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DNA Methylation, Psychostimulant-Induced Addiction and the Position of Cocaine
Chapter 10
DNA Methylation, PsychostimulantInduced Addiction and the Position of Cocaine K. Anier and A. Kalda University of Tartu, Tartu, Estonia
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Addictive substances can be viewed as environmental stimuli that via epigenetic modifications (including DNA methylation) translate to changes in gene expression and long-lasting behavioral phenotypes. Psychostimulants (e.g., cocaine, amphetamine) affect DNA methylation and demethylation, and gene expression that are involved in brain maladaptation. Dynamic changes in methyl-CpG-binding protein 2 expression level and phosphorylation are important homeostatic mechanisms that regulate neuronal adaptations to the psychostimulant. DNA methylation may play an active role in cocaine craving. DNA-modifying enzymes are a potential target for developing new addiction therapies.
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Epigenetics—a series of biochemical processes through which changes in gene expression are achieved without a change in DNA sequence. During the last decade, the field of epigenetics has developed into one of the most influential areas of scientific research. Epigenetic mechanisms are essential for normal cellular development and differentiation and allow the long-term regulation of gene function through nonmutagenic mechanisms. Alterations of epigenetic mechanisms affect the vast majority of nuclear processes including gene transcription and silencing, DNA replication and repair, cell cycle, telomere and centromere function and structure.
The Neuroscience of Cocaine. DOI: http://dx.doi.org/10.1016/B978-0-12-803750-8.00010-5 © 2017 Elsevier Inc. All rights reserved.
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Epigenetic mechanisms, such as DNA methylation and demethylation, contribute to psychostimulant-induced transcriptional and behavioral changes.
LIST OF ABBREVIATIONS 5-caC 5-fC 5-hmC 5-hmU 5-mU BER CpG CPP DNMT H3K4me3 H3K9me2 MeCP2 NAc SAH SAM TDG TET
5-carboxylcytosine 5-formylcytosine 5-hydroxymethylcytosine 5-hydroxymethyluracil 5-methyluracil base excision repair cytosine guanine dinucleotides conditioned place preference DNA methyltransferases trimethylation of histone H3 lysine 4 dimethylation of histone H3 lysine 9 methyl-CpG-binding protein-2 nucleus accumbens S-adenosylhomocysteine S-adenosylmethionine thymine DNA glycosylase ten-eleven translocation family enzyme
10.1 INTRODUCTION Drug addiction is a chronic relapsing disorder that is characterized by compulsive, uncontrollable drug use despite negative consequences (Nestler, Hyman, & Malenka, 2001). Addictive substances can be viewed as environmental stimuli that translate to changes in gene expression and long-lasting behavioral phenotypes. In recent years, epigenetic modifications have come into focus as they may mediate stable and long-term changes in gene expression. 89
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It is hypothesized that epigenetic mechanisms, such as DNA methylation and histone modifications, may be involved in initiating and establishing psychostimulantinduced stable changes at the cellular level by coordinating the expression of gene networks, which then manifests as long-term behavioral changes. An increasing number of reports have provided crucial evidence that aberrant DNA methylation (such as hyper- or hypomethylation compared to normal tissue) in mesolimbic brain areas may alter drug addiction in several ways. Psychostimulants (e.g., cocaine, amphetamine) can directly alter enzymes that modify DNA methylation and demethylation and the expression of genes involved in brain maladaptation. In addition, different environmental factors may alter the subject’s vulnerability to drug abuse via DNA methylation and demethylation. The aim of this review is to describe the role of DNA methylation and demethylation, DNA-modifying enzymes, and DNA methylation interaction with other epigenetic mechanisms, such as histone modifications and noncoding RNAs. We will give an overview of the potential role of DNA methylation and demethylation in the pathogenesis of psychostimulant-induced addiction and during the withdrawal period.
10.2 DNA METHYLATION AND DNA METHYLTRANSFERASES DNA methylation is an important and is the bestcharacterized form of epigenetic modification. Methylation of one of the four DNA bases, cytosine, regulates the transcriptional plasticity of mammalian genomes and plays a pivotal role in cell differentiation and tissue-specific gene expression, repetitive element silencing, imprinting, X-chromosome inactivation, and tumor formation (Bird & Wolffe, 1999; Jaenisch & Bird, 2003). Moreover, DNA methylation dynamically regulates neuronal functions related to memory formation and synaptic plasticity (Day et al., 2013; Feng et al., 2010; Levenson et al., 2006; Szyf, Weaver, Champagne, Diorio, & Meaney, 2005; Zovkic, Guzman-Karlsson, & Sweatt, 2013) and brain plasticity associated with drug addiction (Anier, Malinovskaja, Aonurm-Helm, Zharkovsky, & Kalda, 2010; LaPlant et al., 2010; Lattal & Wood, 2013; Nielsen et al., 2012; Tian et al., 2012). DNA methylation occurs when a methyl group (CH3) is added to the fifth position on the cytosine pyrimidine ring, forming 5-methylcytosine (5-mC). It takes place primarily where a cytosine (C) precedes a guanine (G) in the DNA sequence (C-phosphate link-G, or cytosine guanine dinucleotides, CpG) (Holliday & Pugh, 1975; Klose & Bird, 2006). DNA regions that contain a high frequency of CpG sites are clustered in CpG islands—short regions of 0.5 4 kb in length with a rich (60 70%) cytosine guanine content (Bird, 2002). Approximately 50% of
CpG islands are located in promoter regions, preferentially near transcription start sites, and they are unmethylated in normal cells (Jones & Baylin, 2002; Robertson & Wolffe, 2000). Methylation of DNA cytosine residues at promoter regions generally exerts a repressive effect on gene transcription by preventing binding of transcription factors or by attracting methyl-CpG-binding proteins; e.g., methyl-CpGbinding protein-2 (MeCP2) recruits corepressors of transcription to modify the surrounding chromatin into a silent state (Bird, 1986; Goll & Bestor, 2005; Klose & Bird, 2006). DNA methylation is catalyzed by a family of enzymes called DNA methyltransferases (DNMTs), which mediate the transfer of a methyl group from S-adenosylmethionine (SAM) to cytosine (Goll & Bestor, 2005; Jin, Li, & Robertson, 2011; see also chapter: Cocaine and Epigenetics: An Overview). There are two main DNMT enzyme families: DNMT1 and DNMT3. DNMT1, the first-identified eukaryotic DNMT, methylates hemimethylated cytosines in CpG dinucleotide sequences, thereby maintaining DNA methylation patterns in proliferating cells, and it is also necessary for the de novo methylation of genomic DNA (Bestor, 2000; Egger et al., 2006; Goll & Bestor, 2005). The DNMT3 family includes two active de novo DNMTs, DNMT3A and DNMT3B, which are primarily responsible for establishing new DNA methylation patterns (Holliday & Pugh, 1975; Okano, Bell, Haber, & Li, 1999; see also chapter: Cocaine and Epigenetics: An Overview). The DNMT3 family also includes DNMT3-Like protein (DNMT3L) that has not been shown to possess methyltransferase activity (Bourc’his, Xu, Lin, Bollman, & Bestor, 2001) but instead increases the ability of DNMT3A and DNMT3B to bind to methyl groups (Bird, 2002; Jin et al., 2011). Organisms that contain members of the DNMT1 and DNMT3 families also express DNMT2, which displays weak DNMT activity (Okano, Xie, & Li, 1998; Yoder & Bestor, 1998).
10.3 DNA DEMETHYLATION Several studies have demonstrated that besides DNA cytosine methylation, other chemical modifications of cytosine in DNA exist, including the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) (Kriaucionis & Heintz, 2009; Tahiliani et al., 2009; Wu & Zhang, 2010). 5-hmC may be an intermediate in active DNA demethylation (Tahiliani et al., 2009; Wu & Zhang, 2010); however, in contrast to methylation of DNA at 5-cytosine, which has been relatively well characterized (Feng et al., 2010; Jaenisch & Bird, 2003), the complementary process of DNA demethylation remains poorly understood. Recent discoveries suggest that DNA demethylation processes may be passive or active. Passive DNA demethylation mechanisms generally involve a failure of the repair system to maintain DNA methylation patterns during
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replication or DNA synthesis, and they are associated with the dilution of hemimethylated CpG in subsequent replication cycles. In general, passive DNA demethylation is caused by a reduction in activity of or an absence of DNMTs. Active DNA demethylation involves enzymatic activity that selectively restores an unmodified cytosine base (replacement of 5-mC with C) (Ehrlich & Lacey, 2013; Moore, Le, & Fan, 2013; Ooi & Bestor, 2008). Three enzyme families have been implicated in active DNA demethylation (Fig. 10.1): (1) the ten-eleven translocation (TET1-3) family of enzymes, which modifies methylated cytosines (5-mC) first by hydroxylation to form 5-hmC and then by further oxidation to 5-fC and 5-caC; (2) the AID/ APOBEC (activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme complex) family of enzymes, which deaminate the base 5-mC or 5-hmC to form 5-methyluracil (5-mU) or 5-hydroxymethyluracil (5-hmU); and (3) the family of base excision repair
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glycosylases that initiate DNA repair culminating in the replacement of methylated cytosines (i.e., 5-mU, 5-hmU, or 5-caC) with unmethylated cytosines (Guo, Su, Zhong, Ming, & Song, 2011; Ito et al., 2011; Moore et al., 2013; Tahiliani et al., 2009; see also chapter: Cocaine and Epigenetics: An Overview). It has been observed that 5-hmC is enriched at transcription start sites and gene bodies of a large number of genes with high CpG content (Williams et al., 2011). In vitro studies have revealed that 5-hmC in the gene body prevents the binding of MBD proteins and thus may modify the accessibility of transcriptional machinery to chromatin (Jin, Kadam, & Pfeifer, 2010; Valinluck et al., 2004).
10.4 INTERPLAY BETWEEN DIFFERENT EPIGENETIC MODIFICATIONS Accumulating evidence indicates that DNA methylation and histone modifications are highly intertwined processes
FIGURE 10.1 Schematic representation of DNA methylation/demethylation pathways. Cytosine is converted to 5-methylcytosine (5-mC) by the action of DNA methyltransferases (DNMTs) where S-adenosylmethionine (SAM) is the methyl group donor ( CH3) and is converted to S-adenosylhomocysteine (SAH). The ten-eleven translocation (TET) family of proteins catalyze the oxidation of 5-mC to 5-hydroxymethylcytosine (5-hmC) and further to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). 5-hmC is deaminated to 5-hydroxymethyluracil (5-hmU) in the presence of the activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme complex (AID/APOBEC) family of deaminases. 5-fC and 5-caC are converted to cytosine using thymine DNA glycosylase (TDG) and base excision repair (BER).
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and that DNA methylation affects the interaction between histones and DNA, resulting in either repression or activation of transcription. These coordinated epigenetic mechanisms and the epigenetic marks thus formed may be viewed as the epigenetic code (Turner, 2007). For example, it has been found that interactions between histone methyltransferase G9a, DNMT1, and the replication complex lead to dimethylation of histone H3 lysine 9 (H3K9me2), which is a repressive epigenetic mark (Saitou, Kagiwada, & Kurimoto, 2012; Smallwood, Este`ve, Pradhan, & Carey, 2007). There is also interaction of the H3K9 methyltransferases with DNMT3A and DNMT3B, which cause DNA methylation at H3K9me2 (Fuks, Hurd, Deplus, & Kouzarides, 2003). In addition, several micro-RNAs (miRNAs) are associated with DNA methylation and demethylation processes. For example, miR-143 decreases the expression of DNMT3A and inhibits the proliferation of breast cancer cells (Ng et al., 2014). miR-29 regulates TET1-3 and thymine-DNA glycosylase mRNA levels, and overexpression of miR-29 causes a global decrease in genomic 5-hmC levels (Zhang, Huang, Xu, & Sessa, 2013). Furthermore, recent studies have shown that TET proteins may promote the transcription of target genes by increasing histone modification of H3K4me3 (Shi et al., 2013; Williams et al., 2011). TET1 interacts with histone deacetylases via the transcriptional corepressor SIN3 transcription regulator family member A, thereby promoting transcriptional repression (Williams et al., 2011).
10.5 ROLE OF DNA METHYLATION IN PSYCHOSTIMULANT-INDUCED NEUROPLASTICITY Several studies have demonstrated that DNA methylation, hydroxymethylation, and changes in expression levels of
DNA-modifying enzymes in the nucleus accumbens (NAc, a brain area that contributes to reward and addiction) are altered differentially by acute versus chronic cocaine exposure and during extended withdrawal, suggesting that cocaine and other psychostimulants are capable of dynamic control of DNA methylation and demethylation (Anier et al., 2010; Feng et al., 2015; LaPlant et al., 2010). The expression level of DNMTs in the NAc is the direct link between psychostimulant exposure and DNA methylation. For example, it was shown that acute cocaine administration upregulated DNMT3A and DNMT3B expression in the NAc of mice (Fig. 10.2), increasing DNA hypermethylation at specific gene promoter regions, and that these changes correlated with transcriptional downregulation of this gene (Anier et al., 2010). However, at 24 hours after withdrawal from both acute and repeated cocaine administration, DNMT3A was downregulated in the NAc (LaPlant et al., 2010). The exact mechanism underlying the induction of DNMT expression by cocaine and other psychostimulants is poorly understood. It is possible that psychostimulants act through synaptic targets and alter intracellular signaling cascades leading to the activation or inhibition of transcription factors and other nuclear proteins, which finally alter DNA-modifying enzymes (including DNMTs). A prolonged period of withdrawal after repeated administration of psychostimulants results in heightened craving triggered by acute exposure to psychostimulantassociated cues (Pickens et al., 2011). A recent report suggests that DNA methylation may play an active role in cocaine craving. Massart and colleagues (2015) demonstrated dynamic and time-dependent aberrant DNA methylation in the NAc after a prolonged period of cocaine withdrawal and cue-induced seeking. These methylation changes occurred in gene promoters of selected candidate
FIGURE 10.2 Acute and repeated cocaine treatment altered Dnmt3a and Dnmt3b mRNA levels. Acute (AC) and repeated cocaine (RC) treatment effects on (A) Dnmt3a and (B) Dnmt3b mRNA levels in the nucleus accumbens (NAc) of mice at 1.5 and 24 h after treatment. One-way ANOVA, Bonferroni post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001 compared with respective saline (SAL) group, n 5 11. Error bars indicate SEM. Modified from Anier, K., Malinovskaja, K., Aonurm-Helm, A., Zharkovsky, A., Kalda, A. (2010). DNA methylation regulates cocaine-induced behavioral sensitization in mice. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 35, 2450 2461.
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genes related to drug addiction and were partially negatively correlated with gene expression.
10.6 ROLE OF DNA DEMETHYLATION IN PSYCHOSTIMULANT-INDUCED NEUROPLASTICITY To date, only a few studies have focused on the role of DNA demethylation in psychostimulant-induced addiction. Accumulating data suggest that 5-hmC is a transient intermediate state between 5-mC and 5-fC or 5-caC that ultimately leads to DNA demethylation. However, it has also been speculated that 5-hmC may serve as a stable epigenetic mark associated with prolonged transcriptional activation (Hahn et al., 2013). Recently, Feng and colleagues (2015) demonstrated decreased expression of TET1, but not TET2 or TET3, in mouse NAc at 24 hours after repeated cocaine administration. These changes were associated with increased enrichment of 5-hmC at a large subset of genes involved in drug addiction and correlated with increased expression of those genes (Feng et al., 2015). However, these results are in contrast to previous research that proposed that a decrease in TET1 expression in the adult brain leads to reduced 5-mC conversion to 5-hmC, resulting in promoter hypermethylation and transcriptional repression (Kaas et al., 2013; Rudenko et al., 2013). It is possible that TET2 or TET3 partially compensate for the cocaine-induced downregulation of TET1, resulting in enrichment of 5-hmC at a subset of genes. Therefore, future studies should clarify whether TET2 and TET3 participate in psychostimulant-induced plasticity.
10.7 THE EFFECT OF PSYCHOSTIMULANTS ON WHOLE-GENOME METHYLATION LEVEL One major goal of addiction research is to identify the specific genes that show changes in methylation status in response to repeated psychostimulant exposure that consequently regulate cellular and behavioral adaptations to drugs of abuse. The majority of studies have investigated DNA methylation and demethylation changes at particular genes of interest (Anier et al., 2010; Anier, Zharkovsky, & Kalda, 2013; Nielsen et al., 2012). These reports indicate that cocaine exposure may cause both hyper- and hypomethylation in the promoter of selected genes. To understand the dynamic regulation of DNA methylation and demethylation at particular genomic sites, genome-wide maps of DNA methylation are needed. At present, only a few studies have reported genome-wide mapping of DNA methylation (Feng et al., 2015; Massart et al., 2015). Interestingly, Feng and colleagues (2015) identified numerous cocaine-induced changes in 5-hmC at enhancer
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sites in the NAc. They found that dynamic regulation of 5-hmC correlated with several features of gene expression, including alternative splicing of primary transcripts, alterations in steady-state levels of expression and future inducibility; and indeed, some of the 5-hmC changes were long-lasting (Feng et al., 2015). Whole-genome methylation analysis during cocaine withdrawal and cue-induced cocaine seeking demonstrated that genes are differentially methylated and expressed in the NAc (Massart et al., 2015). These methylation changes occurred in gene promoters and were partially negatively correlated with gene expression changes. Importantly, these findings show that although some early changes in DNA methylation persist and are stable over the prolonged period of withdrawal, other early changes show different types of dynamic alterations over this period. Moreover, many genes change their methylation state only after prolonged withdrawal.
10.8 ROLE OF DNA METHYLATION AND DEMETHYLATION IN PSYCHOSTIMULANTINDUCED BEHAVIOR The most common rodent behavioral procedures to model psychostimulant-induced addiction are locomotor sensitization, conditioned place preference (CPP), and self-administration. Early studies demonstrated that injections of DNMT inhibitors (RG108, zebularine, 5-aza-2-deoxycytidine) into different brain regions differently affected the development of cocaine-induced locomotor sensitization and CPP in mice. For example, LaPlant and colleagues (2010) showed that continuous RG108 intra-NAc infusion over 7 days enhanced cocaine-induced locomotor sensitization. In contrast, we previously found that repeated zebularine intracerebroventricular administration delayed the development of cocaine-induced locomotor sensitization in mice (Fig. 10.3) (Anier et al., 2010), whereas cocaine cotreatment with the methyl donor SAM showed the opposite effect (Fig. 10.4) (Anier et al., 2013). In addition, injections of the DNMT inhibitor 5-aza-2deoxycytidine into the hippocampus CA1 region inhibited cocaine-induced CPP; however, 5-aza had no effect when injected into the prelimbic cortex (Han et al., 2010). The reasons for these discrepancies are unknown, but these data suggest that alterations in DNA methylation in different brain regions may alter cocaine-induced behavior differentially. Mixed results have been obtained following treatment with a DNMT inhibitor after prolonged periods of psychostimulant withdrawal. A prolonged period of withdrawal in cocaine-experienced animals increases DNMT3A expression in mouse NAc (Anier et al., 2013;
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FIGURE 10.3 Repeated DNMT inhibitor zebularine intracerebroventricular (i.c.v.) treatment delayed the development of cocaine-induced locomotor sensitization. Mice were treated with cocaine (15 mg/kg, i.p.) alone or cotreated with zebularine (300 ng per 0.5 mL, i.c.v.) daily for 7 days, and their ambulation was recorded for 1 h immediately after treatment. “S 1 S”—saline (0.5 mL, i.c.v.) 1 saline (0.1 mL per 10 g body weight, i. p.); “Z 1 S”—zebularine (i.c.v.) 1 saline (i.p.); “S 1 C”—saline (i.c.v.) 1 cocaine (i.p.); “Z 1 C”—zebularine (i.c.v.) 1 cocaine (i.p.). Two-way ANOVA with repeated measures, Bonferroni post hoc test, *p , 0.001 S 1 C 1st versus 7th day, n 5 7 12. Error bars indicate SEM. Modified from Anier, K., Malinovskaja, K., Aonurm-Helm, A., Zharkovsky, A., Kalda, A. (2010). DNA methylation regulates cocaine-induced behavioral sensitization in mice. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 35, 2450 2461.
FIGURE 10.4 Repeated S-adenosylmethionine (SAM) pretreatment potentiated the development of cocaine-induced locomotor sensitization. Mice were treated for 7 days intraperitoneally (i.p.) with sterile saline (0.1 mL/10 g body weight) or SAM (10 mmol/kg, i.p.) 20 min before cocaine administration (10 mg/kg, i.p.). Animals (n 5 17 22) were randomly assigned to one of the following treatment groups: “S 1 S”— saline 1 saline; “M 1 S”—SAM 1 saline; “S 1 C”—saline 1 cocaine; “M 1 C”—SAM 1 cocaine. Two-way ANOVA with repeated measures, Bonferroni post hoc test, *p , 0.001 S 1 C 1st versus 7th day; M 1 C 1st versus 7th day. Error bars indicate SEM. Modified from Anier, K., Zharkovsky, A., Kalda, A. (2013). S-adenosylmethionine modifies cocaine-induced DNA methylation and increases locomotor sensitization in mice. The International Journal of Neuropsychopharmacology/ Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP), 16, 2053 2066.
LaPlant et al., 2010). After local infusion of DNMT inhibitor (RG108) or viral-mediated local knockout of DNMT3A, behavioral responses to cocaine are enhanced, whereas DNMT3A overexpression in this region decreases these responses (LaPlant et al., 2010). However, using the cocaine self-administration rat model, Massart and colleagues (2015) demonstrated that DNMT inhibition by intra-NAc injection of RG108 significantly attenuated cue-induced cocaine seeking after prolonged withdrawal. SAM caused the opposite effect and induced a long-term increase in cue-induced cocaine seeking. Recently, the behavioral response to cocaine and the role of TET1 and 5-hmC levels in NAc were evaluated (Feng et al., 2015). Viral-mediated selective TET1 knockdown in the NAc enhanced cocaine-induced CPP robustly, and overexpression of TET1 caused the opposite effect, which indicates that TET1 expression levels in the NAc may be an important factor in the development of cocaine reward. Together, these data suggest that both DNA methylation and demethylation in the NAc are involved in psychostimulant-induced behavioral effects and that DNAmodifying enzymes are a potential target for developing new addiction therapies.
10.9 CONCLUSIONS Epigenetic studies are important for understanding how exposure to a drug of abuse translates to changes in gene expression and long-lasting behavioral phenotypes. Accumulating data now suggest that DNA methylation and demethylation play an important role in the pathogenesis of psychostimulant-induced addiction but also shed light on the complex mechanisms of drug addiction. Because DNA methylation mechanisms are dynamic and reversible, the chemical agents that alter DNA-modifying enzymes may prove to be new therapeutic candidates for addiction treatment. However, it is important to emphasize that only a small number of epigenetic studies on drug addiction have been performed in humans. Therefore, further studies must evaluate the possible role of the epigenetic mechanism in addicted individuals.
MINI-DICTIONARY OF TERMS G
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Aberrant DNA methylation: DNA hyper- or hypomethylation compared to normal tissue. Epigenetics: A series of biochemical processes through which changes in gene expression are achieved throughout the lifecycle of an organism without a change in DNA sequence. Conditioned place preference (CPP): The CPP procedure is a passive drug administration procedure to assess drug-rewarding properties. During the conditioning period, an animal is trained to associate the
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drug of abuse and its vehicle with different environmental cues. During the test trial, the degree to which an animal prefers to spend time in the drug-paired chamber is quantified. Locomotor sensitization: Locomotor sensitization is an intermittent and passive drug administration procedure leading to enhance locomotor response. There are indications that psychostimulant-induced sensitization is one of the main causes of drug-abuse relapse. Self-administration: The self-administration model estimates drug positive reinforcing and rewarding properties. The self-administration model uses the active drug administration paradigm, which serves as a positive reinforcement. Light or tone stimuli are often associated with the drug delivery and reinstate drug-seeking behavior that has been previously extinguished.
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Massart, R., Barnea, R., Dikshtein, Y., Suderman, M., Meir, O., Hallett, M., . . . Yadid, G. (2015). Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving. The Journal of Neuroscience, 35, 8042 8058. Moore, L. D., Le, T., & Fan, G. (2013). DNA methylation and its basic function. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 38, 23 38. Nestler, E. J., Hyman, S. E., & Malenka, R. C. (2001). Molecular neuropharmacology: A foundation for clinical neuroscience (2nd ed., pp. 355 382). New York, NY: McGraw-Hill. Ng, E. K., Li, R., Shin, V. Y., Siu, J. M., Ma, E. S., & Kwong, A. (2014). MicroRNA-143 is downregulated in breast cancer and regulates DNA methyltransferases 3A in breast cancer cells. Tumor Biology, 35, 2591 2598. Nielsen, D. A., Huang, W., Hamon, S. C., Maili, L., Witkin, B. M., Fox, R. G., . . . Moeller, F. G. (2012). Forced abstinence from cocaine selfadministration is associated with DNA methylation changes in myelin genes in the corpus callosum: A preliminary study. Frontiers in Psychiatry, 3, 60. Okano, M., Bell, D. W., Haber, D. A., & Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99, 247 257. Okano, M., Xie, S., & Li, E. (1998). Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Research, 26, 2536 2540. Ooi, S. K., & Bestor, T. H. (2008). The colorful history of active DNA demethylation. Cell, 133, 1145 1148. Pickens, C. L., Airavaara, M., Theberge, F., Fanous, S., Hope, B. T., & Shaham, Y. (2011). Neurobiology of the incubation of drug craving. Trends in Neurosciences, 2011(34), 411 420. Robertson, K. D., & Wolffe, A. P. (2000). DNA methylation in health and disease. Nature Reviews Genetics, 1, 11 19. Rudenko, A., Dawlaty, M. M., Seo, J., Cheng, A. W., Meng, J., Le, T., . . . Tsai, L. H. (2013). Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron, 79, 1109 1122. Saitou, M., Kagiwada, S., & Kurimoto, K. (2012). Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development (Cambridge, England), 139, 15 31. Shi, F. T., Kim, H., Lu, W., He, Q., Liu, D., Goodell, M. A., . . . Songyang, Z. (2013). Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene
repression in mouse embryonic stem cells. The Journal of Biological Chemistry, 288, 20776 20784. Smallwood, A., Este`ve, P. O., Pradhan, S., & Carey, M. (2007). Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes & Development, 21, 1169 1178. Szyf, M., Weaver, I. C., Champagne, F. A., Diorio, J., & Meaney, M. J. (2005). Maternal programming of steroid receptor expression and phenotype through DNA methylation in the rat. Frontiers in Neuroendocrinology, 26, 139 162. Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., . . . Rao, A. (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science (New York, N.Y.), 324, 930 935. Tian, W., Zhao, M., Li, M., Song, T., Zhang, M., Quan, L., . . . Sun, Z. S. (2012). Reversal of cocaine-conditioned place preference through methyl supplementation in mice: Altering global DNA methylation in the prefrontal cortex. PLoS One, 7, e33435. Turner, B. (2007). Defining an epigenetic code. Nature Cell Biology, 9, 2 6. Valinluck, V., Tsai, H. H., Rogstad, D. K., Burdzy, A., Bird, A., & Sowers, L. C. (2004). Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acid Research, 32, 4100 4108. Williams, K., Christensen, J., Pedersen, M. T., Johansen, J. V., Cloos, P. A., Rappsilber, J., & Helin, K. (2011). TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature, 473, 343 348. Wu, S. C., & Zhang, Y. (2010). Active DNA demethylation: Many roads lead to Rome. Nature Reviews. Molecular Cell Biology, 11, 607 620. Yoder, J. A., & Bestor, T. H. (1998). A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast. Human Molecular Genetics, 7, 279 284. Zhang, P., Huang, B., Xu, X., & Sessa, W. C. (2013). Ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG), components of the demethylation pathway, are direct targets of miRNA-29a. Biochemical and Biophysical Research Communications, 437, 368 373. Zovkic, I. B., Guzman-Karlsson, M. C., & Sweatt, J. D. (2013). Epigenetic regulation of memory formation and maintenance. Learning & Memory, 20, 61 74.
DNA Methylation, PsychostimulantInduced Addiction and the Position of Cocaine K. Anier and A. Kalda University of Tartu, Tartu, Estonia
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Addictive substances can be viewed as environmental stimuli that via epigenetic modifications (including DNA methylation) translate to changes in gene expression and long-lasting behavioral phenotypes. Psychostimulants (e.g., cocaine, amphetamine) affect DNA methylation and demethylation, and gene expression that are involved in brain maladaptation. Dynamic changes in methyl-CpG-binding protein 2 expression level and phosphorylation are important homeostatic mechanisms that regulate neuronal adaptations to the psychostimulant. DNA methylation may play an active role in cocaine craving. DNA-modifying enzymes are a potential target for developing new addiction therapies.
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Epigenetics—a series of biochemical processes through which changes in gene expression are achieved without a change in DNA sequence. During the last decade, the field of epigenetics has developed into one of the most influential areas of scientific research. Epigenetic mechanisms are essential for normal cellular development and differentiation and allow the long-term regulation of gene function through nonmutagenic mechanisms. Alterations of epigenetic mechanisms affect the vast majority of nuclear processes including gene transcription and silencing, DNA replication and repair, cell cycle, telomere and centromere function and structure.
The Neuroscience of Cocaine. DOI: http://dx.doi.org/10.1016/B978-0-12-803750-8.00010-5 © 2017 Elsevier Inc. All rights reserved.
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Epigenetic mechanisms, such as DNA methylation and demethylation, contribute to psychostimulant-induced transcriptional and behavioral changes.
LIST OF ABBREVIATIONS 5-caC 5-fC 5-hmC 5-hmU 5-mU BER CpG CPP DNMT H3K4me3 H3K9me2 MeCP2 NAc SAH SAM TDG TET
5-carboxylcytosine 5-formylcytosine 5-hydroxymethylcytosine 5-hydroxymethyluracil 5-methyluracil base excision repair cytosine guanine dinucleotides conditioned place preference DNA methyltransferases trimethylation of histone H3 lysine 4 dimethylation of histone H3 lysine 9 methyl-CpG-binding protein-2 nucleus accumbens S-adenosylhomocysteine S-adenosylmethionine thymine DNA glycosylase ten-eleven translocation family enzyme
10.1 INTRODUCTION Drug addiction is a chronic relapsing disorder that is characterized by compulsive, uncontrollable drug use despite negative consequences (Nestler, Hyman, & Malenka, 2001). Addictive substances can be viewed as environmental stimuli that translate to changes in gene expression and long-lasting behavioral phenotypes. In recent years, epigenetic modifications have come into focus as they may mediate stable and long-term changes in gene expression. 89
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It is hypothesized that epigenetic mechanisms, such as DNA methylation and histone modifications, may be involved in initiating and establishing psychostimulantinduced stable changes at the cellular level by coordinating the expression of gene networks, which then manifests as long-term behavioral changes. An increasing number of reports have provided crucial evidence that aberrant DNA methylation (such as hyper- or hypomethylation compared to normal tissue) in mesolimbic brain areas may alter drug addiction in several ways. Psychostimulants (e.g., cocaine, amphetamine) can directly alter enzymes that modify DNA methylation and demethylation and the expression of genes involved in brain maladaptation. In addition, different environmental factors may alter the subject’s vulnerability to drug abuse via DNA methylation and demethylation. The aim of this review is to describe the role of DNA methylation and demethylation, DNA-modifying enzymes, and DNA methylation interaction with other epigenetic mechanisms, such as histone modifications and noncoding RNAs. We will give an overview of the potential role of DNA methylation and demethylation in the pathogenesis of psychostimulant-induced addiction and during the withdrawal period.
10.2 DNA METHYLATION AND DNA METHYLTRANSFERASES DNA methylation is an important and is the bestcharacterized form of epigenetic modification. Methylation of one of the four DNA bases, cytosine, regulates the transcriptional plasticity of mammalian genomes and plays a pivotal role in cell differentiation and tissue-specific gene expression, repetitive element silencing, imprinting, X-chromosome inactivation, and tumor formation (Bird & Wolffe, 1999; Jaenisch & Bird, 2003). Moreover, DNA methylation dynamically regulates neuronal functions related to memory formation and synaptic plasticity (Day et al., 2013; Feng et al., 2010; Levenson et al., 2006; Szyf, Weaver, Champagne, Diorio, & Meaney, 2005; Zovkic, Guzman-Karlsson, & Sweatt, 2013) and brain plasticity associated with drug addiction (Anier, Malinovskaja, Aonurm-Helm, Zharkovsky, & Kalda, 2010; LaPlant et al., 2010; Lattal & Wood, 2013; Nielsen et al., 2012; Tian et al., 2012). DNA methylation occurs when a methyl group (CH3) is added to the fifth position on the cytosine pyrimidine ring, forming 5-methylcytosine (5-mC). It takes place primarily where a cytosine (C) precedes a guanine (G) in the DNA sequence (C-phosphate link-G, or cytosine guanine dinucleotides, CpG) (Holliday & Pugh, 1975; Klose & Bird, 2006). DNA regions that contain a high frequency of CpG sites are clustered in CpG islands—short regions of 0.5 4 kb in length with a rich (60 70%) cytosine guanine content (Bird, 2002). Approximately 50% of
CpG islands are located in promoter regions, preferentially near transcription start sites, and they are unmethylated in normal cells (Jones & Baylin, 2002; Robertson & Wolffe, 2000). Methylation of DNA cytosine residues at promoter regions generally exerts a repressive effect on gene transcription by preventing binding of transcription factors or by attracting methyl-CpG-binding proteins; e.g., methyl-CpGbinding protein-2 (MeCP2) recruits corepressors of transcription to modify the surrounding chromatin into a silent state (Bird, 1986; Goll & Bestor, 2005; Klose & Bird, 2006). DNA methylation is catalyzed by a family of enzymes called DNA methyltransferases (DNMTs), which mediate the transfer of a methyl group from S-adenosylmethionine (SAM) to cytosine (Goll & Bestor, 2005; Jin, Li, & Robertson, 2011; see also chapter: Cocaine and Epigenetics: An Overview). There are two main DNMT enzyme families: DNMT1 and DNMT3. DNMT1, the first-identified eukaryotic DNMT, methylates hemimethylated cytosines in CpG dinucleotide sequences, thereby maintaining DNA methylation patterns in proliferating cells, and it is also necessary for the de novo methylation of genomic DNA (Bestor, 2000; Egger et al., 2006; Goll & Bestor, 2005). The DNMT3 family includes two active de novo DNMTs, DNMT3A and DNMT3B, which are primarily responsible for establishing new DNA methylation patterns (Holliday & Pugh, 1975; Okano, Bell, Haber, & Li, 1999; see also chapter: Cocaine and Epigenetics: An Overview). The DNMT3 family also includes DNMT3-Like protein (DNMT3L) that has not been shown to possess methyltransferase activity (Bourc’his, Xu, Lin, Bollman, & Bestor, 2001) but instead increases the ability of DNMT3A and DNMT3B to bind to methyl groups (Bird, 2002; Jin et al., 2011). Organisms that contain members of the DNMT1 and DNMT3 families also express DNMT2, which displays weak DNMT activity (Okano, Xie, & Li, 1998; Yoder & Bestor, 1998).
10.3 DNA DEMETHYLATION Several studies have demonstrated that besides DNA cytosine methylation, other chemical modifications of cytosine in DNA exist, including the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) (Kriaucionis & Heintz, 2009; Tahiliani et al., 2009; Wu & Zhang, 2010). 5-hmC may be an intermediate in active DNA demethylation (Tahiliani et al., 2009; Wu & Zhang, 2010); however, in contrast to methylation of DNA at 5-cytosine, which has been relatively well characterized (Feng et al., 2010; Jaenisch & Bird, 2003), the complementary process of DNA demethylation remains poorly understood. Recent discoveries suggest that DNA demethylation processes may be passive or active. Passive DNA demethylation mechanisms generally involve a failure of the repair system to maintain DNA methylation patterns during
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replication or DNA synthesis, and they are associated with the dilution of hemimethylated CpG in subsequent replication cycles. In general, passive DNA demethylation is caused by a reduction in activity of or an absence of DNMTs. Active DNA demethylation involves enzymatic activity that selectively restores an unmodified cytosine base (replacement of 5-mC with C) (Ehrlich & Lacey, 2013; Moore, Le, & Fan, 2013; Ooi & Bestor, 2008). Three enzyme families have been implicated in active DNA demethylation (Fig. 10.1): (1) the ten-eleven translocation (TET1-3) family of enzymes, which modifies methylated cytosines (5-mC) first by hydroxylation to form 5-hmC and then by further oxidation to 5-fC and 5-caC; (2) the AID/ APOBEC (activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme complex) family of enzymes, which deaminate the base 5-mC or 5-hmC to form 5-methyluracil (5-mU) or 5-hydroxymethyluracil (5-hmU); and (3) the family of base excision repair
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glycosylases that initiate DNA repair culminating in the replacement of methylated cytosines (i.e., 5-mU, 5-hmU, or 5-caC) with unmethylated cytosines (Guo, Su, Zhong, Ming, & Song, 2011; Ito et al., 2011; Moore et al., 2013; Tahiliani et al., 2009; see also chapter: Cocaine and Epigenetics: An Overview). It has been observed that 5-hmC is enriched at transcription start sites and gene bodies of a large number of genes with high CpG content (Williams et al., 2011). In vitro studies have revealed that 5-hmC in the gene body prevents the binding of MBD proteins and thus may modify the accessibility of transcriptional machinery to chromatin (Jin, Kadam, & Pfeifer, 2010; Valinluck et al., 2004).
10.4 INTERPLAY BETWEEN DIFFERENT EPIGENETIC MODIFICATIONS Accumulating evidence indicates that DNA methylation and histone modifications are highly intertwined processes
FIGURE 10.1 Schematic representation of DNA methylation/demethylation pathways. Cytosine is converted to 5-methylcytosine (5-mC) by the action of DNA methyltransferases (DNMTs) where S-adenosylmethionine (SAM) is the methyl group donor ( CH3) and is converted to S-adenosylhomocysteine (SAH). The ten-eleven translocation (TET) family of proteins catalyze the oxidation of 5-mC to 5-hydroxymethylcytosine (5-hmC) and further to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). 5-hmC is deaminated to 5-hydroxymethyluracil (5-hmU) in the presence of the activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme complex (AID/APOBEC) family of deaminases. 5-fC and 5-caC are converted to cytosine using thymine DNA glycosylase (TDG) and base excision repair (BER).
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and that DNA methylation affects the interaction between histones and DNA, resulting in either repression or activation of transcription. These coordinated epigenetic mechanisms and the epigenetic marks thus formed may be viewed as the epigenetic code (Turner, 2007). For example, it has been found that interactions between histone methyltransferase G9a, DNMT1, and the replication complex lead to dimethylation of histone H3 lysine 9 (H3K9me2), which is a repressive epigenetic mark (Saitou, Kagiwada, & Kurimoto, 2012; Smallwood, Este`ve, Pradhan, & Carey, 2007). There is also interaction of the H3K9 methyltransferases with DNMT3A and DNMT3B, which cause DNA methylation at H3K9me2 (Fuks, Hurd, Deplus, & Kouzarides, 2003). In addition, several micro-RNAs (miRNAs) are associated with DNA methylation and demethylation processes. For example, miR-143 decreases the expression of DNMT3A and inhibits the proliferation of breast cancer cells (Ng et al., 2014). miR-29 regulates TET1-3 and thymine-DNA glycosylase mRNA levels, and overexpression of miR-29 causes a global decrease in genomic 5-hmC levels (Zhang, Huang, Xu, & Sessa, 2013). Furthermore, recent studies have shown that TET proteins may promote the transcription of target genes by increasing histone modification of H3K4me3 (Shi et al., 2013; Williams et al., 2011). TET1 interacts with histone deacetylases via the transcriptional corepressor SIN3 transcription regulator family member A, thereby promoting transcriptional repression (Williams et al., 2011).
10.5 ROLE OF DNA METHYLATION IN PSYCHOSTIMULANT-INDUCED NEUROPLASTICITY Several studies have demonstrated that DNA methylation, hydroxymethylation, and changes in expression levels of
DNA-modifying enzymes in the nucleus accumbens (NAc, a brain area that contributes to reward and addiction) are altered differentially by acute versus chronic cocaine exposure and during extended withdrawal, suggesting that cocaine and other psychostimulants are capable of dynamic control of DNA methylation and demethylation (Anier et al., 2010; Feng et al., 2015; LaPlant et al., 2010). The expression level of DNMTs in the NAc is the direct link between psychostimulant exposure and DNA methylation. For example, it was shown that acute cocaine administration upregulated DNMT3A and DNMT3B expression in the NAc of mice (Fig. 10.2), increasing DNA hypermethylation at specific gene promoter regions, and that these changes correlated with transcriptional downregulation of this gene (Anier et al., 2010). However, at 24 hours after withdrawal from both acute and repeated cocaine administration, DNMT3A was downregulated in the NAc (LaPlant et al., 2010). The exact mechanism underlying the induction of DNMT expression by cocaine and other psychostimulants is poorly understood. It is possible that psychostimulants act through synaptic targets and alter intracellular signaling cascades leading to the activation or inhibition of transcription factors and other nuclear proteins, which finally alter DNA-modifying enzymes (including DNMTs). A prolonged period of withdrawal after repeated administration of psychostimulants results in heightened craving triggered by acute exposure to psychostimulantassociated cues (Pickens et al., 2011). A recent report suggests that DNA methylation may play an active role in cocaine craving. Massart and colleagues (2015) demonstrated dynamic and time-dependent aberrant DNA methylation in the NAc after a prolonged period of cocaine withdrawal and cue-induced seeking. These methylation changes occurred in gene promoters of selected candidate
FIGURE 10.2 Acute and repeated cocaine treatment altered Dnmt3a and Dnmt3b mRNA levels. Acute (AC) and repeated cocaine (RC) treatment effects on (A) Dnmt3a and (B) Dnmt3b mRNA levels in the nucleus accumbens (NAc) of mice at 1.5 and 24 h after treatment. One-way ANOVA, Bonferroni post hoc test, *p , 0.05, **p , 0.01, ***p , 0.001 compared with respective saline (SAL) group, n 5 11. Error bars indicate SEM. Modified from Anier, K., Malinovskaja, K., Aonurm-Helm, A., Zharkovsky, A., Kalda, A. (2010). DNA methylation regulates cocaine-induced behavioral sensitization in mice. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 35, 2450 2461.
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genes related to drug addiction and were partially negatively correlated with gene expression.
10.6 ROLE OF DNA DEMETHYLATION IN PSYCHOSTIMULANT-INDUCED NEUROPLASTICITY To date, only a few studies have focused on the role of DNA demethylation in psychostimulant-induced addiction. Accumulating data suggest that 5-hmC is a transient intermediate state between 5-mC and 5-fC or 5-caC that ultimately leads to DNA demethylation. However, it has also been speculated that 5-hmC may serve as a stable epigenetic mark associated with prolonged transcriptional activation (Hahn et al., 2013). Recently, Feng and colleagues (2015) demonstrated decreased expression of TET1, but not TET2 or TET3, in mouse NAc at 24 hours after repeated cocaine administration. These changes were associated with increased enrichment of 5-hmC at a large subset of genes involved in drug addiction and correlated with increased expression of those genes (Feng et al., 2015). However, these results are in contrast to previous research that proposed that a decrease in TET1 expression in the adult brain leads to reduced 5-mC conversion to 5-hmC, resulting in promoter hypermethylation and transcriptional repression (Kaas et al., 2013; Rudenko et al., 2013). It is possible that TET2 or TET3 partially compensate for the cocaine-induced downregulation of TET1, resulting in enrichment of 5-hmC at a subset of genes. Therefore, future studies should clarify whether TET2 and TET3 participate in psychostimulant-induced plasticity.
10.7 THE EFFECT OF PSYCHOSTIMULANTS ON WHOLE-GENOME METHYLATION LEVEL One major goal of addiction research is to identify the specific genes that show changes in methylation status in response to repeated psychostimulant exposure that consequently regulate cellular and behavioral adaptations to drugs of abuse. The majority of studies have investigated DNA methylation and demethylation changes at particular genes of interest (Anier et al., 2010; Anier, Zharkovsky, & Kalda, 2013; Nielsen et al., 2012). These reports indicate that cocaine exposure may cause both hyper- and hypomethylation in the promoter of selected genes. To understand the dynamic regulation of DNA methylation and demethylation at particular genomic sites, genome-wide maps of DNA methylation are needed. At present, only a few studies have reported genome-wide mapping of DNA methylation (Feng et al., 2015; Massart et al., 2015). Interestingly, Feng and colleagues (2015) identified numerous cocaine-induced changes in 5-hmC at enhancer
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sites in the NAc. They found that dynamic regulation of 5-hmC correlated with several features of gene expression, including alternative splicing of primary transcripts, alterations in steady-state levels of expression and future inducibility; and indeed, some of the 5-hmC changes were long-lasting (Feng et al., 2015). Whole-genome methylation analysis during cocaine withdrawal and cue-induced cocaine seeking demonstrated that genes are differentially methylated and expressed in the NAc (Massart et al., 2015). These methylation changes occurred in gene promoters and were partially negatively correlated with gene expression changes. Importantly, these findings show that although some early changes in DNA methylation persist and are stable over the prolonged period of withdrawal, other early changes show different types of dynamic alterations over this period. Moreover, many genes change their methylation state only after prolonged withdrawal.
10.8 ROLE OF DNA METHYLATION AND DEMETHYLATION IN PSYCHOSTIMULANTINDUCED BEHAVIOR The most common rodent behavioral procedures to model psychostimulant-induced addiction are locomotor sensitization, conditioned place preference (CPP), and self-administration. Early studies demonstrated that injections of DNMT inhibitors (RG108, zebularine, 5-aza-2-deoxycytidine) into different brain regions differently affected the development of cocaine-induced locomotor sensitization and CPP in mice. For example, LaPlant and colleagues (2010) showed that continuous RG108 intra-NAc infusion over 7 days enhanced cocaine-induced locomotor sensitization. In contrast, we previously found that repeated zebularine intracerebroventricular administration delayed the development of cocaine-induced locomotor sensitization in mice (Fig. 10.3) (Anier et al., 2010), whereas cocaine cotreatment with the methyl donor SAM showed the opposite effect (Fig. 10.4) (Anier et al., 2013). In addition, injections of the DNMT inhibitor 5-aza-2deoxycytidine into the hippocampus CA1 region inhibited cocaine-induced CPP; however, 5-aza had no effect when injected into the prelimbic cortex (Han et al., 2010). The reasons for these discrepancies are unknown, but these data suggest that alterations in DNA methylation in different brain regions may alter cocaine-induced behavior differentially. Mixed results have been obtained following treatment with a DNMT inhibitor after prolonged periods of psychostimulant withdrawal. A prolonged period of withdrawal in cocaine-experienced animals increases DNMT3A expression in mouse NAc (Anier et al., 2013;
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FIGURE 10.3 Repeated DNMT inhibitor zebularine intracerebroventricular (i.c.v.) treatment delayed the development of cocaine-induced locomotor sensitization. Mice were treated with cocaine (15 mg/kg, i.p.) alone or cotreated with zebularine (300 ng per 0.5 mL, i.c.v.) daily for 7 days, and their ambulation was recorded for 1 h immediately after treatment. “S 1 S”—saline (0.5 mL, i.c.v.) 1 saline (0.1 mL per 10 g body weight, i. p.); “Z 1 S”—zebularine (i.c.v.) 1 saline (i.p.); “S 1 C”—saline (i.c.v.) 1 cocaine (i.p.); “Z 1 C”—zebularine (i.c.v.) 1 cocaine (i.p.). Two-way ANOVA with repeated measures, Bonferroni post hoc test, *p , 0.001 S 1 C 1st versus 7th day, n 5 7 12. Error bars indicate SEM. Modified from Anier, K., Malinovskaja, K., Aonurm-Helm, A., Zharkovsky, A., Kalda, A. (2010). DNA methylation regulates cocaine-induced behavioral sensitization in mice. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 35, 2450 2461.
FIGURE 10.4 Repeated S-adenosylmethionine (SAM) pretreatment potentiated the development of cocaine-induced locomotor sensitization. Mice were treated for 7 days intraperitoneally (i.p.) with sterile saline (0.1 mL/10 g body weight) or SAM (10 mmol/kg, i.p.) 20 min before cocaine administration (10 mg/kg, i.p.). Animals (n 5 17 22) were randomly assigned to one of the following treatment groups: “S 1 S”— saline 1 saline; “M 1 S”—SAM 1 saline; “S 1 C”—saline 1 cocaine; “M 1 C”—SAM 1 cocaine. Two-way ANOVA with repeated measures, Bonferroni post hoc test, *p , 0.001 S 1 C 1st versus 7th day; M 1 C 1st versus 7th day. Error bars indicate SEM. Modified from Anier, K., Zharkovsky, A., Kalda, A. (2013). S-adenosylmethionine modifies cocaine-induced DNA methylation and increases locomotor sensitization in mice. The International Journal of Neuropsychopharmacology/ Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP), 16, 2053 2066.
LaPlant et al., 2010). After local infusion of DNMT inhibitor (RG108) or viral-mediated local knockout of DNMT3A, behavioral responses to cocaine are enhanced, whereas DNMT3A overexpression in this region decreases these responses (LaPlant et al., 2010). However, using the cocaine self-administration rat model, Massart and colleagues (2015) demonstrated that DNMT inhibition by intra-NAc injection of RG108 significantly attenuated cue-induced cocaine seeking after prolonged withdrawal. SAM caused the opposite effect and induced a long-term increase in cue-induced cocaine seeking. Recently, the behavioral response to cocaine and the role of TET1 and 5-hmC levels in NAc were evaluated (Feng et al., 2015). Viral-mediated selective TET1 knockdown in the NAc enhanced cocaine-induced CPP robustly, and overexpression of TET1 caused the opposite effect, which indicates that TET1 expression levels in the NAc may be an important factor in the development of cocaine reward. Together, these data suggest that both DNA methylation and demethylation in the NAc are involved in psychostimulant-induced behavioral effects and that DNAmodifying enzymes are a potential target for developing new addiction therapies.
10.9 CONCLUSIONS Epigenetic studies are important for understanding how exposure to a drug of abuse translates to changes in gene expression and long-lasting behavioral phenotypes. Accumulating data now suggest that DNA methylation and demethylation play an important role in the pathogenesis of psychostimulant-induced addiction but also shed light on the complex mechanisms of drug addiction. Because DNA methylation mechanisms are dynamic and reversible, the chemical agents that alter DNA-modifying enzymes may prove to be new therapeutic candidates for addiction treatment. However, it is important to emphasize that only a small number of epigenetic studies on drug addiction have been performed in humans. Therefore, further studies must evaluate the possible role of the epigenetic mechanism in addicted individuals.
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Aberrant DNA methylation: DNA hyper- or hypomethylation compared to normal tissue. Epigenetics: A series of biochemical processes through which changes in gene expression are achieved throughout the lifecycle of an organism without a change in DNA sequence. Conditioned place preference (CPP): The CPP procedure is a passive drug administration procedure to assess drug-rewarding properties. During the conditioning period, an animal is trained to associate the
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drug of abuse and its vehicle with different environmental cues. During the test trial, the degree to which an animal prefers to spend time in the drug-paired chamber is quantified. Locomotor sensitization: Locomotor sensitization is an intermittent and passive drug administration procedure leading to enhance locomotor response. There are indications that psychostimulant-induced sensitization is one of the main causes of drug-abuse relapse. Self-administration: The self-administration model estimates drug positive reinforcing and rewarding properties. The self-administration model uses the active drug administration paradigm, which serves as a positive reinforcement. Light or tone stimuli are often associated with the drug delivery and reinstate drug-seeking behavior that has been previously extinguished.
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