Dorit Ron - US grants

DESCRIPTION (provided by applicant): The goal of the proposed research is to gain understanding of the pathophysiological activities of ethanol in the brain, by studying the effects of ethanol on the NR2 subunits of the ionotropic glutamate N-methyl D-Aspartate (NMDA) receptor channel. The NMDA receptor is a heteromer comprised of the obligatory NR1 subunit and different combination of NR2 subunits (NR2A-D), with the major subunits in the brain being NR2A and NR2B. The cytoplasmic tails of NR2A and NR2B are phosphorylated by the tyrosine kinases Fyn or Src, and phosphorylation modulates the activity of the channel. The NMDA receptor is an important mediator of the activities of ethanol in the brain. Miyakawa and colleagues have reported that systemic administration of ethanol in mice results in tyrosine phosphorylation of hippocampal NR2B by Fyn kinase (Miyakawa et. al., 1997). Similarly, we have found that ethanol leads to phosphorylation of NR2B on tyrosine residues in isolated rat hippocampal slice preparations. We therefore hypothesize that induction of NR2B and possibly NR2A phosphorylation will play an important role in the pathophysiological effects of ethanol. In this study we propose to pursue four specific goals in an effort to better understand the biochemical, physiological and behavioral consequences of ethanol exposure mediated through the NMDA receptor system. First, we will study the spatial and temporal localization of ethanol induced NMDA receptor tyrosine phosphorylation in the brain. Second, we will examine the role of kinases and scaffolding proteins in ethanol induced NMDA receptor phosphorylation. Third, we will characterize the physiological consequences of ethanol induced NMDA receptor phosphorylation. Finally, we will test possible behavioral consequences of ethanol-induced NMDA receptor phosphorylation. Our long term goal is to understand the molecular mechanism of ethanol activities that contribute to the development of disease states associated with alcoholism, in order identify targets for the development of new medications to treat alcoholism.

DESCRIPTION (provided by applicant): Our long-term goal is to test the hypothesis that growth factors such as the glial cell line-derived neurotrophic factor (GDNF) counteract the adverse actions of alcohol and thus prevent or delay the development of alcohol addiction. In the first round of funding we found that the activation of the GDNF pathway in the midbrain dopaminergic ventral tegmental area (VTA) rapidly attenuates the motivation for, and relapse to, ethanol consumption. Using a multidisciplinary approach, we plan to elucidate the mechanism underlying the actions of GDNF to reduce consumption of ethanol. In Aim 1, we will examine whether GDNF in the VTA possesses intrinsic rewarding or stimulant properties. In Aim 2, we will determine whether GDNF adjusts the excitability of VTA neurons in a rat model of long-term excessive ethanol consumption. In Aim 3 we will test if GDNF in the VTA modifies the neuroadaptations in dopamine levels in the nucleus accumbens resulting from excessive ethanol consumption. In Aim 4, we will elucidate the molecular mechanism responsible for GDNF's action to attenuate ethanol-drinking behaviors. Alcoholism is a devastating disease that manifests itself in uncontrolled consumption. Identifying signaling pathways that inhibit this phenotype, such as the ones activated by GDNF, are therefore of great interest, as this will likely lead to the identification of new targets for medication development to treat alcoholism, and may also lead to the identification of genetic risk factors for the disease. PUBLIC HEALTH RELEVANCE: Alcoholism is a devastating disease that affects approximately 14 million people per year in the USA alone. Unfortunately, only limited numbers of drugs are currently approved by the FDA to treat adverse phenotypes associated with the disease. Therefore, there is a great need to identify novel targets for medication development. We found that the growth factor GDNF is a negative regulator of alcohol-drinking behaviors and relapse. Therefore, GDNF and its downstream effector proteins are potential drug targets to treat alcoholism. Here, we plan to elucidate the mechanism by which GDNF regulates alcohol's actions. Results generated from these studies may lead to development of novel, selective agents to treat alcohol abuse.

DESCRIPTION (provided by applicant): Our long-term goal is to test the hypothesis that endogenous systems counteract the adverse actions of alcohol and prevent or delay the development of alcohol addiction. We further hypothesize that malfunction of such pathways increases susceptibility for the development of alcoholism. This hypothesis is based on our recent studies demonstrating a homeostatic brain-derived neurotrophic factor (BDNF)-mediated signaling pathway that is upregulated by acute and intermittent exposure to moderate concentrations of ethanol, both in slices and in vivo, and suppresses sensitivity of mice to ethanol [3, 8]. For example, we observed that voluntary ethanol consumption in mice increases the expression of BDNF specifically in the dorsal striatum, a brain region that controls habit learning, and global reduction of the BDNF gene or inhibition of the BDNF pathway increases ethanol-drinking behaviors [3, 8]. Using a combination of molecular and behavioral approaches, we plan to: 1) Determine whether endogenous dorsal striatal BDNF and its downstream effectors, the dopamine D3 receptor and the neuropeptide, dynorphin, are part of a regulatory mechanism controlling motivation to consume ethanol in rats. 2) Test whether a single nucleotide polymorphism in the BDNF gene that has been shown to impair BDNF function, and is linked to increased risk for various psychiatric disorders and addiction in humans, leads to a breakdown of this protective pathway in the dorsal striatum, and increases sensitivity of mice to the adverse actions of ethanol. 3) Determine whether chronic exposure to excessive levels of ethanol is associated with the inhibition of the BDNF pathway ex vivo and in vivo. Alcoholism is a devastating disease that manifests itself as uncontrolled drinking. Understanding the molecular mechanisms that control this phenotype are therefore of great interest and will likely lead to the identification of new targets for medication development to treat alcoholism, and may lead to the identification of genetic risk factors for the disease. This proposal is aimed to determine whether BDNF and its down-stream effectors, the dopamine D3 receptor and the neuropeptide Dynorphin, are part of an endogenous "anti-addiction" cascade. Our hypothesis further suggests that behavioral adaptations that result in addictive phenotypes, such as compulsive alcohol consumption, occur when this protective pathway is down-regulated, and/or when the BDNF gene is mutated. Alcohol dependence is a widespread problem in our society, and despite decades of research, very few medications exist to treat the disease. Results generated from this study could lead to the identification of new targets for medication development to treat alcoholism and to the identification of genetic risk factors for the disease.

DESCRIPTION (provided by applicant): The long-term goal of our research is to test the hypothesis that post-translational modifications such as phosphorylation are major contributors to brain region-specific responses to alcohol exposure. In the first round of funding we found that the sensitivity of the N-methyl-D-Aspartate receptor (NMDAR) to ethanol in specific brain regions is determined by the activation state of the non-receptor tyrosine kinases Fyn (activation) and Src (inhibition), leading to changes in the phosphorylation state of the NR2B (Fyn) and NR2A (Src) subunits of the NMDAR. We found that these signaling alterations lead to profound changes in the activity of the NMDAR during and after acute exposure to ethanol in slice preparations and in vivo. More recently, we identified an important role for Fyn and NR2B-NMDAR in the dorsal striatum in the long-lasting facilitation of NR2B-containing NMDAR-mediated activity after ethanol exposure and in ethanol drinking behavior. The NMDAR is an important mediator of ethanol's actions in the brain, and our results suggest that changes in the activation state of the Fyn in the dorsal striatum contribute to aberrant synaptic plasticity that may underlie the development of behavioral phenotypes such as the propensity to consume ethanol. Using molecular, biochemical, electrophysiological and behavioral approaches, we propose to elucidate the mechanism leading to, and are the consequences of, ethanol-mediated Fyn activation in the dorsal striatum of rats. As Fyn activity is both positively and negatively regulated by phosphorylation, we will determine the contribution of the tyrosine phosphatases, PTPalpha and STEP, to ethanol's actions on Fyn activation, NR2B phosphorylation and long-term facilitation of NMDAR activity in vivo. Additionally, we plan to investigate possible physiological consequences of dorsal striatal activation of Fyn. Finally, we will determine the contribution of Fyn, PTPalpha, and STEP to operant ethanol self-administration in rats. Our long-term goal is to understand the molecular mechanisms that contribute to the development of disease states associated with alcohol addiction. Identification of new intracellular targets that are mediators of alcohol addiction is an essential future direction for the development of novel interventions for alcohol related disorders.

The first round of funding was aimed at elucidating the role of H-Ras in the nucleus accumbens (NAc) in ethanol-mediated biochemical and behavioral neuroadaptations. We found that acute systemic administration of ethanol, as well excessive consumption of ethanol, results in the activation of H-Ras in the NAc of mice and rats, and that siRNA-mediated knockdown or pharmacologic inhibition of H-Ras in the NAc leads to the attenuation of ethanol-drinking behaviors in rodents. We further showed that ethanol-mediated activation of H-Ras leads to the activation of the PI3K/AKT/mTORC1 pathway in the NAc. Importantly, pharmacological inhibition of each of these kinases, including the FDA-approved mTORC1 inhibitor, Rapamycin, resulted in the inhibition of excessive ethanol consumption and seeking. Based on these results, we hypothesized that signaling pathways downstream of H-Ras in the NAc play a crucial role in the molecular mechanism underlying ethanol-drinking behaviors. Here, we plan to test whether specific pathways that stem from PI3K, AKT and mTORC1 are induced in the NAc of mice and rats in response to repeated cycles of excessive ethanol consumption and withdrawal, and whether these contribute to the development and/or maintenance of ethanol-drinking behaviors. Aim 1 will address the role of SGK1. In Aim 2, we will test the contribution of the transcription factor NFAc4, and the microtuble-associated protein CRMP-2 in ethanol?s actions. In aim 3, we will identify mTORC1-dependent novel mRNAs whose translation is induced by ethanol, resulting in increased levels of proteins that control synaptic functions. HRas/ PI3K/AKT and mTORC1 inhibitors are actively being developed by the pharmaceutical industry as therapies against cancer and autoimmune diseases and as immunosuppressants. Therefore, we believe that results obtained from these experiments could further lead to the identification and rapid development of new medications to treat alcohol abuse disorders. Furthermore, the PI3K/AKTmTORC1 pathway is a focal point in various signaling cascades to be investigated within the ACTG. We will, therefore, be able to integrate our findings, share knowledge and reagents and easily collaborate on these projects.

DESCRIPTION (provided by applicant): The goal of this conference is to foster open discussion of research developments, build new scientific collaborations, and propel the next generation of scientific advances in alcohol neuroscience research. To reach this goal, we plan to accomplish the following four specific aims at the meeting: 1). To provide an international forum that promotes open discussion of cutting edge research at the forefront of alcohol effects on the nervous system and related neuroscience areas. 2) To introduce breakthrough neuroscientific techniques that facilitate the understanding of brain mechanisms driving alcohol drinking and abuse. 3) To promote interaction between young and senior investigators and exchange of ideas that shape the future directions of the alcohol neuroscience community. 4) To foster development of the next generation of alcohol researchers by encouraging the participation of students and postdoctoral fellows both in alcohol and in related research fields. The Alcohol and the Nervous System GRC will focus on presentation and discussion at the frontiers of alcohol research, including studies of acute, chronic and developmental alcohol exposure on neural function and the behavioral consequences of these actions. The scope of the research presented will be restricted to basic and clinical studies involving alcohol and the nervous system and will focus on questions that are important for addressing alcohol actions that contribute to excessive drinking and alcohol addiction. The meeting will also break down barriers to progress by uniting investigators with synergistic and complementary expertise in areas ranging from molecular actions of alcohol, neurocircuits, to translational research. The conference will provide a forum to discuss the latest developments in neuroscientific research on alcohol in an open and highly interactive GRC format, which includes formal talks interspersed with ample discussion time, poster sessions, and informal discussion periods that foster communication, planning and collaboration. This unique conference setup differs from that of other meetings in the field, and will provide a unique opportunity for enhanced communication between investigators at all stages of their careers, as well as between alcohol researchers and invited prominent investigators from outside the alcohol research field. To foster the growth of the next generation of alcohol researchers, we will put concerted efforts to support and encourage participation of students, postdoctoral fellows and young investigators. Thus, the present R13 will foster communication of frontier alcohol research, opportunities for cutting edge scientific collaboration, and entry of new and younger investigators into the alcohol research field. We envision the GRC on Alcohol and the Nervous System will significantly advance current understanding of neurobiology of alcohol addiction and stimulate development of effective therapeutic approaches for the treatment of alcohol use disorders.

DESCRIPTION (provided by applicant): The Alcohol Center for Translational Genetics (ACTG) will identify novel proteins as targets for therapeutics for alcohol use disorders and will determine mechanisms by which they act to regulate excessive ethanol intake. Candidate proteins will be evaluated in ethanol self-administration procedures that model excessive binge drinking in humans, motivation to drink ethanol, and relapse. The anatomical focus will be on 3 brain regions, the nucleus accumbens, the amygdala, and the ventral tegmental area, which all play important roles in ethanol consumption and relapse. Based on findings during the current funding cycle that identified H-Ras/PI3 kinaseAKT/mTORC1 signaling as a key regulator of ethanol consumption, all Research Components will include experiments that test the relationship of the novel proteins with this pathway. Three research projects will focus on proteins new to alcohol research: SGK1, GSK-3 and others whose translation is regulated by mTORC1 (Component 4); PKM? and its direct substrates (Component 5); and orexin/hypocretin receptors (Component 6). An Administrative Core (Component 1) will manage ACTG functions. An Animal Behavior Core (Component 2) will perform studies of intermittent ethanol access in rats and mice and will provide assistance in rat operant self-administration procedures. A Vector and Imaging Core (Component 3) will provide state-of-the art services to generate viral vectors for transgenic expression or gene silencing, and to analyze transcript and protein abundance by laser capture microdissection, high resolution immunofluorescence, and quantitative fluorescent in situ hybridization for detecting mRNAs in dendrites. Two Pilot projects are planned. The first will test the hypothesis that delta opioid receptor mediated inhibition of GABA release decreases alcohol consumption in anxious alcoholics. The second will determine whether up-regulation of NMDA receptor activity, induced by excessive ethanol consumption, facilitates long-term potentiation in the dorsomedial striatum, and thereby enhances ethanol drinking and seeking. Collectively, the ACTG provides a unique opportunity for integrated study of novel proteins that may lead to the development of new treatments for alcohol use disorders in humans.

DESCRIPTION (provided by applicant): This proposal is aimed at studying signaling-dependent neuroadaptations that occur in response to ethanol in subpopulations of neurons within the dorsomedial striatum (DMS). The DMS plays a central role in goal- directed behaviors and addiction, and we found that voluntary and passive exposure of rodents to ethanol activates the tyrosine kinase Fyn specifically in the DMS. We further discovered that in response to ethanol, activated Fyn phosphorylates the NR2B subunit of the NMDARs (GluN2B) resulting in increased synaptic activity of NMDA and AMPA receptors (NMDAR/AMPAR). Finally, we showed that the Fyn-signaling pathway in the DMS plays a crucial role in mechanisms underlying ethanol-drinking behaviors. Neurons in the DMS express either dopamine (DA) D1 or D2 receptors (D1R, D2R) that form the direct Go and indirect No-Go pathways. Thus, DA is thought to have different and possibly opposite effects on D1R and D2R neurons. D1Rs are coupled to G protein ?s (Gs), which activates cAMP/PKA signaling, whereas D2Rs are coupled to Gi, which inhibits cAMP/PKA. We found that Fyn activation depends on cAMP/PKA, and that in vivo administration of a D1R agonist but not D2R agonist activates Fyn signaling in the DMS, and specifically in D1R DMS neurons. Together, these data suggest the DA activates a signaling pathway consisting of D1R/Gs/Fyn/GluN2B/AMPAR in D1R neurons. Ethanol increases DA levels in the striatum, and we hypothesize that ethanol activates Fyn signaling specifically in D1R neurons in the DMS to facilitate neuroadaptations in the direct Go pathway that drive ethanol drinking behaviors. We also hypothesize that remote activation of Gs signaling in D1R DMS neurons is sufficient to produce similar Fyn-dependent cellular and behavioral neuroadaptations. These hypotheses will be tested in vivo using transgenic mice that express Cre in D1R or D2R neurons, and will utilize innovative molecular and chemico- genetic approaches to manipulate single genes (Cre-FLEX), and to remotely activate Gs signaling using the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) method in specific subpopulations of neurons. Aim 1 and Aim 2 will determine whether Fyn-dependent adaptations induced by ethanol occur in D1R, but not D2R, neurons in the DMS, and whether Gs DREADD activation in DMS D1R neurons is sufficient to produce similar outcomes. Aim 3 will investigate the contribution of Fyn signaling in D1R DMS neurons to ethanol drinking behaviors, and whether Gs DREADD activation of Fyn in D1R is sufficient to produce ethanol seeking and drinking. Combining Molecular and Systems Neuroscience methodologies, we will unravel cell- type specific signaling adaptations that underlie the development and maintenance of ethanol drinking and seeking behaviors.

The Pilot Project Component is designed to provide a flexible means for developing and exploring new research activities or directions, and unique opportunities that can evolve into independently funded research projects. During Years 6-10 we propose funding two pilot projects per year with a budget of about $50,000 per project. The expected duration of these projects will be 1 year; projects can be extended to two years upon successful competitive renewal. The Center Scientific Director will manage this Component. Pilot Project applications will be solicited annually from EGCRC and UCSF investigators. Each proposal will be evaluated for scientific merit, innovation, and for relatedness to the Center's overall goals by at least two members of the ACTG Program Advisory Board, the Scientific Director, and the Center Director. Recommendations for funding will be considered for approval by the Center Steering Committee. Two Pilot Projects have been proposed and awarded funding for Year 06. Project 7A (A. Kayser, Director) will examine whether the Delta Opioid Receptor (DOR) agonist, ALK33, can be a potential drug to treat alcohol intake and craving in a preclinical human trial. Experiments in human subjects are also aimed to conducted fMRI studies to determine whether the increase the connectivity between the medial prefrontal cortex and brain regions associated with reward and anxiety. Project 7B (J. Wang, Director) is aimed to study whether excessive ethanol intake leads to the induction of AMPA receptor-mediated synaptic plasticity events in the dorsal striatum of rats by using a combination of slice electrophysiology and the state-of-the-art optogenetics approaches. The projects that were selected by the ACTG directors for funding during years 06. Both projects are expected to contribute to the overall goals of ACTG, which are identify and to validate novel targets for development of therapeutics for alcohol use disorders. The selected pilots will also add additional aspects to the Center (e.g., human clinical and imaging studies, and optogenetics)

The mammalian target of rapamycin in complex 1 (mTORC1) is a kinase that plays a key role in protein translation at dendrites, synaptic plasticity, and learning and memory. We discovered that mTORC1 plays a major role in behaviors associated with alcohol abuse disorders (AUD) including excessive alcohol seeking and intake, as well as memories associated with alcohol reward. We further found that mTORC1 contributes to synaptic and structural plasticity in the nucleus accumbens via the translation of synaptic proteins. More recently we found that repeated cycles of alcohol binge drinking and withdrawal activate mTORC1 signaling in the Orbitofrontal Cortex (OFC), a brain region important for associative learning and reward value. We further showed that inhibition of mTORC1 in the OFC of rats attenuates alcohol seeking and habitual alcohol responding. Finally, we generated data suggesting that NMDA receptors (NMDAR) in the OFC participate in mTORC1 activation and alcohol seeking and habit. The OFC receives glutamatergic inputs from the basolateral amygdala (BLA) and mediodorsal thalamic nucleus (MDmc), and projects to the dorsal striatum (DS), and the BLA. We hypothesize that recruitment of glutamatergic BLA and/or MDmc neurons in response to alcohol activates mTORC1 signaling in the OFC to drive habitual alcohol seeking (Aim 1). We further hypothesize that mTORC1 is activated by alcohol in specific OFC projecting neurons to initiate alcohol seeking and habitual responding (Aim 2). In Aim 3, we will determine the molecular mechanisms underlying the aforementioned behavioral outcomes. Specifically, we will study the contribution of the mTORC1 downstream target, collapsin response mediator protein II (CRMP-2), to alcohol seeking and habit. We will also conduct an RNA sequencing analysis to identify novel downstream targets of mTORC1 in the OFC whose translation is increased by alcohol. To address these hypotheses, and test the contribution of the mTORC1 signaling in the context of OFC circuitries, we plan to use novel molecular tools in combination with behavioral paradigms in rats. Together, this research proposal will allow us for the first time, to elucidate the role of an important molecular target of alcohol in the context of key neural circuits. Furthermore, the contribution of the OFC to AUD has been understudied, and our research effort will greatly expand the knowledge on this topic. Finally, our studies on mTORC1 and its downstream targets will contribute to drug development efforts for the treatment of AUD. RELEVANCE (See instructions): This proposal is aimed to study how the kinase mTORC1 in brain circuitries that centers around the brain region, OFC, contributes to two clinically relevant phenotypes, alcohol seeking and habit. To do so, we will use state of the art molecular approaches in combination with behavioral paradigms in rats. Our study will not only expand our knowledge on the neuroadaptations that underlie alcohol actions in the brain but will also enable the identification of new targets for future drug development to treat AUD.

PROJECT SUMMARY Adolescence marks a period of increased risk taking and exploration. As part of this exploratory behavior, many individuals initiate alcohol use during adolescence, which can lead to increased risk for alcohol use disorders later in life. One major site of adolescent maturation in prefrontal cortex are GABAergic inhibitory neurons, including parvalbumin positive chandelier cells. Chandelier cells synapse directly onto the axon potential initiating sites of neighboring glutamatergic pyramidal cells. They are therefore uniquely poised to regulate pyramidal cell spiking. Here, our goal is to understand how the function of these two inhibitory cell classes are affected by adolescent alcohol consumption, and to determine if manipulations to chloride transporter function alters drinking behavior. We will use a combination of electrophysiology and 2-photon imaging to understand how interneuron function is regulated at the cellular level, and viral manipulation of chloride transporter function paired with behavior to understand how prefrontal inhibitory function regulates drinking. Results of this study will provide insight into how inhibitory circuits contribute to drinking behavior and may elucidate potential therapeutic targets to treat alcohol abuse disorders.

ABSTRACT This proposal is aimed at testing the contribution of the Rho GTPases Rac1 and RhoB in the dorsal striatum to the mechanisms underlying alcohol use disorder (AUD). Rac1 and RhoB play an important role in cytoskeleton rearrangement as well as in the remodeling of dendrites and spines. We recently discovered that excessive alcohol intake and withdrawal activate Rac1 and RhoB in the mouse dorsomedial striatum (DMS), and dorsolateral striatum (DLS), respectively. Based on published and preliminary data, we will test the hypothesis that alcohol activates Rac1 and RhoB, in DMS and DLS neurons, respectively, leading to cellular adaptations which in turn drive the development and/or maintenance of alcohol drinking behaviors including goal directed and habitual alcohol seeking. This hypothesis will be explored by the following aims. Aim 1 will determine the mechanism by which heavy alcohol use actives Rac1 and RhoB in the DMS and DLS. We will also determine whether the small G proteins are activated in subpopulation of striatal neurons. Aim 2 will determine the cellular consequences of Rac1 and RhoB activation in the DMS and DLS, respectively. Specifically, will examine the hypothesis that Rac1 activation by alcohol in the DMS promotes F-Actin formation and dendritic tree branching as well as spine maturation. We will also test the hypothesis that alcohol-dependent RhoB activation in the DLS leads to opposite outcomes e.g. dendritic tree shrinkage and increase in the number of immature spines. Aim 3 will address the behavioral consequences of Rac1 and RhoB activation in the DMS and DLS, and determine whether the small G proteins contribute to the development and/or maintenance of alcohol drinking behaviors. Finally, as the DMS and DLS are central regions in circuitries that drive goal directed and habitual behaviors, respectively, we will determine whether Rac1 and RhoB drive goal directed and habitual alcohol seeking. To achieve these goals, we plan to combine state of the art molecular/genetic tools with mouse alcohol drinking paradigms. If successful, data generated from the studies will: a. shed a light on the contribution of an important group of signaling molecules to AUD; b. will enable us to gain insights on how alcohol exposure produces structural modifications in the dorsal striatum; c. will potentially identify the molecular mechanisms underlying goal-directed and/or habitual alcohol seeking; d. as small molecules targeting Rho GTPases are being developed for other indications, the application has a clear translational trajectory.