Robert Harris - US grants

No Abstract Provided

The overall goal is to identify the neurochemical mechanisms responsible for ethanol and benzodiazepine intoxication, tolerance and dependence. We postulate that the GABA-activated chloride channel is an important site of action for these drugs and the proposed research will focus exclusively on this receptor-channel complex. Of particular importance is the use of molecular biological techniques to define genetic differences in alcohol and benzodiazepine sensitivity at the level of the individual genes coding for the GABA-activated chloride channel complex. The proposal has 4 aims: 1. GABA-activated chloride channels are modulated by endogenous regulators such as calcium, cAMP and protein kinase C. The possibility that these regulators modulate the acute actions of ethanol or benzodiazepines or are responsible for tolerance to these drugs will be studied. 2. The channel complex consists of alpha, beta and perhaps gamma subunits; these distinct subunits have been cloned and sequenced, but it is not known if they confer different ethanol or benzodiazepine sensitivity on the channel. We will answer this question by testing drug sensitivity after expression of the cloned subunits in oocytes and by creation of transgenic mice with over- or under-expression of genes coding for specific subunits. The transgenic mice will allow us to ascribe functional roles to the different subunits. 3. Brain mRNA from Long/Short-Sleep (LS/SS) and Diazepam Sensitive/Resistant (DS/DR) mice and RNA from cloned genes will be expressed in Xenopus oocytes to allow electrophysiological study of channel function. Cloned genes from drug- sensitive mice (LS,DS) will also be injected into embryos of drug- resistant mice (SS,DR) to produce transgenic mice with expression of genes from resistant and sensitive lines. 4. Genetic differences in behavioral sensitivity to ethanol or benzodiazepines may be due to differences in nucleotide sequences of the genes coding for the subunits of the GABA-activated chloride channel or sequences of genes coding for promoter regions. Use of LS/SS and DS/DR mice for gene cloning will identify sequence differences between the lines.

The purpose of this small grant application is to allow an experienced investigator to test new molecular biological techniques for study of effects of ethanol on gene expression. The PI proposes to spend four months in the laboratory of Dr. Michael Miles (Gallo Center, UCSF, San Francisco) to learn new assays and to then return to his laboratory to implement these techniques. In Dr. Miles' laboratory, a beat shock protein (Hsc70) gene will be studied as its expression is known to be regulated by ethanol. Proposed experiments will define the cis-acting elements of this gene that are responsive to ethanol using promoter deletions, point mutations and DNA footprinting of regulatory regions. The long-term goal of this training is to use these techniques to understanding the effects of ethanol on expression of genes coding for subunits of the GABA-A receptor.

The main goal of this project is to use novel anesthetic and non- anesthetic compounds to determine which neurotransmitter receptors are candidates for site(s) of anesthetic action. To accomplish this, we will determine which receptors discriminate between these two classes of compounds. A second goal is to determine what properties are required for modulation of receptor function by correlating the physical properties (derived in part from computer modeling by Dr. Pohorille) of these compounds with their actions on neurotransmitter receptors. To allow comparison of different receptors in the same cell type, all receptors will be produced in Xenopus oocytes by expression of cDNAs, cRNAs, or mRNAs coding for specific receptor subtypes. Receptors to be studied are: GABA/A, glutamate (kainate and NMDA subtypes) and glycine. These receptors can be assembled from different subunits resulting in receptor subtypes with distinct drug sensitivities; we will determine which subunit combinations are anesthetic-sensitive or -resistant. In most cases, human receptors will be studied. Responses to neurotransmitter agonists will be determined electrophysiologically using a two-electrode voltage-clamp recording of membrane currents produced by receptor activation. We propose that these results will distinguish between three hypotheses: i. Action on only a single receptor consistently distinguishes anesthetics from non-anesthetics. ii. All anesthetics but no non-anesthetics affect several receptor system. iii. The effects of anesthetics and non-anesthetics (which can be divided into inactive and convulsant compounds) results from summation of action of excitatory and inhibitory systems.

This application is to continue the University of Colorado Alcohol Research Center. This center has been in formal existence since the NIAAA Centers program was initiated in 1977. The overall goal of this Center is to define the mechanisms (genetic and functional) that determine initial sensitivity to alcohol intoxication and the development of tolerance to these effects. The Center has evolved from animal model development and characterization to the level of specific gene identification. We will utilize existing models for genetic sensitivity and develop new ones for tolerance to ethanol. Center investigators will continue to use the technique of selective breeding for specific behaviors but will also emphasize new models based on genotypic selection (congenic lines) and changes in single genes (transgenic and null mutant mice). There are proposals to isolate and identify specific genes within QTLs for initial sensitivity. We will continue to provide our unique animal models to investigators at other institutions around the world. These genetic models will be used to define the biochemical and electrophysiological mechanisms responsible for the selected behavior and to provide information about candidate genes that may be responsible for these functional changes. In addition, molecular genetic approaches will be used to elucidate differences in gene sequences responsible for behavioral, biochemical, and electrophysiological differences identified by QTL analysis. This multidisciplinary approach provides extensive interaction and synergism between projects. This goals will be accomplished by six research components, an animal production core, and administrative core, and four pilot projects.

neuropharmacology; cerebral cortex; ethanol; cerebellum; quantitative trait loci; neurogenetics; nucleic acid sequence; gene induction /repression; pharmacogenetics; messenger RNA; animal genetic material tag; molecular cloning; laboratory mouse;

adenylate cyclase; neuropharmacology; ethanol; enzyme induction /repression; alcoholism /alcohol abuse; enzyme activity; cyclic AMP; genetic promoter element; isozymes; synapsins; psychopharmacology; behavioral /social science research tag; genetically modified animals; laboratory mouse;

hormone receptor; neurotensin; disease /disorder model; model design /development; alcoholism /alcohol abuse; psychopharmacology; protein structure function; complementary DNA; nucleic acid sequence; behavioral /social science research tag; laboratory mouse; genetically modified animals; animal genetic material tag;

The overall goal of this project is to define the molecular basis for the actions of volatile anesthetics, including novel anesthetics and non- immobilizers, on brain receptors. This will be accomplished at two levels. First, glycine, GABAA, and GluR6 (kainate) receptor subunits will be mutated to determine regions critical for anesthetic action. Receptor function and anesthetic sensitivity will be measured electrophysiologically following expression of receptor cDNAs or cRNAs in Xenopus oocytes. This work will involve collaboration with Trudell and Overduin to test predictions form their structural work by studying the function of receptor mutants. The goal of this part of the project is to define the molecular sites responsible for anesthetic modulation of ligand- gated ion channels. Second, the importance of the glycine receptor in anesthesia will be tested by construction of transgenic mice with mutant receptors. Specifically, we will generate mice with mutations in glycine receptor subunits that will eliminate anesthetic action on these receptors while retaining responsiveness to glycine. This will be done by replacing the normal gene with a mutant gene (knock-in) and by making transgenics that over- express the mutant subunit and crossing them with mutant mice lacking this receptor subunit. Mice will be provided to Eger, Sonner, and Kendig for determination of MAC, abolition by test compounds, and electrophysiological studies, respectively. These mutant mice will provide a critical test of the role of glycine receptors in behavioral and electrophysiological actions of anesthetic agents.

Our hypotheses are that ethanol alters the function of ion channels by binding within protein cavities and that some structural features of these cavities will be similar for related and unrelated ion channels. In addition, we propose that some behavioral actions of ethanol require enhancement of glycine or GABAA receptor function. Our overall goals are (l) to determine the specific protein regions of several brain receptors/channels that are responsible for ethanol action, and (2) to test the importance of two of these receptors in vivo. The first goal will be accomplished by in vitro testing of chimeric and mutated receptors/channels, and the second will use transgenic mice to express mutated receptors that are insensitive to ethanol in vitro. First, we will define the role of specific amino acids in the TM2-3 region of GABAA and glycine receptors in ethanol modulation of receptor function. These studies will be carried out in Xenopus oocytes. To determine if results from GABAA and glycine receptors generalize to another, related, ligand-gated ion channel, we will characterize the response of recombinant neuronal nicotinic acetylcholine receptors to ethanol. Next, we will extend our recent studies showing ethanol activation of G-protein activated inwardly rectifying potassium (GIRK) channels by elucidating the molecular basis of ethanol action on this channel. This will be done by construction of chimeric receptors between GIRK2 and IRK1 channels, followed by mutation of single amino acids. Structural determinants (i.e., amino acid properties and locations) of ethanol sensitivity will be compared for glycine, GABAA, and GIRK channels. Lastly, we will determine the in vivo significance of the glycine and GABA receptors for specific behavioral actions of ethanol by constructing transgenic mice with mutant receptors that are ethanol-resistant. The long-term, health-related, goal of this research is to identify molecular sites of alcohol action that would be useful targets for pharmacotherapies that would reduce alcohol actions such as reinforcement, craving, and dependence.

? DESCRIPTION (provided by applicant): During the current period of support we completed next generation sequencing (RNA-Seq) from cohorts of human brain samples obtained from the NSW Tissue Resource Center-University of Sydney. We also defined expression changes of microRNAs (miRNAs) in both human and mouse brain, proposing that miRNA act as master switches, responsible for many of the changes in gene expression changes and that a single miRNA can alter alcohol consumption. Our previous microarray studies identified potential splice variations in GABAB receptors in human alcoholics and we used RNA-Seq to discover novel, complex splicing of the GABAB1 gene in human brain and showed that chronic alcohol produces additional splicing complexity. We also used our RNA-Seq data to perform a systems network analysis on alcoholics and matched controls across brain regions (prefrontal cortex and basolateral and central amygdala) to define molecular networks based upon lifetime alcohol consumption. We then identified gene networks based on mRNA and microRNA transcriptome profiling that significantly overlap in human alcoholics and mouse models of excessive alcohol consumption. In our proposed studies, we will mine our extensive human RNA-Seq transcriptome profiles in novel and innovative ways to link gene expression changes with genetic differences found in the Collaborative Studies on Genetics of Alcoholism (COGA) studies. We will focus our efforts on identifying candidate FDA approved drugs that can be tested in mouse models of alcohol consumption. Three Specific Aims are proposed: 1) bioinformatics analysis of next generation sequencing and genome-wide association studies will reveal human genes contributing to the risk of alcohol dependence, 2) convergent changes in gene expression between human alcoholics and mouse models of excessive alcohol consumption will be determined using network analysis of synaptoneurosome and microglia genes in the amygdala and prefrontal cortex of both species, and 3) novel therapeutics based on drugs that are in late-phase clinical trials or have existing FDA approval for other purposes will be selected and tested in alcohol drinking models in mice. Identification of effective target compounds in mice will facilitate testing in humans. The repurposing strategy has been used successfully to advance treatment for other diseases but has not been used for alcohol dependence, a disease lacking effective treatment options.

DESCRIPTION (provided by applicant): This Core will have facilities at three sites, the University of Texas at Austin (coordinating site), Indiana University School of Medicine in Indianapolis and the University of Colorado Health Sciences Center at Denver. Efforts at the Indiana site will be supported in part by a subcontract from UT Austin and activities at the Colorado site will be supported by funds available from sources other than the IMA. All three sites have made a considerable investment in gene array technology and the MA proposals benefit from this infrastructure. The overall goal is to use microarray technology to define changes in gene expression that either predict or accompany excessive alcohol consumption. This requires development and validation of arrays that can accurately measure levels of large numbers of RNAs from brain regions of both mice and rats. Of particular importance is reproducibility among sites for all steps of the array process, from tissue dissection to array data informatics. This will provide a measure of molecular neuroadaptation that will be comprehensive (analysis of a large number of brain genes) and consistent across projects. We will use both commercial (Affymetrix) and custom fabricated arrays to provide coverage of mouse, rat and drosophila genomes for NIA projects.

This application requests funds for the partial payment of travel and conference-related expenses of non-US resident scientists (invited speakers and young/new investigators) and for U.S. minority scientists to attend the Eleventh Congress of the International Society of Biomedical Research in Alcoholism (ISBRA). This meeting will be held in San Francisco, California on June 28 through July 3, 2002. The ISBRA Congresses are now the only worldwide scientific meetings for all areas of alcohol research. In addition to biomedical aspects, they also cover biobehavioral and clinical research. The ISBRA Congresses are held biannually and, since the founding of ISBRA, meetings have been held in Germany, USA, Finland, Japan, Canada, UK,. Australia, and Denmark. This will be the third time that the ISBRA Congress will be held in the United States. It will have a large impact on alcohol research not only in North America, but also the European countries, as well as Asian and Pacific Rim countries and Australia. The Congress will meet jointly with the US-based Research Society on Alcoholism (RSA). The program will feature topics of priority interest to the international alcohol research community and provide opportunities for discussion of collaborative research. At present, 43% of the ISBRA membership is non-U.S. based. Accordingly, attendance by non-U.S. scientists is absolutely essential for the success of the meeting; 41 of the 76 proposed invited speakers will be from outside of the U.S. This application requests funding for travel and registration fee for 30 invited speakers, travel registration fee and partial living expenses for 27 young/new investigators and 6 minority students/young investigators and partial coverage of publication costs of the Congress proceedings.

This project will use recombinant ion channels to define the molecular sites of action of inhaled anesthetics. During the past grant cycle, the project demonstrated diverse anesthetic actions on several ion channels--in particular, type A gamma-aminobutyric acid (GABAA) and glycine receptors. The studies prompted and aided Dr. Homanics' construction of mutant mice that were subsequently characterized in vivo by Drs. Sonner and Eger. The results illustrate the utility of a multidisciplinary approach and reveal the complexity of anesthetic actions. Although anesthetics affect many protein targets in ways that plausibly explain anesthesia, the relative importance of each target is not known. Such results prompt the proposed studies of multiple targets including glycine and N-methyI-D-aspartate (NMDA) receptors, and specific sodium channels. We will modify these receptors and channels to provide information on mechanisms, including provision of templates for examination of other ionophores. We propose that inhaled anesthetics bind in, and thereby encroach upon, water-filled cavities formed by transmembrane regions of ion channels. If the cavity volume critically affects channel gating, the presence of anesthetic in the cavity will alter channel function. Specifically, we hypothesize that effects on glycine, NMDA, and some voltage-activated sodium channels mediate primary effects of anesthetics, and that a fuller understanding of anesthesia requires examination of molecular mechanisms of action on these channels, as well as new molecular and genetic tools for the study of these channels in vivo.

DESCRIPTION (provided by applicant): This is a revised proposal for funding of an established graduate training program in neuroscience at the Institute for Neuroscience (INS) at the University of Texas at Austin. Funding is requested for five new predoctoral trainees per year, with students obtaining support from the program for their first two years of graduate training. During this time students will do laboratory rotations and complete coursework requirements before entering thesis laboratories at the end of their second years. A critical mass of neuroscience researchers has been established in Austin, largely due to an emphasis on faculty recruitment in this area. These researchers have a wide variety of interests but share a common goal of strengthening neuroscience training on campus. Thirty-four (34) faculty in the College of Natural Sciences, Department of Psychology and College of Pharmacy will participate in the training. Their research interests range from the molecular, through the biochemical, physiological and electrophysiological, to the behavioral and computational. Each of the training faculty (except for junior faculty who have just been recruited) currently holds at least one major research grant and many have several grants. Students entering this graduate program thus have a wide variety of options for training, and one of the significant strengths of our program is the opportunity for conducting cross-disciplinary research in collaborating laboratories. Incoming students will be expected to choose laboratories in which they will conduct research rotations, after which they will present seminars based on their work. Students will also take a prescribed set of core neuroscience courses as well as choose from a wide variety of elective courses. Graduate students will be highly encouraged to attend, and participate in, a plethora of seminar series offered by the INS or by the different home departments of INS investigators. The responsible conduct of science will also be taught in an ethics course. An effort will be made to 'continue our success in recruiting minority students.

DESCRIPTION (provided by applicant): During the current period of support we have developed a facility for fabrication and analysis of cDNA microarrays and identified patterns of gene expression in different brain regions from alcoholic and non- alcoholic cases. This success of this work has been facilitated by a strong international collaboration with investigators from the University of Queensland, Australia. Several key findings from these studies suggest that distinct patterns of gene expression in the frontal cortex are altered in alcoholics, and that these alcohol- responsive genes belong to functionally diverse families. These results raise several important questions that will be addressed during the next period of support. These include the following: (1) Can these gene clusters be generalized or refined to identify novel functional pathways in a larger and more diverse sample of cases? (2) Similarly, are there clusters of brain proteins that are changed in alcoholic individuals? (3) Can these genomic and proteomic profiles be used to define functionally related clusters that define disruptions of signaling in frontal cortex that are responsible for at least some aspects of alcoholism. (4) Are there patterns of gene expression in nucleated blood cells that can serve as biomarkers for alcohol consumption, dependence, or abstinence? The Specific Aims are: 1) Test the hypothesis that patterns of brain gene expression can identify novel functional pathways that are altered in alcoholics. It is also proposed that brain gene expression profiles will reflect drinking history, and severity of liver and other organ damage, 2) Global proteomics analysis will identify novel proteins that differ between individual alcoholic and non-alcoholic cases, 3) Test the hypothesis that patterns of gene expression in nucleated blood cells can distinguish between non-alcoholics, actively drinking alcoholics, and abstinent alcoholics. cDNA microarrays will be used to test expression of thousands of genes at a time as an initial screening method to identify potential blood-based biomarkers of alcoholism. These studies will provide new targets for addiction pharmacotherapy as well as new diagnostic tools for alcohol abuse and alcoholism.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of this INIA core is to continue to provide robust microarray analysis and informatics capabilities to INIA researchers. During the previous grant period, the INIA Microarray Core at UT Austin enabled the generation of large amounts of microarray data from INIA labs. In the next period, although we plan to generate microarray data, the emphasis of the Core will extend to carrying out novel analyses of consolidated datasets. At the same time we will enhance our microarray capabilities by enabling novel types of microarray profiling experiments such as microRNA (miRNA) profiling and chromatin immunoprecipitation (ChlP-chip) data analysis. The activities of this proposed INIA Core will include updating of our relational microarray database, the Longhorn Array Database (LAD), to accommodate these novel kinds of experimental platforms and datasets, and most importantly, developing completely new analysis capabilities that are not possible with the web browser model that is used by LAD. miRNAs provide a novel mechanism for changing function by changing the levels of brain proteins and we will enable INIA investigators to carry out pioneering neurobiological studies in this area. miRNAs have been shown to be important in post-transcriptionally regulating gene expression in cancer and miRNA profiling is likely to be valuable for understanding INIA models of excessive alcohol consumption. [unreadable] [unreadable] This core will give INIA researchers free access to the analyses tools for DNA microarray data that we have developed and are developing by making these accessible from a web browser. For example, generalized singular value decomposition (GSVD) and pseudoinverse projection, allow construction of predictive models from DNA microarray data. This website will enable researchers without in-depth expertise in mathematics and computer programming to concentrate on the scientific questions they set out to answer by analyzing cDNA, miRNA and oligonucleotide microarray data. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mechanical forces imposed on vascular smooth muscle (VSM) are important modulators of cell structure and function. Much of the interest in mechanically-induced responses of cultured cells stems from their similarity to processes leading to pathological conditions. These responses which include cell migration and reorganization of the cytoskeleton are believed to be mediated through mechanisms involving mechanosensing and transduction originating at the cytoskeleton- extracellular substrate interface. It is further recognized that organizational changes in the actin cytoskeleton are essential for effective contraction, mechanotransduction and signaling in different cell types. However, the exact nature of cytoskeletal reorganization has not been studied and the mechanisms regulating these changes are largely unknown in VSM. We have found that in response to unidirectional stretch, A7r5 smooth muscle cells reorient their position is manner that is dependent on the degree of stretch. Thus, this model provides an endpoint for quantitative assessment of responsiveness to mechanical strain. Utilizing molecular approaches combined with confocal microscopy we will evaluate the role of actin and microtubular components of the cytoskeleton in the cells? response to stretch, determine the role of the mitogen-activated protein kinase signal transduction pathways in the stretch response, and determine the effect of stretch-induced cytoskeletal reorganization on expression of key signal transduction and focal adhesion proteins. The long-term objectives of this project are to understand the role that the cytoskeleton plays in mechanoresponsiveness and to identify changes in expression and regulation of proteins regulating the response, as well as to understand the mechanisms underlying sensing and transduction of mechanical signals. The knowledge gained may be useful in the development of therapeutic agents regulating mechanotransduction mechanisms contributing to cardiovascular pathologies.

DESCRIPTION (provided by applicant): This three-year funding proposal for a P20 Center provides a new paradigm for medication development for the treatment of alcoholism by bringing experienced alcohol researchers together with experts in chemistry, proteomics, gene delivery and Drosophila genetics who are new to the alcohol field. The goal is to develop the technologies and collaborations necessary to define new targets for medication development and translate our knowledge of alcohol targets into selective ligands. The Center consists of an Administrative Core (Adron Harris, PI and Center Director), target identification by neuron-specific transcriptomics (Igor Ponomarev, PI), target identification by proteomics (Dayne Mayfield, PI), ligand selection by high throughput screening (John Mihic, PI), and an Animal Core (Yuri Blednov, PI) to provide mice and behavioral testing to the projects. In addition, we propose two Pilot Projects (two years in duration) to develop technology to test the biological importance of new targets. One will provide tools to deliver inhibitory RNAs (RNAi) to the brain (Maria Croyle, PI) and another will develop knock-in Drosophila carrying mutations in target genes (Nigel Atkinson, PI). The Center has both internal and external advisory boards, providing expert guidance from scientists with a wide range of expertise. A key aim is to provide the collaborations and infrastructure necessary for a future P50 application which will focus new researchers and new technologies on medication development for alcohol dependence.

DESCRIPTION (provided by applicant): This is a renewal of ongoing R01 funding to define molecular sites of alcohol action in brain and to link alcohol effects on these receptors with specific behaviors such as consumption, reward/aversion, intoxication and physical dependence. During the current period of funding we were successful in providing molecular and even atomic level of analysis of alcohol sites on pentameric ligand-gated (pLGIC) ion channels, including the first crystallographic structure of alcohols bound within a channel protein. We also succeeded in constructing mutant mice with GABAa receptor subunits which are resistant to alcohol modulation allowing us to link specific GABAa receptors with discrete behavioral actions of alcohol. We are in the middle of the fourth year of this project period and t date have 30 publications with 7 more submitted or in preparation. Although we and others have made considerable progress, the molecular sites of alcohol action in the brain are not completely defined. Emerging evidence from human and rodent genetics, as well as recombinant receptors, indicates that several targets which have received only limited attention may be important sites of alcohol action in brain. We propose to define the role of two groups of ligand-gated ion channels, the glycine-activated chloride channels (GlyR) and GABAaRs formed from ? subunits (? GABAaRs) in actions of alcohol at the molecular, electrophysiological and behavioral levels. A strength of this proposal is the combined use of knock-out (KO) and knock-in (KI) mice allowing us to define the importance of the presence of a subunit (KO) and the importance of direct alcohol action on the protein (KI) for behavioral actions of alcohol. An innovative aspect is use of a new technology (transcription activator- like effector nuclease; TALEN) to be employed by our collaborator, Dr. Gregg Homanics, that markedly reduces the time and cost required for construction of mutant mice. In addition to our behavioral studies, mutant mice will be used by our collaborator, Dr. Neil Harrison for electrophysiological studies of glycinergic function in accumbal regions. Several GlyRs and ? GABAaRs are genetically linked with human alcohol dependence and understanding their role in alcohol actions may provide new targets for pharmacotherapies of alcohol abuse and alcoholism.

? DESCRIPTION (provided by applicant): This proposal requests continued funding of an established Neuroscience Graduate Training Program in the Institute for Neuroscience (INS) at the University of Texas at Austin (UT-Austin). This program is becoming a nationally recognized center of excellence in neuroscience training with the creation of a new Department of Neuroscience, construction of the new Dell Medical School with Clay Johnston, a neuroscientist, as the inaugural Dean, and recruitment of outstanding Faculty. Faculty have appointments in Neuroscience, Psychology, Pharmacology and Toxicology, Computer Science, Biomedical Engineering, Kinesiology, Communication Sciences and Disorders, and Nutrition and resolutely participate in 8 prestigious centers that are part of the INS, including the Center for Learning and Memory, Center for Perceptual Systems, Institute for Cellular and Molecular Biology, Waggoner Center for Alcohol and Addiction Research, Dell Pediatric Research Institute, Behavioral Neuroscience, Imaging Research Center, and Computational Visualization Center. The program has particular strengths in the neuroscience of perceptual systems, learning and memory, and addiction research, and Faculty provide excellent training across levels of inquiry from molecules to biochemistry, structure, physiology, behavior, neuroethology, and evolution with advanced computation and theory at all levels. This breadth provides graduate students with extensive options for training in cross-disciplinary research, and the training Faculty have substantial funding to foster their research. We propose predoctoral funding for 4 new trainees and 4 second-year trainees (totaling 8 per year). The training program requirements include: a new Boot Camp and laboratory research rotations and seminar presentations, 2 principles of neuroscience courses, a science/ethics course detailing responsible scientific conduct, a course in experimental design and statistics, and 4 electives including a neuroscience of disease course. Students must also complete at least 1 semester of a graduate level course directed by an INS Faculty member that is associated with the neuroscience seminar series, where students present papers from the speaker's work and personally discuss research with the speaker. They also participate in at least 1 specialized journal club related to their project. Students join a research lab by the beginning of year 2 and complete coursework and qualifying exams, including oral defense of their research proposal written in NIH grant format by the end of year 2. Our recruitment strategies have become even more successful as reflected in the quality and diversity of the applicants. The broad interdisciplinary training uniquely prepares our trainees for research success in neuroscience, which is crucial for advancements in the etiology, pathophysiology, and treatment of brain diseases. Furthermore, the founding of the Dell Medical School on the UT-Austin campus will greatly facilitate our translational neuroscience research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A fundamental problem in biology is to understand how cells are able to sense and respond to environment cues. The integration of chemical signals such as growth factors and cytokines with mechanical stimuli is not well understood. The place where cascades involved in solid-state (mechanical) signaling and soluble (chemical) signaling converge and the manner in which they interact is no doubt complex. This research project is designed to investigate signaling events associated with both chemical and mechanical stimuli. Cells of the vascular system are continuously exposed to the effects of mechanical forces such as stretching and fluid shear stress. These forces, which are created by the pulsatile nature of blood flow when the heart contracts and relaxes, have a marked influence on cell structure and function. The adaptations of these cells, including enhanced growth and migration, seem to be important in the pathological conditions that accompany cardiovascular diseases such as atherosclerosis, hypertension, and restenosis. Cardiovascular disease remains a major cause of morbidity and mortality in the U.S. and the economic and human costs associated with these pathologies are enormous. This has resulted in an intense research interest in the mechanisms which regulate contraction, migration, and growth of vascular smooth muscle cells (VSMC). While it is now clear that mechanical forces imposed on cells of the vessel wall are important factors in the initiation and progression of pathological changes, the molecular mechanisms involved in these adaptations are not fully understood. In addition, it is now clear that the basic mechanism of smooth muscle contraction can only be explained in light of actin remodeling. However, the exact nature of cytoskeletal reorganization and the mechanisms regulating these changes are not well known. The overall goal of this project is to elucidate the acute responses in cytoskeletal reorganization that occur during mechanical stress of VSMC and to determine the intracellular signaling mechanisms that are involved. Utilizing molecular approaches combined with fluorescence microscopy, and relying on the precise changes in cell orientation and actin cytoskeletal reorganization as endpoints for quantitative assessment of responsiveness to mechanical strain, we will evaluate the role of various cytoskeletal structures on the response of VSMC to stretch. Further, we will make a systematic determination of the effects of various types of mechanical stress on activation of cell signaling molecules. In addition, we will evaluate the effects of resveratrol, a purported cardioprotective molecule, for its potential effects on stretch-induced cell signaling and receptor mediated cellular hypertrophy. The use of pharmacologic and molecular techniques to stabilize, destabilize or down-regulate specific cytoskeletal components is expected to provide clear answers concerning the role of specific components in mechanotransduction and the cell orientation response. The inhibition or down-regulation of specific signaling proteins is expected to provide information concerning pathways regulating mechanosensing and transduction. The knowledge gained may be useful in the development of therapeutic agents regulating mechanotransduction mechanisms contributing to cardiovascular pathologies.

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.

This Administrative Core will provide the coordination and oversight required for the success of the POl. This will require prompt and efficient appointment of personnel and purchase of supplies and equipment, all of which is handled by this Core. In addition, monthly meetings of all POl investigators to monitor progress and the annual meeting of the Advisory Board to provide a critical evaluation of progress and future directions must be organized. The Advisory Board meetings require considerable planning and coordination to assure that all advisors and POl members can attend and that the logistics are in place for a successful meeting. The assembly and submission of the progress reports and non-competing renewals of the POl will also be the responsibility of this Core. As we encounter new opportunities or new problems in the directions of the POl, we plan to invite scientists with needed expertise to visit the Waggoner Center. We have not requested funds for these visits as we hope to fund their visits through ongoing seminar programs, but coordination of these visits will be handled by this Core.

DESCRIPTION (provided by applicant): This five-year funding proposal for a Program Project Grant on alcohol-related research provides innovative paradigms for medication development for the treatment of alcoholism by bringing together experienced alcohol researchers with expertise in molecular biology, electrophysiology and behavior. The goal is to develop the technologies and collaborations necessary to define new targets for medication development and translate our knowledge of alcohol targets into selective ligands. The PPG consists of an Administrative Core (Adron Harris, PI and Center Director), identification and characterization of specific allosteric modulators of ion channels using phage display (S. John Mihic and Rick Morrisett), molecular and cellular mechanisms of novel therapeutic targets in alcohol reward (Igor Ponomarev and Hitoshi Morikawa), microRNA targets for medication development (Dayne Mayfield) and an animal core (Yuri Blednov and Rueben Gonzales) to provide mice and behavioral testing to the projects. The PPG has both internal and external advisory board members, providing expert guidance from scientists with a wide range of expertise in drug development and therapeutics.

PROJECT SUMMARY This is a competing renewal application for the Integrative Neuroscience Initiative on Alcoholism (INIA)- Neuroimmune consortium (Notice# RFA-AA-16-004/005/006) to identify drug targets based on the genomic, cellular, and behavioral neuroadaptations related to excessive alcohol consumption. INIA-Neuroimmune (INIA- N) will address several NIAAA goals, including: 1) understanding the genomics, electrophysiology, and pharmacology of brain immune pathways and their role in alcohol use disorders (AUDs); 2) using new technologies to study neural circuits involved in excessive alcohol drinking; 3) promoting data reproducibility and translation through testing in multiple species (including humans), multiple laboratories, and multiple assays; 4) using emerging computational resources that connect gene networks to drugs to identify compounds with potential to reduce excessive drinking. A key goal for INIA-N is to probe cross-species genomic datasets, together with novel computational approaches, to predict FDA-approved drugs that can be repurposed to treat AUDs. The overall hypothesis for INIA-N is that excessive alcohol consumption causes genetic changes and neuroadaptations in immune-related pathways that are conserved across multiple species (including humans), allowing for the systematic selection and testing of drug targets from the computational to the clinical level. Ten Research Components, two Scientific Cores, and an Administrative Core comprise the consortium. INIA-N will be directed by the Administrative Core in cooperation with the Executive and Steering Committees and guided by a distinguished Scientific Advisory Board. The Administrative Core will provide leadership, oversight of scientific projects, authentication of study drugs, and integration and translation of project data. INIA-N has four goals: 1) Expand our rodent and human genomic studies to include non-human primates (in collaboration with INIA-Stress) and new rodent models, and integrate these with existing datasets. We will also integrate genetic (genome-wide association studies) and genomic analyses (new human RNA-Seq datasets) to facilitate drug target identification, with an emphasis on neuroimmune targets and the unexplored role of novel non-coding RNAs and splice variants in alcohol consumption; 2) Combine extensive genomic resources with new computational approaches to identify candidate drugs that may be repurposed to treat AUDs. These drugs will be tested in several animal drinking models; 3) Apply systems-level approaches (electrophysiology and live brain imaging) to understand how excessive drinking changes brain function, with an emphasis on our top neuroimmune targets; 4) Select leading candidates that emerge from rigorous behavioral and functional testing to study in the new human laboratory component. INIA-N research encompasses a unique `gene network to pharmacotherapy' approach to apply cutting-edge computational tools to nominate gene targets and drugs from our extensive genomic databases and systematically test these candidates in functional and behavioral assays.