCl2, and 0.5 mM CaCl2) containing a
protease inhibitor cocktail (Sigma–Aldrich, St. Louis, Missouri,
USA). In the HG and MG methods, the tissue was homogenized
on ice in solution A using a hand-operated Teflon-glass grinder
or motor-operated disperser (T10 Basic, IKA, Germany),
respectively. Then, the synaptosome fraction was isolated as
previously described (Carlin et al., 1980). Briefly, the
homogenates were centrifuged at 1400g for 10 min, and the
resultant pellet was then homogenized again. The second
centrifugation was performed at 710g for 10 min. The
supernatants were pooled and centrifuged at 12,000g for 30 min.
The resulting pellet was resuspended in solution B (0.32 M
sucrose and 1 mM NaHCO3) then layered over a 1.2 M/1.0 M/
0.85 M sucrose gradient (10 ml each). After ultracentrifugation
(himac cp 80 wx, Hitachi Koki, Japan) at 82,500g for 2 h, the
synaptosome fraction from the 1.2 M/1.0 M interface was
collected and washed with solution B. All centrifugation was
carried out at 4 �C. Aliquots of the prepared fractions were
dissolved and evaluated by Western blotting using anti-syntaxin
antibody, and fixed with 2.5% glutaraldehyde for transmission
electron microscope (TEM) analysis.
CMS model establishment and synaptosomepreparation
Fifty-eight healthy adult male Sprague–Dawley rats (weighing
160–200 g), purchased from the animal facility of the
Chongqing Medical University (Chongqing, China), were singly-
housed, given access to food and water ad libitum, and
maintained on a 12-h light/datk cycle at a temperature of
22 ± 2 �C unless otherwise noted. The rats were allowed
1 week to acclimate to the environment and then given to both
1% sucrose solution and water for 3 weeks for the sucrose
habituation. At the same time, the sucrose preference (SP) test
was carried out according to the procedure reported by Grippo
et al. with slight modifications (Grippo et al., 2002). Briefly, it
was performed twice weekly in a 1-h test after food and water
deprivation for a 20-h period, which was believed to be the
baseline of SP test. The sucrose and water intakes (g) were
recorded, and the SP was calculated according to the following
formula: SP = sucrose intake (g)/(sucrose intake (g) + water
intake (g)). After 3 weeks, the average sucrose intake of all rats
was stable. Based on the method of randomized block design,
the rats were then divided randomly into CMS and control
groups (n= 29 per group) according to the sucrose intakes of
each rat in the final SP test. Subsequently, the CMS
experiment was performed according to the protocol described
by Lewitus et al. with minor modifications (Lewitus et al., 2009).
In brief, rats were subjected to a 45� cage tilt, stroboscopic
lighting, intermittent white noise (80 dB), soiling of cages, food
and water deprivation followed by restricted food (0.5 g of food
pellets), paired housing and overnight lighting. This series of
stressors was applied for a 1-week period and repeated for
4 weeks (see Table 1). In the meantime, the SP test was
carried out once weekly during the CMS period.
Following these experiments, the rats were sacrificed by
decapitation, and the hippocampi were removed. For differential
analysis of the hippocampal synaptosomal proteins, the fresh
tissue was homogenized using the motor-operated disperser,
and the synaptosome fractions were isolated according to the
aforementioned procedure. The following proteome analysis
was conducted on three sample pools corresponding to the two
groups, and each pool originated from nine or ten rats. The
experiments were performed in accordance with all regional
guidelines and regulations.
Protein sample preparation
The synaposome fractions were suspended in a lysis buffer
containing 7 M urea (Bio-Rad, Hercules, CA, USA), 2 M thiourea
(Bio-Rad), 4% 3-[(3-cholamidopropyl)dim-ethylamm- onio]-
1-propanesulfonate (CHAPS) (Bio-Rad), and 50 mM DTT (Bio-
Rad). After 1 h of incubation on ice, the sample was centrifuged
at 40,000g for 30 min at 4 �C to remove insoluble materials. The
protein concentration was estimated using a Bradford protein
Table 1. CMS schedule
Sunday Lights off overnight Untilt cages 9:00
Dry cages
Strobe light on 16:00
Room light off
Start food and water deprivation
Monday Lights off overnight Strobe light off 9:30
Food ad libitum 11:00
Paired housing 11:20
White noise on 14:20
White noise off
Tuesday Lights on overnight Re-house singly 11:00
Tilt cages 17:00
Untilt cages
Remove food
Wednesday Lights off overnight Strobe light on 10:30
Strobe light off 16:30
Offer restricted food
Add food ad libitum 17:30
Paired housing
Thursday Lights on overnight White noise on 10:00
White noise off 17:00
Re-house singly
Friday Lights off overnight Weigh 13:00
Start food and water deprivation
Saturday Lights off overnight Restore food and water 9:00
Sucrose preference
Tilt cages 10:30
Wet cages
Y. Hu et al. / Neuroscience 230 (2013) 1–12 3
assay kit (Bio-Rad, Hercules, CA, USA) with BSA as a standard.
Before electrophoresis, the protein sample was processed with a
ReadyPrep 2D cleanup kit (Bio-Rad, Hercules, CA, USA)
according to the manufacturer’s instructions. The resultant pellet
was redissolved in 7 M urea, 2 M thiourea and 4% CHAPS, and
the protein concentration was re-determined.
2-D electrophoresis and gel image analysis
Samples containing 120 lg of total protein were mixed with a
rehydration solution containing 7 M urea, 2 M thiourea, 4%
CHAPS, 50 mM DTT, 0.2% Bio-Lyte, and 0.001% bromophenol
blue (Bio-Rad) to a total volume of 350 ll, and used for the
rehydration of 17-cm IPG strips (pH 3–10 NL, Bio-Rad). A
multi-step IEF voltage program was applied to the strips on a
Protean IEF cell (Bio-Rad): 50 V for 12 h, 250 V for 30 min,
1000 V for 1 h, from 1000 V to 10,000 V over a 5-h step-up
period, and 10,000 V for 6 h. Strips were equilibrated in the
reduction buffer (0.375 M Tris–HCl pH 8.8, 6 M urea, 20%
glycerol, 2% sodium dodecyl sulfate (SDS) and 2% DTT), then
in the same buffer but containing 2.5% IAA instead of 2% DTT.
The second dimension was accomplished by running the strips
on 1 mm-thick 10% SDS–polyacylamide vertical slab gels using
a Protean� G xi Multi-Cell (Bio-Rad). The protein condensation
and separation were performed at 12.5 mA/gel for 30 min and
25 mA/gel for 5.0–5.5 h at 20 �C. Protein spots were visualized
by silver staining according to Yan (Yan et al., 2000). In our
experiment, the three samples run in duplicate for technical
replicates.
After visualization, images were obtained using an Epson
10000XL scanner (Epson Co., Ltd. Beijing, China) at an optical
resolution of 600 dpi. Image analysis and spot detection were
accomplished with PDQuest software version 8.0.1 (Bio-Rad
Laboratories, Hercules, CA, USA) using Gaussian spot
modeling. For quantitative comparisons of spots across gels,
six analytical gels (one gel per sample and three gels per
group) were analyzed. To correct for the variability in silver
staining, the individual spot volumes were normalized by
dividing each spot’s optical density (OD) value by the sum total
OD of all the spots in the respective gel. Automated and
manual spot matching were also performed. Spots showing at
least a 1.5-fold change in their integrated intensities in the
replicate gels and with a Student’s t-test p-value of <0.05 were
considered to be statistical differences in protein expression
between the two groups (Kedracka-Krok et al., 2010).
Protein identification by MALDI-TOF/TOF
In-gel protein digestion was performed according to Zhou et al. with
minor modifications (Zhou et al., 2010). The protein spots of interest
were excised from the gels and then destained. After reduction and
alkylation, the gel slices were digested overnight with Sequencing
Grade Modified Trypsin (Promega, Madison, WI, USA). The digested
peptides were extracted with 100 ll 60% CAN (Merck, Darmstadt,
Germany) containing 0.1% TFA (Merck) and concentrated in a Speed
Vac. The peptides were redissolved using a matrix solution and
spotted on a MALDI target plate. Peptides were analyzed using the
4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Foster
City, USA) in the default mode. The data search was conducted on
GPS Explorer (Version 3.6, AB SCIEX) using the search engine
Mascot (Version 2.2, Matrix Science, London, UK), and the
International Protein Index (IPI) rat database (vision 3.64, 39871
sequences, http://www.ebi.ac.uk/IPI) was used for peptide and protein
identification. Search parameters were set as follows: enzyme=
trypsin; allowance=up to one missed cleavage; peptide mass
tolerance= 100 ppm, fragment mass tolerance= 0.4 Da, fixed
Fig. 1. Evaluation of the three classic grindings methods. (A) Electron micrographic examination and (B) Western blotting analysis of synaptosome
fractions prepared by the three classic grinding methods. LNG, liquid nitrogen grinding; HG, hand grinding; MG, mechanical grinding; Tot, total
homogenates; Syn, purified synaptosomes.
4 Y. Hu et al. / Neuroscience 230 (2013) 1–12
modification= carbamidomethylation (Cys), and variable modificat-
ion= oxidation (at Met). General protein identification was based on
two or more peptides whose ion scores surpassed the statistical
threshold (p<0.05). The identified proteins were then matched to
specific processes or functions by searching the Gene Ontology
database (http://www.geneontology.org/).
Western blotting
Western blotting was introduced to evaluate synaptosomal
isolation by the three grinding methods and to confirm the
differential expression of two proteins of interest, clathrin light
chain A and B, by their respective antibodies. Ten micrograms
of protein from the synaptosome fractions prepared were
separated by 10% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) and then transferred onto a
polyvinyldifluoridine membrane. The membrane was blocked
with 5% (w/v) skimmed milk solution for 1 h, and then
incubated overnight with a primary antibody, mouse anti-
syntaxin monoclonal antibody (Stressgen Bioreagents, Victoria,
BC, Canada, dilution 1:1000), rabbit anti-clathrin light chain A/B
(Clta/Cltb) polyclonal antibodies (Santa Cruz Biotechnology,
CA, USA, dilution 1:200), rabbit anti-tubulin b polyclonal
antibody (Bioworld Technology, Louis Park, MN, USA, dilution
1:1000, as protein loading control) in the skimmed milk solution
at 4 �C. After the membrane was washed with Tris–buffer
saline with 0.05% Tween-20 (TBST) (150 mM NaCl, 0.05%
Tween-20, 10 mM Tris–HCl, pH 7.5), anti-mouse or anti-rabbit
IgG horseradish peroxidase-conjugated secondary antibody
(dilution 1:5000) was added to the skimmed milk solution, and
the membrane was incubated for 1 h at 37 �C. The membrane
was washed with TBST, and the blot was developed with ECL
reagents. The chemiluminescence signal was imaged using a
ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). The data were
analyzed with Bio-Rad Quantity One software (Bio-Rad).
Statistical analysis
Using SPSS software, data from the SP test were analyzed by
repeated measurement ANOVAs on an experimental group
(CMS, control) and time point (baseline, week 1, 2, 3, 4) basis.
To detect significant differences between the experimental
groups and time points, multivariate analysis of variance
(MANOVAs) and Bonferroni post hoc tests were used. The
data from Western blotting of Clta and Cltb expression were
compared using Student’s t-tests. A p-value of <0.05 was
considered to be statistically significant. Statistics were
presented as means ± SE.
RESULTS
Evaluation of three classic grindings in hippocampalsynaptosome preparation
Synaptosomal fractions were prepared in parallel by three
methods (LNG, HG and MG), characterized by TEM, and
differentiated by Western blotting (Fig. 1). Comparative
TEM examination demonstrated that the HG and MG
methods resulted in more representative structures than
the LNG method (Fig. 1A). Western blotting indicated
that synaptosomal purity levels in the HG and MG
methods were not significantly different, but both were
approximately 1.7 times higher than that found in the
LNG method (p< 0.05). The results demonstrated that
the HG and MG methods had a similar effect, whereas
both were superior to the LNG method.
CMS model of depression
The CMS rat model employed in this study is one of the
most widely-used animal models for inducing depression-
like behavior. Repeated measurement ANOVAs
displayed no significant differences in water intake.
With respect to sucrose intake, the impact of the
experimental group was considered to be significant
(F(1,56) = 21.89, p< 0.0001), as well as the time point
(F(4,224) = 5.165, p< 0.005). Bonferroni post hoc tests
indicated that following 4 weeks of CMS, the CMS group
consumed significantly less sucrose than its respective
baseline value (p< 0.005). MANOVAs indicated that for
both the following 3- and 4-week time periods of CMS,
the CMS group consumed less sucrose than the control
Fig. 2. Analysis of the fluid intake and SP test. (A) Curve distribution
of mean values (±SEM) of one hour fluid intake and (B) histogram of
mean values (±SEM) of the SP test. ⁄p< 0.005 vs. respective
baseline values (Bonferroni test, n= 29); #p< 0.005 vs. respective
control group values (MANOVAs test, n= 29).
Y. Hu et al. / Neuroscience 230 (2013) 1–12 5
group (F(1,56) = 9.099, p< 0.005, and F(1,56) =
32.105, p< 0.0001, respectively; Fig. 2A).
For the SP test, the impact of the experimental group
was significant (F(1,56) = 5.922, p< 0.05), as well as
that of the time point (F(4,224) = 6.382, p< 0.0001).
Subsequently, Bonferroni post hoc tests indicated that
following 4 weeks of CMS, the CMS group consumed
significantly less sucrose than its respective baseline
value (p< 0.0005). MANOVAs analysis indicated that
following 4 weeks of CMS, the CMS group consumed
less sucrose than control (F(1,56) = 31.166,
p< 0.0001; Fig. 2B). These results indicate that the
CMS significantly reduced sucrose intake and
preference; therefore, the classic CMS rat model of
depression was established.
Fig. 3. Representative 2DE. One hundred and twenty micrograms of
protein from rat hippocampal synaptosomes separated on a 17-cm
immobilized pH gradient strip (pH 3–10, nonlinear) as the first
dimension and a 10% SDS–PAGE as the second dimension.
Nineteen differentially expressed proteins were successfully
identified.
Differential analysis of synaptosomes
To explore the molecular mechanism underlying the
dysfunctional synaptic transmission and plasticity in
MDD, 2DE-based proteomics was utilized to profile
differentially expressed proteins in the CMS and control
groups. As shown in Fig. 3, approximately 1580 protein
spots were detected by silver staining in a single 2DE
map. By differential analysis with PDQuest software, 22
spots were differentially expressed, of which 5 were
upregulated while 17 were downregulated in the CMS
group as compared with the control group.
These 22 spots were then validated byMALDI-TOF/TOF
analysis. The MS and MS/MS data were queried using the
search algorithm GPS 3.6 (mascot 2.2) against the IPI rat
database. Proteins were identified based on a number of
criteria including the MW, pI and MASCOT score (Table 2).
Nineteen differentially expressed proteins were successfully
validated, of which 5 were upregulated and 14 were
downregulated in the CMS group as compared to the
control group. In these identified proteins, Atp6v1e1, Syn2,
Hsp90b1, Hsph1, Dnaja1, Pdia3, Gsn, Actn4, Ina, Pgk1,
Aldoc and Acp6 were all downregulated in the CMS group.
Furthermore, Nsf, Clta, Cltb and Tpi1 were upregulated in
the CMS group. Here it should be noted that of the Dnm1
identified in our study, isoform 2 and 3 of dynamin-1 were
downregulated while isoform 5 of dynamin-1 was
upregulated in the CMS group.
A comprehensive search was undertaken to determine
the subsynaptic location of these 19 validated proteins.
There was a high degree of overlap in localization, as 11
proteins localized to the presynaptic terminal and 17
proteins localized to the postsynaptic terminal.
Interestingly, the proteins involved in synaptic exo/
endocytosis localized to both the presynaptic and
postsynaptic terminals. Through a literature search, many
of these proteins have been reported in different disease
states that can affect mood states such as schizophrenia
and Alzheimer’s disease. As shown in Table 3. Fifteen
proteins have been implicated in these two mental
disorders (4 in schizophrenia, 11 in Alzheimer’s disease)
and 7 in other mental illnesses (e.g., Down’s syndrome,
Huntington’s disease). Furthermore, 4 proteins have
been found to be differentially expressed in depressed
human patients, and 10 proteins in animal models of
depression. The molecular functions of these proteins
Table 2. Hippocampal synaptic proteins perturbed by chronic mild stress (CMS)
ID Accession
No. IPI
Accession
No. Swiss-
Prot
Gene
name
Protein name Mascot
score
Protein
score
CI%
Theoretical
molecular
weight (Da)/PI
Protein
abundance ratio
(CMS/CON)
Presynaptic proteins Postsynaptic proteins
Exocytosis-associated
1 IPI00400615 Q6PCU2 Atp6v1e1 V-type proton
ATPase subunit
E1
61 96.792 26168.9/8.44 0.47 Phillips et al. (2005), Takamori et al.
(2006), Abul-Husn et al. (2009) and
Morciano et al. (2009)
Phillips et al. (2005) and
Collins et al. (2006)
2 IPI00210036 Q63537 Syn2 Isoform IIa of
synapsin-2
129 100 63701.9/8.73 0.43 Phillips et al. (2005), Takamori et al.
(2006), Abul-Husn et al. (2009) and
Morciano et al. (2009)
Phillips et al. (2005), Collins et
al. (2006) and Fernandez et al.
(2009)
3 IPI00210635 Q9QUL6 Nsf Vesicle-fusing
ATPase
138 100 83170.2/6.55 2.41 Takamori et al. (2006) and Abul-Husn et
al. (2009)
Collins et al. (2006) and
Fernandez et al. (2009)
Endocytosis-associated
4 IPI00207305 P08081 Clta Isoform brain of
clathrin light chain
A
111 100 27078/4.41 2.98 Phillips et al. (2005) and Abul-Husn et al.
(2009)
Collins et al. (2006)
5 IPI00207308 P08082 Cltb Isoform brain of
clathrin light chain
B
85 99.987 25216.2/4.56 2.51 Phillips et al. (2005) Collins et al. (2006)
6 IPI00896174 P21575 Dnm1 Isoform 3 of
dynamin-1
256 100 92773.4/6.12 0.31 Phillips et al. (2005), Takamori et al.
(2006), Abul-Husn et al. (2009) and
Morciano et al. (2009)
Collins et al. (2006)
IPI00896174 88 99.994 0.45
IPI00896174 393 100 0.48
7 IPI00782657 P21575 Dnm1 Isoform 2 of
dynamin-1
247 100 96209.4/6.32 0.57 Phillips et al. (2005), Takamori et al.
(2006), Abul-Husn et al. (2009) and
Morciano et al. (2009)
Collins et al. (2006)
8 IPI00558839 P21575 Dnm1 Isoform 5 of
dynamin-1
124 100 97762.1/6.73 1.81 Phillips et al. (2005), Takamori et al.
(2006), Abul-Husn et al. (2009) and
Morciano et al. (2009)
Collins et al. (2006)
Molecular chaperones
9 IPI00365985 Q66HD0 Hsp90b1 Isoform 1 of
endoplasmin
338 100 92998.5/4.72 0.51 Collins et al. (2006)
10 IPI00471835 Q66HA8 Hsph1 Heat shock protein
105 kDa
279 100 97326.7/5.4 0.6 Collins et al. (2006)
11 IPI00210884 P63037 Dnaja1 dnaJ homolog
subfamily A
member 1
109 100 45580.7/6.65 0.64 Collins et al. (2006)
12 gi|1352384 P11598 Pdia3 Protein disulfide-
isomerase A3
62 97.411 57043.9/5.88 0.35
Cytoskeleton-associated
13 IPI00923716 Q68FP1 Gsn Isoform 2 of
Gelsolin
94 99.998 81163.5/5.46 0.63 Collins et al. (2006)
14 IPI00213463 Q9QXQ0 Actn4 Alpha-actinin-4 120 100 105305.6/5.27 0.36 Collins et al. (2006)
84 99.986 0.29
15 IPI00211936 P23565 Ina Alpha-internexin 93 99.998 56394.7/5.17 0.45 Takamori et al. (2006) and Morciano et al.
(2009)
Phillips et al. (2005) and
Collins et al. (2006)
6Y.Huetal./N
euroscience230(2013)1–12
Energymetabolism-associated
16
IPI00231767
P48500
Tpi1
Triosephosphate
isomerase
470
100
27344.9/6.89
1.52
Morcianoetal.(2009)
Collinsetal.(2006)
17
IPI00231426
P16617
Pgk1
Phosphoglycerate
kinase1
102
100
44909.1/8.02
0.6
Morcianoetal.(2009)
Fernandezetal.(2009)
18
IPI00231736
P09117
Aldoc
Fructose-
bisphosphate
aldolaseC
60
96.4
39658.3/6.67
0.56
Phillipsetal.(2005),Collinset
al.(2006)andFernandezetal.
(2009)
19
IPI00372429
Q4FZU0
Acp6
Acid
phosphatase6,
lysophosphatidic
92
99.998
47592.5/7.64
0.58
Y. Hu et al. / Neuroscience 230 (2013) 1–12 7
were classified into five groups according to the GO
database and surveys of relevant literature, namely
(i) synaptic exocytosis, (ii) synaptic endocytosis,
(iii) molecular chaperoning, (iv) cytoskeleton, and (v)
energy metabolism (Table 2).
The expression levels of Clta and Cltb were further
validated by Western blotting as shown in Fig. 4. The
expression levels of Clta and Cltb were significantly
increased in the CMS group (fold change = 1.58,
p< 0.005; and fold change = 2.01, p< 0.005,
respectively). These results were consistent with the
2-D electrophoretic findings.
DISCUSSION
The classical monoamine-deficiency hypothesis of MDD
initially derived from the therapeutic action of
antidepressants. In general, antidepressants have two
pharmacologic actions. One, produced by TCA’s, is to
increase the synaptic levels of 5-HT and NA by blocking
their reuptake. Another, produced by MAOI’s, is to raise
intracellular levels of the two neurotransmitters in
presynaptic neurons through inhibition of the enzyme
monoamine oxidase (Belmaker and Agam, 2008).
Moreover, an increased synthesis of monoamine
neurotransmitters was previously reported in presynaptic
neurons of a chronic-stressed animal model (Anisman
et al., 2008). Holm et al. also discovered a reduction in
GABA release in a depressed rat model (Holm et al.,
2011). Together, these studies show perturbations across
the release of multiple neurotransmitters.
Neurotransmitter release is primarily regulated by
presynaptic exo/endocytosis (Sudhof, 2004). Recently,
LTD has been shown to be facilitated in the
hippocampus of a CMS rat model (Holderbach et al.,
2007). It is well-established that the facilitation of LTD
mainly relies on postsynaptic endocytosis (Wang and
Linden, 2000). Taken together, abnormalities in synaptic
exo/endocytosis may play a substantive role in MDD
pathogenesis.
Therefore, a well-established procedure was used
to purify hippocampal synaptosomes to investigate
potential abnormalities. Synaptosomes are subcellular
membranous structures from the neuronal terminals
produced by in vitro subcellular fractionation of brain
tissue. Shearing forces lead to the severing of neuronal
terminals, and the synaptosomes are generated by
subsequent resealing of the membranes. Synaptosomes
are then isolated from the homogenate by differential and
density-gradient centrifugation. In routine synaptosome
preparation, shearing forces are typically produced by
some type of tissue grinding procedure. The classical
methods include LNG, HG and MG. Aiming to select an
optimal strategy, the effect of these different grinding
methods on the preparation of rat hippocampal
synaptosomes was assessed. Synaptosomal fractions
were prepared in parallel by the three methods,
characterized by TEM, and differentiated by Western
blotting (Fig. 1). Fig. 1A shows that the synaptosomal
fractions typically contain the complete presynaptic
terminal (including mitochondria and synaptic vesicles
Fig. 4. Western blotting analysis confirming 2DE. Western blotting
and quantification of Clta and Cltb was performed relative to b-tublinlevels. Data represented mean values (±SEM). #p< 0.005 relative
to control group (t-test, n= 3). CON, control.
8 Y. Hu et al. / Neuroscience 230 (2013) 1–12
(SVs)) and portions of the postsynaptic side (including the
postsynaptic membrane and postsynaptic density).
However, the proper synaptosomes are the membrane-
bound structures encompassing the SVs, which contain
neurotransmitters. Mitochondria are also usually
enclosed within these membranes, and the thickened
portion of the postsynaptic density is often still attached.
Table 3. Differentially expressed proteins related to mental disorders
Gene Related to depression
Atp6v1e1 Social defeat rat model (Carboni et al., 2006), patient with majo
(Beasley et al., 2006)
Syn2 Electroconvulsive therapy rat model (Elfving et al., 2008), lear
helplessness rat model (Mallei et al., 2011), maternal separati
(Marais et al., 2009), patient with bipolar disorder (Vawter et a
Nsf Gene–environment interaction rat model (Mallei et al., 2008)
Clta
Cltb
Dnm1 Gene–environment rat interaction (Mallei et al., 2008), CMS r
(Kedracka-Krok et al., 2010)
Hsp90b1 Patient with bipolar disorder (Kakiuchi et al., 2007), suicide w
depression (Bown et al., 2000)
Hsph1 Maternal separation rat model (Marais et al., 2009)
Dnaja1
Pdia3 Maternal separation rat model (Marais et al., 2009)
Gsn
Actn4
Ina Social defeat rat model (Carboni et al., 2006)
Tpi1 Maternal separation rat model (Marais et al., 2009)
Pgk1 Social defeat rat model (Carboni et al., 2006)
Aldoc Maternal separation rat model (Marais et al., 2009), social defe
(Carboni et al., 2006), patient with major depression and bipo
(Johnston-Wilson et al., 2000)
Comparative TEM examination demonstrated that the HG
and MG methods resulted in more representative
structures than the LNG method (Fig. 1A). Western
blotting indicated that synaptosomal purity levels in the
HG and MG methods were not significantly different, but
both were higher than that found in the LNG method. The
LNG method may be more disruptive to tissue structures,
thereby affecting synaptosomal enrichment during
density-gradient centrifugation. As to the other two
methods, the HG method is usually laborious and time-
consuming, and thus its applicability to processing a large
number of samples is limited and impractical. Therefore,
the MG method was selected for synaptosome
preparation in the following proteomic analysis.
A 2DE-MS approach was then undertaken that
identified 19 differentially expressed synaptic proteins.
Interestingly, several synaptic endocytosis- (Clta, Cltb,
and Dnm1) and exocytosis-associated proteins
(Atp6v1e1, Syn2, and Nsf) were found to be dysregulated
in the CMS group.
According to literature on disease relevance, some of
these exo/endocytosis-associated proteins have also
been found to be differentially expressed in hippocampal
protein research on other depressed models, e.g., down-
regulation of vacuolar-type H+-ATPase (V-ATPase) in
social defeat stress model (Carboni et al., 2006), down-
regulation of Syn2 in a learned helplessness model
(Mallei et al., 2011), and dysregulation of Nsf and Dnm1
in a gene–environment interaction model (Mallei et al.,
2008), (Table 3). In addition, Syn2 was reduced in the
Related to other mental disorders
r depression Alzheimer’s disease (Haass et al., 1995; Lee et al.,
2010)
ned
on rat model
l., 2002)
Schizophrenia (Imai et al., 2001; Saviouk et al.,
2007)
Schizophrenia (Spellmann et al., 2008),
Huntington’s disease (Morton et al., 2001)
Alzheimer’s disease (Nakamura et al., 1994a),
Pick’s disease (Nakamura et al., 1994b)
Alzheimer’s disease (Nakamura et al., 1994a),
schizophrenia (Vercauteren et al., 2007)
at model Alzheimer disease (Kelly et al., 2005), nicotine
dependence (Xu et al., 2009)
ith major Alzheimer’s disease (Imaizumi et al., 2001),
Morphine dependence (Ammon et al., 2003)
Alzheimer’s disease (Kim et al., 2000), Creutzfeldt–
Jakob disease (Yoo et al., 2002)
Alzheimer’s disease (Chauhan et al., 2008; Carro,
2010), Down’s syndrome (Chauhan et al., 2008)
Alzheimer’s disease (Klaiman et al., 2008)
Alzheimer’s disease (Dickson et al., 2005)
Alzheimer’s disease (Guix et al., 2009)
Mental retardation (Sugie et al., 1989), Down’s
syndrome (Labudova et al., 1999)
at rat model
lar disorder
Alzheimer’s disease (Opii et al., 2008),
schizophrenia (Martins-de-Souza et al., 2009)
Fig. 5. Functional roles of synaptic exo/endocytosis-associated proteins. (A) SV cycle at the presynapse. V-ATPase provides energy by ATP
hydrolysis to pump protons into the SV, which forms a proton gradient across the vesicular membrane; transmitters are transported through specific
channels coupled to this gradient. SVs are tethered to the cytoskeleton by Syn2; its auto-phosphorylation releases vesicles from the cytoskeleton.
The SNARE complex creates a pore in the plasma membrane to allow neurotransmitter release into the synaptic cleft, and Nsf disassembles the
SNARE complex. SVs are recycled through endocytosis, which is mediated by clathrin formation coated along the vesicular membrane. The
GTPase Dnm1 triggers SV fission from the presynaptic membrane. (B) The formation of LTD at the postsynapse. The GluR2-containing synaptic
AMPARs internalized by clathrin-mediated endocytosis that underlie LTD. Dnm1 is involved in the fission of the endocytic AMPARs from the
postsynaptic membrane. AMPARs may be degraded or returned to the membrane surface in a Nsf-dependent manner. The association between
Nsf and GluR2 maintains AMPARs at the postsynaptic membrane.
Y. Hu et al. / Neuroscience 230 (2013) 1–12 9
postmortem hippocampus of bipolar disorder patients
(Vawter et al., 2002), and in turn, its mRNA level in the
hippocampus of electroconvulsive therapeutic rat was
significantly increased (Elfving et al., 2008). It is worth
noting that the dysregulation of Clta and Cltb has not
been reported in any previous MDD studies. This novel
finding provides a new direction for applied research of
potential therapeutic targets for MDD.
Further, the biological functions and disease
relevance of these altered proteins are detailed below.
As visually illustrated in Fig. 5 perturbations in these
proteins could result in the SV cycle disruption and LTD
facilitation at synapses.
Endocytosis-associated proteins
Clta and Cltb, the two light chains of clathrin, regulate the
formation of the clathrin lattice. Notably, the clathrin lattice
mediates the endocytosis of SVs at synapses (Granseth
et al., 2006). Likewise, the GTPase Dnm1 is essential to
endocytotic SV fission at the presynaptic plasma
membrane (Liu et al., 2006) (Fig. 5A). In Dnm1-knockout
mice, SV endocytosis has been found to be severely
impaired during strong exogenous stimulation (Ferguson
et al., 2007). Taken together, the dysregulation of these
three proteins (Clta, Cltb, and Dnm1) potentially creates an
obstacle to proper SV endocytosis, thereby negatively
affecting the rapid clearance of neurotransmitter release
sites, the subsequent priming of SVs, and refilling of the
release-ready SV pool (Kawasaki et al., 2011).
Thus far, the relationship between SV endocytosis and
MDD has not been a subject of published research, and
may be worth further investigation in light of the
aforementioned evidence. LTD requires clathrin-mediated
glutamate receptor subunit 2 (GluR2)-containing AMPARs
endocytosis that is also dependent on Dnm1 (Carroll
et al., 1999; Wang and Linden, 2000) (Fig. 5B). The
up-regulation of Clta and Cltb found in the CMS group
may lead to an increase in this endocytosis. The abnormal
expression of Dnm1 may also result in the difficult fission
10 Y. Hu et al. / Neuroscience 230 (2013) 1–12
of the endocytic AMPARs from the postsynaptic plasma
membrane, thus blocking the recycling of such receptors.
Accordingly, the perturbations in these endocytosis-
associated proteins (Clta, Cltb, and Dnm1) may be
involved in the facilitated LTD mechanism in the
hippocampus of CMS rats (Holderbach et al., 2007).
Exocytosis-associated proteins
Atp6v1e1 is a subunit of the V-ATPase which drives
neurotransmitter accumulation in SVs (Moriyama et al.,
1992; Morel, 2003). By blocking the V-ATPase with
increasing stimulation frequencies, slower SV reuse
increases the rate of synaptic depression in the rat
hippocampus (Ertunc et al., 2007). Accordingly, the
lower expression of Atp6v1e1 found in this study would
create an impairment in neurotransmitter refilling.
Syn2 coats SVs and reversibly tethers them to the
actin-based cytoskeleton, playing a critical role in the
formation, maintenance and regulation of the reserve
SV pool (Humeau et al., 2001; Gitler et al., 2008).
Moreover, Syn2 regulates SV transitioning from the
reserve to the releasable pool through auto-
phosphorylation (Humeau et al., 2001). In Syn2-
knockout mice, Rosahl et al. found that repetitive
stimulation leads to decreased post-tetanic potentiation
and severe synaptic depression (Rosahl et al., 1995). In
the current study, downregulation of Syn2 may
decrease both the reserve pool size and SV mobilization
from the reserve to the releasable pool. Consequently,
the decreased levels of Atp6v1e1 and Syn2 may result
in reduced stimulus-induced neurotransmitter release in
response to prolonged or intense stimulation.
The soluble Nsf attachment protein receptor (SNARE)
complex mediates the fusion of SVs with the presynaptic
plasma membrane, enabling neurotransmitter release.
Subsequently, it is disassembled or otherwise rearranged
by Nsf-dependent ATP hydrolysis (Fig. 5A). Therefore,
Nsf regulates the neurotransmitter release and maintains
the readily releasable SV pool (Tolar and Pallanck,
1998). In this study, the over-expression of Nsf may have
a compensatory upregulating effect on neurotransmitter
release as a response to an underlying deficiency in
synaptic transmission.
To some extent, Nsf also plays an important role in the
exocytotic insertion of the AMPARs in postsynapses
(Carroll et al., 2001). More importantly, the association
between Nsf and GluR2 may maintain the AMPARs in
the postsynaptic plasma membrane, and inhibit the
GluR2–Nsf interaction which would increase clathrin-
mediated endocytosis and obstruct LTD generation
(Collingridge et al., 2004). Herein, the upregulation of
Nsf would increase the expression of GluR2 in the
postsynaptic plasma membrane, which is consistent
with the results found by Huang et al. through Nsf
overexpression (Huang et al., 2005). This event might
be compensatory response to LTD facilitation.
CONCLUSION
In conclusion, the CMS rat hippocampal synaptosome
was analyzed using a comparative proteomic approach.
The CMS-induced dysregulation of several synaptic
proteins involved in exo/endocytosis may interfere with
synaptic neurotransmitter release, SV retrieval and
synaptic LTD expression. The differentially-expressed
proteins found here offers researchers new information
in deciphering the abnormal synaptic transmission and
plasticity found in MDD.
Acknowledgements—We graciously thank Dr. Chenrui Hou of
the Shanghai Institute for Biological Sciences at the Chinese
Academy of Sciences for his assistance with the mass spectro-
metric analysis, Dr. Yongtao Yang for her technical assistance
with the 2DE analysis, and Dr. N.D. Melgiri for his assistance in
editing and proofreading the manuscript. This work was sup-
ported by grants from the National Basic Research Program (or
‘‘973 program’’) of China (2009CB918300) and National Natural
Science Foundation of China (81101009).
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(Accepted 14 August 2012)(Available online 22 August 2012)
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