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: xiepeng@cqmu.edu.cn (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).

REFERENCES

Abul-Husn NS, Bushlin I, Moron JA, Jenkins SL, Dolios G, Wang R,

Iyengar R, Ma’ayan A, Devi LA (2009) Systems approach to

explore components and interactions in the presynapse.

Proteomics 9:3303–3315.

Ammon S, Mayer P, Riechert U, Tischmeyer H, Hollt V (2003)

Microarray analysis of genes expressed in the frontal cortex of

rats chronically treated with morphine and after naloxone

precipitated withdrawal. Brain Res Mol Brain Res 112:113–125.

Anisman H, Merali Z, Hayley S (2008) Neurotransmitter, peptide and

cytokine processes in relation to depressive disorder: comorbidity

between depression and neurodegenerative disorders. Prog

Neurobiol 85:1–74.

Beasley CL, Pennington K, Behan A, Wait R, Dunn MJ, Cotter D

(2006) Proteomic analysis of the anterior cingulate cortex in the

major psychiatric disorders: evidence for disease-associated

changes. Proteomics 6:3414–3425.

Belmaker RH, Agam G (2008) Mechanisms of disease: major

depressive disorder. N Engl J Med 358:55–68.

Bown C, Wang JF, MacQueen G, Young LT (2000) Increased

temporal cortex ER stress proteins in depressed subjects who

died by suicide. Neuropsychopharmacology 22:327–332.

Brambilla P, Perez J, Barale F, Schettini G, Soares JC (2003)

GABAergic dysfunction in mood disorders. Mol Psychiatry

8:721–737. 715.

Campbell S, Macqueen G (2004) The role of the hippocampus in the

pathophysiology of major depression. J Psychiatry Neurosci

29:417–426.

Carboni L, Piubelli C, Pozzato C, Astner H, Arban R, Righetti PG,

HaMDDan M, Domenici E (2006) Proteomic analysis of rat

hippocampus after repeated psychosocial stress. Neuroscience

137:1237–1246.

Carlin RK, Grab DJ, Cohen RS, Siekevitz P (1980) Isolation and

characterization of postsynaptic densities from various brain

regions: enrichment of different types of postsynaptic densities.

J Cell Biol 86:831–845.

Carro E (2010) Gelsolin as therapeutic target in Alzheimer’s disease.

Expert Opin Ther Targets 14:585–592.

Carroll RC, Beattie EC, Xia H, Luscher C, Altschuler Y, Nicoll RA,

Malenka RC, von ZM (1999) Dynamin-dependent endocytosis of

ionotropic glutamate receptors. Proc Natl Acad Sci U S A

96:14112–14117.

Carroll RC, Beattie EC, von ZM, Malenka RC (2001) Role of AMPA

receptor endocytosis in synaptic plasticity. Nat Rev Neurosci

2:315–324.

Chauhan V, Ji L, Chauhan A (2008) Anti-amyloidogenic, anti-oxidant

and anti-apoptotic role of gelsolin in Alzheimer’s disease.

Biogerontology 9:381–389.

Y. Hu et al. / Neuroscience 230 (2013) 1–12 11

Collingridge GL, Isaac JT, Wang YT (2004) Receptor trafficking and

synaptic plasticity. Nat Rev Neurosci 5:952–962.

Collins MO, Husi H, Yu L, Brandon JM, Anderson CN, Blackstock

WP, Choudhary JS, Grant SG (2006) Molecular characterization

and comparison of the components and multiprotein complexes in

the postsynaptic proteome. J Neurochem 97(Suppl. 1):16–23.

Dickson TC, Chuckowree JA, Chuah MI, West AK, Vickers JC (2005)

Alpha-internexin immunoreactivity reflects variable neuronal

vulnerability in Alzheimer’s disease and supports the role of the

beta-amyloid plaques in inducing neuronal injury. Neurobiol Dis

18:286–295.

Elfving B, Bonefeld BE, Rosenberg R, Wegener G (2008) Differential

expression of synaptic vesicle proteins after repeated

electroconvulsive seizures in rat frontal cortex and

hippocampus. Synapse 62:662–670.

Ertunc M, Sara Y, Chung C, Atasoy D, Virmani T, Kavalali ET (2007)

Fast synaptic vesicle reuse slows the rate of synaptic depression

in the CA1 region of hippocampus. J Neurosci 27:341–354.

Ferguson SM, Brasnjo G, Hayashi M, Wolfel M, Collesi C, Giovedi S,

Raimondi A, Gong LW, Ariel P, Paradise S, O’toole E, Flavell R,

Cremona O, Miesenbock G, Ryan TA, De Camilli P (2007) A

selective activity-dependent requirement for dynamin 1 in

synaptic vesicle endocytosis. Science 316:570–574.

Fernandez E, Collins MO, Uren RT, Kopanitsa MV, Komiyama NH,

Croning MDD, Zografos L, Armstrong JD, Choudhary JS, Grant

SG (2009) Targeted tandem affinity purification of PSD-95

recovers core postsynaptic complexes and schizophrenia

susceptibility proteins. Mol Syst Biol 5. http://dx.doi.org/10.1038/

msb.2009.27.

Gitler D, Cheng Q, Greengard P, Augustine GJ (2008) Synapsin IIa

controls the reserve pool of glutamatergic synaptic vesicles. J

Neurosci 28:10835–10843.

Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-

mediated endocytosis is the dominant mechanism of vesicle

retrieval at hippocampal synapses. Neuron 51:773–786.

Grippo AJ, Moffitt JA, Johnson AK (2002) Cardiovascular alterations

and autonomic imbalance in an experimental model of

depression. Am J Physiol Regul Integr Comp Physiol

282:R1333–R1341.

Guix FX, Ill-Raga G, Bravo R, Nakaya T, de Fabritiis G, Coma M,

Miscione GP, Villa-Freixa J, Suzuki T, Fernandez-Busquets X,

Valverde MA, de Strooper B, Munoz FJ (2009) Amyloid-

dependent triosephosphate isomerase nitrotyrosination induces

glycation and tau fibrillation. Brain 132:1335–1345.

Haass C, Capell A, Citron M, Teplow DB, Selkoe DJ (1995) The

vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially

affects proteolytic processing of mutant and wild-type beta-

amyloid precursor protein. J Biol Chem 270:6186–6192.

Holderbach R, Clark K, Moreau JL, Bischofberger J, Normann C

(2007) Enhanced long-term synaptic depression in an animal

model of depression. Biol Psychiatry 62:92–100.

Holm MM, Nieto-Gonzalez JL, Vardya I, Henningsen K, Jayatissa

MN, Wiborg O, Jensen K (2011) Hippocampal GABAergic

dysfunction in a rat chronic mild stress model of depression.

Hippocampus 21:422–433.

Huang Y, Man HY, Sekine-Aizawa Y, Han Y, Juluri K, Luo H, Cheah

J, Lowenstein C, Huganir RL, Snyder SH (2005) S-Nitrosylation of

N-ethylmaleimide sensitive factor mediates surface expression of

AMPA receptors. Neuron 46:533–540.

Humeau Y, Doussau F, Vitiello F, Greengard P, Benfenati F, Poulain

B (2001) Synapsin controls both reserve and releasable synaptic

vesicle pools during neuronal activity and short-term plasticity in

Aplysia. J Neurosci 21:4195–4206.

Imai C, Sugai T, Iritani S, Niizato K, Nakamura R, Makifuchi T, Kakita

A, Takahashi H, Nawa H (2001) A quantitative study on the

expression of synapsin II and N-ethylmaleimide-sensitive fusion

protein in schizophrenic patients. Neurosci Lett 305:185–188.

Imaizumi K, Miyoshi K, Katayama T, Yoneda T, Taniguchi M, Kudo T,

Tohyama M (2001) The unfolded protein response and

Alzheimer’s disease. Biochim Biophys Acta 1536:85–96.

Johnston-Wilson NL, Sims CD, Hofmann JP, Anderson L, Shore AD,

Torrey EF, Yolken RH (2000) Disease-specific alterations in

frontal cortex brain proteins in schizophrenia, bipolar disorder,

and major depressive disorder. The Stanley Neuropathology

Consortium. Mol Psychiatry 5:142–149.

Kakiuchi C, Ishiwata M, Nanko S, Kunugi H, Minabe Y, Nakamura K,

Mori N, Fujii K, Umekage T, Tochigi M, Kohda K, Sasaki T,

Yamada K, Yoshikawa T, Kato T (2007) Association analysis of

HSP90B1 with bipolar disorder. J Hum Genet 52:794–803.

Kawasaki F, Iyer J, Posey LL, Sun CE, Mammen SE, Yan H, Ordway

RW (2011) The DISABLED protein functions in CLATHRIN-

mediated synaptic vesicle endocytosis and exoendocytic coupling

at the active zone. Proc Natl Acad Sci U S A 108:E222–E229.

Kedracka-Krok S, Fic E, Jankowska U, Jaciuk M, Gruca P, Papp M,

Kusmider M, Solich J, Debski J, Dadlez M, Dziedzicka-

Wasylewska M (2010) Effect of chronic mild stress and

imipramine on the proteome of the rat dentate gyrus. J

Neurochem 113:848–859.

Kelly BL, Vassar R, Ferreira A (2005) Beta-amyloid-induced dynamin

1 depletion in hippocampal neurons. A potential mechanism for

early cognitive decline in Alzheimer disease. J Biol Chem

280:31746–31753.

Kim HT, Russell RL, Raina AK, Harris PL, Siedlak SL, Zhu X,

Petersen RB, Shimohama S, Smith MA, Perry G (2000) Protein

disulfide isomerase in Alzheimer disease. Antioxid Redox Signal

2:485–489.

Klaiman G, Petzke TL, Hammond J, Leblanc AC (2008) Targets of

caspase-6 activity in human neurons and Alzheimer disease. Mol

Cell Proteomics 7:1541–1555.

Labudova O, Kitzmueller E, Rink H, Cairns N, Lubec G (1999)

Increased phosphoglycerate kinase in the brains of patients with

Down’s syndrome but not with Alzheimer’s disease. Clin Sci

(Lond) 96:279–285.

Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe

DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y,

Westaway D, Cuervo AM, Nixon RA (2010) Lysosomal proteolysis

and autophagy require presenilin 1 and are disrupted by

Alzheimer-related PS1 mutations. Cell 141:1146–1158.

Lewitus GM, Wilf-Yarkoni A, Ziv Y, Shabat-Simon M, Gersner R,

Zangen A, Schwartz M (2009) Vaccination as a novel

approach for treating depressive behavior. Biol Psychiatry

65:283–288.

Linden DJ (1999) The return of the spike: postsynaptic action

potentials and the induction of LTP and LTD. Neuron 22:661–666.

Liu J, Kaksonen M, Drubin DG, Oster G (2006) Endocytic vesicle

scission by lipid phase boundary forces. Proc Natl Acad Sci U S A

103:10277–10282.

Mallei A, Giambelli R, Barbiero VS, Musazzi L, El Khoury A, Gruber

SH, Mathe AA, Racagni G, Popoli M (2008) Synaptoproteomic

analysis of a rat model of depression with gene–environment

interaction. Eur Neuropsychopharmacol 18(Suppl. 1):S19–S20.

Mallei A, Giambelli R, Gass P, Racagni G, Mathe AA, Vollmayr B,

Popoli M (2011) Synaptoproteomics of learned helpless rats

involve energy metabolism and cellular remodeling pathways in

depressive-like behavior and antidepressant response.

Neuropharmacology 60:1243–1253.

Marais L, Hattingh SM, Stein DJ, Daniels WM (2009) A proteomic

analysis of the ventral hippocampus of rats subjected to maternal

separation and escitalopram treatment. Metab Brain Dis

24:569–586.

Martins-de-Souza D, Gattaz WF, Schmitt A, Novello JC, Marangoni

S, Turck CW, Dias-Neto E (2009) Proteome analysis of

schizophrenia patients Wernicke’s area reveals an energy

metabolism dysregulation. BMC Psychiatry 9:17.

Morciano M, Beckhaus T, Karas M, Zimmermann H, Volknandt W

(2009) The proteome of the presynaptic active zone: from docked

synaptic vesicles to adhesion molecules and maxi-channels. J

Neurochem 108:662–675.

Morel N (2003) Neurotransmitter release: the dark side of the

vacuolar-H+ATPase. Biol Cell 95:453–457.

12 Y. Hu et al. / Neuroscience 230 (2013) 1–12

Moriyama Y, Maeda M, Futai M (1992) The role of V-ATPase in

neuronal and endocrine systems. J Exp Biol 172:171–178.

Moron JA, Abul-Husn NS, Rozenfeld R, Dolios G, Wang R, Devi LA

(2007) Morphine administration alters the profile of hippocampal

postsynaptic density-associated proteins: a proteomics study

focusing on endocytic proteins. Mol Cell Proteomics 6:29–42.

Morton AJ, Faull RL, Edwardson JM (2001) Abnormalities in the

synaptic vesicle fusion machinery in Huntington’s disease. Brain

Res Bull 56:111–117.

Mu J, Xie P, Yang ZS, Yang DL, Lv FJ, Luo TY, Li Y (2007)

Neurogenesis and major depression: implications from proteomic

analyses of hippocampal proteins in a rat depression model.

Neurosci Lett 416:252–256.

Nakamura Y, Takeda M, Yoshimi K, Hattori H, Hariguchi S,

Hashimoto S, Nishimura T (1994a) Involvement of clathrin light

chains in the pathology of Pick’s disease; implication for

impairment of axonal transport. Neurosci Lett 180:25–28.

Nakamura Y, Takeda M, Yoshimi K, Hattori H, Hariguchi S, Kitajima

S, Hashimoto S, Nishimura T (1994b) Involvement of clathrin light

chains in the pathology of Alzheimer’s disease. Acta Neuropathol

87:23–31.

Opii WO, Joshi G, Head E, Milgram NW, Muggenburg BA, Klein JB,

Pierce WM, Cotman CW, Butterfield DA (2008) Proteomic

identification of brain proteins in the canine model of human

aging following a long-term treatment with antioxidants and a

program of behavioral enrichment: relevance to Alzheimer’s

disease. Neurobiol Aging 29:51–70.

Phillips GR, Florens L, Tanaka H, Khaing ZZ, Fidler L, Yates 3rd JR,

Colman DR (2005) Proteomic comparison of two fractions derived

from the transsynaptic scaffold. J Neurosci Res 81:762–775.

Popoli M, Gennarelli M, Racagni G (2002) Modulation of synaptic

plasticity by stress and antidepressants. Bipolar Disord

4:166–182.

Rosahl TW, Spillane D, Missler M, Herz J, Selig DK, Wolff JR,

Hammer RE, Malenka RC, Sudhof TC (1995) Essential functions

of synapsins I and II in synaptic vesicle regulation. Nature

375:488–493.

Sarter M, Bruno JP, Parikh V (2007) Abnormal neurotransmitter

release underlying behavioral and cognitive disorders: toward

concepts of dynamic and function-specific dysregulation.

Neuropsychopharmacology 32:1452–1461.

Saviouk V, Moreau MP, Tereshchenko IV, Brzustowicz LM (2007)

Association of synapsin 2 with schizophrenia in families of

Northern European ancestry. Schizophr Res 96:100–111.

Spellmann I, Muller N, Musil R, Zill P, Douhet A, Dehning S,

Cerovecki A, Bondy B, Moller HJ, Riedel M (2008) Associations of

SNAP-25 polymorphisms with cognitive dysfunctions in

Caucasian patients with schizophrenia during a brief trail of

treatment with atypical antipsychotics. Eur Arch Psychiatry Clin

Neurosci 258:335–344.

Sudhof TC (2004) The synaptic vesicle cycle. Annu Rev Neurosci

27:509–547.

Sugie H, Sugie Y, Nishida M, Ito M, Tsurui S, Suzuki M, Miyamoto R,

Igarashi Y (1989) Recurrent myoglobinuria in a child with mental

retardation: phosphoglycerate kinase deficiency. J Child Neurol

4:95–99.

Takamori S, Holt M, Stenius K, Lemke EA, Gronborg M, Riedel D,

Urlaub H, Schenck S, Brugger B, Ringler P, Muller SA, Rammner

B, Grater F, Hub JS, De Groot BL, Mieskes G, Moriyama Y,

Klingauf J, Grubmuller H, Heuser J, Wieland F, Jahn R (2006)

Molecular anatomy of a trafficking organelle. Cell 127:831–846.

Tolar LA, Pallanck L (1998) NSF function in neurotransmitter release

involves rearrangement of the SNARE complex downstream of

synaptic vesicle docking. J Neurosci 18:10250–10256.

Vawter MP, Thatcher L, Usen N, Hyde TM, Kleinman JE, Freed WJ

(2002) Reduction of synapsin in the hippocampus of patients with

bipolar disorder and schizophrenia. Mol Psychiatry 7:571–578.

Vercauteren FG, Flores G, Ma W, Chabot JG, Geenen L, Clerens S,

Fazel A, Bergeron JJ, Srivastava LK, Arckens L, Quirion R (2007)

An organelle proteomic method to study neurotransmission-

related proteins, applied to a neurodevelopmental model of

schizophrenia. Proteomics 7:3569–3579.

Wang YT, Linden DJ (2000) Expression of cerebellar long-term

depression requires postsynaptic clathrin-mediated endocytosis.

Neuron 25:635–647.

WHO (2001) The World Health Report 2001: Mental health: new

understanding, new hope. Geneva: World Health Organization.

169 p..

Xu Q, Huang W, Payne TJ, Ma JZ, Li MDD (2009) Detection of

genetic association and a functional polymorphism of dynamin 1

gene with nicotine dependence in European and African

Americans. Neuropsychopharmacology 34:1351–1359.

Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH,

Dunn MJ (2000) A modified silver staining protocol for

visualization of proteins compatible with matrix-assisted laser

desorption/ionization and electrospray ionization–mass

spectrometry. Electrophoresis 21:3666–3672.

Yoo BC, Krapfenbauer K, Cairns N, Belay G, Bajo M, Lubec G (2002)

Overexpressed protein disulfide isomerase in brains of patients

with sporadic Creutzfeldt–Jakob disease. Neurosci Lett

334:196–200.

Zhou K, Yang Y, Gao L, He G, Li W, Tang K, Ji B, Zhang M, Li Y,

Yang J, Sun L, Zhang Z, Zhu H, He L, Wan C (2010) NMD: a

receptor hypofunction induces dysfunctions of energy metabolism

and semaphorin signaling in rats: A Synaptic Proteome Study.

Schizophr Bull. http://dx.doi.org/10.1093/schbul/sbq132.

(Accepted 14 August 2012)(Available online 22 August 2012)

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具