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Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats
BBRC Biochemical and Biophysical Research Communications 326 (2005) 321–328 www.elsevier.com/locate/ybbrc
Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats Mijung Yeom, Insop Shim, Hye-Jung Lee, Dae-Hyun Hahm* Department of Medical Science, Graduate School of East-West Medical Science, Kyung Hee University, Gihung-up, Yongin-si, Gyeonggi-do 449-701, Republic of Korea Received 14 October 2004 Available online 24 November 2004
Abstract Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4 mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2 0 3 0 -cyclic nucleotide 3 0 -phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K+ channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine. 2004 Elsevier Inc. All rights reserved. Keywords: Proteome; Nicotine; Addiction; 2-Dimensional electrophoresis; RT-PCR; Striatum; Protein expression; Rat
Tobacco-related diseases are among the worldÕs leading preventable causes of death. They are responsible for approximately 5 million deaths a year. However, total tobacco consumption is still on the rise [1,2]. This is due to the motivating power of tobacco. Although the mechanisms of nicotine addiction are not completely understood, much evidence suggests that nicotine is an active component in tobacco, which drives the continued use despite its detrimental effects [3–6]. Nicotine addiction produces the aversive behavioral consequences that are commonly seen with the uptake of other addictive drugs, such as cocaine and amphetamine, that is, nicotine reinforces self-administration and place preference, increases locomotor activity, and
*
Corresponding author. Fax: +82 31 204 4237. E-mail address: [email protected] (D.-H. Hahm).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.034
enhances rewards from brain stimulation [6,7]. These effects are known to be mediated by the mesocorticolimbic dopamine system, which projects from the ventral tegmental area to the dorsal and ventral (nucleus accumbens) striatum and the prefrontal cortex [8–10]. The many basic research projects that have been conducted in an attempt to remedy nicotine addiction have therefore focused on the drug-induced neuroadaptation underlying sensitization within this pathway and adjacent neural circuitry [7,11–13]. However, as of yet, there is little understanding of the molecular mechanisms that lead to and maintain nicotine addiction. Therefore, the identification of the neurobiological factors that take a part in nicotine addiction is the first step to find the answer. Two-dimensional gel electrophoresis (2-DE), in combination with mass spectrometry and modern bioinformatics tools, is considered to be the most effective tool to detect unexpected changes of protein modulation
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and to identify qualitative and quantitative differences among the expression profiles of the proteins expressed under different conditions [14]. It is also a powerful method that makes it possible to isolate the protein candidates as a biological marker in the complex pathophysiological processes, such as nicotine addiction [15]. The aim of this study was to detect and identify the protein variants related to nicotine addiction in rat brains. These proteins may be useful in understanding the molecular mechanism of nicotine addiction and for isolating the potential drug targets for nicotine addiction-associated disease therapy.
Materials and methods Animals and nicotine treatment. Male Sprague–Dawley rats weighing 250–280 g at the start of experiments were used throughout. Prior to the experimental manipulation, the animals were given a period of 1 week to adjust to the new environment. Animals were cared for and handled in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animals were divided randomly into nicotine-treated (NIC, n = 10) and control (CONT, n = 10) groups. For nicotine injection, ()-nicotine hydrogen tartrate (Sigma–Aldrich, MO) was dissolved in saline and pH of the final solution was adjusted to 7.2. The course of nicotine treatment consisted of three phases; development, withdrawal, and challenge phases. The rats were injected with nicotine (0.4 mg/kg, s.c.) twice daily for 7 consecutive days. After 3 days of withdrawal period, the rats were challenged with the last systemic injection of nicotine on day 11. Normal group was injected with saline instead of nicotine solution. Locomotor activity. After the final nicotine injection in challenge phase, the nicotine-induced locomotor activity was measured in a rectangular container (40 · 40 · 45 cm each) equipped with a video camera above the center of the floor. The wall and floor were made of clear Plexiglas and were painted black. Locomoter activity was monitored by a video-tracking system using S-mart program (PanLab, Barcelona, Spain). The day before the locomotor activity determination, rats were allowed to individually habituate to the test cages for 1 h, during which the activity was monitored. This habituation period was required to overcome the potentially stressful nature of the test apparatus, which may affect the basal levels of locomoter activities of the rats [16]. On the test day, after the baseline determination for 30 min, the animals were challenged with nicotine and locomoter activity was monitored for 30 min. Locomotor activity, expressed as distance travelled (in centimeter), was calculated as the total distance moved during the 30-min period after the drug challenge. Sample preparation. For histological analysis, the striatum of approx. 30–50 mg wet weight was dissected from the brains and stored at 80 C until protein or RNA extraction. For protein extraction, the striatum was homogenized within a detergent lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) Chaps, 0.5% (v/v) Triton X-100, 0.5% (v/v) Pharmalytes pH 3–10 (Amersham Biosciences, NJ), 100 mM DTT, and 1.5 mg/mL complete Protease Inhibitor Cocktail for mammalian tissues (Sigma–Aldrich, MO), sonicated, and incubated for 1 h at room temperature in an orbital shaker. The lysate was then centrifuged at 13,000 rpm for 30 min. The total protein concentration of each sample was analyzed by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, CA). The protein samples were stored in aliquots at 80 C until use. Total RNA from frozen samples was extracted using TRIzol reagent (Invitrogen, CA), in accordance with manufacturerÕs instructions. The RNA extracted was dissolved in DEPC-treated distilled water and stored at 80 C. The concentration and purity of RNA were checked by measuring the
optical density at 260 and 280 nm using GeneQuant pro spectrophotometer (Amersham Biosciences, NJ). Two-dimensional electrophoresis (2-DE) and image analysis. For isoelectric focussing (IEF), 200 lg of total proteins was mixed in a rehydration buffer (7 M urea, 2 M thiourea, 4% Chaps, 0.5% Triton X-100, 100 mM DTT, 0.6% Pharmalytes pH 3–10, and bromophenol blue) in total volume of 340 lL and loaded on 18 cm pH 3–10 NL Immobiline DryStrip (an IPG strip, Amersham biosciences, NJ). After IPG strip rehydration, IEF was carried out for 16 h up to a total of 65,000 V/h using pHaser Isoelectric Focusing System (Genomic Solutions, MI). After the equilibration of rehydrated IPG strips, the second-dimensional gel electrophoresis was subsequently performed on 26 · 24 cm of 10% SDS–polyacrylamide gels using Investigator System (Genomic Solutions, MI). Gels were stained with Coomassie blue G-250 (Bio-Rad Laboratories, CA) staining solution as described by Kang et al. [17]. The stained gels were then analyzed with ImageMaster 2D-Elite (Amersham biosciences, NJ) software for spot detection, quantification, and matching. Protein identification by peptide mass fingerprinting. Selected protein spots were excised from the gels and placed in 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate (NH4HCO3) for 15 min and the supernatant was removed. These steps were repeated until the Coomassie blue G-250 dye was completely removed. The gel pieces were then dried in a vacuum-centrifuge, rehydrated in 15 lL chilled trypsin solution (10 lg/mL of sequencing-grade modified trypsin (Promega Biosciences, CA) in 25mM NH4HCO3 buffer) for 50 min, and incubated at 37 C for 16 h. The supernatant was collected and the peptides were extracted with 5% trifluoroacetic acid (TFA) in 50% ACN twice. Peptide extracts were then dried and stored at 20 C until use. The extracted peptides were desalted using ZipTips C18 resin (Millipore, MA) according to the manufacturerÕs instructions, and eluted (from C18 resin) with matrix, a saturated solution of a-cyano-4hydroxycinnamic acid in 50% CAN/0.1% TFA. MALDI-TOF mass spectra were acquired on Voyager-DE STR MALDI-TOF mass spectrometer (MS) (Applied Biosystems, CA). The peptide mass fingerprints were analyzed by MS-Fit search program using NCBInr and/ or SWISS-PROT protein databases. RT-PCR. One microgram of total RNA was reverse-transcribed with M-MLV reverse transcriptase (Invitrogen, CA) in a 20 lL of reaction mixture containing random hexamers. The cDNA was diluted five times and 5 lL of the resulting diluent was amplified with Taq polymerase in a 20 lL reaction mixture in a PTC-100 programmable thermal controller (MJ Research, MA). All primers in Table 1 were designed from the published cDNA sequences using primer selection software, Primer 3, which is offered through a web site (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). The amplifications were performed as follows: denaturation at 94 C for 30 s, annealing at 53– 58 C for 30 s, and polymerization at 72 C for 30 s. Annealing temperature and the number of PCR cycles to each primer was referred to Table 1. PCR products were separated on 1.2% agarose gels, stained with ethidium bromide, and photographed. The intensities of the bands were measured by using ImageMaster VDS (Amersham Biosciences, NJ) with an image analysis software, ImageMaster TotalLab (Amersham Biosciences, NJ). Data analysis. Data were expressed as means ± standard errors. Differences were analyzed with StudentÕs t test. p values of <0.05 were considered to be significant.
Results and discussion Tobacco smoking still remains a major health problem worldwide. Nicotine is a principal component of tobacco, eliciting addictive behavior in human [1,5,6]. Intermittent nicotine administration brings about the
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Table 1 Primers used in PCRs Name
Primer sequence
Product size (bp)
Annealing temperature (C)
Cycle number
ZBP-89
5 -GACAACCAGACCCTTCCAAA-3 5 0 -CGAAGGTTCACCTGTCCATT-3 0
0
377
58
30
CNP1
5 0 -TCCGAGGAGTACAAGCGTCT-3 0 5 0 -TTGTGATTTCCCAGCTCCTC-3 0
359
58
24
DNase1l3
5 0 -GTGATTAGCTCTCGGCTTGG-3 0 5 0 -CCCAATCAGCCAAACAAAGT-3 0
393
53
40
THIK-2
5 0 -GGGACCATCCTGTTCTTCAA-3 0 5 0 -GAAGTTGCCCAGACGGTAGA-3 0
378
55
35
BRAL-1
5 0 -GGCCGCTACCAGTTCAATTA-3 0 5 0 -CCGACCTTGGCTACTACAGC-3 0
359
58
30
DEDD
5 0 -TCCCCACTATCCTGTGGTGT-3 0 5 0 -AACGCCTTTCAGTGCCTCTA-3 0
388
58
30
BDNF
5 0 -GATGAGGACCAGAAGGTTCG-3 0 5 0 -GATTGGGTAGTTCGGCATTG-3 0
425
58
30
GAPDH
5 0 -ATCCCATCACCATCTTCCAG-3 0 5 0 -CCTGCTTCACCACCTTCTTG-3 0
579
58
30
0
behavioral sensitization, which is defined as an increased behavioral and/or neurochemical response [8]. Locomotor activation and reinforcement may be relevant to the stimulatory effects of nicotine on the mesolimbic dopaminergic system [18]. The striatum and nucleus accumbens are considered as the brain sites relevant to the action of drug rewards, and thus, expected to be associated neuroadaptations, such as sensitization [8,18]. However, the mechanism of nicotine addiction has not yet been completely understood. The goal of this study was therefore to identify the proteins related to the nicotine addiction, by analyzing striatum proteome of nicotine-addictive rat brain as compared to normal one. Behavioral studies indicated that nicotine is an addictive drug which reinforces self-administration, places preference, and increases locomotion [19]. To determine whether the behavioral sensitization will be developed after repeated nicotine injection or not, the locomotor activity was monitored during the first 30 min after every administration of nicotine in this study. Rats in CONT group were injected with saline instead of nicotine. The locomotor activities of NIC group were found to be significantly increased as compared to those of CONT group (p < 0.001). It was confirmed by observing 2.3-fold increase of locomoter activity of NIC group during the challenge period that the rat model of nicotine addiction was successfully developed (Fig. 1). Protein expression patterns of the rat striatum after repeated nicotine exposure were analyzed by using 2DE and MALDI-TOF MS, followed by database searching and protein annotation. Ten animals per each experimental group were randomly spilt in two parts and then used for 2-DE and RT-PCR analyses, respectively. 2-DE was executed three times for each group. At first glance, quite similar expression patterns with lit-
Fig. 1. The effect of nicotine on locomotor activity. Locomotor activity is expressed as the total distance travelled during 30 min before (baseline) and after (test) nicotine injection on the challenge day. (***p < 0.001 as compared to CONT group).
tle differences were shown in both groups (Fig. 2A). The framed area was selected to pick the candidate proteins, which were quantitatively modulated, and thus, estimated to be associated with nicotine addiction. Seven spots, differently expressed between CONT and NIC groups and fulfilled p < 0.05 (t test), were finally isolated within the area (Fig. 2B). These proteins were identified by MALDI-TOF MS and database searching, and thus are summarized in Table 2. The expression of three proteins, ZBP89, CNP1, and DNase1l3, was highly increased in NIC group relative to CONT. And four proteins, THIK-2, BRAL-1, DEDD, and BDNF, mostly disappeared after repeated nicotine exposure, which means that those proteins were detected only in the CONT group. Zinc-finger binding protein-89 (ZBP-89, also known as ZNF148, Zfp148, BFCOL1 and BERF-1) is a Kruppel-type zinc-finger transcription factor that is
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Fig. 2. 2-DE protein patterns of rat striatum in response to repeated nicotine treatment. (A) Full images of 2-DE gels of CONT and NIC group. (B) Close-up of frames a, b, and c of (A). The protein variants which are estimated to be modulated are marked with arrows and numbers. CONT, salineinjected group; NIC, nicotine-injected group.
Table 2 Proteins increased or decreased in rat striatum after repeated nicotine treatment Spot No.
Identified protein
Accession No. NCBI
No. of peptides matched
Sequence coverage (%)
MW (kDa)/pI
Group I 1 2 3
Mowse score
ZBP-89 CNP1 DNase1l3
NP_113803 XP_340905 NP_446359
13 10 6
18 22 26
88.7/6.0 47.3/9.0 35.7/9.1
46,390 4000 25,920
Group II 4 5 6 7
THIK-2 BRAL-1 DEDD BDNF
NP_071628 Q9ESM2a NP_113988 CAA47481
5 4 10 14
10 12 28 38
46.9/9.8 38.0/9.3 36.8/9.2 27.3/8.9
235 140 13,710 164,700
Spots were classified into two groups, groups I and II, exhibited the increasing and decreasing patterns of protein expression, respectively. ZBP-89, zinc-finger binding protein-89; CNP1, 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase 1; DNase1l3, deoxyribonuclease 1-like 3; THIK-2, tandem pore domain halothane inhibited K+ channel; BRAL-1, brain-specific hyaluronan-binding protein; DEDD, death effector domain-containing DNA binding protein; and BDNF, brain-derived neurotrophic factor. a SWISS-PROT protein database was used to evaluate MS data in this case.
universally expressed in living organisms. ZBP-89 has multiple functions including transcriptional regulation of a variety of genes, cell growth arrest, and cell death [20]. Elevated levels of ZBP-89 induce growth arrest and apoptosis through the stabilization of p53 in cells [21]. This means that ZBP-89 induces apoptosis through p53-independent mechanism. The pro-inflammatory
cytokine, IFN-c, is known to induce p53-independent apoptosis. This mechanism seems to be attributed to STAT1 activation, followed by the increased expression of caspase-3, -8, and -9 as well as the inhibition of Bcl-2 expression. In this process, ZBP-89 binds to the GC-rich elements on STAT1 promoter, and thus regulates STAT1 expression and subsequent activation of apopto-
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sis [20]. Repeated administration of addictive drugs induces neuronal apoptosis, in general [22,23]. Although the conflicting experimental results of the apoptotic effects by nicotine exposure have been reported, nicotine administration evokes the apoptosis of neuronal cells in most cases [24–26], which is not consistent in either lung cancer cells [27] or immune cells [28]. Although we did not confirm the effects of nicotine on neural apoptosis, the induction of ZBP-89 after repeated nicotine administration might be related to apoptosis induction by nicotine. 2 0 ,3 0 -Cyclic nucleotide 3 0 -phosphodiesterase 1 (CNP1) is one of the two CNP isoforms. The two CNP isoforms, CNP1 (46 kDa) and CNP2 (48 kDa), are encoded by a single gene. Both CNP mRNAs, abundantly expressed in oligodendrocytes, are differentially expressed during the process of oligodendrocyte differentiation. While CNP2 mRNA is detected in oligodendrocyte progenitor cells, CNP1 mRNA is transcribed only after birth in differentiated oligodendrocytes. And both CNP transcripts are present at much higher levels in myelinating oligodendrocytes [29]. However, the cellular signals that regulate the specific expression of two CNP mRNAs have not been clear yet. Recently, it was reported that cAMP activates CNP1 gene expression via a cis-acting element, located between positions 126 and 102 of the mouse CNP1 promoter in C6 cells. cAMP regulates the development of oligodendrocytes and modulates the expression of myelin-specific proteins including CNP, myelin-associated glycoproteins, myelin basic proteins, and proteolipid proteins [30]. Also, up-regulation of the cAMP pathway is considered as an important molecular mechanism of the withdrawal syndrome by drug abuse. The chronic administration of addictive drugs leads to up-regulation of both adenylyl cyclase and protein kinase-A that control the cAMP cascade [31,32]. Although it can be assumed that cAMP has a role in nicotine addiction by the regulation of CNP1 expression, there has been no report that CNP1 is related to any kind of addiction including nicotine. Deoxyribonuclease 1-like 3 (DNase1l3) is highly homologous to DNase I except for having a basic carboxy-terminal extension and lacking N-linked glycosylation site [33]. DNase1l3 participates in apoptotic DNA fragmentation. The nuclear localization of DNase1l3 is the unique feature that distinguishes it from other suggested apoptotic nucleases. The DNase1l3 activity is regulated by poly(ADP-ribose)polymerase (PARP) during apoptosis, that is, DNase1l3 is inhibited by poly(ADP-ribosyl)ation and activated by the subsequent cleavage of RIPA during apoptosis [34]. Another function of DNase1l3 is an in vivo nuclease barrier to liposomal gene transfection. The main in vivo target of DNase1l3 is not only the apoptotic debris but other membrane-DNA particles such as viruses [33]. DNA1l3
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deficiency is common in systemic lupus erythematosus (SLE). This deficiency may stimulate the characteristic autoimmunity of SLE and increase the susceptibility to polygenic SLE [35]. However, the role of DNase1l3 in the regulation of apoptosis, induced by nicotine administration, has not been investigated yet. Tandem pore domain halothane inhibited K+ channel (THIK-2), identified a few years ago, is the one of two members of a novel 2P K+ channel subfamily. THIK-2 was expressed in several tissues including liver and kidney, and particularly strong in brain [36]. It implies that THIK-2 might have an important function in neurons, even though its physiological function is unknown. Recently, there was a report supporting this. The oxygen sensitivity of glossopharyngeal nerve (GPN) was found to be mediated by THIK [37]. Since GPN neurons were excited by hypoxia via inhibition of background channels such as THIK-2, this excitation could lead to the increase of Ca2+ levels, the activation of nNOS, and the subsequent synthesis and release of NO. The resulting inhibitory effect of NO provided a means of negative feedback modulation of chemoreceptor activity during hypoxia [38]. It was also reported that nicotine exposure attenuated oxygen sensitivity, attributed to hypoxia [37]. However, there has been no report about the effect of nicotine on THIK-2 regulation. Brain-specific hyaluronan-binding protein (BRAL-1) was recently cloned [39]. BRAL-1 was highly concentrated at the nodes of Ranvier in the developing brain and the mature CNS of mouse, where versican V2 was also localized. And, the bral1 mRNA was predominantly expressed in neurons. Oohashi et al. [40] suggested that BRAL-1 played a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes. Death effector domain-containing DNA binding protein (DEDD) contains a death effector domain (DED), the prototypical six a-helical domains which are homologous to the DEDs of the Fas-associated death domain (FADD), caspase-8 and -10, and two nuclear translocation signals (NLS). DEDD is known to weakly induce the apoptosis through its DED. DEDD resides in the cytoplasm in an active form and is translocated to the nucleus upon induction of the CD95-mediated apoptosis which is not associated with the death-inducing signaling complex (DISC). It suggests that DEDD may function in the downstream of CD95-induced apoptosis [41]. DEDD localizes to nucleoli-like structures, activates caspase-6, and inhibits RNA polymerase I-dependent transcription [42]. DEDD also acts as a scaffold that brings caspase-3 to the intermediate filament (IF) for the degradation of IF protein, essential step in the morphological changes during apoptosis [43]. However, the concrete role of DEDD in apoptosis remains unclear.
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Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor (NGF)-related family of neurotrophins and utilizes the Trk receptor-protein tyrosine kinase as primary means of signal transduction [44]. BDNF is extensively expressed throughout the CNS. It is known in general that BDNF promotes cell survival, regulates dendrites and synaptic plasticity, and plays a role in complex behaviors [45]. Also, BDNF contributes to the survival and differentiation of dopaminergic neurons during development [46]. In brain, BDNF expression kinetics by nicotine exposure has been controversial, that is, the high-dose and flash-type exposure of nicotine usually decreased BDNF levels whereas its expression was increased by the long-term exposure [45,47]. The difference between short- and long-term exposure of nicotine seems to be associated with the development of desensitization of neuronal nicotinic acetylcholine receptor [48]. These results did not coincide with our results in which the BDNF expression of both protein and mRNA was decreased by repeated exposure of nicotine. It could be postulated that in repeated injection protocols, the last injection as a challenge after 3-day withdrawal period might produce the acute properties of nicotine addiction in terms of protein expression profiles, which resulted in the down-regulation of BDNF. In order to verify the mRNA expression patterns of the proteins selected, RT-PCR analyses were performed using total RNA of striatum from NIC group. It was found that the transcriptional levels of ZBP89 and DNase1l3 mRNAs were increased by the nicotine exposure and THIK-2, DEDD, and BDNF mRNAs were decreased, which corresponded with expression profiles of these proteins. In cases of CNP1 and BRAL-1, the difference between NIC and CONT groups was not significant (Fig. 3). In summary, we have performed a proteomic analysis of rat striatum after repeated nicotine injection. After development of nicotine addiction, which was verified by analyzing the locomoter activity, seven protein variants were identified to be modulated by MALDI-TOF MS analysis and computer searching. The expression of three proteins such as ZBP-89, CNP1, and DNase1l3 was found to be increased whereas the expression of THIK-2, BRAL-1, DEDD, and BDNF was decreased. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were coincident with those of cognate proteins. These protein candidates might be useful to understand the molecular mechanism of nicotine addiction and isolate the biomarkers or targets for developing the addiction-associated diseases therapy. Further profound studies are needed for understanding the biochemical and physiological roles of every candidate selected, and thus, those proteins are recommended as an useful target for nicotine addiction therapy.
Fig. 3. The gene expression in rat striatum after repeated nicotine treatment. The expression level of each target PCR product was normalized to that of GAPDH, a housekeeping gene. Values are means with standard deviations from at least three independent experiments. (*p < 0.05, **p < 0.01 as compared to CONT group).
Acknowledgments The authors thank Prof. Kang-Duk Choi at Hankyong National University for the technical support and the helpful discussion. This study was supported by the Brain Korea 21 Project from the Ministry of Education, Republic of Korea.
References [1] WHO, The World Health Report, ed., World Health Organization Publications, 2003. [2] M.R. Carrera, J.A. Ashley, T.Z. Hoffman, S. Isomura, P. Wirsching, G.F. Koob, K.D. Janda, Investigations using immunization to attenuate the psychoactive effects of nicotine, Bioorg. Med. Chem. 12 (2004) 563–570. [3] J.A. Dani, D. Ji, F.M. Zhou, Synaptic plasticity and nicotine addiction, Neuron 31 (2001) 349–352. [4] J.Y. Bahk, S. Li, M.S. Park, M.O. Kim, Dopamine D1 and D2 receptor mRNA up-regulation in the caudate-putamen and nucleus accumbens of rat brains by smoking, Prog. Neuropsychopharmacol. Biol. Psychiatry 26 (2002) 1095–1104. [5] P.J. Kenny, A. Markou, Neurobiology of the nicotine withdrawal syndrome, Pharmacol. Biochem. Behav. 70 (2001) 531–549. [6] J.A. Dani, M. De Biasi, Cellular mechanisms of nicotine addiction, Pharmacol. Biochem. Behav. 70 (2001) 439–446. [7] Y. Chae, C.H. Yang, Y.K. Kwon, M.R. Kim, K.H. Pyun, D.H. Hahm, H.J. Lee, I. Shim, Acupuncture attenuates repeated
M. Yeom et al. / Biochemical and Biophysical Research Communications 326 (2005) 321–328
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
nicotine-induced behavioral sensitization and c-Fos expression in the nucleus accumbens and striatum of the rat, Neurosci. Lett. 358 (2004) 87–90. M.S. Todtenkopf, J.R. Stellar, E.A. Williams, D.S. Zahm, Differential distribution of parvalbumin immunoreactive neurons in the striatum of cocaine sensitized rats, Neuroscience 127 (2004) 35–42. W.A. Carlezon Jr., E.J. Nestler, Elevated levels of GluR1 in the midbrain: a trigger for sensitization to drugs of abuse?, Trends Neurosci. 25 (2002) 610–615. F.J. White, P.W. Kalivas, Neuroadaptations involved in amphetamine and cocaine addiction, Drug Alcohol Depend. 51 (1998) 141–153. M.R. Picciotto, M. Zoli, R. Rimondini, C. Lena, L.M. Marubio, E.M. Pich, K. Fuxe, J.P. Changeux, Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine, Nature 391 (1998) 173–177. F.E. Pontieri, G. Tanda, F. Orzi, G. Di Chiara, Effects of nicotine on the nucleus accumbens and similarity to those of addictive drugs, Nature 382 (1996) 255–257. P. Fredrickson, M. Boules, S. Yerbury, E. Richelson, Novel neurotensin analog blocks the initiation and expression of nicotine-induced locomotor sensitization, Brain Res. 979 (2003) 245–248. A. Moller, M. Soldan, U. Volker, E. Maser, Two-dimensional gel electrophoresis: a powerful method to elucidate cellular responses to toxic compounds, Toxicology 160 (2001) 129–138. S. Kageyama, T. Isono, H. Iwaki, Y. Wakabayashi, Y. Okada, K. Kontani, K. Yoshimura, A. Terai, Y. Arai, T. Yoshiki, Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine, Clin. Chem. 50 (2004) 857–866. T.S. Rao, A.I. Sacaan, F.M. Menzaghi, R.T. Reid, P.B. Adams, L.D. Correa, K.T. Whelan, J.M. Vernier, Pharmacological characterization of SIB-1663, a conformationally rigid analog of nicotine, Brain Res. 1003 (2004) 42–53. D. kang, Y.S. Gho, M. Suh, C. Kang, Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Bull. Kor. Chem. Soc. 23 (2002) 1511–1512. V. Di Matteo, M. Pierucci, E. Esposito, Selective stimulation of serotonin2c receptors blocks the enhancement of striatal and accumbal dopamine release induced by nicotine administration, J. Neurochem. 89 (2004) 418–429. V.I. Pidoplichko, M. DeBiasi, J.T. Williams, J.A. Dani, Nicotine activates and desensitizes midbrain dopamine neurons, Nature 390 (1997) 401–404. L. Bai, J.L. Merchant, Transcription factor ZBP-89 is required for STAT1 constitutive expression, Nucleic Acids Res. 31 (2003) 7264–7270. L. Bai, J.L. Merchant, ZBP-89 promotes growth arrest through stabilization of p53, Mol. Cell. Biol. 21 (2001) 4670–4683. Y. Jiang, W. Yang, Y. Zhou, L. Ma, Up-regulation of murine double minute clone 2 (MDM2) gene expression in rat brain after morphine, heroin, and cocaine administrations, Neurosci. Lett. 352 (2003) 216–220. M.J. Garcia-Fuster, M. Ferrer-Alcon, A. Miralles, J.A. GarciaSevilla, Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain, Naunyn Schmiedebergs Arch. Pharmacol. 368 (2003) 421–431. T.A. Slotkin, Developmental cholinotoxicants: nicotine and chlorpyrifos, Environ. Health Perspect. 107 (Suppl. 1) (1999) 71–80. Y.P. Wu, K. Kita, N. Suzuki, Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis, Int. J. Cancer 100 (2002) 37–42.
327
[26] T.S. Roy, J.E. Andrews, F.J. Seidler, T.A. Slotkin, Nicotine evokes cell death in embryonic rat brain during neurulation, J. Pharmacol. Exp. Ther. 287 (1998) 1136–1144. [27] H. Mai, W.S. May, F. Gao, Z. Jin, X. Deng, A functional role for nicotine in Bcl2 phosphorylation and suppression of apoptosis, J. Biol. Chem. 278 (2003) 1886–1891. [28] A. Hakki, K. Pennypacker, S. Eidizadeh, H. Friedman, S. Pross, Nicotine inhibition of apoptosis in murine immune cells, Exp. Biol. Med. (Maywood) 226 (2001) 947–953. [29] S.S. Scherer, P.E. Braun, J. Grinspan, E. Collarini, D.Y. Wang, J. Kamholz, Differential regulation of the 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase gene during oligodendrocyte development, Neuron 12 (1994) 1363–1375. [30] M. Gravel, E. Gao, C. Hervouet-Zeiber, V. Parsons, P.E. Braun, Transcriptional regulation of 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase gene expression by cyclic AMP in C6 cells, J. Neurochem. 75 (2000) 1940–1950. [31] E.T. Tzavara, E. Valjent, C. Firmo, M. Mas, F. Beslot, N. Defer, B.P. Roques, J. Hanoune, R. Maldonado, Cannabinoid withdrawal is dependent upon PKA activation in the cerebellum, Eur. J. Neurosci. 12 (2000) 1038–1046. [32] E.T. Tzavara, K. Monory, J. Hanoune, G.G. Nomikos, Nicotine withdrawal syndrome: behavioural distress and selective upregulation of the cyclic AMP pathway in the amygdala, Eur. J. Neurosci. 16 (2002) 149–153. [33] A. Wilber, M. Lu, M.C. Schneider, Deoxyribonuclease I-like III is an inducible macrophage barrier to liposomal transfection, Mol. Ther. 6 (2002) 35–42. [34] A.G. Yakovlev, G. Wang, B.A. Stoica, C.M. Simbulan-Rosenthal, K. Yoshihara, M.E. Smulson, Role of DNAS1L3 in Ca2+and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments, Nucleic Acids Res. 27 (1999) 1999–2005. [35] A. Wilber, T.P. OÕConnor, M.L. Lu, A. Karimi, M.C. Schneider, Dnase1l3 deficiency in lupus-prone MRL and NZB/W F1 mice, Clin. Exp. Immunol. 134 (2003) 46–52. [36] S. Rajan, E. Wischmeyer, C. Karschin, R. Preisig-Muller, K.H. Grzeschik, J. Daut, A. Karschin, C. Derst, THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels, J. Biol. Chem. 276 (2001) 7302–7311. [37] O. Hafstrom, J. Milerad, N. Asokan, S.D. Poole, H.W. Sundell, Nicotine delays arousal during hypoxemia in lambs, Pediatr. Res. 47 (2000) 646–652. [38] V.A. Campanucci, I.M. Fearon, C.A. Nurse, A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance, J. Physiol. 548 (2003) 731–743. [39] S. Hirakawa, T. Oohashi, W.D. Su, H. Yoshioka, T. Murakami, J. Arata, Y. Ninomiya, The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain, Biochem. Biophys. Res. Commun. 276 (2000) 982–989. [40] T. Oohashi, S. Hirakawa, Y. Bekku, U. Rauch, D.R. Zimmermann, W.D. Su, A. Ohtsuka, T. Murakami, Y. Ninomiya, Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems, Mol. Cell. Neurosci. 19 (2002) 43–57. [41] A.H. Stegh, O. Schickling, A. Ehret, C. Scaffidi, C. Peterhansel, T.G. Hofmann, I. Grummt, P.H. Krammer, M.E. Peter, DEDD, a novel death effector domain-containing protein, targeted to the nucleolus, Embo J. 17 (1998) 5974–5986. [42] M.D. Tibbetts, L. Zheng, M.J. Lenardo, The death effector domain protein family: regulators of cellular homeostasis, Nat. Immunol. 4 (2003) 404–409. [43] J.C. Lee, O. Schickling, A.H. Stegh, R.G. Oshima, D. Dinsdale, G.M. Cohen, M.E. Peter, DEDD regulates degradation of
328
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intermediate filaments during apoptosis, J. Cell Biol. 158 (2002) 1051–1066. [44] M.T. Berhow, D.S. Russell, R.Z. Terwilliger, D. Beitner-Johnson, D.W. Self, R.M. Lindsay, E.J. Nestler, Influence of neurotrophic factors on morphine- and cocaine-induced biochemical changes in the mesolimbic dopamine system, Neuroscience 68 (1995) 969– 979. [45] P.J. Kenny, S.E. File, M. Rattray, Acute nicotine decreases, and chronic nicotine increases the expression of brain-derived neurotrophic factor mRNA in rat hippocampus, Brain Res. Mol. Brain Res. 85 (2000) 234–238.
[46] G.E. Meredith, S. Callen, D.A. Scheuer, Brain-derived neurotrophic factor expression is increased in the rat amygdala, piriform cortex and hypothalamus following repeated amphetamine administration, Brain Res. 949 (2002) 218–227. [47] R. Maggio, M. Riva, F. Vaglini, F. Fornai, R. Molteni, M. Armogida, G. Racagni, G.U. Corsini, Nicotine prevents experimental parkinsonism in rodents and induces striatal increase of neurotrophic factors, J. Neurochem. 71 (1998) 2439–2446. [48] J.L. Galzi, J.P. Changeux, Neuronal nicotinic receptors: molecular organization and regulations, Neuropharmacology 34 (1995) 563–582.
Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats Mijung Yeom, Insop Shim, Hye-Jung Lee, Dae-Hyun Hahm* Department of Medical Science, Graduate School of East-West Medical Science, Kyung Hee University, Gihung-up, Yongin-si, Gyeonggi-do 449-701, Republic of Korea Received 14 October 2004 Available online 24 November 2004
Abstract Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4 mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2 0 3 0 -cyclic nucleotide 3 0 -phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K+ channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine. 2004 Elsevier Inc. All rights reserved. Keywords: Proteome; Nicotine; Addiction; 2-Dimensional electrophoresis; RT-PCR; Striatum; Protein expression; Rat
Tobacco-related diseases are among the worldÕs leading preventable causes of death. They are responsible for approximately 5 million deaths a year. However, total tobacco consumption is still on the rise [1,2]. This is due to the motivating power of tobacco. Although the mechanisms of nicotine addiction are not completely understood, much evidence suggests that nicotine is an active component in tobacco, which drives the continued use despite its detrimental effects [3–6]. Nicotine addiction produces the aversive behavioral consequences that are commonly seen with the uptake of other addictive drugs, such as cocaine and amphetamine, that is, nicotine reinforces self-administration and place preference, increases locomotor activity, and
*
Corresponding author. Fax: +82 31 204 4237. E-mail address: [email protected] (D.-H. Hahm).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.034
enhances rewards from brain stimulation [6,7]. These effects are known to be mediated by the mesocorticolimbic dopamine system, which projects from the ventral tegmental area to the dorsal and ventral (nucleus accumbens) striatum and the prefrontal cortex [8–10]. The many basic research projects that have been conducted in an attempt to remedy nicotine addiction have therefore focused on the drug-induced neuroadaptation underlying sensitization within this pathway and adjacent neural circuitry [7,11–13]. However, as of yet, there is little understanding of the molecular mechanisms that lead to and maintain nicotine addiction. Therefore, the identification of the neurobiological factors that take a part in nicotine addiction is the first step to find the answer. Two-dimensional gel electrophoresis (2-DE), in combination with mass spectrometry and modern bioinformatics tools, is considered to be the most effective tool to detect unexpected changes of protein modulation
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and to identify qualitative and quantitative differences among the expression profiles of the proteins expressed under different conditions [14]. It is also a powerful method that makes it possible to isolate the protein candidates as a biological marker in the complex pathophysiological processes, such as nicotine addiction [15]. The aim of this study was to detect and identify the protein variants related to nicotine addiction in rat brains. These proteins may be useful in understanding the molecular mechanism of nicotine addiction and for isolating the potential drug targets for nicotine addiction-associated disease therapy.
Materials and methods Animals and nicotine treatment. Male Sprague–Dawley rats weighing 250–280 g at the start of experiments were used throughout. Prior to the experimental manipulation, the animals were given a period of 1 week to adjust to the new environment. Animals were cared for and handled in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animals were divided randomly into nicotine-treated (NIC, n = 10) and control (CONT, n = 10) groups. For nicotine injection, ()-nicotine hydrogen tartrate (Sigma–Aldrich, MO) was dissolved in saline and pH of the final solution was adjusted to 7.2. The course of nicotine treatment consisted of three phases; development, withdrawal, and challenge phases. The rats were injected with nicotine (0.4 mg/kg, s.c.) twice daily for 7 consecutive days. After 3 days of withdrawal period, the rats were challenged with the last systemic injection of nicotine on day 11. Normal group was injected with saline instead of nicotine solution. Locomotor activity. After the final nicotine injection in challenge phase, the nicotine-induced locomotor activity was measured in a rectangular container (40 · 40 · 45 cm each) equipped with a video camera above the center of the floor. The wall and floor were made of clear Plexiglas and were painted black. Locomoter activity was monitored by a video-tracking system using S-mart program (PanLab, Barcelona, Spain). The day before the locomotor activity determination, rats were allowed to individually habituate to the test cages for 1 h, during which the activity was monitored. This habituation period was required to overcome the potentially stressful nature of the test apparatus, which may affect the basal levels of locomoter activities of the rats [16]. On the test day, after the baseline determination for 30 min, the animals were challenged with nicotine and locomoter activity was monitored for 30 min. Locomotor activity, expressed as distance travelled (in centimeter), was calculated as the total distance moved during the 30-min period after the drug challenge. Sample preparation. For histological analysis, the striatum of approx. 30–50 mg wet weight was dissected from the brains and stored at 80 C until protein or RNA extraction. For protein extraction, the striatum was homogenized within a detergent lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) Chaps, 0.5% (v/v) Triton X-100, 0.5% (v/v) Pharmalytes pH 3–10 (Amersham Biosciences, NJ), 100 mM DTT, and 1.5 mg/mL complete Protease Inhibitor Cocktail for mammalian tissues (Sigma–Aldrich, MO), sonicated, and incubated for 1 h at room temperature in an orbital shaker. The lysate was then centrifuged at 13,000 rpm for 30 min. The total protein concentration of each sample was analyzed by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, CA). The protein samples were stored in aliquots at 80 C until use. Total RNA from frozen samples was extracted using TRIzol reagent (Invitrogen, CA), in accordance with manufacturerÕs instructions. The RNA extracted was dissolved in DEPC-treated distilled water and stored at 80 C. The concentration and purity of RNA were checked by measuring the
optical density at 260 and 280 nm using GeneQuant pro spectrophotometer (Amersham Biosciences, NJ). Two-dimensional electrophoresis (2-DE) and image analysis. For isoelectric focussing (IEF), 200 lg of total proteins was mixed in a rehydration buffer (7 M urea, 2 M thiourea, 4% Chaps, 0.5% Triton X-100, 100 mM DTT, 0.6% Pharmalytes pH 3–10, and bromophenol blue) in total volume of 340 lL and loaded on 18 cm pH 3–10 NL Immobiline DryStrip (an IPG strip, Amersham biosciences, NJ). After IPG strip rehydration, IEF was carried out for 16 h up to a total of 65,000 V/h using pHaser Isoelectric Focusing System (Genomic Solutions, MI). After the equilibration of rehydrated IPG strips, the second-dimensional gel electrophoresis was subsequently performed on 26 · 24 cm of 10% SDS–polyacrylamide gels using Investigator System (Genomic Solutions, MI). Gels were stained with Coomassie blue G-250 (Bio-Rad Laboratories, CA) staining solution as described by Kang et al. [17]. The stained gels were then analyzed with ImageMaster 2D-Elite (Amersham biosciences, NJ) software for spot detection, quantification, and matching. Protein identification by peptide mass fingerprinting. Selected protein spots were excised from the gels and placed in 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate (NH4HCO3) for 15 min and the supernatant was removed. These steps were repeated until the Coomassie blue G-250 dye was completely removed. The gel pieces were then dried in a vacuum-centrifuge, rehydrated in 15 lL chilled trypsin solution (10 lg/mL of sequencing-grade modified trypsin (Promega Biosciences, CA) in 25mM NH4HCO3 buffer) for 50 min, and incubated at 37 C for 16 h. The supernatant was collected and the peptides were extracted with 5% trifluoroacetic acid (TFA) in 50% ACN twice. Peptide extracts were then dried and stored at 20 C until use. The extracted peptides were desalted using ZipTips C18 resin (Millipore, MA) according to the manufacturerÕs instructions, and eluted (from C18 resin) with matrix, a saturated solution of a-cyano-4hydroxycinnamic acid in 50% CAN/0.1% TFA. MALDI-TOF mass spectra were acquired on Voyager-DE STR MALDI-TOF mass spectrometer (MS) (Applied Biosystems, CA). The peptide mass fingerprints were analyzed by MS-Fit search program using NCBInr and/ or SWISS-PROT protein databases. RT-PCR. One microgram of total RNA was reverse-transcribed with M-MLV reverse transcriptase (Invitrogen, CA) in a 20 lL of reaction mixture containing random hexamers. The cDNA was diluted five times and 5 lL of the resulting diluent was amplified with Taq polymerase in a 20 lL reaction mixture in a PTC-100 programmable thermal controller (MJ Research, MA). All primers in Table 1 were designed from the published cDNA sequences using primer selection software, Primer 3, which is offered through a web site (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). The amplifications were performed as follows: denaturation at 94 C for 30 s, annealing at 53– 58 C for 30 s, and polymerization at 72 C for 30 s. Annealing temperature and the number of PCR cycles to each primer was referred to Table 1. PCR products were separated on 1.2% agarose gels, stained with ethidium bromide, and photographed. The intensities of the bands were measured by using ImageMaster VDS (Amersham Biosciences, NJ) with an image analysis software, ImageMaster TotalLab (Amersham Biosciences, NJ). Data analysis. Data were expressed as means ± standard errors. Differences were analyzed with StudentÕs t test. p values of <0.05 were considered to be significant.
Results and discussion Tobacco smoking still remains a major health problem worldwide. Nicotine is a principal component of tobacco, eliciting addictive behavior in human [1,5,6]. Intermittent nicotine administration brings about the
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Table 1 Primers used in PCRs Name
Primer sequence
Product size (bp)
Annealing temperature (C)
Cycle number
ZBP-89
5 -GACAACCAGACCCTTCCAAA-3 5 0 -CGAAGGTTCACCTGTCCATT-3 0
0
377
58
30
CNP1
5 0 -TCCGAGGAGTACAAGCGTCT-3 0 5 0 -TTGTGATTTCCCAGCTCCTC-3 0
359
58
24
DNase1l3
5 0 -GTGATTAGCTCTCGGCTTGG-3 0 5 0 -CCCAATCAGCCAAACAAAGT-3 0
393
53
40
THIK-2
5 0 -GGGACCATCCTGTTCTTCAA-3 0 5 0 -GAAGTTGCCCAGACGGTAGA-3 0
378
55
35
BRAL-1
5 0 -GGCCGCTACCAGTTCAATTA-3 0 5 0 -CCGACCTTGGCTACTACAGC-3 0
359
58
30
DEDD
5 0 -TCCCCACTATCCTGTGGTGT-3 0 5 0 -AACGCCTTTCAGTGCCTCTA-3 0
388
58
30
BDNF
5 0 -GATGAGGACCAGAAGGTTCG-3 0 5 0 -GATTGGGTAGTTCGGCATTG-3 0
425
58
30
GAPDH
5 0 -ATCCCATCACCATCTTCCAG-3 0 5 0 -CCTGCTTCACCACCTTCTTG-3 0
579
58
30
0
behavioral sensitization, which is defined as an increased behavioral and/or neurochemical response [8]. Locomotor activation and reinforcement may be relevant to the stimulatory effects of nicotine on the mesolimbic dopaminergic system [18]. The striatum and nucleus accumbens are considered as the brain sites relevant to the action of drug rewards, and thus, expected to be associated neuroadaptations, such as sensitization [8,18]. However, the mechanism of nicotine addiction has not yet been completely understood. The goal of this study was therefore to identify the proteins related to the nicotine addiction, by analyzing striatum proteome of nicotine-addictive rat brain as compared to normal one. Behavioral studies indicated that nicotine is an addictive drug which reinforces self-administration, places preference, and increases locomotion [19]. To determine whether the behavioral sensitization will be developed after repeated nicotine injection or not, the locomotor activity was monitored during the first 30 min after every administration of nicotine in this study. Rats in CONT group were injected with saline instead of nicotine. The locomotor activities of NIC group were found to be significantly increased as compared to those of CONT group (p < 0.001). It was confirmed by observing 2.3-fold increase of locomoter activity of NIC group during the challenge period that the rat model of nicotine addiction was successfully developed (Fig. 1). Protein expression patterns of the rat striatum after repeated nicotine exposure were analyzed by using 2DE and MALDI-TOF MS, followed by database searching and protein annotation. Ten animals per each experimental group were randomly spilt in two parts and then used for 2-DE and RT-PCR analyses, respectively. 2-DE was executed three times for each group. At first glance, quite similar expression patterns with lit-
Fig. 1. The effect of nicotine on locomotor activity. Locomotor activity is expressed as the total distance travelled during 30 min before (baseline) and after (test) nicotine injection on the challenge day. (***p < 0.001 as compared to CONT group).
tle differences were shown in both groups (Fig. 2A). The framed area was selected to pick the candidate proteins, which were quantitatively modulated, and thus, estimated to be associated with nicotine addiction. Seven spots, differently expressed between CONT and NIC groups and fulfilled p < 0.05 (t test), were finally isolated within the area (Fig. 2B). These proteins were identified by MALDI-TOF MS and database searching, and thus are summarized in Table 2. The expression of three proteins, ZBP89, CNP1, and DNase1l3, was highly increased in NIC group relative to CONT. And four proteins, THIK-2, BRAL-1, DEDD, and BDNF, mostly disappeared after repeated nicotine exposure, which means that those proteins were detected only in the CONT group. Zinc-finger binding protein-89 (ZBP-89, also known as ZNF148, Zfp148, BFCOL1 and BERF-1) is a Kruppel-type zinc-finger transcription factor that is
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Fig. 2. 2-DE protein patterns of rat striatum in response to repeated nicotine treatment. (A) Full images of 2-DE gels of CONT and NIC group. (B) Close-up of frames a, b, and c of (A). The protein variants which are estimated to be modulated are marked with arrows and numbers. CONT, salineinjected group; NIC, nicotine-injected group.
Table 2 Proteins increased or decreased in rat striatum after repeated nicotine treatment Spot No.
Identified protein
Accession No. NCBI
No. of peptides matched
Sequence coverage (%)
MW (kDa)/pI
Group I 1 2 3
Mowse score
ZBP-89 CNP1 DNase1l3
NP_113803 XP_340905 NP_446359
13 10 6
18 22 26
88.7/6.0 47.3/9.0 35.7/9.1
46,390 4000 25,920
Group II 4 5 6 7
THIK-2 BRAL-1 DEDD BDNF
NP_071628 Q9ESM2a NP_113988 CAA47481
5 4 10 14
10 12 28 38
46.9/9.8 38.0/9.3 36.8/9.2 27.3/8.9
235 140 13,710 164,700
Spots were classified into two groups, groups I and II, exhibited the increasing and decreasing patterns of protein expression, respectively. ZBP-89, zinc-finger binding protein-89; CNP1, 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase 1; DNase1l3, deoxyribonuclease 1-like 3; THIK-2, tandem pore domain halothane inhibited K+ channel; BRAL-1, brain-specific hyaluronan-binding protein; DEDD, death effector domain-containing DNA binding protein; and BDNF, brain-derived neurotrophic factor. a SWISS-PROT protein database was used to evaluate MS data in this case.
universally expressed in living organisms. ZBP-89 has multiple functions including transcriptional regulation of a variety of genes, cell growth arrest, and cell death [20]. Elevated levels of ZBP-89 induce growth arrest and apoptosis through the stabilization of p53 in cells [21]. This means that ZBP-89 induces apoptosis through p53-independent mechanism. The pro-inflammatory
cytokine, IFN-c, is known to induce p53-independent apoptosis. This mechanism seems to be attributed to STAT1 activation, followed by the increased expression of caspase-3, -8, and -9 as well as the inhibition of Bcl-2 expression. In this process, ZBP-89 binds to the GC-rich elements on STAT1 promoter, and thus regulates STAT1 expression and subsequent activation of apopto-
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sis [20]. Repeated administration of addictive drugs induces neuronal apoptosis, in general [22,23]. Although the conflicting experimental results of the apoptotic effects by nicotine exposure have been reported, nicotine administration evokes the apoptosis of neuronal cells in most cases [24–26], which is not consistent in either lung cancer cells [27] or immune cells [28]. Although we did not confirm the effects of nicotine on neural apoptosis, the induction of ZBP-89 after repeated nicotine administration might be related to apoptosis induction by nicotine. 2 0 ,3 0 -Cyclic nucleotide 3 0 -phosphodiesterase 1 (CNP1) is one of the two CNP isoforms. The two CNP isoforms, CNP1 (46 kDa) and CNP2 (48 kDa), are encoded by a single gene. Both CNP mRNAs, abundantly expressed in oligodendrocytes, are differentially expressed during the process of oligodendrocyte differentiation. While CNP2 mRNA is detected in oligodendrocyte progenitor cells, CNP1 mRNA is transcribed only after birth in differentiated oligodendrocytes. And both CNP transcripts are present at much higher levels in myelinating oligodendrocytes [29]. However, the cellular signals that regulate the specific expression of two CNP mRNAs have not been clear yet. Recently, it was reported that cAMP activates CNP1 gene expression via a cis-acting element, located between positions 126 and 102 of the mouse CNP1 promoter in C6 cells. cAMP regulates the development of oligodendrocytes and modulates the expression of myelin-specific proteins including CNP, myelin-associated glycoproteins, myelin basic proteins, and proteolipid proteins [30]. Also, up-regulation of the cAMP pathway is considered as an important molecular mechanism of the withdrawal syndrome by drug abuse. The chronic administration of addictive drugs leads to up-regulation of both adenylyl cyclase and protein kinase-A that control the cAMP cascade [31,32]. Although it can be assumed that cAMP has a role in nicotine addiction by the regulation of CNP1 expression, there has been no report that CNP1 is related to any kind of addiction including nicotine. Deoxyribonuclease 1-like 3 (DNase1l3) is highly homologous to DNase I except for having a basic carboxy-terminal extension and lacking N-linked glycosylation site [33]. DNase1l3 participates in apoptotic DNA fragmentation. The nuclear localization of DNase1l3 is the unique feature that distinguishes it from other suggested apoptotic nucleases. The DNase1l3 activity is regulated by poly(ADP-ribose)polymerase (PARP) during apoptosis, that is, DNase1l3 is inhibited by poly(ADP-ribosyl)ation and activated by the subsequent cleavage of RIPA during apoptosis [34]. Another function of DNase1l3 is an in vivo nuclease barrier to liposomal gene transfection. The main in vivo target of DNase1l3 is not only the apoptotic debris but other membrane-DNA particles such as viruses [33]. DNA1l3
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deficiency is common in systemic lupus erythematosus (SLE). This deficiency may stimulate the characteristic autoimmunity of SLE and increase the susceptibility to polygenic SLE [35]. However, the role of DNase1l3 in the regulation of apoptosis, induced by nicotine administration, has not been investigated yet. Tandem pore domain halothane inhibited K+ channel (THIK-2), identified a few years ago, is the one of two members of a novel 2P K+ channel subfamily. THIK-2 was expressed in several tissues including liver and kidney, and particularly strong in brain [36]. It implies that THIK-2 might have an important function in neurons, even though its physiological function is unknown. Recently, there was a report supporting this. The oxygen sensitivity of glossopharyngeal nerve (GPN) was found to be mediated by THIK [37]. Since GPN neurons were excited by hypoxia via inhibition of background channels such as THIK-2, this excitation could lead to the increase of Ca2+ levels, the activation of nNOS, and the subsequent synthesis and release of NO. The resulting inhibitory effect of NO provided a means of negative feedback modulation of chemoreceptor activity during hypoxia [38]. It was also reported that nicotine exposure attenuated oxygen sensitivity, attributed to hypoxia [37]. However, there has been no report about the effect of nicotine on THIK-2 regulation. Brain-specific hyaluronan-binding protein (BRAL-1) was recently cloned [39]. BRAL-1 was highly concentrated at the nodes of Ranvier in the developing brain and the mature CNS of mouse, where versican V2 was also localized. And, the bral1 mRNA was predominantly expressed in neurons. Oohashi et al. [40] suggested that BRAL-1 played a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes. Death effector domain-containing DNA binding protein (DEDD) contains a death effector domain (DED), the prototypical six a-helical domains which are homologous to the DEDs of the Fas-associated death domain (FADD), caspase-8 and -10, and two nuclear translocation signals (NLS). DEDD is known to weakly induce the apoptosis through its DED. DEDD resides in the cytoplasm in an active form and is translocated to the nucleus upon induction of the CD95-mediated apoptosis which is not associated with the death-inducing signaling complex (DISC). It suggests that DEDD may function in the downstream of CD95-induced apoptosis [41]. DEDD localizes to nucleoli-like structures, activates caspase-6, and inhibits RNA polymerase I-dependent transcription [42]. DEDD also acts as a scaffold that brings caspase-3 to the intermediate filament (IF) for the degradation of IF protein, essential step in the morphological changes during apoptosis [43]. However, the concrete role of DEDD in apoptosis remains unclear.
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Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor (NGF)-related family of neurotrophins and utilizes the Trk receptor-protein tyrosine kinase as primary means of signal transduction [44]. BDNF is extensively expressed throughout the CNS. It is known in general that BDNF promotes cell survival, regulates dendrites and synaptic plasticity, and plays a role in complex behaviors [45]. Also, BDNF contributes to the survival and differentiation of dopaminergic neurons during development [46]. In brain, BDNF expression kinetics by nicotine exposure has been controversial, that is, the high-dose and flash-type exposure of nicotine usually decreased BDNF levels whereas its expression was increased by the long-term exposure [45,47]. The difference between short- and long-term exposure of nicotine seems to be associated with the development of desensitization of neuronal nicotinic acetylcholine receptor [48]. These results did not coincide with our results in which the BDNF expression of both protein and mRNA was decreased by repeated exposure of nicotine. It could be postulated that in repeated injection protocols, the last injection as a challenge after 3-day withdrawal period might produce the acute properties of nicotine addiction in terms of protein expression profiles, which resulted in the down-regulation of BDNF. In order to verify the mRNA expression patterns of the proteins selected, RT-PCR analyses were performed using total RNA of striatum from NIC group. It was found that the transcriptional levels of ZBP89 and DNase1l3 mRNAs were increased by the nicotine exposure and THIK-2, DEDD, and BDNF mRNAs were decreased, which corresponded with expression profiles of these proteins. In cases of CNP1 and BRAL-1, the difference between NIC and CONT groups was not significant (Fig. 3). In summary, we have performed a proteomic analysis of rat striatum after repeated nicotine injection. After development of nicotine addiction, which was verified by analyzing the locomoter activity, seven protein variants were identified to be modulated by MALDI-TOF MS analysis and computer searching. The expression of three proteins such as ZBP-89, CNP1, and DNase1l3 was found to be increased whereas the expression of THIK-2, BRAL-1, DEDD, and BDNF was decreased. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were coincident with those of cognate proteins. These protein candidates might be useful to understand the molecular mechanism of nicotine addiction and isolate the biomarkers or targets for developing the addiction-associated diseases therapy. Further profound studies are needed for understanding the biochemical and physiological roles of every candidate selected, and thus, those proteins are recommended as an useful target for nicotine addiction therapy.
Fig. 3. The gene expression in rat striatum after repeated nicotine treatment. The expression level of each target PCR product was normalized to that of GAPDH, a housekeeping gene. Values are means with standard deviations from at least three independent experiments. (*p < 0.05, **p < 0.01 as compared to CONT group).
Acknowledgments The authors thank Prof. Kang-Duk Choi at Hankyong National University for the technical support and the helpful discussion. This study was supported by the Brain Korea 21 Project from the Ministry of Education, Republic of Korea.
References [1] WHO, The World Health Report, ed., World Health Organization Publications, 2003. [2] M.R. Carrera, J.A. Ashley, T.Z. Hoffman, S. Isomura, P. Wirsching, G.F. Koob, K.D. Janda, Investigations using immunization to attenuate the psychoactive effects of nicotine, Bioorg. Med. Chem. 12 (2004) 563–570. [3] J.A. Dani, D. Ji, F.M. Zhou, Synaptic plasticity and nicotine addiction, Neuron 31 (2001) 349–352. [4] J.Y. Bahk, S. Li, M.S. Park, M.O. Kim, Dopamine D1 and D2 receptor mRNA up-regulation in the caudate-putamen and nucleus accumbens of rat brains by smoking, Prog. Neuropsychopharmacol. Biol. Psychiatry 26 (2002) 1095–1104. [5] P.J. Kenny, A. Markou, Neurobiology of the nicotine withdrawal syndrome, Pharmacol. Biochem. Behav. 70 (2001) 531–549. [6] J.A. Dani, M. De Biasi, Cellular mechanisms of nicotine addiction, Pharmacol. Biochem. Behav. 70 (2001) 439–446. [7] Y. Chae, C.H. Yang, Y.K. Kwon, M.R. Kim, K.H. Pyun, D.H. Hahm, H.J. Lee, I. Shim, Acupuncture attenuates repeated
M. Yeom et al. / Biochemical and Biophysical Research Communications 326 (2005) 321–328
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
nicotine-induced behavioral sensitization and c-Fos expression in the nucleus accumbens and striatum of the rat, Neurosci. Lett. 358 (2004) 87–90. M.S. Todtenkopf, J.R. Stellar, E.A. Williams, D.S. Zahm, Differential distribution of parvalbumin immunoreactive neurons in the striatum of cocaine sensitized rats, Neuroscience 127 (2004) 35–42. W.A. Carlezon Jr., E.J. Nestler, Elevated levels of GluR1 in the midbrain: a trigger for sensitization to drugs of abuse?, Trends Neurosci. 25 (2002) 610–615. F.J. White, P.W. Kalivas, Neuroadaptations involved in amphetamine and cocaine addiction, Drug Alcohol Depend. 51 (1998) 141–153. M.R. Picciotto, M. Zoli, R. Rimondini, C. Lena, L.M. Marubio, E.M. Pich, K. Fuxe, J.P. Changeux, Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine, Nature 391 (1998) 173–177. F.E. Pontieri, G. Tanda, F. Orzi, G. Di Chiara, Effects of nicotine on the nucleus accumbens and similarity to those of addictive drugs, Nature 382 (1996) 255–257. P. Fredrickson, M. Boules, S. Yerbury, E. Richelson, Novel neurotensin analog blocks the initiation and expression of nicotine-induced locomotor sensitization, Brain Res. 979 (2003) 245–248. A. Moller, M. Soldan, U. Volker, E. Maser, Two-dimensional gel electrophoresis: a powerful method to elucidate cellular responses to toxic compounds, Toxicology 160 (2001) 129–138. S. Kageyama, T. Isono, H. Iwaki, Y. Wakabayashi, Y. Okada, K. Kontani, K. Yoshimura, A. Terai, Y. Arai, T. Yoshiki, Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine, Clin. Chem. 50 (2004) 857–866. T.S. Rao, A.I. Sacaan, F.M. Menzaghi, R.T. Reid, P.B. Adams, L.D. Correa, K.T. Whelan, J.M. Vernier, Pharmacological characterization of SIB-1663, a conformationally rigid analog of nicotine, Brain Res. 1003 (2004) 42–53. D. kang, Y.S. Gho, M. Suh, C. Kang, Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Bull. Kor. Chem. Soc. 23 (2002) 1511–1512. V. Di Matteo, M. Pierucci, E. Esposito, Selective stimulation of serotonin2c receptors blocks the enhancement of striatal and accumbal dopamine release induced by nicotine administration, J. Neurochem. 89 (2004) 418–429. V.I. Pidoplichko, M. DeBiasi, J.T. Williams, J.A. Dani, Nicotine activates and desensitizes midbrain dopamine neurons, Nature 390 (1997) 401–404. L. Bai, J.L. Merchant, Transcription factor ZBP-89 is required for STAT1 constitutive expression, Nucleic Acids Res. 31 (2003) 7264–7270. L. Bai, J.L. Merchant, ZBP-89 promotes growth arrest through stabilization of p53, Mol. Cell. Biol. 21 (2001) 4670–4683. Y. Jiang, W. Yang, Y. Zhou, L. Ma, Up-regulation of murine double minute clone 2 (MDM2) gene expression in rat brain after morphine, heroin, and cocaine administrations, Neurosci. Lett. 352 (2003) 216–220. M.J. Garcia-Fuster, M. Ferrer-Alcon, A. Miralles, J.A. GarciaSevilla, Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain, Naunyn Schmiedebergs Arch. Pharmacol. 368 (2003) 421–431. T.A. Slotkin, Developmental cholinotoxicants: nicotine and chlorpyrifos, Environ. Health Perspect. 107 (Suppl. 1) (1999) 71–80. Y.P. Wu, K. Kita, N. Suzuki, Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis, Int. J. Cancer 100 (2002) 37–42.
327
[26] T.S. Roy, J.E. Andrews, F.J. Seidler, T.A. Slotkin, Nicotine evokes cell death in embryonic rat brain during neurulation, J. Pharmacol. Exp. Ther. 287 (1998) 1136–1144. [27] H. Mai, W.S. May, F. Gao, Z. Jin, X. Deng, A functional role for nicotine in Bcl2 phosphorylation and suppression of apoptosis, J. Biol. Chem. 278 (2003) 1886–1891. [28] A. Hakki, K. Pennypacker, S. Eidizadeh, H. Friedman, S. Pross, Nicotine inhibition of apoptosis in murine immune cells, Exp. Biol. Med. (Maywood) 226 (2001) 947–953. [29] S.S. Scherer, P.E. Braun, J. Grinspan, E. Collarini, D.Y. Wang, J. Kamholz, Differential regulation of the 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase gene during oligodendrocyte development, Neuron 12 (1994) 1363–1375. [30] M. Gravel, E. Gao, C. Hervouet-Zeiber, V. Parsons, P.E. Braun, Transcriptional regulation of 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase gene expression by cyclic AMP in C6 cells, J. Neurochem. 75 (2000) 1940–1950. [31] E.T. Tzavara, E. Valjent, C. Firmo, M. Mas, F. Beslot, N. Defer, B.P. Roques, J. Hanoune, R. Maldonado, Cannabinoid withdrawal is dependent upon PKA activation in the cerebellum, Eur. J. Neurosci. 12 (2000) 1038–1046. [32] E.T. Tzavara, K. Monory, J. Hanoune, G.G. Nomikos, Nicotine withdrawal syndrome: behavioural distress and selective upregulation of the cyclic AMP pathway in the amygdala, Eur. J. Neurosci. 16 (2002) 149–153. [33] A. Wilber, M. Lu, M.C. Schneider, Deoxyribonuclease I-like III is an inducible macrophage barrier to liposomal transfection, Mol. Ther. 6 (2002) 35–42. [34] A.G. Yakovlev, G. Wang, B.A. Stoica, C.M. Simbulan-Rosenthal, K. Yoshihara, M.E. Smulson, Role of DNAS1L3 in Ca2+and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments, Nucleic Acids Res. 27 (1999) 1999–2005. [35] A. Wilber, T.P. OÕConnor, M.L. Lu, A. Karimi, M.C. Schneider, Dnase1l3 deficiency in lupus-prone MRL and NZB/W F1 mice, Clin. Exp. Immunol. 134 (2003) 46–52. [36] S. Rajan, E. Wischmeyer, C. Karschin, R. Preisig-Muller, K.H. Grzeschik, J. Daut, A. Karschin, C. Derst, THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels, J. Biol. Chem. 276 (2001) 7302–7311. [37] O. Hafstrom, J. Milerad, N. Asokan, S.D. Poole, H.W. Sundell, Nicotine delays arousal during hypoxemia in lambs, Pediatr. Res. 47 (2000) 646–652. [38] V.A. Campanucci, I.M. Fearon, C.A. Nurse, A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance, J. Physiol. 548 (2003) 731–743. [39] S. Hirakawa, T. Oohashi, W.D. Su, H. Yoshioka, T. Murakami, J. Arata, Y. Ninomiya, The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain, Biochem. Biophys. Res. Commun. 276 (2000) 982–989. [40] T. Oohashi, S. Hirakawa, Y. Bekku, U. Rauch, D.R. Zimmermann, W.D. Su, A. Ohtsuka, T. Murakami, Y. Ninomiya, Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems, Mol. Cell. Neurosci. 19 (2002) 43–57. [41] A.H. Stegh, O. Schickling, A. Ehret, C. Scaffidi, C. Peterhansel, T.G. Hofmann, I. Grummt, P.H. Krammer, M.E. Peter, DEDD, a novel death effector domain-containing protein, targeted to the nucleolus, Embo J. 17 (1998) 5974–5986. [42] M.D. Tibbetts, L. Zheng, M.J. Lenardo, The death effector domain protein family: regulators of cellular homeostasis, Nat. Immunol. 4 (2003) 404–409. [43] J.C. Lee, O. Schickling, A.H. Stegh, R.G. Oshima, D. Dinsdale, G.M. Cohen, M.E. Peter, DEDD regulates degradation of
328
M. Yeom et al. / Biochemical and Biophysical Research Communications 326 (2005) 321–328
intermediate filaments during apoptosis, J. Cell Biol. 158 (2002) 1051–1066. [44] M.T. Berhow, D.S. Russell, R.Z. Terwilliger, D. Beitner-Johnson, D.W. Self, R.M. Lindsay, E.J. Nestler, Influence of neurotrophic factors on morphine- and cocaine-induced biochemical changes in the mesolimbic dopamine system, Neuroscience 68 (1995) 969– 979. [45] P.J. Kenny, S.E. File, M. Rattray, Acute nicotine decreases, and chronic nicotine increases the expression of brain-derived neurotrophic factor mRNA in rat hippocampus, Brain Res. Mol. Brain Res. 85 (2000) 234–238.
[46] G.E. Meredith, S. Callen, D.A. Scheuer, Brain-derived neurotrophic factor expression is increased in the rat amygdala, piriform cortex and hypothalamus following repeated amphetamine administration, Brain Res. 949 (2002) 218–227. [47] R. Maggio, M. Riva, F. Vaglini, F. Fornai, R. Molteni, M. Armogida, G. Racagni, G.U. Corsini, Nicotine prevents experimental parkinsonism in rodents and induces striatal increase of neurotrophic factors, J. Neurochem. 71 (1998) 2439–2446. [48] J.L. Galzi, J.P. Changeux, Neuronal nicotinic receptors: molecular organization and regulations, Neuropharmacology 34 (1995) 563–582.