Jill Bettinger - US grants

DESCRIPTION (provided by applicant): Alcohol abuse is a major social and economic problem for which current drug treatments are inadequate. A primary difficulty with the development of novel treatments for alcoholism is that the molecular nature of the interaction of the nervous system with the drug is incompletely understood. Alcohol is a small, easily diffusible molecule that probably interacts with many proteins in all neurons. A significant challenge for researchers is to determine which interactions are important for altering nervous system function and, ultimately, for the development of addiction. The nervous system mounts a dynamic response to exposure to alcohol that consists of several levels of the development of tolerance. The development of tolerance is important in the etiology of addiction. Variation in the ability to develop tolerance is an important component of the genetic diversity that predicts an individualbs propensity to abuse alcohol. We study the molecular mechanics of acute tolerance to ethanol, which develops during a single ethanol exposure. In mammals, this is observed as a recovery of coordination and cognitive ability at a blood ethanol level that is higher than the level that intoxicated the animal initially. In Caenorhabditis elegans (C. elegans), we observe acute tolerance as a recovery of coordinated locomotion during a single exposure to a constant dose of ethanol. C. elegans is an excellent model for the study of neural function because its extremely simple and well-characterized nervous system (302 neurons) contains at least 118 different neuronal cell types and uses many of the same neurotransmitters as are used by the mammalian brain. C. elegans genetics provides a facile means of dissecting nervous system function, and can be used effectively to address questions of drug effects on neurons. This project is designed to determine the molecular mechanisms of the development of acute tolerance. We will take a forward genetics approach by genetically screening for mutations that disrupt the development of acute tolerance to ethanol. The specific aims of this proposal are: 1- Genetically screen for mutations that disrupt development of acute tolerance to ethanol. 2- Molecularly characterize several of the mutations isolated in the genetic screen. 3- Characterize the function of the genes identified in Specific Aim 2 in acute tolerance. Together, the specific aims of this proposal are designed to thoroughly examine the molecular mechanisms of development of AFT. PUBLIC HEALTH RELEVANCE The project aims to use a forward genetic screen to identify genes in the nematode C. elegans that are required for the development of acute tolerance to ethanol. If the discovered genes are related to human genes then variation in those genes or in genes that regulate their activity or expression is likely to impact on an individual's initial response to alcohol and therefore could predispose that individual to alcoholism.

A more complete picture of the gene networks that regulate alcohol responses will improve our understanding of intoxication, the development of alcohol tolerance and, ultimately, addiction. The overall goal of this project is to use the model organism, Caenorhabditis elegans, to expand our understanding of gene networks that have roles in regulating acute behavioral responses to ethanol. Candidate genes that have been identified by mouse, human and Drosophila studies will be validated in a number of established behavioral assays in C. elegans. Once candidate genes have been confirmed using the worm system, the roles of other, known members of their genetic pathways will be determined. To complement our understanding of the ethanol response pathways in which candidate genes work, novel members of interacting gene networks that regulate ethanol responses in C. elegans will be identified using genetic strategies. Given the link between acute alcohol response and predisposition to alcoholism, the human gene networks homologous to those characterized in this study are likely to be strong candidates for genes that predispose an individual to alcoholism. The following specific aims describe the means by which these gene networks will be identified and assessed. 1. Confirm the involvement in ethanol responses of the candidate genes or gene networks identified by the human, mouse and Drosophila projects. Loss of function of the genes to be validated will be assessed for phenotypic effects on ethanol sensitivity and the development of acute tolerance using standardized behavioral assays. 2. The predicted mechanisms of the gene network-behavior relationships for candidate networks will be confirmed using genetic analysis of double-mutant combinations. 3. Using forward genetic strategies, we will isolate mutations in interacting members of the gene networks for a validated ethanol response gene that has unknown or uncertain network connections. Novel members of a gene network found to regulate ethanol responses will represent candidates for related studies in mice or flies and association studies in humans.

PROJECT SUMMARY An individual's initial level of response (LR) to ethanol (their naïve sensitivity) is highly heritable, and LR strongly predicts lifetime liability to develop alcohol dependence. The genes that regulate LR are therefore important targets for study, but despite intense investigation, they remain poorly understood. One limitation is that it has been difficult to apply what we learn about the genes affecting level of response in model organisms to identifying liability loci in human gene association studies. Here, we solve this problem by beginning with candidate genes initially identified in human studies and by using a genetic model to define the biological mechanisms by which these genes are likely to regulate LR and, subsequently, abuse liability. Recently, allelic variation in members of the SWI/SNF chromatin-remodeling protein complex has been associated with a diagnosis of alcohol dependence in humans. The goal of this proposal is to elucidate the mechanism by which this complex controls ethanol responses, and, because the SWI/SNF complex regulates transcription, to determine the downstream genes that mediate these effects. We study the molecular mechanisms by which SWI/SNF influences the neuronal response to ethanol using the genetic model organism, Caenorhabditis elegans. C. elegans is an excellent model for these studies, because there is striking conservation between the machinery of nervous system function in humans and worms, and there are rich genetic resources available to experimentally manipulate nervous system function in worms. C. elegans behavior is affected by relevant doses of ethanol, and genes that modify ethanol responses in worms also modify ethanol responses, including drinking behavior, in mammals. We have found that altering the function of the SWI/SNF complex in C. elegans alters acute behavioral responses to ethanol, demonstrating that the role of SWI/SNF in modifying the effects of ethanol is conserved. We will determine the specific neurons and neural circuits in which the SWI/SNF complex is required for acute ethanol responses, which will implicate specific neurotransmitter systems in these functions. Second, we will use whole genome expression analysis to identify genes that are regulated by the SWI/SNF complex in differentiated neurons; these will be candidates for mediators of acute ethanol responses. Finally, we will use genetic and behavioral analysis to identify the genes that are responsible for the acute behavioral response to ethanol, and determine the biological mechanisms by which they regulate ethanol responses. Together, these studies will provide novel insight into the biological processes that regulate the level of response to ethanol, a phenotype that is predictive of the development of alcohol dependence. Importantly, this work will also provide new candidate genes for liability loci that can be examined in human populations for association with liability to develop alcohol dependence.

Stem cells have the potential to generate a variety of different cell types and are important for the renewal of cells in adulthood, while differentiated cells are committed to perform a specific function. In recent years, researchers have found that differentiated cells can be converted into stem cells by introducing a few key genetic factors into the differentiated cell. In order to harness this potential for regenerative medicine, it is important to understand the genetic factors that give stem cells their unique potential. The genetic model organism Caenorhabditis elegans has many advantages for studying this process. Importantly, the fates of all cells in the animal are known. This research focuses on two cells that arise through a single cell division and have very different potential: one is capable of making all support cells of the reproductive system, while the other is differentiated. The genetic factors that are different between these two cells will be identified and this will provide insight into what makes a stem cell different from a differentiated cell. In order to train the next generation of STEM professionals, this research will be incorporated into an advanced undergraduate laboratory that gives students a genuine research experience. Students are expected to be from diverse backgrounds, including racial and ethnic groups that are traditionally underrepresented in STEM fields.

Adult stem and progenitor cells are capable of producing a few related cell types. The genes and molecular mechanisms that regulate and determine this capacity are not well understood. This proposal uses progenitors of the C. elegans reproductive system as a model for defining the genetic determinants of multipotency. The somatic gonadal progenitors (SGPs) are multipotent progenitors that generate all somatic tissues of the reproductive system. Each SGP is the product of a cell division that produces one SGP and one differentiated cell, the head mesodermal cell (hmc). Therefore, in this single cell division the potential to generate all of the somatic gonadal types is differentially segregated into one daughter cell. SWI/SNF chromatin remodeling complexes are highly conserved, large protein complexes that regulate chromatin structure and result in gene activation or repression. Molecularly distinct SWI/SNF complexes are expressed in pluripotent stem cells, multipotent progenitors, and differentiated cells, suggesting that different SWI/SNF complexes are likely to play important roles in each of these different cell states. Components of the C. elegans SWI/SNF complex are required to distinguish SGPs from their differentiated hmc sisters. The goals of this proposal are to identify the genes that distinguish multipotent SGPs from their differentiated hmc sisters, and to identify the mechanisms by which SWI/SNF chromatin remodeling complexes regulate these cell fates. These experiments will provide insight into the genes and molecular mechanisms that distinguish multipotent progenitors from differentiated cells, and will identify the role SWI/SNF chromatin remodeling complexes in these cell fates.

PROJECT SUMMARY Genetic variation in humans contributes to an individual's likelihood to develop an alcohol use disorder. The genetic variation that influences alcohol abuse liability is therefore an important target for study, but it has been difficult to identify specific liability genes. Laboratory studies in animal models have been extremely useful in elucidating the molecular pharmacology of alcohol (ethanol), but laboratory derived genetic manipulations rarely model the naturally occurring genetic variation that is observed in wild populations. As such, predicting relevant human allelic variation has been difficult. Here, we study the natural allelic variation found in wild strains of the the nematode worm Caenorhabditis elegans to identify alleles that are tolerated in the wild and can modulate the function of pathways that impact physiological responses to ethanol. C. elegans is an important model species with demonstrated relevance to humans; there is striking conservation between the machinery of nervous system function in worms and humans, and genes that influence ethanol response behaviors in worms also influence the likelihood to develop alcohol use disorders in humans. This collaborative proposal brings together the diverse expertise of two laboratories. We take advantage of a unique resource, recombinant inbred lines (RILs) derived from four genetically diverse wild strains to identify natural allelic variation that can modulate the effects of ethanol. We have shown that the parent wild-type strains and the derived RILs display a range of phenotypes in different behavioral responses ethanol. We will exploit the efficiency and ease of manipulating the C. elegans model to carry out high throughput analyses that will identify genetic variation that alters behavioral and/or transcriptional responses to ethanol. We will directly test the causal nature of candidate ethanol response allelic variants, identified by quantitative trait locus mapping, through the use of gene editing techniques (CRISPR-Cas9) to introduce the allele into strains that carry different alleles. The ability to compare and contrast the influence of genetic variants on different responses to ethanol brings further power to our analyses. We will identify the genes that impact the physiological responses in each behavior uniquely, and second, we will identify genes that affect the behavioral responses across ethanol response phenotypes. We will also assess the impact of alleles that modulate transcription in response to ethanol on behavioral responses. These different analyses can inform us of the molecular mechanisms underlying these different responses to ethanol. Together, these studies will provide both specific and more general novel insights into the neurogenetics of ethanol. We will establish the degree to which genetic variation in known or novel biological pathways that mediate or modulate the effect of ethanol can change responses to ethanol and the degree to which variation in those genes is tolerated in the wild. These data will inform our understanding of human liability to abuse alcohol.

PROJECT SUMMARY There are two, partially overlapping goals of this research: (1) comprehensively define the array of G protein- coupled neuropeptide receptors that act to modulate and mediate acute actions of ethanol in vivo. (2) investigate the impact of specific behavioral states on acute sensitivity to ethanol. We will characterize the roles of all neuropeptide receptors in C. elegans in basal locomotion behaviors and ethanol-induced behavioral effects. This comprehensive assessment will identify receptors that positively and negatively regulate the neuronal circuit that controls locomotion and those receptors that act to promote or negatively regulate ethanol actions. We will assess both the level of initial sensitivity to ethanol and the time-dependent development of acute functional tolerance to the drug. In addition, for those receptors that act to modify responses to ethanol, we will classify any interactions with the neuropeptidase nep-2, which is orthologous to the mammalian neprilysin protein. The mammalian neprilysin protein is involved in the regulation of levels of multiple important signaling peptides, including enkephalins, tachykinin, substance P and others. A mutation in the nep-2 gene produces an ethanol-resistant behavioral phenotype. We hypothesize that a peptide target of NEP-2, which is likely to be elevated in a nep-2 mutant background, acts to counteract acute effects of ethanol via increased signaling through a neuropeptide receptor. The proposed experiments will identify that receptor and test the hypothesis that the receptor acts in a defined neuronal circuit that controls behavioral state decisions. Our preliminary data has identified several mutants that affect both ethanol responses and a behavioral state decision that affects exploratory behavior. The circuit that regulates that decision is well defined, and includes the sites of action of neuropeptides and serotonin. We will define networks of genes that act to regulate that behavioral decision and ethanol responses, and test specific neurons in the controlling circuit for their role in regulating ethanol responses. The relationship between an emotional (or affective) state in humans and the problematic use of drugs of abuse is of significant interest. The successful outcome of this proposed research will provide a better understanding of how specific behavioral states, controlled by a known regulatory circuit, can impact the acute responses to an abused drug. There is a significant correlation between the level of an individual?s initial response to alcohol and their likelihood to develop an alcohol use disorder (AUD). Genetic variation in any of the human orthologs of the C. elegans genes identified in this study has the potential to alter an individual?s level of response to ethanol, and therefore could impact that individual?s predisposition to develop an AUD later in life.

Project Summary ? Project 3 The development of alcohol use disorder (AUD) results from an interaction between both environmental and genetic factors. Studies of the impact of environment on the risk to develop AUD have, to date, focused mainly on psychological characteristics (externalizing phenotypes, etc.) or on developmental defects associated with prenatal or adolescent exposure to alcohol. Here, we will study the role of the dietary omega-3 fatty acid eicosapentaenoic acid (EPA), an environmental factor, in adults for a role in modulating the acute behavioral response to ethanol. Our study of the alcohol-naive acute level of response (LR) is significant because this measure is strongly correlated with the liability to develop AUD. Using two model organisms (C. elegans and mouse), we have previously found that dietary omega-3 fatty acid levels significantly impact the acute LR to alcohol in ethanol-naive animals. In worms, we have found specifically that EPA is essential for the development of acute functional tolerance (AFT) to ethanol. We will use two complementary approaches to identify the mechanisms underlying the effects of EPA on the acute behavioral response to ethanol. In Aim 1 we will identify genes whose expression is regulated in response to different EPA levels (depleted, normal levels, above normal levels) in adult C. elegans. We will correlate gene expression changes and the time course of the effect of dietary EPA on AFT. We will directly test the roles of prioritized candidate genes in the development of AFT to identify the molecular pathways that are important for ethanol response behaviors and are affected by EPA levels. In Aim 2, we will use cutting edge lipidomics to determine what lipid species are derived from the dietary EPA. We will correlate the accumulation of EPA derived lipids to the time course of the effects of dietary EPA on AFT. We will identify candidate lipid mediators of the development of AFT. This will implicate molecular pathways in the machinery underlying AFT. These studies will provide novel insight into the roles of lipids in regulating the level of response to alcohol. Determination of the nature of these roles will enable future identification of the protein targets of ethanol, and the regulatory mechanisms affecting those proteins, that are impacted by these lipid-dependent functions. Finally, in Aim 3, we will continue a successful approach that uses the high-throughput genetic power of C. elegans to test the hypothesized roles of genes that have been implicated by other components of the VCU-ARC in behavioral responses to ethanol.