- Email: [email protected]
iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference
Accepted Manuscript iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference Beibei Zhu, Xiangyu Li, Huan Chen, Hongjuan Wang, Xinchao Zhu, Hongwei Hou, Qingyuan Hu PII:
S0006-291X(17)30617-4
DOI:
10.1016/j.bbrc.2017.03.141
Reference:
YBBRC 37528
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 March 2017 Accepted Date: 26 March 2017
Please cite this article as: B. Zhu, X. Li, H. Chen, H. Wang, X. Zhu, H. Hou, Q. Hu, iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.03.141. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference Beibei Zhu, Xiangyu Li, Huan Chen, Hongjuan Wang, Xinchao Zhu, Hongwei Hou *, Qingyuan Hu *
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Email ID: [email protected], [email protected]
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China National Tobacco Quality Supervision and Test Center, Zhengzhou, 450001, China.
ACCEPTED MANUSCRIPT Abstract Repeated exposures to nicotine are known to result in persistent changes in proteins expression in addiction-related brain regions, such as the striatum, nucleus accumbens and prefrontal cortex, but
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the changes induced in the protein content of the hippocampus remain poorly studied. This study established a rat model of nicotine-induced conditioned place preference (CPP), and screened for proteins that were differentially expressed in the hippocampus of these rats using isobaric tags for
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relative and absolute quantitation labeling (iTRAQ) coupled with 2D-LC MS/MS. The
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nicotine-induced CPP was established by subcutaneously injecting rats with 0.2 mg/kg nicotine. Relative to the control (saline) group, the nicotine group showed 0.67- and 1.5-fold changes in 117 and 10 hippocampal proteins, respectively. These differentially expressed proteins are mainly involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function,
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long-term synaptic potentiation and nervous system development. Furthermore, RT-PCR was used to confirmed the results of the proteomic analysis. Our findings identify several proteins and cellular signaling pathways potentially involved in the molecular mechanisms in the hippocampus
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that underlie nicotine addiction. These results provide insights into the mechanisms of nicotine
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treatment in hippocampus.
Keywords: Nicotine, Conditioned place preference, Hippocampus, iTRAQ, proteomics
ACCEPTED MANUSCRIPT 1. Introduction Nicotine is a primary component in tobacco, endowed with reinforcing properties that is responsible for tobacco addiction [1]: Nicotine binds to nicotinic cholinergic receptors(nAChR),
specific and
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nAChR ultimately become desensitized, which results in long-term tolerance to nicotine in certain critical brain regions, including the ventral tegmental area, nucleus accumbens,
amygdala, and hippocampus. This tolerance is involved in the conversion from casual to
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compulsive drug use[2]. Increasing evidence supports the view that the hippocampus, a key
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modulator of learning and memory, functions as a crucial substrate of addiction[3]. The hippocampus is critical for the transformation of short-term memories into long-term memories in spatial and contextual learning, and it is also connected in a complex manner with several AChR-enriched brain regions in the limbic system [4, 5]. Previous work has demonstrated that
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nicotine exposure can alter multiple aspects of hippocampal function that might play a key role in nicotine addiction[6], but few studies to date have investigated the changes induced by nicotine in the expression of proteins involved in hippocampal function.
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Identification of the proteins that are associated with the development and maintenance of
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drug dependence is not only critical for understanding the molecular mechanisms underlying addiction, but also necessary for the development of new pharmacological means to reduce the use of the drugs [7]. Repeated exposure to an addictive drug can alter the levels or types of proteins expressed in specific brain regions [8], and such alterations have been previously investigated using various molecular techniques. Proteomics, which is characterized by the ability to measure dynamic changes in proteomes on a global scale, has emerged as an extremely powerful tool for discovery previously unrecognized molecular mechanisms underlying drug
ACCEPTED MANUSCRIPT addiction. Proteomics has been used over the past few years to profile the protein expression pattern in response to numerous substances of abuse such as amphetamine, alcohol, cocaine, and morphine [7]. However, very few proteomics studies have been published on nicotine addiction
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during chronic exposure to nicotine. Yeom et al [9] conducted a two dimensional electrophoresis (2DE) analysis and reported that chronic nicotine administration and withdrawal potently regulated the expression of 7 proteins in the striatum of male rats. In other comprehensive study,
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2DE was again used to analyze the protein expression profiles for five brain regions-the
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amygdala, nucleus accumbens, prefrontal cortex, striatum, and ventral tegmental area in rats that received nicotine for 7 days[10]. These results from proteomic analyses have provided insights into the mechanisms of nicotine treatment and withdrawal. However, to our knowledge, no study thus far has determined the changes in hippocampus proteins in the rat by using labeling relative
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and absolute quantification (iTRAQ) proteomics following nicotine-induce CPP. This study established a rat model of nicotine-induced CPP and identified proteins that were differentially expressed in the hippocampus of these rats by using iTRAQ coupled with 2D-LC
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expression.
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MS/MS. Moreover, RT-PCR was used to verified the significantly different changes in protein
2. Materials and Methods 2.1. CPP test
2.1.1. Animals
Male Sprague-Dawley rats (n = 24; Basic Medical College of Zhengzhou University), weighing 280–330g, were individually housed under a reversed 12:12-h light /dark cycle in a temperature and humidity controlled room at 21 ± 1°C and were handled daily. Care of the
ACCEPTED MANUSCRIPT animals was in accordance with National Institutes of Health guidelines and all procedures were approved by our institution’s animal safety committee. 2.1.2. Nicotine CPP training
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The CPP box (Med Associates) used here contained a white chamber and a black chamber featuring different flooring that provided two distinct contexts, and each served as either the white-paired or the black -paired side. A third gray chamber with a distinct floor was also present,
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and it connected the two other chambers and provided a neutral environment. An initial test of
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preference was conducted one day before CPP training by placing rats in the middle chamber of the CPP box for 15 min and allowing them to move freely between chambers, and the time spent in each chamber was recorded using standard MedPC software. Based on this test, 24 male rats were selected that did not exhibit a preferred side, and randomly divided these rats into four
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groups three of the groups received nicotine (N), and one received saline(S). The animals in the four groups were subcutaneously injected with 0.2, 0.4 or 0.6 mg/kg/day nicotine or saline on the first day of training, and all animals that received nicotine injections were placed in the
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nicotine-paired side and all animals that received saline injections were placed in the saline-paired
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side; access to other-paired chamber was blocked by a barrier. On the following day, all animals received saline injections and were placed in the saline-paired side. This procedure was repeated until Day 22. The preference testing was conducted again after the last day of training. All animals had access to both of the paired chambers from the neutral environment for 15 min during the test, and their movement was recorded. 2.2. iTRAQ proteome analysis 2.2.1. Protein preparation
ACCEPTED MANUSCRIPT Immediately after the CPP test, the rats that exhibited a significant difference in preference between N and S sides were sacrificed by means of decapitation and their brains were collected and transferred into ice-cold water. The hippocampus was dissected and stored at -80°C until
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further use. Hippocampus samples were homogenized with N-PRR neuronal protein extraction reagent (Promega) containing protease inhibitor phenylmethanesulfonyl fluoride (Sigma), and the protein concentration in each extracted sample was measured using a bicinchonic acid kit
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(Thermo). Equal amounts of protein from each rat were pooled according to the group, and the
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100 µg of the total protein from each group was processed as the manufacturer,s instruction for the iTRAQ reagent kit (Applied Biosystems, USA). For duplication, samples from N and S were labeled using 114 and 117 tags, 115 and 116 tags, respectively. 2.2.2. High pH reverse phase chromatography
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The pooled samples were subjected to strong cation exchanger fractionation by HPLC (Thermo) with an Extend-C18 analytical column (5 µm, 250×4.6 mm) as follows: flow rate, 0.4 mL/min; Solvent A, 10 mmol/L ammonium formate (pH10); solvent B, 80% acetonitrile (CAN)
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and 10 mmol/L ammonium formate (pH10). A 45 min gradient from 5% to 50% organic solvent
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was used to elute the peptides, after which the organic solvent was increased to 90% to wash the column; 12 fractions were collected and vacuum-dried. 2.2.3. Quantitative proteomic analysis Purified peptide fractions were reconstituted in mobile phase A (0.1% formic acid aqueous
solution) and separated in an analytical column (75 m×50cm, C18, 2 m; Thermo) by using a Nano-LC (Thermo)-Q-TOF MS (Bruker) system with a solvent gradient of 5-90% Buffer B (0.1% formic acid in acetonitrile) in mobile phase A, using flow rate of 0.3 mL/min. Total gradient length
ACCEPTED MANUSCRIPT was 95 min. MS date were acquired in the positive ion mode with a captive spray Nanoflow ES source, at a selected mass range of 50–2200 m/z, and peptide ions featuring +2 to +4 charge states and cycle time of 3s were subjected to MS/ MS. Automatic collision energy and automatic
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MS/MS accumulation were used to activate smart information dependent date acquisition. Data were processed by Mascot software (Version 2.4.1) and compared with the UniProt database (http://www.uniprot.org/). The search was performed using trypsin and allowing for two
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missed cleavages per peptide. Carbamidomethyl alkylation of cysteines was used as the fixed
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modification, and the variable modifications were oxidation of methionine, iTRAQ modifications of lysine, iTRAQ N-terminal and iTRAQ-tyrosine. Peptide and MS/MS tolerance were 20 ppm and 0.1 Da respectively. Proteins featuring unique peptides were identified and are reported with a cumulative confidence >95%.
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2.3. Bioinformatics analyses
Functional classifications were performed using GO (https://david.ncifcrf.gov/), and Pathway Alalysis was performed using KEGG (http://www.genome.jp/kegg/mapper.html). Functional
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protein association networks were constructed using STRING (http://string-db.org/).
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2.4. RT-PCR analyses
RT-PCR was performed on samples from rats in each group. Total RNA was extracted from
the hippocampus samples by using Trizol (Invitrogen) and then reverse transcribed by using Taq polymerase with the PrimeScript™ RT Reagent Kit with gDNA Eraser, according to the manufacturer’s instructions (Takara). All PCR amplifications were performed by using the real-time fluorescence detection method with First Start DNA Master SYBR Green (Roche, Germany) on a LightCycler 96 System (Roche). All primers were designed according to published
ACCEPTED MANUSCRIPT cDNA sequences by using primer selection software (http://tools.thermofisher.com/content.cfm). The specific primers and the annealing temperatures are shown in Table 1. The fold-change values were represented as means ± SEMs of mRNA expression normalized to GAPDH mRNA, and
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values were compared using ANOVA. Table 1 specific primers and annealing temperature
GAPDH-F
gggtgtgaaccacgagaaat
GAPDH-R
actgtggtcatgagcccttc
CHP1-F
gagactggcttttcccacag
CHP1-R
agtgggttgatggcaagttc
Acta2-F
gccctggattttgagaatga
Acta2-R
tgaaagagggctggaagaga
Slc32a1-F
acatcctggtcatcgcctac
Slc32a1-R
gatgccgatggagataggaa
Dst-F
gcgcctctgaagctctctta
Dst-R
tagttgcccgggttacaaag
Gabrg2-F
actcattgtggttctgtcctg
Gabrg2-R
gctgtgacataggagaccttg
Slc6a7-F
gtttccctatcgagcctacac
Slc1a2-F Slc1a2-R
55–65
64
59
60
59
57
63
tgattttccagaagccagg
gccaatacaaccaaggcagt
61
ttcatcccgtccttgaactc
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3. Results
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Slc6a7-R
annealing temperature(℃)
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Specific primers
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gen primer
3.1. Place conditioning
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After conditioning for 22 days, place preference was tested, which revealed significant
preference scores (P< 0.01) for the group conditioned with nicotine at 0.2 mg/kg/day; by contrast, rats that were administered either saline or nicotine at 0.4 or 0.6 mg/kg/day did not demonstrate a place preference. Therefore, CPP was induced in rats by nicotine at a dose of 0.2 mg/kg/day. 3.2. Proteins identified based on iTRAQ In iTRAQ based 2D-LC MS/MS analysis, 4650 non-redundant proteins were identified with >95% confidence (Supplementary Table1). As shown in Fig. 1, the normality test strongly fit
ACCEPTED MANUSCRIPT the frequency distribution of the normalized log-transformed median values of expression ratios of the 4650 proteins. Proteins featuring a change ratio of >1.5-fold or <0.67-fold were regarded as significantly changed proteins (P < 0.05), and our results showed that 117 and 10 proteins were
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significantly downregulated and upregulated, respectively between the nicotine and saline groups (Supplementary Table 2). 3.3. Bioinformatics analysis
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The aforementioned total 127 proteins perform significantly different molecular functions,
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participating in distinct biological processes and pathways (P < 0.05) (Fig. 2 A–C). According to GO annotation, 20 significant molecular functional groups were identified, and the majority function as binding proteins or exhibit activity (Fig. 2A). Molecules associated with calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function, long-term
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synaptic potentiation were enriched in a statistically significant manner in the quantile most expressed in the hippocampus of rats (Fig. 2, B and C). STRING analysis revealed that smooth muscle α-actin 2 (Acta2), dystonin (Dst), and GABA-A receptor 2 long isoform (Gabrg2) were
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present at hub positions of the networks (Fig. 3).
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3.4. Analysis of gene expression of the proteins expressed at significantly different levels To determine whether the significant changes that were observed specific proteins in nicotine
addiction also occurred at the level of gene expression, RT-PCR was performed to evaluate the mRNA levels of 7 significantly proteins whose expression was changed significantly. The expression of the genes was in accordance with proteomics results, except for Gabrg2 which was found to be overexpressed although the proteomics revealed downregulation, and Slc32a1 ,which showed no significant change, although the proteomic data again indicated down-regulation(Fig.
ACCEPTED MANUSCRIPT 4). 4. Discussion In the CPP test, a significant place preference was shown on Day 22 by rats that were
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administered nicotine at 0.2 mg/kg/day. A critical determinant of CPP is the relationship between initial preference and subsequent nicotine stimulation [11]. The stimulus reward of nicotine lead to CPP, and a stronger CPP is indicative of stronger reinforcement. CPP has been elicited by
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mg/kg nicotine -induced CPP is relatively high in adult [13] .
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subcutaneously administering nicotine at doses of 0.06–1.4 mg/kg nicotine [12], The dose of 0.6
This study revealed that 127 proteins showed expression changes. Currently, iTRAQ is the most sensitive proteomic method, because iTRAQ was used in this study, a larger complement of proteins was identified in response to nicotine-induced CPP here than in a previous study
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reporting [14]. Intriguingly, no protein was commonly identified in both studies. The discrepancy in the results can be readily explained by the differences in animals, the nicotine-administration procedures, and gel staining protocols.
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The results of the GO and KEGG analyses indicated that the proteins that were differentially
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expressed are involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function and long-term synaptic potentiation. Our findings suggest that nicotine regulates the calcium signaling pathway by altering the expression of these proteins. Furthermore, given that nicotine-mediated calcium signaling can potently influence neurotransmitter release, GABAergic synapse function, and long-term synaptic potentiation, our findings provide new insights into the molecular mechanisms of nicotine addiction. Our results indicate that nicotine mediates the calcium signaling pathway by altering the
ACCEPTED MANUSCRIPT expression of these proteins: mitochondrial uniporter channel (MCU), visinin-like protein 1(Vsnl1), and calcineurin B homologous protein (CHP1). MCU exhibits calcium channel activity, and MCU expression has been demonstrated to be capable of transcriptionally affecting synaptic
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activity [15]. CHP1 is a calcium-binding protein that is involved in diverse processes, such as regulation of vesicular trafficking, plasma membrane Na+/H+ exchanger activity, and gene transcription [16]. Our results showed that of MCU and CHP1 levels were decreased in the
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nicotine- induced CPP, which indicates that chronic nicotine treatments affects MCU and CHP1
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expression. Vsnl1, which belongs to a highly homologous subfamily of neuronal calcium sensors, is strongly expressed in hippocampal neurons and affects the membrane trafficking of receptors [17]. Vsnl1 not only participates in the regulation of cellular signaling cascades, including cAMP and cGMP signaling cascades, but also interacts with α4β2nAChR and modulates nAChR function
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[18, 19]. The effects of nicotine on the expression of cAMP, and cGMP signaling and α4β2 nAChR function reported in previous studies can explain why nicotine affects Vsnl1 expression in the hippocampus[20], as shown in this study. Therefore, this study suggests that nicotine binding
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to nAChRs activates the receptors leads to a calcium influx, which would raise cytoplasmic levels
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of calcium and increase GABA receptors and inhibit calcium-mediated signaling in the distal apical dendrite[21]. The downstream effects produced by nAChR-dependent calcium signaling potently regulate the transmitter release, neuronal plasticity, gene expression, and neuronal excitability[19].
Most notably, this study revealed that nicotine modulates the expression of three 3 proteins involved in neurotransmitter transport (which is the downstream from signaling of calcium-mediated
signaling):
vesicular
inhibitory
amino
acid
transporter
(Slc32a1),
ACCEPTED MANUSCRIPT sodium-dependent proline transporter (Slc6a7) and amino acid transporter 2 (Slc1a2). Slc32a1, which is localized to vesicles of the inhibitory terminals of GABAergic and symmetric synapses, is a transmembrane ion transporter that packages GABA into vesicles in cholinergic neurons, and
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the protein is primarily involved in the synaptic vesicle cycle, GABAergic synapse function, major fast inhibitory neurotransmitter release, and nicotine addiction [22]. Chronic nicotine administration was shown to enhance Slc32a1 gene expression in rat nucleus accumbens [23]. The
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Slc6a7 encodes a transporter for L-proline, a member of the GABA family. L-proline is an
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excitatory neurotransmitter, and its transporter plays a presynaptic regulatory role in synaptic transmission. Nicotine uptake can potentially be regarded as a co-transport with L-proline [24], and this can explain the regulation of Slc6a7 by nicotine in the hippocampus, as shown here. Lastly, Slc1a2, a high- affinity glutamate transporter, accounts for 90% of hippocampal glutamate
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uptake and is predominantly expressed in astrocytes [25]. Slc1a2 gene expression was reported to be correlated with smoking initiation, current smoking, and smoking cessation[26]. Here, the result showed that Slc32a1 and Slc1a2 expression was decreased and Slc6a7 expression was
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increased, which was a complete expression change after chronic nicotine treatment. The
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significantly different expression of proteins after nicotine-induced CPP might result in an aberrant association between nicotine and nAChR that further influences glutamatergic pathways and neurotransmitter transport. This study also identified changes in the expression of certain proteins related to long-term
synaptic potentiation: glial fibrillary acidic protein (Gfap), neuroligin isoform1 (Nlgn1) and ras-specific guanine nucleotide releasing factor 2 (Rasgrf2). Hippocampal long-term synaptic potentiation is a widely accepted model of synaptic plasticity that is considered to underlie
ACCEPTED MANUSCRIPT learning and memory processes [27]. Nlgn1, which localizes at glutamatergic synapses, is directly associated with synaptic scaffolding molecules, and this might allow Nlgn1 to indirectly influence the strength of cholinergic contacts at cholinergic synapses. Chronic nicotine administration leads
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to long-term changes in Nlgn1 gene expression, which can modulate synapse development and function in the [28]brain. Gfap, a 51-kDa phosphor-protein, is a specific astrocyte marker in the central nervous system. Nicotine-activated Gfap-positive human astrocytes were shown to
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displayed a strongly expression of α7nAChR [29]. Furthermore, chronic nicotine treatment was
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reported to result in an increase in Gfap expression in the CA1 subfield of the rat hippocampus and cerebellum [30], although another in vivo study revealed that nicotine exerted no effect on Gfap expression, as determined using immunohistochemistry [31]. However, Gfap expression was shown to decrease in the rat hippocampus following nicotine-induced CPP [32], which agrees with
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our results. The contrasting changes in Gfap expression observed in the in vivo and in vitro experiments might be due to neuroglial communication [33]. Here, this study found that Gfap expression decreased after nicotine-induced CPP. The reduction in Gfap expression after
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nicotine-induced CPP might result in abnormal communication between the neurons and
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astrocytes involved in nicotine reward. The Rasgrf2 mediates Ca2+-dependent activation of signaling. Rasgrf2 expressed in mice mediates the activation of extracellular-regulated kinases (ERKs) through N-methyl-d-aspartate (NMDA) receptor and contributes primarily to the induction long-term synaptic potentiation [34]. ERK activation was initially recognized to occur downstream of neurotrophic factors, which, coupled with NMDA receptors and nAChR, can produce a response to both glutamate and nicotine [35]. The network analysis performed here revealed cytoskeletal and calcium binding proteins
ACCEPTED MANUSCRIPT formed the largest category and included proteins such as Acta2, Dst and Gabrg2. Gabrg2 is a postsynaptic GABA receptor subunit that can directly or indirectly enhance excitatory neurotransmission, which would cause brain hyperexcitability [36]. The work of Beuten et al. [37]
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revealed an association between nicotine-dependence and Gabrg2 gene expression. Neuronal Dst isoforms are giant cytoskeletal cross-linking proteins that can interact with actin and microtubule networks, protein complexes and cellular membranes. In the neuromuscular system in mice, a loss
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of Dst expression results in a profound sensory ataxia, termed dystonia musculorum, which is
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attributed to the degeneration of sensory nerves [38]. In case of, Acta2, an ATP-binding protein with ATPase activity, the expression of the gene was reported to changes markedly in rats in response to alcohol [39], but how Acta2 functions in nicotine addiction remains unclear. In conclusion, our study identified a large group of differentially expressed proteins in the rat
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hippocampus in nicotine-induced CPP by using iTRAQ based 2D-LC MS/MS. Moreover, RT-PCR analysis was used to confirm the changes in the expression of a subset of the molecules: CHP1 , Slc32a1, Slc6a7, Slc1a2 and Gabrg2. The differentially expressed proteins are involved in
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cytoskeleton organization, calcium-mediated signaling, neurotransmitter transport, GABAergic
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synapse function and long-term synaptic potentiation, which indicates that nicotine might act on nAChRs and thereby directly or indirectly regulate calcium-mediated signaling and neurotransmitter transport and, ultimately, induce long-term synaptic potentiation by modulating neuronal plasticity. Our results clearly showed that a single exposure to nicotine can produce prolonged effects on the hippocampal signaling cascades required for long-term memory formation, which provides new insights into the molecular mechanisms of nicotine reward. However, additional assessments are required to further evaluate this effect by using molecular
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Acknowledgements This work was funded by National Bureau of key projects (110201402037). The authors are grateful to
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the study participants.
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ACCEPTED MANUSCRIPT [37] J. Beuten, J.Z. Ma, T.J. Payne, et al, Single- and Multilocus Allelic Variants within the GABA B Receptor
Subunit 2 ( GABAB2 ) Gene Are Significantly Associated with Nicotine Dependence, Am J Hum Genet, 76 (2005)
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alcohol-preferring rats following chronic ethanol consumption, Pharmacol Biochem Behav, 94 (2009) 131-147.
ACCEPTED MANUSCRIPT Figure Captions： ：
Figure.1 Protein quantification the quantitative ratio histogram
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Figure.2 Classification of identified proteins based on GO. (a) Molecular Function, (b) Biological Processes and (c) Pathways
Figure.3 Differently expressed proteins were predicted to have directive protein-protein
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interactions.
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Figure.4 The expression level of PCR products was normalized to that of GAPDH.
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ACCEPTED MANUSCRIPT Highlights
A rat model of nicotine-induced conditioned place preference (CPP) was established.
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Hippocampal protein changes in the rats were analyzed using iTRAQ-based proteomics.
3.
Nearly 130 proteins were differentially expressed in rats exhibiting nicotine CPP.
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These proteins involved in synaptic potentiation and neurotransmitter transport.
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Conflicts of interest There are no conflicts of interest.
S0006-291X(17)30617-4
DOI:
10.1016/j.bbrc.2017.03.141
Reference:
YBBRC 37528
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 March 2017 Accepted Date: 26 March 2017
Please cite this article as: B. Zhu, X. Li, H. Chen, H. Wang, X. Zhu, H. Hou, Q. Hu, iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.03.141. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference Beibei Zhu, Xiangyu Li, Huan Chen, Hongjuan Wang, Xinchao Zhu, Hongwei Hou *, Qingyuan Hu *
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Email ID: [email protected], [email protected]
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China National Tobacco Quality Supervision and Test Center, Zhengzhou, 450001, China.
ACCEPTED MANUSCRIPT Abstract Repeated exposures to nicotine are known to result in persistent changes in proteins expression in addiction-related brain regions, such as the striatum, nucleus accumbens and prefrontal cortex, but
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the changes induced in the protein content of the hippocampus remain poorly studied. This study established a rat model of nicotine-induced conditioned place preference (CPP), and screened for proteins that were differentially expressed in the hippocampus of these rats using isobaric tags for
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relative and absolute quantitation labeling (iTRAQ) coupled with 2D-LC MS/MS. The
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nicotine-induced CPP was established by subcutaneously injecting rats with 0.2 mg/kg nicotine. Relative to the control (saline) group, the nicotine group showed 0.67- and 1.5-fold changes in 117 and 10 hippocampal proteins, respectively. These differentially expressed proteins are mainly involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function,
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long-term synaptic potentiation and nervous system development. Furthermore, RT-PCR was used to confirmed the results of the proteomic analysis. Our findings identify several proteins and cellular signaling pathways potentially involved in the molecular mechanisms in the hippocampus
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that underlie nicotine addiction. These results provide insights into the mechanisms of nicotine
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treatment in hippocampus.
Keywords: Nicotine, Conditioned place preference, Hippocampus, iTRAQ, proteomics
ACCEPTED MANUSCRIPT 1. Introduction Nicotine is a primary component in tobacco, endowed with reinforcing properties that is responsible for tobacco addiction [1]: Nicotine binds to nicotinic cholinergic receptors(nAChR),
specific and
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nAChR ultimately become desensitized, which results in long-term tolerance to nicotine in certain critical brain regions, including the ventral tegmental area, nucleus accumbens,
amygdala, and hippocampus. This tolerance is involved in the conversion from casual to
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compulsive drug use[2]. Increasing evidence supports the view that the hippocampus, a key
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modulator of learning and memory, functions as a crucial substrate of addiction[3]. The hippocampus is critical for the transformation of short-term memories into long-term memories in spatial and contextual learning, and it is also connected in a complex manner with several AChR-enriched brain regions in the limbic system [4, 5]. Previous work has demonstrated that
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nicotine exposure can alter multiple aspects of hippocampal function that might play a key role in nicotine addiction[6], but few studies to date have investigated the changes induced by nicotine in the expression of proteins involved in hippocampal function.
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Identification of the proteins that are associated with the development and maintenance of
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drug dependence is not only critical for understanding the molecular mechanisms underlying addiction, but also necessary for the development of new pharmacological means to reduce the use of the drugs [7]. Repeated exposure to an addictive drug can alter the levels or types of proteins expressed in specific brain regions [8], and such alterations have been previously investigated using various molecular techniques. Proteomics, which is characterized by the ability to measure dynamic changes in proteomes on a global scale, has emerged as an extremely powerful tool for discovery previously unrecognized molecular mechanisms underlying drug
ACCEPTED MANUSCRIPT addiction. Proteomics has been used over the past few years to profile the protein expression pattern in response to numerous substances of abuse such as amphetamine, alcohol, cocaine, and morphine [7]. However, very few proteomics studies have been published on nicotine addiction
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during chronic exposure to nicotine. Yeom et al [9] conducted a two dimensional electrophoresis (2DE) analysis and reported that chronic nicotine administration and withdrawal potently regulated the expression of 7 proteins in the striatum of male rats. In other comprehensive study,
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2DE was again used to analyze the protein expression profiles for five brain regions-the
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amygdala, nucleus accumbens, prefrontal cortex, striatum, and ventral tegmental area in rats that received nicotine for 7 days[10]. These results from proteomic analyses have provided insights into the mechanisms of nicotine treatment and withdrawal. However, to our knowledge, no study thus far has determined the changes in hippocampus proteins in the rat by using labeling relative
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and absolute quantification (iTRAQ) proteomics following nicotine-induce CPP. This study established a rat model of nicotine-induced CPP and identified proteins that were differentially expressed in the hippocampus of these rats by using iTRAQ coupled with 2D-LC
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expression.
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MS/MS. Moreover, RT-PCR was used to verified the significantly different changes in protein
2. Materials and Methods 2.1. CPP test
2.1.1. Animals
Male Sprague-Dawley rats (n = 24; Basic Medical College of Zhengzhou University), weighing 280–330g, were individually housed under a reversed 12:12-h light /dark cycle in a temperature and humidity controlled room at 21 ± 1°C and were handled daily. Care of the
ACCEPTED MANUSCRIPT animals was in accordance with National Institutes of Health guidelines and all procedures were approved by our institution’s animal safety committee. 2.1.2. Nicotine CPP training
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The CPP box (Med Associates) used here contained a white chamber and a black chamber featuring different flooring that provided two distinct contexts, and each served as either the white-paired or the black -paired side. A third gray chamber with a distinct floor was also present,
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and it connected the two other chambers and provided a neutral environment. An initial test of
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preference was conducted one day before CPP training by placing rats in the middle chamber of the CPP box for 15 min and allowing them to move freely between chambers, and the time spent in each chamber was recorded using standard MedPC software. Based on this test, 24 male rats were selected that did not exhibit a preferred side, and randomly divided these rats into four
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groups three of the groups received nicotine (N), and one received saline(S). The animals in the four groups were subcutaneously injected with 0.2, 0.4 or 0.6 mg/kg/day nicotine or saline on the first day of training, and all animals that received nicotine injections were placed in the
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nicotine-paired side and all animals that received saline injections were placed in the saline-paired
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side; access to other-paired chamber was blocked by a barrier. On the following day, all animals received saline injections and were placed in the saline-paired side. This procedure was repeated until Day 22. The preference testing was conducted again after the last day of training. All animals had access to both of the paired chambers from the neutral environment for 15 min during the test, and their movement was recorded. 2.2. iTRAQ proteome analysis 2.2.1. Protein preparation
ACCEPTED MANUSCRIPT Immediately after the CPP test, the rats that exhibited a significant difference in preference between N and S sides were sacrificed by means of decapitation and their brains were collected and transferred into ice-cold water. The hippocampus was dissected and stored at -80°C until
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further use. Hippocampus samples were homogenized with N-PRR neuronal protein extraction reagent (Promega) containing protease inhibitor phenylmethanesulfonyl fluoride (Sigma), and the protein concentration in each extracted sample was measured using a bicinchonic acid kit
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(Thermo). Equal amounts of protein from each rat were pooled according to the group, and the
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100 µg of the total protein from each group was processed as the manufacturer,s instruction for the iTRAQ reagent kit (Applied Biosystems, USA). For duplication, samples from N and S were labeled using 114 and 117 tags, 115 and 116 tags, respectively. 2.2.2. High pH reverse phase chromatography
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The pooled samples were subjected to strong cation exchanger fractionation by HPLC (Thermo) with an Extend-C18 analytical column (5 µm, 250×4.6 mm) as follows: flow rate, 0.4 mL/min; Solvent A, 10 mmol/L ammonium formate (pH10); solvent B, 80% acetonitrile (CAN)
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and 10 mmol/L ammonium formate (pH10). A 45 min gradient from 5% to 50% organic solvent
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was used to elute the peptides, after which the organic solvent was increased to 90% to wash the column; 12 fractions were collected and vacuum-dried. 2.2.3. Quantitative proteomic analysis Purified peptide fractions were reconstituted in mobile phase A (0.1% formic acid aqueous
solution) and separated in an analytical column (75 m×50cm, C18, 2 m; Thermo) by using a Nano-LC (Thermo)-Q-TOF MS (Bruker) system with a solvent gradient of 5-90% Buffer B (0.1% formic acid in acetonitrile) in mobile phase A, using flow rate of 0.3 mL/min. Total gradient length
ACCEPTED MANUSCRIPT was 95 min. MS date were acquired in the positive ion mode with a captive spray Nanoflow ES source, at a selected mass range of 50–2200 m/z, and peptide ions featuring +2 to +4 charge states and cycle time of 3s were subjected to MS/ MS. Automatic collision energy and automatic
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MS/MS accumulation were used to activate smart information dependent date acquisition. Data were processed by Mascot software (Version 2.4.1) and compared with the UniProt database (http://www.uniprot.org/). The search was performed using trypsin and allowing for two
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missed cleavages per peptide. Carbamidomethyl alkylation of cysteines was used as the fixed
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modification, and the variable modifications were oxidation of methionine, iTRAQ modifications of lysine, iTRAQ N-terminal and iTRAQ-tyrosine. Peptide and MS/MS tolerance were 20 ppm and 0.1 Da respectively. Proteins featuring unique peptides were identified and are reported with a cumulative confidence >95%.
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2.3. Bioinformatics analyses
Functional classifications were performed using GO (https://david.ncifcrf.gov/), and Pathway Alalysis was performed using KEGG (http://www.genome.jp/kegg/mapper.html). Functional
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protein association networks were constructed using STRING (http://string-db.org/).
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2.4. RT-PCR analyses
RT-PCR was performed on samples from rats in each group. Total RNA was extracted from
the hippocampus samples by using Trizol (Invitrogen) and then reverse transcribed by using Taq polymerase with the PrimeScript™ RT Reagent Kit with gDNA Eraser, according to the manufacturer’s instructions (Takara). All PCR amplifications were performed by using the real-time fluorescence detection method with First Start DNA Master SYBR Green (Roche, Germany) on a LightCycler 96 System (Roche). All primers were designed according to published
ACCEPTED MANUSCRIPT cDNA sequences by using primer selection software (http://tools.thermofisher.com/content.cfm). The specific primers and the annealing temperatures are shown in Table 1. The fold-change values were represented as means ± SEMs of mRNA expression normalized to GAPDH mRNA, and
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values were compared using ANOVA. Table 1 specific primers and annealing temperature
GAPDH-F
gggtgtgaaccacgagaaat
GAPDH-R
actgtggtcatgagcccttc
CHP1-F
gagactggcttttcccacag
CHP1-R
agtgggttgatggcaagttc
Acta2-F
gccctggattttgagaatga
Acta2-R
tgaaagagggctggaagaga
Slc32a1-F
acatcctggtcatcgcctac
Slc32a1-R
gatgccgatggagataggaa
Dst-F
gcgcctctgaagctctctta
Dst-R
tagttgcccgggttacaaag
Gabrg2-F
actcattgtggttctgtcctg
Gabrg2-R
gctgtgacataggagaccttg
Slc6a7-F
gtttccctatcgagcctacac
Slc1a2-F Slc1a2-R
55–65
64
59
60
59
57
63
tgattttccagaagccagg
gccaatacaaccaaggcagt
61
ttcatcccgtccttgaactc
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3. Results
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Slc6a7-R
annealing temperature(℃)
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Specific primers
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gen primer
3.1. Place conditioning
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After conditioning for 22 days, place preference was tested, which revealed significant
preference scores (P< 0.01) for the group conditioned with nicotine at 0.2 mg/kg/day; by contrast, rats that were administered either saline or nicotine at 0.4 or 0.6 mg/kg/day did not demonstrate a place preference. Therefore, CPP was induced in rats by nicotine at a dose of 0.2 mg/kg/day. 3.2. Proteins identified based on iTRAQ In iTRAQ based 2D-LC MS/MS analysis, 4650 non-redundant proteins were identified with >95% confidence (Supplementary Table1). As shown in Fig. 1, the normality test strongly fit
ACCEPTED MANUSCRIPT the frequency distribution of the normalized log-transformed median values of expression ratios of the 4650 proteins. Proteins featuring a change ratio of >1.5-fold or <0.67-fold were regarded as significantly changed proteins (P < 0.05), and our results showed that 117 and 10 proteins were
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significantly downregulated and upregulated, respectively between the nicotine and saline groups (Supplementary Table 2). 3.3. Bioinformatics analysis
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The aforementioned total 127 proteins perform significantly different molecular functions,
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participating in distinct biological processes and pathways (P < 0.05) (Fig. 2 A–C). According to GO annotation, 20 significant molecular functional groups were identified, and the majority function as binding proteins or exhibit activity (Fig. 2A). Molecules associated with calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function, long-term
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synaptic potentiation were enriched in a statistically significant manner in the quantile most expressed in the hippocampus of rats (Fig. 2, B and C). STRING analysis revealed that smooth muscle α-actin 2 (Acta2), dystonin (Dst), and GABA-A receptor 2 long isoform (Gabrg2) were
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present at hub positions of the networks (Fig. 3).
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3.4. Analysis of gene expression of the proteins expressed at significantly different levels To determine whether the significant changes that were observed specific proteins in nicotine
addiction also occurred at the level of gene expression, RT-PCR was performed to evaluate the mRNA levels of 7 significantly proteins whose expression was changed significantly. The expression of the genes was in accordance with proteomics results, except for Gabrg2 which was found to be overexpressed although the proteomics revealed downregulation, and Slc32a1 ,which showed no significant change, although the proteomic data again indicated down-regulation(Fig.
ACCEPTED MANUSCRIPT 4). 4. Discussion In the CPP test, a significant place preference was shown on Day 22 by rats that were
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administered nicotine at 0.2 mg/kg/day. A critical determinant of CPP is the relationship between initial preference and subsequent nicotine stimulation [11]. The stimulus reward of nicotine lead to CPP, and a stronger CPP is indicative of stronger reinforcement. CPP has been elicited by
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mg/kg nicotine -induced CPP is relatively high in adult [13] .
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subcutaneously administering nicotine at doses of 0.06–1.4 mg/kg nicotine [12], The dose of 0.6
This study revealed that 127 proteins showed expression changes. Currently, iTRAQ is the most sensitive proteomic method, because iTRAQ was used in this study, a larger complement of proteins was identified in response to nicotine-induced CPP here than in a previous study
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reporting [14]. Intriguingly, no protein was commonly identified in both studies. The discrepancy in the results can be readily explained by the differences in animals, the nicotine-administration procedures, and gel staining protocols.
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The results of the GO and KEGG analyses indicated that the proteins that were differentially
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expressed are involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function and long-term synaptic potentiation. Our findings suggest that nicotine regulates the calcium signaling pathway by altering the expression of these proteins. Furthermore, given that nicotine-mediated calcium signaling can potently influence neurotransmitter release, GABAergic synapse function, and long-term synaptic potentiation, our findings provide new insights into the molecular mechanisms of nicotine addiction. Our results indicate that nicotine mediates the calcium signaling pathway by altering the
ACCEPTED MANUSCRIPT expression of these proteins: mitochondrial uniporter channel (MCU), visinin-like protein 1(Vsnl1), and calcineurin B homologous protein (CHP1). MCU exhibits calcium channel activity, and MCU expression has been demonstrated to be capable of transcriptionally affecting synaptic
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activity [15]. CHP1 is a calcium-binding protein that is involved in diverse processes, such as regulation of vesicular trafficking, plasma membrane Na+/H+ exchanger activity, and gene transcription [16]. Our results showed that of MCU and CHP1 levels were decreased in the
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nicotine- induced CPP, which indicates that chronic nicotine treatments affects MCU and CHP1
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expression. Vsnl1, which belongs to a highly homologous subfamily of neuronal calcium sensors, is strongly expressed in hippocampal neurons and affects the membrane trafficking of receptors [17]. Vsnl1 not only participates in the regulation of cellular signaling cascades, including cAMP and cGMP signaling cascades, but also interacts with α4β2nAChR and modulates nAChR function
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[18, 19]. The effects of nicotine on the expression of cAMP, and cGMP signaling and α4β2 nAChR function reported in previous studies can explain why nicotine affects Vsnl1 expression in the hippocampus[20], as shown in this study. Therefore, this study suggests that nicotine binding
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to nAChRs activates the receptors leads to a calcium influx, which would raise cytoplasmic levels
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of calcium and increase GABA receptors and inhibit calcium-mediated signaling in the distal apical dendrite[21]. The downstream effects produced by nAChR-dependent calcium signaling potently regulate the transmitter release, neuronal plasticity, gene expression, and neuronal excitability[19].
Most notably, this study revealed that nicotine modulates the expression of three 3 proteins involved in neurotransmitter transport (which is the downstream from signaling of calcium-mediated
signaling):
vesicular
inhibitory
amino
acid
transporter
(Slc32a1),
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the protein is primarily involved in the synaptic vesicle cycle, GABAergic synapse function, major fast inhibitory neurotransmitter release, and nicotine addiction [22]. Chronic nicotine administration was shown to enhance Slc32a1 gene expression in rat nucleus accumbens [23]. The
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Slc6a7 encodes a transporter for L-proline, a member of the GABA family. L-proline is an
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excitatory neurotransmitter, and its transporter plays a presynaptic regulatory role in synaptic transmission. Nicotine uptake can potentially be regarded as a co-transport with L-proline [24], and this can explain the regulation of Slc6a7 by nicotine in the hippocampus, as shown here. Lastly, Slc1a2, a high- affinity glutamate transporter, accounts for 90% of hippocampal glutamate
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uptake and is predominantly expressed in astrocytes [25]. Slc1a2 gene expression was reported to be correlated with smoking initiation, current smoking, and smoking cessation[26]. Here, the result showed that Slc32a1 and Slc1a2 expression was decreased and Slc6a7 expression was
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increased, which was a complete expression change after chronic nicotine treatment. The
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significantly different expression of proteins after nicotine-induced CPP might result in an aberrant association between nicotine and nAChR that further influences glutamatergic pathways and neurotransmitter transport. This study also identified changes in the expression of certain proteins related to long-term
synaptic potentiation: glial fibrillary acidic protein (Gfap), neuroligin isoform1 (Nlgn1) and ras-specific guanine nucleotide releasing factor 2 (Rasgrf2). Hippocampal long-term synaptic potentiation is a widely accepted model of synaptic plasticity that is considered to underlie
ACCEPTED MANUSCRIPT learning and memory processes [27]. Nlgn1, which localizes at glutamatergic synapses, is directly associated with synaptic scaffolding molecules, and this might allow Nlgn1 to indirectly influence the strength of cholinergic contacts at cholinergic synapses. Chronic nicotine administration leads
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to long-term changes in Nlgn1 gene expression, which can modulate synapse development and function in the [28]brain. Gfap, a 51-kDa phosphor-protein, is a specific astrocyte marker in the central nervous system. Nicotine-activated Gfap-positive human astrocytes were shown to
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displayed a strongly expression of α7nAChR [29]. Furthermore, chronic nicotine treatment was
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reported to result in an increase in Gfap expression in the CA1 subfield of the rat hippocampus and cerebellum [30], although another in vivo study revealed that nicotine exerted no effect on Gfap expression, as determined using immunohistochemistry [31]. However, Gfap expression was shown to decrease in the rat hippocampus following nicotine-induced CPP [32], which agrees with
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our results. The contrasting changes in Gfap expression observed in the in vivo and in vitro experiments might be due to neuroglial communication [33]. Here, this study found that Gfap expression decreased after nicotine-induced CPP. The reduction in Gfap expression after
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nicotine-induced CPP might result in abnormal communication between the neurons and
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astrocytes involved in nicotine reward. The Rasgrf2 mediates Ca2+-dependent activation of signaling. Rasgrf2 expressed in mice mediates the activation of extracellular-regulated kinases (ERKs) through N-methyl-d-aspartate (NMDA) receptor and contributes primarily to the induction long-term synaptic potentiation [34]. ERK activation was initially recognized to occur downstream of neurotrophic factors, which, coupled with NMDA receptors and nAChR, can produce a response to both glutamate and nicotine [35]. The network analysis performed here revealed cytoskeletal and calcium binding proteins
ACCEPTED MANUSCRIPT formed the largest category and included proteins such as Acta2, Dst and Gabrg2. Gabrg2 is a postsynaptic GABA receptor subunit that can directly or indirectly enhance excitatory neurotransmission, which would cause brain hyperexcitability [36]. The work of Beuten et al. [37]
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revealed an association between nicotine-dependence and Gabrg2 gene expression. Neuronal Dst isoforms are giant cytoskeletal cross-linking proteins that can interact with actin and microtubule networks, protein complexes and cellular membranes. In the neuromuscular system in mice, a loss
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of Dst expression results in a profound sensory ataxia, termed dystonia musculorum, which is
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attributed to the degeneration of sensory nerves [38]. In case of, Acta2, an ATP-binding protein with ATPase activity, the expression of the gene was reported to changes markedly in rats in response to alcohol [39], but how Acta2 functions in nicotine addiction remains unclear. In conclusion, our study identified a large group of differentially expressed proteins in the rat
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hippocampus in nicotine-induced CPP by using iTRAQ based 2D-LC MS/MS. Moreover, RT-PCR analysis was used to confirm the changes in the expression of a subset of the molecules: CHP1 , Slc32a1, Slc6a7, Slc1a2 and Gabrg2. The differentially expressed proteins are involved in
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cytoskeleton organization, calcium-mediated signaling, neurotransmitter transport, GABAergic
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synapse function and long-term synaptic potentiation, which indicates that nicotine might act on nAChRs and thereby directly or indirectly regulate calcium-mediated signaling and neurotransmitter transport and, ultimately, induce long-term synaptic potentiation by modulating neuronal plasticity. Our results clearly showed that a single exposure to nicotine can produce prolonged effects on the hippocampal signaling cascades required for long-term memory formation, which provides new insights into the molecular mechanisms of nicotine reward. However, additional assessments are required to further evaluate this effect by using molecular
ACCEPTED MANUSCRIPT biology tools.
Acknowledgements This work was funded by National Bureau of key projects (110201402037). The authors are grateful to
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the study participants.
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ACCEPTED MANUSCRIPT Figure Captions： ：
Figure.1 Protein quantification the quantitative ratio histogram
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Figure.2 Classification of identified proteins based on GO. (a) Molecular Function, (b) Biological Processes and (c) Pathways
Figure.3 Differently expressed proteins were predicted to have directive protein-protein
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Figure.4 The expression level of PCR products was normalized to that of GAPDH.
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Figure.4
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A rat model of nicotine-induced conditioned place preference (CPP) was established.
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Hippocampal protein changes in the rats were analyzed using iTRAQ-based proteomics.
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Nearly 130 proteins were differentially expressed in rats exhibiting nicotine CPP.
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These proteins involved in synaptic potentiation and neurotransmitter transport.
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Conflicts of interest There are no conflicts of interest.