A genome-wide association study of interhemispheric theta EEG coherence: implications for neural connectivity and alcohol use behavior.

Aberrant connectivity of large-scale brain networks has been observed among individuals with alcohol use disorders (AUDs) as well as in those at risk, suggesting deficits in neural communication between brain regions in the liability to develop AUD. Electroencephalographical (EEG) coherence, which measures the degree of synchrony between brain regions, may be a useful measure of connectivity patterns in neural networks for studying the genetics of AUD. In 8810 individuals (6644 of European and 2166 of African ancestry) from the Collaborative Study on the Genetics of Alcoholism (COGA), we performed a Multi-Trait Analyses of genome-wide association studies (MTAG) on parietal resting-state theta (3-7 Hz) EEG coherence, which previously have been associated with AUD. We also examined developmental effects of GWAS findings on trajectories of neural connectivity in a longitudinal subsample of 2316 adolescent/young adult offspring from COGA families (ages 12-30) and examined the functional and clinical significance of GWAS variants. Six correlated single nucleotide polymorphisms located in a brain-expressed lincRNA (ENSG00000266213) on chromosome 18q23 were associated with posterior interhemispheric low theta EEG coherence (3-5 Hz). These same variants were also associated with alcohol use behavior and posterior corpus callosum volume, both in a subset of COGA and in the UK Biobank. Analyses in the subsample of COGA offspring indicated that the association of rs12954372 with low theta EEG coherence occurred only in females, most prominently between ages 25 and 30 (p < 2 × 10-9). Converging data provide support for the role of genetic variants on chromosome 18q23 in regulating neural connectivity and alcohol use behavior, potentially via dysregulated myelination. While findings were less robust, genome-wide associations were also observed with rs151174000 and parieto-frontal low theta coherence, rs14429078 and parieto-occipital interhemispheric high theta coherence, and rs116445911 with centro-parietal low theta coherence. These novel genetic findings highlight the utility of the endophenotype approach in enhancing our understanding of mechanisms underlying addiction susceptibility.

Targeting fibroblast growth factor 23-responsive pathways uncovers controlling genes in kidney mineral metabolism.

Fibroblast Growth Factor 23 (FGF23) is a bone-derived hormone that reduces kidney phosphate reabsorption and 1,25(OH)2 vitamin D synthesis via its required co-receptor alpha-Klotho. To identify novel genes that could serve as targets to control FGF23-mediated mineral metabolism, gene array and single-cell RNA sequencing were performed in wild type mouse kidneys. Gene array demonstrated that heparin-binding EGF-like growth factor (HBEGF) was significantly up-regulated following one-hour FGF23 treatment of wild type mice. Mice injected with HBEGF had phenotypes consistent with partial FGF23-mimetic activity including robust induction of Egr1, and increased Cyp24a1 mRNAs. Single cell RNA sequencing showed overlapping HBEGF and EGF-receptor expression mostly in the proximal tubule, and alpha-Klotho expression in proximal and distal tubule segments. In alpha-Klotho-null mice devoid of canonical FGF23 signaling, HBEGF injections significantly increased Egr1 and Cyp24a1 with correction of basally elevated Cyp27b1. Additionally, mice placed on a phosphate deficient diet to suppress FGF23 had endogenously increased Cyp27b1 mRNA, which was rescued in mice receiving HBEGF. In HEK293 cells with stable alpha-Klotho expression, FGF23 and HBEGF increased CYP24A1 mRNA expression. HBEGF, but not FGF23 bioactivity was blocked with EGF-receptor inhibition. Thus, our findings support that the paracrine/autocrine factor HBEGF could play novel roles in controlling genes downstream of FGF23 via targeting common signaling pathways.

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.

Endotoxin Preconditioning Reprograms S1 Tubules and Macrophages to Protect the Kidney.

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.

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.

Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes.

Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.

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.

Circulating uromodulin inhibits systemic oxidative stress by inactivating the TRPM2 channel.

High serum concentrations of kidney-derived protein uromodulin [Tamm-Horsfall protein (THP)] have recently been shown to be independently associated with low mortality in both older adults and cardiac patients, but the underlying mechanism remains unclear. Here, we show that THP inhibits the generation of reactive oxygen species (ROS) both in the kidney and systemically. Consistent with this experimental data, the concentration of circulating THP in patients with surgery-induced acute kidney injury (AKI) correlated with systemic oxidative damage. THP in the serum dropped after AKI and was associated with an increase in systemic ROS. The increase in oxidant injury correlated with postsurgical mortality and need for dialysis. Mechanistically, THP inhibited the activation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channel. Furthermore, inhibition of TRPM2 in vivo in a mouse model mitigated the systemic increase in ROS during AKI and THP deficiency. Our results suggest that THP is a key regulator of systemic oxidative stress by suppressing TRPM2 activity, and our findings might help explain how circulating THP deficiency is linked with poor outcomes and increased mortality.

Analysis of whole genome-transcriptomic organization in brain to identify genes associated with alcoholism.

Alcohol exposure triggers changes in gene expression and biological pathways in human brain. We explored alterations in gene expression in the Pre-Frontal Cortex (PFC) of 65 alcoholics and 73 controls of European descent, and identified 129 genes that showed altered expression (FDR < 0.05) in subjects with alcohol dependence. Differentially expressed genes were enriched for pathways related to interferon signaling and Growth Arrest and DNA Damage-inducible 45 (GADD45) signaling. A coexpression module (thistle2) identified by weighted gene co-expression network analysis (WGCNA) was significantly correlated with alcohol dependence, alcohol consumption, and AUDIT scores. Genes in the thistle2 module were enriched with genes related to calcium signaling pathways and showed significant downregulation of these pathways, as well as enrichment for biological processes related to nicotine response and opioid signaling. A second module (brown4) showed significant upregulation of pathways related to immune signaling. Expression quantitative trait loci (eQTLs) for genes in the brown4 module were also enriched for genetic associations with alcohol dependence and alcohol consumption in large genome-wide studies included in the Psychiatric Genetic Consortium and the UK Biobank's alcohol consumption dataset. By leveraging multi-omics data, this transcriptome analysis has identified genes and biological pathways that could provide insight for identifying therapeutic targets for alcohol dependence.