A phenome-wide association and Mendelian randomisation study of alcohol use variants in a diverse cohort comprising over 3 million individuals.

BACKGROUND: Alcohol consumption is associated with numerous negative social and health outcomes. These associations may be direct consequences of drinking, or they may reflect common genetic factors that influence both alcohol consumption and other outcomes. METHODS: We performed exploratory phenome-wide association studies (PheWAS) of three of the best studied protective single nucleotide polymorphisms (SNPs) in genes encoding ethanol metabolising enzymes (ADH1B: rs1229984-T, rs2066702-A; ADH1C: rs698-T) using up to 1109 health outcomes across 28 phenotypic categories (e.g., substance-use, mental health, sleep, immune, cardiovascular, metabolic) from a diverse 23andMe cohort, including European (N ≤ 2,619,939), Latin American (N ≤ 446,646) and African American (N ≤ 146,776) populations to uncover new and perhaps unexpected associations. These SNPs have been consistently implicated by both candidate gene studies and genome-wide association studies of alcohol-related behaviours but have not been investigated in detail for other relevant phenotypes in a hypothesis-free approach in such a large cohort of multiple ancestries. To provide insight into potential causal effects of alcohol consumption on the outcomes significant in the PheWAS, we performed univariable two-sample and one-sample Mendelian randomisation (MR) analyses. FINDINGS: The minor allele rs1229984-T, which is protective against alcohol behaviours, showed the highest number of PheWAS associations across the three cohorts (N = 232, European; N = 29, Latin American; N = 7, African American). rs1229984-T influenced multiple domains of health. We replicated associations with alcohol-related behaviours, mental and sleep conditions, and cardio-metabolic health. We also found associations with understudied traits related to neurological (migraines, epilepsy), immune (allergies), musculoskeletal (fibromyalgia), and reproductive health (preeclampsia). MR analyses identified evidence of causal effects of alcohol consumption on liability for 35 of these outcomes in the European cohort. INTERPRETATION: Our work demonstrates that polymorphisms in genes encoding alcohol metabolising enzymes affect multiple domains of health beyond alcohol-related behaviours. Understanding the underlying mechanisms of these effects could have implications for treatments and preventative medicine. FUNDING: MVJ, NCK, SBB, SSR and AAP were supported by T32IR5226 and 28IR-0070. SSR was also supported by NIDA DP1DA054394. NCK and RBC were also supported by R25MH081482. ASH was supported by funds from NIAAA K01AA030083. JLMO was supported by VA 1IK2CX002095. JLMO and JJMM were also supported by NIDA R21DA050160. JJMM was also supported by the Kavli Postdoctoral Award for Academic Diversity. EGA was supported by K01MH121659 from the NIMH/NIH, the Caroline Wiess Law Fund for Research in Molecular Medicine and the ARCO Foundation Young Teacher-Investigator Fund at Baylor College of Medicine. MSA was supported by the Instituto de Salud Carlos III and co-funded by the European Union Found: Fondo Social Europeo Plus (FSE+) (P19/01224, PI22/00464 and CP22/00128).

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder.

Background: Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5 mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5 hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5 mC and 5 hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Methods: Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5 mC and 5 hmC at the genome-wide level. Differential 5 mC and 5 hmC were evaluated using the methylKit R package and significance was set at false discovery rate < 0.05 and differential methylation > 2. Functional enrichment analyses were performed, and gene-level convergence was evaluated in an independent dataset that assessed 5 mC and 5 hmC of AUD in bulk cortical tissue. Results: We identified 417 5 mC and 363 5hmC significant differential CpG sites associated with AUD, with 59% in gene promoters. Some of the identified genes have been previously implicated in alcohol consumption, including SYK, DNMT3A for 5 mC, GAD1, DLX1, DLX2, for 5 hmC and GATA4 in both. Convergence with a previous AUD 5 mC and 5 hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5 mC and 5 hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5 mC genes. Discussion: This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD, identifying both previously reported and potentially novel gene associations with AUD. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

Identifying novel gene dysregulation associated with opioid overdose death: A meta-analysis of differential gene expression in human prefrontal cortex.

Only recently have human postmortem brain studies of differential gene expression (DGE) associated with opioid overdose death (OOD) been published; sample sizes from these studies have been modest (N = 40-153). To increase statistical power to identify OOD-associated genes, we leveraged human prefrontal cortex RNAseq data from four independent OOD studies and conducted a transcriptome-wide DGE meta-analysis (N = 285). Using a unified gene expression data processing and analysis framework across studies, we meta-analyzed 20 098 genes and found 335 significant differentially expressed genes (DEGs) by OOD status (false discovery rate < 0.05). Of these, 66 DEGs were among the list of 303 genes reported as OOD-associated in prior prefrontal cortex molecular studies, including genes/gene families (e.g., OPRK1, NPAS4, DUSP, EGR). The remaining 269 DEGs were not previously reported (e.g., NR4A2, SYT1, HCRTR2, BDNF). There was little evidence of genetic drivers for the observed differences in gene expression between opioid addiction cases and controls. Enrichment analyses for the DEGs across molecular pathway and biological process databases highlight an interconnected set of genes and pathways from orexin and tyrosine kinase receptors through MEK/ERK/MAPK signaling to affect neuronal plasticity.

Psychosocial moderators of polygenic risk scores of inflammatory biomarkers in relation to GrimAge.

GrimAge acceleration has previously predicted age-related morbidities and mortality. In the current study, we sought to examine how GrimAge is associated with genetic predisposition for systemic inflammation and whether psychosocial factors moderate this association. Military veterans from the National Health and Resilience in Veterans study, which surveyed a nationally representative sample of European American male veterans, provided saliva samples for genotyping (N = 1135). We derived polygenic risk scores (PRS) from the UK Biobank as markers of genetic predisposition to inflammation. Results revealed that PRS for three inflammatory PRS markers-HDL (lower), apolipoprotein B (lower), and gamma-glutamyl transferase (higher)-were associated with accelerated GrimAge. Additionally, these PRS interacted with a range of potentially modifiable psychosocial variables, such as exercise and gratitude, previously identified as associated with accelerated GrimAge. Using gene enrichment, we identified anti-inflammatory and antihistamine drugs that perturbate pathways of genes highly represented in the inflammatory PRS, laying the groundwork for future work to evaluate the potential of these drugs in mitigating epigenetic aging.

Neuronal-specific methylome and hydroxymethylome analysis reveal replicated and novel loci associated with alcohol use disorder.

Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5mC and 5hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5mC and 5hmC at the genome-wide level. Differential 5mC and 5hmC were evaluated using the methylKit R package and significance was set at false discovery rate <0.05 and differential methylation >2. Functional enrichment analyses were performed and replication was evaluated replication in an independent dataset that assessed 5mC and 5hmC of AUD in bulk cortical tissue. We identified 417 5mC and 363 5hmC genome-wide significant differential CpG sites associated with AUD, with 59% in gene promoters. We also identified genes previously implicated in alcohol consumption, such as SYK, CHRM2, DNMT3A, and GATA4, for 5mC and GATA4, and GAD1, GATA4, DLX1 for 5hmC. Replication was observed for 28 CpG sites from a previous AUD 5mC and 5hmC study, including FOXP1. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5mC genes. This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD. We replicated previous findings and identified novel associations with AUD for both 5mC and 5hmC marks within the OFC. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

Positive personality traits moderate persistent high alcohol consumption, determined by polygenic risk in U.S. military veterans: results from a 10-year, population-based, observational cohort study.

BACKGROUND: Understanding the interplay between psychosocial factors and polygenic risk scores (PRS) may help elucidate the biopsychosocial etiology of high alcohol consumption (HAC). This study examined the psychosocial moderators of HAC, determined by polygenic risk in a 10-year longitudinal study of US military veterans. We hypothesized that positive psychosocial traits (e.g. social support, personality traits, optimism, gratitude) may buffer risk of HAC in veterans with greater polygenic liability for alcohol consumption (AC). METHODS: Data were analyzed from 1323 European-American US veterans who participated in the National Health and Resilience in Veterans Study, a 10-year, nationally representative longitudinal study of US military veterans. PRS reflecting genome-wide risk for AC (AUDIT-C) was derived from a Million Veteran Program genome-wide association study (N = 200 680). RESULTS: Among the total sample, 328 (weighted 24.8%) had persistent HAC, 131 (weighted 9.9%) had new-onset HAC, 44 (weighted 3.3%) had remitted HAC, and 820 (weighted 62.0%) had no/low AC over the 10-year study period. AUDIT-C PRS was positively associated with persistent HAC relative to no/low AC [relative risk ratio (RRR) = 1.43, 95% confidence interval (CI) = 1.23-1.67] and remitted HAC (RRR = 1.63, 95% CI = 1.07-2.50). Among veterans with higher AUDIT-C PRS, greater baseline levels of agreeableness and greater dispositional gratitude were inversely associated with persistent HAC. CONCLUSIONS: AUDIT-C PRS was prospectively associated with persistent HAC over a 10-year period, and agreeableness and dispositional gratitude moderated this association. Clinical interventions designed to target these modifiable psychological traits may help mitigate risk of persistent HAC in veterans with greater polygenic liability for persistent HAC.

Cross-Species Convergence of Brain Transcriptomic and Epigenomic Findings in Posttraumatic Stress Disorder: A Systematic Review.

Introduction: Posttraumatic stress disorder (PTSD) is a complex multifactorial disorder influenced by the interaction of genetic and environmental factors. Analyses of epigenomic and transcriptomic modifications may help to dissect the biological factors underlying the gene-environment interplay in PTSD. To date, most human PTSD epigenetics studies have used peripheral tissue, and these findings have complex and poorly understood relationships to brain alterations. Studies examining brain tissue may help characterize the brain-specific transcriptomic and epigenomic profiles of PTSD. In this review, we compiled and integrated brain-specific molecular findings of PTSD from humans and animals. Methods: A systematic literature search according to the PRISMA criteria was performed to identify transcriptomic and epigenomic studies of PTSD, focusing on brain tissue from human postmortem samples or animal-stress paradigms. Results: Gene- and pathway-level convergence analyses revealed PTSD-dysregulated genes and biological pathways across brain regions and species. A total of 243 genes converged across species, with 17 of them significantly enriched for PTSD. Chemical synaptic transmission and signaling by G-protein-coupled receptors were consistently enriched across omics and species. Discussion: Our findings point out dysregulated genes highly replicated across PTSD studies in humans and animal models and suggest a potential role for the corticotropin-releasing hormone/orexin pathway in PTSD's pathophysiology. Further, we highlight current knowledge gaps and limitations and recommend future directions to address them.

Profiling neuronal methylome and hydroxymethylome of opioid use disorder in the human orbitofrontal cortex.

Opioid use disorder (OUD) is influenced by genetic and environmental factors. While recent research suggests epigenetic disturbances in OUD, this is mostly limited to DNA methylation (5mC). DNA hydroxymethylation (5hmC) has been widely understudied. We conducted a multi-omics profiling of OUD in a male cohort, integrating neuronal-specific 5mC and 5hmC as well as gene expression profiles from human postmortem orbitofrontal cortex (OUD = 12; non-OUD = 26). Single locus methylomic analysis and co-methylation analysis showed a higher number of OUD-associated genes and gene networks for 5hmC compared to 5mC; these were enriched for GPCR, Wnt, neurogenesis, and opioid signaling. 5hmC marks also showed a higher correlation with gene expression patterns and enriched for GWAS of psychiatric traits. Drug interaction analysis revealed interactions with opioid-related drugs, some used as OUD treatments. Our multi-omics findings suggest an important role of 5hmC and reveal loci epigenetically dysregulated in OFC neurons of individuals with OUD.

Decoding the role of transcriptomic clocks in the human prefrontal cortex.

Aging is a complex process with interindividual variability, which can be measured by aging biological clocks. Aging clocks are machine-learning algorithms guided by biological information and associated with mortality risk and a wide range of health outcomes. One of these aging clocks are transcriptomic clocks, which uses gene expression data to predict biological age; however, their functional role is unknown. Here, we profiled two transcriptomic clocks (RNAAgeCalc and knowledge-based deep neural network clock) in a large dataset of human postmortem prefrontal cortex (PFC) samples. We identified that deep-learning transcriptomic clock outperforms RNAAgeCalc to predict transcriptomic age in the human PFC. We identified associations of transcriptomic clocks with psychiatric-related traits. Further, we applied system biology algorithms to identify common gene networks among both clocks and performed pathways enrichment analyses to assess its functionality and prioritize genes involved in the aging processes. Identified gene networks showed enrichment for diseases of signal transduction by growth factor receptors and second messenger pathways. We also observed enrichment of genome-wide signals of mental and physical health outcomes and identified genes previously associated with human brain aging. Our findings suggest a link between transcriptomic aging and health disorders, including psychiatric traits. Further, it reveals functional genes within the human PFC that may play an important role in aging and health risk.

Trajectories of alcohol consumption in U.S. military veterans: Results from a 10-year population-based longitudinal study.

BACKGROUND: Alcohol use disorder is a public health problem, especially among US veterans. This study examined the nature and predictors of 10-year trajectories of alcohol consumption in US veterans. METHODS: Data were analyzed from the 2011-2021 National Health and Resilience in Veterans Study, a nationally representative, longitudinal study of 2309 US veterans. RESULTS: Latent growth mixture modeling analyses revealed four trajectories of alcohol consumption (Alcohol Use Disorders Identification Test-Consumption [AUDIT-C]) over a 10-year period: excessive (4.1%; mean [standard deviation] AUDIT-C baseline=8.6 [2.0], slope= -0.33 [0.07]); at-risk (22.1%; baseline=4.1 [1.6], slope=0.02 [0.07]); rare (71.7%; baseline=1.2 [1.3], slope= -0.01 [0.03]); and recovering alcohol consumption (2.1%; baseline=8.4 [1.9], slope= -0.70 [0.14]). The strongest predictors of excessive vs. rare alcohol consumption group were younger age (relative variance explained [RVE]=27.8%), and lower agreeableness (RVE=27.0%); at-risk vs. rare alcohol consumption group were fewer medical comorbidities (RVE=82.3%); recovering vs. rare alcohol consumption group were greater dysphoric arousal symptoms (RVE=46.1%) and current mental health treatment (RVE=26.5%); excessive vs. at-risk alcohol consumption group were younger age (RVE=25.9%), greater dysphoric arousal symptoms of posttraumatic stress disorder (RVE=22.0%), and lower conscientiousness (RVE=19.1%); and excessive vs. recovering alcohol consumption group were current mental health treatment (RVE=61.1%) and secure attachment style (RVE=12.4%). CONCLUSIONS: Over the past decade, more than 1 in 4 US veterans consumed alcohol at the at-risk-to-excessive level. Veterans who are younger, score lower on agreeableness and conscientiousness, endorse greater dysphoric arousal symptoms, and currently not engaged in mental health treatment may require close monitoring and prevention efforts to mitigate the risk of a chronic course of at-risk-to-excessive alcohol consumption.

Dissecting the epigenomic differences between smoking and nicotine dependence in a veteran cohort.

Smoking is a serious public health issue linked to more than 8 million deaths per year worldwide and may lead to nicotine dependence (ND). Although the epigenomic literature on smoking is well established, studies evaluating the role of epigenetics in ND are limited. In this study, we examined the epigenomic signatures of ND and how these differ from smoking exposure to identify biomarkers specific to ND. We investigated the peripheral epigenetic profile of smoking status (SS) and ND in a US male veteran cohort. DNA from saliva was collected from 1135 European American (EA) male US military veterans. DNAm was assessed using the Illumina Infinium Human MethylationEPIC BeadChip array. SS was evaluated as current smokers (n = 137; 12.1%) and non-current smokers (never and former; n = 998; 87.9%). NDFTND was assessed as a continuous variable using the Fagerström Test for ND (FTND; n = 1135; mean = 2.54 ± 2.29). Epigenome-wide association studies (EWAS) and co-methylation analyses were conducted for SS and NDFTND . A total of 450 and 22 genome-wide significant differentially methylated sites (DMS) were associated with SS and NDFTND , respectively (15 overlapped DMS). We identified 97 DMS (43 genes) in SS-EWAS previously reported in the literature, including AHRR and F2RL3 genes (p-value: 1.95 × 10-83 to 4.55 × 10-33 ). NDFTND novel DMS mapped to NEUROG1, ANPEP, and SLC29A1. Co-methylation analysis identified 386 modules (11 SS-related and 19 NDFTND -related). SS-related modules showed enrichment for alcoholism, while NDFTND -related modules were enriched for nicotine addiction. This study confirms previous findings associated with SS and identifies novel and-potentially specific-epigenetic biomarkers of ND that may inform prognosis and novel treatment strategies.

Psychosocial Factors Associated With Accelerated GrimAge in Male U.S. Military Veterans.

OBJECTIVE: Veterans are at high risk for health morbidities linked to premature mortality. Recently developed "epigenetic clock" algorithms, which compute intra-individual differences between biological and chronological aging, can help inform prediction of accelerated biological aging and mortality risk. To date, however, scarce research has examined potentially modifiable correlates of GrimAge, a novel epigenetic clock comprised of DNA methylation surrogates of plasma proteins and smoking pack-years associated with various morbidities and time-to-death. The objective of the study was to examine psychosocial correlates of this novel epigenetic clock. DESIGN: Cross-sectional study. SETTING: U.S. veteran population. PARTICIPANTS: Participants were male, European American (EA), and derived from a nationally representative sample of U.S. veterans (N = 1,135, mean age = 63.3, standard deviation [SD] = 13.0). MEASUREMENTS: We examined the prevalence of accelerated GrimAge and its association with a broad range of health, lifestyle, and psychosocial variables. RESULTS: A total 18.3% of veterans had accelerated GrimAge (≥5 years greater GrimAge than chronological age; mean = 8.4 years acceleration, SD = 2.2). Fewer days of weekly physical exercise (relative variance explained [RVE] = 27%), history of lifetime substance use disorder (RVE = 21%), greater number of lifetime traumas (RVE = 19%), lower gratitude (RVE = 13%), reduced sleep quality (RVE = 7%), lower openness to experience (RVE = 7%), and unmarried/partnered status (RVE = 6%) were independently associated with increased odds of accelerated GrimAge. Increasing numbers of these risk factors were associated with greater odds of accelerated GrimAge, with greatest likelihood of acceleration for veterans with ≥3 risk factors (weighted 21.5%). CONCLUSIONS: These results suggest that nearly 1-of-5 EA male U.S. veterans have accelerated GrimAge, and highlight a broad range of health, lifestyle, and psychosocial variables associated with accelerated GrimAge. Given that many of these factors are modifiable, these findings provide promising leads for risk stratification models of accelerated biological aging and precision medicine-based targets for interventions to mitigate risk for premature mortality in this population.

Central and Peripheral Immune Dysregulation in Posttraumatic Stress Disorder: Convergent Multi-Omics Evidence.

Posttraumatic stress disorder (PTSD) is a chronic and multifactorial disorder with a prevalence ranging between 6-10% in the general population and ~35% in individuals with high lifetime trauma exposure. Growing evidence indicates that the immune system may contribute to the etiology of PTSD, suggesting the inflammatory dysregulation as a hallmark feature of PTSD. However, the potential interplay between the central and peripheral immune system, as well as the biological mechanisms underlying this dysregulation remain poorly understood. The activation of the HPA axis after trauma exposure and the subsequent activation of the inflammatory system mediated by glucocorticoids is the most common mechanism that orchestrates an exacerbated immunological response in PTSD. Recent high-throughput analyses in peripheral and brain tissue from both humans with and animal models of PTSD have found that changes in gene regulation via epigenetic alterations may participate in the impaired inflammatory signaling in PTSD. The goal of this review is to assess the role of the inflammatory system in PTSD across tissue and species, with a particular focus on the genomics, transcriptomics, epigenomics, and proteomics domains. We conducted an integrative multi-omics approach identifying TNF (Tumor Necrosis Factor) signaling, interleukins, chemokines, Toll-like receptors and glucocorticoids among the common dysregulated pathways in both central and peripheral immune systems in PTSD and propose potential novel drug targets for PTSD treatment.

The role of alcohol intake in the pharmacogenetics of treatment with clozapine.

Clozapine (CLZ) is an atypical antipsychotic reserved for patients with refractory psychosis, but it is associated with a significant risk of severe adverse reactions (ADRs) that are potentiated with the concomitant use of alcohol. Additionally, pharmacogenetic studies have explored the influence of several genetic variants in CYP450, receptors and transporters involved in the interindividual response to CLZ. Herein, we systematically review the current multiomics knowledge behind the interaction between CLZ and alcohol intake, and how its concomitant use might modulate the pharmacogenetics. CYP1A2*1F, *1C and other alleles not yet discovered could support a precision medicine approach for better therapeutic effects and fewer CLZ ADRs. CLZ monitoring systems should be amended and include alcohol intake to protect patients from severe CLZ ADRs.

Epigenome-wide association study of posttraumatic stress disorder identifies novel loci in U.S. military veterans.

Posttraumatic stress disorder (PTSD) is a chronic and disabling psychiatric disorder prevalent in military veterans. Epigenetic mechanisms have been implicated in the etiology of PTSD, with DNA methylation being the most studied to identify novel molecular biomarkers associated with this disorder. We performed one of the largest single-sample epigenome-wide association studies (EWAS) of PTSD to date. Our sample included 1135 male European-American U.S. veterans who participated in the National Health and Resilience in Veterans Study (NHRVS). DNA was collected from saliva samples and the Illumina HumanMethylation EPIC BeadChip was used for the methylation analysis. PTSD was assessed using the PTSD Checklist. An EWAS was conducted using linear regression adjusted for age, cell-type proportions, first 10 principal components, and smoking status. After Bonferroni correction, we identified six genome-wide significant (GWS) CpG sites associated with past-month PTSD and three CpGs with lifetime PTSD (prange = 10-10-10-8). These CpG sites map to genes involved in immune function, transcription regulation, axonal guidance, cell signaling, and protein binding. Among these, SENP7, which is involved in transcription regulation and has been linked to risk-taking behavior and alcohol consumption in genome-wide association studies, replicated in an independent veteran cohort and was downregulated in medial orbitofrontal cortex of PTSD postmortem brain tissue. These findings suggest potential epigenetic biomarkers of PTSD that may help inform the pathophysiology of this disorder in veterans and other trauma-affected populations.

CpH methylome analysis in human cortical neurons identifies novel gene pathways and drug targets for opioid use disorder.

Introduction: DNA methylation (DNAm), an epigenetic mechanism, has been associated with opioid use disorder (OUD) in preclinical and human studies. However, most of the studies have focused on DNAm at CpG sites. DNAm at non-CpG sites (mCpHs, where H indicates A, T, or C) has been recently shown to have a role in gene regulation and to be highly abundant in neurons. However, its role in OUD is unknown. This work aims to evaluate mCpHs in the human postmortem orbital frontal cortex (OFC) in the context of OUD. Methods: A total of 38 Postmortem OFC samples were obtained from the VA Brain Bank (OUD = 12; Control = 26). mCpHs were assessed using reduced representation oxidative bisulfite sequencing in neuronal nuclei. Differential analysis was performed using the "methylkit" R package. Age, ancestry, postmortem interval, PTSD, and smoking status were included as covariates. Significant mCpHs were set at q-value < 0.05. Gene Ontology (GO) and KEGG enrichment analyses were performed for the annotated genes of all differential mCpH loci using String, ShinyGO, and amiGO software. Further, all annotated genes were analyzed using the Drug gene interaction database (DGIdb). Results: A total of 2,352 differentially methylated genome-wide significant mCpHs were identified in OUD, mapping to 2,081 genes. GO analysis of genes with differential mCpH loci showed enrichment for nervous system development (p-value = 2.32E-19). KEGG enrichment analysis identified axon guidance and glutamatergic synapse (FDR 9E-4-2.1E-2). Drug interaction analysis found 3,420 interactions between the annotated genes and drugs, identifying interactions with 15 opioid-related drugs, including lofexidine and tizanidine, both previously used for the treatment of OUD-related symptoms. Conclusion: Our findings suggest a role of mCpHs for OUD in cortical neurons and reveal important biological pathways and drug targets associated with the disorder.

Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer's disease.

Antipsychotic drugs are still widely prescribed to control various severe neuropsychiatric symptoms in the elderly and dementia patients although they are off-label use in the United States. However, clinical practice shows greater side effects and lower efficacy of antipsychotics for this vulnerable population and the mechanisms surrounding this aged-related sensitivity are not well understood. Our previous studies have shown that aging-induced epigenetic alterations may be involved in the increasing severity of typical antipsychotic haloperidol induced side effects in aged mice. Still, it is unknown if similar epigenetic mechanisms extend to atypical antipsychotics, which are most often prescribed to dementia patients combined with severe neuropsychiatric symptoms. In this study, we report that atypical antipsychotic risperidone also causes increased motor side effect behaviors in aged mice and 5xFAD mice. Histone deacetylase (HDAC) inhibitor Valproic Acid and Entinostat can mitigate the risperidone induced motor side effects. We further showed besides D2R, reduced expression of 5-HT2A, one of the primary atypical antipsychotic targets in the striatum of aged mice that are also mitigated by HDAC inhibitors. Finally, we demonstrate that specific histone acetylation mark H3K27 is hypoacetylated at the 5htr2a and Drd2 promoters in aged mice and can be reversed with HDAC inhibitors. Our work here establishes evidence for a mechanism where aging reduces expression of 5-HT2A and D2R, the key atypical antipsychotic drug targets through epigenetic alteration. HDAC inhibitors can restore 5-HT2A and D2R expression in aged mice and decrease the motor side effects in aged and 5xFAD mice.