Identification of stress-induced epigenetic methylation onto dopamine D2 gene and neurological and behavioral consequences

The D2 dopamine receptor (DRD2) gene has garnered substantial attention as one of the most extensively studied genes across various neuropsychiatric disorders. Since its initial association with severe alcoholism in 1990, particularly through the identification of the DRD2 Taq A1 allele, numerous international investigations have been conducted to elucidate its role in different conditions. As of February 22, 2024, there are 5485 articles focusing on the DRD2 gene listed in PUBMED. There have been 120 meta-analyses with mixed results. In our opinion, the primary cause of negative reports regarding the association of various DRD2 gene polymorphisms is the inadequate screening of controls, not adequately eliminating many hidden reward deficiency syndrome behaviors. Moreover, pleiotropic effects of DRD2 variants have been identified in neuropsychologic, neurophysiologic, stress response, social stress defeat, maternal deprivation, and gambling disorder, with epigenetic DNA methylation and histone post-translational negative methylation identified as discussed in this article. There are 70 articles listed in PUBMED for DNA methylation and 20 articles listed for histone methylation as of October 19, 2022. For this commentary, we did not denote DNA and/or histone methylation; instead, we provided a brief summary based on behavioral effects. Based on the fact that Blum and Noble characterized the DRD2 Taq A1 allele as a generalized reward gene and not necessarily specific alcoholism, it now behooves the field to find ways to either use effector moieties to edit the neuroepigenetic insults or possibly harness the idea of potentially removing negative mRNA-reduced expression by inducing “dopamine homeostasis.”


Introduction
2][3][4] Their goal was to raise awareness and understanding of the underlying mechanisms driving such behaviors.Moreover, they posed additional questions regarding the millions of individuals who seek out and engage in high-risk novelty situations, pondering the consequences of pleasure-seeking behaviors.They suggested that the answers to these questions may lie within the intricate workings of our brains and perhaps within our genetic makeup. 5,6erica is currently facing the worst opioid epidemic in history.In 2021, the centers for disease control and prevention (CDC) reported 106,699 overdose deaths in the US, with 80,441 of those deaths related to opioids.In addition, provisional data from the CDC indicated that opioid deaths in 2022 would rise to approximately 82,998. 7,83][14][15][16][17] For instance, the D2 dopamine receptor (DRD2) Taq A1 allele has been consistently associated with numerous behavioral phenotypes, including aggression, 18 alcoholism, addictive behaviors, 19 and neuropsychiatric disorders.Both the DRD1 and DRD2 genes are linked to reward pathways and mechanisms. 20The culmination of biochemical processes in mesolimbic regions leads to rewarding phenomena, particularly in the nucleus accumbens (NAc), where increased dopamine levels in synaptic spaces interact with DRD1, DRD2, and other receptor subtypes. 21,22[25][26][27][28] Furthermore, studies have revealed an association between the A1 and B1 minor alleles of the DRD2 gene and cocaine use disorder (CUD). 26These findings suggest that genetic variations in the DRD2 gene, located on chromosome 11 at the q22-q23 region, contribute to an increased susceptibility to psychostimulant use disorder (PUD).Interestingly, these observations align with early research by Gold's group, which hinted at the potential therapeutic efficacy of bromocriptine, a D2 agonist, in combating cocaine abuse and dependence.29 Unlike the DRD2 Taq A1 allele, which is associated with decreased D2 receptor levels, the main variant, DRD2 Taq A2 allele, is characterized by normal D2 receptor levels, possibly offering protection against psychostimulant misuse and abuse. 30storically, Dackis and Gold were among the first to propose the use of the potent D2 agonist bromocriptine as an epigenetic therapy for severe cocaine dependence. 29However, clinical trials revealed that bromocriptine led to the down-regulation of DRD2 receptors and did not prove to be effective for this purpose, resulting in its limited clinical utilization. 31is commentary has uncovered a plethora of studies delineating epigenetic alterations occurring within the DRD2 gene across both substance-related and non-substance-related RDS behaviors.

Epigenetics and addictive behaviors
Epigenetics refers to the molecular modifications imposed on chromatin within the nucleus of a cell, playing a crucial role in regulating various DNA-related processes, including chromatin organization, DNA repair, RNA transcription, and splicing, among other essential cellular functions. 32Substance use disorder (SUD) serves as a prime example of how environmental factors can influence gene expression. 33In this regard, a person's experiences, particularly volitional repetitive drug use, can modify the epigenome in the brain in a way that is specific to certain brain regions and cell types. 34It is hypothesized that dysregulation and modification of DNA-related processes induced by drugs can lead to epigenetic alterations, which may contribute to aberrant cellular functions that facilitate the pathogenesis of psychoactive substance dependence.Nestler's group has previously proposed that gaining insights into epigenetic processes holds therapeutic promise. 35argeting significant drug-induced epigenetic alterations within the brain could potentially disrupt the cycle of drug dependence, thereby preventing individuals from succumbing to a relentless cycle of addiction. 35mprehending the intricacies of the neuroepigenetic landscape necessitates acknowledging the array of epigenetic modifications. 35Among these, a significant epigenetic change occurring in adverse environments involves histone post-translational modifications (PTMs), such as methylation and even dopaminylation. 36,37Essentially, chromatin consists of DNA that is intricately wound around histone protein octamers and forms nucleosomes, which subsequently allows for compact packaging within the cell nucleus.This structural arrangement serves as an adaptable scaffold that responds to external stimuli.Notably, histones are rich in arginine and lysine residues, contributing to their highly basic nature.PTMs of these residues and others on histone N-terminal tails, extending from the nucleosome core, modulate the physical properties and charge distribution of chromatin, thereby regulating DNA-associated processes.

DNA methylation
A biomarker is a quantitative, measurable indicator of a biological molecule, state, or condition. 53DNA methylation is the most researched epigenetic biomarker due to its chemical stability, role in mammalian development and disease, and its significant role in modulating gene expression across a wide array of biological processes. 54,55Although DNA methylation induces physical changes to gene structure, it is reversible in nature.Its primary function is to impede DNA transcription, thereby suppressing the expression of specific genes.DNA methylation tests have become more affordable and accessible and require only a small amount of DNA, which can be obtained from body fluids, cells, or tissues. 19In addition, DNA can be isolated from bacteria, viruses, plants, or mammals.DNA methylation is a biochemical process characterized by the addition of a methyl group to DNA molecules.A common occurrence is the addition of a methyl group to the 5-carbon position of a cytosine ring, forming 5-methylcytosine (5-mC). 56DNA methylation assays are techniques employed to quantify the levels of 5-mC within DNA samples.The enzyme DNA methyltransferase (DNMT) plays a pivotal role in catalyzing DNA methylation, particularly at CpG dinucleotide sites. 57Notably, DNMT-1 is primarily responsible for DNA replication in the mitotic cells of the brain, which exhibit the highest levels of DNA methylation in the body. 58DNMT-3A and DNMT-3B, on the other hand, regulate methylation patterns during early developmental stages. 59 methylation plays a critical role in early brain development and the specification of regions through gene expression. 571][62][63][64] For instance, exposure to environmental carcinogens, such as pollution, can induce mutations in genes responsible for DNA methylation, leading to altered cellular states such as proliferation or differentiation and ultimately resulting in cancer. 64reover, DNA methylation is involved in genomic imprinting, a process where genes are silenced or inactivated through DNA methylation.Imprinting occurs when one allele from either the father or mother is silenced, resulting in an imprinted gene. 65These parentof-origin effects can be inherited by gametes and passed down to offspring, giving rise to various diseases such as Prader-Willi syndrome and Angelman syndrome. 66,67This mode of inheritance also aids in understanding the role of DNA methylation in psychiatric disorders such as major depressive disorder, bipolar disorder, schizophrenia, autism, and related conditions.This finding is significant as it provides crucial insights into how abnormalities in this mechanism contribute to the pathophysiology of diverse disorders, and it suggests the potential of DNA methylation as a therapeutic target. 67

DNA demethylation
DNA demethylation is a process that occurs alongside DNA methylation but is not as widely understood. 68It serves as a biomarker for DNA damage, involving the removal of a methyl group from DNA, which can occur actively or passively in both dividing and non-dividing cells.Passive demethylation entails the loss of 5mC during DNA replication, while active demethylation involves the alteration or removal of a methyl group from 5 mC.Notably, 5 mC, a methylated form of cytosine, is commonly utilized as a point of interest for gene mutations and as an epigenetic marker due to its regulatory role in gene transcription. 55,69rthermore, a derivative of 5mC known as 5-hydroxymethylcytosine (5hmC) is abundantly present in various organ tissues, particularly in the brain.DNA demethylation acts as a marker for DNA damage and facilitates repair processes by identifying potentially mutated sites. 70Primordial germ cells of an embryo and developing zygotes are the primary sites where demethylation occurs, emphasizing its key role in differentiation mechanisms. 71A demethylation is mediated by enzymes from the ten-eleven translocation (TET) family. 72,73The TET enzymes function as tumor suppressors in various malignancies, and their loss or dysfunction is closely associated with rapidly mutating cancers. 74In addition, thymine DNA glycosylase plays a crucial role in DNA demethylation and normal development by initiating base excision repair, which is essential for repairing damaged DNA throughout the cell cycle. 75 summary, DNA demethylation serves as a mechanism for epigenetic reprogramming of genes, influenced by environmental risk factors such as injuries and substance use.

Histone modification
Histones, a class of proteins, facilitate the packaging of DNA into structural units known as nucleosomes.This packaging provides structural support and ensures that DNA fits appropriately within the nucleus of the cell.The modification of histones exemplifies epigenetic regulation, as it influences transcription and alters phenotypes in response to environmental stimuli and stressors.Histone modifications achieve this process by tightly compacting DNA, hindering its accessibility to the cellular machinery. 76Conversely, histone relaxation facilitates increased access of proteins to DNA, thereby enhancing its susceptibility to analysis by the cell.Histone alterations have also been found to affect DNA repair and replication, in addition to cell state modifications. 77They are also utilized to synthesize macromolecules like lipids and carbohydrates, as well as to regulate cell metabolism and energy outputs. 78It is crucial to note that histone alterations frequently have an indirect effect by activating downstream signaling cascades through proteins.

Histone acetylation
Histone acetylation represents a reversible epigenetic mechanism often associated with enhanced gene transcription and implicated in processes underlying memory formation and drug addiction.It serves as a conduit for environmental cues, particularly those from drugs of abuse, to elicit targeted modifications in gene expression.The enzymatic regulation of acetylation involves histone deacetylases (HDACs) and histone acetyltransferases (HATs). 79uring histone acetylation, a negatively charged acetyl group is affixed to lysine residues on histone proteins, 80 resulting in a relaxed chromatin structure conducive to transcription factor binding and heightened gene expression.
Among histones, H3 and H4 are highly conserved and have garnered significant attention due to their pivotal roles in chromatin organization. 81Histone acetylation often occurs concomitantly with other chromatin modifications.Similar to DNA methylation and demethylation, histone acetylation is anticipated to function as a biomarker for various diseases.A reduction in histone acetylation has been associated with neurodevelopment disorders, neural degeneration, plasticity, and memory impairment.The CLOCK protein exemplifies HAT and stands as a prominent transcription factor integral to circadian rhythms and cellular homeostasis.Disruptions in this HAT cause a number of changes associated with sleep deprivation and bipolar disorder manic-like behaviors. 82A thorough investigation into histones will be essential for the development of numerous therapeutic strategies for psychiatric disorders, neurodegenerative diseases, and tumor cells.

Chromatin remodeling
Changes to histone proteins can influence the structure of chromatin, encompassing modifications such as acetylation, deacetylation, methylation, and demethylation. 83These modifications ultimately dictate whether transcription is activated or repressed.Chromatin that is tightly condensed and transcriptionally inactive is termed heterochromatin, where genes are typically silenced or inactivated.Conversely, euchromatin refers to loosely condensed chromatin that is more accessible for transcription.In euchromatin, DNA is readily accessible for binding by transcription factors and other DNA-binding proteins, facilitating the regulation of gene expression. 84The process of transitioning chromatin from a condensed to a more accessible state is referred to as chromatin remodeling, as discussed in earlier sections detailing histone modifications.
Chromatin plays a pivotal role in a multitude of cellular functions, encompassing DNA repair, DNA replication, chromosome segregation, and signal transduction, among others.Chromatin remodelers typically consist of multiprotein complexes and enzymes that harness adenosine triphosphate hydrolysis to modify histones and reconfigure nucleosomes.An example of chromatin remodeling is evident in the development of an embryonic heart.Mutations or defects resulting from these remodelers can lead to a variety of cardiovascular disorders in adults. 85

Non-coding RNA (ncRNA) expression
ncRNA refers to RNA molecules transcribed from DNA but not translated into proteins.There are four major types of ncRNA involved in regulating gene expression: MicroRNAs (miRNA), long ncRNAs (lncRNA), Piwi-interacting RNAs, and short interfering RNAs, all of which have been extensively investigated. 86These regulatory mechanisms play pivotal roles in managing transcription and translation, influencing chromatin remodeling, and are integral to physiological processes as well as disease states. 87MiRNAs function by binding to target RNAs, thereby suppressing gene expression and inhibiting translation.They facilitate the transfer of genetic information within cells, between different cells and tissues, and even across body fluids such as breast milk, sweat, urine, and blood.Due to their involvement in cardiovascular disorders and tumor suppression, miRNAs are considered excellent diagnostic markers. 60,64In contrast, lncRNAs are more celltype specific and are expressed at lower levels compared to miRNAs.They primarily regulate transcription, which may occur within or outside the nucleus.In addition, lncRNAs exhibit diverse functions, including regulation of mRNA within the cytoplasm of a cell. 86

The DRD2 gene as a therapeutic target for RDS
In a blinded study, Blum et al., 88 demonstrated the initial allelic association of the DRD2 gene with alcoholism.The study employed 70 brain samples collected from both nonalcoholics and individuals diagnosed with alcoholism.DNA samples underwent digestion with restriction endonucleases and were probed with a clone containing the complete 3' coding exon, the polyadenylation signal, and approximately 16.4 kilobases (kb) of noncoding 3' sequence of the human DRD2 gene (lambda hD2G1).The findings revealed that the presence of the A1 allele of the DRD2 gene accurately classified 77% of the individuals with alcoholism, while its absence correctly classified 72% of the nonalcoholics.This polymorphic pattern of the DRD2 gene suggested the presence of a susceptibility gene for at least one form of alcoholism on the q22-q23 region of chromosome 11.
Using similar brain tissue, Noble et al. 23 conducted a study investigating the allelic association of the human DRD2 gene with the binding characteristics of the DRD2 receptor in 66 brains obtained from both non-alcoholic and alcoholic subjects.In this blinded experiment, DNA extracted from the cerebral cortex underwent treatment with the restriction endonuclease Taq and was probed with a 1.5-kb digest of a clone (XhD2G1) of the human DRD2 gene.The binding characteristics, including the binding affinity (K d ) and the number of binding sites (B max ), of DRD2 were determined in the caudate nuclei of these brains using tritiated spiperone as the ligand.The results revealed that compared to nonalcoholic subjects, the adjusted K d was significantly lower in alcoholic subjects, indicating a higher binding affinity of DRD2 in the latter group.In addition, B max was found to be lower in subjects carrying the A1 allele, which exhibited a strong association with alcoholism, whereas no significant changes in B max were observed in subjects carrying the A2 allele.Moreover, a progressively reduced B max was found in subjects with A2/A2, A1/A2, and A1/A1 alleles, with subjects carrying A2/A2 alleles exhibiting the highest mean values and those with A1/A1 alleles exhibiting the lowest.The observed polymorphic pattern of the DRD2 gene and its variation in receptor expression strongly suggests the involvement of the dopaminergic system in predisposing individuals to at least one subtype of severe alcoholism.Subsequent studies have corroborated these findings, [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104] and as of February 22, 2024, a PubMed search using the term "Dopamine D2 Receptor Gene" yielded a total of 5485 listings.
While we are cognizant of epigenetic insults affecting at least seven major neurotransmitter pathways, including cannabinoidergic, cholinergic, serotonergic, opioidergic, gluconergic, GABAergic, and glutaminergic, our focus lies on the dopaminergic system, specifically the DRD2 gene.The primary review, which is based on PUBMED listings using the search term "Methylation and the Dopamine D2 Receptor Gene," yielded 378 listings as of February 22, 2024.

Epigenetic modifications and DRD2 gene
While we recognize that many reward genes and associated loci have been shown to undergo epigenetic modifications after the consumption of drugs of abuse (i.e., alcohol), the purpose of this commentary is to present a narrative that focuses on identifying epigenetic modifications, specifically in the DRD2 gene.In this commentary, we chose not to denote DNA and/or histone methylation, but instead, we provide a summary based on behavioral effects.
A recent study by Bohnsack et al. provides an example of targeting and editing epigenetic histone modifications to attenuate unwanted behaviors. 104Adolescent binge drinking is widely recognized to induce epigenetic modifications at the enhancer region of the activityregulated cytoskeleton-associated protein (ARC) immediate-early gene, specifically known as the synaptic activity response element (SARE). 104These epigenetic modifications lead to a decrease in ARC expression in the amygdala, a phenomenon observed in both rodent models and human studies. 104n an experiment conducted by Pandey's group, it was demonstrated that the use of dCas9-P300 resulted in increased histone acetylation at the ARC SARE.This intervention effectively normalized deficits in ARC expression and consequently led to a reduction in adult anxiety and excessive alcohol consumption in a rat model that examined alcohol exposure during adolescence. 104Interestingly, dCas9-Kruppel-associated box (KRAB), in contrast, was found to promote repressive histone methylation at the ARC SARE, resulting in decreased ARC expression, the manifestation of anxiety-like behaviors, and increased alcohol consumption in the control group.

SUD
A summary of studies focusing on the epigenetics SUD is presented in Table 1.Detailed descriptions of SUD are provided in the following subsections.

5.1.1.
Cannabis-In a study conducted by Oyaci et al., 105 DNA methylation levels at the DRD2 gene were compared among patients based on clinical parameters and DRD2 genotype distribution.Their findings revealed significant differences in methylation status between groups, particularly concerning the presence of a family history of CUD or synthetic cannabinoid use disorder (SCUD).One notable limitation of the study is the utilization of inaccurate controls (the authors did not screen the controls for other potentially addictive behaviors such as gambling and overeating).It is known that without such screening, the presence of the DRD2 A1 allele, for example, could be as high as 40%.
This flaw could have prevented the investigators from establishing direct evidence for high DNA methylation with both CUD and SCUD.In a separate work conducted by Gerra et al., 106 a genetic and sociodemographic analysis involving both CUD patients and control groups was performed to explore DNA methylation in the DRD2-ANKKI gene region.They identified significant hypermethylation at exon 8 of the DRD2 gene.Interestingly, the study also uncovered a correlation between higher education levels and a reduced risk of CUD.The authors astutely proposed that their findings of differentially methylated regions (DMRs) at exon 8 of the DRD2 gene could serve as potential biomarkers or targets for the development of pharmacological therapeutic agents.
In a study by DiNieri et al., 107 utilizing an animal model of prenatal exposure to delta-9tetrahydrocannabinol (THC), it was observed that prenatal cannabis exposure led to a reduction in DRD2 messenger RNA expression in the NAc, a crucial brain reward region.
Furthermore, Hurd's group 107 identified histone methylation differences in the offspring of pregnant rats exposed to THC.Chromatin immunoprecipitation of the adult NAc revealed an increase in the 2meH3K9 repressive mark and a decrease in 3meH3K4 and RNA polymerase II at the DRD2 gene locus in the THC-exposed offspring.This diminished DRD2 expression correlates with reduced DRD2 binding sites and heightened sensitivity to opiate reward in adulthood.Moreover, the study suggested that maternal cannabis use alters the epigenetic mechanisms governing histone lysine methylation, thereby influencing the developmental regulation of mesolimbic DRD2 in offspring.This subsequent reduction in DRD2 may increase the susceptibility of the offspring to addiction in the future.Of interest, earlier work from Blum's laboratory 108 demonstrated significant alteration of the vas deferens from prenatal exposure to THC in that there was an increased sensitivity to enkephalins, in agreement with the findings of DiNieri et al. 107 5.1.2.Tobacco-As highlighted by Liu et al., 109 previous research has underscored the importance of the NCAM1-TTC12-ANKK1-DRD2 gene cluster in addiction susceptibility among individuals of European and African descent.Building on this foundation, Liu and colleagues conducted next-generation bisulfite sequencing to uncover smoking-associated DMRs.Through haplotype-based association analysis, they identified a significant association between cigarettes per day and the C-T-A-G haplotype in DRD2, formed by rs4245148, rs4581480, rs4648317, and rs11214613.Furthermore, the study identified four significant smoking-associated DMRs, with three residing in the DRD2/ANKK1 region.
These findings elucidate a notable association of variants and haplotypes within the ANKK1/DRD2 region with nicotine dependence among Chinese male smokers.Importantly, the results also underscore the pivotal role of DNA methylation in facilitating such associations.

Alcohol-Strong correlations have been observed between DRD2
][91][92][93][94][95][96][97][98][99][100][101][102][103][104] Consequently, disruptions in DRD2 function constitute a critical component of theoretical models elucidating the pathophysiology of substance dependence. 110Moreover, as proposed by Pandey et al., 111 epigenetic modifications may serve as fundamental molecular mechanisms influencing how alcohol exposure impacts the brain.This idea has been investigated by Bidwell et al., who demonstrated that, after controlling for age, DRD2 promoter DNA methylation was positively associated with responses to alcohol cues in the left caudate, right caudate, left putamen, right putamen, and right Nac. 77,112 This finding indicated that robust striatal activation, in response to reward cues, was linked with DNA methylation at the DRD2 gene.In addition, DRD2 methylation was linked to alcohol use disorder (AUD) severity.Specifically, DRD2 methylation was linked to scores on the AUDs Identification Test, Impaired Control Scale, and Alcohol Dependence Scale.
Chronic consumption of alcohol and various other illicit drugs has been associated with adverse consequences, including functional connectivity deficits within neural networks linked to executive control. 113These deficits may indirectly contribute to the development of aberrant alcohol-seeking behavior. 113Hagerty et al. 114 found that, specifically, average DNA methylation at the DRD2 gene was negatively correlated with left and right executive control network connectivity, but no correlation was found with the other networks that were tested.In addition, DRD2 DNA methylation was found to be linked with the severity of alcohol problems.These findings bolster a theoretical framework linking the neurobiological markers of alcohol consumption among polysubstance users to epigenetic influences.Individuals with AUD tend to have dopaminergic alteration, and those with a family history of AUD may experience influences on brain development. 115Hill and Sharma discovered a significant positive correlation between familial high-risk status and DNA methylation at the DRD2 gene. 116In fact, significant differences in the volume of the left inferior temporal, left fusiform, and left insula regions were observed between high-risk and low-risk familial risk groups.Previously, these regions have been associated with social cognition.In addition, the DNA methylation of the DRD2 gene was inversely correlated with the volumes of grey matter in these regions.
Along similar lines of thinking, Hillemacher et al., 117 concerned about the role of epigenetics on dopaminergic neurotransmission and its influence on alcohol dependence, found a significant increase of DNA methylation at the DRD2 gene during alcohol withdrawal/early abstinence.Furthermore, they discovered a significant association between craving, as measured by the obsessive-compulsive drinking scale, and the DNA methylation of the DRD2 gene.Their findings regarding this association with alcohol craving underscore the pivotal role of DRD2 gene DNA methylation in the neurobiology of addictive behavior.
It is worth noting that the inability to demonstrate significant alterations in DNA methylation between controls and patients may stem from an inadequate screening of the control group. 118reover, as previously pointed out by several researchers, [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104] reduced ligand binding of DRD2 has consistently been observed in the striatum of individuals with AUD and other RDS behaviors.The reduced DRD2 binding has been suggested to indicate diminished DRD2 density, which, in turn, has been proposed to trigger cravings and increase the likelihood of relapse.Accordingly, Feltmann et al., 119 surprisingly, unlike others, did not find DNA methylation differences in the investigated regions of the DRD2 gene.However, in Wistar rats, chronic alcohol drinking significantly decreased mRNA levels of the long isoform of the DRD2 gene in the NAc.In addition, alcohol drinking also decreased the striatal density of DRD2-DRD2 homoreceptor complexes, increased the density of A2AR-DRD2 heteroreceptor complexes in the NAc shell and the dorsal striatum, and decreased the density of sigma1R-DRD2 heteroreceptor complexes in the dorsal striatum.Interestingly, the chronic alcohol consumption in these rats appears to fit well with earlier findings from Blum's laboratory involving Golden Syrian hamsters. 120In particular, compared to the control hamsters who only drank water, the experimental hamsters who freely drank ethanol after a year had a noticeably lower concentration of a leucine-enkephalin-like immunoreactive substance in their basal ganglia. 120is discovery suggests that ethanol's effects involve the synthesis of endogenous peptidyl opiates, which appears akin to the findings of long-term drinking, as presented in the Feltmann et al. study.Thus, although we cannot rule out the possibility of DNA methylation differences occurring in other regions and/or other epigenetic modifications not studied in the Feltmann et al. 119 study, it begs the question as to the possibility that mRNA expression suppression by alcohol drinking also reduces the synthesis of DRD2.Feltmann et al. 119 also provided support for the hypothesis that AUD was associated with a hypodopaminergic system and proposed the A2AR-DRD2 heteroreceptor complex as a promising novel target for treatment.Blum et al. 30 proposed that mild D2 receptor stimulation achieved by certain therapeutic modalities can cause dopamine release, which, in turn, can change D2-directed mRNA and improve DRD2 function in humans.This increase in DRD2 activity is thought to reduce craving behaviors, particularly in high-risk, genetically compromised populations.Accordingly, Cadet's group has conducted eloquent experiments comparing methamphetamine (METH) and modafinil in terms of epigenetic insults. 121nzález et al., 121 demonstrated that repeated administration of either METH or modafinil to mice resulted in cognitive effects.In the medial prefrontal cortex, there were observed inductions in histone acetylation and DNA methylation profiles.Mice subjected to repeated METH exposure, but not modafinil, exhibited impaired cognitive memory, as revealed by the novel object recognition test, with a notable decrease in memory recognition.In addition, METH-treated mice demonstrated (i) decreased levels of histone H3 and H4 acetylation and increased levels of 5-mC and (ii) reduced histone H3 acetylation enrichment at promoters of the DRD2 gene.These findings suggest that epigenetic dysregulation, particularly at the DRD2 gene, is associated with the long-term cognitive decline effects of METH and its adverse impacts on medial prefrontal cortex function.Further studies are warranted to elucidate the specific mechanisms of epigenetic regulation affected by psychostimulant abuse.

Psychostimulant abuse-Recently,
Changes in histone methylation and acetylation on DNA within the NAc and lysine residues have been observed with repeated cocaine administration.Nestler's group investigated histone arginine (R) methylation in models related to reward processing.Specifically, Damez-Werno et al. 122 found that in both animal and human self-administration experiments, the histone mark protein-R-methyltransferase-6 (PRMT6) and asymmetric demethylation of R2 on histone H3 (H3R2me2a) were reduced in the rodent and cocainedependent human NAc.In fact, while PRMT6 overexpression in medium spiny neurons (MSNs) expressing DRD1 (D1-MSNs) is protective against cocaine-seeking behaviors, PRMT6 overexpression in D2-MSNs in all NAc neurons has been associated with increased cocaine-seeking behaviors.Along these lines, Blum et al. hypothesized that dopaminylation (H3R2me2a binding) occurs in PUD, and the binding inhibitor Srcin1, like the major DRD2 A2 allelic polymorphism, protects against psychostimulant-seeking behavior by normalizing NAc dopamine expression.Moreover, numerous studies have verified the association between the DRD2 Taq A1 allele (30 -40 lower DRD2 numbers) and severe cocaine dependence.According to Lepack et al., 123 acute cocaine increases dopamine in NAc synapses and causes histone H3 glutamine 5 dopaminylation and subsequent inhibition of DRD2 expression.With prolonged cocaine use, the inhibition of DRD2 expression increases and accompanies cocaine withdrawal.Furthermore, they have reported that during cocaine withdrawal, the Src kinase signaling inhibitor 1 (Srcin1 or p140CAP) decreased H3R2me2a binding.Thus, this inhibited dopaminylation induced a "homeostatic brake."Blum's group 37 suggested that the reduction in Src signaling in NAc D2-MSNs, like the DRD2 Taq A2 allele, a well-known genetic mechanism protective against SUD, normalizes the NAc dopamine expression and decreases cocaine cravings and cocaine-seeking behaviors.Therefore, Srcin1 could be an important target for therapeutic interventions.
Another important facet related to METH-induced psychosis may involve both genetic DNA antecedents and epigenetic post-translational changes in mRNA expression, for example, on the DRD2 gene.Nohesara et al. 124 found that METH induces a decrease in DNA methylation of a number of dopamine-related genes (i.e., DRD3, DRD4, and COMT) in patients with METH psychosis but not in non-METH psychosis patients.The suggestion here is that, in general, METH dependency is linked with a reduction in DNA methylation and an open chromatin conformation, leading to increased expression of several important genes involved in the pathogenesis of psychotic disorders. 121Nohesara et al. 124 suggested that these epigenetic modifications may serve as valuable diagnostic biomarkers for diagnosing psychosis in METH abusers.In addition, they support the use of a methylrich diet for the suppression or prevention of psychosis in these patients, thus encouraging further association and interventional studies involving larger populations.

5.1.5.
Opioid abuse-Opioid use disorder (OUD) and other reward-dysregulated disorders have a high degree of heritability.Zhang et al. 125 demonstrated that various methylation quantitative trait loci (mQTLs) in the DRD1 and DRD2 genes were identified in both the healthy control and heroin use disorder groups.Specifically, rs4867798-CpG_174872884 and rs5326-CpG_174872884 in the DRD1 gene were the unique singlenucleotide polymorphism-CpG pairs observed in patients suffering from heroin use disorder.This groundbreaking research suggests that certain dopaminergic mQTLs may be linked with characteristics of OUD by implicating DNA methylation and gene expression.However, it underscores the necessity for improved screening of controls to reassess the findings regarding DRD2. 1201.6.Eating disorders-Similar to SUD, anorexia nervosa is a multifaceted and highly heritable disease.[126][127][128] Rask-Andersen et al. 128 reviewed the relevant literature and discovered that 175 association studies had been conducted on an anorexia nervosa cohort, examining 128 different polymorphisms related to 43 genes.The strongest correlations indicate that certain dopaminergic genes play a key role in regulating body mass index.129 Moreover, findings by Frieling et al. 130 revealed an increase in the expression of dopamine transporter (DAT) mRNA and a decrease in DRD2 expression.Frieling et al. suggested that the upregulation of the DAT gene was accompanied by a hypermethylation of the gene's promoter in the anorexia nervosa and bulimia nervosa group, while significant hypermethylation of the DRD2 promoter was only present in the anorexia nervosa group.
No discrepancies in methylation or expression were detected in the various other dopamine receptors that were examined.In addition, Groleau et al. 131 found that women diagnosed with bulimia spectrum disorder, particularly those with borderline personality disorder, demonstrated significant elevations in DRD2 DNA methylation levels than women with no eating disorders.Moreover, women with a history of bulimia spectrum disorder and childhood sexual abuse showed increased DRD2 DNA methylation compared to the group with no eating disorder history.These results suggest that individuals with bulimia spectrum disorder experience an increase in DNA methylation of the DRD2 gene promoter, which may serve as a stronger marker of comorbid psychopathology rather than being a board correlate of eating disorders in general.

5.1.7.
3][134] Hillemacher et al. 134 reported that DNA methylation patterns in the DRD2-gene were modified with respect to abstinence over a 12-month or a 30-month period.In addition, they observed increased levels of DNA methylation in individuals who were non-abstinent and those who did not seek treatment.These findings suggest that altered DRD2 expression, resulting from variations in DNA methylation, holds pathophysiological significance in lifetime pathologic gambling behavior.5.1.8.Personality disorders-Utilizing chimpanzees, which are known to exhibit five dimensions of personality (agreeableness, dominance, extraversion, openness, and reactivity/ undependability), Staes et al. 135 observed that DRD2 DNA methylation is most strongly linked to extraversion.In addition, they found that varying DNA methylation levels at specific DRD2 sites were linked to changes in extraversion in nursery-reared, but not mother-reared, individuals.These findings provide further support for the importance of biological mother-related rearing in early life.This work further suggests that early-life experiences can influence long-lasting behavioral effects, theoretically through epigenomic modification.[138][139][140] 5.1.9.Maternal deprivation (MD)-Gondre-Lewis's group correctly points out that stress experienced early in life can trigger a complex neurochemical cascade, affecting the emotional and addictive behaviors of adolescents and adults later in life. 141Moreover, research indicates that drug-seeking behavior and excessive alcohol drinking are often comorbid with depressive-like symptoms and behaviors. 142These behaviors are frequently observed in individuals who faced adversity in their early life and are important features found in animal models of early life stress exposure. 143In fact, Guo et al. 144 reported that, in rats, the rats subjected to MD, chronic unpredictable stress (CUS), and a combination of both (MD/CUS) displayed increased levels of DRD2 promoter DNA methylation compared to normal controls.The authors concluded that early-life MD increased vulnerability to stress-induced depressive-like behavior in adult rats.In line with the findings of Gondre-Lewis's group, [141][142][143] increased methylation of the DRD2 promoter gene in the ventral tegmental area may raise the risk of depression and alcohol-seeking behavior.Furthermore, Zhu et al. 145 explored the effects of MD on adult rats' spatial learning and memory, exploratory, and limbic activity, along with their association with the expression of DAT, DRD1, DRD2, and DRD3 in the NAc.In addition, Zhu et al. 145 investigated the potential involvement of DNA methylation in the regulation of DRD2 gene expression.Based on their work, only the DRD2 mRNA level was associated with total distance.However, the expression of DNMT1 and 3 alpha (DNMT3A) and the methylated CpG levels in the promoter region of the DRD2 gene were not significantly altered in the MD group compared to the control group. 120Functional differences may have occurred in other sites at the DRD2 genes not included in the study, and/or other epigenetic modifications were associated with DRD2 mRNA expression.Notwithstanding, it is possible that deficient control screening and interpretation may have impacted the association study, and further research with carefully selected controls, not just MD, may alter these results.ΔFosB is selectively induced in D1-MSNs in the NAc of resilient mice and in D2-MSNs of susceptible mice. 146In this regard, Hamilton et al. reported that FOSB-targeted histone acetylation in D2-MSNs or histone methylation in D1-MSNs promotes a stress-susceptible, depressive-like phenotype, while histone methylation in D2-MSNs or histone acetylation in D1-MSNs enhance resilience to social stress as measured by social interaction behavior and sucrose preference. 147Importantly, the study presented the first evidence of targeting histone modifications specifically to genes and cells, simulating real-world transcriptional processes that regulate social defeat stress behavior.

Effector moieties
The effector moieties are molecules that selectively bind to proteins to modify their biological activities.These molecules, essentially ligands, can modify enzyme activity, genomic activity, cell signaling, and other protein functions. 35While identifying epigeneticinduced post-transcriptional methylation or acetylation on chromosome histones is crucial, one of the first and most successful effector moieties in editing transcriptional activator domains was VP64, which is derived from the herpes simplex virus.VP64 recruits RNA polymerase ll directly for gene-targeted transcriptional activation. 148It is now understood that by targeting VP64 to the promoter region of a single gene using DNA-binding domains such as ZFP, 149 TALE, 150,151 or CRISPR/dCas9, 152 laboratories can consistently induce transcription of targeted genes in vitro and even in vivo. 153,154rthermore, to attain bidirectional control over gene expression, the suppression of endogenous genes is typically achieved using KRAB effector moieties.KRAB represents a transcriptional repression domain present in human zinc finger transcription factors.It functions by recruiting heterochromatin-forming complexes that subsequently deposit H3K9me3 repressive marks, resulting in transcriptional suppression. 155Similar to VP64, the KRAB domain modifies the epigenome by enlisting the secondary factors rather than utilizing enzymatic activity.In addition, promoter-or enhancer-targeted KRAB has been used in cell culture and in the brain. 156,157In addition, research has identified specific transcription factors and epigenetic readers, erasers, and writers associated with the addiction pathogenesis.More recently, a number of effector moieties have been developed for neuroepigenetic editing.These effector moieties include DNA-modifying enzymes such as DNMT3A (responsible for CpG methylation). 158,159and TET1 (which catalyzes hydroxymethylation of CpG). 160,161They also encompass proteins involved in regulating histone PTMs such as NFκB subunit p65 (which recruits HATs to acetylate core histones). 162histone methyltransferase G9a (which catalyzes H3K9me2), 162,163 p300 HAT (which acetylates all four core histones), 164 Sin3-interaction domain (which recruits HDACs), 165,166 lysine-specific demethylase 1 (which demethylates H3K4 and H3K9), 167 PRDM9 (which methylates H3K4 and H3K36), 168 and DOT1L (which methylates H3K79). 169,170Furthermore, transcription factors such as CREB have also been utilized, thus mimicking known mechanisms of transcriptional regulation. 171

Research outlook in epigenetics of SUD
The evidence presented in this commentary strongly indicates the significant role of epigenetic (mis) regulation in SUD.Numerous associations and interventional studies demonstrate that epigenetic mechanisms are responsible for the complex regulation of dopamine homeostasis, particularly concerning the expression of the DRD2 gene.Epigenetic regulatory mechanisms (i.e., histone modifications, ncRNA, and DNA methylation) interact in an orchestrated fashion to establish phenotypes influenced by genetic background (e.g., the presence of mutations) and environmental factors (e.g., the presence of active compounds from drugs, alcohol, and tobacco).Importantly, these interactions occur in a tissue-and cell-specific manner.Hence, a detailed mapping of epigenetic modifications across various presentations of SUD will enable the understanding of the precise regulatory mechanisms involved in each case and identify potential targets for therapeutic interventions.
The utilization of effector moieties emerges as a promising intervention.However, caution is warranted due to the pleiotropic effects of epigenetic modifiers (e.g., DNMTs, HDACs, and HTAs), and systemic inactivation of these enzymes may lead to undesirable side effects, hindering their clinical translation.Further research is needed to elucidate mechanisms for cell-specific activation or delivery of the effector moieties to mitigate side effects.Moreover, given that prenatal SUD may impact offspring health, mechanistic and interventional studies are imperative to determine and target epigenetic modifications, aiming to mitigate the effects of prenatal SUD on fetal health.

Summary
SUD often entails persistent behavioral abnormalities observed in susceptible individuals following repeated exposure to psychoactive drugs of abuse.The enduring nature of these behavioral alterations suggests potential long-lasting changes in gene expression within specific brain regions, which may contribute to the addiction phenotype.Advanced research over the past decade has revealed the pivotal involvement of epigenetic mechanisms in orchestrating lasting alterations, whether positive or negative, in gene expression across various tissues, particularly in the brain.This understanding has spurred investigations aimed at elucidating the impact of epigenetic regulatory processes in mediating the enduring effects of psychoactive substances of abuse on the brain, predominantly using animal models of drug addiction.However, more recently, human neuroepigenetic research has been rapidly emerging.Compelling evidence indicates that repeated exposure to drugs of abuse induces alterations within the brain's reward regions through three primary modes of epigenetic regulation: Histone modifications such as acetylation and methylation, DNA methylation, and ncRNAs.In this commentary, our focus lies on investigating epigenetic modifications to the DRD2 gene to directly illustrate the role of these epigenetic changes in addiction-related behavioral abnormalities.At present, there is a growing awareness regarding the utilization of various effector moieties to counteract these undesirable negative epigenetic changes, as recently observed by Pandey's group 104 in mitigating alcohol-induced anxiety.
Experimentally, effector moieties are providing unprecedented information relevant to not only mechanistic insights but also to demonstrate the importance of removing these negative epigenetic insults, leading to the concomitant attenuation of specific mRNA transcriptional expression, such as the DRD2 gene.One important unanswered question is whether induction, for example, of dopamine homeostasis, would increase the conversion of posttranscriptional histone methylation to acetylation.Our laboratory has been developing a nutraceutical complex to gently induce pro-dopamine regulation, potentially in both animal models and humans.To answer this profoundly important question, we advocate for the scientific community to perform the necessary research in this arena.

Conclusion
The DRD2 gene has been extensively investigated in various neuropsychiatric disorders.
Numerous international studies have been performed since the initial association of the DRD2 Taq A1 allele with severe alcoholism in 1990.In our opinion, the primary cause of negative reports regarding the association of various DRD2 gene polymorphisms is the inadequate screening of controls, failing to eliminate many hidden RDS behaviors.Moreover, pleiotropic effects of DRD2 variants have been observed in neurophysiological, neuropsychological, stress response, social stress defeat, MD, and gambling disorder contexts, where epigenetic DNA methylation and negative histone post-translational methylation have been identified, as discussed in this commentary.As of October 19, 2022, there are 70 articles focusing on DNA methylation and 20 articles on histone methylation are listed in PUBMED.Importantly, Blum and Noble characterized the DRD2 Taq A1 allele not as specific to alcoholism but as a generalized reward gene.Therefore, it now behooves the field to find ways to either use effector moieties to edit the neuroepigenetic insults or possibly harness the idea of potentially removing negative mRNA-reduced expression by inducing dopamine homeostasis.This represents a futuristic laudable goal.

5. 1 . 10 .
Social defeat stress-In terms of social defeat stress, Zhu et al. reported that chronic social defeat stress regulates FOSB expression in the NAc, which promotes the cell-type-specific accumulation of ΔFosB in the two MSN subtypes in this region.
methylation was positively associated with responses to alcohol severity Bidwell et al. 112 DRD2 DNA methylation was significantly associated with alcohol problem severity Hagerty et al. 114 DNA methylation at the DRD2 gene was significantly increased in association with familial high-risk status Hill and Sharma 116 Craving was significantly associated with DRD2-gene methylation Hillemacher et al. 117 Psychostimulants METH-treated mice also showed (i) decreased global levels of total H3ac and H4ac and increased global levels of 5-methylcytosine (5-mC) and (ii) decreased H3ac enrichment at promoters of DRD2 González et al. 121In both human and animal self-administration experiments, the histone mark protein-R-methyltransferase-6 (PRMT6) and asymmetric demethylation of R2 on histone H3 (H3R2me2a) decreased in the rodent and cocaine-dependent human NAc.Damez-Werno et al. 122 METH induced a decrease in DNA methylation of a number of dopamine-related genes (i.e., DRD3, DRD4, and COMT) in patients with METH psychosis but not in non-METH psychosis patients.Nohesara et al. 124 Opioids Specifically, rs4867798-CpG_174872884 and rs5326-CpG_174872884 in the DRD1 gene showed hypermethylation Zhang et al. 125