Eric J. Nestler - US grants

The mesolimbic dopamine system, which consists of dopaminergic neurons in the ventral tegmental area (VTA) and their projections to the nucleus accumbens (NAc) and other forebrain regions, is an important neural substrate for the reinforcing and locomotor activating properties of several types of drugs of abuse, including opiates, cocaine and other stimulants, and ethanol. We have demonstrated a series of common, chronic biochemical actions of these drugs in the VTA and NAc, which could underlie part of the long-term changes in mesolimbic dopamine function that characterize drug addiction. These changes include an upregulation of tyrosine hydroxylase (the rate limiting enzyme in dopamine biosynthesis) in the VTA and an upregulation in the cAMP pathway in the NAc. Moreover, chronic administration of these drugs of abuse reduces levels of neurofilament proteins and increases levels of glial filament proteins specifically in the VTA, changes suggestive of neural insult or injury. These findings raise the possibility that neurotrophic factors, which exert a neuroprotective effect in many systems, may counter these and other actions of drugs of abuse. We have recently obtained direct support for this possibility. Infusion of certain neurotrophic factors directly into the VTA can prevent and reverse the ability of morphine and of cocaine to produce many of their typical biochemical actions in the mesolimbic dopamine system. One major objective of this Project is to further characterize such novel pharmacological actions of neurotrophic factors in this neural pathway. A second major objective of this Project is to investigate the related possibility that some of the previously identified biochemical changes that drugs of abuse produce in the mesolimbic dopamine system occur via drug regulation of neurotrophic factor signaling pathways in these brain regions. Support for this possibility comes from preliminary investigations wherein chronfc administration of morphine or cocaine has been shown to regulate levels of specific proteins involved in the neurotrophic factor signaling cascades, including ERK (also referred to as MAP kinase), phospholipase C-gamma-l, and JAK (a cytoplasmic protein tyrosine kinase). The proposed studies will further investigate drug regulation of these proteins, as well as their possible role in mediating morphine and cocaine regulation of some of the other biochemical adaptations identified in the VTA and NAc. For example, recent experiments in which levels of ERK1 were selectively reduced in the VTA by an antisense oligonucleotide strategy support the hypothesis that morphine regulation of ERK1 may mediate its upregulation of tyrosine hydroxylase in this brain region. Together, the proposed studies have the potential to reveal important new information concerning the molecular mechanisms of action of drugs of abuse on the mesolimbic dopamine system, which could contribute ultimately to the development of novel pharmacotherapies for addictive disorders.

DESCRIPTION (provided by applicant): While neurotrophic factors are best characterized for their role in nervous system development and differentiation, increasing evidence supports their involvement in the regulation of neural plasticity in adult animals. The major objective of this proposal is to continue our characterization of such a role for neurotrophic factors in the neural plasticity that accompanies chronic exposure to drugs of abuse. This competing renewal focuses on interactions between one prominent neurotrophic factor, BDNF, and the actions of 2 drugs of abuse, morphine and cocaine, at the level of the mesolimbic dopamine system, a neural pathway important for the reinforcing and addicting actions of these and other drugs of abuse. Work over the past 5 years of this grant supports three major types of interactions between BDNF and drugs of abuse. First, we (and others) have shown that exogenous BDNF, applied directly into the mesolimbic dopamine system, modifies the rewarding and locomotor-activating effects of these drugs. Exogenous BDNF also modifies the ability of drugs of abuse to produce certain characteristic biochemical and morphological adaptations in the mesolimbic dopamine system. Second, we have demonstrated that chronic exposure to morphine or cocaine causes adaptations in specific BDNF signaling proteins, and have recently obtained direct evidence that such alterations may contribute to the behavioral effects of these drugs. Third, by use of mutant mice and other approaches, which allow inducible and region-specific knockouts of BDNF or its TrkB receptor from the mesolimbic dopamine system or its afferent inputs, we have provided compelling evidence that endogenous BDNF signaling is critically involved in controlling an animal's responses to drug exposure. The goal of the proposed studies is to further characterize these interactions between BDNF and drugs of abuse, and to increasingly relate specific molecular phenomena to behavioral responses to the drugs by use of viral-mediated gene transfer and genetic mutations in mice. These proposed studies will improve our understanding of the role played by BDNF in regulating an animal's adaptations to drugs of abuse. The studies have the potential not only of shedding new light on mechanisms underlying drug addiction, but also in directing novel approaches toward the development of new treatments for addictive disorders. Moreover, in a more general sense, the proposed studies will utilize models of addiction to better understand the potent influence of BDNF on mesolimbic dopamine function in the fully differentiated, adult brain.

[unreadable] DESCRIPTION (provided by applicant): We have made significant progress, through support from this RO1/R37 grant, in identifying adaptations in signal transduction proteins induced in the mesolimbic dopamine system by chronic exposure to cocaine or other drugs of abuse. This neural system, which consists of dopaminergic neurons in the ventral tegmental area (VTA) and their projection regions such as the nucleus accumbens (NAc), is implicated in the reinforcing and addicting actions of these drugs. Work over the past 5 years has focused largely on two transcription factors, deltaFosB and CREB, as important mediators of the longer-lasting effects of drugs on the VTA-NAc pathway. CREB activation by drugs of abuse, which is relatively short-lived after cessation of drug use, appears to represent a mechanism of drug tolerance and dependence, and could mediate a negative emotional state seen in many addicts during early phases of drug withdrawal. The situation for deltaFosB is very different. deltaFosB, like CREB, is induced in the NAc and certain other regions by chronic exposure to drugs of abuse, however, unlike CREB, deltaFosB is long-lived in that it persists in brain for as long as 6-8 weeks after cessation of drug administration. Also unlike CREB, deltaFosB appears to mediate enhanced sensitivity to the rewarding effects of drugs of abuse. Thus, deltaFosB could represent a mechanism of relatively prolonged sensitization that contributes to features of addiction long after the last drug exposure. In formulating a plan for this competitive renewal of this R01 grant, we have decided to focus our efforts on characterizing the deltaFosB system in greater detail. (Our studies of CREB will continue with support from other grants.) Our focus on deltaFosB is justified by several considerations. First, deltaFosB is induced by virtually all drugs of abuse in the NAc and certain other regions, but there has to date been no systematic characterization of precisely where such induction occurs in brain and in which cell type. Second, deltaFosB's unusual stability provides a novel potential mechanism underlying some of the long-term effects of drugs of abuse on the brain. Yet, there has been no information available as to the biochemical mechanisms responsible for this stability. Third, virtually nothing is known about regulation of the fosB gene, the induction of which is also required for deltaFosB accumulation with drugs of abuse. Fourth, as a transcription factor, deltaFosB's interesting effects on behavior are presumably mediated via alterations in the expression of target genes, but the regulation of gene expression by deltaFosB in the NAc and other brain regions in vivo remains incompletely understood. The goal of the proposed studies, then, is to make significant progress in these several critical areas of investigation: to better understand, at the precise molecular level, exactly how and where deltaFosB is induced by chronic drug exposure, why deltaFosB persists in brain for so long, and the target genes through which deltaFosB mediates aspects of the drug-addicted phenotype. This work will contribute to our growing knowledge of drug-induced neuroadaptations in the brain at the molecular level. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The objective of this new Conte Center is to study the role of the brain's appetitive neural circuits in the regulation of mood and motivation as they relate to depression and antidepressant action. The program of research contains three major strengths. First, is the multidisciplinary nature of our ongoing and proposed research. Each research area represents an integration of molecular, cellular, pharmacological, and behavioral levels of analysis aimed at obtaining a more complete understanding of the neurobiology of mood. Second, is the integration of our basic science research with the investigation of clinical populations. Third, is the extraordinary degree of integration of the various Projects to examine highly related aspects of the central hypothesis of the Center. While research in depression has focused largely on hippocampus and cerebral cortex, other neural circuits possibly involved have received much less attention. This Center focuses on one of these other circuits, namely, appetitive regions of brain, including nucleus accumbens and hypothalamus, important for motivation, reward, appetite, sleep, psychomotor activity, and circadian rhythms. A focus on these appetitive circuits makes sense given the degree to which abnormalities in these various domains are seen in patients with depression and other mood disorders. Indeed, we have obtained substantial evidence, presented in this application, for the importance of these appetitive circuits in animal models of depression. The Center is organized into a small Coordination Core, two scientific Cores, and five Projects. The Transgenic Core is responsible for providing state-of-the-art molecular tools to Center investigators, while the Behavioral Core is responsible for providing to individual Projects an extremely broad battery of behavioral tests in mice and rats relevant to mood and motivation. Project 1 is focused on the transcription factor CREB as a key regulator of appetitive circuits and its role in mood and motivation. Subsequent Projects evaluate a series of other molecular signals in these neural pathways that are both important regulators of CREB function and important effectors in mediating CREB's behavioral phenotype. Project 2 focuses on BDNF; Project 3 focuses on three hypothalamic peptides, MSH, orexin (hypocretin), and MCH; Project 4 focuses on NPAS2 and other circadian genes; Project 5 focuses on RGS proteins, in particular, RGS9 and RGS16.

DESCRIPTION (provided by applicant): The objective of this grant is to contribute to a better understanding of the regulation of mood and the mechanisms underlying depression and antidepressant treatments. For the first nine years of the grant, our focus was quite broad. We contributed to an appreciation of the role of several classes of signaling proteins in these phenomena. Over the past year, we have greatly focused the grant on one of these signaling proteins, namely, the transcription factor DeltaFosB. DeltaFosB, a member of the Fos family, is induced in a region- and cell type-specific manner in brain by many types of chronic perturbations, including chronic stress and chronic antidepressant treatments. Unlike all other Fos family proteins, which exhibit a very short half-life, DeltaFosB is a highly stable protein. As a result, once it accumulates in brain it can persist for weeks or months and thereby mediate relatively long-lived plasticity to the original perturbation. This grant focuses on the consequences of DeltaFosB induction in four brain areas, the nucleus accumbens (NAc), ventral periaqueductal gray (vPAG), medial prefrontal cortex (mPFC), and basolateral amygdala (BLA). In NAc, chronic stress induces DeltaFosB selectively in dynorphin+ neurons. Using a combination of inducible transgenic mice and viral mediated gene transfer to overexpress DeltaFosB or a dominant negative antagonist, we have evidence that stress induction of DeltaFosB in this brain region mediates an "antidepressant-like" response to stress in the forced swim and learned helplessness tests. In the vPAG, chronic stress as well as chronic antidepressant treatments induce DeltaFosB primarily in substance P+ neurons, which also appears to mediate an antidepressant-like response. Work is currently underway to better understand the behavioral phenotype mediated by DeltaFosB in these two regions, as well as to establish the cellular localization and functional significance of DeltaFosB induction by stress or antidepressant treatments in the mPFC and BLA. As a transcription factor, DeltaFosB presumably produces these behavioral effects through the regulation of other genes. Accordingly, a related goal of this grant is to identify putative target genes for DeltaFosB in each of these brain regions. Our work to date thus suggests that induction of DeltaFosB, at least in the NAc and vPAG, may represent a positive adaptation to stress, which contributes to an individual's ability to actively cope with adverse circumstances. In the vPAG, where DeltaFosB is also induced by antidepressants, the protein may contribute to the mechanism of action of these treatments. The proposed studies will further evaluate these hypotheses, and explore the behavioral consequences of DeltaFosB induction in the mPFC and BLA. Together, the proposed research will improve our understanding of the ways in which the brain adapts to chronic stress and antidepressant treatments.

[unreadable] DESCRIPTION (provided by applicant): Project 1 focuses on the ability of the transcription factor CREB in the NAc (nucleus accumbens) to regulate mood and motivational state. We have considerable evidence that increased CREB function in this brain region, which occurs under several conditions of active stress, causes a decrease in an animal's sensitivity to emotional stimuli, regardless of whether the stimulus is aversive or rewarding. Conversely, reduced CREB function, caused by a lack of emotional stimulation (e.g., prolonged social isolation), has the opposite effect. These findings suggest that CREB in the NAc may function as a key molecular gate between emotional stimuli and their behavioral responses. A possible relationship between both extremes in CREB activity and the different range of symptoms seen in various subtypes of human depression will be further explored in animal models. Preliminary data, for example, show that either extreme change in CREB activity in the NAc (either excessively high or excessively low activity), and its behavioral consequences, can be corrected by chronic, not acute, antidepressant treatment. Related studies will characterize target genes through which CREB produces this behavioral phenotype in the NAc. The genes encoding the opioid peptide dynorphin and the AMPA glutamate receptor subunit GluR1 are examples of such targets of CREB that will be examined in this Project, as will additional targets identified with DNA expression arrays and ChIP on chip (chromatin immunoprecipitation x promoter) arrays. ChIP will also be used to characterize the molecular mechanisms by which CREB, at the level of chromatin remodeling, regulates its target genes. In addition, the Project will investigate the role played by CREB in the VTA (ventral tegmental area) in regulating depression-like behavior, as well as establish the behavioral phenotype of several other CREB family proteins, which we have shown recently subserve very different functions in the VTA-NAc pathway. CREB function is a major theme of this Center. All of the subsequent Projects of this Grant represent extensions of our central hypothesis that CREB in the VTA-NAc is a key regulator of hedonic and affective state. Subsequent Projects extend this theme by examining other molecular constituents of VTA and NAc neurons, which are regulated by CREB (e.g., BDNF in Project 2, CCK in Project 4) and help control CREB activity (e.g., MCH in Project 3), and by characterizing their role in mediating CREB's complex behavioral phenotype related to depression and its treatment. [unreadable] [unreadable] [unreadable]

2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-, (4S-(4alpha,4aalpha,5aalpha,6beta,12aalpha))-; AAV vector; Achievement; Achievement Attainment; Achromycin; Animal Model; Animal Models and Related Studies; Antidepressant Agent; Antidepressant Drugs; Antidepressants; Antidepressive Agents; Arts; Brain; Breeding; Cells; Cost Savings; Depression; Encephalon; Encephalons; Ensure; Gene Action Regulation; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Gene Targeting; Gene Transfer; Genes; Genetic Alteration; Genetic Change; Genetic defect; Genetics-Mutagenesis; Genotype; Grant; Herpes Simplex; Herpes Simplex Infections; Herpes simplex disease; Herpesvirus hominis disease; Individual; Investigators; Knock-out; Knockout; Knockout Mice; Laboratories; Localized; Mammals, Mice; Mediating; Mental Depression; Methods; Mice; Mice, Knock-out; Mice, Knockout; Molecular Biology, Mutagenesis; Murine; Mus; Mutagenesis; Mutation; Nervous System, Brain; Null Mouse; Play; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Programs (PT); Programs [Publication Type]; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNAi; Research; Research Personnel; Researchers; Rewards; Role; Saving, Cost; Sequence-Specific Posttranscriptional Gene Silencing; System; System, LOINC Axis 4; TCN; Targetings, Gene; Tetracycline; Tetracycline Antibiotic; Tetracyclines; Transgenic Organisms; Viral; Viral Vector; Work; adeno-associated viral vector; adeno-associated virus vector; genome mutation; innovate; innovation; innovative; interest; knockout gene; model organism; pre-clinical; preclinical; programs; shRNA; short hairpin RNA; small hairpin RNA; social role; tool; transfer of a gene; transgenic

PROJECT SUMMARY ? ADMINISTRATIVE CORE The Administrative Core ensures the effective integration and interaction of the scientists and other personnel working on the Projects and Scientific Cores that comprise this Center. First, the Core establishes the scientific priorities and directions of the research through regular meetings of the Center's Executive Committee. Second, the Core is responsible for administering the day-to-day activities of the Center, including the monitoring of all budgets, submission of progress reports, and adherence to regulatory requirements associated with our research. Third, the Core coordinates the many modes of communication among Center investigators, including regular face-to-face meetings that ensure our integrated research program. Likewise, the Core supports visits to New York by members of our External Advisory Committee, and many key consultants, to meet with Center faculty and trainees and review the progress of our research. Fourth, the Core maintains a creative Center website to foster communication not only among Center investigators, but also with the scientific community and general public at large. Such communication includes resource and data sharing and the dissemination of vast amounts of genome-wide chromatin and gene expression data as rapidly as possible as well as enabling the free download of novel software packages designed to analyze these complex datasets. Fifth, the Core fosters several additional outreach efforts to patient advocacy and community organizations. Sixth, the Core, through the Executive Committee, works to ensure the successful career paths of numerous junior faculty as well as student and postdoctoral trainees. Numerous individual R and K grants and NRSAs have been generated by the Center's research, which we expect will continue. Such career development focuses in particular on the recruitment and retention of women and minority scientists; we are proud of our track record in this regard. Seventh, the Core is responsible, with our several training programs, to ensure the safe and ethical conduct of research. We are very proud of our track record of joining together effectively as a unified research team to drive this large undertaking, and we are confident in our ability to further these advances over the next four years.

The newly formed Chromatin and Gene Regulation Core provides state-of-the-art tools for analysis of gene expression, including alterations in chromatin structure, in the brain's reward regions in depression. Core services are utilized by all of the Center's five Projects, since these molecular tools have been validated not only for study of rodent tissue, but also for human brain tissue obtained at autopsy. The parallel analysis of brain reward regions in animal models of depression, and in human depressed patients, offers a particularly powerful translational dimension to our Center. All of the Center's Projects utilize DMAexpression arrays to study differences in mRNA levels within brain reward regions of experimental animals and humans. This has been expanded recently to include arrays for miRNAs (microRNAs), endogenous inhibitors of mRNA expression. The Core provides the arraying and array analysis for Project investigators. A major advance over the first 4 years of the Center has been the development of experimental protocols to study changes in chromatin structure within brain reward regions in rodent models and in human tissue. This includes chromatin immunoprecipitation (ChIP) assays as well as ChIP combined with DMA promoter microarrays, so-called "ChIP on chip" assays. These exciting techniques are now being applied to Project studies as a major new initiative for our Center. By centralizing these activities into this new Core, we ensure a high quality of data and the ready ability to compare and contrast findings across the various Projects. Indeed, the array data already accumulated have demonstrated many common changes in gene expression in mice with mutations in the various genes of interest to the four preclinical Projects and in depressed human tissue. Economic savings are also achieved. We should mention that DNA expression array analyses were included in the Transgenic Core of our original Conte Center. However, given the expanded use of these arrays, expansion to analysis of miRNAs, and our new initiatives in chromatin remodeling, it makes sense to create this new Core, which supports highly cohesive studies of gene regulation. Together, the advanced technologies provided by this new Core offer an unprecedented evaluation of regulation of gene expression in brain reward regions in depression.

This newly formed Project 3 utilizes state-of-the-art tools to analyze regulation of gene expression, including alterations in chromatin structure, in the brain's reward regions by opiates and cocaine. One objective is to characterize changes in mRNA levels in the NAc (nucleus accumbens) and ventral tegmental area (VTA) after chronic administration of opiates or cocaine and after varying periods of withdrawal. This endeavor will also include comparison of the effects of self-administered vs forced drug exposure. These analyses, which are ongoing, are already providing unique insight into the shifting patterns of gene expression changes that occur in the VTA-NAc during a course of drug treatment and recovery during withdrawal. A major advance over the past 4 years of the PPG has been the development of experimental protocols to study changes in chromatin structure within brain reward regions in rodent models. This includes chromatin immunoprecipitation (ChIP) assays as well as ChIP combined with DNA promoter microarrays, socalled "ChIP on chip" assays. Such studies of chromatin remodeling offer two major advances over conventional DNA expression arrays. First, studies of chromatin modifications provide the first ever look at regulation of gene expression within a particular brain region of a behaving animal. Second, this work has the potential of revealing fundamentally new information about the molecular mechanisms by which drugs of abuse induce particularly long-lasting changes in brain reward regions. We are using a unique strategy to overlay findings from DNA expression arrays on those from ChIP on chip arrays. The result has been a much more complete and reliable view of changes in gene expression induced in the VTA-NAc by chronic drug exposure. We are also analyzing arrays for miRNA's (microRNA's), endogenous inhibitors of mRNA expression. Drug regulation of miRNA's may explain some of the changes in steady-state mRNA levels which do not appear to be mediated via transcriptional mechanisms. The Project has recruited two biostatistic faculty who are experts in the analysis of gene array data to apply the most rigorous analysis to our large volume of array gene sets. A final effort of the Project is to investigate drug regulation of chromatin remodeling mechanisms themselves for their involvement in the process of drug addiction. Together, the advanced technologies used by this new Project offer an unprecedented evaluation of the regulation of gene expression in brain reward regions by opiates and cocaine and will guide work in the other Projects of this PPG to explore molecular and cellular mechanisms of addiction.

Project 1 concentrates on the role of the transcription factor CREB in mediating the long-term actions of opiates and cocaine. The major focus is the NAc (nucleus accumbens). We have considerable evidence that increased CREB function in this brain region, which occurs in response to chronic opiate or stimulant exposure, represents one mechanism of drug tolerance and dependence. Thus, CREB activation in the NAc decreases an animal's sensitivity to subsequent drug exposure, and induces a negative emotional state, which suggests that it may contribute to aversive symptoms during drug withdrawal. We will further characterize the CREB behavioral phenotype in the NAc in additional behavioral models and by use of more advanced tools which selectively knockout CREB from this brain region. We will also establish the behavioral phenotype of several other CREB family proteins, which we have shown recently are also regulated in the NAc by drugs of abuse and subserve very different functions in the NAc. Related studies will characterize target genes through which CREB produces this behavioral phenotype in the NAc. The genes encoding the opioid peptide dynorphin, the AMPA glutamate receptor subunit GluR1, adenylyl cyclase type 8, and BDNF (brain-derived neurotrophic factor) are examples of such targets of CREB that will be examined in this Project, as will additional targets identified with DNA expression arrays and ChIP on chip (chromatin mmunoprecipitation x promoter) arrays. ChIP will also be used to characterize the molecular mechanisms by which CREB, at the level of chromatin remodeling, regulates its target genes. In addition, the Project will carry out a smaller number of highly targeted investigations of the role played by CREB in the VTA (ventral tegmental area) and LC (locus coeruleus) in mediating long-term drug action in these regions. Our studies of the LC, in particular, continue to provide novel insight into the molecular and cellular basis of drug action, which informs our studies of the VTA-NAc reward circuit. CREB function is one of the major themes of this PPG. All of the subsequent Projects of this Grant represent, in part, extensions of our central hypothesis that CREB in the VTA-NAc is a key regulator of drug reward and addiction. Subsequent Projects extend this theme by characterizing the influence of CREB, and related transcriptional mechanisms (i.e., AFosB), on the neurophysiology of VTA and NAc neurons (Project 2), on the detailed molecular mechanisms of gene regulation including modifications in chromatin structure n the VTA-NAc (Project 3), and by further establishing the complex behavioral phenotype mediated by CREB and related transcriptional mechanisms in drug reinforcement and craving (Project 4).

Description (provided by applicant): We greatly appreciate the opportunity to submit this Challenge Grant to NIMH under: (15) Translational Science, 15-MH-108, Screening approaches to identify pharmacologic treatments for mental disorders. The objective of this grant is to implement a high throughput screen for small molecules that potentiate the activity of FosB, a transcription factor that promotes resilience and antidepressant-like responses in several animal models and is deficient in the brains of depressed humans. FosB, which is induced in certain brain regions by chronic stress, represents a positive, coping mechanism that promotes positive adaptation to stress. 1) FosB induction in brain by chronic stress correlates with an animal's resilience to the deleterious effects of the stress. 2) Using viral-mediated gene transfer or inducible and brain region-specific bitransgenic mouse models, overexpression of FosB in these specific brain areas is sufficient to render animals resistant to subsequent stress and to reverse behavioral abnormalities induced by chronic stress. 3) Conversely, overexpression of a dominant negative antagonist of FosB in these brain regions makes animals more vulnerable to stress. 4) Chronic administration of standard antidepressant medications also induces FosB in these same brain regions, and overexpression of the dominant negative antagonists of FosB block the antidepressant-like behavioral effects of these medications in several behavioral assays. 5) Depressed humans have lower levels of FosB in these brain regions compared to extensively matched control subjects. This is the ideal time to submit this Challenge Grant, since we are poised to initiate the proposed studies immediately and they can be completed within two years. Thus, Drs. Eric Nestler and Gabby Rudenko have developed a novel high throughput screen to identify small molecule potentiators of FosB. An initial screen of ~50,000 compounds has identified ~500 hits that now require substantial validation. A series of validation assays, first in vitro and then in vivo, have also been developed. While targeting a transcription factor for psychiatric drug development is highly novel, several factors suggest that it represents a viable, albeit high risk, approach. For example, FosB is expressed at low levels throughout brain and peripheral tissues under normal conditions, suggesting considerable tissue specificity. Moreover, even if small molecule FosB activators prove unsuitable for the treatment of depression, high affinity, small molecule ligands for FosB would be invaluable tools to help us better understand FosB action in brain. Such molecules also could potentially be used to image FosB in the living human brain by use of PET or related brain imaging technologies, which would be a major boon to clinical investigations in depression and perhaps represent a novel tool to diagnose depression or other stress-related disorders or to track a patient's progress during treatment. We have recently shown that induction of the transcription factor, DeltaFosB, in brain represents a positive adaptation that helps individuals cope with chronic stress. Interestingly, the protein is also induced in brain by standard antidepressnts treatments, and depressed humans show lower levels of DeltaFosB in brain. We now propose to identify small molecule activators of DeltaFosB as novel treatment agents for depression.

DESCRIPTION (provided by applicant): The objective of this grant is to contribute to a better understanding of the regulation of mood under normal circumstances as well as the mechanisms underlying depression and antidepressant treatment. For the first 14 years of the grant, our focus was quite broad. We contributed to an appreciation of the role of several classes of signaling proteins in these phenomena. Over the past 4 years, we have greatly focused the grant on one of these signaling proteins, namely, the transcription factor DFosB. DFosB, a member of the Fos family, is induced in a region- and cell type-specific manner in brain by many types of chronic stimuli, including chronic consumption of natural rewards, chronic stress, or chronic antidepressant treatments. Unlike all other Fos family proteins, which exhibit a very short half-life, DFosB is a highly stable protein. As a result, once it accumulates in brain it can persist for weeks or months and thereby mediate relatively long-lived plasticity to the original chronic stimulus. This grant focuses on the consequences of DFosB induction in the nucleus accumbens (NAc) and certain other brain areas. In the NAc, chronic exposure to stress or antidepressants induces DFosB in both dynorphin+ and enkephalin+ neurons, while natural rewards do so selectively in dynorphin+ neurons. Using a combination of inducible bitransgenic mice and viral-mediated gene transfer to overexpress DFosB or a dominant negative antagonist, we have evidence that DFosB induction in this brain region mediates an antidepressant-like response in the social defeat, forced swim, and learned helplessness tests. DFosB mediates a similar antidepressant-like effect in other brain areas of interest. Work is currently underway to better understand the behavioral phenotype mediated by DFosB in these regions in models of natural rewards, stress, and antidepressant responses. As a transcription factor, DFosB presumably produces these behavioral effects through the regulation of other genes. Accordingly, a related major goal of this grant is to identify putative target genes for DFosB in these brain regions, including the use of chromatin remodeling assays to help identify DFosB targets as well as to provide detailed insight into the underlying molecular mechanisms involved. Our work to date thus suggests that induction of DFosB in the NAc and other brain areas may represent a positive adaptation to stress, which enhances motivation for natural rewards and contributes to an individual's ability to actively cope with adverse circumstances. In some of these regions, where DFosB is also induced by antidepressants, the protein may contribute to the therapeutic mechanism of action of these treatments. The proposed studies will further evaluate these hypotheses and improve our understanding of stress and antidepressant responses.

DESCRIPTION (provided by applicant): This proposal continues our characterization of the role of neurotrophic signaling pathways in the neural plasticity induced in the ventral tegmental area (VTA)-nucleus accumbens (NAc) reward circuit by chronic exposure to opiate or stimulant drugs of abuse. The proposal has three Aims: 1) to delineate the cell-type specific actions of BDNF-TrkB signaling in the VTA-NAc in regulating molecular, cellular, and behavioral responses to opiates and cocaine;2) to delineate the adaptations in BDNF-TrkB signaling cascades that mediate the ability of opiates to induce morphological changes in VTA dopamine neurons;and 3) to delineate the adaptations in BDNF-TrkB signaling cascades that mediate the ability of cocaine and opiates to induce opposite morphological changes in NAc medium spiny neurons. We have made important progress in each of these Aims over the past 5 years, and have substantial preliminary data to support highly penetrating and mechanistic studies over the proposed new grant period. Using genetic mutant mice and viral-mediated gene transfer, which has enabled highly localized knockouts of BDNF or TrkB within the VTA or NAc of adult animals, we have demonstrated distinct roles for BDNF-TrkB signaling in regulating responses to opiates and cocaine. Using more advanced tools, which now enable such knockouts in specific neuronal cell types within these brain regions, we will characterize the specific influence of D1 vs. D2 containing NAc medium spiny neurons, and VTA dopamine neurons, in these phenomena. One of the most dramatic changes that opiates induce in VTA dopamine neurons is a decrease in their overall size. This decrease is mediated by an impairment in BDNF signaling within these neurons, specifically, downregulation of the IRS-AKT pathway. We now have evidence that this downregulation is part of overall opiate-induced adaptations in AKT-RHEB-mTOR signaling. The proposed experiments are designed to further establish the detailed molecular events that underlie this morphological adaptation. In contrast, cocaine and opiates induce distinct changes in NAc medium spiny neurons: cocaine increases, whereas opiates decrease, the neurons'dendritic arborizations and spine density. We have shown that these adaptations too involve altered BDNF signaling, and now propose to further characterize the precise changes in neurotrophic signaling pathways, including changes in NF?B signaling that mediate these adaptations. Together, the proposed experiments will provide fundamentally new insight into the dramatic ways in which opiates and stimulants change VTA-NAc neurons, information which could be mined in future years to define improved diagnostic tests and treatments for drug addiction. PUBLIC HEALTH RELEVANCE: The objective of this grant is to study the role of neurotrophic signaling pathways, acting in the brain's reward circuits, in drug abuse models in mice. We investigate the influence of chronic exposure to drugs of abuse on levels and activity of these complex signaling pathways within brain reward regions. We then manipulate the activity of specific signaling proteins in reward regions by use of genetic mutant mice or viral mediated gene transfer, and study the molecular, cellular, and behavioral consequences of these manipulations. Together, these studies promise to provide fundamentally novel information concerning how drugs of abuse alter the brain's reward circuits after chronic administration, and to offer new leads toward the development of improved diagnostic tests and more effective treatments of drug addiction.

PROJECT SUMMARY ? OVERALL The objective of this Conte Center is to take maximal advantage of recent advances in chromatin biology, so- called epigenetics, to fundamentally increase our understanding of the long-lasting abnormalities in the brain that cause depression. Our work focuses on key limbic brain regions, such as nucleus accumbens and prefrontal cortex, which have been implicated directly in the control of mood in health and disease. The Center is composed of four Projects led by Eric Nestler (Mount Sinai), Schahram Akbarian (Mount Sinai), David Allis (Rockefeller), and Carol Tamminga (UT Southwestern), and two Scientific Cores?the Animal Models Core led by Venetia Zachariou (Mount Sinai) and Chromatin and Gene Analysis Core led by Li Shen (Mount Sinai). The PIs, along with several Co-PIs, are leaders in their fields who use their complementary expertise and approaches to execute a multidisciplinary program of research focused on transcriptional and chromatin abnormalities both in mouse models of depression and in postmortem brains of depressed humans. A defining feature of the Center is bidirectional translation, with findings from mice validated in humans, and with discoveries in humans put back into animal models to study underlying mechanisms. The Center's research is defined by four themes. First, we study a broad range of epigenetic mechanisms, including histone and DNA modifications, nucleosome turnover, and the 3D structure of chromatin, which work in concert to control gene transcription. This involves the use of several next generation sequencing methods and advanced bioinformatics to analyze the resulting complex datasets. Second, we use this insight to understand how exposure to stress early in life controls an individual's susceptibility vs. resilience to stress-related disorders for a lifetime through long-lasting epigenetic mechanisms. Third, we focus on sex differences in this epigenetic regulation, having defined shared as well as many distinct mechanisms operating in male vs. female brain. Fourth, these studies are identifying numerous key target genes and molecular pathways that are defining novel mechanisms underlying depression and other stress-related disorders, which will help drive the field toward improved treatments and diagnostic tests.

The main objective of the Chromatin and Gene Analysis Core is to provide the technical and bioinformatic infrastructure to optimally mine the vast amounts of genome-wide gene expression and chromatin data that will be generated from the Center's work. Center investigators have lead the field in several aspects of genome-wide chromatin analyses, including pioneering these approaches in brain, which offers several unique technical challenges. Together, we have defined optimal methods of chromatin immunoprecipitation (ChlP) for mouse and human brain. As well, this Core has established expertise in analyzing the rich ChlP- Seq and RNA-Seq datasets obtained, and will work to continually improve the tools available. Much of the genome-wide data obtained by our Center will be generated by the individual Projects and analyzed by the Core. In parallel, the Core will run more routine genome-wide assays on defined animal models and thereby provide a foundation for the more specific and sophisticated measures in the individual Projects. This will include screening families of chromatin regulatory proteins for alterations in mouse depression models, which will drive research in the individual Projects. Additionally, the Core will pilot several novel technologies and approaches, including testing whether any potent trans-generational transmission of behavioral abnormalities can be mediated via sperm or ova from stressed mice. All four Projects will be served by this Core; Projects 1-3 for the analysis of animal models and Project 4 for postmortem human brain tissue, which offers an additional set of unique technical challenges. By consolidating the analytical work and some routine genome-wide analyses within a centralized Core, we ensure rigorous control over the data and facilitate comparisons of experimental findings across the individual Projects. This consolidation also makes financial sense, since we concentrate and maximize efficient use of our analytical expertise. The Core is also responsible, with the Administrative Core, in developing and maintaining the multiple ways in which these highly complex and large datasets, and analytical tools, are both shared across the multiple Projects and laboratories that comprise the Center as well as shared with the scientific community and lay public at large.

The objective of Project 1 is to characterize the important role of repressive histone methylation in nucleus accumbens (NAc) and prefrontal cortex (PFC) in mediating depression- and antidepressant-like responses in animal models. We have demonstrated downregulation of di-methylation of Lys 9 of histone H3 (H3K9me2) in NAc and PFC after several forms of chronic stress, which is mediated by repression of G9a and GLP, the histone methyltransferases which catalyze this epigenetic state. We have also demonstrated that such adaptations mediate pro-depression-like effects, while augmenting H3K9me2 exerts potent antidepressant actions. We will now extend our considerable preliminary findings in several ways. We will utilize ChlP-Seq to map H3K9me2 binding genome-wide. While global levels of H3K9me2 are reduced in NAc by chronic stress, many genes show induction of G9a/H3K9me2 binding, indicating the existence of complex mechanisms that control this repressive histone methylation mark at particular genes. Our hypothesis is that the chromatin state at individual genes helps determine which genes show reduced vs. increased H3K9me2 binding overlaid on global decreases in the enzymatic machinery involved. In conjunction with the Chromatin and Gene Analysis Core, we will also gain crucial information concerning the relationship between H3K9me2 and several other chromatin modifications in the brain in vivo, and why certain genes show stable chromatin changes over the life cycle of the animal. By exploring the involvement of the transcription factor CREB, which we have found interacts with G9a/H3K9me2 as part of a pathological molecular circuit in NAc to drive susceptibility to stress, we expect to gain unprecedented insight into mechanisms of gene regulation in depression models. We will then focus on a small number of target genes for H3K9me2 and characterize their involvement in depression-related phenomena. Many of our key findings observed to date in animal models have already been validated in the NAc and PFC of depressed humans, as discussed in Project 4, emphasizing the translational nature of our Center. These studies provide a template for the analysis of the roles played by other key histone modifications in depression.

PROJECT SUMMARY (See instructions): A main objective of the Animal Models Core is to provide a broad range of behavioral models of stimulant and opiate addiction in mice and rats to support the PPG's overall goal to establish molecular, cellular, and circuit mechanisms of addiction. Such models include several routine behavioral assays as well as more sophisticated self-administration, self-stimulation, and cognition paradigms. The imperative to employ this broad behavioral battery is that it is difficult to infer something about such a complex behavioral syndrome as addiction from a single model or even a limited number of models. The Core then utilizes these behavioral resources in two main ways. First, the Core provides microdissections of brain reward regions from carefully defined mouse models for molecular characterization in each Project and in the Chromatin and Gene Analysis Core. Second, the Core works with each of the four Projects to generate causal evidence that directly links specific molecular, cellular, and circuit-level adaptations to particular behavioral abnormalities that define a state of addiction. The Core accomplishes this goal by providing a range of genetic mutant mice as well as a large number of vectors for viral-mediated gene transfer, all of which are extensively validated by the Core. The mice and vectors are often generated initially to meet the specific needs of an individual Project, but then are provided to other Projects to broaden their application and thereby promote PPG integration. This Core has led the field in generating mutant mice and viral vectors, which make it possible to selectively manipulate a given gene of interest within a particular neuronal cell type or brain region of adult animals, thus avoiding confounds with more traditional approaches. Finally, the Core provides advanced optogenetic tools to Project investigators to directly implicate altered neuronal and circuit function to addiction-related behavioral abnormalities. By consolidating this behavioral, mouse mutagenesis, viral vector, and optogenetic work within a centralized Core, we ensure rigorous control over the data and facilitate comparisons and contrasts of experimental results across the individual Projects. This consolidation also makes financial sense, since we concentrate and maximize efficient use of the required expertise.

The objective of Project 1 is to characterize the important role of repressive histone methylation in nucleus accumbens (NAc) and prefrontal cortex (PFC) in mediating stimulant and opiate addiction. We have demonstrated downregulation of dimethylation of Lys 9 of histone H3 (H3K9me2) in NAc and PFC after stimulant or opiate exposure, effects mediated via the repression of G9a, a histone methyltransferase which catalyzes this epigenetic mark. We have also demonstrated that such adaptations, which occur with investigator- or self-administered drug, increase behavioral responses to both drugs of abuse. We will now extend our considerable preliminary findings in several ways. We will utilize ChlP-Seq to map H3K9me2 binding genome-wide in response to stimulant or opiate self-administration over a broad time course. While global levels of H3K9me2 are reduced in NAc by drug exposure, many individual genes show induction of G9a/H3K9me2 binding, indicating the existence of complex mechanisms that control chromatin modifications at particular genes. Our hypothesis is that the chromatin landscape at individual genes helps determine which genes show reduced vs. increased H3K9me2 binding overlaid on global decreases in the enzymatic machinery involved. In conjunction with the Chromatin and Gene Analysis Core, we will also gain crucial information concerning the relationship between H3K9me2 and several other chromatin modifications in the NAc and PFC in vivo, including the role of DNA methylation and of several histone deacetylases, and better understand why these various chromatin changes are long-lasting only at a subset of genes after a course of drug administration. Another major effort is to define the molecular mechanisms by which chronic stimulants or opiates repress G9a expression in NAc and PFC; we have highly novel evidence implicating the beta- catenin transcriptional network in this phenomenon. Finally, we will focus on a small number of target genes- which encode synaptic proteins-for H3K9me2 and related chromatin modifications and characterize their involvement in addiction-related phenomena. Working with Project 4, we will validate key findings from animal models in the NAc and PFC of addicted humans, emphasizing the translational nature of our PPG. RELEVANCE (See instmctions): Addiction remains one of the worid's greatest public health problems, yet its pathophysiology remains incompletely understood and available treatments for addictions to various drugs of abuse are inadequately effective for most people. We believe that the most effective way of eventually developing definitive treatments and cures for addiction rests in part in a better understanding of its underiying neurobiology.

? DESCRIPTION (provided by applicant): Earlier versions of this R01 grant focused on the role of neurotrophic signaling pathways in the neural plasticity induced in the ventral tegmental area (VTA) nucleus accumbens (NAc) reward circuit by chronic exposure to stimulant or opiate drugs of abuse, with a particular focus on the molecular mechanisms by which these drugs alter the morphology of VTA and NAc neurons. In this renewal application, we propose to focus on one major aspect of this effort: stimulant and opiate regulation of the morphology of NAc medium spiny neurons (MSNs). While there has been considerable work on this topic, major questions persist, given different laboratories' use of contingent vs. noncontingent drug exposure and of different methods to quantify dendritic morphology, and their focus on different times after drug withdrawal and on different subregions of NAc (core vs. shell). A further complicating factor is that few studies have considered different effects of drug exposure on the two main subtypes of NAc MSNs, those expressing predominantly the D1 vs. D2 dopamine receptor. The goal of this grant is to characterize, in a comprehensive cell type and subregion specific manner, the effect of cocaine or heroin self-administration, over the life cycle of self-administration behavior (acquisition, withdrawal, extinction, and relapse), on the number and morphology of NAc dendritic spines. We will also gain insight into input specificity of such dendritic plasticity as ell as the postsynaptic strength of spines under control and drug self-administration conditions by use of optogenetic and 2 photon imaging approaches. In parallel, we will continue our innovative studies that have provided novel insight into the molecular basis by which chronic drug self-administration induces this postsynaptic plasticity of NAc MSN dendritic spines. This work, in concert with ongoing studies in many other laboratories focused largely on complementary aspects of glutamatergic synaptic plasticity in drug abuse models, will help establish the ways in which such dendritic plasticity contributes to a state of addiction. As well, these studies will hep us capitalize on our improved understanding of drug-induced dendritic plasticity in NAc MSNs by identifying target genes suitable for future drug discovery efforts.

PROJECT SUMMARY There is an urgent need for novel therapeutics to treat drug addiction. One potential novel drug target that plays a key role in this devastating disorder is ?FosB. ?FosB accumulates in highly specific regions of the brain (in particular the nucleus accumbens) in response to cocaine or other drugs of abuse. ?FosB mediates increases in drug-seeking behavior seen after prior drug exposure. As a transcription factor, ?FosB regulates the expression of many genes crucial to drug addiction, including the AMPA glutamate receptor subunit GluA2 and cyclin-dependent kinase 5 (Cdk5). ?FosB can both repress and activate gene transcription, but the molecular basis of this dual action is not known. One explanation is that ?FosB forms both heterodimers with JunD as well as homodimers with itself, and that these two ?FosB-containing species differentially regulate gene transcription. We seek to validate the therapeutic potential of ?FosB, and to delineate its molecular mechanisms. To this end, our goal is to leverage compounds that target ?FosB species in vivo. We hypothesize that, by regulating ?FosB with small molecules, we can exploit ?FosB to strategically regulate key genes and overcome harmful neuronal and behavioral adaptations induced by chronic cocaine. Our approach is to develop potent in vivo chemical probes that target ?FosB and discriminate between ?FosB homodimers and heterodimers. We demonstrated with first generation scaffolds that pharmacologically targeting ?FosB elicited biological and behavioral responses in mice chronically treated with cocaine. We have now identified new scaffolds with more drug-like properties, but low micromolar activity, which we have validated in vitro. We propose to: 1) improve the potency of our probes through chemical optimization and iterative testing, 2) demonstrate that our compounds directly bind ?FosB and reveal their mechanism-of-action using structural biology, and 3) measure the impact of our probes in vivo both on the behavioral responses to cocaine, and on the transcription of GluA2 and Cdk5. The rationale for this proposal is that improved probes will enable us to test the therapeutic potential of ?FosB as a viable drug target to ameliorate aspects of drug addiction. In addition, our improved probes will enable us to delineate aspects of ?FosB in vivo (in particular the role of ?FosB homodimers vs. heterodimers). This proposal is innovative because it will yield chemical tools for a novel and non-traditional putative therapeutic target for which no probes are currently available. Importantly, we will be able to test whether ?FosB can be used as a conduit to safely regulate specific genes that maintain the addicted state by harnessing the highly region-specific accumulation of ?FosB in the nucleus accumbens in response to drugs of abuse. Furthermore, our probes will enable us to reveal completely new mechanistic information on ?FosB which cannot be easily gained using current techniques. This information is very significant because it could reveal completely novel strategies to treat drug addiction.

? DESCRIPTION (provided by applicant): In many brain regions, glial cells substantially outnumber nerve cells, but their role in physiological and pathophysiological conditions remains poorly understood. Astrocytes are the most widely distributed glia with intimate anatomical interactions with excitatory synapses. Recent studies reveal that, in addition to providing structural and nutritional support, astrocytes dictate synapse formation and subsequent synapse refinement- elimination in the developing CNS. Our preliminary results show that, after chronic exposure to cocaine or morphine, some of these glia-based developmental mechanisms re-emerge in the adult nucleus accumbens (NAc), a forebrain region essential for addiction-related behavioral abnormalities. These drug-induced, glia- mediated synaptic remodeling processes may profoundly rewire the neurocircuits involving the NAc, and critically contribute to the pathophysiology of drug addiction. Focusing on this unique angle, the objectives of this application are: 1) To characterize the molecular and cellular mechanisms underlying glia-mediated synaptogenesis and synaptodegeneration in the NAc in mice after cocaine or morphine self-administration and withdrawal; 2) To determine the circuitry consequences of drug-induced, glia-mediated synaptic remodeling, particularly, how NAc excitatory synapses are refashioned in cocaine- and morphine-exposed mice by glia- mediated synaptogenesis or synaptodegeneration; and 3) To determine the behavioral consequences of drug- induced, glia-mediated synapse and circuitry remodeling using the mouse model of incubation of cue-induced drug craving, a drug relapse model that depends on NAc excitatory circuits. To achieve these goals, we will use a multidisciplinary approach, across the Dong and Nestler laboratories, including confocal imaging, slice electrophysiology, optogenetics, in vivo viral-mediated gene transfer, RNA interference, transgenic mouse lines, and mouse models of drug self-administration. By targeting the previously unexplored glia-mediated synapse and circuitry remodeling in drug-exposed mice, the proposed experiments promise to open new avenues toward understanding cellular and circuitry mechanisms underlying drug addiction and providing new strategies for anti-addiction treatments.

PROJECT SUMMARY ? PROJECT 1 Project 1 focuses on transcriptional and epigenetic mechanisms in the nucleus accumbens (NAc) and ventral tegmental area (VTA), key brain reward regions implicated in depression and other stress-related disorders. Over the past four years, we made significant progress in demonstrating the direct relevance of several modes of chromatin regulation to depression-related phenomena in VTA and NAc both in animal models and, with Project 4, in postmortem human brain. One of the primary rationales for focusing on chromatin mechanisms is their heuristic importance in mediating lifelong consequences of prior behavioral experience. Accordingly, with the Animal Models Core, we optimized a protocol where maternal separation (MS) during postnatal days P11- 20, but not other time windows, increases the susceptibility of male and female mice to subsequent stress in adulthood. This increase in stress susceptibility is latent: without subsequent stress the animals appear normal. This establishes an ideal system in which to study the chromatin mechanisms that mediate a lifelong increase in stress susceptibility, which we refer to colloquially as ?chromatin scars.? RNA-seq, with the Chromatin and Gene Analysis Core, revealed lasting gene expression abnormalities in VTA and NAc in MS-exposed mice before adult stress exposure. We focus here on two of the inferred top upstream regulators of these gene expression abnormalities, two transcription factors, OTX2 in dopamine neurons (DA) of VTA and ESR1 in D2- type medium spiny neurons (MSNs) of NAc. Indeed, we have found that both genes, upon their manipulation in the respective brain area and cell type, drive stress susceptibility. We are now characterizing the underlying mechanisms involved by defining chromatin changes that are induced at specific target genes in concert with alterations in OTX2 or ESR1 to control their lifelong expression in response to early life stress. We are also analyzing a small number of these target genes to understand how their lasting regulation controls the functioning of VTA DA neurons and D2 NAc MSNs to regulate stress susceptibility for a lifetime. Together, these studies are providing new insight into how early life stress controls stress susceptibility in males and females over the life cycle.

Project Summary / Abstract In this R13 application we request support for a new Gordon Research Conference (GRC) on ?Neurobiology of Drug Addiction,? which will take place in July 2017 in Hong Kong, China. The objectives of this GRC are to foster open discussion of novel research developments, build new collaborations, and propel the next generation of scientific advances in addiction research. To achieve these objectives, we will pursue the following four specific aims at the meeting: 1) Provide an international forum to brainstorm about cutting edge, multidisciplinary research at the forefront of drug addiction; 2) Introduce breakthrough neuroscientific techniques that facilitate the understanding of brain mechanisms driving compulsive drug use and relapse; 3) Promote interactions among young and senior investigators, and exchange of ideas that will shape the future directions of addiction neuroscience; 4) Foster development of the next generation of addiction researchers by encouraging the participation of students and postdoctoral fellows who are committed to addiction research. The conference focuses on presentations and discussions at the frontiers of addiction research. The scope of the lectures will be confined to basic and clinical studies involving addictive drugs (including alcohol) and the nervous system, and will focus on mechanisms of compulsive drug use and relapse. The meeting will also break down barriers to progress by uniting investigators with synergistic and complementary expertise in areas ranging from molecular mechanisms to translational clinical research. The conference will provide a forum to address the latest developments in addiction research in an open and highly interactive GRC format, which includes formal talks interspersed with ample discussion time, poster sessions, and informal discussion periods that cultivate communication and collaborations. This conference setup differs from that of other meetings in the field, and will provide a unique opportunity for close interactions among investigators at all stages of their careers and among investigators with different research approaches and cultural perspectives. Concerted efforts to support participation of students, postdoctoral fellows, young investigators, and under-represented minorities will nurture the growth of the next generation of addiction researchers. We envision that the Neurobiology of Drug Addiction GRC will significantly advance our understanding of the neural mechanisms underlying drug addiction and stimulate the development of effective therapeutic approaches for addiction treatment.

PROJECT SUMMARY/ABSTRACT? PROJECT 1 Project 1's objective is to characterize several novel transcription factors, not previously implicated in addiction, in mediating the lasting actions of stimulant and opiate drugs of abuse in nucleus accumbens (NAc) and prefrontal cortex (PFC). Virtually all prior studies of transcription factors in addiction have taken a candidate approach. This is in marked contrast to an unbiased approach of using ?big data? to deduce, in an open-ended manner, those factors that are most important in mediating specific aspects of the complex addiction phenotype. We utilize this more powerful approach by taking advantage of large-scale RNA-seq and related datasets from rodent addiction models and humans with substance use disorders to identify those factors that appear to play particularly critical roles in drug action. We focus on cell type-specific actions of three of the most highly ranked transcription factors from our PPG?s datasets: E2F3, ZFP189, and RXR?. For example, ~25% of all genes that display primed or desensitized changes in expression in NAc as a result of past cocaine self-administration are predicted to be direct targets of E2F3. Based solely on these bioinformatics predictions, we have generated robust preliminary data to validate the importance of each of these transcription factors in drug addiction and now propose to better understand their actions. We will complete characterization of their role in NAc in controlling behavioral responses to cocaine in self- administration assays as well as map their target genes on a genome-wide basis. Both of these efforts will be performed in a cell type-specific manner, as we have evidence for some of the factors playing different roles in different neuronal types in this brain region. This work in animal models will be complemented by studies of humans with cocaine use disorders, where we already have preliminary evidence for their abnormal regulation. We will extend these studies to PFC, where we have found that a different E2F3 splice isoform controls behavioral and genomic responses to cocaine, as well as to opiate models where we know that these factors also show prominent dysregulation. Together, this work will reveal new transcriptional mechanisms underlying cocaine and opiate addiction.

PROJECT SUMMARY/ABSTRACT? OVERALL PPG This new Program Project Grant (PPG) utilizes recent advances in transcriptional biology to fundamentally increase our knowledge of the long-lasting abnormalities in brain that underlie stimulant and opiate addiction. Our work focuses on several specific cell types in key addiction-related brain regions: nucleus accumbens, dorsal striatum, and prefrontal cortex. The PPG is composed of four Projects and three Cores all at Mount Sinai. The PIs are leaders in their fields who have an established history of effective collaboration and use their complementary expertise and approaches to chart a multidisciplinary course in the proposed research. Project 1 (Eric Nestler) focuses on novel transcription factors induced in brain reward regions by self-administered stimulants and opiates. Project 2 (Paul Kenny) mines the PPG?s complex datasets to understand the role played by circular RNAs in addiction; these are a newly discovered class of non-coding RNAs some of which, within brain, are concentrated at synapses. Project 3 (Anne Schaefer) focuses on the influence of microglia in controlling transcriptional responses to drugs of abuse within brain reward neurons and their behavioral consequences. Project 4 (Yasmin Hurd) concentrates on the influence of enhancer regions, and their transcriptional and chromatin mediators, in controlling molecular and behavioral adaptations to drugs of abuse. All four projects validate findings from animals in human postmortem brain tissue, while discoveries in human substance use disorders are fed back to animal models to explicate the underlying mechanisms involved. The PPG is supported by three Cores, an Administrative Core (Eric Nestler) to oversee and coordinate PPG operations; an Animal Models Core (Vanna Zachariou) to provide animal models of addiction and other advanced tools (e.g., viral gene transfer, inducible mutant mice) to manipulate individual genes of interest in specific cell types of the targeted brain regions and thereby provide causal evidence linking molecular-cellular plasticity to addiction-related phenomena; and a Gene and Chromatin Analysis Core (Li Shen) to provide state- of-the-art methods and bioinformatics to characterize genome-wide regulation of gene expression and chromatin modifications in addiction. This pioneering investigation of transcriptional mechanisms of drug addiction will help drive major advances in the field.

PROJECT SUMMARY There is an urgent need to develop effective strategies to combat drug addiction, a major public health burden. Unfortunately, current efforts are hampered by a focus on a very limited range of drug targets. Decades of work have established that the transcription factor DFosB plays a critical role in drug addictive behaviors in rodent models, with validation in humans available as well. In response to chronic cocaine or opioids (e.g., heroin) administration, DFosB mediates aspects of drug seeking, reward, self-administration, and relapse. Due to its unusual stability, DFosB accumulates to very high levels in the brain in regions critical for reward, making it an attractive target for addiction therapies. However, critical mechanistic aspects of DFosB function are not known, making it difficult to pursue DFosB as a therapeutic target. It is not known how DFosB molecules are arranged in vivo, what molecular features control their ability to bind to DNA and turn genes on or off, and whether these features can be targeted strategically with small molecules in order to regulate DFosB function in vivo to combat drug use disorders. We hypothesize that, by modulating ?FosB with small molecules, we can selectively regulate key strategic DFosB gene targets, and thereby the long-term neural and behavioral adaptations that DFosB triggers in response to chronic drug use. To test our hypotheses, we propose to 1) optimize a series of validated lead compounds into high-affinity chemical probes targeting DFosB in vitro and in vivo; 2) unravel how key molecular features in DFosB regulate its actions; and 3) determine how targeting these features either with our chemical probes or novel genetic tools alters behaviors in animal models of addiction. To this end, we have an outstanding translational research team overseeing a robust and effective experimental platform that draws on our prior combined work. We have already achieved important milestones. First, we have discovered that DFosB partners not only with JunD but also with itself in order to bind DNA, and these two species are structurally and functionally very different. Second, we have uncovered a molecular switch in ?FosB that controls its binding to DNA and that works differently in heteromeric vs. homomeric ?FosB complexes. Third, we have developed a large panel of lead compounds that target DFosB and that we can leverage to gain both fundamental mechanistic insight into DFosB function, as well as assess their in vivo effects on addictive behaviors. Together, our work creates a powerful, previously unavailable, and highly actionable platform to test the utility of DFosB as a therapeutic target. The positive impact of this work will be to further de-risk DFosB as a therapeutic target by creating comprehensive, mechanism-based knowledge onto which a drug discovery program will be anchored, focused on a completely novel target to combat addiction. This innovative proposal will provide novel insight into the pathophysiology of drug addiction, and novel chemical probes validated in cells and animal models, which together will serve as a strong platform from which to target DFosB for therapeutic or diagnostic purposes.