A neuropeptide signal confers ethanol state dependency during olfactory learning in Caenorhabditis elegans.

Alcohol intoxication can impact learning and this may contribute to the development of problematic alcohol use. In alcohol (ethanol)-induced state-dependent learning (SDL), information learned while an animal is intoxicated is recalled more effectively when the subject is tested while similarly intoxicated than if tested while not intoxicated. When Caenorhabditis elegans undergoes olfactory learning (OL) while intoxicated, the learning becomes state dependent such that recall of OL is only apparent if the animals are tested while intoxicated. We found that two genes known to be required for signal integration, the secreted peptide HEN-1 and its receptor tyrosine kinase, SCD-2, are required for SDL. Expression of hen-1 in the ASER neuron and scd-2 in the AIA neurons was sufficient for their functions in SDL. Optogenetic activation of ASER in the absence of ethanol during learning could confer ethanol state dependency, indicating that ASER activation is sufficient to signal ethanol intoxication to the OL circuit. To our surprise, ASER activation during testing did not substitute for ethanol intoxication, demonstrating that the effects of ethanol on learning and recall rely on distinct signals. Additionally, intoxication-state information could be added to already established OL, but state-dependent OL did not lose state information when the intoxication signal was removed. Finally, dopamine is required for state-dependent OL, and we found that the activation of ASER cannot bypass this requirement. Our findings provide a window into the modulation of learning by ethanol and suggest that ethanol acts to modify learning using mechanisms distinct from those used during memory access.

Genes regulating levels of ω-3 long-chain polyunsaturated fatty acids are associated with alcohol use disorder and consumption, and broader externalizing behavior in humans.

BACKGROUND: Individual variation in the physiological response to alcohol is predictive of an individual's likelihood to develop alcohol use disorder (AUD). Evidence from diverse model organisms indicates that the levels of long-chain polyunsaturated omega-3 fatty acids (ω-3 LC-PUFAs) can modulate the behavioral response to ethanol and therefore may impact the propensity to develop AUD. While most ω-3 LC-PUFAs come from diet, humans can produce these fatty acids from shorter chain precursors through a series of enzymatic steps. Natural variation in the genes encoding these enzymes has been shown to affect ω-3 LC-PUFA levels. We hypothesized that variation in these genes could contribute to the susceptibility to develop AUD. METHODS: We identified nine genes (FADS1, FADS2, FADS3, ELOVL2, GCKR, ELOVL1, ACOX1, APOE, and PPARA) that are required to generate ω-3 LC-PUFAs and/or have been shown or predicted to affect ω-3 LC-PUFA levels. Using both set-based and gene-based analyses we examined their association with AUD and two AUD-related phenotypes, alcohol consumption, and an externalizing phenotype. RESULTS: We found that the set of nine genes is associated with all three phenotypes. When examined individually, GCKR, FADS2, and ACOX1 showed significant association signals with alcohol consumption. GCKR was significantly associated with AUD. ELOVL1 and APOE were associated with externalizing. CONCLUSIONS: Taken together with observations that dietary ω-3 LC-PUFAs can affect ethanol-related phenotypes, this work suggests that these fatty acids provide a link between the environmental and genetic influences on the risk of developing AUD.

SWI/SNF complexes act through CBP-1 histone acetyltransferase to regulate acute functional tolerance to alcohol.

BACKGROUND: SWI/SNF chromatin remodeling genes are required for normal acute responses to alcohol in C. elegans and are associated with alcohol use disorder in two human populations. In an effort to discover the downstream genes that are mediating this effect, we identified SWI/SNF-regulated genes in C. elegans. RESULTS: To identify SWI/SNF-regulated genes in adults, we compared mRNA expression in wild type and swsn-1(os22ts) worms under conditions that produce inactive swsn-1 in mature cells. To identify SWI/SNF-regulated genes in neurons, we compared gene expression in swsn-9(ok1354) null mutant worms that harbor a neuronal rescue or a control construct. RNA sequencing was performed to an average depth of 25 million reads per sample using 50-base, paired-end reads. We found that 6813 transcripts were significantly differentially expressed between swsn-1(os22ts) mutants and wild-type worms and 2412 transcripts were significantly differentially expressed between swsn-9(ok1354) mutants and swsn-9(ok1354) mutants with neuronal rescue. We examined the intersection between these two datasets and identified 603 genes that were differentially expressed in the same direction in both comparisons; we defined these as SWI/SNF-regulated genes in neurons and in adults. Among the differentially expressed genes was cbp-1, a C. elegans homolog of the mammalian CBP/p300 family of histone acetyltransferases. CBP has been implicated in the epigenetic regulation in response to alcohol in animal models and a polymorphism in the human CBP gene, CREBBP, has been associated with alcohol-related phenotypes. We found that cbp-1 is required for the development of acute functional tolerance to alcohol in C. elegans. CONCLUSIONS: We identified 603 transcripts that were regulated by two different SWI/SNF complex subunits in adults and in neurons. The SWI/SNF-regulated genes were highly enriched for genes involved in membrane rafts, suggesting an important role for this membrane microdomain in the acute alcohol response. Among the differentially expressed genes was cbp-1; CBP-1 homologs have been implicated in alcohol responses across phyla and we found that C. elegans cbp-1 was required for the acute alcohol response in worms.

mRNA profiling reveals significant transcriptional differences between a multipotent progenitor and its differentiated sister.

BACKGROUND: The two Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic tissues of the adult reproductive system. The sister cells of the SGPs are two head mesodermal cells (hmcs); one hmc dies by programmed cell death and the other terminally differentiates. Thus, a single cell division gives rise to one multipotent progenitor and one differentiated cell with identical lineage histories. We compared the transcriptomes of SGPs and hmcs in order to learn the determinants of multipotency and differentiation in this lineage. RESULTS: We generated a strain that expressed fluorescent markers specifically in SGPs (ehn-3A::tdTomato) and hmcs (bgal-1::GFP). We dissociated cells from animals after the SGP/hmc cell division, but before the SGPs had further divided, and subjected the dissociated cells to fluorescence-activated cell sorting to collect isolated SGPs and hmcs. We analyzed the transcriptomes of these cells and found that 5912 transcripts were significantly differentially expressed, with at least two-fold change in expression, between the two cell types. The hmc-biased genes were enriched with those that are characteristic of neurons. The SGP-biased genes were enriched with those indicative of cell proliferation and development. We assessed the validity of our differentially expressed genes by examining existing reporters for five of the 10 genes with the most significantly biased expression in SGPs and found that two showed expression in SGPs. For one reporter that did not show expression in SGPs, we generated a GFP knock-in using CRISPR/Cas9. This reporter, in the native genomic context, was expressed in SGPs. CONCLUSIONS: We found that the transcriptional profiles of SGPs and hmcs are strikingly different. The hmc-biased genes are enriched with those that encode synaptic transmission machinery, which strongly suggests that it has neuron-like signaling properties. In contrast, the SGP-biased genes are enriched with genes that encode factors involved in transcription and translation, as would be expected from a cell preparing to undergo proliferative divisions. Mediators of multipotency are likely to be among the genes differentially expressed in SGPs.

Long-Chain ω-3 Levels Are Associated With Increased Alcohol Sensitivity in a Population-Based Sample of Adolescents.

BACKGROUND: The levels of the ω-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been associated with alcohol sensitivity in vertebrate and invertebrate model systems, but prior studies have not examined this association in human samples despite evidence of associations between ω-3 LC-PUFA levels and alcohol-related phenotypes. Both alcohol sensitivity and ω-3 LC-PUFA levels are impacted by genetic factors, and these influences may contribute to observed associations between phenotypes. Given the potential for using EPA and DHA supplementation in adjuvant care for alcohol misuse and other outcomes, it is important to clarify how ω-3 LC-PUFA levels relate to alcohol sensitivity. METHODS: Analyses were conducted using data from the Avon Longitudinal Study of Parents and Children. Plasma ω-3 LC-PUFA levels were measured at ages 15.5 and 17.5. Participants reported on their initial alcohol sensitivity using the early drinking Self-Rating of the Effects of Alcohol (SRE-5) scale, for which more drinks needed for effects indicates lower levels of response per drink, at ages 15.5, 16.5, and 17.5. Polygenic liability for alcohol consumption, alcohol problems, EPA levels, and DHA levels was derived using summary statistics from large, publicly available datasets. Linear regressions were used to examine the cross-sectional and longitudinal associations between ω-3 LC-PUFA levels and SRE scores. RESULTS: Age 15.5 ω-3 LC-PUFA levels were negatively associated with contemporaneous SRE scores and with age 17.5 SRE scores. One modest association (p = 0.02) between polygenic liability and SRE scores was observed, between alcohol problems-based polygenic risk scores (PRS) and age 16.5 SRE scores. Tests of moderation by genetic liability were not warranted. CONCLUSIONS: Plasma ω-3 LC-PUFA levels may be related to initial sensitivity to alcohol during adolescence. These data indicate that diet-related factors have the potential to impact humans' earliest responses to alcohol exposure.

Dietary Omega-3 Fatty Acids Differentially Impact Acute Ethanol-Responsive Behaviors and Ethanol Consumption in DBA/2J Versus C57BL/6J Mice.

BACKGROUND: Complex interactions between environmental and genetic factors influence the risk of developing alcohol use disorder (AUD) in humans. To date, studies of the impact of environment on AUD risk have primarily focused on psychological characteristics or on the effects of developmental exposure to ethanol (EtOH). We recently observed that modifying levels of the long-chain ω-3 (LC ω-3) fatty acid, eicosapentaenoic acid (EPA), alters acute physiological responses to EtOH in Caenorhabditis elegans. Because mammals derive ω-3 fatty acids from their diet, here we asked if manipulating dietary levels of LC ω-3 fatty acids can affect EtOH-responsive behaviors in mice. METHODS: We used 2 well-characterized inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), which differ in their responses to EtOH. Age-matched young adult male mice were maintained on isocaloric diets that differed only by being enriched or depleted in LC ω-3 fatty acids. Animals were subsequently tested for acute EtOH sensitivity (locomotor activation and sedation), voluntary consumption, and metabolism. Fat deposition was also determined. RESULTS: We found that dietary levels of LC ω-3s altered EtOH sensitivity and consumption in a genotype-specific manner. Both B6 and D2 animals fed high LC ω-3 diets demonstrated lower EtOH-induced locomotor stimulation than those fed low LC ω-3 diets. EtOH sedation and EtOH metabolism were greater in D2, but not B6 mice on the high LC ω-3 diet. Conversely, LC ω-3 dietary manipulation altered EtOH consumption in B6, but not in D2 mice. B6 mice on a high LC ω-3 diet consumed more EtOH in a 2-bottle choice intermittent access model than B6 mice on a low LC ω-3 diet. CONCLUSIONS: Because EtOH sensitivity is predictive of risk of developing AUD in humans, our data indicate that dietary LC ω-3 levels should be evaluated for their impact on AUD risk in humans. Further, these studies indicate that genetic background can interact with fatty acids in the diet to significantly alter EtOH-responsive behaviors.

Voltage-Sensitive Potassium Channels of the BK Type and Their Coding Genes Are Alcohol Targets in Neurons.

Among all members of the voltage-gated, TM6 ion channel superfamily, the proteins that constitute calcium- and voltage-gated potassium channels of large conductance (BK) and their coding genes are unique for their involvement in ethanol-induced disruption of normal physiology and behavior. Moreover, in vitro studies document that BK activity is modified by ethanol with an EC50~23 mM, which is near blood alcohol levels considered legal intoxication in most states of the USA (0.08 g/dL = 17.4 mM). Following a succinct introduction to our current understanding of BK structure and function in central neurons, with a focus on neural circuits that contribute to the neurobiology of alcohol use disorders (AUD), we review the modifications in organ physiology by alcohol exposure via BK and the different molecular elements that determine the ethanol response of BK in alcohol-naïve systems, including the role of an ethanol-recognizing site in the BK-forming slo1 protein, modulation of accessory BK subunits, and their coding genes. The participation of these and additional elements in determining the response of a system or an organism to protracted ethanol exposure is consequently analyzed, with insights obtained from invertebrate and vertebrate models. Particular emphasis is put on the role of BK and coding genes in different forms of tolerance to alcohol exposure. We finally discuss genetic results on BK obtained in invertebrate organisms and rodents in light of possible extrapolation to human AUD.

SWI/SNF chromatin remodeling regulates alcohol response behaviors in Caenorhabditis elegans and is associated with alcohol dependence in humans.

Alcohol abuse is a widespread and serious problem. Understanding the factors that influence the likelihood of abuse is important for the development of effective therapies. There are both genetic and environmental influences on the development of abuse, but it has been difficult to identify specific liability factors, in part because of both the complex genetic architecture of liability and the influences of environmental stimuli on the expression of that genetic liability. Epigenetic modification of gene expression can underlie both genetic and environmentally sensitive variation in expression, and epigenetic regulation has been implicated in the progression to addiction. Here, we identify a role for the switching defective/sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex in regulating the behavioral response to alcohol in the nematode Caenorhabditis elegans. We found that SWI/SNF components are required in adults for the normal behavioral response to ethanol and that different SWI/SNF complexes regulate different aspects of the acute response to ethanol. We showed that the SWI/SNF subunits SWSN-9 and SWSN-7 are required in neurons and muscle for the development of acute functional tolerance to ethanol. Examination of the members of the SWI/SNF complex for association with a diagnosis of alcohol dependence in a human population identified allelic variation in a member of the SWI/SNF complex, suggesting that variation in the regulation of SWI/SNF targets may influence the propensity to develop abuse disorders. Together, these data strongly implicate the chromatin remodeling associated with SWI/SNF complex members in the behavioral responses to alcohol across phyla.

Variation in SWI/SNF Chromatin Remodeling Complex Proteins is Associated with Alcohol Dependence and Antisocial Behavior in Human Populations.

BACKGROUND: Testing for direct gene or single nucleotide polymorphism replication of association across studies may not capture the true importance of a candidate locus; rather, we suggest that relevant replication across studies may be found at the level of a biological process. We previously observed that variation in 2 members of the switching defective/sucrose nonfermenting (SWI/SNF) chromatin remodeling complex is associated with alcohol dependence (AD) in the Irish Affected Sib Pair Study for Alcohol Dependence. Here, we tested for association with alcohol-related outcomes using a set of genes functioning in the SWI/SNF complex in 2 independent samples. METHODS: We used a set-based analysis to examine the 29 genes of the SWI/SNF complex for evidence of association with (i) AD in the adult Collaborative Study on the Genetics of Alcoholism (COGA) case-control sample and (ii) antisocial behavior, hypothesized to be a genetically related developmental precursor, in a younger population sample (Spit for Science [S4S]). RESULTS: We found evidence for association of the SWI/SNF complex with AD in COGA (p = 0.0435) and more general antisocial behavior in S4S (p = 0.00026). The genes that contributed most strongly to the signal in COGA were SS18L1, SMARCD1, BRD7, BCL7B, SMARCB1, and BCL11A. In the S4S sample, ACTB, ARID2, BCL11A, BCL11B, BCL7B, BCL7C, DPF2, and DPF3 all contributed strongly to the signal. CONCLUSIONS: We detected associations between the SWI/SNF complex and AD in an adult population selected from treatment-seeking probands and antisocial behavior in an adolescent population sample. This provides strong support for a role for SWI/SNF in the development of alcohol-related problems.

Nicotine Enhances the Hypnotic and Hypothermic Effects of Alcohol in the Mouse.

BACKGROUND: Ethanol (EtOH) and nicotine abuse are 2 leading causes of preventable mortality in the world, but little is known about the pharmacological mechanisms mediating co-abuse. Few studies have examined the interaction of the acute effects of EtOH and nicotine. Here, we examine the effects of nicotine administration on the duration of EtOH-induced loss of righting reflex (LORR) and characterize the nature of their pharmacological interactions in C57BL/6J mice. METHODS: We assessed the effects of EtOH and nicotine and the nature of their interaction in the LORR test using isobolographic analysis after acute injection in C57BL/6J male mice. Next, we examined the importance of receptor efficacy using nicotinic partial agonists varenicline and sazetidine. We evaluated the involvement of major nicotinic acetylcholine receptor (nAChR) subtypes using nicotinic antagonist mecamylamine and nicotinic α4- and α7-knockout mice. The selectivity of nicotine's actions on EtOH-induced LORR was examined by testing nicotine's effects on the hypnotic properties of ketamine and pentobarbital. We also assessed the development of tolerance after repeated nicotine exposure. Last, we assessed whether the effects of nicotine on EtOH-induced LORR extend to hypothermia and EtOH intake in the drinking in the dark (DID) paradigm. RESULTS: We found that acute nicotine injection enhances EtOH's hypnotic effects in a synergistic manner and that receptor efficacy plays an important role in this interaction. Furthermore, tolerance developed to the enhancement of EtOH's hypnotic effects by nicotine after repeated exposure of the drug. α4* and α7 nAChRs seem to play an important role in nicotine-EtOH interaction in the LORR test. In addition, the magnitude of EtOH-induced LORR enhancement by nicotine was more pronounced in C57BL/6J than DBA/2J mice. Furthermore, acute nicotine enhanced ketamine and pentobarbital hypnotic effects in the mouse. Finally, nicotine enhanced EtOH-induced hypothermia but decreased EtOH intake in the DID test. CONCLUSIONS: Our results demonstrate that nicotine synergistically enhances EtOH-induced LORR in the mouse.

Drosophila and Caenorhabditis elegans as Discovery Platforms for Genes Involved in Human Alcohol Use Disorder.

BACKGROUND: Despite the profound clinical significance and strong heritability of alcohol use disorder (AUD), we do not yet have a comprehensive understanding of the naturally occurring genetic variance within the human genome that drives its development. This lack of understanding is likely to be due in part to the large phenotypic and genetic heterogeneities that underlie human AUD. As a complement to genetic studies in humans, many laboratories are using the invertebrate model organisms (iMOs) Drosophila melanogaster (fruit fly) and Caenorhabditis elegans (nematode worm) to identify genetic mechanisms that influence the effects of alcohol (ethanol) on behavior. While these extremely powerful models have identified many genes that influence the behavioral responses to alcohol, in most cases it has remained unclear whether results from behavioral-genetic studies in iMOs are directly applicable to understanding the genetic basis of human AUD. METHODS: In this review, we critically evaluate the utility of the fly and worm models for identifying genes that influence AUD in humans. RESULTS: Based on results published through early 2015, studies in flies and worms have identified 91 and 50 genes, respectively, that influence 1 or more aspects of behavioral responses to alcohol. Collectively, these fly and worm genes correspond to 293 orthologous genes in humans. Intriguingly, 51 of these 293 human genes have been implicated in AUD by at least 1 study in human populations. CONCLUSIONS: Our analyses strongly suggest that the Drosophila and C. elegans models have considerable utility for identifying orthologs of genes that influence human AUD.

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays.

BACKGROUND: The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white(mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated. METHODS: We manipulated mini-w and w expression via (i) transposons marked with mini-w, (ii) RNAi against mini-w and w, and (iii) a null allele of w. We assessed EtOH sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation. RESULTS: In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies. CONCLUSIONS: EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described here is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w.

PBRM-1/PBAF-regulated genes in a multipotent progenitor in Caenorhabditis elegans.

The Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic cells of the adult reproductive system. The 2 SGPs originate in the mesodermal layer and are born through a division that produces one SGP and one head mesodermal cell (hmc). One hmc terminally differentiates, and the other dies by programmed cell death. The polybromo-associated BAF (PBAF) chromatin remodeling complex promotes the multipotent SGP fate. The complete loss of PBAF causes lethality, so we used a combination of Cre/lox recombination and GFP nanobody-directed protein degradation to eliminate PBRM-1, the signature subunit of the PBAF complex, from 83 mesodermal cells, including SGPs, body muscles, and the hmc. We used RNA sequencing to identify genes acting downstream of PBAF in these cells and identified 1,955 transcripts that were significantly differentially expressed between pbrm-1(-) and pbrm-1(+) in the mesoderm of L1 larvae. We found that genes involved in muscle cell function were overrepresented; most of these genes had lower expression in the absence of PBRM-1, suggesting that PBAF promotes muscle differentiation. Among the differentially expressed genes were 125 that are normally expressed at higher levels in SGP vs hmc and positively regulated by pbrm-1 and 53 that are normally expressed at higher levels in hmc vs SGP and are negatively regulated by pbrm-1; these are candidate regulators of the SGP/hmc fate decision. We validated one candidate gene using a fluorescent reporter; the hsp-12.3 reporter was derepressed in SGPs in pbrm-1 mutants, suggesting that hsp-12.3 expression is normally repressed by pbrm-1 in SGPs.

Natural allelic variation modifies acute ethanol response phenotypes in wild strains of C. elegans.

BACKGROUND: Genetic variation contributes to the likelihood that an individual will develop an alcohol use disorder (AUD). Traditional laboratory studies in animal models have elucidated the molecular pharmacology of ethanol, but laboratory-derived genetic manipulations rarely model the naturally occurring genetic variation observed in wild populations. Rather, these manipulations are biased toward identifying genes of central importance in the phenotypes. Because changes in such genes can confer selective disadvantages, they are not ideal candidates for carrying AUD risk alleles in humans. We sought to exploit Caenorhabditis elegans to identify allelic variation existing in the wild that modulates ethanol response behaviors. METHODS: We tested the acute ethanol responses of four strains recently isolated from the wild (JU1511, JU1926, JU1931, and JU1941) and 41 multiparental recombinant inbred lines (mpRILs) derived from them. We assessed locomotion at 10, 30, and 50 min on low and high ethanol concentrations. We performed principal component analyses (PCA) on the different phenotypes, tested for transgressive behavior, calculated heritability, and determined the correlations between behavioral responses. RESULTS: We observed a range of responses to ethanol across the strains. We detected a low-concentration locomotor activation effect in some of the mpRILs not seen in the laboratory wild-type strain. PCA showed different ethanol response behaviors to be independent. We observed transgressive behavior for many of the measured phenotypes and found that multiple behaviors were uncorrelated. The average broad-sense heritability for all phenotypes was 23.2%. CONCLUSIONS: Genetic variation significantly affects multiple acute ethanol response behaviors, many of which are independent of one another. This suggests that the genetic variation captured by these strains likely affects multiple biological mechanisms through which ethanol acts. Further study of these strains may allow these distinct mechanisms to be identified.

Case-only exome variation analysis of severe alcohol dependence using a multivariate hierarchical gene clustering approach.

BACKGROUND: Variation in genes involved in ethanol metabolism has been shown to influence risk for alcohol dependence (AD) including protective loss of function alleles in ethanol metabolizing genes. We therefore hypothesized that people with severe AD would exhibit different patterns of rare functional variation in genes with strong prior evidence for influencing ethanol metabolism and response when compared to genes not meeting these criteria. OBJECTIVE: Leverage a novel case only design and Whole Exome Sequencing (WES) of severe AD cases from the island of Ireland to quantify differences in functional variation between genes associated with ethanol metabolism and/or response and their matched control genes. METHODS: First, three sets of ethanol related genes were identified including those a) involved in alcohol metabolism in humans b) showing altered expression in mouse brain after alcohol exposure, and altering ethanol behavioral responses in invertebrate models. These genes of interest (GOI) sets were matched to control gene sets using multivariate hierarchical clustering of gene-level summary features from gnomAD. Using WES data from 190 individuals with severe AD, GOI were compared to matched control genes using logistic regression to detect aggregate differences in abundance of loss of function, missense, and synonymous variants, respectively. RESULTS: Three non-independent sets of 10, 117, and 359 genes were queried against control gene sets of 139, 1522, and 3360 matched genes, respectively. Significant differences were not detected in the number of functional variants in the primary set of ethanol-metabolizing genes. In both the mouse expression and invertebrate sets, we observed an increased number of synonymous variants in GOI over matched control genes. Post-hoc simulations showed the estimated effects sizes observed are unlikely to be under-estimated. CONCLUSION: The proposed method demonstrates a computationally viable and statistically appropriate approach for genetic analysis of case-only data for hypothesized gene sets supported by empirical evidence.

Multi-species data integration and gene ranking enrich significant results in an alcoholism genome-wide association study.

BACKGROUND: A variety of species and experimental designs have been used to study genetic influences on alcohol dependence, ethanol response, and related traits. Integration of these heterogeneous data can be used to produce a ranked target gene list for additional investigation. RESULTS: In this study, we performed a unique multi-species evidence-based data integration using three microarray experiments in mice or humans that generated an initial alcohol dependence (AD) related genes list, human linkage and association results, and gene sets implicated in C. elegans and Drosophila. We then used permutation and false discovery rate (FDR) analyses on the genome-wide association studies (GWAS) dataset from the Collaborative Study on the Genetics of Alcoholism (COGA) to evaluate the ranking results and weighting matrices. We found one weighting score matrix could increase FDR based q-values for a list of 47 genes with a score greater than 2. Our follow up functional enrichment tests revealed these genes were primarily involved in brain responses to ethanol and neural adaptations occurring with alcoholism. CONCLUSIONS: These results, along with our experimental validation of specific genes in mice, C. elegans and Drosophila, suggest that a cross-species evidence-based approach is useful to identify candidate genes contributing to alcoholism.

Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.

BACKGROUND: Ethanol (EtOH) is metabolized by a 2-step process in which alcohol dehydrogenase (ADH) oxidizes EtOH to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase (ALDH). Although variation in EtOH metabolism in humans strongly influences the propensity to chronically abuse alcohol, few data exist on the behavioral effects of altered EtOH metabolism. Here, we used the nematode Caenorhabditis elegans to directly examine how changes in EtOH metabolism alter behavioral responses to alcohol during an acute exposure. Additionally, we investigated EtOH solution osmolarity as a potential explanation for contrasting published data on C. elegans EtOH sensitivity. METHODS: We developed a gas chromatography assay and validated a spectrophotometric method to measure internal EtOH in EtOH-exposed worms. Further, we tested the effects of mutations in ADH and ALDH genes on EtOH tissue accumulation and behavioral sensitivity to the drug. Finally, we tested the effects of EtOH solution osmolarity on behavioral responses and tissue EtOH accumulation. RESULTS: Only a small amount of exogenously applied EtOH accumulated in the tissues of C. elegans and consequently their tissue concentrations were similar to those that intoxicate humans. Independent inactivation of an ADH-encoding gene (sodh-1) or an ALDH-encoding gene (alh-6 or alh-13) increased the EtOH concentration in worms and caused hypersensitivity to the acute sedative effects of EtOH on locomotion. We also found that the sensitivity to the depressive effects of EtOH on locomotion is strongly influenced by the osmolarity of the exogenous EtOH solution. CONCLUSIONS: Our results indicate that EtOH metabolism via ADH and ALDH has a statistically discernable but surprisingly minor influence on EtOH sedation and internal EtOH accumulation in worms. In contrast, the osmolarity of the medium in which EtOH is delivered to the animals has a more substantial effect on the observed sensitivity to EtOH.

Transcription factors regulating the fate and developmental potential of a multipotent progenitor in Caenorhabditis elegans.

Multipotent stem and progenitor cells have the capacity to generate a limited array of related cell types. The Caenorhabditis elegans somatic gonadal precursors are multipotent progenitors that generate all 143 cells of the somatic gonad, including complex tissues and specialized signaling cells. To screen for candidate regulators of cell fate and multipotency, we identified transcription factor genes with higher expression in somatic gonadal precursors than in their differentiated sister, the head mesodermal cell. We used RNA interference or genetic mutants to reduce the function of 183 of these genes and examined the worms for defects in the somatic gonadal precursor cell fate or the ability to generate gonadal tissue types. We identify 8 genes that regulate somatic gonadal precursor fate, including the SWI/SNF chromatin remodeling complex gene swsn-3 and the Ci/GLI homolog tra-1, which is the terminal regulator of sex determination. Four genes are necessary for somatic gonadal precursors to generate the correct number and type of descendant cells. We show that the E2F homolog, efl-3, regulates the cell fate decision between distal tip cells and the sheath/spermathecal precursor. We find that the FACT complex gene hmg-4 is required for the generation of the correct number of somatic gonadal precursor descendants, and we define an earlier role for the nhr-25 nuclear hormone receptor-encoding gene, in addition to its previously described role in regulating the asymmetric division of somatic gonadal precursors. Overall, our data show that genes regulating cell fate are largely different from genes regulating developmental potential, demonstrating that these processes are genetically separable.

Transcriptional analysis of the response of C. elegans to ethanol exposure.

Ethanol-induced transcriptional changes underlie important physiological responses to ethanol that are likely to contribute to the addictive properties of the drug. We examined the transcriptional responses of Caenorhabditis elegans across a timecourse of ethanol exposure, between 30 min and 8 h, to determine what genes and genetic pathways are regulated in response to ethanol in this model. We found that short exposures to ethanol (up to 2 h) induced expression of metabolic enzymes involved in metabolizing ethanol and retinol, while longer exposure (8 h) had much more profound effects on the transcriptome. Several genes that are known to be involved in the physiological response to ethanol, including direct ethanol targets, were regulated at 8 h of exposure. This longer exposure to ethanol also resulted in the regulation of genes involved in cilia function, which is consistent with an important role for the effects of ethanol on cilia in the deleterious effects of chronic ethanol consumption in humans. Finally, we found that food deprivation for an 8-h period induced gene expression changes that were somewhat ameliorated by the presence of ethanol, supporting previous observations that worms can use ethanol as a calorie source.

An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.

BACKGROUND: Ethanol induces similar behavioral responses in mammals and the fruit fly, Drosophila melanogaster. By coupling assays for ethanol-related behavior to the genetic tools available in flies, a number of genes have been identified that influence physiological responses to ethanol. To enhance the utility of the Drosophila model for investigating genes involved in ethanol-related behavior, we explored the value of an assay that measures the sedative effects of ethanol on negative geotaxis, an evoked locomotor response. METHODS: We established eRING (ethanol Rapid Iterative Negative Geotaxis) as an assay for quantitating the sedative effects of ethanol on negative geotaxis (i.e., startle-induced climbing). We validated the assay by assessing acute sensitivity to ethanol and rapid ethanol tolerance in several different control strains and in flies with mutations known to disrupt these behaviors. We also used eRING in a candidate screen to identify mutants with altered ethanol-related behaviors. RESULTS: Negative geotaxis measured in eRING assays was dose-dependently impaired by ethanol exposure. Flies developed tolerance to the intoxicating effects of ethanol when tested during a second exposure. Ethanol sensitivity and rapid ethanol tolerance varied across 4 control strains, but internal ethanol concentrations were indistinguishable in the 4 strains during a first and second challenge with ethanol. Ethanol sensitivity and rapid ethanol tolerance, respectively, were altered in flies with mutations in amnesiac and hangover, genes known to influence these traits. Additionally, mutations in the beta integrin gene myospheroid and the alpha integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations. CONCLUSIONS: The eRING assay is suitable for investigating genetic mechanisms that influence ethanol sensitivity and rapid ethanol tolerance. Ethanol sensitivity and rapid ethanol tolerance depend on the function of alpha and beta integrins in flies.