Christopher W. Cowan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Identifying key molecules that mediate brain reward plasticity remains an important goal of current drug abuse research. We recently identified a key role for the Fragile X Mental Retardation Protein (FMRP), a RNA binding protein involved in regulating protein synthesis in dendrites, as an essential downstream component of MEF2-dependent synapse elimination. Moreover, we observed significant deficits in cocaine-induced behavioral plasticity in FMRP-deficient mice. Taken together with the recently documented role for MEF2 in repeated cocaine-induced structural and functional plasticity, our findings in the Fmr1 KO mice suggest an important role for synapse elimination as a process that promotes long-lasting behavioral plasticity associated with chronic cocaine use. In this proposal, we will test the hypothesis that FMRP mediates an acute process of synapse elimination/remodeling in the NAc that is required for behavioral plasticity associated with repeated cocaine exposure. To this end, we propose the following: Specific Aim 1: Our preliminary findings indicate that Fmr1-/- mice have significantly reduced cocaine sensitization and place preference to cocaine. As these are developmental knockout mice, the precise populations of cells where FMRP exerts its function on cocaine-induced behaviors is not clear. To this end, we will take a two-pronged approach: 1) we will express WT FMRP in the nucleus accumbens (NAc) using viral- mediated gene delivery in the Fmr1 KO mice and test for functional rescue of cocaine behaviors, and 2) we will selectively knockout the Fmr1 gene in the NAc using viral-mediated gene delivery of Cre recombinase in adult conditional Fmr1 knockout mice and test the requirement for FMRP in the adult NAc during cocaine-induced behavioral plasticity. Specific Aim 2: Our recent findings revealed that FMRP is strictly required for MEF2-dependent regulation of synapse number in hippocampal pyramidal neurons, and NAc expression of active MEF2 enhances both sensitized locomotion and place preference to cocaine. Therefore, we will test the hypothesis that FMRP functions in the NAc to mediate MEF2-induced sensitized behavioral responses to cocaine. To this end, we will use established viral-mediated gene delivery to determine whether MEF2-modulated behavioral responses to cocaine require FMRP function in vivo using Fmr1-/- mice and established behavioral assays. Specific Aim 3: We previously demonstrated that inhibition of MEF2 activity is necessary and sufficient to regulate the chronic cocaine-induced increase in NAc MSN spine density. Since we found that FMRP is required for MEF2-regulated excitatory synapse density in hippocampal neurons, we will test the hypothesis that the cocaine-induced increase in NAc spine density requires FMRP in vivo. Using established spine imaging methods, we will assess the basal and chronic cocaine-regulated NAc spine density in Fmr1 KO mice.

DESCRIPTION (provided by applicant): Identifying key molecules that mediate brain reward plasticity remains an important goal of current drug abuse research. We recently identified a key role for MEF2 transcription factors as regulators of cocaine-induced synaptic and behavioral plasticity associated with repeated cocaine exposure. We find that cocaine-dependent inhibition of MEF2 in the NAc is required for increased MSN spine density, and that enhanced synaptic connectivity in the NAc after chronic cocaine exposure represents a compensatory mechanism that limits maladaptive behavioral responses associated with addiction, rather than supporting them. In this grant, we will elucidate cocaine- and cAMP-induced signaling events that control MEF2 activity in the striatum, including an exciting new regulatory mechanism for class IIa histone deacetylases that could have important implications for epigenetic regulation of drug addiction. To this end, we propose the following: Specific Aim 1: Our preliminary findings suggest that chronic cocaine exposure inhibits MEF2 activity by a cAMP-dependent process involving inhibitory phosphorylation of MEF2 (P-S408/444). In this aim, we will test the hypothesis that cocaine and cAMP signaling regulates MEF2 activity through control of P-S408/444 levels. In doing so, we will also characterize the spatial and temporal regulation of P-MEF2 in vivo after cocaine exposure. To these ends, we will use established experimental approaches, such as immunohistochemistry, RNAi-based protein replacement and transcriptional reporter assays, to test in vivo and in cultured primary striatal neurons the importance of P-S408/444 MEF2 for cocaine and cAMP-dependent regulation of MEF2. Specific Aim 2: Our preliminary findings revealed that overexpression of the regulator of calmodulin signaling (RCS) negatively regulates MEF2 activity in cultured striatal neurons in a Ser55 dependent manner (PKA site). In this aim, we will test the hypothesis that P-S55 RCS is required for cAMP-dependent inhibition of basal and calcium-activated MEF2-dependent transcription, and test the hypothesis that RCS functions in vivo to limit sensitized behavioral responses to cocaine. To this end, we will analyze existing RCS knockout mice using established experimental approaches to test in vivo, and in cultured striatal neurons, the importance of RCS as a MEF2 negative regulator and as a cocaine-induced regulator of behavioral plasticity in vivo. Specific Aim 3: Our preliminary studies indicate that activation of cAMP/PKA signaling promotes the nuclear import of HDAC5 in striatal neurons via dephosphorylation of HDAC5 at a novel Cdk5 site. In this aim, we will test the hypothesis that chronic cocaine exposure, via PKA-dependent dephosphorylation of HDAC5, promotes enhanced HDAC5 nuclear localization, which serves to reduce transcriptional responses and limit sensitized behavioral responses to repeated cocaine exposure. We will test this idea using novel P-HDAC5 antibodies with NAc tissues of cocaine exposed mice, mechanistic analysis of HDAC5 nucleocytoplasmic shuttling, and behavioral analysis of HDAC5 phospho-site mutant-expressing mice. PUBLIC HEALTH RELEVANCE: Drug addiction is a chronic human diseases characterized by uncontrolled drug use despite severe adverse consequences to the addict. The results of the proposed studies will provide valuable new insights into the genes and brain mechanisms that control long- lasting behavioral responses to chronic drug use, and could generate new therapeutic targets for the treatment of drug addiction, for which there are currently very limited treatment options.

DESCRIPTION (provided by applicant): Identifying key molecules that modulate drug experience-induced brain reward plasticity remains an important goal of current drug abuse research. We recently identified a novel regulatory mechanism by which cocaine stimulates the transient dephosphorylation of HDAC5 at S279, which induces nuclear accumulation of this chromatin-modifying enzyme. We also find that dephosphorylation of this site is critical for HDAC5 to limit cocaine reward and to reduce cocaine-primed reinstatement of drug seeking. In addition, we find that the closely-related enzyme, HDAC4, is oppositely regulated by chronic cocaine exposure, suggesting an important role for this regulation in the development of cocaine-induced behavioral plasticity. In this grant we seek to characterize the differential regulation of HDAC4 and HDAC5 and to test the importance of this regulation for cocaine reward and drug relapse behaviors in vivo. To this end, we propose the following: Specific Aim 1: In this aim, we will characterize the differential phosphorylation of HDAC4 and HDAC5 in response to cocaine exposure, in order to better understand the potential role of this regulation for cocaine reward and drug seeking behaviors. We will use established conditions and novel reagents generated in our lab to assess phosphorylation and subcellular distribution of these HDACs. Specific Aim 2: In this aim, we will utilize viral-mediated gene delivery to express phosphorylation site mutants of HDAC4 in the mouse NAc. In addition, we will use novel floxed HDAC4 conditional KO mice and viral-mediated cre expression in the adult NAc to test the necessity of HDAC4 for limiting cocaine-induced behavioral adaptations. Specific Aim 3: In this aim, we will extend our findings that cocaine triggers transient nuclear accumulation of HDAC5 to limit cocaine reward by testing the effects of the dephosphorylated HDAC5 in the most relevant model for cocaine addiction, intravenous self-administration of cocaine. Our initial findings reveal a suppression of cocaine-prime reinstatement when the dephosphorylated form of HDAC5 is expressed. We seek to expand upon these studies to determine the effect of nuclear HDAC5 on cocaine taking and seeking, motivation to work for cocaine, and propensity to reinstate drug seeking behaviors.

Project Summary/Abstract Autism Spectrum Disorders (ASDs) are thought to result from in brain synapse dysfunction. Mutations or deletions of the MEF2C gene have been linked recently to several neurodevelopmental disorders, including autism, intellectual disability (ID) and schizophrenia (SCZ). Mef2 transcription factors promote activity- dependent, glutamatergic synapse elimination ? a possible dysregulated process in autism. We find that Mef2c conditional forebrain knockout (Mef2c cKO) mice display behavioral abnormalities similar to the 3 core features used to diagnose autism in humans, including abnormalities in communication, social interaction, and repetitive motor behaviors. Surprisingly, we observed a significant increase in inhibitory (GABAergic) and a decrease in excitatory (glutamatergic) synapse density and strength, which suggests the hypothesis that Mef2c functions as an activity-regulated transcriptional repressor to control the balance of inhibitory and excitatory synapses in developing cortical neurons. Loss of Mef2c repressor function promotes excitatory synapse elimination and increases inhibitory synapse density, which results in numerous behaviors with potential relevance to autism, ID and SCZ. In this revised grant proposal, we seek to extend our extensive preliminary findings to explore the role and regulation of Mef2c in cortical synapse development and in behaviors associated with intellectual and developmental disabilities. Aim 1. In this aim, we will use well-established behavioral assays in the lab to analyze social interaction, communication, repetitive behaviors, reward, anxiety, and learning and memory tests in Mef2c cKO mice. Aim 2. In this aim, we will take a multi-pronged approach in culture and in vivo to analyze the role of Mef2c in the regulation of excitatory and inhibitory synapse density and function. We will test the hypothesis that Mef2c functions cell-autonomously as a transcriptional repressor to regulate glutamatergic synapse elimination and GABAergic synapse formation/stabilization, and it does so in an activity-dependent, homeostatic, negative feedback mechanism that promotes inhibition and reduces excitation. Aim 3. In this aim, we will extend our preliminary findings with HTS-CLIP and RNA-seq to assess the role of Pcdh17, an newly identified common MEF2/FMRP target gene, in Mef2c-induced excitatory synapse elimination and inhibitory synapse formation in cortical pyramidal neurons in culture and in vivo.

SPECIFIC AIMS External and internal cues associated with the drug use environment often develop into persistent and powerful triggers for relapse to drug seeking. Many studies have reported that epigenetic enzymes, like histone deacetylases (HDACs), that alter chromatin structure and influence gene expression play an important role in addiction-relevant behaviors in animal models. In the previous grant period, we showed that the class IIa HDACs, HDAC5, plays important roles in the regulation of multiple cocaine-induced behaviors, including drug related learning and memory. The HDAC5 target gene, Npas4, emerged as an important, novel transcription factor required in the adult NAc for cocaine conditioned place preference and operant drug discrimination learning in the mouse intravenous self-administration. Our preliminary findings indicate that NPAS4 is induced in DARPP-32-positive medium spiny neurons (MSNs) and parvalbumin-positive fast spiking interneurons (PV- FSIs), but the relevant population(s) in which NPAS4 regulates drug behaviors remains unknown. Our long-term goal is to understand the role and regulation of NPAS4 in the development and/or persistence of drug addiction-related behaviors. Our central hypothesis is that NPAS4 induction within one or more subpopulations of activated NAc neurons promotes glutamatergic synaptic plasticity events that underlie long-lasting drug context/cue learning and memory. We will employ a series of multi-disciplinary, hypothesis testing experiments and cutting-edge molecular genetic approaches that will interrogate the in vivo cell type- specific role and regulation of NPAS4 and NPAS4-expressing cells in the NAc for drug addiction-related learning and memory. We will test and refine our central hypothesis with the following specific aims: Specific Aim 1: Determine the cell-type specific roles for NPAS4 during acquisition and extinction of drug self-administration. In this aim, we will take a cell-type specific loss- and gain-of-function approaches to study the function of NPAS4 during operant discrimination and extinction learning in the mouse drug self- administration assay. Specific Aim 2: Determine whether NPAS4-positive NAc neurons define a functional ?engram? population linking external cues to drug reward and seeking. Using our newly developed Npas4 enhancer-driven, tamoxifen-dependent cre virus, we will use a chemogenetic approach to manipulate the activity of the NPAS4-positive population and test its role in durg taking and seeking behaviors. Specific Aim 3: Determine the cellular function of NPAS4 in the NAc during drug SA. We will use cell- type specific loss- and gain-of-function strategies to evaluate the effects of NPAS4 on excitatory ad inhibitory synapse plasticity in mice self-administering cocaine or heroin.

PROJECT SUMMARY - Overall The personal, social and criminal consequences of opioid and cocaine abuse are enormous problems in North America. This is most tragically seen in rising morbidity due to heroin, prescription opioids and fentanyl overdose in the USA. Addiction to drugs typically cycles between three phases, active drug use, withdrawal from drug use and relapse to drug use. A point in the cycle of addiction where pharmacological intervention can be particularly beneficial is to interfere with the overwhelming motivation by addicts to relapse to drug use, even after extended periods of abstinence when acute withdrawal symptoms have dissipated. However, the enduring state of relapse vulnerability arises from interdependent brain adaptations produced during all three phases of addiction. Thus, in order to develop biological rationales for treating relapse, it is necessary to understand not only the neurobiology of relapse itself, but to determine which changes produced by drug administration and drug withdrawal contribute to the final enduring state of relapse vulnerability. The overarching goal of the Center for Opioid and Cocaine Addiction (COCA) is to create and maintain mechanisms of scientific synergy that will facilitate discovering the neuropathologies that underpin the enduring and uncontrollable drive to seek opioids and cocaine, and thereby advance biological rationales needed to efficiently generate pharmacotherapies that inhibit drug relapse. This goal will be achieved through a bidirectional translational strategy that involves 3 Cores and 4 research Projects. In addition to the Administrative and Pilot Cores, the Animal & Validation Core makes available transgenic rodents that have been trained to self-administer heroin or cocaine, and have been instrumented with intracranial cannulae, fiber optics or GRIN lens. This Core will also validate all viral reagents and transgenic animals shared by the COCA Cores and Projects. The 4 Projects range from determining the epigenetic substrates of long- lasting drug-induced alterations to understanding the molecular and brain circuit mechanisms of cue-induced drug seeking in rodents and humans. The Projects are designed to be highly integrated and form a bidirectional translation strategy for providing biological rationales for new therapeutic approaches to relapse prevention.

PROJECT SUMMARY ? Project 1 Cowan A major challenge for treating drug addiction is the poor understanding of the molecular mechanisms by which drug use produces persistent changes in brain function that facilitate drug seeking even after long periods of abstinence. Chronic drug use engages an epigenetic process, involving the chromatin-remodeling enzyme, histone deacetylase 5 (HDAC5), in striatal medium spiny neurons that functions to limit the development of multiple addiction-related behaviors, including reinstatement of cocaine and heroin seeking. However, HDAC5?s regulation and function during and after drug taking is complex and poorly understood. Cocaine and heroin produce reactive oxygen and nitrogen species (ROS/RNS) that alter the redox state of the cell, and redox-mediated cysteine thiol modifications alter the structure/function of target proteins, including class IIa HDACs. Repeated treatment of animals or humans with the antioxidant and glutathione precursor, N- acetylcysteine (NAC), reduces the vulnerability to reinstatement of cocaine and heroin seeking, and NAC blocks ROS-promoted HDAC5 nuclear export. The long-term goal of Project 1 is to understand the redox protein signaling and epigenetic mechanisms by which HDAC5 and NAC regulate heroin and cocaine relapse behaviors in hopes of identifying better therapeutic strategies for the treatment of drug addiction. We hypothesize that: (1) cocaine- and heroin-produced oxidative stress modifies HDAC5 cysteine thiol groups in the nucleus accumbens, which promotes nuclear export and reduces the anti-relapse actions of HDAC5, and (2) the anti-relapse effects of repeated NAC treatment are due, at least in part, to its ability to protect HDAC5 from ROS signaling events that promote nuclear exclusion and changes in gene expression. We will use cutting-edge mouse genetics and cre-dependent viral tools that allow for time-delimited and cell-type specific manipulation of nucleus accumbens core neurons during drug taking and relapse events. Aim 1. Molecular and Cellular Mechanisms: In this aim, we will determine how cocaine and heroin SA regulate HDAC5?s redox-sensitive posttranslational modifications, subcellular distribution, genomic binding and regulation of gene expression. Aim 2. Circuit Specificity: In this aim, we will take a cutting-edge molecular genetic approach to dissect the cell-type specific roles for HDAC5 to negatively-regulate cocaine and heroin drug seeking. Aim 3. Treatment: In this aim, we will analyze the regulation of HDAC5 by NAC, and test the role of HDAC5 in the long-lasting ability of NAC to reduce reinstatement of heroin and cocaine seeking.