Yuri A. Blednov - US grants

DESCRIPTION (provided by applicant): Due to the differences among the genetic animals models being maintained, used and developed in 5 of the 6 IMA sites, a separate U-24 application to support genetic animal models is being submitted from each site. Dr. John Crabbe, Professor of Behavioral Neuroscience, Oregon Health Sciences University (OHSU), will serve as coordinator of the overall Genetic Animal Models Core described in this application. The major goal of the GAMC is to integrate animal model development availability and usage across sites. Genetic animal models have been a major staple of alcohol research since the first were developed in the late 1940?s. it has been known for many years that animals experienced with alcohol self-administration and/or dependent on alcohol will increase their intake for a relatively short period of time after a period of withdrawal. However, these and other existing models are not yet optimal. Generally, the magnitude and architecture of the response does not convincingly display either gross excess or obvious loss of control that extends for a long time after the initial period of intoxicating self-administration. Virtually nothing is known about the genetic predisposition to self-administration potentiated by any of the above manipulations. Little to nothing has been done to characterize any of these phenomena in existing mouse genetic models of high or low ethanol drinking or high or low withdrawal. The general goals of the GAMC are to facilitate the development of more robust phenotypes and genotypes; facilitate the exploration of gene X environment interactions and facilitate development of novel genetic technology to explore the two-hit hypothesis, i.e. that at least two clusters of genes must be dysregulated to produce abusive self-administration; to achieve coordinated genetic animal model utilization and development across IMA sites and Cores; and to provide relevant data to the Informatics Core. At the Austin site, specific emphasis is placed on development of novel mutant mice with deletions or mutations of single genes. Construction of mutant mice required by other INIA projects will use an inbred C57 background. Mutant mice will be tested for alcohol phenotypes at the Austin site.

The overall objective of this INIA grant is to define mechanisms of alcohol avoidance and provide expertise in behavioral testing to other INIA investigators to evaluate ethanol-related behaviors in new mutant mice. Based on our data from the current period of INIA funding, we propose that the avoidance of high concentrations of ethanol by rodents is caused by increased release and/or production of proinflammatory cytokines in liver and brain and that these cytokines cause long-lasting changes in gene expression in the brain. We also found that initial high acceptance of alcohol can decrease when alcohol is presented continuously with intervening weekly abstinence periods, and this decrease depends upon the genetic background. We developed two genetic mouse models which maintain consistent high levels of preference and consumption (FVBxB6F1 mice;Sustained Alcohol Preference, SAP) or develop avoidance of alcohol (NZBxB6F1 mice;Reduced Alcohol Preference, RAP). These animal models provide the opportunity to compare brain gene expression profiles formed by initial alcohol consumption, sustained consumption and development of alcohol avoidance. To test the cytokine hypothesis, we will study: 1) ethanol consumption in knockout mice lacking genes for some proinflammatory cytokines and/or their receptors;2) the effect of systemic treatment with proinflammatory cytokines (particularly a-TNF) and antagonists of cytokine receptors on voluntary ethanol consumption;3) brain gene expression profiles after cytokine treatment to allow comparison with array data obtained from mice with ethanol avoidance;4) treatment of FVBxB6F1 and NZBxB6F1 hybrid mice via 3 cycles of alcohol consumption and abstinence and identification of key genes by microarray analysis;5) generation of brain regional knock-down mice of key genes by administration of RNAi. This work will use 3 INIA Cores (Texas Array and Informatics Core, RA Harris -PI;Colorado RNAi core, W.Zawada - PI;California Mouse Animal Model Core, A. Roberts - PI)andcollaborations withsix INIA investigators (A. Alcantara - Austin, Texas;S. Bergeson - Austin, Texas;R. D. Mayfield - Austin, Texas;R. Davis - Houston, Texas;A. Ryabinin - Portland, Oregon;B. Tabakoff - Aurora, Colorado).

DESCRIPTION (provided by applicant): This project is based on INIA-West studies showing changes in neuroimmune gene expression in animal models of alcohol intake and in brain of human alcoholics. We found that deletion of any of six INIA candidate neuroinflammatory genes decreased alcohol consumption and activation of immune signaling increased alcohol consumption. These data suggest that in human alcoholism and in our genetic animal models there is a misregulation of pro-inflammatory signaling in brain. Several of our candidate genes are part of a specific toll-like receptor (TLR4) signaling pathway that we will study behaviorally and biochemically. Specific Aim 1 will: Define the molecular components of TLR4 signaling that are responsible for promotion of excessive alcohol consumption. These studies will use null mutant mice lacking key components of this system. Neuroinflammatory signaling is also a potential target for medication development for alcoholism and we will test three anti-inflammatory drugs: Minocycline, Pioglitazone and AE1-329. Specific Aim 2 will: Define the gene networks that are perturbed by excessive alcohol consumption and neuroimmune activation in mouse and compare these to gene expression changes in human alcoholism. This aim will also define changes in brain cytokines related to regulation of alcohol consumption by measuring cytokine levels in brain of mice treated with anti-inflammatory drugs which reduce alcohol consumption. Specific Aim 3 is a Core function that will provide behavioral testing of new INIA candidate genes for other INIA projects using RNAi, conditional null mutant mice and pharmacological approaches. INIA Interactions: Genetic manipulation In mice will use RNAi and null mutant mice from the Lasek and Homanics INIA cores. We will provide behavioral testing for the Heberlein and Ponomarev projects and treated mice to Ponomarev. We will collaborate with the Mayfield and Ponomarev projects to compare our data for gene expression profiling (human and mouse), the Roberts/Kosten cores for medication testing and the Siggins and Morrisett projects for electrophysiology.

PROJECT SUMMARY (See instructions): This Shared Resource Core is as part of a P01 grant to provide animal testing. The Program Project Director is Dr. R. A. Harris, and the P.I. of this Project is Yuri Blednov and the Co-Investigator is Rueben Gonazles. This Animal Core will provide control mice and mice treated with alcohol in a limited access binge consumption model (Drinking in the Dark, DID) for the analysis of miRNA levels (Mayfield Project) as well as mice pre-treated with drugs nominated by the other projects and tested using DID or CPP for gene expression and electrophysiology (Ponomarev/Morikawa Project). This Core will identify alcohol-sensitive therapeutic targets by behavioral testing of new peptides generated in Mihic/Morriset Project, epigenetic drugs for Ponomarev/Morikawa project and miRNAs found in the Mayfield project. This behavioral testing will use the DID model of alcohol consumption and Conditioned Place Preference (CPP) in mice and operant self-administration of alcohol in rats.

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.