Adrian Rothenfluh - US grants

DESCRIPTION (provided by applicant): Alcohol abuse disorders are a major health care concern that affect millions of people every year in the United States. Alcoholism has a significant genetic etiology, but few genes are known that significantly contribute to the development of the disease. The goal of this proposal is to study genes regulating the actin cytoskeleton, and the molecular mechanisms by which they do so, to regulate ethanol-induced behaviors in Drosophila. This idea emerged in parallel in two model organisms. First, previous genetic screens for Drosophila mutants with altered behavioral responses to alcohol identified mutations in multiple genes regulating the actin cytoskeleton. Second, mice with a knock-out of an actin regulatory protein also showed altered responses to alcohol, including increased voluntary drinking. We therefore postulate that the dynamic regulation of the actin cytoskeleton is a major determinant of behavioral responses to ethanol. We propose genetic, molecular, biochemical, and cell culture approaches to define the roles of two small GTPase pathways, the Arf6, and Rho-family of GTPases in regulating alcohol-induced behaviors. First, we will study how two distinct isoforms of a regulatory protein of Rho-GTPases, RhoGAP18B, are differentially involved in the regulation of both ethanol-induced hyperactivity, and sedation. Second, we will test mutations in members of the Arf6 signaling pathway for their responses to alcohol. Arf6 regulates membrane traffic and actin dynamics at the plasma membrane. We will use genetic, and biochemical approaches to define what the molecular links are between the Rho and Arf6 signaling pathways. Third, we will test the contribution of other actin regulatory genes to ethanol-induced behaviors, notably the role of cofilin, an actin-severing protein that controls the balance between free globular and filamentous actin protein. The members of these small GTPase signaling pathways are highly conserved from Drosophila to mammals, and some are known to participate in complex behaviors such as learning and memory, in vertebrates and invertebrates. We predict that these pathways are also functionally conserved in their regulation of ethanol-induced behaviors. Alcoholism is a devastating disease that severely impacts personal, and public health. The proposed research will advance our understanding of the genetic basis for the development of alcoholism. This in turn, will result in the identification of new risk factors and potential therapeutic targets for the treatment of alcohol abuse disorders. PUBLIC HEALTH RELEVANCE: Alcoholism is a devastating disorder that significantly contributes to mortality, disability, and to healthcare costs. The goal of this research is to understand the genes that determine, and mediate the behavioral responses to alcohol. In conducting this research, we hope to identify risk factors for the development of alcoholism, as well as provide leads for the development of new therapeutic strategies aiding in the treatment of alcohol use disorders.

DESCRIPTION (provided by applicant): This R21 grant proposal aims to characterize the molecular mechanisms that transform initial alcohol exposures into long-lasting changes in the brain. Our work focuses on a family of chromatin modification enzymes, the histone demethylases (HDM). Epigenetic changes are increasingly being recognized as relevant in addiction and modulation of histone methylation at specific genomic loci often governs changes in gene transcription in response to environmental cues. Because of its ease of genetic manipulation, Drosophila represents a powerful platform on which to study how alterations in chromatin programming result in alcohol- induced changes in behavior. We seek to test the hypothesis that specific HDMs regulate the experience- dependent behavioral changes that accompany exposure to ethanol. To understand the in vivo role of these chromatin modification enzymes in alcohol tolerance and consumption preference, we have begun to systematically knock out every one of the 14 Drosophila HDM genes, all of which have closely related mammalian orthologs. To accomplish this goal, we are employing cutting-edge molecular techniques such a recombineering and homologous recombination. In this exploratory/developmental research grant we propose the following two aims: Aim1 seeks to systematically determine the ethanol tolerance and consumption preference phenotypes of Drosophila strains carrying genetic modifications in all 14 HDM genes. We will accomplish this aim by testing knock-out and overexpression lines for each HDM gene, and by quantitatively assessing their ethanol tolerance and consumption preference. In our preference assay, naive flies show a tendency to avoid food containing 15% ethanol, while ethanol pre-exposure transforms this avoidance into a significant preference. Our preliminary data suggests that at least two HDM knock-out lines show altered ethanol consumption preference. In Aim2, we propose to determine where functionally relevant HDMs bind throughout the genome, by performing chromatin immunoprecipitation coupled with massive parallel deep sequencing (ChIP-seq) using HA-tagged HDM proteins we have in hand. This work will yield a comprehensive map of genes differentially bound by functionally relevant HDMs upon a behavior-changing ethanol exposure. Determining the in vivo functions of HDMs in experience-dependent ethanol plasticity has the potential for high impact on understanding the development of human addiction. These proteins are highly conserved and represent a class of druggable enzymes that hold promise for the future development of therapeutic intervention.

???? PROJECT SUMMARY / ABSTRACT ??????????????????????????????????????????????????????? Millions of Americans abuse illicit drugs, including the stimulant cocaine. The annual economic burden this creates is in the hundreds of billions of dollars. There is a substantial genetic contribution to the development of substance abuse disorders (SUD), but many molecular pathways remain to be elucidated. The vinegar fly, Drosophila melanogaster, has been a genetic model organism for more than a hundred years. Major strides have been made in the last 10 years studying the behavioral responses to alcohol in flies, both in characterizing novel conserved genes and pathways, but also in the development of new assays that resemble addiction more closely. The goal of this proposal is to engineer Drosophila flies to self-administer cocaine. This compound normally acts as an aversive antiherbivore chemical, and we hypothesize that this is because cocaine amplifies dopaminergic signaling, which in flies is mainly, but not exclusively, aversive. First, we will determine whether flies self-administer cocaine. We will establish dose response curves, and test the effects of cocaine pre-exposure on cocaine preference/aversion. We will also test the impact of dopamine signaling on cocaine consumption. Second, we will engineer flies that contain cocaine-sensitive dopamine transporters only in these dopamine neurons required for the development of self-administration. In the other, aversive dopamine neurons, these engineered flies will contain a cocaine-insensitive dopamine transporter. The proposed experiments will yield a novel cocaine self-administration assay in a model organism that has historically demonstrated great economy of scale and success in elucidating the molecular mechanisms of numerous basic physiological, and behavioral processes.

???? PROJECT SUMMARY / ABSTRACT ??????????????????????????????????????????????????????? Alcohol abuse disorders (AUD) are a major health hazard that affects millions of people every year in the United States. Repeat alcohol exposure can lead to tolerance, increased preference and consumption. Such behavioral changes, and the accompanying neuronal plasticity, underlie addiction, yet many molecular mechanisms governing these changes remain to be elucidated. Signaling through the insulin receptor (InR) in neurons is known to modify plasticity, but its effects on alcohol-induced behavioral responses are not known. Our goal is to understand the cellular, neural, and circuit mechanisms of Arf6-dependent nervous system InR signaling in alcohol-induced plasticity. This is based on our published data that Arf6 mediates InR signaling in vivo and in cell culture. First, we will determine the mechanisms of Arf6-mediated InR signaling and endocytosis. We will systematically, and comprehensively test the role of two gene families known to directly regulate the function of Arf6. Second, we will determine neural circuits that specifically affect ethanol-induced tolerance and consumption preference, as well as the naïve aversive reactions to alcohol. We will test the role of Arf6 regulators in these circuits in alcohol-induced behavioral responses. Third, we will determine in vivo mechanisms how signaling from the InR pathway can affect distinct alcohol-induced behavioral changes: tolerance, and consumption preference. The genes we propose to investigate are all conserved from Drosophila to humans, and the proposed research will advance our molecular understanding of the mechanisms regulating alcohol-induced behavioral changes. This in turn, will result in the identification of new risk factors and therapeutic targets for the treatment of alcohol abuse disorders.

Dopamine neurons are a critical component of reward pathways in the brain. Historically, dopamine neurons were considered a relatively homogeneous population mediating association of reinforcement signals from food, water, and reproduction with coinciding sensory stimuli. However, recent studies have shown that these neurons form subpopulations with distinct physiological profiles, neural projections, and biological functions. These distinct subpopulations also have different roles in the progression of the addiction cycle, from use to abuse, abstinence and relapse. The goal of this application is to engineer novel genetic tools that will allow precise manipulation of distinct subpopulations of dopamine circuits, with specificity down to single pairs of neurons. To this end we will first apply a new technique, single-cell ATAC-seq, to determine all open chromatin/accessible DNA enhancer elements of every one of the ~250 dopamine neurons in the Drosophila brain. Second, we will determine which open enhancer fragments, or combinations thereof, will uniquely identify single dopamine cells/cell-types. And third, based on this analysis, we will engineer numerous new genetic tools and test them for their in vivo efficacy and specificity. While these Aims are linear and fully interdependent, the grant overall applies the very new technique of single-cell ATAC-seq to ?break new ground? and ?accelerate the pace of discoveries to advance addiction research?. The proposal essentially tests the hypothesis that this unbiased, genome-wide approach can be harnessed to generate new tools for precision intervention. Because the approach itself can be scaled, applied to any cell type, and translated to mammals, this application is fully consistent with the spirit of the Cutting-Edge Basic Research Awards (CEBRA) mechanism, which will ?support high-risk, high impact research? (PAR-18-437).