David De Sa Nogueira

Karine Merienne

Dr Katia Befort
email: katia.befort@unistra.fr

Affiliation

Neuroepigenetics and addictive behaviors: where do we stand?

Keywords: Neuroepigenetics, Transcriptomics, RNA-seq, Addiction, brain 5hmC, 5-hydroxymethylcytosine, 5mC, 5-methylcytosine, CPP, conditioned place preference, DNAhm, DNA hydroxymethylation, DNAm, DNA methylation, DNMT, DNA

Substance use disorders involve long-term changes in the brain that lead to compulsive drug seeking, craving, and a high probability of relapse. Recent findings have highlighted the role of epigenetic regulations in controlling chromatin access and regulation of gene expression following exposure to drugs of abuse. In the present review, we focus on data investigating genome-wide epigenetic modifications in the brain of addicted patients or in rodent models exposed to drugs of abuse, with a particular focus on DNA methylation and histone modifications associated with transcriptional studies. We highlight critical factors for epigenomic studies in addiction. We discuss new findings related to psychostimulants, alcohol, opiate, nicotine and cannabinoids. We examine the possible transmission of these changes across generations. We highlight developing tools, specifically those that allow investigation of structural reorganization of the chromatin. These have the potential to increase our understanding of alteration of chromatin architecture at gene regulatory regions.

Neuroepigenetic mechanisms involved in addictive behaviors could explain persistent phenotypic effects of drugs and, in particular, vulnerability to relapse.

methyltransferase; DS, dorsal striatum; HDAC, histone deacetylase; MeCP2, methylated DNA-binding protein 2; miR, microRNAs; NAc, nucleus accumbens; PFC, prefrontal cortex; SUD, substance use disorder;

1-Introduction

Drug addiction or substance use disorder (SUD) is a complex brain disease involving long-term changes that lead to compulsive drug seeking, craving, and a high probability of relapse [START_REF] Berridge | Is Addiction a Brain Disease?[END_REF][START_REF] Volkow | Neurobiologic Advances from the Brain Disease Model of Addiction[END_REF]. Uncontrolled drug intake is associated with negative emotional states like anxiety when the drug is not available, and occurs despite individual and social negative consequences [START_REF] Everitt | Neural systems of reinforcement for drug addiction: from actions to habits to compulsion[END_REF][START_REF] Koob | Neurobiological substrates for the dark side of compulsivity in addiction[END_REF]. The neuronal circuits involved in addictive behaviors are complex [START_REF] Koob | Neurocircuitry of addiction[END_REF], and imaging tools have identified disrupted brain structures in addicted humans that are implicated in addiction vulnerability [START_REF] Volkow | The Brain on Drugs: From Reward to Addiction[END_REF]. Key structures include the mesolimbic dopaminergic system, with the ventral tegmental area (VTA) neurons projecting to the nucleus accumbens (NAc part of the ventral striatum). Other regions involved in motivational and emotional processes or in memory association such as the dorsal striatum (DS), prefrontal cortex (PFC), insula, extended amygdala and hippocampus (hipp), participate in this reward circuit. The rostromedial tegmental nucleus (RMTg) has been recently described to play a role in the control of the mesolimbic pathway [START_REF] Bourdy | A new control center for dopaminergic systems: pulling the VTA by the tail[END_REF]. Also, the lateral habenula is disrupted by drugs of abuse and proposed to be a hub in addictive responses (see Mathis & Kenny, in this special issue, and [START_REF] Velasquez | The role of the habenula in drug addiction[END_REF];). Only a proportion of individuals become addicted and this vulnerability, meaning the transition from recreational to uncontrolled and compulsive intake, is largely impacted by the nature of the drug, genetic factors, in addition to the developmental, social and the psychological context of the individual (EMCDDA, 2017;[START_REF] Juli | Genetic of addiction: common and uncommon factors[END_REF][START_REF] Kreek | Genetic influences on impulsivity, risk taking, stress responsivity and vulnerability to drug abuse and addiction[END_REF].

Knowledge of molecular mechanisms involved in behavioral adaptations observed following repeated drug exposure has greatly improved over the last decade. Modifications at the level of neuron connectivity signaling, dendritic spine morphology or synaptic plasticity [START_REF] Robinson | Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats[END_REF][START_REF] Ron | The "ups and downs" of signaling cascades in addiction[END_REF] have been described together with transcriptional regulations (reviewed in [START_REF] Contet | Mu opioid receptor: a gateway to drug addiction[END_REF][START_REF] Mcclung | Regulation of gene expression by chronic morphine and morphine withdrawal in the locus ceruleus and ventral tegmental area[END_REF][START_REF] Przewlocki | Opioid abuse and brain gene expression[END_REF][START_REF] Rhodes | Gene expression induced by drugs of abuse[END_REF][START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF][START_REF] Spanagel | Addiction and its brain science[END_REF].

Increasing evidence is now emerging that epigenetic modulations participate in vulnerability to addiction, as well as in the maintenance of behavioral adaptations induced by substance abuse. Epigenetic mechanisms involve chromatin modifications (chemical or conformational) that alter gene function without changing the DNA sequence. These mechanisms are often initial responses to a dynamic environment and regulate several processes including gene expression, DNA replication and repair, growth, cell cycle and, development. In the last years, such dynamic modifications have been specifically studied in neurons, in relationship to neurodevelopmental, psychiatric and neurodegenerative disorders [START_REF] Cholewa-Waclaw | The Role of Epigenetic Mechanisms in the Regulation of Gene Expression in the Nervous System[END_REF][START_REF] Francelle | Contribution of Neuroepigenetics to Huntington's Disease[END_REF][START_REF] Nestler | Epigenetic Basis of Mental Illness[END_REF]. As a result, the concept of neuroepigenetics emerged to specifically describe epigenetic regulations occurring in post-mitotic neurons as opposed to epigenetic mechanisms involved in developmental processes [START_REF] Day | DNA methylation and memory formation[END_REF]. Several neuroepigenetic regulations occur throughout reward circuitry to regulate gene expression changes following exposure to drugs of abuse [START_REF] Walker | Regulation of chromatin states by drugs of abuse[END_REF], which include addition of epigenetic marks on histone proteins or DNA sequence (Figure 1 and see below).

Additional modifications involve changes in non-coding RNA levels that impact gene expression and function and have been recently described in cocaine addiction [START_REF] Smith | MicroRNAs regulate synaptic plasticity underlying drug addiction[END_REF]. They do play a critical role in synaptic plasticity underlying addiction and will not be detailed here [START_REF] Mayfield | Emerging roles for ncRNAs in alcohol use disorders[END_REF][START_REF] Smith | MicroRNAs regulate synaptic plasticity underlying drug addiction[END_REF]. As the epigenetic field is rapidly evolving, recent reviews dedicated to specific drugs like alcohol [START_REF] Farris | Epigenetic modulation of brain gene networks for cocaine and alcohol abuse[END_REF][START_REF] Pandey | Epigenetic basis of the dark side of alcohol addiction[END_REF], cannabinoids [START_REF] Szutorisz | High times for cannabis: Epigenetic imprint and its legacy on brain and behavior[END_REF] or cocaine [START_REF] Sadri-Vakili | Cocaine triggers epigenetic alterations in the corticostriatal circuit[END_REF][START_REF] Vaillancourt | DNA Methylation Dynamics and Cocaine in the Brain: Progress and Prospects[END_REF][START_REF] Zwiller | Epigenetics and drug addiction: a focus on MeCP2 and on histone acetylation[END_REF], and to a more global view of addiction [START_REF] Nestler | Epigenetic mechanisms of drug addiction[END_REF] have been published to highlight the importance of epigenetic dysregulations in addictive behaviors.

In the present review, we focus our attention on recent data interrogating global epigenetic modifications (genome-wide) in the brain following drugs of abuse in humans, non-human primates or rodent models, with a particular focus on DNA methylation and histone modifications, associated with transcriptional studies. We specifically highlight critical factors that should be considered when evaluating neuroepigenetics reprogramming.

We discuss whether shared mechanisms or biomarkers have been identified within epigenetic programming following drug exposure. We highlight how developing tools, including investigation of structural reorganization of the chromatin, will increase our understanding of these molecular adaptations in addictive behaviors, that could explain persistent phenotypic effects of drugs and, in particular, vulnerability to relapse.

2-Critical factors for epigenome analysis in addiction

Recent studies in the addiction field have focused on epigenomic approaches, with the analysis of global changes at the level of chromatin for DNA methylation or histone modifications. These data are listed for psychostimulants (Table 1), alcohol (Table 2), opiate (Table 3), nicotine or cannabinoid (Table 4), the latter still being scarce. Recent data are emerging from genome-wide analysis, using chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq). This allows quantification of the amount of immunoprecipitated fragmented chromatin for all genomic regions by high-throughput sequencing, using an antibody specific for a given targeted histone mark. Also, for global analysis of epigenetic dysregulations, a concomitant analysis of the gene expression profile represents a powerful tool to correlate the observed adaptations. Transcriptional analysis within brain reward circuitry has been widely performed in the field of addiction using either candidate approaches or microarray technologies (for reviews see [START_REF] Contet | Mu opioid receptor: a gateway to drug addiction[END_REF][START_REF] Mcclung | Regulation of gene expression by chronic morphine and morphine withdrawal in the locus ceruleus and ventral tegmental area[END_REF][START_REF] Przewlocki | Opioid abuse and brain gene expression[END_REF][START_REF] Rhodes | Gene expression induced by drugs of abuse[END_REF][START_REF] Russo | The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens[END_REF][START_REF] Spanagel | Addiction and its brain science[END_REF]. The emergence of high-throughput sequencing with the development of RNA-seq allows powerful transcriptional profiling. This method, based on the creation of cDNA libraries from isolated RNA, provides a reliable analysis of the transcriptome, at a higher base-pair resolution than classical microarray, and allows detection of non-coding RNAs, or alternative splice variants (BOX). We therefore also examined studies, which also provided transcriptomic analysis using RNA microarray or RNA-seq approaches, enabling to assess drug-induced epigenetic changes on gene expression programs. Specific results will be discussed below but several factors have to be considered when examining such genome-wide studies and we highlight the most critical ones here (Figure 2).

2.1-Animal models

Most of the studies investigating modifications in the brain have been performed in rodents, with the exception of a few ethanol studies, that examined human or rhesus macaque samples (see Table 2). Also, we have not included studies investigating epigenetic adaptations from human blood samples. Using rodent animals, one expects to obtain less variability compared with human studies performed on large cohorts of persons with distinct and often unknown histories of addiction. In contrast, various behavioral responses to drugs of abuse, like alcohol, are also observed in animal models within a homogenous population. This vulnerability may be partly explained by subtle environmental changes that can also induce epigenetic modifications. Also, differences may be identified when comparing drug effect on distinct strains at the level of behavior [START_REF] Ayranci | Dissociation of heroininduced emotional dysfunction from psychomotor activation and physical dependence among inbred mouse strains[END_REF] or transcriptomic adaptations [START_REF] Grice | Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine[END_REF][START_REF] Korostynski | Morphine effects on striatal transcriptome in mice[END_REF]. Still, rodent models are very useful to study drug effects under more controlled conditions.

2.2.Diversity of paradigms

Among the rodent studies, it is interesting to note that various protocols have been developed, from acute drug treatment (A) to subchronic or chronic (C) administrations and sometimes following withdrawal periods (W). Doses, route of administration as well as duration of treatments vary extensively across studies making it difficult to describe a global modification scheme. Also, most studies analyzed passive drug administration with very few studies investigating voluntary consumption in rodent models (see below, [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF][START_REF] Cadet | Genome-wide DNA hydroxymethylation identifies potassium channels in the nucleus accumbens as discriminators of methamphetamine addiction and abstinence[END_REF][START_REF] Fernandez-Castillo | Active and passive MDMA ('ecstasy') intake induces differential transcriptional changes in the mouse brain[END_REF][START_REF] Fonteneau | Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study[END_REF][START_REF] Freeman | Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-administration[END_REF][START_REF] Massart | Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving[END_REF]. When more studies are available, it will be useful to compare passive versus voluntary intake to evaluate possible transcriptomic or epigenetic signatures of voluntary consumption of drugs of abuse.

In SUD, increasing research to investigate mechanisms that could explain long term adaptations leading to relapse have been developed. Nevertheless, the neurobiological mechanisms involved in long-term drug abstinence are still poorly understood. Very few studies investigating whole-genome changes have focused their attention on abstinent conditions. Earlier studies have investigated drug abstinence at the transcriptome level [START_REF] Kuntz-Melcavage | Gene expression changes following extinction testing in a heroin behavioral incubation model[END_REF][START_REF] Spijker | Morphine exposure and abstinence define specific stages of gene expression in the rat nucleus accumbens[END_REF] and even fewer studies directly compared adaptations across drugs. Interestingly, in an open-ended approach using microarray, chronic morphine treatment revealed a collection of genes in the extended amygdala which represented promising candidates potentially involved in drug dependence and craving [START_REF] Befort | Mu-opioid receptor activation induces transcriptional plasticity in the central extended amygdala[END_REF]. Expression of these candidate genes were evaluated following four weeks of cessation of drug treatment (morphine, nicotine, THC and alcohol) and a common transcriptional signature of protracted abstinence was identified (Le [START_REF] Merrer | Protracted abstinence from distinct drugs of abuse shows regulation of a common gene network[END_REF].

Interestingly, unlike other drugs, cocaine differentially altered some behavioral responses in abstinent conditions and, oppositely modified expression of several genes [START_REF] Becker | Differential behavioral and molecular alterations upon protracted abstinence from cocaine versus morphine, nicotine, THC and alcohol[END_REF]. These findings emphasize differences between cocaine addiction and addiction to other drugs, though highlighting commonalities between opiate, alcohol, THC and nicotine abuse. These data complete accumulating evidence that specific adaptations in terms of behavior, connectivity, morphology or transcription can occur depending on the type of drug and questioned the unitary theory of addiction proposing common mechanisms for dependence and relapse processes [START_REF] Badiani | Opiate versus psychostimulant addiction: the differences do matter[END_REF][START_REF] Ozburn | Commonalities and Distinctions Among Mechanisms of Addiction to Alcohol and Other Drugs[END_REF]. Altogether, while wholegenome approaches for epigenetic adaptations are still needed to explore this hypothesis, one has to keep in mind that generalizing conclusions from these studies mostly obtained with psychostimulants may not be always pertinent for the other drugs of abuse.

In summary, time-dependent effects of drugs of abuse on transcriptional and epigenetic modifications represent a critical factor, as emotional deficits are still detectable after long periods of withdrawal [START_REF] Goeldner | Impaired emotional-like behavior and serotonergic function during protracted abstinence from chronic morphine[END_REF]. Several time-points should be included in future studies, and as proposed by Baker-Andersen [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF], including animals with a history of drug seeking will bring insight into the memory process altered by drug exposure. This will be particularly important to decipher specific mechanisms involved in states facilitating relapse vulnerability. In addition, the adolescent period represents a time when drug exposure increases the risk of addiction. There is limited research examining epigenetic changes in normal brain maturation during adolescence [START_REF] Mychasiuk | Epigenetic and gene expression changes in the adolescent brain: What have we learned from animal models[END_REF], but these data will further our understanding of these complex processes for a more complete comprehension of how the effects of drug exposure changes across time.

2.3-Brain structure or cell type targets

The complexity of the reward-circuit makes the comparison across studies challenging as targeted brain structures vary across studies. Interestingly, most epigenetic studies focused on the NAc and PFC (see Tables), which represent the structures where most initial transcriptomic studies were performed [START_REF] Nestler | Epigenetic mechanisms of drug addiction[END_REF]. Among key reward-related structures, the NAc has been widely studied as the motivational center in reward related responses and is a critical structure for the initial rewarding effects of psychostimulants, whereas the PFC was investigated, primarily, for its role in goal-directed behavior and decision-making.

Adaptations within the dorsal striatum (DS) have received far less attention than those occurring in the NAc, while changes in this structure would better inform epigenetic regulations in link with expression of compulsive drug-seeking traits, including relapse [START_REF] Belin | Cocaine seeking habits depend upon dopamine-dependent serial connectivity linking the ventral with the dorsal striatum[END_REF]. Brown and colleagues showed decreased expression of synaptic plasticityassociated genes in the DS of animals categorized as relapse-vulnerable [START_REF] Brown | Down-regulated striatal gene expression for synaptic plasticity-associated proteins in addiction and relapse vulnerable animals[END_REF], a regulation related to decreased expression of specific miRNA in subregions of the DS [START_REF] Quinn | Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats[END_REF]. It seems critical to evaluate changes in homogenous structures with few different cell types as there is now accumulating evidence that epigenetic modifications may be specific to particular brain structures and even to cell-types. Even subtypes of medium spiny neurons of the NAc are differently affected by epigenetic modifications [START_REF] Hamilton | Cell-Type-Specific Epigenetic Editing at the Fosb Gene Controls Susceptibility to Social Defeat Stress[END_REF]. Several techniques have been developed to enrich or isolate specific cells from brain tissue, including immunopanning, laser capture micro-dissection, fluorescence-activated (FAC) sorting and magnetically labeled antibodies (see [START_REF] Holt | Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System[END_REF]. The latter technique allows neuronal, astrocytic, and microglia cell populations to be sorted in adult rodent brain at a low cost. Distinguishing between neurons and glial cells, as recently performed using FAC sorting in human samples of heroin addicts, appears now to be critical for a deep analysis of epigenetic mechanisms (see below, §3.2 [START_REF] Kozlenkov | DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper-and Hypomethylation and a Younger Epigenetic Age[END_REF].

Noteworthy, few whole-genome studies directly compared transcriptomic changes occurring across several structures following nicotine and methamphetamine [START_REF] Mychasiuk | Persistent gene expression changes in NAc, mPFC, and OFC associated with previous nicotine or amphetamine exposure[END_REF] or alcohol [START_REF] Mulligan | Genetic divergence in the transcriptional engram of chronic alcohol abuse: A laser-capture RNA-seq study of the mouse mesocorticolimbic system[END_REF] exposure (see Tables 123), but no specific or global epigenetic signatures were established.

2.4-Sex differences

Risk taking is often higher in males, but effects of many drugs appear more deleterious in females. Indeed, anxiety and depression are higher in females and these represent strong predictors of addiction. One study explored epigenetic changes in both sexes for DNAm following ethanol exposure in the human cortex (Wang et al., 2016a). Interestingly, no adaptations could be observed in females. Whether this was due to a low number of female individuals under study or really reflects a sex difference is not clear. Interestingly, the study by Engmann on 3D chromatin conformation showed a specific increase in Auts2 gene only in male mice (see below § 6.1, [START_REF] Engmann | Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2[END_REF]. More studies are needed to explore these aspects to better understand epigenetic mechanisms in the future and clarify how gene expression influences individual heterogeneity in vulnerability to addiction.

3-Changes in DNA methylation and hydroxymethylation

DNA methylation (DNAm) and hydroxymethylation (DNAhm) have been predominantly studied in the mammalian developing brain and described as dynamic processes, as they play a critical role in the establishment and maintenance of cell identity [START_REF] Bogdanovic | DNA methylation and the preservation of cell identity[END_REF]. DNA methylation corresponds to the addition of a methyl group, usually on the 5' position of the carbon of the pyrimidine ring of cytosine, and occurs at dinucleotides CpG and CpH (H=A/C/T) (see Figure 1) [START_REF] Kinde | Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2[END_REF][START_REF] Lister | Global epigenomic reconfiguration during mammalian brain development[END_REF]. This modification (5-mC), when occurring in the promoter region, is mostly associated with transcriptional repression and is catalyzed by DNA methyltransferases involved in the maintenance of methylation (DNMT1) or in de novo methylation (DNMT3 a and b).

Demethylation processes are conducted by ten-eleven translocation enzyme which catalyzes the hydroxylation of the methylated cytosine (5hmC). Other forms are also produced, with 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are processed by DNA repair mechanisms and further generate unmethylated cytosine [START_REF] Auclair | Mechanisms of DNA methylation and demethylation in mammals[END_REF]. In contrast, hydroxymethylation in intragenic regions is associated with higher gene expression [START_REF] Guibert | Functions of DNA methylation and hydroxymethylation in mammalian development[END_REF][START_REF] Kato | Comprehensive DNA methylation and hydroxymethylation analysis in the human brain and its implication in mental disorders[END_REF]. Proteins with methylated DNA binding domain (MBD) play a critical role in DNA methylation, with the protein methyl CpG-binding protein 2 (MeCP2) being one critical reader of DNA methylation in the brain [START_REF] Kinde | Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2[END_REF]. MeCP2 protein level is increased by cocaine and contributes to behavioral responses to psychostimulants [START_REF] Cassel | Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain[END_REF][START_REF] Deng | MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psychostimulants[END_REF][START_REF] Im | MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212[END_REF]. However, additional studies are needed to better understand the implications of this process. Methods for comprehensive analysis of DNA methylation and hydroxymethylation include bisulfite sequencing, or collection of methylated, hydroxymethylated, or unmethylated DNA by specific binding proteins, antibodies, or restriction enzymes, followed by sequencing or microarray analysis. These techniques allow measurement of DNA methylation at a singlebase resolution (see BOX).

3.1-Correlation of DNAm with transcriptional adaptations

The possible correlation of DNAm changes induced by different drugs of abuse and gene expression at a genome-wide scale has been examined in a number of studies. In one, psychostimulant exposure induced differentially methylated regulations which were correlated with gene expression adaptations. In a study examining the effect of cocaine selfadministration, combination of whole-genome and candidate approaches allowed identifying DNAm regulations in the NAc, which were partly negatively correlated with gene expression changes [START_REF] Massart | Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving[END_REF]. Analysis of 5hmC (hMeDIP-Seq) in the NAc following a model of compulsive methamphetamine self-administration showed similar correlation [START_REF] Cadet | Genome-wide DNA hydroxymethylation identifies potassium channels in the nucleus accumbens as discriminators of methamphetamine addiction and abstinence[END_REF]. Hydroxymethylation peak changes were found in intergenic sites while only few modifications were present at transcription start sites, exons and, introns. Interestingly, potassium channel genes with increased DNA hydroxymethylation peaks at intergenic sites were associated with an increased expression of potassium channel coding genes in exposed but non-compulsive rats [START_REF] Cadet | Genome-wide DNA hydroxymethylation identifies potassium channels in the nucleus accumbens as discriminators of methamphetamine addiction and abstinence[END_REF]. In another study using methylome analysis in the PFC following self-administration of cocaine in rats, the authors have revealed that increased DNAm were inversely correlated to transcriptional activation when methylation occurred in the gene promoter. Interestingly, this was not observed when methylation took place inside gene bodies [START_REF] Fonteneau | Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study[END_REF], where a strong variation was observed in the tested genes with both up-or down-regulations associated with hypermethylation. Alternative promoters in gene bodies or the presence of alternative splice sites could explain this observation. Also, similar methylation levels can occur in active and inactive gene bodies across brain tissue [START_REF] Aran | Replication timing-related and gene body-specific methylation of active human genes[END_REF]. A better understanding of these modifications will be provided with a more complete genome annotation and improved description of alternative promotor or splice sites. Others have described more mixed results in a cocaine selfadministration paradigm, with both validation of transcriptional regulation associated with DNAm and non-regulated transcripts [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF]. Whether these correlations are specific to the type of drugs is not clear. Indeed, following alcohol exposure, Wang et al. investigated DNAm changes in PFC postmortem samples from individuals with alcohol use disorders (Wang et al., 2016a). They found 1812 differentially methylated CpGs among which the majority were hypermethylated (1201 CpGs), and preferentially found in promotor regions and gene bodies. When analyzing these data together with previous transcriptomic data from the same samples [START_REF] Zhang | Differentially coexpressed genes in postmortem prefrontal cortex of individuals with alcohol use disorders: influence on alcohol metabolism-related pathways[END_REF], the authors did not observe any correlation between DNAm changes and gene expression. The authors argue that this could be due to single nucleotide polymorphism on DNAm affecting gene expression. A similar finding with no correlation between DNAm and gene expression was obtained in a transgenerational study in rats using a binge alcohol protocol at adolescence (see §5.2, [START_REF] Asimes | Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naive male offspring[END_REF]. One can-not exclude the fact that the complexity of data in genomewide analysis and the accuracy of genome annotation make bioinformatic analysis difficult for such correlations. Also, double analysis of DNAm and hydroxymethylation in future studies may help to clarify these aspects. Together, these data indicate more complex mechanisms for DNAm processes than initially described, which may specifically differ depending on the methylated gene loci or the drug abused.

3.2-Cellular specificity of DNAm

In a global DNA methylation profiling (DNAm microarray) of orbito frontal cortex (OFC) from heroin addicts who died of overdose, the authors investigated heroin effects on DNAm specifically in neuronal nuclei separated by FAC sorting [START_REF] Kozlenkov | DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper-and Hypomethylation and a Younger Epigenetic Age[END_REF]. Hypermethylated regions were preferentially found in exons and gene bodies and mostly depleted from promoter regions, in genes involved in synaptic plasticity. In contrast, hypomethylated regions were preferentially found in promoter regions. To complete their analysis, they performed H3K27Ac ChIP-seq and showed that only hypomethylated regions were enriched in enhancers, at both distal (putative active enhancers) and proximal (active promoters) regions from the transcription start site, while hypermethylated regions were depleted. Investigating modification specifically in neurons reveals enhanced hypermethylated gene regions in genes enriched preferentially in glutamatergic, but not GABAergic, neurons. Altogether, these results are in accordance with reduced glutamatergic transmission in the frontal cortex observed following drug exposure and particularly highlight that DNA methylation changes in neurons are specific to targeted gene regions. Other studies have recently analyzed transcriptomic profiling using RNA-seq in targeted cell-types. These studies were conducted in rodent paradigms with prolonged nicotine exposure, using laser dissected Pomc neurons, known to be involved in nicotine-anorectic effects [START_REF] Silva | Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring[END_REF], or SNc neurons, implicated in Parkinson disease and expressing high levels of nicotinic acetylcholine receptors [START_REF] Henley | Transcriptional regulation by nicotine in dopaminergic neurons[END_REF]. A recent study targeted microglial cells isolated from the prefrontal cortex of alcohol exposed mice, a population of cells involved in the neuroinflammatory response of alcohol [START_REF] Mccarthy | Microglialspecific transcriptome changes following chronic alcohol consumption[END_REF]. The results revealed subtle changes in gene expression following nicotine and a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption. Targeting specific cell-types demonstrated the critical importance of studying DNAm changes in a highly homogenous cell population. Altogether, further studies dissecting cell-type specificity for epigenetic regulations should focus on more precise mechanisms involved in adaptations following drug abuse. Such approaches may encounter difficulties due to limitation in sorting cell-types or in DNA amount needed from specific brain structures. Nevertheless, technical advances are emerging very rapidly with approaches conducted with limited DNA amount or performing single-cell epigenome sequencing [START_REF] Farlik | Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics[END_REF][START_REF] Wen | Single cell epigenome sequencing technologies[END_REF],

which will surely open more precise investigation of these neuroadaptations.

3.3-Dynamics of DNAm changes

Few studies have investigated the dynamics of DNA modifications at the transcriptomic or epigenetic levels. In the NAc of rats self-administering cocaine over 10 days, DNAm changes were examined either a day following the treatment (short) or 3 weeks after (prolonged withdrawal). The results revealed stable changes in DNAm at the two timepoints illustrating a persistent adaptation. Interestingly, several modifications were timedependent, indicating a more dynamic process associated with DNAm in link with behavioral adaptations induced by cocaine [START_REF] Massart | Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving[END_REF]. Using a similar cocaine selfadministration model in rats, others have compared gene expression profiles (RNA microarray) following 1, 10 or 100 days of withdrawal [START_REF] Freeman | Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-administration[END_REF]. Their analysis also revealed complex adaptations, with several categories of time-dependent changes in gene expression in NAc and PFC, including persistent or unstable changes throughout withdrawal.

In an elegant study investigating the effect of cocaine at several steps of dependence in a mouse model of self-administration, the authors questioned the implication of adaptations from simple drug exposure versus learned cocaine-seeking [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF]. The authors used methyl-binding protein immunoprecipitation followed by high throughput sequencing (MBD-seq) to measure DNAm in isolated neurons of mouse PFC. DNAm patterns were compared after acute and prolonged withdrawal or in a situation of relapse. Passive cocaine exposure and cocaine self-administration produced distinct patterns of 5mC enrichment. Persistent methylations were observed across the time-points, mostly hypermethylation, which were embedded within genes or located distal. Hypermethylation induced by cocaine is in line with increased expression of DNMT3A and 3B observed following repeated administration [START_REF] Anier | DNA methylation regulates cocaine-induced behavioral sensitization in mice[END_REF][START_REF] Laplant | Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens[END_REF][START_REF] Pol Bodetto | Cocaine represses protein phosphatase-1Cbeta through DNA methylation and Methyl-CpG Binding Protein-2 recruitment in adult rat brain[END_REF]. Also, some methylation profiles were specific to mice subjected to 3 weeks of abstinence following cocaine self-administration but not in animals following one day of withdrawal or in yoked cocaine controls [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF]. These data highlight the complexity of dynamic changes in DNAm following drug exposure, with implications of changes related to withdrawal or to learning processes with the maintenance of cocainerelated memories. Further studies investigating such time-dependent changes in DNAm are needed to clarify epigenetic adaptations underlying the cognitive adaptations that lead to addictive behaviors. In particular, exploring drug-seeking and relapse represent a major challenge to understanding long-term process observed in human addiction. Whether these adaptations are specific to psychostimulants or share common mechanisms across drugs is still an open question. In addition to methylation, hydroxymethylation also plays a major role in this dynamic process [START_REF] Bachman | 5-Hydroxymethylcytosine is a predominantly stable DNA modification[END_REF], and this adds an additional level of complexity to DNAm mechanisms in the context of SUD.

3.4-DNAm at splicing sites

Recent studies have highlighted that DNAm may occur at specific splicing sites in the gene and therefore not directly impact gene expression levels, but rather the expression of distinct variants. Consequently, this may differentially modulate synaptic plasticity or signaling process and thus have specific phenotypic impacts. In the study by [START_REF] Baker-Andresen | Persistent variations in neuronal DNA methylation following cocaine selfadministration and protracted abstinence in mice[END_REF] (see above), 12 of 15 persistent gene-associated DMRs were located within intronic regions or non-coding loci, whereas principal promoter regions and exons were relatively devoid of changes. These DNAm changes induced by cocaine self-administration were able to modify alternative splicing and therefore expression of isoforms. Therefore, they demonstrated that the absence of an overall change in a gene expression did not exclude alteration of splice variant expression. In Nestler's group, using ChIP and RNA-seq in the NAc of cocaine-treated mice, they established chromatin and transcriptional profiles with the identification, for the first time, of the transcription factor E2F3 as a regulator of cocaineinduced gene expression and alternative splicing [START_REF] Feng | Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens[END_REF]. In a follow up experiment, they assayed the effects of NAc overexpression of E2F3 isoforms on cocaine behavioral response and transcriptomic profiling (RNA-seq) [START_REF] Cates | Transcription Factor E2F3a in Nucleus Accumbens Affects Cocaine Action via Transcription and Alternative Splicing[END_REF]. Interestingly, overexpression of E2F3a was sufficient to mediate cocaine-induced locomotor sensitization and cocaine CPP, and the knockdown prevented these responses. E2F3a overexpression induced differentially expressed genes and differential alternative splicing events in a similar manner as cocaine, and both cocaine and E2F3a overexpression increased E2F3 binding at consensus sequences near alternative splicing sites [START_REF] Cates | Transcription Factor E2F3a in Nucleus Accumbens Affects Cocaine Action via Transcription and Alternative Splicing[END_REF]. These findings reveal a crucial role for E2F3a as a key regulator of cocaine-elicited molecular actions with both transcriptional and splicing profile changes leading to behavioral adaptations to drugs of abuse. In a study analyzing previously published data using an innovative bioinformatics approach to model association between splicing sites and histone marks [START_REF] Feng | Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens[END_REF], Hu et al found two marks (H3K36me3 and H3K4me1) with the strongest association with alternative splicing [START_REF] Hu | Histone posttranslational modifications predict specific alternative exon subtypes in mammalian brain[END_REF], indicating a major role for histone methylation in this process. In another study from the same group, repeated cocaine administration effects on DNA hydroxymethylation (5hmC-seq) in the same brain structure revealed a majority of 5hmC distributed in gene bodies and intergenic regions [START_REF] Feng | Role of Tet1 and 5-hydroxymethylcytosine in cocaine action[END_REF]. Combining these results with analysis of both Chip-seq (H3K27Ac and H3K4Me1) and RNA-seq, the authors observed 24 genes showing an increase in both 5hmC and mRNA 24 hours after the last cocaine injection. They demonstrated that 5hmC dynamic modulation correlated with putative enhancers. Moreover, 5hmC regulations were found at exon boundaries which indicated the involvement of 5hmC at splicing sites. Consequently, hydroxymethylation correlated with splicing isoforms up-regulation. Together, these results indicate that 5hmC alterations represent a mechanism inducing long lasting changes and play a major role in cocaineinduced adaptations. This may potentially be extended to other drugs of abuse.

Interestingly, altered splicing sites have been studied in neurodevelopmental disorders such as schizophrenia or depression. For example, an alternative splicing of GAD1 encoding glutamic acid decarboxylase was altered by DNA methylation at a CpG island close to the putative promoter of GAD1 in cortex and hippocampus of patients with schizophrenia [START_REF] Tao | GAD1 alternative transcripts and DNA methylation in human prefrontal cortex and hippocampus in brain development, schizophrenia[END_REF]. Whether this adaptation was specific to neurons is still not clear. Another study revealed potential alteration of splicing sites for the glutamate ionotropic kainate receptor GRIK2 induced by hypomethylation at intronic sites in isolated astrocytes cells from PFC of individuals who died during a depressive episode [START_REF] Nagy | Astrocytic abnormalities and global DNA methylation patterns in depression and suicide[END_REF]. Taken together, it underlines possible general mechanisms involved in psychiatric disorders, that would be common with addiction.

In summary, a combined analysis of methylome (both methylation and hydroxymethylation) and transcriptome will be informative in assessing splice variants expression and understanding time-dependent methylation changes following drug exposure.

The current studies have highlighted a dynamic process of methylation and hydroxymethylation following psychostimulant exposure. Whether similar conclusion can be proposed for the other drugs of abuse has still not been explored. The human studies are confronted with the constraint of analysis of DNAm at a unique time point in postmortem tissue (Cecil et al., 2016b). Whether extrapolation of data from studies examining DNA methylation in blood samples would be feasible is still questionable.

4-Modifications of Histones

Another epigenetic mechanism that influences chromatin structure and thus the interaction between DNA and histones is the direct occurrence of modifications at the level of the N-terminal part of the histone tail [START_REF] Walker | Regulation of chromatin states by drugs of abuse[END_REF]. For example, acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation modulate in a reversible manner the degree of compaction of the chromatin. Specific enzymes are implicated in these processes to establish the marks (writers) or erase them (erasers). Histone acetyltransferases and histone deacetylases (HDAC) target specific histone residues for acetylation and deacetylation, respectively. Histone methyltransferases and demethylase act in a similar fashion for methyl groups. This represents a dynamic process. Typically, amino-acid acetylation promotes an open chromatin state associated with transcriptional activation.

Histone methylations, usually on Lys and Arg, lead to activation or repression depending on the targeted residue. Methylated Lys 9 of Histone 3 (H3K9me) and H3K27me are associated with transcription repression, while H3K4me enhances transcription. In addition, the location of these modifications, at the promoter or regulatory regions, increases the complexity level of these regulations. These posttranslational mechanisms in response to psychostimulants have been widely studied using candidate gene approaches [START_REF] Rogge | The role of histone acetylation in cocaine-induced neural plasticity and behavior[END_REF][START_REF] Sadri-Vakili | Cocaine triggers epigenetic alterations in the corticostriatal circuit[END_REF][START_REF] Walker | Regulation of chromatin states by drugs of abuse[END_REF]. In addition, new tools allow studying the chromatin accessibility with DNaseI-Seq/ATAC-Seq to profile open chromatin or the spatial organization with chromosome conformation capture (3C)-based techniques (see BOX). Studies on DNAm and hydroxymethylation pointed to modifications at the level of enhancers, suggesting that their activity may be modulated by drugs of abuse by DNA methylation processes. Studying modifications of histones, in particular the ones associated with enhancers, will provide insights into the role of DNA methylation.

4.1-Histone acetylations

Most studies examining histone modifications focused on the effects of passive cocaine exposure (see Table 1). A first study from Nestler's group has examined the role of histone modification in addictive behaviors and demonstrated that chronic cocaine treatment induced histone deacetylase 5 (HDAC5) gene expression in the NAc [START_REF] Renthal | Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli[END_REF]. This enzyme usually represses gene expression by deacetylating histones. Interestingly, overexpression of HDAC5 in the NAc attenuated cocaine CPP while HDAC5 deficient mice showed a behavioral enhancement. This epigenetic enzyme has also been implicated in methamphetamine craving in the DS [START_REF] Li | Role of Dorsal Striatum Histone Deacetylase 5 in Incubation of Methamphetamine Craving[END_REF][START_REF] Li | Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons[END_REF]. The genomic effect of repeated cocaine in the NAc was further studied with chromatin immunoprecipitation coupled with promoter microarray analysis (ChIP-chip) for acetylated and methylated histone [START_REF] Renthal | Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins[END_REF]. Cocaine increased both acetylation and methylation at many more genes than it induced decreases. There was no systematic correlation between chromatin modifications and gene expression. The results also revealed concomitant hyperacetylation of H3 and H4 in very few genes, suggesting that the two marks play distinct roles [START_REF] Renthal | Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins[END_REF]. The authors used ChIP for FosB to identify direct targets for this transcription factor known to be regulated by cocaine [START_REF] Renthal | Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins[END_REF]. They identified a particularly interesting gene family, the sirtuins or Sirt (silent information regulator of transcription) which are histone deacetylases whose function in the nervous system is still not clearly understood. The authors identified a significant enrichment of the transcription factor on the Sirt2 promoter together with an increase of acetylated H3, associated with an increase of Sirt2 transcript in the NAc.

The same finding was obtained for Sirt1. Also, only chronic cocaine was able to increase both Sirt1 and Sirt2 activity in the NAc, compared to a single drug administration. Interestingly, pharmacological activation and inhibition of sirtuins were able to respectively enhance and diminish cocaine CPP as well as electrical excitability of NAc neurons [START_REF] Renthal | Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins[END_REF].

In addition, a dramatic reduction in cocaine self-administration was observed following inhibition of sirtuins. Altogether, these findings implicate sirtuins in regulating behavioral effects of cocaine but whether this effect is due to histone modification or to sirt modulation of signaling process is not fully established. With a similar protocol in mice, a ChIp-seq approach was performed and the authors demonstrated that Sirt1 activation induced the transcription factor forkhead box O3 in the NAc. Interestingly, the overexpression of this factor in the NAc enhanced cocaine CPP [START_REF] Ferguson | SIRT1-FOXO3a regulate cocaine actions in the nucleus accumbens[END_REF]. Altogether, these studies highlighted a role for sirtuins in cocaine-induced adaptations at the molecular and behavioral levels but the precise mechanism involved is still not fully established. Interestingly, Sirt1 overexpression in the NAc increased cocaine or morphine rewarding effects [START_REF] Ferguson | Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action[END_REF], suggesting a role for the sirtuin family across drugs of abuse. Whether similar implication of these HDAC is also observed in self-administration paradigm would be critical to better understand underlying adaptations for long term drug exposure and relapse events. Noticeably, sirtuins have been implicated in depressive-like state, a disorder often associated with SUD [START_REF] Kim | SIRT1 Mediates Depression-Like Behaviors in the Nucleus Accumbens[END_REF].

Interestingly, histone acetylation in the striatum was also observed in a model of methamphetamine exposure using a combination of microarray and ChIP-seq analysis [START_REF] Cadet | Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum[END_REF]. Particularly, methamphetamine caused significant increases in H4K5Ac binding which correlated with levels of gene expression. Many regulated pathways were identified such as cell death and survival and nucleic acid metabolism. As in Martin et al. [START_REF] Martin | Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens[END_REF], the authors found an upregulation of several transcription factors like cFos, Egr1 and Egr2. In addition, their findings revealed that acute and chronic administration of methamphetamine induced differential regulations in striatal gene expression, with more increased gene expression after an acute injection and mostly decreased gene expression following chronic treatment. Also, acute treatment induced additional H4K5Ac binding sites in more genes than in the chronic situation. These observations suggest that epigenetic adaptations to single drug exposure may be distinct from adaptions to long-term exposure, which may have triggered other epigenetic factors to differentially impact gene expression. This highlights potential specific epigenetic modifications in situation of chronic treatment with psychostimulant and, moreover, in conditions involving drug-seeking associated learning process.

4.1-Histone methylations

Histone methylations seem to play a major role in cocaine effects, with activation or repression of gene expression depending on location and number of methyl groups involved.

Nestler's group investigated the relationship between two histone lysine dimethyltransferase G9a and GLP (G9a-like protein) and cocaine treatment. The authors observed a down regulation of both proteins in the NAc, induced by chronic cocaine exposure, which was associated with a decrease of the repressive mark H3K9me2 [START_REF] Maze | Essential role of the histone methyltransferase G9a in cocaine-induced plasticity[END_REF]. Using genetic animal model and viral approaches, the authors demonstrated that G9a down regulation was able to modify neuron morphology in the NAc and enhance preference for cocaine. However, G9a overexpression in the NAc shell of rats increased H3K9me2, which surprisingly enhanced motivation for cocaine self-administration associated with increased anxiety [START_REF] Anderson | Overexpression of the Histone Dimethyltransferase G9a in Nucleus Accumbens Shell Increases Cocaine Self-Administration, Stress-Induced Reinstatement, and Anxiety[END_REF]. Repeated cocaine decreased both H3K9me2 and G9a in the NAc of mice and, infusions of an HDAC inhibitor increased global levels of histone acetylation but also, of repressive histone methylation and G9A expression, illustrating cross talk among different types of histone modifications in the adult brain [START_REF] Kennedy | Class I HDAC inhibition blocks cocaine-induced plasticity by targeted changes in histone methylation[END_REF].

Also, in a study modulating G9a expression specifically in Drd1 or Drd2 expressing neurons using genetic models or overexpression, distinct behavioral effects in response to cocaine were highlighted [START_REF] Maze | G9a influences neuronal subtype specification in striatum[END_REF]. In particular, selective deletion of G9a in Drd2 neurons resulted in the unsilencing of transcriptional programs normally specific to Drd1 neurons, coupled with acquisition of Drd1-associated projection and electrophysiological properties.

Therefore, the authors proposed a new role for G9a in contributing to neuronal subtype identity. In another study from the same group, H3K9me3 was shown to be altered by repeated cocaine in the NAc [START_REF] Maze | Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens[END_REF]. ChIp-seq analysis revealed that H3K9me3 was predominantly within intergenic regions and at repetitive genomic sequences. Similar finding was described following morphine treatment, with a specific decrease of G9a associated with diminished levels of H3K9me2 in the NAc mostly in intergenic regions and repetitive sequences [START_REF] Sun | Morphine epigenomically regulates behavior through alterations in histone H3 lysine 9 dimethylation in the nucleus accumbens[END_REF]. A ChIP-seq analysis was performed to further identify targets of G9a and, data confirmed previously described genes associated with morphine effects as well as novel targets, including glutamatergic signaling genes like Grin2a, Grip1, Grm5, and Grm8. Together, these findings suggest a critical role for histone methyltransferase, whose control of basal patterns of gene expression is altered by exposure to cocaine and morphine.

Interestingly, a recent study investigated the role of histone Arg (R) methylation in cocaine action and showed a decreased expression of protein-R-methyltransferase-6 (PRMT6) and its associated histone mark (H3R2me2a) in the NAc of mice or rats exposed to cocaine or in human addicts [START_REF] Damez-Werno | Histone arginine methylation in cocaine action in the nucleus accumbens[END_REF]. This down-regulation is specifically observed in Drd2 expressing neurons, and opposite in Drd1. Using ChIP-seq, they identified Src kinase signaling inhibitor 1 (Srcin1) as a target for reduced H3R2me2 binding, highlighting the effect of cocaine on another histone mark.

Altogether, histone acetylation and methylation are playing an important role in drug adaptations in the NAc, however, more studies are necessary to propose a molecular mechanism for such adaptations, particularly with distinct drugs of abuse.

5-Epigenetic transmission

Exposure to drugs of abuse during a sensitive period for brain development may be critical for epigenetic changes as they could be exacerbated later on in the adulthood or even become transgenerational [START_REF] Bale | Epigenetic and transgenerational reprogramming of brain development[END_REF]. Such studies are highlighted in the tables (see * in Tables) and some of them discussed below.

5.1-Gestational exposure

Genome-wide approaches have revealed that in utero drug exposure could induce epigenetic modifications. Most of these focus on nicotine effects as numerous studies showed fetal brain development alterations due to maternal smoking [START_REF] Banderali | Short and long term health effects of parental tobacco smoking during pregnancy and lactation: a descriptive review[END_REF].

Transcriptomic analysis was performed on offspring cortical samples from mice exposed to nicotine (200µg/ml) from parental mating to weaning, followed by a three-month withdrawal [START_REF] Jung | An epigenetic mechanism mediates developmental nicotine effects on neuronal structure and behavior[END_REF]. The authors identified Ash21, a gene involved in histone methylation, as the most up-regulated transcripts by nicotine exposure during development. They further examined genome-wide changes in H3K4me3 by ChIP-seq analysis and identified most alterations induced by developmental nicotine exposure at promoter regions of genes involved in glutamate neurotransmission. Interestingly, knockdown of Ash2l abolished nicotinemediated alterations of dendritic complexity and decreased nicotine-dependent changes in passive avoidance behavior. These data highlighted Ash2l, forming a complex together with the transcription factor Mef2c, as critical targets for nicotine to alter neuron morphology and alter persistent behavior during development. Altogether, these genome-wide approaches point to a novel mechanism of gene regulation during brain development involving H3K4me3 epigenetic mark.

In a study examining the impact of e-cigarette smoke exposure during early life development (e-cigarette aerosol, 3 h/day; 5 day/week until postnatal day 4-6, with or without 13-16 mg/ml nicotine), the authors analyzed gene expression alterations using RNA-seq in the mouse offspring frontal cortex [START_REF] Lauterstein | Frontal Cortex Transcriptome Analysis of Mice Exposed to Electronic Cigarettes During Early Life Stages[END_REF]. They described pathways with common gene networks related to cancer, organismal injury and abnormalities, and gastrointestinal disease. Interestingly, sex-dependent transcriptional changes were observed following aerosol exposure, with or without nicotine. In another study examining maternal nicotine exposure (nicotine in drinking water during gestation, 200µg/ml), the authors examined transcriptional profiling (RNA-seq) on laser dissected Pomc neurons from the offspring [START_REF] Silva | Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring[END_REF]. This approach revealed one consistent change, the upregulation of Gm15851, a lncRNA of yet unidentified function. In a global DNA methylation profiling (DNAm microarray) of human dorsolateral prefrontal cortex of nicotine exposed fetuses, the authors investigated the role of DNAm in adaptations to nicotine effects and gestational age [START_REF] Chatterton | In utero exposure to maternal smoking is associated with DNA methylation alterations and reduced neuronal content in the developing fetal brain[END_REF]. Two hypomethylated regions within SDHAP3 (involved in mitochondrial membrane function) and GNA15 (encodes a G protein subunit) promoters have been identified specifically in exposed -fetuses. Interestingly, the authors observed an increase of SDHAP3 transcript only in exposed -fetuses between early and late development. A similar finding was noted for GNA15, with a larger increase in exposed versus unexposed -fetuses. These data suggest that maternal nicotine exposure induces both gene expression and DNA methylation modifications during cortical development [START_REF] Chatterton | In utero exposure to maternal smoking is associated with DNA methylation alterations and reduced neuronal content in the developing fetal brain[END_REF]. Interestingly, modification of the differentially methylated region (DMR) levels of these two genes has been also observed in autism and schizophrenia studies in cerebellum and cortical areas [START_REF] Ladd-Acosta | Common DNA methylation alterations in multiple brain regions in autism[END_REF][START_REF] Nardone | DNA methylation analysis of the autistic brain reveals multiple dysregulated biological pathways[END_REF][START_REF] Wockner | Brain-specific epigenetic markers of schizophrenia[END_REF]. Altogether, this highlight a link between DNA methylation and neurodevelopmental diseases, and such implication may also apply to SUD.

Prenatal ethanol effects have been examined in rats using RNA microarray [START_REF] Middleton | Molecular substrates of social avoidance seen following prenatal ethanol exposure and its reversal by social enrichment[END_REF] or RNA-seq [START_REF] Ignacio | Effects of Acute Prenatal Exposure to Ethanol on microRNA Expression are Ameliorated by Social Enrichment[END_REF], and specific genes or miRNA have been identified in amygdala and NAc. Interestingly, these regulations involving signaling pathways could participate in the social motivation deficits seen in adolescent rats produced by prenatal ethanol, an effect that was reversed by a social enrichment in the offspring.

Another study using genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and 3 miRNAs in a gestational ethanol-exposure model [START_REF] Marjonen | Early maternal alcohol consumption alters hippocampal DNA methylation, gene expression and volume in a mouse model[END_REF], including MiR138-2 also detected in the previous study, but with an opposite regulation. These distinct results may reflect differences in duration and timing of exposure. Interestingly, the authors identified a Histone cluster 1 H2ai, which was down regulated by ethanol exposure, indicative of altered-chromatin conformation by alcohol, across generation.

5.2-Transgenerational inheritance

Transgenerational inheritance, reflecting transmission to unexposed offspring, has also been recently explored in the addiction field. Initial studies investigated candidate genes following alcohol, morphine or cocaine exposure. For example, Pomc gene hypermethylation caused by fetal alcohol exposure was transmitted to offspring through male germline [START_REF] Govorko | Male germline transmits fetal alcohol adverse effect on hypothalamic proopiomelanocortin gene across generations[END_REF]. Analysis of the progeny of adolescent female rats exposed to morphine showed an increase of D2 and kappa opioid receptor expression in two generations of mice, suggesting epigenetic adaptations [START_REF] Byrnes | Multigenerational effects of adolescent morphine exposure on dopamine D2 receptor function[END_REF]. In another study, reduced cocaine intake was observed specifically in male offspring of cocaine-experienced males, coupled with an increased association of acetylated histone H3 (H3K9K14ac2) with BDNF promoters [START_REF] Vassoler | Epigenetic inheritance of a cocaine-resistance phenotype[END_REF]. This epigenetic adaptation was associated with increased baseline anxiety in these animals, which was unaltered by subsequent cocaine exposure [START_REF] White | Enhanced anxiety in the male offspring of sires that self-administered cocaine[END_REF].

More recent studies explored epigenetic inheritance using genome-wide approaches to better investigate molecular targets or pathways modulated in progeny. In order to evaluate the impact of smoking marijuana on subsequent offspring, one study examined the effects of parental THC exposure (during rat adolescence) on DNA methylation in the NAc of adult F1 unexposed rats using a bisulfite-sequencing approach. The authors identified 406 hypermethylated and 621 hypomethylated DMRs across the genome. Interestingly, the majority of DMRs were detected in gene bodies and downstream of transcriptional start sites, particularly within genes involved in the glutamatergic synaptic regulation [START_REF] Watson | Genome-Wide DNA Methylation Profiling Reveals Epigenetic Changes in the Rat Nucleus Accumbens Associated With Cross-Generational Effects of Adolescent THC Exposure[END_REF]. The DMR-associated genes exhibited altered mRNA expression in the NAc. A gene network centered on Dlg4 coding for PSD95, a scaffolding protein involved in synaptic plasticity was identified, together with genes involved in glutamatergic neurotransmission.

Dlg4 gene is regulated by epigenetic factors in aging and neurodegenerative diseases like Alzheimer's disease, Huntington's disease and schizophrenia [START_REF] Bustos | Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice[END_REF]. Notably, PSD95 plays a major role in the formation of synapses during specific time points of neurodevelopment and regulates synaptic function that influences behavioral phenotypes in SCZ [START_REF] Coley | PSD95: A synaptic protein implicated in schizophrenia or autism?[END_REF]. Alteration of DNAm of Dlg4 by parental THC exposure could therefore be associated with an increased risk of schizophrenia in cannabis consumers [START_REF] Hudson | Phytocannabinoids modulate emotional memory processing through interactions with the ventral hippocampus and mesolimbic dopamine system: implications for neuropsychiatric pathology[END_REF]. In a similar approach, other authors have analyzed the effect of parental methamphetamine exposure on hippocampal samples of offspring using DNAm microarray targeting annotated CpG Islands and promoter regions [START_REF] Itzhak | Long-term parental methamphetamine exposure of mice influences behavior and hippocampal DNA methylation of the offspring[END_REF]. 545 CpG islands and 156 promoter region hypermethylated DMRs induced by in utero methamphetamine exposure have been identified. Gene ontology analysis in the DMR highlighted "cerebral cortex GABAergic interneuron differentiation" for hypermethylated DMRs and "embryonic development" for hypomethylated ones. These results suggested that the observed phenotypes in F1 generation, including enhanced response to cocaine-conditioned reward and hyperlocomotion, and reduced fear conditioning, could be the result of abnormal brain development.

Another study investigated modification of DNAm profile (Bis-Seq) in hypothalamus of unexposed offspring of rats, whose parents were exposed using a binge alcohol protocol at adolescence [START_REF] Asimes | Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naive male offspring[END_REF]. The authors showed altered epigenetic modifications in these naïve rats which varied depending on parental exposure. Indeed, differentially methylated cytosines were distinct between offspring, depending on which parent was exposed to ethanol. In addition, the highest number of hypermethylated and hypomethylated regions identified was observed when both parents were exposed to ethanol. Finally, less is known about morphine exposure on offspring and only one study described transcriptomic adaptations (RNA-seq) using maternal morphine exposure [START_REF] Vassoler | Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure[END_REF]. The authors mainly observed altered gene expression in the NAc for genes specifically involved in synaptic plasticity and neural development associated with a higher resistance to selfadminister morphine in the offspring.

In summary, all these studies highlight that prenatal exposure to drugs of abuse alters neural development and synaptic plasticity, as well as phenotypic behavioral responses. The transmission to the offspring seems to involve epigenetic modifications, but the mechanistic process of these regulations at the chromatin level still needs to be further addressed, including with comparison to transcriptomic profiling. Investigation of the persistence of these regulations by examining regulations in subsequent generations (F2 and F3) would be helpful for a more comprehensive interpretation of these mechanisms. Human studies have examined regulation in blood samples (Cecil et al., 2016a;[START_REF] Markunas | Identification of DNA methylation changes in newborns related to maternal smoking during pregnancy[END_REF] for alcohol or nicotine early exposure and highlighted epigenetic changes but direct correlations with modifications occurring in brain structures may be difficult to establish.

6-Developping tools for neuroepigenetic studies

6.1-Chromatin 3D structure and psychostimulant addiction

More recent studies have used sophisticated approaches to decipher the architecture of the chromatin in response to drugs of abuse. One study raised the question of the alteration of 3D architecture of chromatin induced by cocaine treatment, at genes showing epigenetic changes [START_REF] Engmann | Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2[END_REF]. Using FAC sorting and viral mediated approaches, the authors found that Auts2, a gene linked to cognitive disorders, was enriched in Drd2 medium spiny neurons in mice. An increased expression was also detected in cocaine-addict postmortem NAc samples. In mouse NAc, using chromosome conformation capture-on-chip (4Cseq) investigating one specific locus vs all loci, the authors found that chromatin looping connected Auts2 predominantly with a brain-specific calcium binding gene (Caln1) in basal conditions, an interaction that was disrupted following chronic cocaine treatment. Using bisulfite sequencing, they showed increased DNAm at the vicinity of Auts2 interaction site. They further manipulated DNAm using CRISPR-genome editing in Neuro2a cells, targeting DNMT activity at the interaction site between Auts2 and Caln1. This brought the evidence that DNAm alteration on Auts2 gene could modify gene expression of a distant chromatin interaction partner, Caln1 [START_REF] Engmann | Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2[END_REF]. Even if this was shown in vitro due to current technical limitation in vivo, it demonstrated a causality between interaction and expression. Future technical improvement will certainly allow testing this mechanism directly in brain structures. These new findings provide novel insight for explaining the impact of epigenetic modifications on gene expression. Modification of expression in response to drugs may involve alteration of the 3D structure of chromatin at gene regulatory regions.

6.2-Epigenetic-based therapeutic interventions

Epigenetic modifications have been proposed as potential biomarkers for SUD (Cecil et al., 2016b). DNAm patterns in human blood cells may be used to identify vulnerability towards addiction in individuals. Hypermethylation in the 3'-protein-phosphatase-1G gene locus has been associated with alcohol use disorder [START_REF] Ruggeri | Association of Protein Phosphatase PPM1G With Alcohol Use Disorder and Brain Activity During Behavioral Control in a Genome-Wide Methylation Analysis[END_REF]. But whether these modifications are specific to blood cells or to a particular drug is still not clear.

Epigenetic regulations may explain the maintenance of long-term adaptation and a better understanding of the mechanisms across drugs of abuse will help highlight targets for relapse treatment. Manipulation of histone acetylation levels using pharmacological inhibitors of HDACs was assessed in preclinical cocaine studies. Such HDAC inhibitors reduced cocaine intake and motivation in self-administering rats while no effect of the inhibitor was observed on sucrose intake [START_REF] Romieu | Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats[END_REF]. Reduced reinstatement of cocaine-seeking behavior was also observed following 3 weeks of abstinence [START_REF] Romieu | The inhibition of histone deacetylases reduces the reinstatement of cocaine-seeking behavior in rats[END_REF]. Extinction of cocaine-induced CPP was accelerated following a similar treatment [START_REF] Malvaez | Modulation of chromatin modification facilitates extinction of cocaine-induced conditioned place preference[END_REF].

In contrast, opposite results were obtained in other studies on cocaine intake [START_REF] Sun | The effects of sodium butyrate, an inhibitor of histone deacetylase, on the cocaine-and sucrose-maintained selfadministration in rats[END_REF] and cocaine-induced hyperlocomotion [START_REF] Kumar | Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum[END_REF]. These results point to the importance of the specificity of these inhibitors or the type of treatment used, that may explain distinct effects on addictive responses. Therefore, the need for selective inhibitors of HDAC and controlled paradigms seem crucial to specifically target subtypes of HDAC in therapeutic approaches. Also, differential regulations were observed, depending on the targeted brain structure or the type of drug, as illustrated for HDAC5 with distinct regulations following methamphetamine in DS compared to cocaine in NAc (see above [START_REF] Li | Role of Dorsal Striatum Histone Deacetylase 5 in Incubation of Methamphetamine Craving[END_REF].

These observations reveal the complexity of such therapeutic strategies. Another approach could be to target histone acetyltransferase. These enzymes, like CBP, play a role in cocaine action in the NAc [START_REF] Malvaez | CBP in the nucleus accumbens regulates cocaine-induced histone acetylation and is critical for cocaine-associated behaviors[END_REF] but no study has evaluated their potential efficacy in drug-induced responses. Activation of CBP has recently been shown to reverse deficits in memory processes and promote neurogenesis [START_REF] Chatterjee | A novel activator of CBP/p300 acetyltransferases promotes neurogenesis and extends memory duration in adult mice[END_REF]. Effects of activation of histone acetyltransferase on drug-induced phenotypes could be proposed as a therapeutic approach.

Several tools are currently available to edit epigenetic factors, like ZFP, TALENs and CRISPR (reviewed in [START_REF] Waryah | Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing[END_REF]. Such approaches provide accurate modification of a given type of epigenetic marks at a precise gene locus, allowing subtle changes to be detected. In vivo, such tools are able to reverse memory deficits in a mouse model of Alzheimer's disease [START_REF] Bustos | Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice[END_REF]. In addictive disorders, one recent study by Nestler's group examined the role of cocaine-induced histone modifications at the gene locus for cyclin-dependent kinase 5 cdk5, a critical gene in reward related behaviors [START_REF] Heller | Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine-and Stress-Evoked Behavior[END_REF]. The authors used zinc finger proteins-genome editing targeting H3K9/14ac, an active mark or H3K9me2, a repressive mark, in vivo and demonstrated a causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression as well as in reward and stress behaviors. Their results revealed novel indicators of a role of this gene in addictive behaviors and, highlighted that targeted epigenetic remodeling approaches represent useful tools for studying molecular changes involved in addictive responses. Neuroepigenetic editing thus represents an emerging tool that will be developed to further investigate cell-type and gene specific adaptations.

7-Concluding remarks

In summary, genome-wide studies have highlighted the implication of several types of neuroepigenetic modifications associated with drugs of abuse, which may have an impact on addictive behaviors. Histone acetylation and methylation are clearly altered by psychostimulants. DNAm and hydroxymethylation represent dynamic mechanisms that may affect enhancers, promoters, splice-sites with different outcome on gene expression. Further, analysis of 3D structure of chromatin brings new insight into the mechanism by which drugs alter gene expression. Combining chromatin architecture analyses with epigenetic and transcriptomic analyses is crucial for a better understanding of these adaptations.

BOX

Transcriptomic profiling (see [START_REF] Hitzemann | Genes, behavior and next-generation RNA sequencing[END_REF][START_REF] Wang | RNA-Seq: a revolutionary tool for transcriptomics[END_REF] RT-qPCR, gene expression analysis for candidate genes RNA microarrays, initial genome-wide gene expression profiling using oligonucleotide arrays and hybridization-based approaches. RNA-Seq, a high throughput sequence-based method. RNA are converted into libraries of cDNA and each molecule is further sequenced with read between 30 to 400 bp. Reads are then aligned to a reference genome. It allows to analyze alternative splicing , the expression of coding and non-coding RNAs with a higher base-pair resolution DNA methylation profiling see [START_REF] Kurdyukov | DNA Methylation Analysis: Choosing the Right Method[END_REF] Whole-genome methylation, approaches to quantify the amount of deoxycytidine (dC) and methylated cytosines (5mC) in a hydrolysed DNA sample using high performance liquid chromatography (ultraviolet), liquid chromatography coupled with mass spectrometry, pyrosequencing LINE-1. These methods do no offer a precise quantification of methylation levels. Bisulfite sequencing, conversion by bisulfite of cytosine into uracil while 5-methylcytosines are not affected. Following conversion, either whole genome bisulfite sequencing or reduced representation bisulfite sequencing where CpG regions are enriched can be processed. Methylcytosine binding proteins (MBD) and antibodies targeting 5mC (MeDIP), approaches allowing enrichment of differentially methylated regions (DMR), can be performed before bisulfite treatment. DNAm microarrays, following bisulfite conversion, allows using specific microarrays to interrogate regionmethylation levels such as promoters or gene bodies. DNA digestion, particular endonucleases are able to digest CCGG sequence to analyze either whole-genome or specific gene methylation levels.

Chromatin profiling see [START_REF] Park | ChIP-seq: advantages and challenges of a maturing technology[END_REF] ChIp, Chromatin immunoprecipitation, state of the art technique to study chromatin alterations such as DNAbinding proteins, histone modifications and nucleosomes alterations. qChIP, investigats a single locus by qPCR following chromatin immunoprecipitation. ChIP-ChIP, interrogates selected-region modifications using DNA microarrays. ChIP-Seq, whole-genome sequencing following chromatin precipitation with high base-pair resolution. DamID-Seq, indentification of DNA adenine methyltransferase without immunoprecipitation step, based on a fusion protein and E. coli DNA adenine methyltransferase; limited to in vitro models. ATAC-Seq / DNA-Seq, addresses chromatin accessibility, providing similar information to RNA-seq but focusing on DNA; used for profiling enhancer regions.

Chromosome conformation capture (see [START_REF] Davies | How best to identify chromosomal interactions: a comparison of approaches[END_REF][START_REF] Dekker | The 4D nucleome project[END_REF] 3C, analysis of interactions between a single pair of genomic loci. 4C, identification of interactions between one locus and all other genomic loci. 5C, study of functional contacts between all the genes within a given region. Hi-C, identification of functional contacts through all parts of the genome. Imaging techniques, mapping of chromatin interactions.

Figure legends Figure 1 .

 legends1 Figure legendsFigure1. A) Histone proteins (H2A, H2B, H3 and H4) form an octamer with two copies of each, which is wrapped around 147bp of DNA to form a functional unit of chromatin, the nucleosome. Modifications such as methylation (Me), bimethylation (2Me) , trimethylation (3Me) or acetylation (Ac) can alter the N-terminal tails of these histones (here, H3 is depicted). These modifications lead to either an open (enable gene expression) or close chromatin state (repress gene expression). B) Addition of a methyl group to a cytosine (Me, position 5) can alter the DNA segment activity. When a promoter is highly methylated, this modification often leads to decrease gene expression. The methyl groups can be oxidized by the ten-eleven translocation enzymes family to form a hydroxymethyl group (hMe).

Figure 2 .

 2 Figure 2. Representative scheme illustrating some critical factors for the analysis of neuroepigenetic modifications by drugs of abuse (cocaine, MDMA, alcohol, cannabinoids and opiates). Species, sex and age of the individuals can modify observed effects. Several paradigms of drug administration are classically used, with distinct timing (acute, chronic, abstinent) to study direct drug effects or relapse, various modes of administration (oral, vapor, intraperitoneal or intravenous), with passive or voluntary access to the drugs (choice or operant system). They all enable to study addiction-related behavioral responses. The genome-wide analysis can be performed on whole brain or microdissected samples to focus on specific brain structures. Recent technologies allow now to lower cellular heterogeneity and focus on specific cell types. A: acute, C: chronic, W: withdrawal.

Table 1 :

 1 Whole-genome epigenetic studies following psychostimulant exposure?

	Analysis	Species	Administration paradigm	Dose	Region	Reference

Table 2 :

 2 Whole-genome epigenetic studies following alcohol exposure : transgenerational study; A: acute; C: chronic; W: withdrawal; E: extinction; R: reinstatement; e.o.d: every other day; i.p: intraperitoneal; i.v: intravenous; d: day; y: year; PD: postnatal day; GD: gestational day; CeA: central amygdala)

	Analysis	Species	Administration	Dose	Region	Reference
			paradigm

*

Table 3 :

 3 Whole-genome epigenetic studies following opiate exposure (*: transgenerational study; A: acute; C: chronic; W: withdrawal; E: extinction; R: reinstatement; e.o.d: every other day; i.p: intraperitoneal; i.v: intravenous; d: day; y: year;)

		Analysis Species Administration	Dose	Region	Reference
				paradigm
		RNA	Mouse	A, i.p	20mg/kg	Striatum (Korostynski
		microarray		C, 5d ; 3/d i.p	10,20,40mg/kg		et al., 2007)
		RNA	Mouse	C, 5d s.c	25mg pellet	NAc	(Grice et al.,
	Morphine	microarray RNA microarray RNA microarray	Mouse Mouse	C, i.p 6d S, 6h s.c C, 4d s.c	20-100mg/kg 25mg pellet	Extended amygdala Hypo	2007) (Befort et al., 2008) (Anghel et al., 2010)
		* RNA-seq Rat	C, 21d i.v E, 10d	0.25,0.75,1.25mg/kg	NAc	(Vassoler et al., 2017)
				R, i.p	1 mg/kg
		* DNAm microarray Human			DLPFC	(Chatterton et al., 2017)
	Heroine	DNAm microarray RNA microarray RNA	Human Human Rat	C, 7d i.p	1mg/kg	OFC NAc NAc	(Kozlenkov et al., 2017) (Egervari et al., 2017) (Yan et al.,
		microarray					2017)
		RNA-seq	Rat	C, 19d i.v	0.06mg/kg-19d	NAc	(Imperio et
				E,1d			al., 2016)

Table 4 :

 4 Whole-genome epigenetic studies following cannabinoid or nicotine exposure (*: transgenerational study; A: acute; C: chronic; W: withdrawal; E: extinction; R: reinstatement; e.o.d: every other day; i.p: intraperitoneal; i.v: intravenous; d: day; y: year; PD: postnatal day)

			Analysis	Species Administration	Dose	Region	Reference
					paradigm
	THC	*	Bis-seq RNA microarray	Rat Mouse	C, PD28 to PD49 e.o.3d i.p C, 4.5d i.p	1.5mg/kg 10mg/kg	NAc Cerebellum	(Watson et al., 2015) (Colombo et al., 2009)
		*	RNA microarray	Mouse	C, 21d oral W, 90d	200µg/ml	Cerebral cortex	(Jung et al., 2016)
			RNA-seq	Human			DLFC	(Tao et al., 2017)
	Nicotine	RNA-seq * RNA-seq Mouse C, 5d by Mouse C, 14d s.c inhalation	2mg/kg/h mini osmotic pumps 13-16mg/ml	SNc neurons FC	(Henley et al., 2013) (Lauterstein et al., 2016)
		* RNA-seq Mouse C, 28d oral 200µg/ml	Pomc neurons	(Silva et al., 2016)
			RNA-seq	Rat	C, 14d i.p	0.3mg/kg	PFC, OFC, NAc	(Mychasiuk et al., 2013)

Acknowledgments

We thank Dr D Massotte and Dr MC Olmstead for fruitful discussions and constant support. We would like to thank the "Centre National de la Recherche Scientifique", and the "Université de Strasbourg".


What is gained or "lost in translation" in Huntington's disease

Emmanuel Brouillet 1,2 and Karine Merienne 3 1 CEA, DRF, Institut de Biology François Jacob, Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France 2 CNRS, CEA, Paris-Sud Univ., Univ. Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265, Fontenay-aux-Roses, France Robert Frost, the famous American poet , defined poetry as what is "lost in translation".

But there is also complexity, and indeed poetry, in the process of "translation" in biology. Translation, through its subprocesses of initiation, elongation and termination, allows a cell to read the genetic code to synthesize proteins. Even in healthy cells, approximately 30% of newly synthetized polypeptides are lost during translation as a result of mistakes (defective ribosomal products -DriPs).

This suggests that translation is tightly controlled and that failure rates are likely to be even higher in diseased cells [START_REF] Alvarez-Castelao | A critical appraisal of quantitative studies of protein degradation in the framework of cellular proteostasis[END_REF]. In neurons, most proteins are produced in the rough endoplasmic reticulum and the soma. However, translation also occurs in the dendrites and the axon to allow neurons to swiftly adapt to changes in demand or in local energy requirements. It is thus conceivable that defects in translation may contribute to neurodegeneration, as has recently been shown for genetic forms of Parkinson's disease associated with LRRK2 mutations [START_REF] Martin | Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease[END_REF].

In this issue of Brain, Creus-Muncunill and co-workers present data suggesting that disruption of translation may also contribute to pathology in Huntington's disease [START_REF] Creus-Muncunill | Increased translation as a novel pathogenic mechanisms in Huntington's disease[END_REF]).

Huntington's disease is an inherited neurodegenerative disorder caused by mutation of the huntingtin gene. Expansion of a triplet CAG repeat leads to expansion of a polyglutamine tract in the huntingtin protein (HTT). This leads to early loss of striatal projection neurons. Mutant huntingtin (mHTT) is neurotoxic to neurons and astrocytes, through gain-and loss-of-function mechanisms affecting many cellular functions [START_REF] Saudou | The Biology of Huntingtin[END_REF]. Creus-Muncunill et al. discovered that levels of 4E-BP1, a protein that inhibits translation, are reduced in a mouse model of Huntington's disease. 4E-BP1 is also hyper-phosphorylated, which prevents it from interacting with the regulatory protein eIF4E. This suggests that translation may be activated inappropriately in Huntington's disease, and indeed, Creus-Muncunill et al. found an increase in translation in the brain of patients post mortem. The group, led by Esther Pérez-Navarro, also used mass spectrometry to obtain a global view of several hundred proteins with altered expression in Huntington's disease mice. While levels of proteins related to ribosomal function and energy production were increased, there was decreased translation of proteins related to neuronal function. Pharmacological blockade of translation using 4EGI-1, a selective inhibitor of 4E-BP1, restored ribosomal and mitochondrial proteins to control levels in Huntington's disease mice. Furthermore, injecting 4EGI-1 into the brain improved motor signs in the mice.

But as with many discoveries, these findings raise more questions than they provide definitive answers.

Is an increase in translation "logical" in neurodegeneration? At first sight, it might appear to be a sensible reaction of cells that are under stress. And indeed, the proteins that show increased translation in Huntington's disease are linked to regulation of translation and energy metabolism.

Thus, increasing their levels could in principle make cells healthier. But this is not the case, as blocking translation with 4EGI-1 is neuroprotective. Transcriptomic data related to Huntington's disease have not shown major dysregulation of genes controlling translation [START_REF] Seredenina | What have we learned from gene expression profiles in Huntington's disease?[END_REF]. Increased translation seems to be linked to increased phosphorylation of 4E-BP1. This may result from increased activity of a kinase and/or a decreased activity of a phosphate both of which remains to be identified.

Moreover, the increase in translation does not affect all striatal proteins indiscriminately. Levels of proteins preferentially expressed by striatal projection neurons (e.g. DARPP32) are down-regulated in Huntington's disease, and do not recover under 4EGI-1 treatment. This is consistent with known reductions in mRNA levels of striatal identity genes [START_REF] Seredenina | What have we learned from gene expression profiles in Huntington's disease?[END_REF]. For these genes, transcription and translation are both reduced. Another intriguing question is how can mHTT have different effects on translation depending on a gene's function? One possibility is that specific molecular machinery might control the translation of different types of genes, with mutant HTT having differential effects on these machineries. This issue remains to be resolved. Cellular power sources (i.e. mitochondria and glycolysis) and translation machinery have evolved simultaneously and are remarkably conserved in eukaryotes, likely because the energy cost of translation is extremely high [START_REF] Alvarez-Castelao | A critical appraisal of quantitative studies of protein degradation in the framework of cellular proteostasis[END_REF]. Are translation and energy metabolism "in tune" in Huntington's disease? Increased translation should increase cellular demand for ATP, and Creus-Muncunill et al. identified a consistent increase in translation of proteins related to energy metabolism. But why is this increase in translation detrimental? Energy metabolism and production of reactive oxygen species are abnormal in Huntington's disease [START_REF] Liot | Energy defects in Huntington's disease: Why "in vivo" evidence matters[END_REF]. Thus it is conceivable that increased expression of proteins related to energy production may augment oxidative stress in unhealthy cells.

The mechanisms underlying the improvement in motor function in Huntington's disease mice treated with 4EGI-1 remain to be fully elucidated. 4EGI-1 does not change the number of mHTT-containing aggregates, indicating no direct effect on mHTT aggregation. 4EGI-1 may act through unexpected targets, as shown for cycloheximide which blocks apoptosis by mechanisms other than just translation inhibition [START_REF] Chow | Reevaluation of the role of de novo protein synthesis in rat thymocyte apoptosis[END_REF]. The improved motor function of Huntington's disease mice receiving 4EGI-1 could also result from changes in cell signalling. In the data from Creus-Muncunill et al., it is quite remarkable to see that 20% of the down-regulated proteins in R6/1 mice are kinases (20%) while 10% are GTPases, including Rab proteins. Restoring expression of these proteins with 4EGI-1 may also be protective, improving cell signalling and trafficking of vesicles.

The results with 4EGI-1 provide a convincing proof-of-concept of the potential value of targeting translation. Inhibition of translation is also being explored in cancer therapy [START_REF] Blagden | The biological and therapeutic relevance of mRNA translation in cancer[END_REF].

3

  CNRS/Strasbourg University UMR 7364, Laboratory of Adaptive and Cognitive Neuroscience (LNCA), Strasbourg F-67000, France

For

  Huntington's disease, a selective molecular tool that restores expression of ribosomal and energyrelated proteins could be a good option. Creus-Muncunill et al. illustrate perfectly how basic research feeds into medicine by laying the foundations for translational research. The value of basic research is too often neglected by local and governmental funding agencies that exclusively favour applied approaches. The origins of research into translation can be traced back to the beginning of the 20 th century. Since that time, many basic scientists, including more than 20 Nobel prize winners, have contributed to our knowledge of translation. Thanks to this work, "translational" research on translation in Huntington's disease can finally begin.

Figure 1

 1 Figure1The discovery that mRNA translation is inappropriately increased in Huntington's disease raises several questions. In the healthy brain (control), translation regulation involves an equilibrium in the formation of the molecular complex (comprising 4E-BP, EIF4E, EIF4G and EIF4A) that initiates translation. While 4E-BP tends to inhibit translation through binding to EIF4E, its phosphorylated form (P-4E-BP) has a low affinity for EIF4E, losing its inhibitory effect. Creus-Muncunill et al. show that elevated phosphorylation of 4E-BP in Huntington's disease could produce a disinhibition of translation leading to increased levels of proteins involved in ribosomal function and energy metabolism. Intracerebral injection of the inhibitor of translation 4EGI-1 in a mouse model of Huntington's disease, restores ribosomal and energy production proteins to normal levels and improves motor performance. This indicates that increased translation in Huntington's disease is detrimental, and as such represents an interesting target or new therapeutic strategies. Questions remaining to be answered are shown in red (see main text for details).