Leveraging genome-wide data to investigate differences between opioid use vs. opioid dependence in 41,176 individuals from the Psychiatric Genomics Consortium.

To provide insights into the biology of opioid dependence (OD) and opioid use (i.e., exposure, OE), we completed a genome-wide analysis comparing 4503 OD cases, 4173 opioid-exposed controls, and 32,500 opioid-unexposed controls, including participants of European and African descent (EUR and AFR, respectively). Among the variants identified, rs9291211 was associated with OE (exposed vs. unexposed controls; EUR z = -5.39, p = 7.2 × 10-8). This variant regulates the transcriptomic profiles of SLC30A9 and BEND4 in multiple brain tissues and was previously associated with depression, alcohol consumption, and neuroticism. A phenome-wide scan of rs9291211 in the UK Biobank (N > 360,000) found association of this variant with propensity to use dietary supplements (p = 1.68 × 10-8). With respect to the same OE phenotype in the gene-based analysis, we identified SDCCAG8 (EUR + AFR z = 4.69, p = 10-6), which was previously associated with educational attainment, risk-taking behaviors, and schizophrenia. In addition, rs201123820 showed a genome-wide significant difference between OD cases and unexposed controls (AFR z = 5.55, p = 2.9 × 10-8) and a significant association with musculoskeletal disorders in the UK Biobank (p = 4.88 × 10-7). A polygenic risk score (PRS) based on a GWAS of risk-tolerance (n = 466,571) was positively associated with OD (OD vs. unexposed controls, p = 8.1 × 10-5; OD cases vs. exposed controls, p = 0.054) and OE (exposed vs. unexposed controls, p = 3.6 × 10-5). A PRS based on a GWAS of neuroticism (n = 390,278) was positively associated with OD (OD vs. unexposed controls, p = 3.2 × 10-5; OD vs. exposed controls, p = 0.002) but not with OE (p = 0.67). Our analyses highlight the difference between dependence and exposure and the importance of considering the definition of controls in studies of addiction.

Alcohol Dehydrogenases, Aldehyde Dehydrogenases, and Alcohol Use Disorders: A Critical Review.

Alcohol use disorders (AUDs) are complex traits, meaning that variations in many genes contribute to the risk, as does the environment. Although the total genetic contribution to risk is substantial, most individual variations make only very small contributions. By far the strongest contributors are functional variations in 2 genes involved in alcohol (ethanol [EtOH]) metabolism. A functional variant in alcohol dehydrogenase 1B (ADH1B) is protective in people of European and Asian descent, and a different functional variant in the same gene is protective in those of African descent. A strongly protective variant in aldehyde dehydrogenase 2 (ALDH2) is essentially only found in Asians. This highlights the need to study a wide range of populations. The likely mechanism of protection against heavy drinking and AUDs in both cases is alteration in the rate of metabolism of EtOH that at least transiently elevates acetaldehyde. Other ADH and ALDH variants, including functional variations in ADH1C, have also been implicated in affecting drinking behavior and risk for alcoholism. The pattern of linkage disequilibrium in the ADH region and the differences among populations complicate analyses, particularly of regulatory variants. This critical review focuses upon the ADH and ALDH genes as they affect AUDs.

Inflammation and vascular smooth muscle cell dedifferentiation following carotid artery ligation.

Following vascular injury medial smooth muscle cells dedifferentiate and migrate through the internal elastic lamina where they form a neointima. The goal of the current study was to identify changes in gene expression that occur before the development of neointima and are associated with the early response to injury. Vascular injury was induced in C57BL/6 mice and in Myh11-creER(T2) mTmG reporter mice by complete ligation of the left carotid artery. Reporter mice were used to visualize cellular changes in the injured vessels. Total RNA was isolated from control carotid arteries or from carotid arteries 3 days following ligation of C57BL/6 mice and analyzed by Affymetrix microarray and quantitative RT-PCR. This analysis revealed decreased expression of mRNAs encoding smooth muscle-specific contractile proteins that was accompanied by a marked increase in a host of mRNAs encoding inflammatory cytokines following injury. There was also marked decrease in molecules associated with BMP, Wnt, and Hedgehog signaling and an increase in those associated with B cell, T cell, and macrophage signaling. Expression of a number of noncoding RNAs were also altered following injury with microRNAs 143/145 being dramatically downregulated and microRNAs 1949 and 142 upregulated. Several long noncoding RNAs showed altered expression that mirrored the expression of their nearest coding genes. These data demonstrate that following carotid artery ligation an inflammatory cascade is initiated that is associated with the downregulation of coding and noncoding RNAs that are normally required to maintain smooth muscle cells in a differentiated state.

Obesity alters molecular and functional cardiac responses to ischemia/reperfusion and glucagon-like peptide-1 receptor agonism.

This study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miRNA) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-minutes coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect the blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca(2+) binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion.

Gene Expression Changes in Glutamate and GABA-A Receptors, Neuropeptides, Ion Channels, and Cholesterol Synthesis in the Periaqueductal Gray Following Binge-Like Alcohol Drinking by Adolescent Alcohol-Preferring (P) Rats.

BACKGROUND: Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including increased pain, fear, and anxiety. The periaqueductal gray (PAG) is involved in processing pain, fear, and anxiety. The effects of adolescent binge drinking on gene expression in this region have yet to be studied. METHODS: Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/wk for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). We used RNA sequencing to assess the effects of ethanol intake on gene expression. RESULTS: Ethanol significantly altered the expression of 1,670 of the 12,123 detected genes: 877 (53%) decreased. In the glutamate system, 23 genes were found to be altered, including reduction in 7 of 10 genes for metabotropic and NMDA receptors. Subunit changes in the NMDA receptor may make it less sensitive to ethanol. Changes in GABAA genes would most likely increase the ability of the PAG to produce tonic inhibition. Five serotonin receptor genes, 6 acetylcholine receptor genes, and 4 glycine receptor genes showed decreased expression in the alcohol-drinking rats. Opioid genes (e.g., Oprk1, Oprm1) and genes for neuropeptides linked to anxiety and panic behaviors (e.g., Npy1r) had mostly decreased expression. Genes for 27 potassium, 10 sodium, and 5 calcium ion channels were found to be differentially expressed. Nine genes in the cholesterol synthesis pathway had decreased expression, including Hmgcr, encoding the rate-limiting enzyme. Genes involved in the production of myelin also had decreased expression. CONCLUSIONS: The results demonstrate that binge alcohol drinking during adolescence produces developmental changes in the expression of key genes within the PAG; many of these changes point to increased susceptibility to pain, fear, and anxiety, which could contribute to excessive drinking to relieve these negative effects.

Integration of general amino acid control and target of rapamycin (TOR) regulatory pathways in nitrogen assimilation in yeast.

Two important nutrient-sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism during changes in nutrient availability. Amino acid starvation activates the GAAC through Gcn2p phosphorylation of translation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. Although Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p in conjunction with Gln3p activates genes required for the assimilation of secondary nitrogen sources such as gamma-aminobutyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to the GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.

Alteration of gene expression by alcohol exposure at early neurulation.

BACKGROUND: We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. RESULT: Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. CONCLUSION: This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.

Genome-wide association study of alcohol dependence implicates a region on chromosome 11.

BACKGROUND: Alcohol dependence is a complex disease, and although linkage and candidate gene studies have identified several genes associated with the risk for alcoholism, these explain only a portion of the risk. METHODS: We carried out a genome-wide association study (GWAS) on a case-control sample drawn from the families in the Collaborative Study on the Genetics of Alcoholism. The cases all met diagnostic criteria for alcohol dependence according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition; controls all consumed alcohol but were not dependent on alcohol or illicit drugs. To prioritize among the strongest candidates, we genotyped most of the top 199 single nucleotide polymorphisms (SNPs) (p < or = 2.1 x 10(-4)) in a sample of alcohol-dependent families and performed pedigree-based association analysis. We also examined whether the genes harboring the top SNPs were expressed in human brain or were differentially expressed in the presence of ethanol in lymphoblastoid cells. RESULTS: Although no single SNP met genome-wide criteria for significance, there were several clusters of SNPs that provided mutual support. Combining evidence from the case-control study, the follow-up in families, and gene expression provided strongest support for the association of a cluster of genes on chromosome 11 (SLC22A18, PHLDA2, NAP1L4, SNORA54, CARS, and OSBPL5) with alcohol dependence. Several SNPs nominated as candidates in earlier GWAS studies replicated in ours, including CPE, DNASE2B, SLC10A2, ARL6IP5, ID4, GATA4, SYNE1, and ADCY3. CONCLUSIONS: We have identified several promising associations that warrant further examination in independent samples.

Effects of chronic intermittent ethanol exposure and withdrawal on neuroblastoma cell transcriptome.

Cycles of heavy drinking and abstinence can lead to alcohol use disorder. We studied the effects of chronic intermittent ethanol exposure (CIE) over 3 weeks on neuroblastoma cells, using an ethanol concentration frequently attained in binge drinking (40 mM, 184 mg/dL). There were many changes in gene expression but most were small. CIE affected pathways instrumental in the development or plasticity of neurons, including axonal guidance, reelin signaling, and synaptogenesis. Genes involved in dopamine and serotonin signaling were also affected. Changes in transporters and receptors could dampen both NMDA and norepinephrine transmissions. Decreased expression of the GABA transporter SLC6A11 could increase GABA transmission and has been associated with a switch from sweet drinking to ethanol consumption in rats. Ethanol increased stress responses such as the unfolded protein response. TGF-ß and NFκB signaling were increased. Most of the genes involved in cholesterol biosynthesis were decreased in expression. Withdrawal for 24 h after CIE caused most of the CIE-induced expression changes to move back toward unexposed levels.

Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects.

Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.

The Access Technology Program of the Indiana Clinical Translational Sciences Institute (CTSI): A model to facilitate access to cutting-edge technologies across a state.

INTRODUCTION: Access to cutting-edge technologies is essential for investigators to advance translational research. The Indiana Clinical and Translational Sciences Institute (CTSI) spans three major and preeminent universities, four large academic campuses across the state of Indiana, and is mandate to provide best practices to a whole state. METHODS: To address the need to facilitate the availability of innovative technologies to its investigators, the Indiana CTSI implemented the Access Technology Program (ATP). The activities of the ATP, or any program of the Indiana CTSI, are challenged to connect technologies and investigators on the multiple Indiana CTSI campuses by the geographical distances between campuses (1-4 hr driving time). RESULTS: Herein, we describe the initiatives developed by the ATP to increase the availability of state-of-the-art technologies to its investigators on all Indiana CTSI campuses, and the methods developed by the ATP to bridge the distance between campuses, technologies, and investigators for the advancement of clinical translational research. CONCLUSIONS: The methods and practices described in this publication may inform other approaches to enhance translational research, dissemination, and usage of innovative technologies by translational investigators, especially when distance or multi-campus cultural differences are factors to efficient application.

A large-scale genome-wide association study meta-analysis of cannabis use disorder.

BACKGROUND: Variation in liability to cannabis use disorder has a strong genetic component (estimated twin and family heritability about 50-70%) and is associated with negative outcomes, including increased risk of psychopathology. The aim of the study was to conduct a large genome-wide association study (GWAS) to identify novel genetic variants associated with cannabis use disorder. METHODS: To conduct this GWAS meta-analysis of cannabis use disorder and identify associations with genetic loci, we used samples from the Psychiatric Genomics Consortium Substance Use Disorders working group, iPSYCH, and deCODE (20 916 case samples, 363 116 control samples in total), contrasting cannabis use disorder cases with controls. To examine the genetic overlap between cannabis use disorder and 22 traits of interest (chosen because of previously published phenotypic correlations [eg, psychiatric disorders] or hypothesised associations [eg, chronotype] with cannabis use disorder), we used linkage disequilibrium score regression to calculate genetic correlations. FINDINGS: We identified two genome-wide significant loci: a novel chromosome 7 locus (FOXP2, lead single-nucleotide polymorphism [SNP] rs7783012; odds ratio [OR] 1·11, 95% CI 1·07-1·15, p=1·84 × 10-9) and the previously identified chromosome 8 locus (near CHRNA2 and EPHX2, lead SNP rs4732724; OR 0·89, 95% CI 0·86-0·93, p=6·46 × 10-9). Cannabis use disorder and cannabis use were genetically correlated (rg 0·50, p=1·50 × 10-21), but they showed significantly different genetic correlations with 12 of the 22 traits we tested, suggesting at least partially different genetic underpinnings of cannabis use and cannabis use disorder. Cannabis use disorder was positively genetically correlated with other psychopathology, including ADHD, major depression, and schizophrenia. INTERPRETATION: These findings support the theory that cannabis use disorder has shared genetic liability with other psychopathology, and there is a distinction between genetic liability to cannabis use and cannabis use disorder. FUNDING: National Institute of Mental Health; National Institute on Alcohol Abuse and Alcoholism; National Institute on Drug Abuse; Center for Genomics and Personalized Medicine and the Centre for Integrative Sequencing; The European Commission, Horizon 2020; National Institute of Child Health and Human Development; Health Research Council of New Zealand; National Institute on Aging; Wellcome Trust Case Control Consortium; UK Research and Innovation Medical Research Council (UKRI MRC); The Brain & Behavior Research Foundation; National Institute on Deafness and Other Communication Disorders; Substance Abuse and Mental Health Services Administration (SAMHSA); National Institute of Biomedical Imaging and Bioengineering; National Health and Medical Research Council (NHMRC) Australia; Tobacco-Related Disease Research Program of the University of California; Families for Borderline Personality Disorder Research (Beth and Rob Elliott) 2018 NARSAD Young Investigator Grant; The National Child Health Research Foundation (Cure Kids); The Canterbury Medical Research Foundation; The New Zealand Lottery Grants Board; The University of Otago; The Carney Centre for Pharmacogenomics; The James Hume Bequest Fund; National Institutes of Health: Genes, Environment and Health Initiative; National Institutes of Health; National Cancer Institute; The William T Grant Foundation; Australian Research Council; The Virginia Tobacco Settlement Foundation; The VISN 1 and VISN 4 Mental Illness Research, Education, and Clinical Centers of the US Department of Veterans Affairs; The 5th Framework Programme (FP-5) GenomEUtwin Project; The Lundbeck Foundation; NIH-funded Shared Instrumentation Grant S10RR025141; Clinical Translational Sciences Award grants; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; National Institute of General Medical Sciences.

Differential effects of ethanol in the nucleus accumbens shell of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar rats: a proteomics study.

The objective of this study was to determine the effects of ethanol injections on protein expression in the nucleus accumbens shell (ACB-sh) of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar (W) rats. Rats were injected for 5 consecutive days with either saline or 1 g/kg ethanol; 24 h after the last injection, rats were killed and brains obtained. Micro-punch samples of the ACB-sh were homogenized; extracted proteins were subjected to trypsin digestion and analyzed with a liquid chromatography-mass spectrometer procedure. Ethanol changed expression levels (1.15-fold or higher) of 128 proteins in NP rats, 22 proteins in P, and 28 proteins in W rats. Few of the changes observed with ethanol treatment for NP rats were observed for P and W rats. Many of the changes occurred in calcium-calmodulin signaling systems, G-protein signaling systems, synaptic structure and histones. Approximately half the changes observed in the ACB-sh of P rats were also observed for W rats. Overall, the results indicate a unique response to ethanol of the ACB-sh of NP rats compared to P and W rats; this unique response may reflect changes in neuronal function in the ACB-sh that could contribute to the low alcohol drinking behavior of the NP line.

Ethanol activates immune response in lymphoblastoid cells.

The short-term effects of alcohol on gene expression in brain tissue cannot directly be studied in humans. Because neuroimmune signaling is altered by alcohol, immune cells are a logical, accessible choice to study and may provide biomarkers. RNAseq was used to study the effects of 48-h exposure to ethanol on lymphoblastoid cell lines (LCLs) from 20 alcoholic subjects and 20 control subjects. Ethanol exposure resulted in differential expression of 4456 of the 12,503 genes detectably expressed in the LCLs (FDR [false discovery rate] ≤ 0.05); 52% of these showed increased expression. Cells from alcoholic subjects and control subjects responded similarly. The genes whose expression changed fell into many pathways: NFκB, neuroinflammation, IL6, IL2, IL8, and dendritic cell maturation pathways were activated, consistent with increased signaling by NFκB, TNF, IL1, IL4, IL18, TLR4, and LPS. Signaling by Interferons A and B decreased, as did EIF2 signaling, phospholipase C signaling, and glycolysis. Baseline gene expression patterns were similar in LCLs from alcoholic subjects and control subjects. At relaxed stringency (p < 0.05), 465 genes differed, 230 of which were also affected by ethanol. There was a suggestion of compensation because baseline differences (no ethanol) were in the opposite direction of differences due to ethanol exposure in 78% of these genes. Pathways with IL8, phospholipase C, and α-adrenergic signaling were significant. The pattern of expression was consistent with increased signaling by several cytokines, including interferons, TLR2, and TLR3 in alcoholics. Expression of genes in the cholesterol biosynthesis pathway, including the rate-limiting enzyme HMGCR, was lower in alcoholic subjects. LCLs show many effects of ethanol exposure, some of which might provide biomarkers for alcohol use disorders. Identifying genes and pathways altered by ethanol can aid in interpreting which genes within loci identified by GWAS might play functional roles.

Cyclic changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10 weeks of treatment.

BACKGROUND AND AIMS: This study determined the kinetics of gene expression during the first 10 weeks of therapy with Pegylated-interferon-alfa2b (PegIntron) and ribavirin (administered by weight) in HCV patients and compared it with the recently completed Virahep C study 12 in which Peginterferon-alfa2a (Pegasys) and ribavirin were administered. METHODS: RNA was isolated from peripheral blood monocytes (PBMC) from twenty treatment-naïve patients just before treatment (day 1) and at days 3, 6, 10, 13, 27, 42 and 70 days after treatment. Gene expression at each time was measured using Affymetrix microarrays and compared to that of day 1. RESULTS: The expression of many genes differed significantly (p

Differential gene expression in the nucleus accumbens with ethanol self-administration in inbred alcohol-preferring rats.

The current study examined the effects of operant ethanol (EtOH) self-administration on gene expression kin the nucleus accumbens (ACB) and amygdala (AMYG) of inbred alcohol-preferring (iP) rats. Rats self-trained on a standard two-lever operant paradigm to administer either water-water, EtOH (15% v/v)-water, or saccharin (SAC; 0.0125% g/v)-water. Animals were killed 24 h after the last operant session, and the ACB and AMYG dissected; RNA was extracted and purified for microarray analysis. For the ACB, there were 513 significant differences at the p<0.01 level in named genes: 55 between SAC and water; 215 between EtOH and water, and 243 between EtOH and SAC. In the case of the AMYG (p<0.01), there were 48 between SAC and water, 23 between EtOH and water, and 63 between EtOH and SAC group. Gene Ontology (GO) analysis indicated that differences in the ACB between the EtOH and SAC groups could be grouped into 15 significant (p<0.05) categories, which included major categories such as synaptic transmission, cell and ion homeostasis, and neurogenesis, whereas differences between the EtOH and water groups had only 4 categories, which also included homeostasis and synaptic transmission. Several genes were in common between the EtOH and both the SAC and water groups in the synaptic transmission (e.g., Cav2, Nrxn3, Gabrb2, Gad1, Homer1) and homeostasis (S100b, Prkca, Ftl1) categories. Overall, the results suggest that changes in gene expression in the ACB of iP rats are associated with the reinforcing effects of EtOH.

Gene expression changes in the ventral hippocampus and medial prefrontal cortex of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking.

Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including decreased hippocampal and prefrontal cortex volume and deficits in memory. We used RNA sequencing to assess the effects of adolescent binge drinking on gene expression in these regions. Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-h sessions/day during the dark/cycle, 5 days/week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session). Ethanol significantly altered the expression of 416 of 11,727 genes expressed in the ventral hippocampus. Genes and pathways involved in neurogenesis, long-term potentiation, and axonal guidance were decreased, which could relate to the impaired memory function found in subjects with adolescent alcohol binge-like exposure. The decreased expression of myelin and cholesterol genes and apparent decrease in oligodendrocytes in P rats could result in decreased myelination. In the medial prefrontal cortex, 638 of 11,579 genes were altered; genes in cellular stress and inflammatory pathways were increased, as were genes involved in oxidative phosphorylation. Overall, the results of this study suggest that adolescent binge-like alcohol drinking may alter the development of the ventral hippocampus and medial prefrontal cortex and produce long-term consequences on learning and memory, and on control of impulsive behaviors.

Transancestral GWAS of alcohol dependence reveals common genetic underpinnings with psychiatric disorders.

Liability to alcohol dependence (AD) is heritable, but little is known about its complex polygenic architecture or its genetic relationship with other disorders. To discover loci associated with AD and characterize the relationship between AD and other psychiatric and behavioral outcomes, we carried out the largest genome-wide association study to date of DSM-IV-diagnosed AD. Genome-wide data on 14,904 individuals with AD and 37,944 controls from 28 case-control and family-based studies were meta-analyzed, stratified by genetic ancestry (European, n = 46,568; African, n = 6,280). Independent, genome-wide significant effects of different ADH1B variants were identified in European (rs1229984; P = 9.8 × 10-13) and African ancestries (rs2066702; P = 2.2 × 10-9). Significant genetic correlations were observed with 17 phenotypes, including schizophrenia, attention deficit-hyperactivity disorder, depression, and use of cigarettes and cannabis. The genetic underpinnings of AD only partially overlap with those for alcohol consumption, underscoring the genetic distinction between pathological and nonpathological drinking behaviors.

Functional gene expression differences between inbred alcohol-preferring and -non-preferring rats in five brain regions.

The objective of this study was to determine if there are innate differences in gene expression in selected CNS regions between inbred alcohol-preferring (iP) and -non-preferring (iNP) rats. Gene expression was determined in the nucleus accumbens (ACB), amygdala (AMYG), frontal cortex (FC), caudate-putamen (CPU), and hippocampus (HIPP) of alcohol-naïve adult male iP and iNP rats, using Affymetrix Rat Genome U34A microarrays (n = 6/strain). Using Linear Modeling for Microarray Analysis with a false discovery rate threshold of 0.1, there were 16 genes with differential expression in the ACB, 54 in the AMYG, 8 in the FC, 24 in the CPU, and 21 in the HIPP. When examining the main effect of strain across regions, 296 genes were differentially expressed. Although the relatively small number of genes found significant within individual regions precluded a powerful analysis for over-represented Gene Ontology categories, the much larger list resulting from the main effect of strain analysis produced 17 over-represented categories (P < .05), including axon guidance, gliogenesis, negative regulation of programmed cell death, regulation of programmed cell death, regulation of synapse structure function, and transmission of nerve impulse. Co-citation analysis and graphing of significant genes revealed a network involved in the neuropeptide Y (NPY) transmitter system. Correlation of all significant genes with those located within previously established rat alcohol QTLs revealed that of the total of 313 significant genes, 71 are located within such QTLs. The many regional and overall gene expression differences between the iP and iNP rat lines may contribute to the divergent alcohol drinking phenotypes of these rats.

Estrogen receptor-dependent attenuation of hypoxia-induced changes in the lung genome of pulmonary hypertension rats.

17ß-estradiol (E2) exerts complex and context-dependent effects in pulmonary hypertension. In hypoxia-induced pulmonary hypertension (HPH), E2 attenuates lung vascular remodeling through estrogen receptor (ER)-dependent effects; however, ER target genes in the hypoxic lung remain unknown. In order to identify the genome regulated by the E2-ER axis in the hypoxic lung, we performed a microarray analysis in lungs from HPH rats treated with E2 (75 mcg/kg/day) ± ER-antagonist ICI182,780 (3 mg/kg/day). Untreated HPH rats and normoxic rats served as controls. Using a false discovery rate of 10%, we identified a significantly differentially regulated genome in E2-treated versus untreated hypoxia rats. Genes most upregulated by E2 encoded matrix metalloproteinase 8, S100 calcium binding protein A8, and IgA Fc receptor; genes most downregulated by E2 encoded olfactory receptor 63, secreted frizzled-related protein 2, and thrombospondin 2. Several genes affected by E2 changed in the opposite direction after ICI182,780 co-treatment, indicating an ER-regulated genome in HPH lungs. The bone morphogenetic protein antagonist Grem1 (gremlin 1) was upregulated by hypoxia, but found to be among the most downregulated genes after E2 treatment. Gremlin 1 protein was reduced in E2-treated versus untreated hypoxic animals, and ER-blockade abolished the inhibitory effect of E2 on Grem1 mRNA and protein. In conclusion, E2 ER-dependently regulates several genes involved in proliferative and inflammatory processes during hypoxia. Gremlin 1 is a novel target of the E2-ER axis in HPH. Understanding the mechanisms of E2 gene regulation in HPH may allow for selectively harnessing beneficial transcriptional activities of E2 for therapeutic purposes.