Drugs and addiction

Drugs and addiction: an introduction to epigeneticsadd_3321 480..489

Chloe C. Y. Wong1, Jonathan Mill1 & Cathy Fernandes2

Institute of Psychiatry, SGDP Research Centre1 and Department of Psychosis Studies,2 King’s College London, De Crespigny Park, Denmark Hill, London, UK

ABSTRACT

Addiction is a debilitating psychiatric disorder, with a complex aetiology involving the interaction of inherited predis- positions and environmental factors. Emerging evidence suggests that epigenetic alterations to the genome, including DNA methylation and histone modifications, are important mechanisms underlying addiction and the neurobiological response to addictive substances. In this review, we introduce the reader to epigenetic mechanisms and describe a potential role for dynamic epigenetic changes in mediating addictive behaviours via long-lasting changes in gene expression. We summarize recent findings from both molecular and behavioural experiments elucidating the role of epigenetic changes in mediating the addictive potential of various drugs of abuse, including cocaine, amphetamine and alcohol. The implications of these findings for molecular studies of addiction and the future development of novel therapeutic interventions are also discussed.

Keywords Addiction, alcohol, DNA methylation, drugs, epigenetics, genetics.

Correspondence to: Jonathan Mill, Institute of Psychiatry, SGDP Research Centre, King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. E-mail: jonathan.mill@kcl.ac.uk Submitted 6 November 2009; initial review completed 22 December 2009; final version accepted 22 November 2010

INTRODUCTION

Drug and alcohol dependence are debilitating psychiatric disorders that are common and associated with high morbidity and mortality rates [1]. Like other common complex diseases, addiction is a multi-factorial and polygenic disorder that does not conform to a simple Mendelian transmission pattern [2,3]. Family, twin and adoption studies suggest that the heritability of substance use disorders is moderate to high (ranging from 0.30 to 0.70), with shared and non-shared environmental factors also important [4]. Considerable research effort has been expended on elucidating the underlying neuro- biological causes of addiction. Most aetiological studies have focused on identifying genetic variants that predis- pose an individual to the spectrum of addictive disorders. There has been some success in the search for underlying genetic variation predisposing to addiction, and poly- morphisms in a number of candidate neurotransmitter system genes have been associated with susceptibility [5]. However, given the high heritability of substance use disorders, it is notable that the currently identified risk loci for addiction account only for a modest proportion (less than 5%) of the total genetic variance [6]. Moreover, findings from molecular genetic studies are generally

characterized by non-replication, small effect sizes and significant heterogeneity. It is clear that neither genetic nor environmental factors act in isolation to increase sus- ceptibility to addiction. For example, an approximately fivefold increase in genetic influence on alcohol consump- tion is observed in urban environments where there are a greater number of youths and higher alcohol sales compared to rural settings [7].

At a more basic level, the onset of addiction clearly results from the interplay between inherited predisposi- tion (e.g. via genetic variants mediating the personality traits associated with drug-seeking behaviour and de- pendence) and the environment (e.g. actual exposure to drugs of abuse). Because addiction manifests only following repeated exposure to an addictive substance, identification of both the predisposing factors (inherited and environmental) and the dynamic neurobiological changes that take place in response to chronic drug exposure is key to elucidating the aetiology of addiction. Drug-taking behaviour is characterized by a three- stage cycle, including preoccupation–anticipation, binge–intoxication and withdrawal–negative affect [8]. Impulsive drug use is often associated with positive rein- forcement where drug-seeking behaviour is mediated by the pleasurable effects of the drug. Following repeated

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drug exposure, however, drug use becomes compulsive and negatively reinforcing, where drug-seeking behav- iour is mediated by the ability of the drug to eliminate aversive emotional states associated with prolonged intoxication. Understanding the mechanism(s) that pre- dispose individuals to the environmental factors associ- ated with drug-taking behaviour and the systems that translate the response to environmental stimuli (e.g. drug exposure) into long-lasting cellular memories in the brain are thus fundamental to unlocking the neurobiological changes that are implicated causally in addiction.

Emerging evidence has shown that epigenetic factors are important contributors to disease susceptibility for a number of common neuropsychiatric phenotypes related to addiction, including schizophrenia [9] and depressive disorders [10]. Of particular relevance to addiction, it has been shown that epigenetic processes can change dynamically in response to external factors, providing a key mechanism by which the environment can influence gene expression and, hence, phenotype. The aim of this review is to introduce the potential role that epigenetic mechanisms play in mediating addictive behaviours by triggering long-lasting changes in gene expression in response to addictive substances. Following a brief description of epigenetic processes, we discuss the rel- evance of epigenetics to addiction and illustrate how enduring epigenetic changes can occur in response to repeated drug exposure, potentially underlying the onset of addiction.

EPIGENETICS AND THE REGULATION OF GENE EXPRESSION

With the exception of a few rare somatic mutation events, the sequence of nucleotides comprising an individual’s genome is identical across all cells in the body and remains unchanged from the moment of conception onwards. However, DNA is structurally much more complex than a simple string of nucleotides, and at a functional level the genome is anything but static. While every cell in our bodies contains the same DNA sequence, each has its own unique phenotype characterized by a specific pattern of gene expression that is in a constant state of flux. It is not only the gene-encoding DNA sequence that is important in determining the phenotype of a cell, but also the degree to which specific genes are functionally active at any particular time in development. Sequencing the genome was therefore only the first step in our quest to understand how genes are expressed and regulated. Sitting above the DNA sequence is a second layer of information (the ‘epigenome’) that regulates several genomic functions, including when and where genes are turned on or off.

The British biologist, Conrad Waddington, first coined the term ‘epigenetics’ (literally meaning ‘above genetics’) in the mid-20th century, introducing the concept of an epigenetic landscape to describe the ways in which cell- fates are established during development, enabling the tissues and organs of complex organisms to develop from an initially undifferentiated mass of cells. A contempo- rary definition regards epigenetics as the reversible regu- lation of gene expression, occurring independently of DNA sequence, mediated principally through changes in DNA methylation and chromatin structure [11]. Epigenetic processes are essential for normal cellular development and differentiation, and allow the long-term regulation of gene function through non-mutagenic mechanisms [12]. For a glossary of epigenetic mecha- nisms and a basic description of the genomic functions they perform, see Table 1.

DNA methylation

The methylation of one of the four DNA bases, cytosine, is the best understood and the most stable epigenetic modification, regulating the transcriptional plasticity of mammalian genomes (Figs 1 and 2). DNA methylation occurs when a methyl group is added to position 5 of the cytosine pyrimidine ring in a reaction catalysed by a group of enzymes called DNA methyltransferases (DNMTs). This occurs primarily where a cytosine (C) occurs next to guanine (G) in the DNA sequence [‘C—phosphate link—G—’, or cytosine-guanine dinucle- otides (CpG)], although cytosine methylation at non-CpG positions has also been reported. The intrinsic link between DNA methylation and the regulation of gene expression is demonstrated by the observed inverse corre- lation between the level of promoter DNA methylation and the degree of expression of many genes [11], even though the reality is more complex, with diverse patterns across different genomic regions. The addition of a methyl group to CpG sites, over-represented in ‘CpG- islands’ in the promoter regulatory regions of many genes, displaces the binding of transcription factors and attracts methyl-binding proteins that instigate chromatin compaction of DNA and gene silencing. The pivotal role of DNA methylation during development is illustrated by phenotypic analyses of mice with mutations in various DNA methyltransferase (Dnmt) genes [13–16].

Histone modification

Another form of epigenetic mechanism acting to modulate gene expression is the post-translational modification, or changes occurring after the biosynthe- sis, of histones. Histones are the basic proteins around which DNA is wrapped to form nucleosomes [17]. There are various covalent histone modifications, including

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Table 1 Glossary of epigenetic terms used in this paper.

Term Definition

Chromatin The complex of DNA, histones and other proteins that make up chromosomes. Chemical modifications to both DNA and histone proteins are important in regulating the structure of chromatin, which in turn regulates gene expression

DNA methylation The addition of a methyl group at position 5 of the cytosine pyrimidine ring in CpG dinucleotides, catalysed by DNA methyltransferase (Fig. 2). Cytosine DNA methylation occurring at CpG sites, over-represented in CpG islands in the promoter regulatory regions of many genes, often displaces the binding of transcription factors and attracts methyl-binding proteins that instigate chromatin compaction and gene silencing (Fig. 1)

Epiallele An allele that can stably exist in more than one epigenetic state and gives a distribution of phenotypes from genetically identical cells

Epigenetics The reversible regulation of gene expression mediated principally through changes in DNA methylation, chromatin structure and small interfering RNA (siRNA)

Euchromatin Chromatin existing in an activated open state that permits the access of the cells’ transcriptional machinery to DNA, promoting gene expression

Heterochromatin Condensed chromatin represented by the tight packaging of the DNA and histone proteins that is associated with repressed transcription. Heterochromatin obstructs the access of transcription factors and other instigators of gene expression to DNA

Histone modifications

Histones are the basic proteins around which DNA is wrapped to form nucleosome, the basic repeat unit of chromatin (Fig. 1). Covalent histone modifications, including acetylation, phosphorylation, methylation, sumoylation and ubiquitylation, modulate gene expression via alterations in chromatin structure. Histone modifications are established key components in the overall regulation of gene expression in the genome and are mediated via histone-modifying enzymes such as histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation of the lysine residues at the N terminus of histones proteins removes positive charges, which reduces the affinity between histones and DNA. This allows an easier access of RNA polymerase and transcription factors to the promoter region. Histone acetylation catalysed by histone acetyltransferases (HATs) often enhances transcription while histone deacetylation catalysed by histone deacetylases (HDAC) is associated with transcriptional repression

Metastable epialleles Epialleles that can be epigenetically modified in a variable and reversible manner in response to external or stochastic factors. The epigenetic state of the epialleles is transgenerationally stable once established

siRNA (small interfering RNA)

A class of short (21–26 nucleotides in length), double-stranded RNA which is involved in the RNA interference (RNAi) pathway and suppresses the expression of specific genes. RNAi is known to cause epigenetic changes in gene transcription, mediated by both DNA methylation and histone modifications

CpG: cytosine-guanine dinucleotide.

Figure 1 Epigenetic modifications to DNA and histones are associated with transcription. DNA is wrapped around a cluster of histone proteins to form nucleosomes. Changes in chromatin structure via DNA methylation (M) and histone modification including phosphory- lation (P), acetylation (A) and sumoylation (S) can affect the access of transcription factors, hence gene expression (adapted from [49])

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acetylation, phosphorylation, methylation, sumoylation and ubiquitylation, which together constitute a complex ‘histone code’ modulating gene expression via altera- tions in chromatin structure [18]. Histone modification mediated via histone-modifying enzymes such as his- tone acetyltransferases (HATs) and histone deacetylases (HDACs) are established key components in the overall regulation of gene expression in the genome [19]. Con- densed chromatin (heterochromatin) is associated with repressed transcription as the tight packaging of the DNA and histone proteins obstructs the access of tran- scription factors and other instigators of gene expression to DNA. In contrast, when chromatin is in an activated open state (euchromatin), access of the cells’ transcrip- tional machinery to DNA is permitted and hence gene expression can occur (Fig. 1).

While often investigated independently, epigenetic modifications to DNA and histones are not mutually exclusive, but interact in several ways. The methyl- binding protein MeCP2, for example, binds specifically to methylated cytosines, attracting histone deacetylases which hypoacetylate histones and inhibits access of tran- scription factors [20]. Another recent study has shown that unmethylated histone H3 residues at the lysine 4 position recruit DNA methyltransferases resulting in de novo DNA methylation [21].

ENVIRONMENTAL MEDIATION OF THE EPIGENOME

Mounting evidence suggests that epigenetic processes can be influenced by exposure to a range of external envi- ronmental factors, either globally or at specific loci [22]. DNA methylation, for example, has been shown to vary as a function of nutritional, chemical, physical and even

psychosocial factors (e.g. stress exposure). As epigenetic changes are inherited mitotically in somatic cells, they provide a possible mechanism by which the effects of external environmental factors at specific stages in devel- opment can produce long-term changes in behaviour. The role of epigenetic mechanisms in mediating phenotypic effects of environmental stimuli is supported by evidence that the environment can induce epigenetic variation in genetically identical animals [23,24] and between iden- tical twins [25,26]. Alleles that can exist stably in more than one epigenetic state and give a distribution of pheno- types from genetically identical cells are termed ‘epialle- les’. Loci that can be modified epigenetically in a variable and reversible manner in response to external or stochas- tic factors are defined as ‘metastable epialleles’ [27].

Given that the epigenetic state of metastable epialleles can be transmitted stably via cell division, the effects of external environmental factors during specific stages in development can manifest in long-term phenotypic changes via such epialleles. A classic example of the interplay between the genome, the environment and epi- genetic processes is illustrated by the Agouti viable yellow allele (Avy) inbred mouse strain, which demonstrates a range of coat-colour phenotypes, depending upon the epigenetic state of a large transposable element inserted upstream of the Agouti gene. The transposon contains a cryptic promoter, which expresses a phenotype charac- terized by yellow fur and various detrimental metabolic features, including obesity [28]. When the transposon is methylated, this phenotype is not expressed; the mice have agouti (brown) fur and are metabolically healthy. Interestingly, DNA methylation across this region, and thus phenotype, can be manipulated in the offspring by altering the diet of pregnant mothers [29,30]. Maternal dietary methyl-donor supplementation of pregnant mice

Figure 2 DNA methylation involves the addition of a methyl group at position 5 of the cytosine pyrimidine ring in cytosine-guanine dinucleotides (CpG). This reaction uses S-adenosylmethionine as a methyl donor and is catalysed by a group of enzymes called DNA methyltransferases

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with folic acid, vitamin B12, choline, betaine and genistein (the major phytostrogen in soy) were shown to increase offspring DNA methylation, leading to gene expression associated with brown fur and good metabolic health.

Environmental mediation of the epigenome thus pro- vides a mechanism for the gene–environment interac- tions currently being uncovered in psychiatry, including those interactions underlying predisposition to addiction [17]. Of particular interest to addictive behaviour, there is mounting evidence that alcohol and drugs of abuse can themselves have a direct effect on epigenetic processes, and that these alterations may contribute to the mole- cular basis of addiction.

EPIGENETICS AND DRUG ADDICTION

Long-lasting behavioural abnormalities are classic char- acteristics of psychiatric disorders, particularly where the development of mental illness is gradual, and often shows a chronic relapsing course over a life-time [31]. The rever- sal of symptoms in response to treatment is slow, occur- ring over weeks to months, and chronic administration of psychiatric medications is often required for their full clinical effect to be achieved. In the field of addiction, for example, drug relapse is one of the core features and is perhaps the most complicated clinical problem for effective treatment [32]. Drug-induced neuronal plasticity medi- ated via alterations in gene expression has long been viewed as a major molecular mechanism for the develop- ment of drug addiction and relapse [33–35]. Recreational drugs exert rapid effects upon behaviour by activating the reward centres, such as the mesolimbic dopamine system projecting dopaminergic neurones of the ventral tegmen- tal area (VTA) to the nucleus accumbens (NAc). Transient increases in members of the transcription factor Fos family, including c-fos, FosB Fra-1 and Fra-2, in the NAc and dorsal striatum are observed upon acute exposure to drugs of abuse [36]. Following repeated exposure, rec- reational drug use can lead to addiction, as encouraged by persistent drug reward-related memories [37]. The expression of transcription factor DFosB is increased sev- eralfold in the striatum during repeated drug exposure, and often persists long after drug exposure ceases. The extraordinary stability of DFosB in neurones has led to the theory that it plays a significant role in the onset of addiction [33,38,39]. In addition, the expression of several other genes, including BDNF and activator of G-protein signalling 3 (AGS3), are known to be altered persistently for several weeks after the last drug adminis- tration, and manipulation of these genes in rodent models has been shown to be associated with the regulation of drug relapse behaviour [38,40–42].

Emerging evidence has demonstrated that epigenetic changes may partially mediate persistent neuroplasticity

changes, as observed in drug addiction [43]. For example, the activity of DNA methyltransferases, the key enzymes in DNA methylation, has a significant role in regulating the induction of synaptic plasticity in the hippocampus [44,45]. Inhibition of DNMT activity was shown to alter the methylation states of the promoter of reelin and BDNF, two genes implicated in the induction of synaptic plasticity in the adult hippocampus [44]. DNMT inhibi- tors such as zebularine and 5-aza-2-deoxycytidine can also block the induction of long-term potentiation (LTP) [45]. Because DNMTs are involved in the silencing of the memory suppressor gene, Pp1, their inhibition during the memory consolidation period prevents the increase of Pp1 methylation and leads to aberrant transcription of the gene and a reduction in memory formation. In addi- tion to DNA methylation, histone acetylation has also been associated with the establishment of long-term memory. During the initial stages of consolidation of long-term association memories in a contextual fear- conditioning paradigm, a significantly enhanced level of histone H3, but not H4 acetylation, was observed in animals that underwent contextual fear conditioning, compared to controls [46]. Moreover deficits in memory formation and long-term potentiation in mice with a heterozygous mutation in CREB-binding protein (CBP) were ameliorated by treatment with histone deacetylase inhibitors [47]. These studies provide evidence that epi- genetic modifications, including both DNA methylation and histone modifications such as acetylation, play an essential regulatory role in synaptic plasticity and long- term behavioural adaptation in the nervous system.

Given that neuronal plasticity has a recognized role in regulating drug addiction, and epigenetic mechanisms are major mediators of long-term plasticity, epigenetic changes are probable candidates for maintaining drug addiction. Indeed, emerging evidence suggests that epige- netic mechanisms may be the molecular basis of drug- induced changes in gene expression in brain reward regions, contributing to the lasting neural and behav- ioural plasticity that underlies addiction [48]. Figure 3 illustrates how such epigenetic changes mediated by the interaction of inherited predispositions, environmental stimuli and exposure to drugs can trigger the long-lasting alterations in gene expression that influence susceptibil- ity to addictive behaviours.

The role of epigenetic mechanisms in drug-related behaviours is supported by an increasing body of evi- dence from molecular and behavioural experiments in both animals and humans. Selected examples of epige- netic changes induced by various drugs of abuse, includ- ing alcohol, amphetamine, cocaine, nicotine and opiates, are illustrated in Table 2. Evidence from these studies not only demonstrate the imperative role of epigenetic modi- fications in regulating behavioural responses to drug

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exposure, but also shed light on the complex mechanism of drug addiction. However, it is worth noting that only a few epigenetic studies on addiction have been performed in humans, and the majority of findings presented in Table 2 are from studies in animals.

FUTURE DIRECTIONS: IMPLICATIONS FOR MOLECULAR STUDIES OF ADDICTION AND THE DEVELOPMENT OF NOVEL THERAPEUTIC INTERVENTIONS

In this paper, we have introduced the importance of epigenetic processes in regulating gene expression and described the role that dynamic epigenetic changes may play in mediating addictive behaviours via long-lasting transcriptional changes following repeated drug expo- sure. There is emerging evidence for the role of epigenetic mechanisms underlying addictive behaviours to various types of drugs of abuse, including cocaine, amphet- amine, alcohol, nicotine and opiates. The current rapid growth in epigenetic research, combined with new high-throughput technologies for epigenomic profiling, could potentially revolutionize our understanding of the molecular changes associated with addiction and exposure to drugs of abuse.

Epigenetic studies of brain-specific processes such as addiction and psychiatric disease are subject to

several limitations that make them particularly hard to undertake successfully [49]. A major limitation to the optimal study of addiction-associated epigenetic changes is that researchers may require post-mortem human brain tissue. This is because epigenetic changes are often tissue- (and cell-) specific, and thus most likely to be apparent in the brain areas in which addiction is primarily manifest. Unfortunately, high-quality post-mortem brain samples that have been well- characterized for addiction and/or drug exposure are a limited resource, but it is likely that peripheral tissues can still provide other valuable information. Epigenetic studies of human neurobiological processes such as addiction are also limited by insufficient knowledge about the ‘normal’ epigenetic patterns that characterize different brain regions and cell types. However, it is hoped that current initiatives such as the NIH Epige- nomic Roadmap [50], which aims to catalogue patterns of epigenetic variation across different cell- and tissue- types, will be useful in this regard. Additionally, little is known about how factors such as age, sex and envi- ronmental exposure influence epigenetic patterns, so samples should be matched as carefully as possible for all potential confounding variables. For these reasons, the use of rodent models, as exemplified by many of the examples discussed in this paper, will be of great value in furthering our understanding of the role of epigenetic processes in addiction.

Figure 3 The proposed relationship between inherited predispositions, environmental factors, exposure to addictive substances and vulnerability to addictive disorders. When exposed to adverse environmental stimuli, individuals carrying susceptibility genes or epialleles predisposing to addictive behaviours may have an increased risk of developing addiction. Acute drug use may produce enduring alterations in gene expression via epigenetic changes that influence susceptibility to addictive disorders. Enhanced vulnerability to drugs of abuse will then feed back into increased risk of future drug use (as shown by the dashed arrow) that bring about further modifications to the epigenome and gene expression

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Table 2 Selected examples of drug-induced epigenetic changes.

Type of drug Species Reference Key findings

Alcohol Human [55] • A significant increase in global DNA methylation has been reported in alcoholic patients compared to normal controls

Rat [56] • Rats exposed to excessive ethanol, at levels equivalent to human binge drinking, demonstrated tissue-specific alteration in histone H3 acetylation at lysine9

Mouse [57] • Exposure to chronic ethanol treatment altered DNA methylation at the NMDA receptor Nr2b gene in mice Rat [58] • Enhanced levels of histone H3 and H4 acetylation, CBP and neuropeptide Y (NPY), as well as decreased

levels of HDAC activity, in rat amygdala were found to be involved in the anxiolytic effects of acute ethanol • Moreover, the development of anxiety in alcohol withdrawal was associated with decreased histone

acetylation and increased HDAC activity in the amygdala • The administration of a known HDAC inhibitor, trichostatin A, prevented the development of alcohol

withdrawal-related anxiety in rats by recovering the diminished levels of histone acetylation and inhibiting the augmented HDAC activity following chronic alcohol exposure

Drosophila [59] • Behavioural tolerance observed in drosophila following the administration of a single dose of benzyl alcohol, for example, was shown to be mediated via histone H4 acetylation across the promoter region of Slo, a gene with an established role in the induction of tolerance to sedative drugs

Alcohol and nicotine

Human [60] • DNA methylation at the monoamine oxidase-A (MAOA) gene was shown to be associated significantly with alcohol and nicotine dependence in women

Amphetamine Mouse [61] • Alteration in expression of Mecp2 in the nucleus accumbens (NAc) altered both amphetamine-induced locomotion and the rewarding properties of amphetamine in conditional place preference

Mouse [62] • Following chronic amphetamine administration, DFosB, a key transcription factor involved in the behavioural responses to cocaine, has been shown to accumulate in the striatum in mice

• The up-regulation of DFosB desensitizes c-fos mRNA induction to a subsequent drug exposure by binding to the c-fos promoter and recruits histone deacetylase 1 (HDAC1), which deacetylases nearby histones and attenuates gene activity

Mouse [63] • The administration of HDAC inhibitors butyric acid (BA) and valproic acid (VPA) was shown to potentiate amphetamine-induced behavioural sensitisation in mice

• An increased level of H4 hyperacetylation in the striatum was observed upon administration of VPA and amphetamine, while their co-treatment induced an additive effect on histone H4 acetylation

• The occurrence of the chronic amphetamine induced-sensitized response following amphetamine challenge was blocked by repeated administration of butyric acid (BA) and VPA

• The enhanced amphetamine-induced behavioural sensitization caused by HDAC inhibitors has also been shown to be associated with increased associative learning and memory, suggesting that histone deacetylation accounts at least partially for the induction and maintenance of the behavioural responses to amphetamine

Rat [64] • Rats exposed to subchronic amphetamine treatment showed differential DNA methylation and mRNA expression of Dnmt1

Cocaine Rat [65] • MeCP2, a key transcriptional repressor, was shown to control the effects of cocaine on striatal brain-derived neurotrophic factor (BDNF) levels via homeostasis interactions with microRNA-212

Mouse [66] • Repeated cocaine administration reduced global levels of histone 3 lysine 9 (H3K9) demethylation in the nucleus accumbens of mice

• This reduction in H3K9 level was mediated by down-regulation of histone methyltransferase G9a, one of the key histone modification enzymes

• Repression of G9a after repeated cocaine administration increased the plasticity of nucleus accumbens neurones and enhanced the preference for cocaine

Mouse [67] • Cocaine sensitivity was mediated via histone acetylation at the c-fos gene promoter in the striatum in mice Mouse [68] • Decreased cerebral volume and significantly lower sustained visual–spatial attention and spatial working

memory were observed in the offspring of male mice exposed to cocaine • Such phenotypical changes might be caused by the observed altered expression levels of DNMT1 and

DNMT3 in the germ-cell rich seminiferous tubular tissue of the male mice exposed to cocaine Mouse [69] • The levels of global DNA methylation and expression of selected genes in hippocampal neurones were

altered significantly in the male offspring of cocaine-exposed mothers at 3 and 30 days postnatum, compared to the offspring of non-exposed mothers

Rat [70] • Acute cocaine administration induced transient H4 hyperacetylation of various genes, e.g. cFos and FosB • Chronic cocaine injection did not have an effect on the c-fos promoter, but resulted in H3 hyperacetylation

of the FosB promoter, as well as the Cdk5 and Bdnf genes • These histone modifications were shown to be long-lasting, persisting for at least 1 week after the last

cocaine injection Rat [52] • Administration of HDAC inhibitors extinguished cocaine-induced conditioned place preference in mice Rat [53] • Administration of HDAC inhibitors decreased cocaine self-administration in rats

Nicotine Human [71] • A DNA methylation status signature at the monoamine oxidase-A (MAOA) gene is associated with smoking status

Human and mouse

[72] • Significant and persistent demethylation of MAOB promoter was found in former smokers (abstinent for over 10 years) and current smokers when compared to non-smokers

• The role of tobacco smoke in mediating DNA methylation change is supported by significantly higher level of nucleic acid demethylase activity in mice exposed to cigarette smoke compared to controls

Mouse [73] • Injection of nicotine down-regulated Dnmt1 mRNA and protein expression in mice, as well as decreasing DNA methylation level of the promoter region of glutamic acid decarboxylase 67 (GAD67), one of the two enzymes that synthesize GABA in the brain

Opiate Human [74] • Increased DNA methylation in the promoter of opioid receptor mu 1(OPRM1) gene was seen in methadone-maintained former heroin addicts compared to controls

GABA: gamma aminobutyric acid; HDAC: histone deacetylase; NMDA: N-methyl-D-aspartate.

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Because epigenetic mechanisms are dynamic and reversible, chemical agents that alter the modification of histones or the methylation of DNA might prove to be potent candidates for therapeutic interventions. More- over, the identification of specific epigenetic patterns asso- ciated with specific disease phenotypes might be useful biomarkers for early disease diagnosis and preventive intervention. Promising results have been seen in the cancer field, where epigenetic drugs and therapies are used as effective anti-cancer therapeutics [51]. Although epigenetic research is still largely unexplored in the field of addiction, recent findings demonstrating that administra- tion of HDAC inhibitors extinguished cocaine-induced conditioned place preference in mice [52] and decreased cocaine self-administration in rats [53] highlight the potential therapeutic use of drugs that target epigenetic processes. Recent technological advances in epigenomic profiling have made it feasible to screen the entire DNA methylome with individual base-pair resolution [54]. We are now at the exciting stage where it is possible to assess the ways by which drugs of abuse alter specific epigenetic processes in the brain with the aim of identifying the molecular mechanisms underlying addiction.

Declarations of interest

None.

References

1. Goldman D., Oroszi G., Ducci F. The genetics of addictions: uncovering the genes. Focus 2006; 4: 401.

2. Goldman D. Recent developments in alcoholism: genetic transmission. Recent Dev Alcohol 1993; 11: 231–48.

3. Enoch M. A., Goldman D. Genetics of alcoholism and sub- stance abuse. Psychiatr Clin North Am 1999; 22: 289–99.

4. Dick D. M., Foroud T. Candidate genes for alcohol depen- dence: a review of genetic evidence from human studies. Alcohol Clin Exp Res 2003; 27: 868–79.

5. Wong C. C., Schumann G. Genetics of addictions: strategies for addressing heterogeneity and polygenicity of substance use disorders [Review]. Phil Trans R Soc Lond B Biol Sci 2008; 363: 3213–22.

6. Agrawal A., Lynskey M. T. Are there genetic influences on addiction: evidence from family, adoption and twin studies. Addiction 2008; 103: 1069–81.

7. Dick D. M., Rose R. J., Viken R. J., Kaprio J., Koskenvuo M. Exploring gene–environment interactions: socioregional moderation of alcohol use. J Abnorm Psychol 2001; 110: 625–32.

8. Koob G. F., Le M. M. Plasticity of reward neurocircuitry and the ‘dark side’ of drug addiction. Nat Neurosci 2005; 8: 1442–4.

9. Petronis A. The origin of schizophrenia: genetic thesis, epi- genetic antithesis, and resolving synthesis. Biol Psychiatry 2004; 55: 965–70.

10. Mill J., Petronis A. Molecular studies of major depressive disorder: the epigenetic perspective. Mol Psychiatry 2007; 12: 799–814.

11. Jaenisch R., Bird A. Epigenetic regulation of gene expres- sion: how the genome integrates intrinsic and environmen- tal signals. Nat Genet 2003; 33: 245–54.

12. Henikoff S., Matzke M. A. Exploring and explaining epige- netic effects. Trends Genet 1997; 13: 293–5.

13. Okano M., Bell D. W., Haber D. A., Li E. DNA methyltrans- ferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99: 247–57.

14. Lyko F., Ramsahoye B. H., Kashevsky H., Tudor M., Mastrangelo M. A., Orr-Weaver T. L. et al. Mammalian (cytosine-5) methyltransferases cause genomic DNA methy- lation and lethality in Drosophila. Nat Genet 1999; 23: 363–6.

15. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992; 69: 915–26.

16. Lei H., Oh S. P., Okano M., Juttermann R., Goss K. A., Jaenisch R. et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 1996; 122: 3195–205.

17. Docherty S., Mill J. Epigenetic mechanisms as mediators of environmental risks for psychiatric disorders. Psychiatry 2008; 7: 500–06.

18. Berger S. L. The complex language of chromatin regulation during transcription. Nature 2007; 447: 407–12.

19. Nusinzon I., Horvath C. M. Histone deacetylases as tran- scriptional activators? Role reversal in inducible gene regu- lation. Sci STKE 2005; 2005: re11.

20. Robertson K. D., Wolffe A. P. DNA methylation in health and disease. Nat Rev Genet 2000; 1: 11–9.

21. Ooi S. K., Qiu C., Bernstein E., Li K., Jia D., Yang Z. et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 2007; 448: 714–7.

22. Sutherland J. E., Costa M. A. X. Epigenetics and the environ- ment. Ann NY Acad Sci 2003; 983: 151–60.

23. Cibelli J. B., Campbell K. H., Seidel G. E., West M. D., Lanza R. P. The health profile of cloned animals. Nat Biotechnol 2002; 20: 13–4.

24. Tamashiro K. L. K., Wakayama T., Yamazaki Y., Akutsu H., Woods S. C., Kondo S. et al. Phenotype of cloned mice: devel- opment, behavior, and physiology. Exp Biol Med 2003; 228: 1193.

25. Fraga M. F., Ballestar E., Paz M. F., Ropero S., Setien F., Ballestar M. L. et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA 2005; 102: 10604–9.

26. Kaminsky Z. A., Tang T., Wang S. C., Ptak C., Oh G. H. T., Wong A. H. C. et al. DNA methylation profiles in monozy- gotic and dizygotic twins. Nat Genet 2009; 41: 240–5.

27. Rakyan V. K., Blewitt M. E., Druker R., Preis J. I., Whitelaw E. Metastable epialleles in mammals. Trends Genet 2002; 18: 348–51.

28. Yen T. T., Gill A. M., Frigeri L. G., Barsh G. S., Wolff G. L. Obesity, diabetes, and neoplasia in yellow A(vy)/- mice: ectopic expression of the agouti gene. FASEB J 1994; 8: 479–88.

29. Dolinoy D. C., Weidman J. R., Waterland R. A., Jirtle R. L. Maternal genistein alters coat color and protects avy mouse offspring from obesity by modifying the fetal epigenome. Environ Health Perspect 2006; 114: 567.

30. Waterland R. A., Jirtle R. L. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003; 23: 5293–300.

An introduction to epigenetics in addiction 487

© 2011 The Authors, Addiction © 2011 Society for the Study of Addiction Addiction, 106, 480–489

31. Tsankova N., Renthal W., Kumar A., Nestler E. J. Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci 2007; 8: 355–67.

32. O’Brien C. P. Anticraving medications for relapse preven- tion: a possible new class of psychoactive medications. Am J Psychiatry 2005; 162: 1423–31.

33. Kalivas P. W., O’Brien C. Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacology 2008; 33: 166–80.

34. Nestler E. J. Molecular basis of long-term plasticity underly- ing addiction. Nat Rev Neurosci 2001; 2: 119–28.

35. Shaham Y., Hope B. T. The role of neuroadaptations in relapse to drug seeking. Nat Neurosci 2005; 8: 1437–9.

36. Nestler E. J., Barrot M., Self D. W. DeltaFosB: a sustained molecular switch for addiction. Proc Natl Acad Sci USA 2001; 98: 11042–6.

37. Hyman S. E., Malenka R. C., Nestler E. J. Neural mechanisms of addiction: the role of reward-related learning and memory. Annu Rev Neurosci 2006; 29: 565–98.

38. Bowers M. S., McFarland K., Lake R. W., Peterson Y. K., Lapish C. C., Gregory M. L. et al. Activator of G protein sig- naling 3: a gatekeeper of cocaine sensitization and drug seeking. Neuron 2004; 42: 269–81.

39. McClung C. A., Ulery P. G., Perrotti L. I., Zachariou V., Berton O., Nestler E. J. DeltaFosB: a molecular switch for long-term adaptation in the brain. Brain Res Mol Brain Res 2004; 132: 146–54.

40. Graham D. L., Edwards S., Bachtell R. K., DiLeone R. J., Rios M., Self D. W. Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-administration and relapse. Nat Neurosci 2007; 10: 1029–37.

41. Grimm J. W., Lu L., Hayashi T., Hope B. T., Su T. P., Shaham Y. Time-dependent increases in brain-derived neurotrophic factor protein levels within the mesolimbic dopamine system after withdrawal from cocaine: implications for incubation of cocaine craving. J Neurosci 2003; 23: 742– 7.

42. Lu L., Dempsey J., Liu S. Y., Bossert J. M., Shaham Y. A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal. J Neurosci 2004; 24: 1604–11.

43. Levenson J. M., Sweatt J. D. Epigenetic mechanisms in memory formation. Nat Rev Neurosci 2005; 6: 108–18.

44. Levenson J. M., Roth T. L., Lubin F. D., Miller C. A., Huang I. C., Desai P. et al. Evidence that DNA (cytosine-5) methyl- transferase regulates synaptic plasticity in the hippocam- pus. J Biol Chem 2006; 281: 15763–73.

45. Miller C. A., Sweatt J. D. Covalent modification of DNA regu- lates memory formation. Neuron 2007; 53: 857–69.

46. Levenson J. M., O’Riordan K. J., Brown K. D., Trinh M. A., Molfese D. L., Sweatt J. D. Regulation of histone acetylation during memory formation in the hippocampus. J Biol Chem 2004; 279: 40545–59.

47. Alarcon J. M., Malleret G., Touzani K., Vronskaya S., Ishii S., Kandel E. R. et al. Chromatin acetylation, memory, and LTP are impaired in CBP+/- mice: a model for the cognitive deficit in Rubinstein–Taybi syndrome and its amelioration. Neuron 2004; 42: 947–59.

48. Colvis C. M., Pollock J. D., Goodman R. H., Impey S., Dunn J., Mandel G. et al. Epigenetic mechanisms and gene networks in the nervous system. J Neurosci 2005; 25: 10379–89.

49. Pidsley R. M., Mill J. Epigenetic studies of psychosis: current findings, methodological approaches and implications

for post-mortem research. Biol Psychiatry 2010; Epub ahead of print.

50. Bernstein B. E., Stamatoyannopoulos J. A., Costello J. F., Ren B., Milosavljevic A., Meissner A. et al. The NIH Roadmap Epigenomics Mapping Consortium. Nat Biotechnol 2010; 28: 1045–8.

51. Yoo C. B., Jones P. A. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 2006; 5: 37–50.

52. Malvaez M., Sanchis-Segura C., Vo D., Lattal K. M., Wood M. A. Modulation of chromatin modification facilitates extinction of cocaine-induced conditioned place preference. Biol Psychiatry 2009; 67: 36–43.

53. Romieu P., Host L., Gobaille S., Sandner G., Aunis D., Zwiller J. Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats. J Neurosci 2008; 28: 9342.

54. Lister R., Pelizzola M., Dowen R. H., Hawkins R. D., Hon G., Tonti-Filippini J. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009; 462: 315–22.

55. Bonsch D., Lenz B., Reulbach U., Kornhuber J., Bleich S. Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism. J Neural Transm 2004; 111: 1611–6.

56. Kim J. S., Shukla S. D. Acute in vivo effect of ethanol (binge drinking) on histone H3 modifications in rat tissues. Alcohol Alcohol 2006; 41: 126–32.

57. Marutha Ravindran C. R., Ticku M. K. Changes in methyla- tion pattern of NMDA receptor NR2B gene in cortical neurons after chronic ethanol treatment in mice. Mol Brain Res 2004; 121: 19–27.

58. Pandey S. C., Ugale R., Zhang H., Tang L., Prakash A. Brain chromatin remodeling: a novel mechanism of alcoholism. J Neurosci 2008; 28: 3729–37.

59. Wang Y., Krishnan H. R., Ghezzi A., Yin J. C., Atkinson N. S. Drug-induced epigenetic changes produce drug tolerance. PLoS Biol 2007; 5: e265.

60. Philibert R. A., Gunter T. D., Beach S. R., Brody G. H., Madan A. MAOA methylation is associated with nicotine and alcohol dependence in women. Am J Med Genet B Neuropsy- chiatr Genet 2008; 147B: 565–70.

61. Deng J. V., Rodriguiz R. M., Hutchinson A. N., Kim I. H., Wetsel W. C., West A. E. MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psycho- stimulants. Nat Neurosci 2010; 13: 1128–36.

62. Renthal W., Carle T. L., Maze I., Covington H. E., III, Truong H. T., Alibhai I. et al. Delta FosB mediates epigenetic desen- sitization of the c-fos gene after chronic amphetamine exposure. J Neurosci 2008; 28: 7344–9.

63. Kalda A., Heidmets L. T., Shen H. Y., Zharkovsky A., Chen J. F. Histone deacetylase inhibitors modulates the induction and expression of amphetamine-induced behavioral sensi- tization partially through an associated learning of the environment in mice. Behav Brain Res 2007; 181: 76–84.

64. Numachi Y., Shen H., Yoshida S., Fujiyama K., Toda S., Matsuoka H. et al. Methamphetamine alters expression of DNA methyltransferase 1 mRNA in rat brain. Neurosci lett 2007; 414: 213–7.

65. Im H. I., Hollander J. A., Bali P., Kenny P. J. MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212. Nat Neurosci 2010; 13: 1120–27.

66. Maze I., Covington H. E. III, Dietz D. M., LaPlant Q., Renthal W., Russo S. J. et al. Essential role of the histone

488 Chloe C. Y. Wong et al.

© 2011 The Authors, Addiction © 2011 Society for the Study of Addiction Addiction, 106, 480–489

methyltransferase G9a in cocaine-induced plasticity. Science 2010; 327: 213–6.

67. Levine A. A., Guan Z., Barco A., Xu S., Kandel E. R., Schwartz J. H. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum. Proc Natl Acad Sci USA 2005; 102: 19186–91.

68. He F., Lidow I. A., Lidow M. S. Consequences of paternal cocaine exposure in mice. Neurotoxicol Teratol 2006; 28: 198–209.

69. Novikova S. I., He F., Bai J., Cutrufello N. J., Lidow M. S., Undieh A. S. Maternal cocaine administration in mice alters DNA methylation and gene expression in hippocampal neurons of neonatal and prepubertal offspring. PLoS ONE 2008; 3: e1919.

70. Kumar A., Choi K. H., Renthal W., Tsankova N. M., Theobald D. E., Truong H. T. et al. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in stria- tum. Neuron 2005; 48: 303–14.

71. Philibert R. A., Beach S. R., Gunter T. D., Brody G. H., Madan A., Gerrard M. The effect of smoking on MAOA promoter methylation in DNA prepared from lymphoblasts and whole blood. Am J Med Genet B Neuropsychiatr Genet 2009; 153B: 619–28.

72. Launay J. M., Del Pino M., Chironi G., Callebert J., Peoc’h K., Mégnien J. L. et al. Smoking induces long-lasting effects through a monoamine-oxidase epigenetic regulation. PloS One 2009; 4: 182–95.

73. Satta R., Maloku E., Zhubi A., Pibiri F., Hajos M., Costa E. et al. Nicotine decreases DNA methyltransferase 1 expres- sion and glutamic acid decarboxylase 67 promoter methy- lation in GABAergic interneurons. Proc Natl Acad Sci USA 2008; 105: 16356.

74. Nielsen D. A., Yuferov V., Hamon S., Jackson C., Ho A., Ott J. et al. Increased OPRM1 DNA methylation in lymphocytes of methadone-maintained former heroin addicts. Neuropsy- chopharmacology 2008; 34: 867–73.

An introduction to epigenetics in addiction 489

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