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DNA METHYLATION AND ITS CATALYZING DNMT ENZYMES IN ADDICTION

Studies of cocaine and other psychostimulants

As noted in the previous section, there remain relatively few studies of DNA meth- ylation in addiction, with most studies to date focused on the role of DNMTs or the methyl-CpG-binding protein 2 (MeCP2). A study from our group demonstrated that repeated cocaine exposure regulated Dnmt3a transcription in mouse NAc (LaPlant et al., 2010). Dnmt3a, but not other Dnmts, was upregulated at an early time point of withdrawal (4 h after the last cocaine dose), followed by downregulation after 24 h. Whether this surprisingly complicated pattern of Dnmt3a regulation is associated with fluctuations in DNA methylation requires further investigation. Importantly; however, after 28 days of withdrawal, after either cocaine IP injections or cocaine self-administration, Dnmt3a was upregulated in the NAc (LaPlant et al., 2010). This long-lasting induction of Dnmt3a is of particular interest given its potential influence on downstream regulation of target genes, a possibility that requires direct examination. In contrast, different effects of cocaine were reported by Anier, Malinovskaja, Aonurm-Helm, Zharkovsky, and Kalda (2010), who found induction of both Dnmt3a and Dnmt3b in mouse NAc, but only after acute (not chronic) cocaine administration. The reasons for these discrepancies are not known, but they could be due to the different experimental paradigms used. Furthermore, it has been shown that drug regulation of Dnmtl expression in NAc and other regions was dependent on genetic background: chronic methamphetamine treatment increased Dnmtl in these regions of Fischer 344/N rats, but exerted the opposite effect in Lewis/N rats (Numachi et al., 2007). This opposite regulation of Dnmtl expression was associated with contrasting behavioral susceptibilities to methamphetamine in the two rat lines.

Pharmacological and viral-mediated gene transfer approaches have been used to examine the behavioral influence of DNMTs on drug addiction. Overexpression of DNMT3a in the NAc attenuated the rewarding effects of cocaine (LaPlant et al., 2010). Moreover, DNMT3a overexpression was sufficient to increase dendritic spine density of NAc neurons to comparable levels seen in response to cocaine administration. Conversely, viral-mediated knockdown of DNMT3a in the NAc, or inhibition of DNMTs via local infusion of the DNMT inhibitor RG108, had the opposite effect. These findings establish an important role of DNMT3a in cocaine-induced neural and behavioral plasticity.

MeCP2 is an X-linked methyl-DNA binding protein that is best studied for its involvement in Rett syndrome, an autism spectrum disorder. More recently, MeCP2 has been implicated in neural and behavioral responses to psychostimulants. In one study, MeCP2 was induced in the dorsal striatum of rats with extended access to IV cocaine self-administration.Viral-mediated MeCP2 knockdown in this region decreased the rats’ cocaine intake (Im, Hollander, Bali, & Kenny, 2010). In parallel, chronic amphetamine administration was shown to increase MeCP2 phosphorylation at Ser421 in the mouse NAc, and viral-mediated knockdown of MeCP2 in this region increased amphetamine place conditioning, whereas local MeCP2 overexpression had the opposite effect (Deng et al., 2010). Importantly, Deng et al. (2014) subsequently showed that mice with a Ser421Ala mutation in MeCP2 displayed greater locomotor sensitization to experimenter-administered cocaine as well as greater self-administration of the drug. The mutant MeCP2 mice also displayed reduced electrical excitability of NAc medium spiny neurons and altered transcriptional responses to cocaine. These exciting studies together link MeCP2 function in NAc and dorsal striatum with psychostimulant addiction.

The next step in these investigations is to study the effect of drug exposure on DNA methylation itself. Mass spectrometry-based measurements showed that chronic cocaine decreased total levels of methylated DNA in the PFC (Tian et al., 2012). However, there was no such change in the NAc (Feng et al., 2015) or in response to other drugs of abuse, for example, morphine (Tian et al., 2012). A major need in the field is to obtain genomewide maps of DNA methylation in the NAc and other brain reward regions after chronic drug administration (see later). In the absence of such genome-wide studies, a few candidate genes have been shown to exhibit altered methylation in addiction models (Table 8.1). For example, chronic cocaine administration induced DNA hypermethylation and increased binding of MeCP2 at the protein phosphatase-1 catalytic subunit (Pp1c) gene promoter in the NAc, which was associated with transcriptional repression (Anier et al., 2010). In contrast, cocaine administration induced hypomethylation and decreased binding of MeCP2 at the FosB promoter in the NAc, associated with induction of FosB (Anier et al., 2010). Wright et al. (2015) showed that chronic cocaine was shown to induce c-Fos expression in the NAc, which was associated with reduced methylation at CpG dinucleotides in the c-Fos gene promoter. Outside of brain reward pathways, significant hypomethylation at multiple CpG sites of the Sox10 promoter region was observed in the corpus callosum of rats at 30 days of forced abstinence from cocaine self-administration (Nielsen, Huang, et al., 2012). As Sox10 expression is enriched in oligodendrocytes, this finding highlights the need to study cocaine regulation of DNA methylation in nonneuronal cell types.

The influence of DNA methylation in addiction models raises the possibility that methylation manipulations might provide a plausible path for addiction therapy. Indeed, methyl supplementation through administration of the methyl donor methionine significantly inhibited cocaine reward in mice (LaPlant & Nestler, 2011; Tian et al., 2012). Also, rats receiving methionine underwent either a sensitization regimen of intermittent cocaine injections or intravenous cocaine self-administration, followed by cue-induced and drug-primed reinstatement. Methionine not only blocked locomotor sensitization but also attenuated drug-primed reinstatement (Wright et al., 2015). Systemic methionine administration was also associated with reversal of DNA hypomethylation at the c-Fos gene in the NAc (Wright et al., 2015). In contrast, using a similar cocaine self-administration approach, intra-NAc injection of a methyl donor promoted cue-induced cocaine seeking after prolonged withdrawal, whereas injection of the DNMT inhibitor RG108 had the opposite effect (Massart et al., 2015). These seemingly opposite effects of a methyl donor on drug behavior may be due to differences in route of administration (systemic vs intra-NAc). Further research is needed to investigate this and alternative possibilities and to test the effect of methyl supplementation in human addicts.

Gene name

Drug of abuse

Differential

methylation

region

Direction of change

Associated

mRNA/protein

change

Species/tissue or cell type

DNA

methylation

methodology

References

ALDH1A2

Alcohol

Promoter

Hypermethylation

NA

Human/saliva

Illumina bead chip/

pyrosequencing

Harlaar et al. (2014)

ANP

Alcohol

Promoter

Hypomethylation

mRNA

increase

Human/blood

Methylation specific qPCR

Hillemacher, Frieling, Luber, et al. (2009)

AVP

Alcohol

Promoter

Hypermethylation

mRNA no change

Human/blood

Methylation- specific qPCR

Hillemacher, Frieling, Luber, et al.

(2009)

c-Fos

Cocaine

Promoter

Hypomethylation

mRNA

increase

Rat/nucleus

accumbens

Bisulfite

sequencing

Wright et al. (2015)

DAT

Alcohol

Promoter

Hypermethylation

NA

Human/blood

Methylation- specific qPCR

Hillemacher, Frieling, Hartl, et al.

(2009)

DLK1

Alcohol

Intergenic

region

Hypomethylation

NA

Human/

sperm

Bisulfite

sequencing

Ouko et al.

(2009)

FosB

Cocaine

Promoter

Hypomethylation

mRNA

increase

Mouse/

nucleus

accumbens

MeDIP/ methylation- specific qPCR

Anier et al. (2010)

GluAl

Methamphetamine

Promoter

Hypomethylation

Both decrease

Rat/striatum

MeDIP

Jayanthi et al. (2014)

GluA2

Methamphetamine

Promoter

Hypomethylation

Both decrease

Rat/striatum

MeDIP

Jayanthi et al. (2014)

HERP

Alcohol

Promoter

Hypermethylation

mRNA

decrease

Human/blood

Methylation- specific qPCR

Bleich et al.

(2006)

HTR3A

Alcohol

Promoter

Hypermethylation

NA

Human/blood

Methylation array, Sequenom

H. Zhang et al. (2013)

MAOA

Alcohol

Promoter

Hypermethylation

Undetectable

mRNA

Woman/

lymphoblast

lines

Sequenom

Philibert, Gunter, Beach, Brody, and Madan (2008)

NGF

Alcohol

Promoter

Hypermethylation

Protein

decrease

Human/blood

Bisulfite

sequencing

Heberlein et al. (2013)

NR2B

Alcohol

Promoter

Hypomethylation

mRNA

increase

Human/

blood;

Mouse/

neuronal

culture

Bisulfite

sequencing

Biermann et al. (2009) and Marutha Ravindran and Ticku (2005)

OPRM1

Alcohol

Promoter

Hypermethylation

NA

Human/blood

Bisulfite

sequencing

Zhang et al. (2012)

OPRM1

Opioids

Promoter

Hypermethylation

NA

Human/ blood, sperm

Pyrosequencing;

bisulfite

sequencing

Chorbov, Todorov, Lynskey, and Cicero (2011) and Nielsen et al. (2009)

PDYN

Alcohol

3' UTR CpG-SNP

Hypermethylation

mRNA

increase

Human/

prefrontal

cortex

Pyrosequencing

Taqi et al. (2011)

PP1c

Cocaine

Promoter

Hypermethylation

mRNA

decrease

Mouse/

nucleus

accumbens

MeDIP/ methylation- specific qPCR

Anier et al. (2010)

PPM1G

Alcohol

3' of gene sequence

Hypermethylation

mRNA

decrease

Human/blood

Methylation array, Sequenom

Ruggeri et al. (2015)

SNCA

Alcohol

Promoter

Hypermethylation

Both decrease

Human/blood

Methylation- specific qPCR

Bonsch et al. (2005)

Sox10

Cocaine

Promoter

Hypomethylation

NA

Rat/white

matter

Bisulfite

sequencing

Nielsen, Huang, et al. (2012)

Sty2

Alcohol

First exon

Hypermethylation

mRNA

decrease

Rat/prefrontal

cortex

Pyrosequencing

Barbier et al. (2015)

MeDIP, Methylated DNA immune-precipitation; NA, not available; qPCR, quantitative PCR; SNP, single-nucleotide polymorphism; UTR, untranslated region.

 
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