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Neuroscience xxx (2015) xxx–xxx

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ITRAQ-BASED QUANTITATIVE ANALYSIS OF HIPPOCAMPAL POSTSYNAPTIC DENSITY-ASSOCIATED PROTEINS IN A RAT CHRONIC MILD STRESS MODEL OF DEPRESSION

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X. HAN, a,b,c  W. SHAO, a,b,c  Z. LIU, a,b,c S. FAN, a,b,c J. YU, b,c J. CHEN, b,c R. QIAO, b,c J. ZHOU b,c* AND P. XIE a,b,c,d*

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a Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China

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b Institute of Neuroscience, Chongqing, China

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c

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d

Department of Neurology, Yongchuan Hospital, Chongqing Medical University, Chongqing, China

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Abstract—Major depressive disorder (MDD) is a prevalent psychiatric mood illness and a major cause of disability and suicide worldwide. However, the underlying pathophysiology of MDD remains poorly understood due to its heterogenic nature. Extensive pre-clinical research suggests that many molecular alterations associated with MDD preferentially localize to the postsynaptic density (PSD). Here, we used a rodent chronic mild stress (CMS) model to generate susceptible and unsusceptible subpopulations. Proteomic analysis using an isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry was performed to identify differentially expressed proteins in enriched PSD preparations from the hippocampi of different groups. More than 1500 proteins were identified and quantified, and 74 membrane proteins were differentially expressed. Of these membrane proteins, 51 (69%) were identified by SynaptomeDB search as having a predicted PSD localization. The unbiased profiles identified several PSD candidate proteins that may be related to CMS vulnerability or insusceptibility, and these two CMS phenotypes

Chongqing

Medical

displayed differences in the abundance of several types of proteins. A detailed protein functional analysis pointed to a role for PSD-associated proteins involved in signaling and regulatory functions. Within the PSD, the N-methyl-D-aspartate (NMDA) receptor subunit NR2A and its downstream targets contribute to CMS susceptibility. Further analysis of disease relevance indicated that the PSD contains a complex set of proteins of known relevance to mental illnesses including depression. In sum, these findings provide novel insights into the contribution of PSD-associated proteins to stress susceptibility and further advance our understanding of the role of hippocampal synaptic plasticity in MDD. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

University,

Chongqing Key Laboratory of Neurobiology, Chongqing, China

Key words: major depressive disorder, chronic mild stress, hippocampus, postsynaptic density, iTRAQ, proteomics. 16

*Correspondence to: J. Zhou, Institute of Neuroscience, Chongqing Medical University, No. 1 Yixue Road, Yuzhong District, Chongqing 400016, China. P. Xie, Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, No. 1 Yixue Road, Yuzhong District, Chongqing 400016, China. Tel: +86-23-68485490; fax: +86-23-68485111. E-mail addresses: [email protected] (J. Zhou), xiepeng@ cqmu.edu.cn (P. Xie).   These authors contributed equally to this work. Abbreviations: 2DE, two-dimensional gel-electrophoresis; ANOVA, analysis of variance; BCA, bicinchoninic acid; BSA, bovine serum albumin; CMS, chronic mild stress; DAVID, database for annotation, visualization and integrated discovery; FASP, filter-aided sample preparation method; FDR, false discovery rate; FST, forced swim test; GO, gene ontology; GRAVY, grand average hydropathicity; HCD, higher energy collisional dissociation; iTRAQ, isobaric tag for relative and absolute quantitation; MDD, major depressive disorder; MW, molecular weight; NMDA, N-methyl-D-aspartate; Nrgn, neurogranin; pI, isoelectric point; PSD, postsynaptic density; SCX, strong cation exchange; SPT, sucrose preference test; TMDs, transmembrane domains; UniProt, universal protein resource. http://dx.doi.org/10.1016/j.neuroscience.2015.04.006 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Major depressive disorder (MDD) is a common and debilitating mental illness affecting approximately 2.5% of the general population and is predicted to be the number two cause of illness worldwide by 2020 (Lecrubier, 2001). Symptoms of the illness include cognitive impairment and memory loss, implicating synaptic dysfunction in the pathophysiology of MDD (Levens and Gotlib, 2009; Barabassy et al., 2010; Boyle et al., 2010). This possibility is supported by animal studies demonstrating a reduction of dendritic spine numbers and hippocampal neuronal function (Radley et al., 2006; Liu and Aghajanian, 2008). Postmortem studies have suggested that somatodendritic, axonal, synaptic, and glial cell number changes are all involved in the inhibition of adult hippocampal neurogenesis (D’Sa and Duman, 2002; Sheline et al., 2003; Pittenger and Duman, 2008; Lorenzetti et al., 2009). This is also consistent with brain imaging studies that have reported a reduction in hippocampal volume in postmortem animal models of either stress or depression (D’Sa and Duman, 2002; Pittenger and Duman, 2008). In animal models, stress-induced hippocampal neuropathological changes may be summarized as follows: loss of dendritic spines, decrease in the number and length of dendrites, loss of synapses, loss of glia, and the impairment of neurogenesis (D’Sa and Duman, 2002; Sheline et al., 2003; Pittenger and Duman, 2008). To date, however, the protein substrates involved in synaptic plasticity in the hippocampus of

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MDD have not been well-explored (Czeh and Lucassen, 2007). Recent studies have used molecular profiling technologies to examine the brains of postmortem depressed patients and rodent models of depression. Several groups have used microarray approaches to demonstrate abnormalities in the expression of transcripts related to synaptic transmission, particularly glutamate and GABAergic signaling, in the brains of depressed patients with or without suicide, especially in the frontal and limbic regions (Choudary et al., 2005; Sequeira et al., 2007, 2009; Klempan et al., 2009). A number of proteomic studies investigating the overall level of hippocampal tissue from various stress systems have shown that stressful events alter the expression of proteins with roles in energy metabolism, neurogenesis, synaptic plasticity, and neurotransmission (Martins-de-Souza et al., 2010; Martins-de-Souza et al., 2012a,b). Although it is unclear whether altering these molecular dysregulations can affect overall hippocampal structure and function, there is evidence that significant changes occur at the subcellular level, particularly at neuronal synapses. Because of the complexity of the central nervous system, the uses of fractionation enrichment techniques are usually necessary to achieve a reliable quantitation of low-abundance proteins. Within the synapse, the postsynaptic site contains a high concentration of proteins, including receptors and their intracellular signaling components that receive and transduce synaptic information. This postsynaptic electron-dense structure, termed the postsynaptic density (PSD), is a highly-organized structure attached to the postsynaptic neuronal terminal and is comprised of a complex network of cytoskeletal scaffolding and signaling proteins. These proteins facilitate the movement of receptor and signaling complexes. The PSD is critical to normal neurotransmission but is also critical to adaptive behaviors such as learning and memory. It has been strongly implicated in neuropsychiatric disorders (Jay et al., 2004), such as MDD, through its roles in synaptic plasticity and cognitive function (Duric et al., 2013). Although constituents of the PSD have been implicated in MDD using conventional detection techniques (Kinnunen et al., 2003; Duric et al., 2013), no study has yet focused on exploring global changes in the hippocampal PSD fraction through the use of a high-throughput proteomic screen. In this study, using quantitative proteomics, we examined changes in the expression levels of hippocampal PSD-associated proteins in a chronic mild stress (CMS) rat model of depression. This model generates CMS-susceptible and unsusceptible subpopulations, reflecting the two hedonic responses to CMS. To measure the relative changes in protein expression, we used isobaric tag for relative and absolute quantitation (iTRAQ) followed by tandem mass spectrometric (LC-MS/MS) analysis. Our large-scale subcellular proteome profiles identified several differential PSD-associated proteins that may be specifically related to stress vulnerability. These data should provide a valuable resource for deciphering the

molecular mechanism(s) underlying synaptic plasticity observed in MDD.

the

abnormal

EXPERIMENTAL PROCEDURES

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Ethics statement

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This study was approved by the Ethics Committee of the Chongqing Medical University (Chongqing, China), and all procedures of animal care and treatment were in accordance with the National Institutes of Health Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals). Special care was taken to minimize the number and suffering of animals.

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Animals and CMS treatment

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Eighty-one healthy adult male Sprague–Dawley rats, purchased from the animal facility at the Chongqing Medical University (Chongqing, China), were housed individually, given access to food and water ad libitum, and maintained on a 12-h light/dark cycle (light from 7:30 to 19:30) at a constant temperature of 21–22 °C and humidity of 55 ± 5% (unless otherwise noted). Animal weight was approximately 165 g when adaptation for sucrose consumption was initiated and approximately 250 g at the start of the stress regime. The rats were allowed one week to acclimate to the environment and then 1% sucrose solution was given to them, as well as water for five weeks for sucrose habituation. During this period, sucrose preference of all rats was tested twice weekly during the first three weeks and once weekly during the last two weeks. According to the sucrose intakes of the three final baseline tests, the rats were divided randomly into two groups and placed in separate rooms. Stress groups were exposed to a four-week CMS procedure, whereas the control group was left undisturbed. The CMS protocol was performed according to the procedure described in our previous studies (Hu et al., 2013; Yang et al., 2013). In short, rats were subjected to a variety of mild stress factors: paired housing, a 45° cage tilt along the vertical axis, a soiled cage (with 300 ml water spilled into bedding), exposure to an empty water bottle immediately following a period of acute water deprivation, stroboscopic illumination (300 flashes/min), continuous overnight illumination, and white noise. The procedure including the time and length of stressors are described in Appendix Table 1. At the end of every week, the sucrose preference of all of the rats was assessed, and the forced swim test (FST) was carried out in the final week.

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Sucrose preference test (SPT)

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The SPT was conducted as a measure of the anhedonic effect of CMS. A two-bottle preference test was used, where the rats had access to both water and a 1% sucrose solution for 24 h during a no-stress period. The positions of the two bottles (on the left and right sides of the cages) were randomly varied. All fluid consumption was recorded by weighing the two bottles before testing and after 24 h. The sucrose preference was calculated

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according to the preference = sucrose (g) + water intake (g)).

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FST

Extraction and digestion of proteins

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The FST was performed as described previously with slight modifications (Porsolt et al., 1977). The rats were placed individually in a cylinder (40 cm in height  20 cm in diameter) filled with water (24 ± 1 °C) to a height of 30 cm. A 15-min pretest period was followed 24 h later by a 5-min test period during which the total immobility time was recorded. The test was monitored by a video surveillance system. Water in the cylinders was changed before each trial.

The obtained PSD pellet was dissolved using a sample buffer (4% SDS, 10 mM DTT, 150 mM Tris–HCl, pH 8.0). The lysate was boiled in water for 5 min and then centrifuged at 40,000g for 15 min. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions (Pierce, USA). Bovine serum albumin (BSA) was used as the standard. Subsequent protein digestion was performed using the filter-aided sample preparation method (FASP) in a 10-kDa molecular weight cut-off centrifugation filter (Sartorius) (Wisniewski et al., 2009a,b). The sample was diluted with 200 ll of UA buffer (8 M urea, 150 mM Tris–HCl, pH 8.0) and then centrifuged at 14,000g for 30 min. This step was repeated once. Then, 100 ll of 50 mM iodoacetamide in UA buffer was added to the filters, and the samples were incubated in darkness for 30 min. Filters were washed twice with 100 ll of UA buffer followed by two washes with 100 ll of dissolution buffer (50 mM triethylammonium bicarbonate, pH 8.5). Proteins were digested overnight in a 40 ll dissolution buffer using trypsin (Promega) at an enzyme-to-protein ratio of 1:50 at 37 °C. The released peptides were collected by centrifugation at 14,000g for 10 min followed by two washes with dissolution buffer.

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iTRAQ labeling and strong cation exchange (SCX) fractionation

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The tryptic peptides were labeled with an iTRAQ-8plex kit (Applied Biosystems) according to the manufacturer’s protocol. Samples from the control group were labeled with reagents 115 and 118, samples from the susceptible group were labeled with reagents 114 and 117, and samples from the unsusceptible group were labeled with reagents 113 and 116. The incubation was allowed to proceed at room temperature for 2 h and then stopped by the addition of 10 mM KH2PO4, 25% ACN, pH 3.0. Subsequently, all the three labeledsamples were pooled, vacuum-dried and further fractionated offline using SCX chromatography. Briefly, the peptides were dissolved and loaded onto a Polysulfoethyl 4.6  100 mm column (5 lm, 200 A˚, PolyLC Inc., MD, USA) at a flow rate of 1 ml/min. A suitable gradient elution was applied to separate peptides at a flow rate of 1 ml/min with elution buffer (10 mM KH2PO4, 500 mM KCl in 25% acetonitrile, pH 3.0). Eluted peptides were collected and desalted by an offline fraction collector. The resulting 30 fractions were combined to 10 pools and desalted on C18 Cartridges (Emporeä SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma, St. Louis, MO, USA). Each final fraction was concentrated by a vacuum concentrator and reconstituted with 40 ll of 0.1% formic acid for LC-MS/MS analysis.

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following formula: sucrose intake (g)/(sucrose intake

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Statistical analysis of SPT and FST data

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Statistical analyses were carried out using SPSS 16.0. Data from the SPT and FST were analyzed by a oneway repeated measured analyses of variance (ANOVA) followed by a post hoc LSD test. A p-value of less than 0.05 was considered to be statistically significant. Statistics were presented as mean ± standard errors (SE).

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Preparation of hippocampal PSD fractions

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PSD fractions were obtained using a biomedical method essentially as previously reported (Phillips et al., 2001; Abul-Husn et al., 2009; Hu et al., 2013; Focking et al., 2014). Rats were sacrificed by decapitation and the brains rapidly removed. The fresh hippocampal tissue samples were collected and pooled. Using a motor-operated Teflon-glass grinder, the pooled tissues were homogenized on icein solution A (0.32 M sucrose, 1 mM MgCl2, and 0.1 mM CaCl2) containing a protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA), respectively. All the following fractionation steps were carried out at 4 °C unless otherwise specified. 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. Supernatants were pooled and centrifuged at 12,000g for 30 min. The resulting pellet was suspended for a second time in solution B (0.32 M sucrose and 0.1 mM CaCl2) then layered over the 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. To obtain PSD fractions, an aliquot solution (synaptosomal fraction) was diluted with ice-cold 0.1 mM CaCl2 and brought to an equal volume of 2hypotonic buffer (40 mM Tris–HCl pH 8, 2% Triton X-100, 0.2 mM CaCl2) containing protease and phosphatase inhibitor cocktails. This solution was mixed by inversion and incubated on a shaker for 20 min at 4 °C. The PSD pellet was collected by centrifugation at 40,000g for 20 min at 4 °C and then stored at 80 °C before use in the proteomic analysis. The following iTRAQ-labeling proteomic experiment was conducted on six sample pools corresponding to the three groups

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with each pool originating from three or four rats (Henningsen et al., 2012; Hu et al., 2013).

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LC-MS/MS analysis

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SCX fractions were used for LC-MS/MS analysis and loaded on a pre-column (20 mm  100 lm; 5 lm-C18). Reversed phase chromatography was performed using the Thermo EASY-nLC 1000 (Thermo Fisher Scientific, San Jose, CA, USA) with a binary buffer system consisting of 0.1% formic acid (buffer A) and 84% ACN in 0.1% formic acid (buffer B). An analytical column (100 mm  75 lm; 3 lm-C18) was used. The online LC separation used a gradient from 0% to 35% buffer B for 100 min, then 35–100% buffer B for 8 min, and then 100% buffer B for 12 min. The flow rate was 250 nl/min. The column was operated at a constant temperature of 35 °C regulated by an in-house designed oven with a Peltier element. Peptides eluted from LC were directly injected into the coupled Q-Exactive mass spectrometer (Thermo Fisher Scientific) via the nanoelectrospray source (Thermo Fisher Scientific). A full MS scan was operated in the data-dependent mode and acquired over the range m/z 300–1800 with a mass resolution of 70,000 at m/z 200. Up to the top ten most abundant isotope patterns with a charge of P2 from the survey scan were selected with an isolation window of 1.6 Th and fragmented by higher energy collisional dissociation (HCD) with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 60 ms, respectively, and the ion target value for both scan modes was set to 106. In this mode of operation, MS/MS scans were acquired with a mass resolution of 17,500 at m/z 200 and an isolation window of 2 m/z. Furthermore, since virtually all scans ‘‘time out’’ at 60 ms due to the high target value, the maximum ion signal in the fragmentation spectra is obtained.

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Data analysis

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Mascot 2.2 (Matrix Science, Boston, MA, USA) and Proteome Discoverer 1.3 (Thermo Fisher Scientific) software were used to simultaneously identify and quantify proteins based on the UniProt_Rat release 2013_03 database of 41,766 protein sequences. To calculate the false discovery rate (FDR) of peptide identification, the search was performed using the ‘‘decoy’’ option in Mascot. The user-defined search parameters were as follows: sample type as iTRAQ 8-plex (peptide-labeled), enzyme as trypsin, selection only of tryptic peptides with two missed cleavages, variable modifications of methionine oxidation, fixed modification of carbamidomethyl cysteine, peptide mass tolerance of ±20 ppm, and fragment mass tolerance of 0.1 Da. Proteome Discoverer 1.3 software was used to extract the peak intensity of each expected iTRAQ reporter ion from each analyzed fragmentation spectrum. The search parameters were as follows: peptide FDR

iTRAQ-based quantitative analysis of hippocampal postsynaptic density-associated proteins in a rat chronic mild stress model of depression.

Major depressive disorder (MDD) is a prevalent psychiatric mood illness and a major cause of disability and suicide worldwide. However, the underlying...
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