Paul J. Kenny - US grants

DESCRIPTION (provided by applicant): This application is in response to RFA-DA-07-006 to design, synthesize and test in relevant preclinical models, potential treatments for drug addiction. The pharmacological approach proposed is to develop new classes of potent and selective orexin-1 (OX1) receptor antagonists that may prove efficacious in preventing relapse in abstinent human cocaine addicts. To accomplish this aim, we have assembled a team of highly motivated researchers with expertise in every aspect of the drug discovery process and in animal models of addiction. Specific Aim I will involve establishing a robust in vitro cell-based functional assays for OX1 receptors (and appropriate counter-screens) which can support a medicinal chemistry program aimed at developing new classes of potent and selective OX1 receptor antagonists. Specifically, we will examine changes in agonist-induced increases of intracellular calcium signaling via membrane potential dyes and a fluorescent plate reader (FLIPR) in CHO cells stably expressing human OX1 receptors (OX1 receptors couple to Gq).In addition, we will establish a novel agonist-induced, G-protein-independent, receptor internalization assay coupled with high content imaging to make quantitative measurements of 0X1 receptor internalization upon agonist stimulation in U20S cells stably expressing OX1 receptors. This internalization assay will provide a counter-screen to the calcium signaling assays. Specific Aim II will utilize an iterative medicinal chemistry program based on structure-activity relationships (SAR) to discover new classes of potent and selective OX1 receptor antagonists. Structure-activity relationships will be employed to optimize new classes of OX1 receptor antagonists for drug metabolism and pharmacokinetics (DMPK), and brain penetration properties. Specific Aim III will test new classes of potent and selective OX1 receptor antagonists with favorable DMPK and brain penetration in the rat stress-induced reinstatement model of relapse to cocaine seeking. In addition, the intrinsic rewarding properties of OX1 receptor antagonists that block relapse-like behavior will be assessed in the intracranial self-stimulation (ICSS) thresholds procedure in rats in order to triage compounds with potential abuse liability. Such an integrated multidisciplinary research plan will capitalize on the unique drug discovery capabilities at Scripps Florida, and promises to yield novel therapeutic entities for the prevention of relapse in human addicts.

DESCRIPTION (provided by applicant): The persistence of the tobacco smoking habit can be attributed in large part to the addictive actions of nicotine. Similar to all major drugs of abuse, nicotine excites brain reward systems. This stimulatory action of nicotine on reward systems underlies, or at least contributes to, it's positive reinforcing effects that maintain nicotine intake. In contrast, withdrawal from nicotine after chronic exposure decreases the activity of brain reward systems. Avoidance and alleviation of this withdrawal-associated reward deficit may also contribute to the persistence of the tobacco habit. An understanding of the mechanisms by which nicotine stimulates brain reward systems, and generates reward deficits during withdrawal, may lead to development of novel therapies for the treatment of habitual tobacco smoking. The overall hypothesis guiding this proposal is that nicotine acts at specific subtypes of nicotinic acetylcholine receptors (nAChRs), comprised of discrete nAChR subunits, to stimulate reward systems and generate reward deficits during withdrawal. Specific Aim I of this proposal will identify the nAChR subtypes that regulate nicotine consumption in mice. This goal will be achieved by identifying mice with targeted deletion of specific nAChR subunits (nAChR-/- mice) that demonstrate attenuated intravenous nicotine self- administration (SA) compared with wildtype (WT) littermates. Specific Aim II will identify nAChR subunits that regulate the stimulatory effects of nicotine on brain reward systems. This goal will be achieved by identifying nAChR-/- mice with attenuated nicotine-induced lowering of intracranial self-stimulation (ICSS) thresholds compared with WT mice. Convergent data from nicotine SA and ICSS experiments will provide compelling evidence for a role of specific nAChR subunits in nicotine reinforcement. Specific Aim III will identify nAChR subunits at which chronically administered nicotine acts to induce nicotine dependence and the expression of reward deficits during withdrawal. This goal will be achieved by identifying nAChR-/- mice with attenuated elevations of ICSS thresholds compared with WT mice during spontaneous withdrawal from chronically administered nicotine. These studies promise to yield significant new insights into the neurobiological mechanisms of nicotine addiction, with relevance for the treatment of the tobacco habit in human smokers.

DESCRIPTION (provided by applicant): This is a new RO1 grant application seeking to gain a better understanding of the role of noncoding RNA regulatory networks relating to the NMDA receptor (NMDA-R) hypofunction hypothesis in schizophrenia. The underlying hypothesis of this application is that specific miRNAs or families of miRNAs play a role in regulating schizophrenia-like behavioral deficits in mice. The goals of this proposal are threefold: First, we will investigate expression patterns of brain-specific microRNAs (miRNAs) in NMDA-R- related animal models of schizophrenia. Specifically, we will examine in mice the effects of acute or chronic exposure to the non-competitive NMDA-R antagonist and schizomimetic agent, MK-801, on miRNA expression in brain regions implicated in schizophrenia in human patients, with a focus on prefrontal cortex (PFC). It is predicted that pharmacological and genetic models of schizophrenia in mice will be associated with distinct patterns of dysregulated miRNA expression. Importantly, convergent data between pharmacological and genetic models of schizophrenia will provide convergent support for the participation of particular miRNAs or families of miRNAs in schizophrenia-related deficits. Importantly, we will examine the effects of agents with known beneficial effects on schizophrenia-associated behavioral deficits (haloperidol and clozapine) on the expression of miRNAs shown to be dysregulated in the schizomimetic mouse models. It is predicted that antipsychotic agents with known clinical utility will reverse, at least in part, dysregulated patterns of miRNA expression in the mouse models of schizophrenia. To more directly assess the roles of identified miRNAs in schizophrenia-like behavioral deficits in mice, we will modulate the expression of targeted miRNAs by direct intracerebral infusion of locked nucleic acid (LNA)-modified antagomiRs into the brains of mice. The effects of modulating targeted miRNAs in this manner will be assessed on baseline and MK-801-induced behaviors in three procedures that model aspects of schizophrenia-like deficits in mice: hyperlocomotion with stereotyped behaviors;decreases in social interaction;and elevations of intracranial self-stimulation thresholds. It is predicted that miRNAs mediate the expression of schizophrenia-like deficits in mice and that decreasing the expression of targeted miRNAs may attenuate the expression of schizophrenia-like deficits. The experiments proposed in this application promise to yield significant new insights into the pathophysiology of schizophrenia, and may reveal novel treatment and/or biomarker approaches for schizophrenia-associated behavioral deficits. RELEVANCE TO PUBLIC HEALTH: Schizophrenia is a chronic psychiatric disorder characterized by impairments in perception or expression of reality, by significant social or occupational dysfunction and by profound cognitive impairment. Thus, schizophrenia results in tremendous human suffering and negative economic impact on society. Approximately 1% of the population of the United States, around 3 million people, suffers from schizophrenia. Importantly, the underlying etiology of schizophrenia remains largely unknown and there is a marked need for improved treatment paradigms. The proposed work has the potential to aid in the development of completely novel therapeutics.

This grant application seeks to better understand the role for microRNAs in the molecular mechanisms by which cocaine remodels the striatum and thereby drives the emergence of addiction-relevant behavioral abnormalities. A major accomplishment during the previous funding period was showing that disruption of the microRNA biogenic machinery in striatum profoundly decreases cocaine intake in genetically modified mice. This finding supports a key role for striatal microRNAs in regulating the reinforcing properties of cocaine. Subsequently, we established that two closely related microRNAs, miR-212 and miR-132, are induced in the striatum of rats demonstrating compulsive-like cocaine-taking behaviors. miR-212 exerts an inhibitory influence on cocaine-taking behavior through a mechanism involving enhanced striatal CREB signaling and diminished MeCP2/BDNF signaling. By contrast, our recent data suggests that miR-132 may enhance the motivational properties of cocaine through, as yet, unclear mechanisms. We hypothesize that the balance between miR-212 and miR-132 signaling influences resilience vs. vulnerability to addiction. In this competitive renewal, we will use cutting edge molecular, cellular and behavioral approaches to define the precise mechanisms by which miR-212 and miR-132 control drug intake. The research plan builds logically and innovatively on the progress made during the last funding period. In Specific Aim I, the cellular mechanisms by which miR-212 and miR-132 act in striatum will be investigated. To accomplish this aim we have successfully constructed two new lines of transgenic mice: those with ?floxed? alleles for the miR-212 or the miR-132 gene. Using these mice, we will conditionally delete miR-212 or miR-132 in D1 receptor-expressing medium spiny neurons (D1-MSNs) or in D2-MSNs, the two major cell populations that together represent ~95% of total neurons in striatum. The effects of these cell-specific lesions on cocaine self-administration, and other addiction-relevant behaviors, will be investigated. In Specific Aim II, the molecular mechanisms by which miR-212 and miR-132 control cocaine intake will be investigated. We will use High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) to identify genes targeted by miR-212 and miR-132 in D1-MSNs and D2- MSNs. We will verify that identified genes are direct targets for miR-212 and/or miR-132 using 3?UTR luciferase reporter assays, RNA expression analysis and protein immunoblotting. Under Specific Aim III, we will use in vivo CRISPR technology to investigate the contribution of the most promising gene targets whose expression is controlled by miR-212 and/or miR-132 in regulating the motivational properties of cocaine. These studies promise to yield significant new insights into molecular and cellular mechanisms of cocaine addiction.

DESCRIPTION (provided by applicant): This application is in response to RFA-DA-10-018, Medications Development for Substance Related Disorders (R01). We aim to design, synthesize and test in relevant preclinical models, potential treatments for tobacco addiction. The pharmacological approach proposed is to develop new classes of potent and selective positive allosteric modulators (PAMs) at a5* nicotinic acetylcholine receptors (nAChRs), that may prove efficacious in facilitating smoking cessation efforts. To accomplish this aim, we have assembled a team of highly motivated researchers with expertise in every aspect of the drug discovery process and in animal models of addiction. Under Specific Aim I, we will utilize in vitro cell-based functional assays for a5*nAChRs receptors (and related nAChR subtypes for counter-screens) to drive an iterative medicinal chemistry program aimed at developing new classes of small molecule drugs that amplify a5* nAChR signaling. Specifically, as a primary screening strategy we will examine changes in agonist-induced increases of intracellular calcium signaling via membrane potential dyes and a fluorescent plate reader (FLIPR) in HEK cells stably expressing a5* nAChRs. As a secondary screening strategy, we will assess the effects of novel PAMs on voltage-clamp recordings in Xenopus oocytes expressing a5* nAChRs. Oocyte will provide important insights into the mode of action of PAMs. In addition, we will also test novel a5* nAChR PAMs in a rubidium (86Rb+) efflux assay using synaptosomes from a brain region in which nAChRmediated currents are almost exlusively from a5* nAChRs. Specific Aim II will utilize an iterative medicinal chemistry program based on structure-activity relationships (SAR) to develop new classes of potent and selective a5* nAChR PAMs. Starting points for chemistry will be known pharmacophores from the literature and hits from a HTS campaign. SAR will be employed to optimize new classes of a5* nAChR PAMs for drug metabolism and pharmacokinetics (DMPK), and brain penetration properties. Specific Aim III will test new classes of potent and selective a5* nAChR PAMs with favorable DMPK and brain penetration in the mouse intravenous nicotine self-administration procedure, using wildtype mice and mice with null mutations in a5 nAChR subunits. In addition, the intrinsic rewarding properties of a5* nAChR PAMs and their effects on nicotine reward will be assessed in the brain-stimulation reward (BSR) procedure in rats. This highly integrated multidisciplinary program will capitalize on the unique drug discovery capabilities at Scripps Florida, and promises to yield novel therapeutic entities for smoking cessation. PUBLIC HEALTH RELEVANCE: Tobacco smoking results in greater than 5 million deaths each year even when using the most clinically efficacious smoking cessation agents available, approximately 80% of smoking attempting to quit will relapse within one year, highlighting the need to develop safe yet more clinically effective smoking cessation agents. Here, we plan to develop novel therapeutics tofacilitate smoking cessation efforts based on an entirely new mechanism of action, the rationale for which is based on new genetic and behavioral evidence. If successful, this program of research promises to have a significant positive impact on human health.

DESCRIPTION (provided by applicant): The burden of disease and negative economic impact of tobacco addiction on society is staggering. It is predicted that ~0.6 billion current smokers worldwide will die from smoking-related illnesses, such as chronic obstructive pulmonary disorder (COPD), lung cancer, and cardiovascular disease. The development of efficacious smoking cessation aids is considered the most cost-effective intervention possible within a modern health-care system. Although clinically efficacious, current smoking cessation agents have very limited utility. Indeed, there is considerable risk of relapse even when treated with the most efficacious medications currently available, Chantix and Zyban, which must also now carry "black box" warnings on their labeling because of reported serious mental-health events associated with their use. Pharmacotherapy is therefore an effective strategy to aid smoking cessation efforts, but there is a pressing need to develop safer and more effective therapeutics. Product Development Partnerships (PDPs) are a collaborative effort between academia, the pharmaceutical industry and charitable organizations that seek to discover, develop and deliver new medicines. PDPs bridge the gap between research and development for diseases for which there has traditionally been low priority in industry to develop effective therapeutics. The major advantage of the PDP mechanism for therapeutic development for smoking cessation with Scripps Florida as the Managing Partner (MP) is that it takes full advantage of our core mission at TSRI-Scripps Florida to develop medications to aid smoking cessation and combines this expertise with the scientific and financial resources of PDP partners. Therapeutics will include small molecules (novel and re-purposed, RNAs, peptides, antibodies, and novel formulations and delivery mechanisms). During the UH2 phase of the MITD, we will thoroughly explore the feasibility of establishing a successful PDP for the generation of new smoking cessation products. During the UH3 phase we will execute on the Milestones described below. The Milestones achieved during the UH2 phase of the MITD PDP are;1) Construct a searchable bioinformatic database to prioritize targets for therapeutic development. We will then report our findings using the database, along with all available scientific and strategic information on drug discovery in tobacco addiction, as a most comprehensive review of the nicotine addiction discovery process;and 2) We will organize a symposium, in conjunction with NIDA program staff, at a major scientific meeting (SRNT or CPDD) and FDA to discuss current clinical endpoints, and the potential and utility for developing biomarkers. We will also host a 2-day summit at TSRI-Scripps Florida to which all awardees of UH2 phase grants, NIDA program officials, leading academic scientists, established and perspective industry partners who may join the PDP, representatives from charitable foundations charged with combating tobacco addiction and smoking- associated disorders, and officials from the State of Florida Department of Health. At the end of this summit, we will identify the optimal partnerships to successfully drive the mission of the MITD PDP. The milestones achieved during the UH3 phase of the MITD PDP are: 1) We will fill a robust preclinical/clinical discovery pipeline based on findings during the UH2 phase;2) By year 3 of the UH3, we will have 1 compound entering Phase II clinical trials;and 3) By year 5 of the UH3, we will have 1 compound entering Phase III clinical trials, and another 1-2 compounds entering Phase II clinical trials. PUBLIC HEALTH RELEVANCE: The burden of disease and negative economic impact of tobacco addiction on society is staggering. It is predicted that ~0.6 billion current smokers worldwide will die from smoking-related illnesses, such as chronic obstructive pulmonary disorder (COPD), lung cancer, and cardiovascular disease. Indeed, if current trends in tobacco use persist, by 2020 smoking will become the largest single health problem worldwide, causing ~8.4 million deaths annually. The World Bank estimates that in high-income countries, smoking-related healthcare accounts for between 6-15% of all healthcare costs, ~$160 billion annually. Smokers who quit before the onset of tobacco-related illness can largely avoid the increased mortality risk. Nevertheless, ~80% of smokers currently attempting to quit will relapse within the first month of abstinence. The development of efficacious smoking cessation aids is considered the most cost-effective intervention possible within a modern health-care system. Product Development Partnerships (PDPs) are a collaborative effort between academia, the pharmaceutical industry and charitable organizations that seek to discover, develop and deliver new medicines. PDPs bridge the gap between research and development for diseases for which there has traditionally been low priority in industry to develop effective therapeutics (small market size etc).

DESCRIPTION (provided by applicant): This application describes a highly integrated series of projects aiming to develop orexin-1 (OX1) receptor antagonists for treatment of tobacco dependence. Project 1 will utilize iterative medicinal chemistry based on structure-activity relationships (SAR) to discover new classes of potent and selective OX1 receptor antagonists. SAR will be used to optimize new classes of OX1 receptor antagonists for drug metabolism and pharmacokinetics (DMPK), and brain penetration properties. We have already generated excellent OX1 receptor antagonists as starting points for SAR based on our ongoing medicinal chemistry efforts. Project 2 will support SAR by testing new compounds in in vitro cell-based functional assays for OX1 receptors (and appropriate counter-screens) to determine potency and selectivity. We have already established and validated a range of OX1 receptor cell-based assays, and have successfully used these assays to identify starting points for SAR. Project 3 will test new classes of potent and selective OX1 receptor antagonists to identify those with the most favorable physiochemical and brain penetration profiles, and identify those least likely to have toxicity in humans. Project 4 will test the in vivo efficacy of novel OX1 receptor antagonists in cutting-edge behavioral procedures that assess addiction-related behavioral effects of nicotine. Importantly, have developed a new IV nicotine self-administration procedure for mice, and will assess novel OX1 receptor antagonists in wild type and OX1 receptor knockout mice to determine their behavioral selectivity. This integrated multidisciplinary research plan will capitalize on the unique drug discovery capabilities at Scripps Florida, and promises to yield novel therapeutic entities for the prevention of relapse in human tobacco smokers.

DESCRIPTION (provided by applicant): This revised application is a request for renewal of R01 DA020686. A major accomplishment of the previous funding period was identifying a key for ? 5-containing nicotinic acetylcholine receptors (?5* nAChRs) in the habenulo-interpeduncular tract in regulating nicotine intake. We found that nicotine-induced activation of ? 5* nAChRs in the habenulo-interpeduncular tract triggered a negative motivational signal that served to reduce further nicotine consumption. Genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the ? 5, ? 3 and ?4 nAChR subunits respectively, increases vulnerability to tobacco addiction and smoking- associated diseases including lung cancer. Here, we will fully define the role for ? 5*, ? 3* and ? 4* nAChRs in the MHb-IPN tract regulating nicotine reinforcement. Under Specific Aim I, we will assess nicotine self-administration behavior in knock-in mice in which the ? 5 nAChR subunit gene has been genetically modified to express a major risk allele for tobacco dependence in humans. Second, we will use an elegant combination of Double-floxed Inverted (DiO) adeno- associated viruses in Cre-expressing transgenic mice or in mice treated with Cre-expression virus to selectively re-express ? 5 nAChR subunits in discrete neuronal populations and pathways in the brains of ? 5 subunit knockout mice. We will then assess the impact on nicotine self-administration behavior in these rescued mice. Under Specific Aim II we will investigate the role for ? 3* nAChRs in the habenulo-interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that ? 3 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. Under Specific Aim III we will investigate the role for ? 4* nAChRs in the habenulo- interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that ? 4 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will similarly confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. These studies promise to yield significantly new advances in our understanding of nicotine dependence and tobacco addiction.

The goals of Project 4 are to assess the therapeutic potential of novel 0X1 receptor antagonists with the most desirable pharmacological and physiochemical properties. In Specific Aim I we will test the effects of novel 0X1 receptor antagonists on intravenous (IV) nicotine self-administration behavior in rats. We will will test their effects on cue-induced reinstatement of extinguished nicotine-seeking behavior, and in attenuating the incubation of craving for nicotine. In complementary experiments we will also examine their effects on food-motivated behaviors using almost identical procedures. In this manner we can identify novel 0X1 receptor antagonists that selectively decrease nicotine addiction-related behaviors in rats, and also those that act in a non-specific manner through generalized disruption of behavioral performance. Nicotine stimulates brain reward circuitries, reflected in lowering of intracranial self-stimulation (ICSS) thresholds in rats, and obtaining this action of nicotine may motivate the initiation and persistence of the tobacco habit. Under Specific Aim 11, we will test the effects of novel 0X1 receptor antagonists on nicotine-induced lowering of ICSS thresholds in rats. Using the ICSS procedure we will also be able to identify 0X1 receptor antagonists with intrinsic abuse potential as those compounds that induce nicotine-like lowering ICSS thresholds. Finally, a major concern when developing novel antagonists for 0X1 receptors as potential therapeutics, or for any other potential drug target in brain, is that they may have unknown off-target actions that contribute to their behavioral effects. As such, positive data generated in animal mode s of nicotine addiction using such compounds would be entirely confounded and have little clinical utility. In order to test for the behavioral selectivity of novel 0X1 receptor antagonists we will test their effects on intravenous nicotine self-administration behavior in wildtype mice and in mice with null mutation in the 0X1 receptor (i.e., knockout mice). It is predicted that selective 0X1 receptor antagonists will decrease nicotine intake in wildtype mice without altering intake in knockout mice.

DESCRIPTION (provided by applicant): This application is in response to RFA-NS-13-003, NIH Blueprint for Neuroscience Research Grand Challenge: Discovering Novel Drugs for Disorders of the Nervous System (U01). Orexin- A (OXA) and orexin-B (OXB), also known as hypocretin-1 and hypocretin-2, are lateral hypothalamic (LH) neuropeptides that stimulate orexin-1 (OX1) and orexin-2 (OX2) receptors. Recently, our laboratory and others have generated compelling evidence that OXA peptide, acting through OX1 receptors, regulates the stimulatory effects of nicotine on brain reward systems and thereby controls nicotine self-administration behavior in rats and mice. In addition, OX1 receptors regulate the relapse-like reinstatement of extinguished drug-seeking responses in abstinent rats and mice. Indeed, the development of OX1 receptor antagonists that are safe for use in humans is considered perhaps the most promising approach to developing novel therapeutic agents for tobacco dependence and other substance abuse disorders in humans. We aim to facilitate the design, synthesis and testing in relevant preclinical models, of novel OX1 receptor antagonists through the Blueprint Neurotherapeutics Network. We have identified novel chemical scaffolds that are yielding selective OX1 receptor antagonists that should be suitable for development through the Blueprint Network. We have developed robust cell-based assays to reliably OX1 receptor antagonist actions (and appropriate counterscreens). In addition, we have established the most relevant animal model of nicotine addiction currently available, the intravenous nicotine self-administration procedure, in mice. We can now assess the effects of novel OX1 receptor antagonists on nicotine reinforcement in wildtype mice to determine if the compounds demonstrate in vivo efficacy. Also, the effects of novel OX1 receptor antagonists on responding for nicotine in OX1 receptor knockout mice can be assessed, thereby determining if the compounds are behaviorally selective. This exciting drug development program capitalizes on the unique capabilities of Blueprint Neurotherapeutics Network. It will leverage our progress to date in identifying novel chemical scaffolds and take advantage of our highly relevant in vitro and in vivo assays. Hence, this program promises to yield OX1 receptor antagonists as novel therapeutic for smoking cessation.

PROJECT SUMMARY This application is submitted in response to PA-13-194: Mechanisms of alcohol and nicotine co-addiction. Allelic variation in CHRNA5, the gene encoding the ?5 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to alcohol and tobacco dependence. Here, we will use cutting-edge molecular, genetic and behavioral techniques to investigate the role for ?5* nAChRs, particularly those in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway where ?5* nAChRs are densely expressed, in regulating the motivational properties of alcohol. In Specific Aim I, we will assess alcohol drinking in two lines of mice with deficient ?5* nAChR signaling: ?5 subunit knockout mice and ?humanized? knock-in mice in which the ?5 nAChR subunit gene has been genetically modified to express the major risk allele for tobacco and alcohol dependence in humans. Second, we will use the intracranial self-stimulation (ICSS) procedure to assess the rewarding and aversive effects of alcohol in these lines of mutant mice. We predict that alcohol intake will be increased, and aversive effects of alcohol decreased, in mice with deficient ?5* nAChR signaling. In Specific Aim II, we will investigate the effects of alcohol on the MHb-IPN system. First, we will use optogenetics coupled with electrophysiological recordings to examine the effects of alcohol on excitatory and inhibitory transmission at the MHb-IPN synapse and determine the role for ?5* nAChRs in these effects. Second, we will use rubidium efflux assays to determine the effects of alcohol drinking on the activity of ?5* nAChRs in the MHb-IPN system. We predict that alcohol stimulates excitatory MHb inputs to IPN ? an ?aversion? signal ? and that this effect is attenuated by deficient ?5* nAChR signaling. We further predict that prolonged alcohol intake results in diminished activity of ?5* nAChRs in MHb-IPN system, which may contribute to the development of the alcohol drinking habit. In Specific Aim III, we will investigate the involvement of the MHb-IPN system, and ?5* nAChRs in this system, in regulating alcohol drinking. First, we will use an elegant Cre recombinase-dependent chemogenetics (DREADD) approach to stimulate or inhibit neurons in the MHb-IPN system, or more selectively only those MHb-IPN neurons that express ?5* nAChRs, and examine effects on alcohol drinking. Second, we will use virus-mediated gene transfer to re-express otherwise absent ?5 nAChR subunits in MHb or IPN neurons of the ?5 KO mice and examine the effects on alcohol drinking in these mice. We predict that the MHb-IPN system, and ?5* nAChRs in this system, play a key role in regulating alcohol intake. This innovative program of research may yield novel insights into the mechanisms of alcohol dependence that supports development of entirely new classes of therapeutic agents.

DESCRIPTION (provided by applicant): This application is in response to RFA-NS-13-003, NIH Blueprint for Neuroscience Research Grand Challenge: Discovering Novel Drugs for Disorders of the Nervous System (U01). Orexin- A (OXA) and orexin-B (OXB), also known as hypocretin-1 and hypocretin-2, are lateral hypothalamic (LH) neuropeptides that stimulate orexin-1 (OX1) and orexin-2 (OX2) receptors. Recently, our laboratory and others have generated compelling evidence that OXA peptide, acting through OX1 receptors, regulates the stimulatory effects of nicotine on brain reward systems and thereby controls nicotine self-administration behavior in rats and mice. In addition, OX1 receptors regulate the relapse-like reinstatement of extinguished drug-seeking responses in abstinent rats and mice. Indeed, the development of OX1 receptor antagonists that are safe for use in humans is considered perhaps the most promising approach to developing novel therapeutic agents for tobacco dependence and other substance abuse disorders in humans. We aim to facilitate the design, synthesis and testing in relevant preclinical models, of novel OX1 receptor antagonists through the Blueprint Neurotherapeutics Network. We have identified novel chemical scaffolds that are yielding selective OX1 receptor antagonists that should be suitable for development through the Blueprint Network. We have developed robust cell-based assays to reliably OX1 receptor antagonist actions (and appropriate counterscreens). In addition, we have established the most relevant animal model of nicotine addiction currently available, the intravenous nicotine self-administration procedure, in mice. We can now assess the effects of novel OX1 receptor antagonists on nicotine reinforcement in wildtype mice to determine if the compounds demonstrate in vivo efficacy. Also, the effects of novel OX1 receptor antagonists on responding for nicotine in OX1 receptor knockout mice can be assessed, thereby determining if the compounds are behaviorally selective. This exciting drug development program capitalizes on the unique capabilities of Blueprint Neurotherapeutics Network. It will leverage our progress to date in identifying novel chemical scaffolds and take advantage of our highly relevant in vitro and in vivo assays. Hence, this program promises to yield OX1 receptor antagonists as novel therapeutic for smoking cessation.

PROJECT SUMMARY This revised R01 application is submitted in response to PAR-14-309 and seeks to understand the mechanisms by which a microRNA enriched in cortical parvalbumin-expressing (PV) GABAergic interneurons regulates the activity of these cells and behaviors under their control. In prefrontal cortex (PFC), microRNA-206 (miR-206) is highly enriched in PV interneurons and its expression levels in post-mortem PFC correlate with psychosis in schizophrenia and bipolar patients. In exciting preliminary data, we show that newly generated miR-206 knockout mice demonstrate cellular and cognitive deficits consistent with the hypothesis that this microRNA regulates PV interneuron function in PFC. The goals of this proposal are threefold: First, cellular and behavioral abnormalities in miR-206 KO mice, and in KO mice in which miR-206 expression has been ?rescued? in cortical PV interneurons, will be assessed. To accomplish this Aim we have generated miR-206 knockout mice that express Cre recombinase in PV neurons. We will use a Cre-dependent expression vector to re-express the otherwise deleted miRNA in cortical PV interneurons. We will then assess the strength of synaptic connectivity between cortical PV interneurons and neighboring pyramidal neurons in PFC of miR-206 KO mice, KO mice with rescued miR-206 expression in cortical PV interneurons, and appropriate control groups. We will also characterize aspects of cognition and emotionality controlled by cortical PV interneurons in these mice. Second, the intracellular mechanisms by which miR-206 controls activity of PV interneurons, and hence mPFC function, will be investigated. We will use High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) to identify genes targeted by miR-206 in cortical PV interneurons. We will verify that identified genes are direct targets for miR-206 using 3'UTR luciferase reporter assays, RNA expression analysis and protein immunoblotting. Third, the molecular mechanisms by which miR- 206 controls activity of PV interneurons, and hence mPFC function, will be investigated. Specifically, we will use in vivo CRISPR technology to delete the most promising genes targeted by miR-206 in PV interneurons and assess the behavioral consequences. The cutting-edge experiments proposed in this application will facilitate greater understanding of the molecular mechanisms by which cortical PV interneuron activity is regulated and may yield new insights into the pathophysiology of PV interneuron-related psychiatric disorders.

PROJECT SUMMARY The GPCR superfamily of ligand regulated receptors has proven to be a rich source of targets for development of therapeutics for a myriad of human diseases. The orexin 1 and orexin 2 receptors are class A GPCR?s differentially expressed in the central nervous system. The orexin 1 receptor is most abundantly expressed in the locus coeruleus and is thought to control aspects of emotion, reward, and the autonomic nervous system, whereas the orexin 2 receptor is expressed in regions controlling arousal such as the tubermammillary nucleus, an important site for the regulation of sleep/wakefulness. Almost 20 years after their initial discovery, the first potent dual orexin 1 / orexin 2 receptor antagonist therapeutic has been brought to market for insomnia (Belsomera ®, suvorexant, Merck). Despite a massive effort to identify antagonists of the the orexin receptor, almost no reports of small molecule agonists appear in the primary or patent literature. Elegant genetic ablation experiments using orexin knock-out animals and experiments with intracerebroventricular dosing of orexin A or orexin B peptides suggests a role for orexin receptor agonists or potentiators for a number of potential indications including: 1) depression; 2) learning and memory (cognition); 3) attention deficit hyperactivity disorder (ADHD); 4) treatment for colon cancer; and 5) sleep disorders including narcolepsy. While the orexin peptides are potent agonists of both orexin receptors, they are not selective nor do they cross the blood brain barrier well, making them poor probe substrates for in vivo pharmacology studies. This provides the impetus to identify small molecule, brain penetrant activators of the orexin receptors to interrogate the receptors function in the context of disease states. We have optimized a cell-based high-throughput screening compatible primary assay that specifically measures the functional activity of orexin 1 and orexin 2. A preliminary 10k pilot screen was performed leading to the identification of a number of small molecule agonists of these receptors demonstrating this assay to be robust and reproducible. A full HTS screening campaign of the Scripps Institutional Drug Discovery Library (~640k compounds) will lead to the identification of multiple classes of orexin receptor agonists and potentiators for further development. In an iterative process, these validated hits will be characterized through a cascade of in vitro cell-based assays to determine potency, selectivity and mechanism of action. Preliminary medicinal chemistry lead optimization will identify early structure activity relationships and in vitro drug metabolism will be assessed to confirm tractibility of early leads. These efforts will provide first in-class chemical tools to be used to further probe orexin receptor function in animal models of disease.

PROJECT SUMMARY This application is submitted in response to RFA-DA-19-002, Development of Medications to Prevent and Treat Opioid Use Disorders and Overdose (UG3/UH3). The application describes a highly integrated project aimed at developing novel Gpr151 antagonists to facilitate long-term abstinence in opioid-dependent individuals. Gpr151 is an orphan G-protein coupled receptor (GPCR) that is expressed almost exclusively in the medial habenula. In exciting new data, we demonstrate that Gpr151 co-localizes with ? opioid receptors in the medial habenula and that this orphan receptor regulates the inhibitory effects of opioids on habenular neurons. Moreover, we show that Gpr151 plays a critical role in regulating the motivational properties of opioid drugs such as morphine and oxycodone in mice. Specifically, we find mice with null mutation in Gpr151 (Gpr151-/- mice) are resistant to the stimulant and rewarding effects of these opioids and self-administer lower quantities of oxycodone. Here, we propose to leverage this new knowledge, in conjunction with our expertise in small molecule drug development, to identify and optimize novel Gpr151 antagonists for the treatment of opioid dependence in humans. As Gpr151 is an orphan receptor for which there are no known ligands, the lack of potent agonists can hamper the identification of novel antagonists. However, we have now developed robust cell-based assays (and appropriate counter-screens) to reliably monitor Gpr151 function and used these assays to identify novel Gpr151 agonists. During the UG3 phase of this application, we will optimize this series of agonists to increase their potency and selectivity for Gpr151 (12 months). Once optimized, we will use potent and selective agonists from this series to facilitate the identification of novel antagonists at Gpr151 derived from the same or related chemical series (primary strategy) or via high-throughput screening (backup strategy) (12 months). Identification of novel Gpr151 antagonists will trigger the transition to the UH3 phase of the project. During the UH3 phase, we will employ an iterative medicinal chemistry based on structure-activity relationships (SAR) to optimize the potency and selectivity of novel Gpr151 receptor antagonists. SAR will also be used to optimize these antagonists for drug metabolism and pharmacokinetics (DMPK) and brain penetration properties, and identify those that are least likely to have toxicity in humans. We will assess the effects of those with the most favorable drug-like physiochemical properties on electrophysiological responses of medial habenula to opioid drugs. In addition, we will assess the in vivo efficacy these novel antagonists using the intravenous oxycodone self-administration and reinstatement of extinguished responding procedures in wild-type and Gpr151-/- mice. This multidisciplinary research plan will capitalize on the unique relevant scientific and drug discovery expertise of our team of committed investigators to develop novel therapeutics to facilitate abstinence in opioid-dependent individuals.

PROJECT SUMMARY/ABSTRACT? PROJECT 2 Project 2's objective is to understand the role for circular RNAs (circRNAs) in the mechanisms by which cocaine remodels the dorsal striatum (DS) and nucleus accumbens (NAc) to precipitate compulsive cocaine- seeking behaviors. circRNAs are highly conserved single-stranded ?back-spliced? RNAs in which the 5? and 3? ends of the transcript are covalently joined. Emerging evidence suggests that circRNAs are a major class of regulatory noncoding RNAs that are enriched in brain and that play key roles in basic aspects of neuronal function, but their involvement in the molecular, cellular, and behavioral actions of cocaine (and other drugs of abuse) has yet not been investigated. We will characterize patterns of circRNA expression in DS and NAc of mice that show compulsive-like cocaine consumption using paired-end ribominus RNA-sequencing. We will also assess circRNA expression in postmortem striatal tissues from humans with cocaine use disorders. We already have robust evidence for prominent cocaine regulation of several circRNAs in these brain regions. Those cocaine-responsive circRNAs that show similar abnormal expression in mice and humans will be prioritized for further investigation. We will determine whether prioritized cocaine-responsive circRNAs are regulated specifically in different types of DS and NAc neurons. We will then investigate the role played by these prioritized cocaine-responsive circRNAs in regulating the molecular and cellular responses to cocaine within these cell types of DS and NAc. This will be accomplished by use of in vivo CRISPR technology or RNAi to knockdown prioritized circRNAs in a cell type-specific manner and assess the influence of these circrRNAs in controlling baseline and cocaine-induced alterations in the intrinsic excitability of the neurons as well as their transcriptional responses to cocaine. Finally, we will investigate the ways in which cocaine-responsive circRNAs control compulsive-like cocaine self-administration behavior including relapse in mice. These highly innovative studies promise to yield fundamentally new insights into the molecular and cellular mechanisms of cocaine addiction, with parallel future studies planned for opiate drugs of abuse.

Medial habenula (mHb) neurons that project to the interpeduncular nucleus (IPn) regulate the ?set-point? for nicotine aversion and play a critical role in regulating nicotine intake. Nevertheless, little is known about the consequences of nicotine consumption on the function of this circuit. We have collected compelling preliminary data in rodents showing that nicotine reduces habenular volume, induces loss of mHb cholinergic neurons, and triggers degeneration of projection fibers from mHb to IPn. We detect similar reductions of habenular volume in human cigarette smokers compared with non-smokers. Selective lesion of the habenula-IPn circuit markedly increases nicotine intake in rats, suggesting that nicotine-induced damage to the habenula-IPn circuit is likely to increase the motivational significance of the drug. Uniquely, neurons in the mHb express interleukin-18 (IL- 18), a cytokine that regulates the function of microglia and other components of the innate immune system. Microglia are known to control the strength of excitatory and inhibitory transmission by pruning neurons and synapses to sculpt circuits in the brain. We find that nicotine has greater stimulatory effects on the habenula- IPn circuit of Il18-/- mice than wild-type mice. Preliminary findings suggest that nicotine induces far greater damage to mHb neurons in Il18-/- mice than wild-type littermates. Finally, Il18-/- mice demonstrate lower numbers of microglia and reduced levels of microglia-derived cytokines in the habenula than wild-type mice. Based on these findings, we hypothesize that nicotine triggers habenular degeneration and that IL-18, produced by habenular neurons, protects against this response. We further hypothesize that IL-18 acts by recruiting local microglia to prune habenular neurons, thereby opposing the excitatory actions of nicotine on these cells. Here, we will use state-of-the-art molecular, cellular and behavioral approaches to test these hypotheses. In Specific Aim I, we will use iDISCO tissue clearing coupled with light-sheet microscopy for super 3D resolution to fully characterize the role for IL-18 and microglia in regulating excitotoxic effects of nicotine on mHb neurons. In Specific Aim II, we will investigate the role for which IL-18 and microglia in regulating the stimulatory actions of nicotine on mHb neurons. In Specific Aim III, we will investigate the relevance of nicotine- induced damage to the mHb, and the involvement of IL-18 and microglia in this process, to the motivational properties of nicotine. This highly innovative program of research may yield fundamental new insights into the links between brain immune function, habenular plasticity and nicotine addiction.

Project Summary: Opioid drugs such as oxycodone are thought to act on addiction-relevant brain circuits exclusively through opioid receptors, most notably the ? opioid receptors (MORs). In common with other addictive drugs, morphine is a plant-derived alkaloid with an innately aversive bitter taste, a sensory modality that evolved to protect against ingestion of potentially noxious substances. Synthetic and semisynthetic opioids such as oxycodone also have alkaloid structures and a bitter taste. Taste 2 receptors (T2Rs) evolved to detect potentially poisonous alkaloids, but their potential involvement in the addiction-related behavioral actions of opioids is unknown to nicotine. Recently, we established a key role for T2Rs in regulating the aversive behavioral actions of nicotine that motivate nicotine avoidance and protect against addiction. Here, we will explore the role for T2Rs in regulating avoidance of opioids. T2Rs signal through a specialized G protein called ?-gustducin, which is derived from the Gnat3 gene. In Experiment 1, we will assess the rewarding and aversive properties of oxycodone in wild-type and Gnat3-/- mice using conditioned place preference and avoidance conditioning. In Experiment 2, we will assess the reinforcing properties of oxycodone in wild-type and Gnat3-/- mice using the intravenous self-administration procedure. In Experiment 3, we will assess physical and affective components of opioid withdrawal in oxycodone-dependent wild-type and Gnat3-/- mice. In Experiment 4, we will determine which subtypes of T2Rs `cmehosense' oxycodone and other opioid drugs. These experiments have the potential to establish an entirely new class of receptors that can be targeted for the development of novel addiction therapeutics.

PROJECT SUMMARY This application is submitted in response to RFA-D-21-019: Single Cell Opioid Responses in the Context of HIV (SCORCH) Program Expansion: CNS Data Generation for Chronic Opioid, Methamphetamine, and/or Cocaine Exposures. Human immunodeficiency virus (HIV) can infect nonneuronal cells in the brain, particularly microglia, with these cells acting as a reservoir of latent infection. HIV infection has deletions effects on the function of nonneuronal and neuronal cells, including those cells located in brain sites relevant to cognition, emotion and motivation. The same brain sites impacted by HIV are known to regulate the actions of opioids and other classes of addictive drugs, and opioid use disorder (OUD) is more prevalent in HIV-infected individuals than the general population. Moreover, HIV infection and OUD reciprocally interact, with each condition potentially exacerbating the severity of the other. Mice infected with EcoHIV, a modified HIV strain that targets CD4+ T cells, macrophages and microglia, recapitulates the major pathobiological features of chronic HIV infection in individuals on antiretroviral therapy (ART). Here, we will leverage state-of-the-art single cell sequencing (scSeq), molecular, cellular and behavioral approaches to define cell type-specific interactions between HIV and opioids in the brains of EcoHIV-infected mice. In Specific Aim I, we optimize the intravenous (IV) heroin self-administration procedure, already well-established in our laboratories, for use in EcoHIV- infected mice. We will then examine the effects of EcoHIV infection on the expression of opioid addiction-relevant behaviors. Conversely, we will examine the effects of heroin consumption on the expression of cognitive abnormalities in EcoHIV-infected mice relevant to HIV-associated neurocognitive impairment (HIV-NCI) in humans. Finally, will examine the effects of ART on the expression of addiction- and HIV-NCI-relevant behavioral abnormalities in EcoHIV-infected mice. In Specific Aim II, we will perform scSeq on brain regions relevant to opioid addiction and HIV-NCI, collected from EcoHIV mice with our without a history of intravenous opioid self- administration behavior. We will investigate the effects of ART on scSeq patterns in these mice. The scSeq data will be mined to identify cells in the brain infected by EcoHIV, and determine which cells show the most robust transcriptional responses to EcoHIV in opioid-naïve and opioid-experienced mice. In situ hybridization and immunohistochemistry will be used to validate key findings and prioritize candidate genes for further investigation. In Specific Aim III, we will use in vivo CRISPR-Cas9 to target prioritized genes identified in Aim II in a cell type- and brain region-specific manner. The impact of CRISPR cleavage of prioritized genes on the expression of addiction- and HIV-NCI-relevant behavioral abnormalities in EcoHIV-infected mice will be examined. This innovative program of research may yield fundamental new insights into disease-relevant interactions between HIV infection and opioid drugs.

SUMMARY The Training Program in Substance Use Disorders at the Icahn School of Medicine at Mount Sinai (ISMMS) will offer late-stage predoctoral (PhD) students and early-stage postdoctoral fellows an integrated program of training in substance use disorders (SUDs) research that leverages the exceptional expertise in cellular, molecular and behavioral neuroscience, translational neuroscience, neurology, psychiatry, genomic science and pharmacological sciences at ISMMS. The overarching goal of the program is to provide rigorous, broad- based, individualized, and multidisciplinary training with enhanced opportunities for mentoring, collaboration, and career development designed to prepare the next generation of independent investigators in SUDs research. At the heart of this new Training Program will be a superb training faculty representing remarkable diversity in basic and clinically relevant topics; from synaptic plasticity and chromatin remodeling; cognitive, social, and emotional disorders; disorders of mood, motivation, and anxiety; addiction; human neuropsychology; and cognitive neuroscience ? all relevant to SUDs. The Training Program comprises inter- related components: academic coursework, laboratory training, non-curricular training activities (seminars, retreats, etc.), testing/evaluation, teaching opportunities, mentoring, and career development activities. The diverse research environment at ISMMS will provide for laboratory opportunities in core areas of strength, including in translational neuroscience, computational neuroscience, neuropsychiatric genomics, neuroimaging, epigenetics, and synaptic and behavioral plasticity, and the study of diverse model systems. The Training Program includes more than a dozen initiatives that have been newly created or adapted from existing programs to support training in SUDs, which emphasize multidisciplinary collaborations, and the promotion of exceptional rigor and creativity. Pre- and post-doctoral trainees will participate in required and optional training activities to ensure strong grounding in basic neuroscience and opportunities to learn from and with peers and faculty from across ISMMS, including those in the Graduate School of Biomedical Sciences, the medical school, interdisciplinary Centers and Institutes, and the Friedman Brain Institute. Creating a structured culture of mentoring across the career continuum of participants is a hallmark feature of the Training Program. Formal and informal advising and mentoring will be integrated into training across roles and levels, including mentoring opportunities for training faculty and sponsors (preceptors). Integrating predoctoral and postdoctoral training through formal mechanisms will provide greater continuity in the overall training experience, enhanced opportunities for collaboration among trainees interested in pursuing careers in SUDs research, and will benefit the neuroscience research effort at ISMMS. The Training Program in Substance Use Disorders will prepare the most promising trainees for productive, independent careers in SUD research at all levels ? from preclinical genetics, cellular and molecular mechanisms of the disorders, to clinical research and interventions.