iTRAQ-based proteomics analysis of hippocampus in spatial memory deficiency rats induced by simulated microgravity

Accepted Manuscript iTRAQ-based proteomics analysis of hippocampus in spatial memory deficiency rats induced by simulated microgravity

Tingmei Wang, Hailong Chen, Ke Lv, Guohua Ji, Yongliang Zhang, Yanli Wang, Yinghui Li, Lina Qu PII: DOI: Reference:

S1874-3919(17)30097-0 doi: 10.1016/j.jprot.2017.03.013 JPROT 2801

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

10 November 2016 2 March 2017 17 March 2017

Please cite this article as: Tingmei Wang, Hailong Chen, Ke Lv, Guohua Ji, Yongliang Zhang, Yanli Wang, Yinghui Li, Lina Qu , iTRAQ-based proteomics analysis of hippocampus in spatial memory deficiency rats induced by simulated microgravity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jprot(2017), doi: 10.1016/j.jprot.2017.03.013

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iTRAQ-based proteomics analysis of hippocampus in spatial memory deficiency rats induced by simulated microgravity Tingmei Wang1, 2, Hailong Chen1, 3, Ke Lv1, Guohua Ji1, Yongliang Zhang1, 2, Yanli Wang1, 2, Yinghui Li1, 2, and Lina Qu1,* 1

State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, 100094, China 2

Space Institute of Southern China (Shenzhen), Shenzhen, 518117, China

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School of Life Sciences, Northwestern Polytechnical University, Xi’an, 710072, China

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*Corresponding author: Lina Qu:

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State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, No.26 Beiqing Road, Haidian District, Beijing, 100094, P. R.

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China.

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E-mail: [email protected]

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iTRAQ-based proteomics analysis of hippocampus in spatial memory deficiency rats induced by simulated microgravity Tingmei Wang1, 2, Hailong Chen1, 3, Ke Lv1, Guohua Ji1, Yongliang Zhang1, 2, Yanli Wang1, 2, Yinghui Li1, 2 , Lina Qu1,* 1

State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut

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Research and Training Center, Beijing, 100094, China 2

School of Life Sciences, Northwestern Polytechnical University, Xi’an, 710072, China Space Institute of Southern China (Shenzhen), Shenzhen, 518117, China

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Abstract:

It has been demonstrated that simulated microgravity (SM) may lead to cognitive dysfunction.

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However, the underlying mechanism remains unclear. In present study, tail-suspension (30°) rat was employed to explore the effects of 28 days of SM on hippocampus-dependent learning and

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memory capability and the underlying mechanisms. We found that 28-day tail-suspension rats displayed decline of learning and memory ability in Morris water maze (MWM) test. Using

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iTRAQ-based proteomics analysis, a total of 4774 proteins were quantified in hippocampus. Of

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these identified proteins, 147 proteins were differentially expressed between SM and control group. Further analysis showed these differentially expressed proteins (DEPs) involved in different molecular function categories, and participated in many biological processes. Based on

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the results of PANTHER pathway analysis and further western blot verification, we observed the expression of glutamate receptor 1 (GluR1) and glutamate receptor 4 (GluR4) which involved in

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metabotropic glutamate receptor group III pathway and ionotropic glutamate receptor pathway were significantly induced by SM. Moreover, an increased concentration of glutamic acid (Glu) was also found in hippocampus while the concentrations of 5-hydroxytryptamine (5-HT), dopamine (DA), γ-amino acid butyric acid (GABA) and epinephrine (E) were decreased. Our finding confirms that 28-day SM exposure can cause degrading of the spatial learning and memory capability and the possible mechanisms might be related with glutamate excitotoxicity and imbalances in specific neurotransmitters. Key

words:

Excitotoxicity

Simulated

microgravity;

iTRAQ;

Cognitive

dysfunction;

Hippocampus;

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Significance statement： The goal of sending astronauts farther into space and extending the duration of spaceflight missions from months to years will challenge the current capabilities of bioastronautics. The investigation of the physiological and pathological changes induced by spaceflight will be critical in developing countermeasures to ensure astronauts to complete spaceflight mission accurately

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and effectively and return to earth safely. It has been demonstrated that spaceflight may lead to impairments in cognitive function which is crucial for mission success. Here we show that

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long-term simulated microgravity, the most potent environment risk factor during spaceflight, impairs the spatial learning and memory of rats and the underlying mechanism may be involved in

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glutamate excitotoxicity and imbalances in specific neurotransmitters release in hippocampus, which may provide new insight for the countermeasures of cognitive impairment during

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spaceflight.

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Introduction The plan of future space exploration mission requires astronauts to spend months or even years in space. It was reported that spaceflight can have many detrimental effects on physiological function of astronauts, due to various harmful stressors such as microgravity, radiation, physical isolation and high workload[1]. Among all these aeronautic environmental stressors, microgravity is

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considered to be one of the main hazards threatening human health [1-3]. Actual (spaceflight) or simulated microgravity can directly induce various physiological adaptations, including those in

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the central nervous system [4]. Previous studies have suggested that microgravity affects several aspects of brain functions, such as cognitive performance, posture control, locomotion, manual

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control [5-10]. Since the success of the spaceflight is highly dependent on astronauts’ mental health, the investigation of the underlying molecular mechanisms involved in the initiation and

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progression of psychopathologies is one of the main challenges of the space neurobiology. Hippocampus is part of the limbic system and plays important roles in learning and memory

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formation, and specifically spatial learning and episodic memory. Any malfunction and/or structural changes of the hippocampus could have substantial consequences on learning and

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memory consolidation as well as general cognitive performance. According to the previous studies,

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7 days of SM exposure significantly changed the expression of cytoskeleton, metabolism, mitochondrial function related proteins in hippocampus [11, 12]. 21 days of SM induced changes in neurotransmitters (GABA and Glu), and metabolism related proteins [2, 12]. Moreover,

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different period (7/14/21/28 days) SM-induced oxidative stress was found in many areas of brain, including hippocampus [13-15]. It has been suggested that behavioral alterations induced by 7 or

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14 days of SM are likely to be associated with cytomorphological changes and loss of neurons in hippocampus of rats [10, 16]. So far, studies of cognitive impairment induced by SM are still relatively small and existing evidences are far from enough to clarify its inherent mechanisms. On the other hand, the effect of microgravity on hippocampus (brain) might be correlated with exposing time although this hasn’t been explored. As the duration of flights has increased, the effect of microgravity, especially long-term microgravity, on the cognitive function and the underlying mechanisms need to be explored imperatively. Therefore, the current study was performed to investigate the effects of long-term exposure to SM environment on the spatial

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learning and memory ability. Specifically, comprehensive analyses of protein expression were

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performed to explore the underlying mechanisms of spatial memory deficiency.

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Experimental Procedures Animals. Adult male Sprague-Dawley (SD) rats of SPF grade (8 weeks old, Beijing Vital River Laboratory Animal Technology Co. Ltd. China, Certification No: SCXK 2012-0001) were used in the study. All rats were housed individually in the cages, kept on a 23–25°C room temperature, 12-h light/12-h dark cycle and free access to food and water. All experimental procedures were

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approved by China Astronaut Research and Training Center Animal Care and Use Committee, and performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments)

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guidelines (http://www.nc3rs.org.uk/sites/default/files/documents/Guidelines/ARRIVE%20in%20 mandarin%20v3. pdf).

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Tail-suspension model (30°). Rats were randomly divided into 2 groups, including the control group and SM group (8 rats for each group). Tail-suspension rats have been widely used as a

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model to simulate microgravity effects [14, 17]. Briefly, the rats were individually housed in a special tail-suspension cage with their tail suspended in the center of the cage and allowed only

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the forelimbs to touch the bottom of the cage; and the rat’s body was inclined at approximately 30° from the ground. For the control group, rats were kept exactly at the same condition with the

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tail-suspensions except their tails do not need to hang up. All animals had free access to water and

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food during entire process. On the 28th day, the rats were sacrificed, and the hippocampus were removed immediately. The samples were placed in liquid nitrogen for later determination. Morris water maze (MWM) test. The MWM was adapted for rats from the prototype originally

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described by Morris with some modifications [18, 19]. Briefly, The MWM test was performed after 28 days tail-suspension. The maze consisted of a circular pool filled with water (~24°C) that

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was made opaque with non-toxic black pigment. An escape platform was placed in the third quadrant of the maze and 5 cm below the water surface. Colorful, distinguishing, geometric clues with magnetic were affixed at particular visible locations to the rats while in the maze. Rats were trained on a total of 12 trials for 4 days, with 3 trials per session and 1 session per day. Prior to the first training trial, rats were given an ordinary habituation trial without the platform. Trials were 120s and rats that did not find the platform within that time were guided to the destination by the experimenter. Once on the top of the platform, rats were left for an extra 10s before being removed. Start locations (the first, second and fourth quadrant) were pseudo-randomized so that each outset was used fair-minded. On the fifth day, cognitive function was assessed in probe trials

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wherein the platform was removed and rats searched the empty pool for 90s. Behavioral tests were performed during the daytime and recorded by a video camera. iTRAQ(isobaric tags for relative and absolute quantitation)-based proteomics analysis. Protein extraction. Hippocampus sample of each animal was ground into powder in liquid nitrogen and extracted with lysis buffer (8 M urea, 1% DTT, PMSF) and sonicated at 400 W for 30

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times (1s for each time). After centrifugation at 4℃, 40000g for 30min, the supernatant was collected and mixed well with 6×volume of chilled acetone and incubated at -20℃ for at least 1h.

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After centrifugation at 4℃, 40000g for 5min, the supernatant was discarded, and the precipitate was dissolved with 20l dissolution buffer (iTRAQ Reagent Multi-Plex Kit, Applied Biosystems).

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Protein concentrations were quantified by Bradford method (Pierce™ Coomassie (Bradford) Protein Assay Kit, ThermoFisher). Protein processing and iTRAQ labeling. Total protein (100g)

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was taken out of each sample and mixed well with 1% SDS (1l), and 2l reducing reagent was added and incubated at 60℃ for 1h. Subsequently, cysteine blocking reagent was added and

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incubated for 10min under room temperature, and then digested overnight at 37℃ by trypsin with an enzyme/protein ratio of 1:50. The sample was then labeled with iTRAQ reagent as follows: the

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control group, labeled with iTRAQ reagent 114 and 115; the tail-suspension group, labeled with

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iTRAQ reagent 116 and 117. First dimensional high-PH RPLC separation. The iTRAQ labelled peptide mixture was fractionated using a high-pH RPLC column from Agela (250×4.6 mm i.d, C18, 5μm). The samples were loaded onto the column in buffer A1 (2% ACN+98% H2O, pH = 10)

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and eluted by buffer B1 (98% ACN+2% H2O, pH = 10, flow rate = 0.7 mL/min) with a gradient of 5%-35% B1 for 30min, 35%-95% B1 for 2min, 95% B1 for 5min, 95%-5% B1 for 2min, 5% B1

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for 6min. The absorbance at 214 nm was monitored, and a total of 24 fractions were collected and dried by a rotary vacuum concentrator. LC-MS/MS analysis. Each sample was resuspended in buffer A2 (1.9% ACN+98% H2O+0.1% FA) and centrifuged at 12000g for 3min. 10l supernatant was loaded onto an Eksigent Nano LC 2D plus HPLC by the autosampler onto a 5μm C18 trap column (ID100μm, 20mm length). The peptides were then eluted onto a 3μm analytical C18 column (ID75μm, 120mm length) packed in-house. Separation was run at 330 nl/min starting from 5% B2 (98% ACN+1.9% H 2O+0.1% FA), followed by stepwise gradient (8% B2 for 5min, 22% B2 for 34min, 32% B2 for 41min, 90% B2 for 42min), maintenance at 90% B2 for another 46min, and finally a return to 5% B2 for 1min. Data acquisition was performed

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with a TripleTOF™ 5600 mass spectrometer (AB Sciex). Data were acquired using an ion spray voltage of 2.0kV, mass range 300-1400Da, and capillary temperature of 320°C. Raw data files acquired from the TripleTOF 5600 System were converted into MGF files using Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, USA). Proteins were identified using Mascot server engine (Matrix Science, London, UK; version 2.3.02) uniprot-rat database.

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Bioinformatics analysis. To determine possible biological functions of DEPs, Gene Ontology (GO) analysis was performed in biological process, cellular component, and molecular function

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using DAVID Bioinformatics Resources. The protein-protein network analysis of the DEPs was performed by the “Analyze Network Algorithm” provided by MetacoreTM sofware. Pathway

identify significantly enriched pathways in DEPs.

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maps of MetaCore and PANTHER classification system (http://www.pantherdb.org/) are used to

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Western blot. Hippocampus were collected and lysed in RIPA Lysis Buffer (Cat. #C1053). The lysates were incubated for 10min on ice, centrifuged for 10min at 12000g at 4℃, and supernatant

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was collected. The total protein concentration was determined using the Pierce○BCA Protein R

Assay Kit (Cat. #OC185139.). The resolved proteins were transferred to a pvdf membrane

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(Millipore). After blocking in 5% skimmed milk at room temperature for 1h, the membranes were

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incubated in 5% BSA with primary antibodies against GluR1 (CST, #13185, 1:1000), GluR4 (CST, #3824, 1:1000), β-Actin (CST, #3700, 1:1000) at the lysates were subjected to 10% SDS-PAGE

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followed by immunoblotting.

Biochemical analysis of neurotransmitters. The level of Glu, GABA, 5-HT, DA, acetylcholine

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(Ach), noradrenalin (NE) and E were determined by using LC-MS/MS system (Shimadzu Prominence UFLC connected with an Applied Biosystem 5500 Q-Trap mass detector). Briefly, hippocampus samples were homogenized in 0.1ml precooled normal saline (NS) containing 0.2% formic acid (FA), followed by mixed with 0.2ml ice-cold acetonitrile and incubated at 4℃ for 30min. After centrifuged at 4℃, 12000g for 15min, the supernatant was collected and injected into LC-MS/MS system for analysis. The neurotransmitters and internal standard were detected in multiple reactions monitoring (MRM) mode. The MRM transitions and the related optimized declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) for the analytes are shown in Table1. Ratios of the peak areas of the analyte vs.

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the internal standard were used to quantify the neurotransmitter concentrations. Statistical analysis. All data were presented as mean ± SEM per experimental condition, and P<0.05 was considered statistically significant. The statistical analyses were performed using SigmaPlot 12.0. The MWM data were evaluated with two-way analysis of the variance (ANOVA) followed by Bonferroni post-test. For all other experiments, statistical significances were tested by

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the one-way ANOVA or the Student’s t-test.

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Results Long-term SM impairs spatial learning and memory To evaluate the effects of long-term SM on hippocampus-dependent learning and memory function, we measured 28-day tail-suspension rats using the MWM, a test of spatial learning and memory. The results indicated that both SM and control rats learned the task over this 4 days

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training period, manifested by spending less time and swimming shorter distances to find the hidden platform (Figure1.A-B). Interestingly, analysis of latency to platform on day 3 and day 4 of

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training showed that SM rats needed more time than control rats to find the hidden platform (p<0.05; Figure1.A). SM rats swam significantly longer distances than control rats to find the

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hidden platform on day 3 and day 4 (p<0.05; Figure1.B). In addition, we also found there was no significant difference in the swimming speeds between tail-suspension rats and the control rats

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(Figure1.C). In the probe test, 1day after training (day5), SM rats exhibited significantly poorer memory to locate the original position of the removed platform, because they spent less time in

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the target Q3 quadrant searching the hidden platform compared with the controls (p<0.05; Figure1.D-E). These data suggest that long-term simulated microgravity may impair spatial

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learning and memory.

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Overview of proteome changes in hippocampus of SM rats We next investigated the effect of SM on the expression of proteins in hippocampus of rats using iTRAQ-based proteomics analysis. In both control and SM condition, a total of 4774 proteins

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were quantified (with FDR<0.01). To determine DEPs between SM rats and control rats, the correlation coefficient between two biological replicates was evaluated firstly. The two controls

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(i.e. tags 114 and 115) were used as denominators, respectively. Only the proteins that were quantified with iTRAQ ratios were used to calculate correlation coefficient, and the ratios (SM vs. control) were then log-transformed and plotted against each other. As shown in Figure2, the correlation coefficients between two biological replicates were 0.84 (P<2.2E-16) and 0.87 (P<2.2E-16), thereby indicating the biological reproducibility of SM-regulated protein expression. Further, the biological variations between two replicates were estimated to set an optimal cut-off value of fold change for determining significantly altered differential protein expression levels. A fold change cutoff of ≥1.5 or ≤0.67 was defined as indicating up- or down-regulation, and only proteins, which were identified by three or more peptides and had >1.5-fold change in at least two

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of four comparisons (i.e. tag 116/114, 117/114, 116/115 and 117/115 for SM samples vs. the controls), were considered to be DEPs. The up-regulated and down-regulated DEPs were summarized in Table 2 and Table 3, respectively. For further understanding the mechanisms of spatial learning and memory impairment induced by SM, Gene Ontology (GO) classification and pathway enrichment of DEPs were performed. The

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result indicated that 28 days of SM might affect the metabolism function, cytoskeleton system, synaptic transmission, apoptosis, etc. According to GO classification, the DEPs involved in

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oxidative phosphorylation (GO: 0006119), generation of precursor metabolites and energy (GO: 0006091), negative regulation of cytoskeleton organization (GO: 0051494), regulation of action

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potential in neuron (GO: 0019228), regulation of cytoskeleton organization (GO: 0051493), negative regulation of actin filament polymerization (GO: 0030837), regulation of action potential

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(GO: 0001508), transmission of nerve impulse (GO: 0019226), regulation of membrane potential (GO: 0042391), regulation of synaptic transmission (GO: 0050804) and other biological processed

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(Table 4, more details see supplemental material). DEPs were mainly located in proton-transporting two-sector ATPase complex (GO: 0016469), proton-transporting V-type

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ATPase complex (GO: 0033176), respiratory chain (GO: 0070469), mitochondrion (GO:

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0005739), cytoskeleton (GO: 0005856), cytoskeletal part (GO: 0044430), microtubule (GO: 0005874), intermediate filament cytoskeleton (GO: 0045111), microtubule associated complex (GO: 0005875), voltage-gated sodium channel complex (GO: 0001518) (Table 5, more details see

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supplemental material). DEPs in molecular function are embodied in ATPase binding (GO: 0051117), ATPase activity (GO: 0016887), voltage-gated sodium channel activity (GO: 0005248)

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(Table 6, more details see supplemental material). The MetaCore pathway map analysis of DEPs was used to identify the 10 most statistically significant pathways, based on calculated P values (Table 7). As shown in Table 7, the DEPs involved in oxidative phosphorylation, ATP metabolism, and ATP/ITP metabolism. Moreover, two of ten MetaCore pathways were found to be directly associated with neurophysiological process, which is the neurophysiological process GABA-A receptor life cycle and neurophysiological process_dynein-dynactin motor complex in axonal transport in neurons (Figure 3A&B.). Additionally, protein-protein interactions were noted among all the DEPs (Figure 3C). SM exposure increases GluR1 and GluR4 expression in hippocampus

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Glutamate receptors (GluRs) play an important role in synaptic plasticity (long-term potentiation & long-term depression), which is directly involved in the process of learning and memory [20, 21]. The memory impairment observed in tail-suspension rats might be related with abnormal expression of GluRs, at least in part. Firstly, based on the preliminary PANTHER pathway analysis, DEPs were significantly enriched in metabotropic glutamate receptor group III pathway

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(P00039) and ionotropic glutamate receptor pathway (P00037) (Table 8). Secondly, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid selective glutamate receptor complex

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(GO: 0032281) were found to be enriched in cellular component of DEPs (Table 5.). We therefore validated the expression of P19490 (Gria1 or GluR1) and P19493 (Gria4 or GluR4) which were

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functionally involved in metabotropic glutamate receptor group III pathway and ionotropic glutamate receptor pathway (Table 8) by western blot. Considered with the result of proteome, the

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higher levels of GluR1 and GluR4 were observed in hippocampus after 28 days of tail-suspension (Figure 4).

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SM exposure disturbs neurotransmitter release

Next, the concentrations of Glu, 5-HT, DA, GABA, Ach, E and NE in SM group and control

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group were analyzed (Figure 5). This metabolites analysis was carried out using student-t tests. A

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significant difference was observed after exposure to SM for 28 days, suggesting that exposure to SM may lead to neurotransmitters variations in rat hippocampus. As shown in Figure 5A, the concentration of Glu was significantly up-regulated after SM exposure (p<0.05) (Figure 5A). On

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the contrary, concentrations of 5-HT, DA, GABA, and E were significantly down-regulated by SM (p<0.05) (Figure 5B-E). In addition, there is non-significant change in concentrations of Ach and

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NE (Figure 5F-G).

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Discussion The investigation of the physiological and pathological changes induced by spaceflight or microgravity will be critical in developing countermeasures to ensure astronauts to complete spaceflight mission effectively and return to earth safely. It has been demonstrated the spaceflight may lead to impairments in cognitive function [3, 22, 23]. Human cognitive performance is an

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important factor for the success of the spaceflight mission. How microgravity affects cognitive function need to be investigated since highly effective countermeasures might be required.

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In present study, 30° tail-suspension rat model was typically employed to simulate microgravity, and we observed 28-day tail-suspension induced spatial learning and memory deficit in rats during

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MWM test. Spatial memory refers to the part of the memory system that encodes stores, recognizes and recalls spatial information about one's environment and spatial orientation. The

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MWM was adapted for rats from the prototype formerly described by Morris[18]. It was also reported that 14 days of SM significantly decreased the escape latency and swimming distance of

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rats compared with the controls[24], suggesting that SM might lead to impairment in spatial learning and memory. This finding is consistent with our present study as shown in Figure 1A&B.

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Swimming speed is related to escape latency and swimming distance. It was established that SM

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model (rat hindlimb uploading) causes rat hindlimb muscle atrophy[25]. In order to eliminate the interference of muscle atrophy on swimming in tail-suspension rats, we therefore compared the swimming speed of the two groups, so as to make more accurate evaluation of spatial learning and

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memory ability. Our results showed there was no significant change in swimming speed after 28 days of SM exposure, indicating the muscle atrophy of rat hindlimb could not disturb the results of

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the escape latency and swimming distance during MWM test . Hippocampus is closely related to spatial learning and memory function in the brain. We therefore investigate the effect of 28 days SM on the protein expression in hippocampus. Based on iTRAQ-based proteomics analysis, 75 proteins were found overexpressed and 72 proteins were found lower expressed in hippocampus of rats after 28 days of SM exposure in comparison to the controls (Table 2&3). Learning and memory are encoded within the mammalian brain as biochemical and physical changes at synapses that alter synaptic transmission, a process known as synaptic plasticity. Synaptic plasticity could be regulated by altering the efficacy of neurotransmitter release or by changing the numbers and properties of neurotransmitter

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receptors[26]. Therefore, neurotransmitters and its specific receptors mediated pathways play an essential role in the processes of learning and memory [27-29]. Disturbance of neurotransmitter metabolism plays an important role in cognitive deficits induced by variety of neurological diseases or noxious stimuli [28-33]. Further bioinformatics analysis of proteomics data showed many DEPs were specifically associated with synaptic transmission, and the results of LC-MS/MS displayed the concentration of Glu was significantly increased after SM exposure while the

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concentrations of 5-HT, DA, GABA, and E were significantly decreased. These results suggested

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that spatial learning and memory impairment induced by 28 days of SM might be related to specific neurotransmitters level.

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Glu acting on Amino-acid-3-hydroxy-5-methyl-isoxazol-4-propionic acid (AMPA) receptors mediates most fast excitatory synaptic transmission in the brain [26, 34, 35]. Regulating the

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numbers or properties of AMPA receptors is crucial to excitatory synaptic transmission and the consequent formation of appropriated neural connections during learning and memory[36].

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Normally, Glu released into the synapse binds to its GluRs, which results in induction of long-term potentiation (LTP) and synaptic strengthening, and then contributes to learning and

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memory formation. However, excessive production and release of Glu was reported to cause

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excitotoxic cell death due to overstimulation GluRs [37-40]. AMPA receptor is a hetero-oligomeric protein complex consisting of combinations of four different kinds of subunits GluR1- GluR4, also known as GluRA-GluRD, each encoded by a separate gene: gria1-gria4[41,

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42]. The important role of AMPA on learning and memory has been studied in numerous studies [21, 43-45]. As well, the excessive activation of AMPA receptors has been demonstrated to be

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potently toxic for the brain [46, 47], including GluR1 and GluR4 [42, 48, 49]. In present study, we found the expression of GluR1 and GluR4 were significantly up-regulated by SM exposure (Figure 4). Excitotoxicity is a major cause of neuronal death in numerous neurological disorders including ischemia, traumatic brain injury and neurodegenerative disease [39, 50, 51]. Studies have reported that increased apoptosis can be found in hippocampus after 7 or 14 days of SM [10, 16]. We previously investigated the effects of 28-day SM on neuronal morphology, expression of apoptosis-related molecules in rat hippocampus. The results indicated that the numbers of hippocampal neurons were decreased after 28 days of simulated microgravity compared with the controls, accompanied by elevated expression of apoptosis-related proteins[52]. Furthermore, the

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pathways analysis in the present study showed the DEPs exerted functions in apoptosis signaling pathway (P00006) (Table 8). All these results implied that 28 days of SM exposure might result in excitotoxicity in hippocampus, which might be one of mechanisms of cognitive dysfunction induced by SM. DEPs were involved in a wide range of biological processes, suggesting the impact of SM on

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learning and memory was complex and multifaceted. The malfunction of other biological process may also be involved in learning and memory processes during SM exposure. The cytoskeleton is

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the main structure giving shape and mechanical strength to cells. It is now widely accepted that cytoskeleton plays a role in sensing changes in gravity[53]. Cytoskeleton changes induced by

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microgravity were found in various cell types, including hippocampus neuron cell [11, 53]. Cytoskeleton is associated with the changes of cell shape, function and signaling. Emerging

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evidence suggested that various high level brain functions depend on the generation of neuronal networks involved the establishment, maintenance, and repairing of synaptic contacts, such as

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cognitive function [54-56]. Neuronal cytoskeleton is not only required for establishment and maintenance of synaptic contacts but also provides the structural basis for synaptic transmission

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[54-56]. In addition, disruption of synaptic function has been implicated in a wide range of

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neurodevelopmental and neurodegenerative diseases, such as AD [54-56]. In present study, based on GO cellular component analysis, most DEPs were located in cytoskeleton, such as cytoskeleton (GO: 0005856), cytoskeletal part (GO: 0044430), and microtubule (GO: 0005874), et al. More

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details see Figure 5 and supplemental material. These results imply that hippocampus dysfunction induced by SM may be related to the SM-associated cytoskeleton changes.

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In summary, our data demonstrated that long-term SM exposure impaired the spatial learning and memory function. Based on proteomics analysis, a total of 147 proteins were found to be differentially expressed in 28-day SM rats’ hippocampus. GO function ontology classification indicated that DEPs were enriched in 41 GO biological processes, 11 GO molecular functions, 25 GO cellular component and 16 PANTHER pathways. It was also shown the expression of GluR1 and GluR4 were up-regulated, and the level of Glu was increased, while the concentration of 5-HT, DA, GABA, and E were declined after28 days of SM exposure. All of these results suggest that SM exposure may lead to comprehensive biological effects on hippocampus. The impairment of learning and memory function induced by SM is a complicated process, and one of the possible

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mechanisms might be related with glutamate excitotoxicity and imbalances in specific

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neurotransmitters.

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Acknowledgements: This work was supported by the National Major Scientific Instrument and Equipment Development Project (2013YQ19046706, 2012YQ0401400901); Foundation of China Space Medicine Engineering Advanced Research (2015SY54A0501); National Natural Science Fund (31401010); Shenzhen Science & Technology Institute (20150629164441050); and Foundation of

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State Key Laboratory of Space Medicine Fundamentals and Application (SMFA15B01). Conflict of Interest:

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We declare that we have no conflict of interest.

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Medicine& Medical Engineering. 2016;29:235-9. [53] Vorselen D, Roos WH, MacKintosh FC, Wuite GJ, van Loon JJ. The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2014;28:536-47. [54] Stefen H, Chaichim C, Power J. Regulation of the Postsynaptic Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its Disruption in Disease. 2016;2016:2371970. [55] Kevenaar JT, Hoogenraad CC. The axonal cytoskeleton: from organization to function. Neural plasticity. 2015;8:44. [56] Bodaleo FJ, Gonzalez-Billault C. The Presynaptic Microtubule Cytoskeleton in Physiological and

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Pathological Conditions: Lessons from Drosophila Fragile X Syndrome and Hereditary Spastic

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Paraplegias. Frontiers in molecular neuroscience. 2016;9:60.

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Figure legends: Figure1. Tail-suspension rats show spatial learning and memory deficits in the Morris water maze test. A, Escape latencies of tail-suspension and control rats. B, Swimming distance of tail-suspension and control rats. C, swimming speed did not differ among the SM and control group during the four sessions. D, Path length in target quadrant (%) of tail-suspension and control

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tail-suspension and control rats in the probe tests. (n=6, *p<0.05).

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rats. E, Representative Morris water maze program generated-swimming tracing pattern of

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Figure2. Correlation coefficients of biological replicates. The ratios of quantified proteins were

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log-transformed and plotted between two biological replicates.

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Figure3. Overview of proteomics in hippocampus of SM rats. A, Neurophysiological process: dynein-dynactin motor complex in axonal transport in neurons. B, Neurophysiological process:

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GABA-A receptor life cycle. C, Protein-protein interaction network of the DEPs.

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Figure4. GluR1 and GluR4 expression in rat hippocampus was induced by SM. A, GluR1 and GluR4 level in hippocampus of control and SM rats was quantified by western blot normalized to β-actin level (GluR1 180KD, GluR4 200 KD, β-actin 40KD). B&C. Quantitative analysis of

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western blot (n=4, *p<0.05, **p<0.01).

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Figure5. Contents of neurotransmitters in SM and control groups. A. Increases in the content of Glu under SM (g/g). B. Decreases in the content of GABA under SM (g/g). C. Decreases in the content of 5-HT under SM (ng/g). D. Decreases in the content of DA under SM (ng/g). E. Decreases in the content of epinephrine under SM (ng/g). F. non-significant change in the content of Ach under SM (ng/g). G. non-significant change in the content of noradrenaline under SM

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(ng/g). (n=5,*p<0.05, **p<0.01).

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Table 1. Multiple reaction monitoring (MRM) transitions, DP (declustering potential), EP (entrance potential), CE (collision energy) and CXP (collision cell exit potential) of the analytes. Q1 → Q3 (m/z)

DP(V)

EP(V)

CE(eV)

CXP(V)

DA E NE 5-HT Ach GABA Glu

154 → 137 184 → 166 170 → 152 177 → 160 146 → 87 104 → 87 148 → 84

30 40 95 65 160 20 20

12 10 12 12 10 10 10

15 12 15 10 20 25 40

40 15 10 8 30 8 9

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Compounds

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Table 2. Up-regulated proteins identified by iTRAQ technology after 28 days of SM exposure in hippocampus. Accession

Protein names Mannose-P-dolichol utilization defect 1

D3Z8Z7

Protein Ankdd1a (Fragment)

D3ZM97

Olfactory receptor

D3ZZQ8

Protein RGD1561381

E9PSV8

Neuronal membrane glycoprotein M6-b

F1LZG6

Protein Lrba (Fragment)

M0R451

Glyceraldehyde-3-phosphate dehydrogenase

M0R8M1

Uncharacterized protein (Fragment)

M0R907

Protein Snrpd3

P19493

Glutamate receptor 4

P35467

Protein S100-A1

P54900

Sodium channel subunit beta-2

P63081

V-type proton ATPase 16 kDa proteolipid subunit

P63155

Crooked neck-like protein 1

P80431

Cytochrome c oxidase subunit 7B, mitochondrial

Q1HL14

Longevity assurance-like protein 1

Q5FVN2

Transmembrane protein 41B

Q66H18

Protein Sypl1

Q6IRG7

Claudin

Q7M730

Sodium channel subunit beta-4

Q7TNK0

Serine incorporator 1

Q80W89

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11

D4AC70

Procollagen, type VIII, alpha 1 (Predicted)

F1LNN1

Proteasome subunit beta type

F1MAM6

Protein Dnah8

M0R5G5

Protein Pkd1l2

P00159

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D

PT E

CE

O54715

PT

D3Z865

V-type proton ATPase subunit S1 Cytochrome b Solute carrier family 2, facilitated glucose transporter member 1

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P11167 Q05140

Clathrin coat assembly protein AP180

Q09426

2-hydroxyacylsphingosine 1-beta-galactosyltransferase

Q64244

ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1

A0A0A0MY37

Leukocyte surface antigen CD47

D3ZPU3

Estradiol 17-beta-dehydrogenase 12

D3ZXG2

Protein Armc4

D4ABE8

Uncharacterized protein

D4ABI7

Protein LOC100365676

D4ABK1

Protein Syngr3

D4ADV4

Sodium-driven chloride bicarbonate exchanger

F1LMZ4

Ribosome-releasing factor 2, mitochondrial

F1LPA7

Tetraspanin (Fragment)

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Continued Table 2 Accession

Protein names Choline transporter-like protein 1 (Fragment)

F1LRV4

Heat shock 70 kDa protein 4

F1M9V3

Enolase (Fragment)

G3V879

Ubiquinone biosynthesis protein COQ7 homolog

G3V8Y3

Kinesin-like protein

G3V912

Protein Tmx4

G3V9W6

Aldehyde dehydrogenase

M0R8J4

Protein Wdr87 (Fragment)

M0R9I6

Aminomethyltransferase

M0RBE5

Neuritin (Fragment)

O35795

Ectonucleoside triphosphate diphosphohydrolase 2

P01048

T-kininogen 1

P05508

NADH-ubiquinone oxidoreductase chain 4

P09006

Serine protease inhibitor A3N

P13852

Major prion protein

P19490

Glutamate receptor 1

P34926

Microtubule-associated protein 1A

P38650

Cytoplasmic dynein 1 heavy chain 1

P47863

Aquaporin-4

P61805

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1

Q3SWT7

Nuclear receptor binding protein

Q498C8

Protein RER1

Q4QQW8

Putative phospholipase B-like 2

Q568Z4

Signal peptidase complex subunit 3

Q5EBD0

SEC14-like 2 (S. cerevisiae)

Q5U2M9

Protein Rfx3

Q63910

Alpha globin

Q68FW4

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D

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Protein Samd8 Syntaxin-18 Biliverdin reductase A

AC

Q6AZ33

CE

Q641X0

PT

F1LPF3

Q6EIX2

Mitochondrial import inner membrane translocase subunit TIM16

Q8CFD0

Sideroflexin-5

Q8SEZ0

NADH-ubiquinone oxidoreductase chain 5

Q99MS0

SEC14-like protein 2

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Table 3. Down regulated proteins identified by iTRAQ technology after 28 days of SM exposure in hippocampus. Accession

Protein names Protein Fsip2

A0JN27

General transcription factor IIH subunit 2

B0K010

Protein Txndc17

B2GUV5

ATPase, H transporting, lysosomal V1 subunit G1

D3ZPI7

Glyceraldehyde-3-phosphate dehydrogenase

D3ZS15

Uncharacterized protein

D3ZWL9

Protein Dido1

D4A6P0

Protein LOC100359747

D4A783

Uncharacterized protein

D4A7I6

Protein RGD1309995

D4A7Q3

Protein LOC685888

E9PTN4

Protein Srpk1 (Fragment)

F1LMQ2

Farnesyl pyrophosphate synthase

F1LPU4

Choline O-acetyltransferase

F1LWR6

Protein Mlip (Fragment)

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Positive cofactor 2, multiprotein complex, glutamine/Q-rich-associated protein (Predicted), isoform CRA_a

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G3V684

PT

A0A096MJX4

Uncharacterized protein (Fragment)

O35314

Secretogranin-1

P02625

Parvalbumin alpha

P19132

Ferritin heavy chain

P60841

Alpha-endosulfine

P62329

Thymosin beta-4

Q03344

ATPase inhibitor, mitochondrial

Q1KQ07

Signal transducer and activator of transcription

Q505I4

UPF0235 protein C15orf40 homolog

Q5RKI9

Protein FRA10AC1 homolog Ribosome-recycling factor, mitochondrial 28 kDa heat- and acid-stable phosphoprotein

AC

Q62785

CE

Q5FVF1

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D

M0RCM1

Q64350

Translation initiation factor eIF-2B subunit epsilon

Q66H40

High mobility group nucleosome-binding domain-containing protein 3

Q6AZ25

Tropomyosin 1, alpha

Q6P756

Adaptin ear-binding coat-associated protein 2

Q925G0

RNA-binding protein 3

Q9EPJ0

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1

Q9WV63

Kinesin-like protein KIF2A

B1WBQ7

DNA mismatch repair protein Msh2

D3ZC13

Olfactory receptor

D3ZFJ6

Lactamase, beta (Predicted)

D3ZUZ5

Ferritin

D4AB12

Uncharacterized protein

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Continued Table 3 Accession

Protein names Angel homolog 2 (Drosophila) (Predicted), isoform CRA_a

F1M9F6

Protein Gbp5

F1M9F9

Jouberin

G3V8R0

Protein RGD1311703

M0RBL8

Protein Tceal6

P62634

Cellular nucleic acid-binding protein

Q63638

Striated muscle-specific serine/threonine-protein kinase

Q6IG03

Keratin, type II cytoskeletal 73

Q6J2U6

E3 ubiquitin-protein ligase RNF114

Q8K3F3

Protein phosphatase 1 regulatory subunit 14B

B1WC50

Ewsr1 protein

D4ADP2

Protein Ube2q2l

F1LSK5

Protein Heatr5a

F1LUC1

Protein Fam13c

F1M208

Protein Piezo2

F1M2K7

Enolase (Fragment)

F1MAQ5

Microtubule-associated protein

F7FC39

Protein Spta1

M0RCH6

Protein Chmp4b

O35820

2'-deoxynucleoside 5'-phosphate N-hydrolase 1

P15865

Histone H1.4

P35745

Acylphosphatase-2

P46844

Biliverdin reductase A

P50617

Dendrin

Q4V898

RNA-binding motif protein, X chromosome

Q6ED65

Echinoderm microtubule-associated protein-like 5

Q6IFW6

Keratin, type I cytoskeletal 10

Q6IG04

Keratin, type II cytoskeletal 2 epidermal Keratin, type II cytoskeletal 72 FK506-binding protein-like

AC

Q6MG81

CE

Q6IG02

PT E

D

MA

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SC

RI

PT

F1LT13

Q76MV3

Cytochrome C oxidase assembly protein COX17

Q9Z339

Glutathione S-transferase omega-1

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%

PValue

GO:0045785~positive regulation of cell adhesion

4.31

6.96E-04

GO:0030155~regulation of cell adhesion

5.17

1.18E-03

GO:0051494~negative regulation of cytoskeleton organization

3.45

2.84E-03

GO:0010639~negative regulation of organelle organization

3.45

8.33E-03

GO:0019228~regulation of action potential in neuron

3.45

9.86E-03

GO:0006119~oxidative phosphorylation

3.45

1.15E-02

GO:0006811~ion transport

9.48

1.24E-02

7.76

1.58E-02

6.90

1.69E-02

3.45

1.70E-02

2.59

1.78E-02

2.59

2.35E-02

6.90

3.00E-02

3.45

3.55E-02

6.03

3.56E-02

GO:0043933~macromolecular complex subunit organization

7.76

3.62E-02

GO:0055082~cellular chemical homeostasis

6.03

3.76E-02

5.17

3.93E-02

2.59

4.01E-02

2.59

4.01E-02

GO:0015672~monovalent inorganic cation transport

5.17

4.30E-02

GO:0048878~chemical homeostasis

6.90

4.39E-02

4.31

4.41E-02

GO:0051129~negative regulation of cellular component organization

3.45

5.09E-02

GO:0000041~transition metal ion transport

2.59

5.46E-02

GO:0044271~nitrogen compound biosynthetic process

5.17

6.67E-02

GO:0065003~macromolecular complex assembly

6.90

6.80E-02

GO:0051249~regulation of lymphocyte activation

3.45

7.01E-02

GO:0009150~purine ribonucleotide metabolic process

3.45

7.12E-02

GO:0006091~generation of precursor metabolites and energy

4.31

7.28E-02

GO:0015992~proton transport

2.59

7.63E-02

GO:0009259~ribonucleotide metabolic process

3.45

7.71E-02

GO:0006818~hydrogen transport

2.59

7.82E-02

GO:0042391~regulation of membrane potential

3.45

8.08E-02

GO:0006164~purine nucleotide biosynthetic process

3.45

8.70E-02

GO:0030837~negative regulation of actin filament polymerization

1.72

8.88E-02

GO:0002694~regulation of leukocyte activation

3.45

9.08E-02

GO:0044270~nitrogen compound catabolic process

2.59

9.21E-02

GO:0050804~regulation of synaptic transmission

3.45

9.60E-02

GO:0034621~cellular macromolecular complex subunit organization

4.31

9.85E-02

GO:0042592~homeostatic process

7.76

9.90E-02

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Term

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Table 4. GO biological process of DEPs (DAVID).

GO:0006812~cation transport GO:0050801~ion homeostasis GO:0043242~negative regulation of protein complex disassembly GO:0019725~cellular homeostasis GO:0051493~regulation of cytoskeleton organization

GO:0008366~axon ensheathment

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GO:0007272~ensheathment of neurons

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GO:0019226~transmission of nerve impulse

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GO:0006873~cellular ion homeostasis

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GO:0043244~regulation of protein complex disassembly

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GO:0001508~regulation of action potential

AC

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GO:0006163~purine nucleotide metabolic process

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Table 5. GO cellular component of DEPs (DAVID). %

PValue

GO:0016469~proton-transporting two-sector ATPase complex

3.45

6.94E-03

GO:0005856~cytoskeleton

12.93

7.00E-03

GO:0033176~proton-transporting V-type ATPase complex

2.59

9.03E-03

GO:0045095~keratin filament

3.45

9.29E-03

GO:0044430~cytoskeletal part

10.34

1.11E-02

GO:0031090~organelle membrane

12.07

1.69E-02

GO:0032281~alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

1.72

2.86E-02

4.31

3.09E-02

5.17

3.62E-02

3.45

3.95E-02

3.45

4.24E-02

2.59

4.53E-02

9.48

5.13E-02

5.17

5.40E-02

4.31

5.43E-02

6.03

5.80E-02

6.03

6.10E-02

12.93

6.32E-02

7.76

6.52E-02

5.17

6.68E-02

GO:0005875~microtubule associated complex

2.59

7.82E-02

GO:0016023~cytoplasmic membrane-bounded vesicle

6.90

8.17E-02

GO:0001518~voltage-gated sodium channel complex

1.72

8.35E-02

GO:0031982~vesicle

7.76

8.52E-02

6.90

9.74E-02

PT

Term

selective glutamate receptor complex GO:0005874~microtubule

RI

GO:0043025~cell soma GO:0005882~intermediate filament

SC

GO:0045111~intermediate filament cytoskeleton GO:0070469~respiratory chain GO:0005743~mitochondrial inner membrane GO:0005773~vacuole GO:0005740~mitochondrial envelope GO:0005739~mitochondrion GO:0031410~cytoplasmic vesicle

PT E

D

GO:0019866~organelle inner membrane

MA

GO:0031226~intrinsic to plasma membrane

NU

GO:0005783~endoplasmic reticulum

AC

CE

GO:0031988~membrane-bounded vesicle

ACCEPTED MANUSCRIPT 34

Table 6. GO molecular function of DEPs (DAVID). %

PValue

GO:0051117~ATPase binding

2.59

3.72E-03

GO:0016887~ATPase activity

6.03

8.63E-03

GO:0015078~hydrogen ion transmembrane transporter activity

3.45

2.29E-02

GO:0015077~monovalent inorganic cation transmembrane transporter activity

3.45

2.62E-02

GO:0003697~single-stranded DNA binding

2.59

3.84E-02

GO:0042803~protein homodimerization activity

5.17

5.90E-02

GO:0022890~inorganic cation transmembrane transporter activity

3.45

6.64E-02

2.59

7.42E-02

GO:0042625~ATPase activity, coupled to transmembrane movement of ions

2.59

7.61E-02

GO:0005248~voltage-gated sodium channel activity

1.72

8.26E-02

6.90

9.29E-02

PT

Term

RI

GO:0003777~microtubule motor activity

AC

CE

PT E

D

MA

NU

SC

GO:0042802~identical protein binding

ACCEPTED MANUSCRIPT 35

Table 7. Top ten maps given by MetaCore. Maps

Proteins

pValue

Oxidative phosphorylation

NDUFA11, COX VIIb-1, MT-ND5

1.033E-03

Neurophysiological process_Dynein-dynactin

PRNP, Dynein 1

4.847E-03

Ubiquinone metabolism

NDUFA11, MT-ND5

8.940E-03

Heme metabolism

BVRA, FTH1

1.682E-02

ATP metabolism

CD38, ENTPD2-alpha

1.775E-02

ATP/ITP metabolism

ACYP2, ENTPD2-alpha

2.383E-02

wtCFTR and deltaF508-CFTR traffic / Clathrin

AP180

3.835E-02

coated vesicles formation (normal and CF) SERPINA3 (ACT)

JAK-STAT pathway GluR1

SC

Translation_Role of Retinoic acid signaling in the initiation of translation Neurophysiological process_GABA-A receptor

Dynein 1

AC

CE

PT E

D

MA

NU

life cycle

RI

Development_Thrombopoetin signaling via

PT

motor complex in axonal transport in neurons

4.211E-02 4.211E-02 5.144E-02

ACCEPTED MANUSCRIPT 36

Table 8. Pathway analysis by PANTHER (http://www.pantherdb.org) Pathway

Proteins

Huntington disease (P00029)

F1MAM6,

% D3ZPI7,

21.40

D4AB12, 3ZPI7, F1M2K7, M0R451,

21.40

D4AB12,

M0R451, P38650,M0RCM1

Glycolysis (P00024)

M0RCM1, F1M9V3 P19490, P19493

7.10

Ionotropic glutamate receptor pathway (P00037)

P19490, P19493

7.10

Gonadotropin-releasing hormone receptor pathway (P06664)

P11167

3.60

JAK/STAT signaling pathway (P00038)

Q1KQ07

Blood coagulation (P00011)

P01048

Interleukin signaling pathway (P00036)

Q1KQ07

Apoptosis signaling pathway (P00006)

D3ZS15

3.60

Integrin signalling pathway (P00034)

D4AC70

3.60

RI

SC

Inflammation mediated by chemokine and cytokine signaling pathway (P00031)

NU

Vasopressin synthesis (P04395) ATP synthesis (P02721) CCKR signaling map (P06959)

MA

PDGF signaling pathway (P00047)

CE

PT E

D

EGF receptor signaling pathway (P00018)

AC

PT

Metabotropic glutamate receptor group III pathway (P00039)

3.60 3.60 3.60

Q1KQ07

3.60

Q568Z4

3.60

D3ZS15

3.60

Q64244

3.60

Q1KQ07

3.60

Q1KQ07

3.60

ACCEPTED MANUSCRIPT

NU

SC

RI

PT

37

AC

CE

PT E

D

MA

Fig. 1

ACCEPTED MANUSCRIPT

PT

38

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 2

ACCEPTED MANUSCRIPT

PT

39

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 3

ACCEPTED MANUSCRIPT

PT

40

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 4

ACCEPTED MANUSCRIPT

NU

SC

RI

PT

41

AC

CE

PT E

D

MA

Fig. 5

ACCEPTED MANUSCRIPT

SC

RI

PT

42

AC

CE

PT E

D

MA

NU

Graphical abstract

ACCEPTED MANUSCRIPT 43

Significance statement： The goal of sending astronauts farther into space and extending the duration of spaceflight missions from months to years will challenge the current capabilities of bioastronautics. The investigation of the physiological and pathological changes induced by spaceflight will be critical in developing countermeasures to ensure

PT

astronauts complete spaceflight mission accurately and effectively and return to earth safely. It has been demonstrated the spaceflight may lead to impairments in cognitive

RI

function which is crucial for mission success. Here we show that long-term simulated microgravity, the most potent environment risk factor during spaceflight, impairs the

SC

spatial learning and memory of rats and the underlying mechanism may be involved in glutamate excitotoxicity and imbalances in specific neurotransmitters release in

NU

hippocampus, providing new insight for the countermeasures of cognitive impairment

AC

CE

PT E

D

MA

during spaceflight.

ACCEPTED MANUSCRIPT 44

Highlights Long-term simulated microgravity(SM) impairs spatial learning and memory function of rats. 149 proteins were differentially expressed between SM group and the controls. SM up-regulated GluR1 and GluR4 expression and Glu release.

AC

CE

PT E

D

MA

NU

SC

RI

PT

The concentration of 5-HT, DA, GABA and E were down-regulated by SM.