Robert O. Messing - US grants

NINCDS support is requested for the study of voltage-dependent Ca2+ channel regulation by protein kinase C. Phorbol ester tumor promoters, which activate protein kinase C, inhibit Ca2+ influx through voltage-dependent Ca2+ channels in the cultured neural cell line, PC12. The interaction between phorbol esters and Ca2+ channels will be examined in detail by studying K+-stimulated 45Ca2+ uptake and 3H-Ca2+ channel antagonist binding in PC12 cells. Muscarinic agonists also activate protein kinase C, by stimulating phosphoinositide hydrolysis and diacylglycerol production, and the effect of these agents on K+-stimulated 45Ca2+ uptake will be studied as well. Ca2+ channel antagonist receptors, which are functionally linked to Ca2+ channels in PC12, will be isolated by extensive purification or affinity labeling, and receptors will be phosphorylated by protein kinase C in vitro. In vivo phosphorylation by protein kinase C will be studied by treating 32P-labeled cells with phorbol esters or muscarinic agonists, and by measuring 32P incorporation into Ca2+ channel antagonist receptors isolated from these cells. Finally, the autoradiographic distribution of phorbol ester and Ca2+ channel antagonist receptors will be studied in brain slices from epileptic mutant mice to investigate changes in Ca2+ channel density or protein kinase C content that may predispose to epilepsy. Seizure-induced changes in Ca2+ channel number and protein kinase C levels will be examined by autoradiography of brain slices from mice subjected to chemically- or electrically-induced convulsions. The ultimate goal of this proposal is to delineate molecular events that underlie regulation of voltage-dependent Ca2+ channels by protein kinase C and to investigate whether disturbances in regulation might be involved in susceptibility to epilepsy, or in neuronal injury following repeated seizures. These studies may reveal information that will lead to better understanding and more effective treatment of epileptic disorders.

A major challenge in alcohol research is to determine the molecular events that underlie alcohol abuse. Much evidence implicates ethanol- induced increases n voltage-dependent calcium channels in alcohol dependence and manifestations of alcohol withdrawal in animals. Studies in our laboratory, using the neural cell line PC12, demonstrated that exposure to 25-200 mM ethanol for 2-6 days increases depolarization- stimulated calcium uptake, which remains elevated for several hours following removal of ethanol. This is associated with an increase in the number of binding sites for dihydropyridine calcium channel antagonists, indicating that ethanol increases the number of functional L-type calcium channels. The process by which this occurs requires protein kinase C (PKC) and chronic ethanol exposure appears to activate PKC by increasing levels of two isozymes, deltaPKC and epsilon PKC. The first goal of this project is to determine which of these isozymes mediates up-regulation of L-channels by ethanol. Since L-channels are composed of multiple subunits, a second goal is to identify which subunits are regulated by ethanol. The final goal is to determine whether similar PKC isozymes and L-channel subunits are regulated by ethanol in experimental animals. Studies are planned to use antisense DNA and RNA to inhibit PKC isozyme expression and block ethanol's effects on L-channels, and to make stable transfectants of PC12 cells that over-express PKC isozymes to mimic or enhance ethanol's effects. Antibodies will be made against L-channel subunits which will be analyzed for ethanol-induced changes in abundance by Northern analysis, Western analysis, and immunoassay. Finally, strains of mice bred for susceptibility or resistance to alcohol withdrawal seizures will be provide specific information about which PKC isozymes and L-channel subunits are involved in up-regulation of calcium channels by ethanol. In addition, they may uncover differences in regulations of L-channels by PCK that underlie genetic differences in the development of alcohol dependence and susceptibility to withdrawal seizures. This will advance the search for genes involved in the development of dependence on alcohol.

Chronic ethanol exposure can damage the nervous system, in part, by altering the growth of neural processes (neurites). In some regions of the brain, ethanol increases neurite growth. This could harm the nervous system by slowing conduction down dendrites to the cell body, and by disturbing the balanced development and organization of synapses. PC12 cells, which form neurites in response to nerve growth factor (NGF), have been used to study mechanisms involved in neurite outgrowth. Ethanol enhances NGF-induced neurite outgrowth by a process that is dependent on protein kinase C (PKC). Chronic ethanol exposure increases levels of two PKC isozymes, delta and epsilon, and increases PKC activity in PC12 cells. Studies outlined in this proposal will examine whether these PKC isozymes mediate enhancement of neurite outgrowth by ethanol in PC12 cells and primary cultures of cerebellar Purkinje cells which are known to respond to NGF and ethanol with increased neurite growth. In addition to increasing NGF-induced neurite outgrowth, ethanol also enhances NGF- induced stimulation of mitogen-activated protein kinases, suggesting that ethanol regulates NGF signal transduction. Studies are planned to investigate whether ethanol enhances NGF signaling by promoting the activity of the GTP-binding protein Ras or the kinase Raf-1, which are critical for NGF-induced neural differentiation. Methods to be used include PKC assay in cell homogenates and permeabilized cells, MAP kinase assays, assays for Ras-GTP formation, Ras GTPase activity, and Raf-1 activity, Western analyses, and stable transfection of PC12 cells to over- express PKC isozymes and to express antisense RNA and dominant-negative mutants. In addition, oligodeoxynucleotides and new PKC antagonists will be studied for isozyme-specific activity in PC12 cells. These will then be used to inhibit specific PKC isozymes in Purkinje cell cultures. These studies will provide new information about a biochemical mechanism that may be important in the pathogenesis of brain damage in alcoholics and of neurologic abnormalities in children born to alcoholic mothers. In addition, these studies will identify and test PKC isozyme-selective agents that may eventually prove useful as drugs in the treatment of ethanol neurotoxicity.

DESCRIPTION (provided by applicant): Studies with protein kinase C epsilon (PKC Epsilon) null mice indicate that PKC Epsilon regulates anxiety and alcohol consumption, dependence and reward. This appears to be related to actions at GABAA receptors since these mice are supersensitive to allosteric GABAA receptor agonists, including ethanol, in vivo and in vitro. The overall goal of this project is to understand how PKC Epsilon regulates GABAA receptor function. Studies will use immunohistochemistry and receptor binding autoradiography to compare the distribution of specific GABAA receptor subunits in wild type and PKC Epsilon null mice: Autoradiography will also be used to determine the affinity and density of binding sites for ligands that bind to benzodiazepine sites on specific receptor subpopulations. Western analysis will be used to measure the abundance of receptor subunits in limbic brain regions that express PKC Epsilon. L(tk-) fibroblast cell lines expressing different types of receptors will be examined to determine if PKC Epsilon regulation of allosteric sensitivity is specific for a particular subunit combination. Finally a PKC Epsilon mutant that can utilize novel ATP analogs as phosphate donors will be used to identify immediate substrates of PKC Epsilon in L(tk-) cells and in synaptoneurosomes, using immunoprecipitations with antibodies to GABAA receptor subunits and associated proteins, and 2D gel electrophoresis and mass spectrometry for unknown substrates. The hope is to define a PKC Epsilon pathway to further our understanding of GABAA receptor function as it relates to alcoholism.

Discoveries during the prior period of support demonstrated that PKCe is a key enzyme that down-regulates GABAA receptor function through three mechanisms: (1) modulation of allosteric sensitivity via phosphorylation of receptor subunits;(2) down-regulation of cell surface abundance, possibly through phosphorylation of the N-ethylmaleimide-sensitive factor (NSF), and (3) down-regulation of GABAA receptor function. In this continuation, we will extend this work by examining PKCe phosphorylation of 32 and y2S GABAA receptor subunits, the regulation of NSF by PKCe, the role of cofilin-1 and cyclophilin A as possible PKCe substrates that regulate GABAA receptor sensitivity to allosteric drugs, and the role of increased a2 subunit abundance in the striatum in mediating PKCe null phenotypes. Studies will use transfected cell lines expressing specific receptor subunit combinations and an analog-sensitive mutant of PKCs. Other studies will use primary neurons cultured from PKCe null and wild type mice. Phosphorylation sites will be identified by mass spectrometry and phosphorylation site alanine mutants will be constructed to determine if these mutations prevent phosphorylation by PKCe. GABAA receptor currents in intact cells will be examined by whole cell patch clamp. a2 subunits will be overexpressed in the striatum to see if overexpression reduces alcohol consumption in wild type mice. An RNAi vector will be developed to knock down ot2in the striatum to determine if decreasing expressing of a2 subunits increases alcohol consumption in PKCe null mice. These studies will provide novel molecular information about how PKCe regulates the sensitivity of GABAA receptors to ethanol and will provide new information about how PKCe regulates GABAA receptor cell surface s1 ability and function. PERFORMANCE SITE(S) (organization, city, state) Ernest Gallo Clinic and Research Center Emeryville, CA PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Messing, Robert O. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Messing, Robert O. ROBERM Ernest Gallo Clinic and Principal Investigator Research Center (EGCRQ Chou, Wen-Hai N/A EGCRC Assoc. Investigator Qi, Zhan-Heng N/A EGCRC Postdoctoral Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Shokat, Kevan M. UCSF Collaborator Human Embryonic Stem Cells ^ No (H Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list http://stemcells.nih.qov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable toSBIR/STTR Only. See SBIR/STTR instructions. Cl Yes d No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Messing, Robert O. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,

Protein kinase C epsilon (PKCe) modulates nociceptor sensitization by inflammatory mediators. The overall goal of this project is to identify PKCe substrates that mediate this process. Three candidates are proposed: The alpha subunit of the tetrodotoxin-insensitive sodium channel Nav1.8a, the alpha subunit of the N-type calcium channel Cav2.2a, and the polymodal, vanilloid receptor TRPV1. The first aim will use a peptide activator of PKCe, the tyeRACK peptide, in gene- targeted mice that have a null mutation in one of these channels to determine if absence of these channels impairs PKCe-mediated nociceptor sensitization. In the second aim we will generate knock-in mice expressing an analog-sensitive mutant of PKCe, PKCe-as, which can be specifically inhibited by an analog of the general kinase inhibitor PP1, 1-Na, and can use benzyl-ATP as a phosphate donor. The third aim will examine whether channels found to contribute to PKCe- mediated nociceptor sensitization are phosphorylated in a PKCe-dependent pathway using recombinant PKCe-as for in vitro studies and DRG neurons from PKCe-as knock-in mice for cell- based studies. Phosphorylated residues will be identified by mass spectrometry. In the fourth aim identified phosphorylation sites will be mutated and expressed in heterologous systems and in DRG neurons to determine if PKCe regulation of these channels is impaired. These studies will advance our knowledge about mechanisms underlying nociceptor sensitization, identify downstream substrates of PKCe in this process, and provide new potential targets for development of therapeutic agents to treat pain.

DESCRIPTION (Provided by applicant): The Administrative Core will perform the centralized administrative functions for all 4 Research Components (Components 5-8) and the Pilot Projects (Component 9). It also will coordinate the activities of the Scientific Support Cores (Components 2-4). The Program Advisory Board will provide scientific oversight, and the Executive Committee will make executive decisions regarding ACTG activities. The Executive Committee will set goals, evaluate progress toward general scientific research directions, and oversee quality control mechanisms in all areas of Center activity. Oversight also will be provided for transgenic animal production, behavioral assays, production of viral vectors for gene silencing and transgenic expression, imaging and histological analyses, and genomics services. The Core will also sponsor enrichment activities, including guest speakers, a new course on the Neurobiology of Addiction for the UCSF Neuroscience Graduate Program, and an ACTG website to publicize ACTG-sponsored events, staff openings and links to other organizations'websites. There will be two lines of administrative responsibility within the Administrative Core. Dr. Messing, the Center Director, will supervise the general coordination of all Center administrative activities and the Center's budget. Dr. Heberlein, the Scientific Director, will supervise the coordination of and future planning for scientific directions.

The Animal and Behavior Core will provide services, technical assistance and training needed for the breeding and behavioral testing of the knockout and knock-in mice that will be examined by the investigators within the research components. Centralized breeding and genotyping will maximize efficiency and allow for the development and use of a centralized system for tracking the production and use of all mice created under this center. In addition, the Animal and Behavior Core will standardize the methods and the analyses for alcohol-related behavioral testing for mice and rats. A series of behavioral tests will evaluate the acute sedating and ataxic effects of alcohol, as well as the reinforcing and rewarding properties of alcohol, in mice generated by the research components. The Core will instruct research personnel in the conduct of other behavioral studies, as needed. By conducting the bulk of the behavioral testing within the Core, we ensure that the procedures are performed, and the data are analyzed, in a consistent manner, allowing for maximal comparability of the effects of different genetic manipulations across center projects. The Core will additionally seek to develop a useful and reliable protocol for operant self-administration of alcohol by mice that produces a maximal level of alcohol intake for future use by the research components.

The Administrative Core will perform the centralized administrative functions for all 4 Research Components (Components 5-8) and the Pilot Projects (Component 9). It also will coordinate the activities of the Scientific Support Cores (Components 2-4). The Program Advisory Board will provide scientific oversight, and the Executive Committee will make executive decisions regarding ACTG activities. The Executive Committee will set goals, evaluate progress toward general scientific research directions, and oversee quality control mechanisms in all areas of Center activity. Oversight also will be provided for transgenic animal production, behavioral assays, production of viral vectors for gene silencing and transgenic expression, imaging and histological analyses, and genomics services. The Core will also sponsor enrichment activities, including guest speakers, a new course on the Neurobiology of Addiction for the UCSF Neuroscience Graduate Program, and an ACTG website to publicize ACTG-sponsored events, staff openings and links to other organizations'websites. There will be two lines of administrative responsibility within the Administrative Core. Dr. Messing, the Center Director, will supervise the general coordination of all Center administrative activities and the Center's budget. Dr. Heberlein, the Scientific Director, will supervise the coordination of and future planning for scientific directions.

DESCRIPTION (provided by applicant): Studies with protein kinase C delta knockout (PKC4-/-) mice indicate that PKC4 regulates ethanol intoxication and self-administration. These effects may be related to PKC4 actions at extrasynaptic GABAA receptors, since thalamic and hippocampal neurons from PKC4-/- mice lack ethanol enhancement of tonic inhibitory GABA currents. The main hypothesis of this project is that PKC4 regulates ethanol intoxication and self-administration by phosphorylating proteins that alter the function of extrasynaptic GABAA receptors in brain regions controlling these behaviors. This hypothesis will be tested using a novel mouse model, a knock-in mouse that expresses an ATP analog-sensitive mutant of PKC4 (AS-PKC4). AS-PKC4 can be selectively and potently inhibited by analogs of the general kinase inhibitor PP1 that cross the blood brain barrier and can be administered orally or by i.p. injection. Studies will determine if inhibition of AS-PKC4 mice increases signs of ethanol intoxication and enhances ethanol self-administration in adult mice. Electrophysiological studies in brain slices will investigate whether inhibiting AS-PKC4 blocks ethanol potentiation of tonic GABA currents in hippocampal, thalamic, and amygdala neurons. In vitro kinase assays will determine if 22, 23, and 4 subunits are possible substrates of PKC4. Novel ATP analogs will be used with tissues from AS-PKC4 mice to identify PKC4 phosphorylation sites on GABAA receptor-associated proteins. Studies in cells that heterologously express 1422/34 receptors and receptor-associated proteins with alanine substitutions at PKC4 phosphorylation sites, will determine if these sites regulate the ethanol sensitivity of 1422/34 receptors. The overall goal of this project is to identify a novel PKC4 signaling pathway that regulates the ethanol sensitivity of extrasynaptic GABAA receptors and may contain targets for the development of new therapeutics to treat alcohol use disorders.

DESCRIPTION (provided by applicant): Alcohol use disorders are common and incur a huge cost to society. Unfortunately, pharmacotherapy of alcohol use disorders is restricted to three FDA approved drugs: disulfuram, naltrexone, and acamprosate. Therefore, there is considerable need for the development of new agents to treat these disorders. Mounting evidence suggests that protein kinase C epsilon (PKC¿) is a good target for development of drugs to treat alcohol use disorders, based mainly on studies using gene-targeted mice that lack this enzyme (Prkce-/- mice). These mice drink substantially less ethanol than wild type mice, show heightened aversion to ethanol, are more easily intoxicated by low doses of ethanol, and recover from intoxication very slowly compared with wild type mice. These behaviors are associated with enhanced function of GABAA receptors, which are the major mediators of inhibitory neurotransmission in the nervous system. PKC¿ regulates GABAA receptors by phosphorylating GABAA2 subunits at Ser-327, which reduces their sensitivity to benzodiazepines and ethanol. PKC¿ also phosphorylates the N-ethylmaleimide sensitive factor (NSF), at Ser-460 and Thr-461, which stimulates removal of GABAA receptors from synapses. The long-term goal of this project is to identify targets for the development of new drugs to treat alcohol use disorders. The objective of this application is to specifically validate PKC¿ as a drug target and identify additional drug targets within neuronal PKC¿ signaling pathways. The main hypothesis to be tested is that selective inhibitors of PKC¿ signaling can reduce voluntary ethanol consumption in mice. Three aims are proposed. The first aim will determine the consequences of administering a highly selective PKC¿ inhibitor and validate PKC¿ as a drug target to decrease alcohol consumption. This will be achieved by using a new line of mutant mice in which the ATP binding pocket of PKC¿ has been modified to allow highly specific inhibition by small molecules that do not inhibit native kinases. In the second aim, three novel, related compounds that are potent inhibitors of PKC¿ will be administered to wild type mice to determine if this class of compounds should be developed further as drugs to treat alcohol use disorders. The third aim will use a newly developed proteomics approach to identify direct substrates of PKC¿ for study of their role in regulating GABAA receptors and behavioral responses to ethanol. Because of its focus on identifying new drug targets and pharmacological agents to treat alcohol use disorders, this project has high translational value for serving public health. PUBLIC HEALTH RELEVANCE: Alcohol use disorders are extremely common and very costly to society. There are few therapeutic options available to treat afflicted persons. The studies outlined in this proposal will serve the public health by providing important information that can be used to develop inhibitors of the enzyme protein kinase C epsilon as safe and effective treatments.

Alcohol use disorders (AUDS) impact millions of individuals and constitute one of the most serious public health problems worldwide. Despite its devastating impact on society, only a few effective medications are currently available. Orexins/hypocretins are hypothalamic peptides that act via orexin receptors that have been linked to regulating feeding and sleep. In addition, the orexin system has been shown to play an important role in alcohol self-administration and in stress-induced reinstatement. These effects are thought to be mediated by orexin containing neurons that project from the lateral hypothalamus to the ventral tegmental area (VTA) and nucleus accumbens (NAc) or from the perifornical regions to the amygdala respectively. However, the role of orexins through orexin receptors and their signaling pathway in driving ethanolmediated behaviors is less explored. The overall goal of this research project, component 6 of the NIAAAGallo Center application is to determine the role of orexins, orexin receptors and their downstream signaling pathways in different brain regions in ethanol-mediated behaviors. The Center provides a unique opportunity to apply a multidisciplinary approach that integrates behavioral, biochemical and electrophysiological techniques in the latest animal models of binge drinking, ethanol selfadministration and stress-induced reinstatement. The research aims to understand the role of orexinmediated synaptic and cellular mechanisms in the central amygdala, VTA and NAc using behavioral models of alcohol addiction. In Aim 6.1, we will determine whether Ox-Rs in the central amygdala drive stress-induced reinstatement of ethanol seeking. In Aim 6.2, we will apply electrophysiological and biochemical techniques to dissect the signaling pathways underlying orexins effects in the central amygdala. In Aim 6.3, we will determine the mechanism of action of orexins in the VTA and the NAc in regulating binge ethanol consumption and ethanol self-administration. Orexins/hypocretins play a crucial role in addiction, the sleep-wake cycle, motivation and stress, and this provides a strong rationale for our proposed studies, the experiments outlined have been designed specifically to lead to clinical studies aimed at determining the efficacy of orexin receptor antagonists in human subjects with AUDs.

The Administrative Core will perform the centralized administrative functions for all 3 Research Components (Components 4-6) and the Pilot Projects (Component 7). It also will coordinate the activities of the Scientific Support Cores (Components 2-3). The Program Advisory Board will provide scientific oversight, and the Steering Committee will make executive decisions regarding ACTG activities. The Steering Committee will set goals, evaluate progress toward general scientific research directions, and oversee quality control mechanisms in all areas of Center activity. Oversight also will be provided for behavioral assays, production of viral vectors for gene silencing and transgenic expression, and imaging and histological analyses. The Administrative Core will also sponsor enrichment activities, including guest speakers, a course on the Neurobiology of Addiction for the UCSF Neuroscience Graduate Program, and an ACTG website to publicize ACTG-sponsored events, staff openings and links to other organizations¿ websites. There will be two lines of administrative responsibility within the Administrative Core. Dr. Messing, the Center Director, will supervise the general coordination of all Center administrative activities and the Center¿s budget. Dr. Ron, the Scientific Director, will supervise the coordination of and future planning for scientific directions

PROJECT SUMMARY Neuroadaptive changes in gene expression due to repeated ethanol consumption is a major mechanism that underlies the transition from moderate to excessive drinking. Gene expression profiling studies of human alcoholics and ethanol dependent rodents have identified a multitude of ethanol-responsive gene networks. Little is known about how these networks are recruited into a neuroadaptive response. One mechanism could involve ethanol regulation of transcriptional co-regulators that bind and modulate the activity of several transcription factors, thereby orchestrating a diverse neuroadaptive transcriptional network. However, very few studies have identified transcriptional co-regulators in the brain that respond to ethanol and in turn regulate ethanol consumption. In ongoing experiments we found evidence that the transcriptional co-regulator Lim-only 4 (LMO4) regulates ethanol consumption. LMO4 is expressed in brain regions important for addiction and recent work indicates that it plays an important role in cocaine sensitization and conditioned reward for sucrose. Our initial results show that LMO4 levels in the nucleus accumbens decrease during intermittent, binge-like ethanol consumption, and a gene trap mutation in the Lmo4 gene, which reduces LMO4 levels by 50%, increases the development of excessive ethanol consumption in male C57BL/6J mice. Given these findings we propose that LMO4 is an ethanol-responsive, neuroadaptive transcriptional co-regulator that regulates ethanol consumption. This hypothesis will be addressed in two Specific Aims: (1) to determine whether LMO4 regulates ethanol consumption in both sexes, and (2) to identify brain regions in which LMO4 acts to moderate ethanol consumption. Should the results show that LMO4 in specific regions of the adult brain regulates ethanol consumption in male and female mice, future studies aimed at determining how ethanol regulates LMO4 and how LMO4 regulates ethanol consumption could shed new light on a fundamental mechanism by which ethanol provokes a neuroadaptive response that leads to excessive drinking.

PROJECT SUMMARY Alcohol use disorder is a major public health problem. To date, available treatment options are limited to psychosocial intervention, three FDA approved medications (disulfuram, acamprosate, and naltrexone) and a few drugs approved for other indications, all with relatively small effect sizes. There is a clear need for additional treatments based on a deeper understanding of neurobiological mechanisms. Corticotrophin releasing factor (CRF) is a 41-amino acid neuropeptide produced mainly by neurons of the paraventricular hypothalamus, central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) where it plays an important role in behavioral and physiological responses to stress. CRF has been long implicated in driving excessive ethanol consumption through prior studies that used CRF receptor antagonists in rodents. However, recent attempts to test CRF receptor antagonists as treatments for alcohol craving in humans have been disappointing. Part of this lack of success may be due to inadequate drug-like properties of some compounds and by a need for different human laboratory models that test drug effects on withdrawal and negative reinforcement in dependent subjects. Another reason may be due to the fact that CRF is released from neurons with other peptides and neurotransmitters that may act in synergy to drive excessive drinking. Understanding which of these co-released factors is important necessitates a different strategy that focuses on the CRF neurons themselves rather than on CRF receptors. The lack of genetic access to subpopulations of CRF neurons has made it difficult to study the biology of CRF neurons, the sources of CRF in different brain regions, and the circuitry underlying CRF-regulated behaviors. To fill this gap, we generated a BAC transgenic Wistar rat line in which Cre recombinase is expressed from the Crh gene promoter to enable genetic access to CRF neurons. In this project, we will use these rats to pursue the hypothesis that as animals develop ethanol dependence, CRF neurons in the CeA and BNST promote ethanol consumption through the coordinated release of GABA, CRF, and other neuropeptides. We will examine this hypothesis by selectively activating or inhibiting these neuronal populations and their projections using chemogenetic tools, and we will address the relative importance of the different transmitters and modulators released from these neurons using Cre-dependent RNA interference. Finally, we will investigate the role of repeated ethanol consumption on the transcriptome of these CRF neuronal populations to understand how ethanol changes their phenotype, which should provide us with important new clues as to how they drive excessive drinking. !

PROJECT SUMMARY This project is a continuation and further development of a previous INIA project, which was based on studies showing ethanol-induced changes in brain neuroimmune gene expression in animal models and humans. Those data suggested that ethanol dysregulates proinflammatory, Toll-like receptor (TLR) signaling in the brain. During the previous period of funding, we found that pharmacological or genetic manipulation of molecules that signal through the myeloid differentiation primary response gene 88 (MyD88) alters ethanol intake and preference. The proposed renewal has three Specific Aims. Specific Aim 1 seeks to repurpose three existing FDA-approved drugs with proven anti-inflammatory activity: apremilast (a phosphodiesterase-4 inhibitor recently approved for treating psoriasis), sulfasalazine (an inhibitor of IKK? used in the treatment of inflammatory bowel disease), and gemfibrozil (a PPAR? activator used in the treatment of hyperlipidemia). These drugs will be tested for their effectiveness in reducing ethanol intake and altering other ethanol- dependent behaviors in mice. Specific Aim 2 will explore another branch of TLR inflammatory signaling that depends on signaling via the TIR-domain-containing adapter-inducing interferon-? (TRIF) protein. This aim will examine the role of brain regional and cell-specific knockdown of TRIF-dependent signaling proteins on ethanol intake using lentiviral-mediated RNA interference in collaboration with the Lasek project. Specific Aim 3 serves a collaborative function to provide behavioral testing of new drugs and candidate genes for other INIA projects. The Mayfield project will analyze gene expression networks to predict drugs that will normalize the network, and we will test these drugs for their ability to reduce ethanol consumption in mice. The Hitzemann and Mayfield projects will elect candidate long non-coding RNAs based on transcriptome analyses of high drinking-in-the-dark (HDID) mouse lines and of rhesus macaques chronically exposed to ethanol (in collaboration with INIA-Stress) and human postmortem brain samples. The Homanics project will generate mutant mice for the candidate long non-coding RNAs, and we will perform behavioral testing with these mice. We will also continue to provide mutant mice for the Roberto project and provide Crh-Cre rats for the Pfefferbaum/Zahr project. We anticipate that other INIA projects will identify additional drugs or targets that require behavioral testing, which we will carry out as part of this collaborative function.