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The National Institute on Drug Abuse Genetics and Epigenetics Cross-Cutting Research Team convened a diverse group of researchers, clinicians, and healthcare providers on the campus of the University of California, San Diego, in June 2018. The goal was to develop strategies to integrate genetics and phenotypes across species to achieve a better understanding of substance use disorders through associations between genotypes and addictive behaviors. This conference (a) discussed progress in harmonizing large opioid genetics cohorts, (b) discussed phenotypes that are used for genetics studies in humans, (c) examined phenotypes that are used for genetics studies in animal models, (d) identified synergies and gaps in phenotypic analyses of human and animal models and (e) identified strategies to integrate genetics and genomics data with phenotypes across species. The meeting consisted of panels that focused on phenotype harmonization (Dr. Laura Bierut, Dr. Olivier George, Dr. Dan Larach and Dr. Sesh Mudumbai), translating genetic findings between species (Dr. Elissa Chesler, Dr. Gary Peltz and Dr. Abraham Palmer), interpreting and understanding allelic variations (Dr. Vanessa Troiani and Dr. Tamara Richards) and pathway conservation in animal models and human studies (Dr. Robert Hitzemann, Dr. Huda Akil and Dr. Laura Saba). There were also updates that were provided by large consortia (Dr. Susan Tapert, Dr. Danielle Dick, Dr. Howard Edenberg and Dr. Eric Johnson). Collectively, the conference was convened to discuss progress and changes in genome-wide association studies.

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors., Although MDD is one of the most prevalent and debilitating disorders worldwide, it has been difficult to understand its pathophysiology and to develop more effective treatments (1). Epidemiological studies have [...]

Paternal stress can induce long-lasting changes in germ cells potentially propagating heritable changes across generations. To date, no studies have investigated differences in transmission patterns between stress-resilient and stress-susceptible mice. We tested the hypothesis that transcriptional alterations in sperm during chronic social defeat stress (CSDS) transmit increased susceptibility to stress phenotypes to the next generation. We demonstrate differences in offspring from stressed fathers that depend on paternal category (resilient vs susceptible) and offspring sex. Importantly, artificial insemination (AI) reveals that sperm mediates some of the behavioral phenotypes seen in offspring. Using RNA-sequencing (RNA-seq), we report substantial and distinct changes in the transcriptomic profiles of sperm following CSDS in susceptible versus resilient fathers, with alterations in long noncoding RNAs (lncRNAs) predominating especially in susceptibility. Correlation analysis revealed that these alterations were accompanied by a loss of regulation of proteincoding genes by lncRNAs in sperm of susceptible males. We also identify several co-expression gene modules that are enriched in differentially expressed genes (DEGs) in sperm from either resilient or susceptible fathers. Taken together, these studies advance our understanding of intergenerational epigenetic transmission of behavioral experience. [ABSTRACT FROM AUTHOR]

Background Examining transcriptional regulation by antidepressants in key neural circuits implicated in depression and understanding the relation to transcriptional mechanisms of susceptibility and natural resilience may help in the search for new therapeutic agents. Given the heterogeneity of treatment response in human populations, examining both treatment response and nonresponse is critical. Methods We compared the effects of a conventional monoamine-based tricyclic antidepressant, imipramine, and a rapidly acting, non–monoamine-based antidepressant, ketamine, in mice subjected to chronic social defeat stress, a validated depression model, and used RNA sequencing to analyze transcriptional profiles associated with susceptibility, resilience, and antidepressant response and nonresponse in the prefrontal cortex (PFC), nucleus accumbens, hippocampus, and amygdala. Results We identified similar numbers of responders and nonresponders after ketamine or imipramine treatment. Ketamine induced more expression changes in the hippocampus; imipramine induced more expression changes in the nucleus accumbens and amygdala. Transcriptional profiles in treatment responders were most similar in the PFC. Nonresponse reflected both the lack of response-associated gene expression changes and unique gene regulation. In responders, both drugs reversed susceptibility-associated transcriptional changes and induced resilience-associated transcription in the PFC. Conclusions We generated a uniquely large resource of gene expression data in four interconnected limbic brain regions implicated in depression and its treatment with imipramine or ketamine. Our analyses highlight the PFC as a key site of common transcriptional regulation by antidepressant drugs and in both reversing susceptibility– and inducing resilience–associated molecular adaptations. In addition, we found region-specific effects of each drug, suggesting both common and unique effects of imipramine versus ketamine. [ABSTRACT FROM AUTHOR]

Adult women are twice as likely as men to suffer from affective and anxiety disorders, although the mechanisms underlying heightened female stress susceptibility are incompletely understood. Recent findings in mouse Nucleus Accumbens (NAc) suggest a role for DNA methylation-driven sex differences in genome-wide transcriptional profiles. However, the role of another epigenetic process-microRNA (miR) regulation-has yet to be explored. We exposed male and female mice to Subchronic Variable Stress (SCVS), a stress paradigm that produces depression-like behavior in female, but not male, mice, and performed next generation mRNA and miR sequencing on NAc tissue. We applied a combination of differential expression, miR-mRNA network and functional enrichment analyses to characterize the transcriptional and post-transcriptional landscape of sex differences in NAc stress response. We find that male and female mice exhibit largely non-overlapping miR and mRNA profiles following SCVS. The two sexes also show enrichment of different molecular pathways and functions. Collectively, our results suggest that males and females mount fundamentally different transcriptional and post-transcriptional responses to SCVS and engage sex-specific molecular processes following stress. These findings have implications for the pathophysiology and treatment of stress-related disorders in women. [ABSTRACT FROM AUTHOR]

Summary Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here, we performed RNA sequencing on four brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery. [ABSTRACT FROM AUTHOR]

Circadian rhythm disruptions are prominently associated with bipolar disorder (BD). Circadian rhythms are regulated by the molecular clock, a family of proteins that function together in a transcriptional-translational feedback loop. The CLOCK protein is a key transcription factor of this feedback loop, and previous studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice (1). The CLOCK 3111T/C single-nucleotide polymorphism (SNP; rs1801260) is a genetic variation of the human CLOCK gene that is significantly associated with increased frequency of manic episodes in BD patients (2). The 3111T/C SNP is located in the 3'-untranslated region of the CLOCK gene. In this study, we sought to examine the functional implications of the human CLOCK 3111T/C SNP by transfecting a mammalian cell line (mouse embryonic fibroblasts isolated from Clock-/- knockout mice) with pcDNA plasmids containing the human CLOCK gene with either the T or C SNP at position 3111. We then measured circadian gene expression over a 24-h time period. We found that the CLOCK3111C SNP resulted in higher mRNA levels than the CLOCK 3111T SNP. Furthermore, we found that Per2, a transcriptional target of CLOCK, was also more highly expressed with CLOCK 3111C expression, indicating that the 3'-UTR SNP affects the expression, function, and stability of CLOCK mRNA. [ABSTRACT FROM AUTHOR]

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown.Wedetermined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d).Wefound minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during "incubated" cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) inthe Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons,wemeasured colabeling of Fos with Drd1 and Drd2 in threeDSsubregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons. [ABSTRACT FROM AUTHOR]

Drug addiction involves long-term behavioral abnormalities and gene expression changes throughout the mesolimbic dopamine system. Epigenetic mechanisms establish/maintain alterations in gene expression in the brain, providing the impetus for investigations characterizing how epigenetic processes mediate the effects of drugs of abuse. This review focuses on evidence that epigenetic events, specifically histone modifications, regulate gene expression changes throughout the reward circuitry. Drugs of abuse induce changes in histone modifications throughout the reward circuitry by altering histone-modifying enzymes, manipulation of which reveals a role for histone modification in addiction-related behaviors. There is a complex interplay between these enzymes, resulting in a histone signature of the addicted phenotype. Insights gained from these studies are key to identifying novel targets for diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior. [ABSTRACT FROM AUTHOR]

Stable changes in neuronal gene expression have been studied as mediators of addicted states. Of particular interest is the transcription factor ΔFosB, a truncated and stable FosB gene product whose expression in nucleus accumbens (NAc), a key reward region, is induced by chronic exposure to virtually all drugs of abuse and regulates their psychomotor and rewarding effects. Phosphorylation at Ser27 contributes to ΔFosB's stability and accumulation following repeated exposure to drugs, and our recent work demonstrates that the protein kinase CaMKIIα phosphorylates ΔFosB at Ser27 and regulates its stability in vivo. Here, we identify two additional sites on ΔFosB that are phosphorylated in vitro by CaMKIIα, Thr149 and Thr180, and demonstrate their regulation in vivo by chronic cocaine. We show that phosphomimetic mutation of Thr149 (T149D) dramatically increases AP-1 transcriptional activity while alanine mutation does not affect transcriptional activity when compared with wild-type (WT) ΔFosB. Using in vivo viral-mediated gene transfer of ΔFosB-T149D or ΔFosB-T149A in mouse NAc, we determined that overexpression of ΔFosB-T149D in NAc leads to greater locomotor activity in response to an initial low dose of cocaine than does WT ΔFosB, while overexpression of ΔFosB-T149A does not produce the psychomotor sensitization to chronic low-dose cocaine seen after overexpression of WT ΔFosB and abrogates the sensitization seen in control animals at higher cocaine doses. We further demonstrate that mutation of Thr149 does not affect the stability of ΔFosB overexpressed in mouse NAc, suggesting that the behavioral effects of these mutations are driven by their altered transcriptional properties. [ABSTRACT FROM AUTHOR]

Antidepressants (ADs) are the most common treatment for major depressive disorder (MDD). However, only ∼30% of patients experience adequate response after a single AD trial, and this variability remains poorly understood. Here, we investigated microRNAs (miRNAs) as biomarkers of AD response using small RNA-sequencing in paired samples from MDD patients enrolled in a large, randomized placebo-controlled trial of duloxetine collected before and 8 weeks after treatment. Our results revealed differential expression of miR-146a-5p, miR-146b-5p, miR-425-3p and miR-24-3p according to treatment response. These results were replicated in two independent clinical trials of MDD, a well-characterized animal model of depression, and post-mortem human brains. Furthermore, using a combination of bioinformatics, mRNA studies and functional in vitro experiments, we showed significant dysregulation of genes involved in MAPK/Wnt signalling pathways. Together, our results indicate that these miRNAs are consistent markers of treatment response and regulators of the MAPK/Wnt systems. [ABSTRACT FROM AUTHOR]

Sex differences in addiction have been described in humans and animal models. A key factor that influences addiction in both males and females is adolescent experience. Adolescence is associated with higher vulnerability to substance use disorders, and male rodents subjected to adolescent social isolation (SI) stress form stronger preferences for drugs of abuse in adulthood. However, little is known about how females respond to SI, and few studies have investigated the transcriptional changes induced by SI in the brain's reward circuitry. We tested the hypothesis that SI alters the transcriptome in a persistent and sex-specific manner in prefrontal cortex, nucleus accumbens, and ventral tegmental area. Mice were isolated or group housed from postnatal day P22 to P42, then group housed until ∼P90. Transcriptome-wide changes were investigated by RNA sequencing after acute or chronic cocaine or saline administration. We found that SI disrupts sex-specific transcriptional responses to cocaine and reduces sex differences in gene expression across all three brain regions. Furthermore, SI induces gene expression profiles in males that more closely resemble group-housed females, suggesting that SI "feminizes" the male transcriptome. Coexpression analysis reveals that such disruption of sex differences in gene expression alters sex-specific gene networks and identifies potential sex-specific key drivers of these transcriptional changes. Together, these data show that SI has region-specific effects on sex-specific transcriptional responses to cocaine and provide a better understanding of reward-associated transcription that differs in males and females. [ABSTRACT FROM AUTHOR]

Summary Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a primary brain reward region, are seen at early versus late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce “metaplasticity” in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner. [ABSTRACT FROM AUTHOR]

The transcription factor ΔFosB has been proposed as a molecular switch for the transition from casual, volitional drug use into a chronically addicted state, but the upstream regulatory mechanisms governing ΔFosB expression are incompletely understood. In this study, we find a novel regulatory role for the transcription factor E2F3, recently implicated in transcriptional regulation by cocaine, in controlling ΔFosB induction in the mouse nucleus accumbens (NAc) following cocaine administration. We find that an E2F consensus sequence 500 bp upstream of the Fosb transcription start site is enriched for E2F3 specifically over other E2F isoforms. We further conclude that ΔFosB expression is regulated specifically by E2F3a, not E2F3b, that E2f3a expression is specific to D1 receptor-expressing medium spiny neurons, and that E2F3a overexpression in NAc recapitulates the induction of Fosb and ΔFosb mRNA expression observed after chronic cocaine exposure. E2F3a knockdown in NAc does not abolish ΔFosb induction by cocaine, a result consistent with previously published data showing that singular knockdown of upstream regulators of ΔFosB is insufficient to block cocaine-induced expression. Finally, to elucidate potential combinatorial epigenetic mechanisms involved in E2F3a's regulation of Fosb , we explore H3K4me3 enrichment at the Fosb promoter and find that it is not enhanced by E2F3a overexpression, suggesting that it may instead be a pre-existing permissive mark allowing for E2F3a to interact with Fosb . Together, these findings support a role for E2F3a as a novel, upstream regulator of the addiction-mediating transcription factor ΔFosB in NAc.

Circadian rhythm disruptions are prominently associated with bipolar disorder (BD). Circadian rhythms are regulated by the molecular clock, a family of proteins that function together in a transcriptional-translational feedback loop. The CLOCK protein is a key transcription factor of this feedback loop, and previous studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice (1). The CLOCK 3111T/C single-nucleotide polymorphism (SNP; rs1801260) is a genetic variation of the human CLOCK gene that is significantly associated with increased frequency of manic episodes in BD patients (2). The 3111T/C SNP is located in the 3'-untranslated region of the CLOCK gene. In this study, we sought to examine the functional implications of the human CLOCK 3111T/C SNP by transfecting a mammalian cell line (mouse embryonic fibroblasts isolated from Clock(-/-) knockout mice) with pcDNA plasmids containing the human CLOCK gene with either the T or C SNP at position 3111. We then measured circadian gene expression over a 24-h time period. We found that the CLOCK3111C SNP resulted in higher mRNA levels than the CLOCK 3111T SNP. Furthermore, we found that Per2, a transcriptional target of CLOCK, was also more highly expressed with CLOCK 3111C expression, indicating that the 3'-UTR SNP affects the expression, function, and stability of CLOCK mRNA.

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during "incubated" cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling of Fos with Drd1 and Drd2 in three DS subregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons., (Copyright © 2015 the authors 0270-6474/15/358232-13$15.00/0.)