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Changes in the expression of transthyretin and protein kinase Cγ genes in the prefrontal cortex in response to naltrexone
Neuroscience Letters 488 (2011) 288–293
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Changes in the expression of transthyretin and protein kinase C␥ genes in the prefrontal cortex in response to naltrexone Jaehak Yu a,1 , Debasish Halder b,1 , Mi Na Baek b , Nando Dulal Das b , Mi Ran Choi b , Dong Yul Oh c , Ihn Geun Choi d , Kyoung Hwa Jung b,e,∗ , Young Gyu Chai b,∗ a
Department of Neuropsychiatry, Konkuk University School of Medicine, Seoul, Republic of Korea Division of Molecular and Life Sciences, Hanyang University, 1271 Sa-dong, Ansan, Gyeonggi-do 426-791, Republic of Korea Department of Psychiatry, Kwandong University, Gangneung, Republic of Korea d Department of Neuropsychiatry, College of Medicine, Hallym University, Seoul, Republic of Korea e Institute of Natural Science and Technology, Hanyang University, Ansan, Republic of Korea b c
a r t i c l e
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Article history: Received 18 August 2010 Received in revised form 17 November 2010 Accepted 17 November 2010 Keywords: Naltrexone Ethanol Prefrontal cortex Hippocampus Transthyretin (TTR) Protein kinase C (PKC)␥
a b s t r a c t Naltrexone, an opioid receptor antagonist, has been approved for clinical use in the treatment of alcohol dependence. In the present study, we examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200 l (16 mg/kg) of naltrexone for 4 weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity Pathway Analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and hippocampus. We found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the expression of transthyretin (TTR) and protein kinase C (PKC)␥ were increased in the PFC but not in the hippocampus of naltrexonetreated mice. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice. These findings provide a basis for conducting pharmacogenetic research on the effect of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders. © 2010 Elsevier Ireland Ltd. All rights reserved.
Among the neurotransmitter systems, the endogenous opioid system is the most important for controlling alcohol-seeking behavior. Prolonged alcohol administration generally decreases endogenous opioid activity, which in turn, causes opioid withdrawal and promotes alcohol consumption through negative reinforcement [27]. The therapeutic efficacy of opioid receptor antagonists in the treatment of alcohol dependence suggests the existence of an alcohol–opioid interaction that may be regulated directly or through interactions with other neurotransmitters [12]. Naltrexone is an opioid antagonist that interferes with the rewarding effects of drinking alcohol or taking other drugs. Naltrexone blocks -opioid receptors, which reduces the reinforcing effects of alcohol leading to decreased feelings of intoxication and fewer cravings [25,30,31].
∗ Corresponding authors. Tel.: +82 31 400 5513; fax: +82 31 406 6316. E-mail addresses: [email protected] (K.H. Jung), [email protected] (Y.G. Chai). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.049
Although naltrexone has been approved for the treatment of alcoholism, there is great individual variation in the efficacy of naltrexone among different patients. Some patients with alcoholism respond very well, while others report no benefit. Drinkers who have a large endogenous opioid response to ethanol would have a large response to the blocking effect of naltrexone. However, those who lack a strong endogenous opioid response to alcohol would not notice any beneficial effects of naltrexone. Recently, Anton et al. focused on several factors that can modulate the response to naltrexone, including genetic variants that may predispose individuals to respond more positively to this pharmacotherapy [1]. Furthermore, naltrexone may act on enzymes within the cell that are involved in the neuroadaptation to alcohol. It has been shown that chronic ethanol intake alters the distribution of these enzymes within the cells and can change the way brain cells respond to alcohol [15,16,24]. Alcohol intake increases the release of opioid peptides and subsequently increases gene expression in the brain regions related to reinforcement and reward, such as the hippocampus or prefrontal cortex (PFC). The PFC is thought to be
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Fig. 1. Naltrexone reduced alcohol consumption in the C57BL/6 mice. Mice were injected with naltrexone (16 mg/kg) every 3 days for 1 month. (B) Volumetric alcohol consumption was markedly reduced in naltrexone-treated mice compared to NTC mice. (A) However, the level of water consumption was increased in naltrexone-treated mice. (C) qRT-PCR analysis revealed that the expression levels of PKC␥, Ttr and Ndst4 mRNA were increased in the PFC of naltrexone-treated mice. Here, values represent the mean ± standard error (SE) of three independent experiments. (D) However, the expressions of PKC␥ and Ttr were reduced in the hippocampus of naltrexone treated mice.
vulnerable to the neurotoxic effects of alcohol use [10]. The PFC shows major alcohol-related damage, including a loss of grey and white matter in chronic alcoholics. Morphometric studies have revealed smaller volumes of gray matter [5] and total tissue [18] in the PFCs of adults with alcohol use disorders compared with controls. The direct toxicity of ethanol and its first metabolite, acetaldehyde, accounts for some of these detrimental effects. Due to the susceptibility of the frontal cortex to alcohol-induced damage and its importance in judgment, decision-making, and other executive functions, we chose to analyze genetic changes in this region. We also looked at changes in the hippocampus. It has been reported that the hippocampal formation is one of the brain regions most sensitive to prolonged ethanol ingestion [3]. Alcohol intake increases the release of opioid peptides and subsequently increases the expression of genes related to reinforcement and reward in the hippocampus. Previous studies has found that the hippocampus is involved in the learning of ethanol-associated cues and may play a critical role in the cue-induced reinstatement of ethanol-seeking, a process dependent on the - and ␦-opioid receptors [22]. In this study, we used cDNA microarray analysis to profile gene expression changes in the hippocampus and PFC of naltrexoneinjected C57BL/6 mice following ethanol treatment. We found that treatment with naltrexone led to alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in response to ethanol compared to non-treated control (NTC) mice. Among the differentially expressed genes, we detected two candidate genes uniquely expressed in the PFC that may be involved in the naltrexone-induced reduction of alcohol consumption. Twelve male C57BL/6 mice weighing 20–30 g (Orient Bio, Gyeonggi-do, Korea) were group housed with ad libitum access to food and water until naltrexone injection. One week later mice were randomly separated for naltrexone and non-treated control (NTC) groups (n = 6 per group). Naltrexone group mice were treated with naltexone + ethanol and the NTC group mice were only ethanol treated. After the naltrexone injection, mice were housed individually in a temperature- and humidity-controlled room on a 12 h
light/dark cycle. Mice were injected intraperitoneally with 200 l (16 mg/kg) of naltrexone every 3 days [14] and were monitored for 4 weeks. A modified version of the two-bottle choice experiment from a previous publication was used [9,19]. Mice were placed in cages containing a 15% alcohol bottle and a water bottle. The positions of the bottles were changed every 3 days. The consumption of alcohol and water were measured every 3 days. Four weeks after the naltrexone injection, the mice were anesthetized with Zoletil 50 (Virbac, Carros, France). The brains were rapidly removed and placed into ice-cold phosphate-buffered saline (PBS). The regions of interest (hippocampus and PFC) were carefully dissected following the method described in Chiu et al. [7]. Hippocampal and PFC tissue collected pooled from four from mice of each treatment group were homogenized and then total RNA was isolated using the TRIzol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions. The quality of the RNA was verified by the presence of prominent 18S and 28S bands on 1% agarose formaldehyde gels and an A260 /A280 ratio between 1.9 and 2.1 (data not shown). mRNA expression was measured using qRT-PCR. The primer sequences used for the qRT-PCR were as follows: PKC␥ – Forward = 5 -CTCGTTTCTTCAAGCAGCCAA-3 , Reverse = 5 GTGAACCACAAAGCTACAGACT-3 ; Ttr – Forward = 5 -TTGCCTCGCTGGACTGGTA-3 , Reverse = 5 -TTACAGCCACGTCTACAGCAG3 ; Ndst4 – Forward = 5 -ACTTTTTGCTTGGTGAGCATCC-3 , Reverse = 5 -CCGATAAGGGAGGTCTTTGATGT-3 . For the microarray analysis, we used the Affymetrix mouse gene 1.0 ST GeneChip containing 770317 probes sets (genes). Total RNA (8 g) pooled from hippocampus and PFC of each treatment group mice was hybridized for three individual GeneChip analysis using the manufacturer’s procedure (Affymetrix, Santa Clara, CA). Microarray data were scanned using a laser confocal scanner (Affymetrix, Clara, CA, USA). All genes showing at least a 2.0-fold change were selected and subsequently classified into functional subgroups using the Protein ANalysis THrough Evolutionary Relationships program (PANTHER; http://www.pantherdb.org). All
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Table 1 Up/down regulated genes in the PFC of naltrexone-treated mice. Gene symbol Up regulated genes Ttr Ndst4 Npy2r Spink8 Kcnj13 Dcn Gm879 Pkp2 Cpne7 Pcdh20 Down regulated genes Cux2 Fhod3 Exph5 Satb2 6330527O06Rik Myl4 Cckbr Rasgef1b 4930578G10Rik Dkkl1
FC
Gene accession
123.25 19.64 12.94 11.07 9.43 9.35 8.00 7.40 7.22 7.03
NM NM NM NM NM NM NM NM NM NM
−4.60 −4.63 −4.83 −4.91 −4.93 −4.95 −5.17 −5.98 −6.88 −7.75
013697 022565 008731 183136 001110227 007833 001034874 026163 170684 178685
ENSMUST00000111752 NM 175276 NM 176846 NM 139146 NM 029530 NM 010858 NM 007627 NM 145839 ENSMUST00000068158 NM 015789
Gene description Transthyretin N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 4 Neuropeptide Y receptor Y2 Serine peptidase inhibitor, Kazal type 8 Potassium inwardly-rectifying channel, subfamily J, member 13 Decorin Gene model 879 (NCBI) Plakophilin 2 Copine VII Protocadherin 20 Cut-like homeobox 2 Formin homology 2 domain containing 3 Exophilin 5 Special AT-rich sequence binding protein 2 RIKEN cDNA 6330527O06 gene Myosin, light polypeptide 4 Cholecystokinin B receptor RasGEF domain family, member 1B RIKEN cDNA 4930578G10 gene dickkopf-like 1
FC, fold change (microarray analysis). Genes were selected by using significance analysis of microarray data with a p value, p < 0.05 and an average fold-change 2.0.
genes were categorized into functional groups at p-value (EASE Score) p < 0.05. Using the selected genes, Ingenuity Pathway Analysis (IPA) version 7.6 (Ingenuity® Systems, www.ingenuity.Com, CA, USA) was used to search for biological pathways and inter-relationship between network genes in the subsets of candidate genes regulated in the different brain regions. The graphical representation of the molecular relationships between genes/gene products were based on the following criteria: genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line); all edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base; the intensity of the node color indicates the degree of up- (red) or down- (green) regulation; and nodes are displayed using various shapes that represent the functional class of the gene product. Microarray data were analyzed by using the GeneChip Operating software (GCOS)/Microarray Suite Version 5.0 (MAS 5.0, Affymetrix, Santa Clara, CA). The detection p-value (p < 0.5) was used to statistically determine whether a transcript is expressed on a chip. All values are expressed as the mean ± standard error (SE). All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data were tested using a one-way ANOVA followed by the Tukey’s HSD post hoc test. p < 0.05 were considered to be significant. To examine the effect of naltrexone on alcohol consumption, one group of mice was injected with naltrexone and another group was used as a non-treated control (NTC). Mice were allowed free access to 15% alcohol for 30 days. The consumption of alcohol and water were measured every 3 days. After day 3, naltrexone-injected mice showed a reduction in voluntary alcohol consumption compared to NTC mice. The reduced alcohol consumption was measured after 4 weeks of treatment (Fig. 1B). However, water consumption in the naltrexone-injected mice was higher than that of the NTC mice (Fig. 1A). This suggests that the decreased ethanol consumption was not caused by an overall decrease in the total amount of fluid consumed. To determine changes in the gene expression of naltrexonetreated mice, we compared the gene expression profile of two specific brain regions, the hippocampus and PFC, to that of NTC mice. We detected 566 genes differentially expressed in the PFC of naltrexone-treated mice. Of these, 265 genes were up-regulated
and 301 were down-regulated. Moreover, in the hippocampus of naltrexone-treated mice, we found 394 differentially expressed genes. 216 genes were up-regulated and 178 genes were downregulated. To identify the Gene Ontology (GO) terms and the biological pathways associated with the differentially expressed genes, we employed both the GO and pathway mapping tools. The major GO represented in naltrexone and ethanol regulated genes were Biological Processes and Molecular Function (Supplementary Figs. 1 and 2). Using IPA, we identified a network associated with cell morphology, cell death, and organismal development that was highly up-regulated in the PFC. Associated network functions in the hippocampus were cell death, nervous system development and function, and neurological disease. The 10 genes showing the greatest change in the naltrexone-treated PFC were selected for a confirmation study (Table 1). Among these genes, transthyretin (TTR) was the most up-regulated gene in the PFC. We also selected the 10 genes showing the greatest changes in the hippocampus of naltrexone-treated mice (Table 2). qRT-PCR was used to confirm the expression of these genes. Consistent with the microarray data, TTR expression was increased in the PFC of naltrexone-treated mice compared to NTC mice (Fig. 1C). However, the expression of Ndst4 was up-regulated in both the PFC and hippocampus of naltrexoneinjected mice (Fig. 1C and D). Additionally, we studied the cAMP response element-binding (CREB) pathway from IPA analysis in PFC and hippocampus of naltrexone-treated mice. It is reported that CREB pathway is vital for ethanol dependence by which gene expression is modulated in the brain during the course of adaptation to chronic ethanol exposure and its withdrawal [28]. From the IPA analysis of CREB pathway, we found the up-regulated protein kinase C (PKC) in PFC of naltrexone-treated mice (Fig. 2). However, PKC expression was not changed in the hippocampus region (Supplementary Fig. 3). To confirm the PKC expression in PFC, we selected major isoform, PKC␥ and qRT-PCR analysis revealed that PKC␥ expression was increased in the PFC but not in the hippocampus of naltrexone-treated mice (Fig. 1C and D). However, we did not check other isoform of PKC specially PKC due to its suppressive effect on naltrexone and alcohol [26]. Interactions between alcohol and the opioid signaling pathway have been well documented in both basic research and clinical practice. The effectiveness of the opioid-receptor antagonist naltrexone
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Fig. 2. IPA network analysis of the CREB signaling pathway in the PFC of naltrexone-treated mice. The network is displayed as nodes (gene or gene products) and edges (lines: biological relationships between nodes). Solid lines denote direct interactions, while dotted lines represent indirect interactions between genes represented in this network. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
as an adjunct in the treatment of alcoholism has placed a spotlight on interactions between ethanol and opioid signaling [11,27]. The blockade of opioid receptors with naltrexone decreases alcohol intake in alcohol-preferring rats and reduces the alcohol-induced increase in opioidergic activity, thereby inhibiting the reinforcing properties of alcohol [8]. In our previous study, we examined naltrexone injected mice reduces alcohol consumption in a similar pattern with the amiRNA-based silencing of the neurokinin 1 receptor in the hippocampus [2]. Here we reported that naltrexoneinjected mice consumed less ethanol than NTC mice. In addition, we observed different patterns of genes expression in the hippocampus and PFC in response to naltrexone and ethanol. Among these differentially expressed genes, TTR and PKC␥ expression were distinct in the PFC but not in the hippocampus. Recently it has reported that blocking the opioid receptors of specific brain regions with naltrexone mediates alcohol seeking in a model of relapse. However, opioid receptor blockade in the dorsal hippocampus did not produce a significant attenuation of alcohol seeking [21]. PKC is an important family of enzymes that regulate numerous intracellular functions, neurotransmitter synthesis, receptor
and ion channel function, neuronal excitability, development, and gene expression [20]. Studies utilizing null mutant mice have shed light on the roles of PKC␥ and PKC in responses to ethanol. It has reported that inhibitors of PKC might be useful in reducing ethanol consumption, anxiety, and alcohol induced hyperalgesia [15]. A previous study from our group showed that PKC decreased as time increased in SH-SY5Y neuroblastoma cells treated with naltrexone and alcohol [26]. In addition, PKC and PKC␥ null mice demonstrate opposite phenotypes with regards to ethanol sensitivity. PKC␥ null mice are less sensitive to ethanol. The reduced ethanol sensitivity phenotype of PKC␥ null mice was associated with increased ethanol consumption in a two-bottle choice paradigm [4]. Here we found that PKC␥ was up-regulated in the PFC of naltrexone-injected mice. These data are in agreement with the previous finding that chronic ethanol consumption increases membrane bound PKC␥ in limbic forebrain regions while decreasing the total level of this isozyme in the frontal cortex [23]. In addition, stress-induced opioid craving has been significantly associated with increased anxiety, fear, and sadness. Naltrexone therapies that target both the reinforcement and the stress- and
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Table 2 Up/down regulated genes in the hippocampus of naltrexone-treated mice. Gene symbol Up regulated genes Ndst4 Gm879 Cpne7 Spink8 Npy2r Cabp7 LOC100046859 Nnat Il16 Pkp2 Down regulated genes Herc5 Tmem196 Rasgef1b Coch Pou3f2 Sema3a Kcnh5 Dkkl1 6330527O06Rik Exph5
FC
Gene accession
Gene description
16.27 9.21 8.79 8.22 8.05 6.99 6.66 6.20 5.83 5.76
NM 022565 NM 001034874 NM 170684 NM 183136 NM 008731 NM 138948 XR 032810 NM 010923 NM 010551 NM 026163
N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 4 Gene model 879 (NCBI) Copine VII Serine peptidase inhibitor, Kazal type 8 Neuropeptide Y receptor Y2 Calcium binding protein 7 Hypothetical protein LOC100046859 Neuronatin Interleukin 16 Plakophilin 2
−4.05 −4.06 −4.28 −4.43 −4.47 −4.55 −4.90 −5.53 −5.87 −5.89
ENSMUST00000031817 ENSMUST00000058644 NM 145839 NM 007728 ENSMUST00000098234 NM 009152 NM 172805 NM 015789 NM 029530 NM 176846
Hect domain and RLD 5 Transmembrane protein 196 RasGEF domain family, member 1B Coagulation factor C homolog (Limulus polyphemus) POU domain, class 3, transcription factor 2 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3A Potassium voltage-gated channel, subfamily H (eag-related), member 5 dickkopf-like 1 RIKEN cDNA 6330527O06 gene Exophilin 5
FC, fold change (microarray analysis). Genes were selected by using significance analysis of microarray data with a p value, p < 0.05 and an average fold-change 2.0.
cue-related aspects of drug seeking could be beneficial for relapse prevention. TTR was originally characterized as a blood plasma and cerebrospinal fluid (CSF) transporter of thyroxine (T4) and vitamin A [13]. TTR expression is associated with depression and anxietylike behavior. Decreased levels of TTR has found in the CSF of patients with depression [29]. Here we reported the increased level of TTR expression in the PFC of naltrexone-treated mice. Among the differentially expressed genes, TTR showed the greatest increase in expression in the PFC. Previous study showed that TTR mRNA expression is decreased in a chronic stress-specific manner, and protein levels were reduced in the cortex, but not in the choroid plexus [17]. In addition, TTR expression in the rat hippocampus was shown to increase in response to the chronic administration of anti-psychotic drugs, which include antidepressants and antianxiety drugs [6]. Our data suggest that the reduction of alcohol consumption in naltrexone treated mice may be due to a decrease level of anxiety caused by the increased expression of TTR in the PFC. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in specific regions of the mouse brain. We observed changes in a wide range of genes expressed in the PFC and hippocampus of naltrexone-injected mice following ethanol treatment. In addition, we reported two candidate genes that were up-regulated in the PFC of naltrexone-injected mice. These findings provide a basis for conducting pharmacogenetic research on the effects of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders.
Acknowledgement This study was supported by the grant (A080906) of Korea Healthcare Technology Research and Development Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.11.049.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Changes in the expression of transthyretin and protein kinase C␥ genes in the prefrontal cortex in response to naltrexone Jaehak Yu a,1 , Debasish Halder b,1 , Mi Na Baek b , Nando Dulal Das b , Mi Ran Choi b , Dong Yul Oh c , Ihn Geun Choi d , Kyoung Hwa Jung b,e,∗ , Young Gyu Chai b,∗ a
Department of Neuropsychiatry, Konkuk University School of Medicine, Seoul, Republic of Korea Division of Molecular and Life Sciences, Hanyang University, 1271 Sa-dong, Ansan, Gyeonggi-do 426-791, Republic of Korea Department of Psychiatry, Kwandong University, Gangneung, Republic of Korea d Department of Neuropsychiatry, College of Medicine, Hallym University, Seoul, Republic of Korea e Institute of Natural Science and Technology, Hanyang University, Ansan, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 18 August 2010 Received in revised form 17 November 2010 Accepted 17 November 2010 Keywords: Naltrexone Ethanol Prefrontal cortex Hippocampus Transthyretin (TTR) Protein kinase C (PKC)␥
a b s t r a c t Naltrexone, an opioid receptor antagonist, has been approved for clinical use in the treatment of alcohol dependence. In the present study, we examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200 l (16 mg/kg) of naltrexone for 4 weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity Pathway Analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and hippocampus. We found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the expression of transthyretin (TTR) and protein kinase C (PKC)␥ were increased in the PFC but not in the hippocampus of naltrexonetreated mice. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice. These findings provide a basis for conducting pharmacogenetic research on the effect of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders. © 2010 Elsevier Ireland Ltd. All rights reserved.
Among the neurotransmitter systems, the endogenous opioid system is the most important for controlling alcohol-seeking behavior. Prolonged alcohol administration generally decreases endogenous opioid activity, which in turn, causes opioid withdrawal and promotes alcohol consumption through negative reinforcement [27]. The therapeutic efficacy of opioid receptor antagonists in the treatment of alcohol dependence suggests the existence of an alcohol–opioid interaction that may be regulated directly or through interactions with other neurotransmitters [12]. Naltrexone is an opioid antagonist that interferes with the rewarding effects of drinking alcohol or taking other drugs. Naltrexone blocks -opioid receptors, which reduces the reinforcing effects of alcohol leading to decreased feelings of intoxication and fewer cravings [25,30,31].
∗ Corresponding authors. Tel.: +82 31 400 5513; fax: +82 31 406 6316. E-mail addresses: [email protected] (K.H. Jung), [email protected] (Y.G. Chai). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.049
Although naltrexone has been approved for the treatment of alcoholism, there is great individual variation in the efficacy of naltrexone among different patients. Some patients with alcoholism respond very well, while others report no benefit. Drinkers who have a large endogenous opioid response to ethanol would have a large response to the blocking effect of naltrexone. However, those who lack a strong endogenous opioid response to alcohol would not notice any beneficial effects of naltrexone. Recently, Anton et al. focused on several factors that can modulate the response to naltrexone, including genetic variants that may predispose individuals to respond more positively to this pharmacotherapy [1]. Furthermore, naltrexone may act on enzymes within the cell that are involved in the neuroadaptation to alcohol. It has been shown that chronic ethanol intake alters the distribution of these enzymes within the cells and can change the way brain cells respond to alcohol [15,16,24]. Alcohol intake increases the release of opioid peptides and subsequently increases gene expression in the brain regions related to reinforcement and reward, such as the hippocampus or prefrontal cortex (PFC). The PFC is thought to be
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Fig. 1. Naltrexone reduced alcohol consumption in the C57BL/6 mice. Mice were injected with naltrexone (16 mg/kg) every 3 days for 1 month. (B) Volumetric alcohol consumption was markedly reduced in naltrexone-treated mice compared to NTC mice. (A) However, the level of water consumption was increased in naltrexone-treated mice. (C) qRT-PCR analysis revealed that the expression levels of PKC␥, Ttr and Ndst4 mRNA were increased in the PFC of naltrexone-treated mice. Here, values represent the mean ± standard error (SE) of three independent experiments. (D) However, the expressions of PKC␥ and Ttr were reduced in the hippocampus of naltrexone treated mice.
vulnerable to the neurotoxic effects of alcohol use [10]. The PFC shows major alcohol-related damage, including a loss of grey and white matter in chronic alcoholics. Morphometric studies have revealed smaller volumes of gray matter [5] and total tissue [18] in the PFCs of adults with alcohol use disorders compared with controls. The direct toxicity of ethanol and its first metabolite, acetaldehyde, accounts for some of these detrimental effects. Due to the susceptibility of the frontal cortex to alcohol-induced damage and its importance in judgment, decision-making, and other executive functions, we chose to analyze genetic changes in this region. We also looked at changes in the hippocampus. It has been reported that the hippocampal formation is one of the brain regions most sensitive to prolonged ethanol ingestion [3]. Alcohol intake increases the release of opioid peptides and subsequently increases the expression of genes related to reinforcement and reward in the hippocampus. Previous studies has found that the hippocampus is involved in the learning of ethanol-associated cues and may play a critical role in the cue-induced reinstatement of ethanol-seeking, a process dependent on the - and ␦-opioid receptors [22]. In this study, we used cDNA microarray analysis to profile gene expression changes in the hippocampus and PFC of naltrexoneinjected C57BL/6 mice following ethanol treatment. We found that treatment with naltrexone led to alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in response to ethanol compared to non-treated control (NTC) mice. Among the differentially expressed genes, we detected two candidate genes uniquely expressed in the PFC that may be involved in the naltrexone-induced reduction of alcohol consumption. Twelve male C57BL/6 mice weighing 20–30 g (Orient Bio, Gyeonggi-do, Korea) were group housed with ad libitum access to food and water until naltrexone injection. One week later mice were randomly separated for naltrexone and non-treated control (NTC) groups (n = 6 per group). Naltrexone group mice were treated with naltexone + ethanol and the NTC group mice were only ethanol treated. After the naltrexone injection, mice were housed individually in a temperature- and humidity-controlled room on a 12 h
light/dark cycle. Mice were injected intraperitoneally with 200 l (16 mg/kg) of naltrexone every 3 days [14] and were monitored for 4 weeks. A modified version of the two-bottle choice experiment from a previous publication was used [9,19]. Mice were placed in cages containing a 15% alcohol bottle and a water bottle. The positions of the bottles were changed every 3 days. The consumption of alcohol and water were measured every 3 days. Four weeks after the naltrexone injection, the mice were anesthetized with Zoletil 50 (Virbac, Carros, France). The brains were rapidly removed and placed into ice-cold phosphate-buffered saline (PBS). The regions of interest (hippocampus and PFC) were carefully dissected following the method described in Chiu et al. [7]. Hippocampal and PFC tissue collected pooled from four from mice of each treatment group were homogenized and then total RNA was isolated using the TRIzol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions. The quality of the RNA was verified by the presence of prominent 18S and 28S bands on 1% agarose formaldehyde gels and an A260 /A280 ratio between 1.9 and 2.1 (data not shown). mRNA expression was measured using qRT-PCR. The primer sequences used for the qRT-PCR were as follows: PKC␥ – Forward = 5 -CTCGTTTCTTCAAGCAGCCAA-3 , Reverse = 5 GTGAACCACAAAGCTACAGACT-3 ; Ttr – Forward = 5 -TTGCCTCGCTGGACTGGTA-3 , Reverse = 5 -TTACAGCCACGTCTACAGCAG3 ; Ndst4 – Forward = 5 -ACTTTTTGCTTGGTGAGCATCC-3 , Reverse = 5 -CCGATAAGGGAGGTCTTTGATGT-3 . For the microarray analysis, we used the Affymetrix mouse gene 1.0 ST GeneChip containing 770317 probes sets (genes). Total RNA (8 g) pooled from hippocampus and PFC of each treatment group mice was hybridized for three individual GeneChip analysis using the manufacturer’s procedure (Affymetrix, Santa Clara, CA). Microarray data were scanned using a laser confocal scanner (Affymetrix, Clara, CA, USA). All genes showing at least a 2.0-fold change were selected and subsequently classified into functional subgroups using the Protein ANalysis THrough Evolutionary Relationships program (PANTHER; http://www.pantherdb.org). All
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Table 1 Up/down regulated genes in the PFC of naltrexone-treated mice. Gene symbol Up regulated genes Ttr Ndst4 Npy2r Spink8 Kcnj13 Dcn Gm879 Pkp2 Cpne7 Pcdh20 Down regulated genes Cux2 Fhod3 Exph5 Satb2 6330527O06Rik Myl4 Cckbr Rasgef1b 4930578G10Rik Dkkl1
FC
Gene accession
123.25 19.64 12.94 11.07 9.43 9.35 8.00 7.40 7.22 7.03
NM NM NM NM NM NM NM NM NM NM
−4.60 −4.63 −4.83 −4.91 −4.93 −4.95 −5.17 −5.98 −6.88 −7.75
013697 022565 008731 183136 001110227 007833 001034874 026163 170684 178685
ENSMUST00000111752 NM 175276 NM 176846 NM 139146 NM 029530 NM 010858 NM 007627 NM 145839 ENSMUST00000068158 NM 015789
Gene description Transthyretin N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 4 Neuropeptide Y receptor Y2 Serine peptidase inhibitor, Kazal type 8 Potassium inwardly-rectifying channel, subfamily J, member 13 Decorin Gene model 879 (NCBI) Plakophilin 2 Copine VII Protocadherin 20 Cut-like homeobox 2 Formin homology 2 domain containing 3 Exophilin 5 Special AT-rich sequence binding protein 2 RIKEN cDNA 6330527O06 gene Myosin, light polypeptide 4 Cholecystokinin B receptor RasGEF domain family, member 1B RIKEN cDNA 4930578G10 gene dickkopf-like 1
FC, fold change (microarray analysis). Genes were selected by using significance analysis of microarray data with a p value, p < 0.05 and an average fold-change 2.0.
genes were categorized into functional groups at p-value (EASE Score) p < 0.05. Using the selected genes, Ingenuity Pathway Analysis (IPA) version 7.6 (Ingenuity® Systems, www.ingenuity.Com, CA, USA) was used to search for biological pathways and inter-relationship between network genes in the subsets of candidate genes regulated in the different brain regions. The graphical representation of the molecular relationships between genes/gene products were based on the following criteria: genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line); all edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base; the intensity of the node color indicates the degree of up- (red) or down- (green) regulation; and nodes are displayed using various shapes that represent the functional class of the gene product. Microarray data were analyzed by using the GeneChip Operating software (GCOS)/Microarray Suite Version 5.0 (MAS 5.0, Affymetrix, Santa Clara, CA). The detection p-value (p < 0.5) was used to statistically determine whether a transcript is expressed on a chip. All values are expressed as the mean ± standard error (SE). All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data were tested using a one-way ANOVA followed by the Tukey’s HSD post hoc test. p < 0.05 were considered to be significant. To examine the effect of naltrexone on alcohol consumption, one group of mice was injected with naltrexone and another group was used as a non-treated control (NTC). Mice were allowed free access to 15% alcohol for 30 days. The consumption of alcohol and water were measured every 3 days. After day 3, naltrexone-injected mice showed a reduction in voluntary alcohol consumption compared to NTC mice. The reduced alcohol consumption was measured after 4 weeks of treatment (Fig. 1B). However, water consumption in the naltrexone-injected mice was higher than that of the NTC mice (Fig. 1A). This suggests that the decreased ethanol consumption was not caused by an overall decrease in the total amount of fluid consumed. To determine changes in the gene expression of naltrexonetreated mice, we compared the gene expression profile of two specific brain regions, the hippocampus and PFC, to that of NTC mice. We detected 566 genes differentially expressed in the PFC of naltrexone-treated mice. Of these, 265 genes were up-regulated
and 301 were down-regulated. Moreover, in the hippocampus of naltrexone-treated mice, we found 394 differentially expressed genes. 216 genes were up-regulated and 178 genes were downregulated. To identify the Gene Ontology (GO) terms and the biological pathways associated with the differentially expressed genes, we employed both the GO and pathway mapping tools. The major GO represented in naltrexone and ethanol regulated genes were Biological Processes and Molecular Function (Supplementary Figs. 1 and 2). Using IPA, we identified a network associated with cell morphology, cell death, and organismal development that was highly up-regulated in the PFC. Associated network functions in the hippocampus were cell death, nervous system development and function, and neurological disease. The 10 genes showing the greatest change in the naltrexone-treated PFC were selected for a confirmation study (Table 1). Among these genes, transthyretin (TTR) was the most up-regulated gene in the PFC. We also selected the 10 genes showing the greatest changes in the hippocampus of naltrexone-treated mice (Table 2). qRT-PCR was used to confirm the expression of these genes. Consistent with the microarray data, TTR expression was increased in the PFC of naltrexone-treated mice compared to NTC mice (Fig. 1C). However, the expression of Ndst4 was up-regulated in both the PFC and hippocampus of naltrexoneinjected mice (Fig. 1C and D). Additionally, we studied the cAMP response element-binding (CREB) pathway from IPA analysis in PFC and hippocampus of naltrexone-treated mice. It is reported that CREB pathway is vital for ethanol dependence by which gene expression is modulated in the brain during the course of adaptation to chronic ethanol exposure and its withdrawal [28]. From the IPA analysis of CREB pathway, we found the up-regulated protein kinase C (PKC) in PFC of naltrexone-treated mice (Fig. 2). However, PKC expression was not changed in the hippocampus region (Supplementary Fig. 3). To confirm the PKC expression in PFC, we selected major isoform, PKC␥ and qRT-PCR analysis revealed that PKC␥ expression was increased in the PFC but not in the hippocampus of naltrexone-treated mice (Fig. 1C and D). However, we did not check other isoform of PKC specially PKC due to its suppressive effect on naltrexone and alcohol [26]. Interactions between alcohol and the opioid signaling pathway have been well documented in both basic research and clinical practice. The effectiveness of the opioid-receptor antagonist naltrexone
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Fig. 2. IPA network analysis of the CREB signaling pathway in the PFC of naltrexone-treated mice. The network is displayed as nodes (gene or gene products) and edges (lines: biological relationships between nodes). Solid lines denote direct interactions, while dotted lines represent indirect interactions between genes represented in this network. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
as an adjunct in the treatment of alcoholism has placed a spotlight on interactions between ethanol and opioid signaling [11,27]. The blockade of opioid receptors with naltrexone decreases alcohol intake in alcohol-preferring rats and reduces the alcohol-induced increase in opioidergic activity, thereby inhibiting the reinforcing properties of alcohol [8]. In our previous study, we examined naltrexone injected mice reduces alcohol consumption in a similar pattern with the amiRNA-based silencing of the neurokinin 1 receptor in the hippocampus [2]. Here we reported that naltrexoneinjected mice consumed less ethanol than NTC mice. In addition, we observed different patterns of genes expression in the hippocampus and PFC in response to naltrexone and ethanol. Among these differentially expressed genes, TTR and PKC␥ expression were distinct in the PFC but not in the hippocampus. Recently it has reported that blocking the opioid receptors of specific brain regions with naltrexone mediates alcohol seeking in a model of relapse. However, opioid receptor blockade in the dorsal hippocampus did not produce a significant attenuation of alcohol seeking [21]. PKC is an important family of enzymes that regulate numerous intracellular functions, neurotransmitter synthesis, receptor
and ion channel function, neuronal excitability, development, and gene expression [20]. Studies utilizing null mutant mice have shed light on the roles of PKC␥ and PKC in responses to ethanol. It has reported that inhibitors of PKC might be useful in reducing ethanol consumption, anxiety, and alcohol induced hyperalgesia [15]. A previous study from our group showed that PKC decreased as time increased in SH-SY5Y neuroblastoma cells treated with naltrexone and alcohol [26]. In addition, PKC and PKC␥ null mice demonstrate opposite phenotypes with regards to ethanol sensitivity. PKC␥ null mice are less sensitive to ethanol. The reduced ethanol sensitivity phenotype of PKC␥ null mice was associated with increased ethanol consumption in a two-bottle choice paradigm [4]. Here we found that PKC␥ was up-regulated in the PFC of naltrexone-injected mice. These data are in agreement with the previous finding that chronic ethanol consumption increases membrane bound PKC␥ in limbic forebrain regions while decreasing the total level of this isozyme in the frontal cortex [23]. In addition, stress-induced opioid craving has been significantly associated with increased anxiety, fear, and sadness. Naltrexone therapies that target both the reinforcement and the stress- and
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Table 2 Up/down regulated genes in the hippocampus of naltrexone-treated mice. Gene symbol Up regulated genes Ndst4 Gm879 Cpne7 Spink8 Npy2r Cabp7 LOC100046859 Nnat Il16 Pkp2 Down regulated genes Herc5 Tmem196 Rasgef1b Coch Pou3f2 Sema3a Kcnh5 Dkkl1 6330527O06Rik Exph5
FC
Gene accession
Gene description
16.27 9.21 8.79 8.22 8.05 6.99 6.66 6.20 5.83 5.76
NM 022565 NM 001034874 NM 170684 NM 183136 NM 008731 NM 138948 XR 032810 NM 010923 NM 010551 NM 026163
N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 4 Gene model 879 (NCBI) Copine VII Serine peptidase inhibitor, Kazal type 8 Neuropeptide Y receptor Y2 Calcium binding protein 7 Hypothetical protein LOC100046859 Neuronatin Interleukin 16 Plakophilin 2
−4.05 −4.06 −4.28 −4.43 −4.47 −4.55 −4.90 −5.53 −5.87 −5.89
ENSMUST00000031817 ENSMUST00000058644 NM 145839 NM 007728 ENSMUST00000098234 NM 009152 NM 172805 NM 015789 NM 029530 NM 176846
Hect domain and RLD 5 Transmembrane protein 196 RasGEF domain family, member 1B Coagulation factor C homolog (Limulus polyphemus) POU domain, class 3, transcription factor 2 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3A Potassium voltage-gated channel, subfamily H (eag-related), member 5 dickkopf-like 1 RIKEN cDNA 6330527O06 gene Exophilin 5
FC, fold change (microarray analysis). Genes were selected by using significance analysis of microarray data with a p value, p < 0.05 and an average fold-change 2.0.
cue-related aspects of drug seeking could be beneficial for relapse prevention. TTR was originally characterized as a blood plasma and cerebrospinal fluid (CSF) transporter of thyroxine (T4) and vitamin A [13]. TTR expression is associated with depression and anxietylike behavior. Decreased levels of TTR has found in the CSF of patients with depression [29]. Here we reported the increased level of TTR expression in the PFC of naltrexone-treated mice. Among the differentially expressed genes, TTR showed the greatest increase in expression in the PFC. Previous study showed that TTR mRNA expression is decreased in a chronic stress-specific manner, and protein levels were reduced in the cortex, but not in the choroid plexus [17]. In addition, TTR expression in the rat hippocampus was shown to increase in response to the chronic administration of anti-psychotic drugs, which include antidepressants and antianxiety drugs [6]. Our data suggest that the reduction of alcohol consumption in naltrexone treated mice may be due to a decrease level of anxiety caused by the increased expression of TTR in the PFC. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in specific regions of the mouse brain. We observed changes in a wide range of genes expressed in the PFC and hippocampus of naltrexone-injected mice following ethanol treatment. In addition, we reported two candidate genes that were up-regulated in the PFC of naltrexone-injected mice. These findings provide a basis for conducting pharmacogenetic research on the effects of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders.
Acknowledgement This study was supported by the grant (A080906) of Korea Healthcare Technology Research and Development Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.11.049.
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