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Mechanisms of Epigenetic memory and addiction


Overview

Originally Published: 04/23/2014

Post Date: 04/23/2015

by Luis M Tuesta & Yi Zhang | The EMBO Journal Vol 33 | No 10 | 2014


Summary/Abstract

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

Content

Epigenetic mechanisms underlying brain reward and drug addiction

 

 

Reward processing is an integral neural event that ensures survival in an organism, as it reinforces positive behaviors and experiences. Perception of a reward is mediated by the brain reward system and uses dopamine as its principal neurotransmitter. The ventral tegmental area (VTA) is a midbrain region crucial to reward processing and represents a major dopaminergic output in the brain. Dopaminergic output from the VTA primarily projects to the nucleus accumbens (NAc), a principal center for reward processing, while the dorsal striatum (DST), another region heavily implicated in addictive disorders, receives most of its dopaminergic input from midbrain neurons in the substantia nigra pars compacta (SNpc) (Fig 3) (Hyman et al, 2006; Russo & Nestler, 2013). Food reward has been shown to activate dopaminergic neurons in the VTA. Indeed, feeding behavior is heavily influenced by the expectation of pleasure and reward, and this proves to be a very powerful motiva­tor of consumption (Saper et al, 2002; Zheng & Berthoud, 2007). A recent report suggests a role for DNA methylation in food reward. The study shows that associative learning for sucrose rewards increases methylation of learning-associated genes within dopami-nergic neurons in the VTA and that inhibition of DNA methylation in this brain region, but not the NAc, prevents acquisition of the behavior (Day et al, 2013). This is the first report to link activity-dependent DNA methylation and demethylation to appetitive behav­ior such as volitional food reward consumption. Interestingly, it has also been reported that chronic overeating of a highly palatable diet leads to obesity that results in brain reward deficits as well as reduced D2R availability (Johnson & Kenny, 2010)—a phenomenon also seen in individuals with obesity (Volkow et al, 2008) and indi­viduals with a history of cocaine abuse (Volkow et al, 1993). Just as the midbrain dopamine system plays a major role in regulating food reward, drugs of abuse such as cocaine and nicotine have a very powerful effect on dopaminergic output (Kalivas et al, 1988; Nisell et al, 1994; Picciotto et al, 1998).

While natural rewards reinforce behaviors that help ensure survival in an organism, drugs of abuse do not. As opposed to food, drugs of abuse act directly on dopaminergic neurons, resulting in overactivation of brain reward circuitry. Indeed, development of addiction can be characterized by increased consumption, followed by a persistent desire for, and to acquire, the drug. As consumption further increases and becomes habitual, tolerance may develop, and a negative affective state akin to withdrawal will be established when the drug is unavailable. Finally, this cycle of behavior may not only affect physiological reward processing, but also severely compromise social and occupational activities of the individual (Koob & Le Moal, 2001). It should be noted, however, that casual drug use does not always result in transition to compulsive drug use and addiction. In fact, only about 17% of cocaine users eventually become addicted (Anthony et al, 1994).

Drug addiction is a complex and devastating disease. It incor­porates genetic and environmental factors that when combined, can hijack neural pathways involved in normal reward memory processing, increase an individual’s propensity to abuse drugs, and severely compromise their ability to stop. Fundamentally, drug addiction can be seen as an aberrant learning disorder, as it shares common mechanisms seen in memory acquisition and maintenance (Everitt et al, 2001; Hyman et al, 2006; Kelley, 2004; Thomas et al, 2008; Torregrossa et al, 2011). However, while our knowledge of the epigenetic mechanisms governing learning and memory has greatly increased over the last decade, we are only beginning to understand the epigenetic mechanisms underlying drug addiction.

Histone modifications in drug reward

The term ‘addiction’ refers to a confluence of genetic, physiological, and environmental factors that lead to uncontrolled drug intake and significant lifestyle disruption; however, current behavioral models of addiction focus primarily on the rewarding and motivational aspects of drug intake. More specifically, animal models in the field of addiction epigenetics primarily focus on behavioral responses to administration of drugs of abuse as a way to measure drug reward. While more comprehensive behavioral models would benefit the interpretation of epigenetic studies, assessment of drug-induced molecular changes and how they contribute to the behavior changes are critical for understanding the development of addiction.

Histone acetylation Most of our knowledge on epigenetic regulation of addiction has focused on the effects of psychostimulants such as cocaine and amphetamine on histone modifications. Among these, the most common modifications studied involve histone acetylation and methylation. Cocaine exposure alters acetylated H3 and H4 levels in the NAc (Kumar et al, 2005; Schroeder et al, 2008; Shen et al, 2008). For instance, acute cocaine exposure increases H4 acetylation at the promoter of c-Fos, an immediate early gene and a marker of neuronal activation, while chronic exposure results in no such a change (Kumar et al, 2005; Renthal et al, 2008). Neverthe­less, chronic cocaine exposure can also result in gene activation that is not induced by acute treatment. One such example is the acetyl-ation of H3 at the BDNF and CDK5 promoter regions (Kumar et al, 2005). While cocaine administration can alter histone acetylation at many gene promoters, it does not necessarily result in altered tran­scription in the NAc (Renthal et al, 2009). It is worth mentioning that the lack of correlation does not imply that a similar changes in BDNF promoter acetylation have been detected following cocaine exposure (Sadri-Vakili et al, 2010), but rather highlights the complexity of transcriptional output resulting from changes in histone acetylation.

Consistent with the above studies, behavioral tests measuring the effect of HDAC deletion on cocaine sensitivity and reward have also resulted in mixed outcomes. For instance, while deletion of HDAC1 in the NAc attenuates behavioral responses to cocaine, deletion of HDAC2 or HDAC3 in the NAc does not (Kennedy et al, 2013). Inter­estingly, inhibition of HDAC3, the most highly expressed HDAC in the brain (Broide et al, 2007), enhances extinction and prevents reinstatement of cocaine seeking in a conditioned place preference paradigm (Malvaez et al, 2013). To date, most behavioral studies investigate the effects of psychostimulants on drug seeking and loco-motor sensitization. However, to obtain a more complete picture on the role of epigenetic modifications in drug addiction, behavioral models of addiction more similar to the human condition, such as intravenous drug self-administration, should be considered.

Histone methylation Several recent studies have investigated the effects of drugs of abuse on histone methylation states. While drug exposure fails to have a general effect on HMTs and HDMs, chronic cocaine treatment represses G9a in the nucleus accumbens, as evidenced by decreases in H3K9 dimethylation (Maze et al, 2010). Additionally, G9a inhibition in NAc, either genetically or pharmaco­logically, increases behavioral responses to cocaine and opiates, and overexpressing G9a can reverse these effects (Maze et al, 2010; Sun et al, 2012). Furthermore, Cre-dependent knockout of G9a in the NAc increases dendritic arborization (Maze et al, 2010), suggesting that H3K9 dimethylation by G9a may play a role in drug-dependent synaptic plasticity. Mechanistically, G9a appears to play a central role in a negative feedback loop with DFosB, a long-lasting transcrip­tion factor central to drug addiction (Feng & Nestler, 2013; Robison & Nestler, 2011). G9a inhibits induction of DFosB, and in turn, DFosB inhibits expression of G9a (Maze et al, 2010; Sun et al, 2012). Additionally, prolonged HDAC inhibition not only inhibits behav­ioral responses to cocaine, but also induces G9a expression, a finding consistent with the ability of G9a overexpression to inhibit such behavioral responses to psychostimulants (Kennedy et al, 2013).

While these findings support the involvement of epigenetic regu­lation in drug reward, one also cannot undermine the role of tran­scription factors in the recruitment and modulation of epigenetic modifying enzymes. Indeed, transcription factors such as DFosB, myocyte enhancer factor 2 (MEF2), and CREB are all known to recruit epigenetic modifying enzymes (Robison & Nestler, 2011). DFosB can drive CDK5 transcription by recruiting CBP (Levine et al, 2005) and, conversely, inhibit c-Fos transcription by recruiting HDAC1 (Renthal et al, 2008). MEF2 can recruit the class II HDAC, p300, while CREB also binds CBP (He et al, 2011a; Robison & Nestler, 2011). It is therefore likely that transcription factors and epigenetic enzymes work in concert to mediate the transcriptional regulation of drug reward.

DNA methylation in drug reward

There are relatively few studies to date that focus on the role of DNA methylation in drug reward and addiction. It is known that acute and chronic cocaine exposure promotes Dnmt3a expression in the NAc (Anier et al, 2010; LaPlant et al, 2010). More specifically, 28-day cocaine withdrawal increases Dnmt3a levels in the NAc regardless of whether the cocaine is self-administered or delivered in a non-contingent manner. With regard to a causal relationship, inhibition of Dnmt3a in the NAc via knockdown or via pharmaco­logical administration of RG108 increases behavioral responses to cocaine. Conversely, overexpression of Dnmt3a shows the opposite, blunting cocaine reward. Plasticity is also affected, as chronic cocaine use increases accumbal thin dendritic spine density, an effect mimicked by local overexpression of Dnmt3a (LaPlant et al, 2010). In addition to Dnmt3a, the methyl-CpG binding protein MeCP2 has also been linked to addiction. MeCP2 contributes to gene silencing by recruiting HDACs to methylated DNA (Amir et al, 1999; Van Esch et al, 2005). Chronic cocaine self-administration in rats increases striatal MeCP2 levels. Interestingly, when MeCP2 is locally knocked down in the striatum, rats decrease their cocaine intake levels (Im et al, 2010). Conversely, genetic ablation of MeCP2 in the NAc enhances amphetamine reward (Deng et al, 2010). While Dnmt3a and MeCP2 appear to regulate aspects of drug reward, no direct evidence of differential methylation of addiction-related genes has been shown. Regardless, available evidence suggests a cocaine reward-blunting role for Dnmt3a in the NAc and, in the case of MeCP2, a paradoxical pattern of epigenetic regulation of drug reward that is anatomically discrete.

Until recently, DNA demethylation has been a topic of much speculation. It is clear now that TET enzymes regulate DNA deme-thylation and likely play a central role in learning and memory (Guo et al, 2011a,b; Kaas et al, 2013; Ma et al, 2009; Rudenko et al, 2013; Zhang et al, 2013). Similarly, DNA demethylation is likely involved in adaptive and maladaptive changes in gene expression that contribute to the addiction phenotype. As mentioned earlier, 5hmC is a DNA demethylation intermediate highly enriched in the brain, and intragenic 5hmc is associated with gene transcription (Szulwach et al, 2011). To date, studies that aim to profile genomic methylation states utilize bisulfite sequencing that fails to distin­guish 5mC from 5hmC. This limitation disguises genomic regions of active demethylation in favor of transcriptionally repressive methy­lated DNA. However, TET-assisted bisulfite sequencing (TAB-Seq) allows for 5hmC detection at single-base resolution (Yu et al, 2012) and may provide a useful tool to more adequately investigate the effects of drugs of abuse on brain DNA methylation states.

MicroRNAs in drug reward

Recent studies suggest that some of the epigenetic events can be mediated by microRNAs (Bali & Kenny, 2013). Interestingly, cocaine exposure can modulate microRNA (miRNA) levels in the NAc, such as upregulation of miR-181 and downregulation of miR-124 and let-7d. Importantly, modulation of miRNA levels corresponding to the changes seen following cocaine exposure can potentiate behavioral responses to the drug (Chandrasekar & Dreyer, 2009, 2011), suggest­ing that transcriptional regulation of addiction-related genes by miRNA is sufficient to increase susceptibility to drug reward. This also implies that opposite directional manipulation of striatal miRNA can curb cocaine reward and consumption. Indeed, overexpression of striatal miR-212 reduces cocaine intake in rats through increasing activity of CREB, a transcription factor that opposes cocaine reward (Hollander et al, 2010; Robison & Nestler, 2011). These results further suggest that in addition to histone and DNA modifications, miRNAs can also play a significant role in the development of addiction.

Transgenerational inheritance of drug phenotypes

A classical interpretation of an epigenetic change dictates that it must be heritable, and recent evidence suggests that preference for drugs of abuse can also be inherited to subsequent generations. For example, offspring of alcohol-preferring rats, when compared to offspring of non-alcohol-preferring rats, show increased nicotine intake and reinstatement following extinction, yet remarkably, do not show any difference in cocaine intake (Le et al, 2006). In the case of psychostimulants, male offspring from cocaine-experienced sires display a cocaine-resistant phenotype, but normalize intake when BDNF signaling is pharmacologically inhibited (Vassoler et al, 2013). Interestingly, cocaine-experienced sires show increased H3 acetylation and BDNF expression in sperm, indicating germline epigenetic reprogramming (Vassoler et al, 2013). It is also important to note that while the amount of drug consumed is a reliable reflec­tion of the reinforcing properties of a drug, this is only one metric of addiction and does not encompass the complete behavioral spec­trum commensurate with drug addiction. Regardless, prolonged drug use shows the potential to promote heritable epigenetic modifi­cations that could place progeny at increased vulnerability for drug abuse later in life.

Open questions and future directions

The examples presented above suggest that epigenetic regulation is part of the mechanism underlying addiction. It is thus possible that epigenetic modifications in dopaminergic VTA neurons by local TET/TDG-driven demethylation could regulate synaptic plasticity in the hippocampus, promoting consolidation of long-term reward memories in the PFC (Fig 3). Of course, this hypothesis focuses solely on dopaminergic output, discounting other local cell types that may contribute to the phenotype. As such, one area in the study of epigenetic mechanisms of addiction that remains largely unad­dressed is the neurochemical resolution at which epigenetic changes occur to effect behavioral changes. Most of the studies presented in this review focus on individual brain regions and not cell types. Just as there are transcriptional repressors and silencing markers that maintain gene expression under control, heterogeneity of neuron types within a given brain region may have a role in regulating neuronal activity. For instance, in addition to dopamine neurons, the VTA is also populated by GABA neurons. Indeed, activation of VTA GABA neurons is known to suppress excitation of dopamine neurons (van Zessen et al, 2012), yet repeated exposure to cocaine has been shown to disinhibit dopamine neurons via reduced activity of GABA neurons within the VTA (Bocklisch et al, 2013). This suggests that we cannot make absolute claims about the role of epigenetic mechanisms on behavior based solely on a single brain region unless we are able to precisely select a homogeneous neuro­nal population and focus our manipulations and analysis on said group of cells. Furthermore, as discussed earlier, caution should be taken when attributing findings from pharmacological studies on epigenetic modifying enzymes to addiction and learning behavior as these may lack target specificity or be ineffective in postmitotic neurons. However, recent advances in developing genome-editing tools will soon allow us to investigate the role of specific epigenetic modifying enzymes in the development of the addictive process within distinct neuronal populations. For instance, the CRISPR/Cas9 system can facilitate this process by excising multiple genetic targets with remarkable precision (Cong et al, 2013; Ran et al, 2013a,b). This technology may permit the study of various epigenetic modify­ing enzymes within specific subsets of neurons in vivo, thus addressing a major challenge in the field. Additionally, some of the studies discussed in this review correlate subtle changes in epige­netic states with distinct behavioral phenotypes without addressing the cause-and-effect relationship. In this regard, CRISPR/Cas9 tech­nology will be very useful in addressing this issue as it may allow precise manipulation of epigenetic states at specific genomic loci.

As mentioned earlier in this review, the study of addiction epige-netics could benefit from behavioral models that more accurately mirror drug intake in humans, such as intravenous drug self-administration. This behavioral paradigm assesses the reinforcing properties of a drug, where animals perform a learned operant task (lever pressing) in order to receive an intravenous infusion of a drug (Fowler & Kenny, 2011; Tuesta et al, 2011). This is a particularly important distinction because the experimental animal has absolute control over its drug intake, as opposed to reacting to a non-contingent drug challenge that can result in altered stress hormone transmission in the brain (Palamarchouk et al, 2009). While techni­cally challenging, drug self-administration in mice yields valuable insights into acquisition behavior, compulsivity, and relapse to drug seeking (Fowler & Kenny, 2011). Thus, it can provide a more complete behavioral model of drug addiction, especially given that a majority of genetic manipulations are currently performed in mice.

There are relatively few studies that focus on the role of DNA methylation and drug addiction, and to date, there are no studies that look at the role of DNA demethylation. The process of DNA methylation has long been considered to be a stable, static process, but with our recent understanding of the molecular mechanisms of DNA demethylation catalyzed by the TET and TDG enzymes, DNA methylation appears not to be as stable as previously thought. It will therefore be necessary to address the specific role that DNA deme-thylation machinery plays in the addiction process. Despite the chal­lenges, regulation of DNA methylation states has the potential to serve as a molecular switch that can drive memory formation and shape vulnerability to substance abuse disorders.

The role of epigenetic mechanisms in learning and memory still remains a nascent field of study; yet accumulating evidence suggests that epigenetic mechanisms can regulate the ability to store long­term memories. Maintenance of these memories can last for the life­time of an individual and it is intriguing to speculate how drugs of abuse can potentially induce similar lasting changes in reward path­ways that may predispose a person to addiction. Drug addiction is an exciting new frontier for investigation, as it is a behavior affected by numerous genetic and environmental factors. The multifactorial nature of the disease requires interdisciplinary contributions. In order to develop a molecular understanding of addiction, we will need to use genomic tools, such as RNA-Seq, whole-genome bisulfite sequencing (WGBS), and Tet-assisted bisulfite sequencing (TAB-Seq) to determine the effects of drugs of abuse on global epigenetic modifications and gene expression. When combined with powerful behavioral techniques such as intravenous self-administration throughout various stages of the addictive process and cell-specific manipulation of gene expression, our under­standing of epigenetic mechanisms of addiction may yield exciting new avenues for therapeutic intervention.

Acknowledgement Work in the Zhang laboratory was supported by NIH Grants GM68804 and U01DK089565. Y.Z. is an Investigator of the Howard Hughes Medical Institute.

 

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