Ryan W. Logan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Basic and clinical research suggests there are extensive bidirectional interactions between circadian rhythms and addiction. Disruptions to the circadian system, either by environmental or genetic perturbation, may increase the vulnerability to addiction, while chronic drug use leads to circadian disruptions that persist during abstinence and may contribute to relapse. Although these relationships are intriguing, very little is known about the molecular mechanisms underlying the relationship between the circadian system and the transition to addiction. Animal studies have demonstrated that chronic exposure to cocaine leads to alterations in the expression and function of specific circadian genes (i.e., components of the molecular clock) in the mesolimbic dopamine reward system. A major region of convergence for reward circuitry and a key substrate that regulates drug reward and motivation is the nucleus accumbens (NAc). The NAc is comprised of mostly two specific subtypes of medium spiny neurons (MSNs) that predominantly express either dopamine 1 or 2 receptors (D1+ or D2+). These two subtypes of MSNs have distinct roles in the regulation of cocaine reward behaviors, although the molecular mechanisms underlying these differences remain unclear. We have identified a novel role of the circadian transcription factor, neuronal PAS domain protein 2 (NPAS2) in the regulation of cocaine reward, potentially via its interactions with the transcription factor nuclear factor kappa B (NF?B) and its selective expression in D1+ MSNs of the NAc. The aims of the K01 are as follows: 1) to investigate the functional role of NPAS2 in the regulation of different cocaine reward behaviors, (i.e., conditioned place preference and self-administration), via expression in D1+ or D2+ MSNs of the NAc; 2) to examine the effects of acute and chronic cocaine exposure on the expression of Npas2 and NF?B, along with the interactions between these transcription factors in the NAc; and 3) to investigate whether NPAS2 positively regulates NF?B-mediated transcription in D1+ and D2+ MSNs of the NAc. Therefore, the proposed K01 award will characterize the role of these novel cell type specific molecular mechanisms in the NAc that regulate cocaine reward. These studies will also further clarify the role of the molecular clock in the transition to addiction. These research goals are aligned with the strategic goals of NIDA and the NIH BRAIN Initiative to identify the functional significance of specific cell types in the brain by integrating innovative molecular and behavioral approaches. The candidate's long-term career goal is to develop an independent research program investigating the role of the circadian system in the regulation of reward circuitry that underlies the transition to drug addiction. The candidate has a strong background in circadian rhythms research and requires additional training to develop expertise in translational addiction research and the combination of advanced molecular approaches and complex behavioral assays. The proposed K01 Career Development Award will ensure the candidate's successful transition to an independent investigator by providing extensive conceptual and methodological training in the following areas: 1) conceptual expertise on the neurobiology of addiction for basic and translational research; 2) methodological proficiency in the combination of advanced molecular techniques, including protein immunoprecipitation, immunohistochemistry, and brain cell type specific viral-mediated gene transfer, with complex behavioral assays, including cocaine conditioned place preference and intravenous cocaine self-administration in mice; and 3) to integrate previous conceptual and research abilities with newly acquired neurobiological techniques and approaches, and conceptual expertise in addiction research, to become an independent investigator. The candidate will be mentored and advised by a team of faculty with expertise in the cellular and molecular mechanisms of addiction, reward neural circuitry and drug-induced neural plasticity, and circadian rhythms. The individualized career development plan includes a combination of regular meetings with mentors and consultants, grantsmanship skills training, formal coursework, conference attendance, formal research presentations, and technical and methodological training. The University of Pittsburgh and the Department of Psychiatry provides the necessary infrastructure to become a successful independent addiction researcher, including exceptional core and mentor research facilities, individualized career development programs and training opportunities, and a long-standing commitment by the institution and the departmental chair to develop promising young investigators.

? DESCRIPTION: Basic and clinical research suggests there are extensive bidirectional interactions between circadian rhythms and addiction. Disruptions to the circadian system, either by environmental or genetic perturbation, may increase the vulnerability to addiction, while chronic drug use leads to circadian disruptions that persist during abstinence and may contribute to relapse. Although these relationships are intriguing, very little is known about the molecular mechanisms underlying the relationship between the circadian system and the transition to addiction. Animal studies have demonstrated that chronic exposure to cocaine leads to alterations in the expression and function of specific circadian genes (i.e., components of the molecular clock) in the mesolimbic dopamine reward system. A major region of convergence for reward circuitry and a key substrate that regulates drug reward and motivation is the nucleus accumbens (NAc). The NAc is comprised of mostly two specific subtypes of medium spiny neurons (MSNs) that predominantly express either dopamine 1 or 2 receptors (D1+ or D2+). These two subtypes of MSNs have distinct roles in the regulation of cocaine reward behaviors, although the molecular mechanisms underlying these differences remain unclear. Recent human genetics studies have identified variants in the gene coding for the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) associated with psychiatric disorders that are highly comorbid with addiction disorders. We have identified a novel role of NPAS2 in the regulation of cocaine reward via activity in D1+ MSNs of the NAc. The R21 aims are as follows: 1) Leverage CRISPR/Cas9 technology to generate split Cre mice that enable us to use intersectional genetics approaches to target D1+ or D2+ MSNs specifically in the striatumR and 2) Generate NPAS2Qdeficient mice exclusively in striatal D1+ or D2+ MSNs to investigate the cell type specific molecular mechanisms regulating reward and motivation (R33 phase). The R33 aims will characterize these mice by 1) investigating the role of Npas2 in the regulation of circadian regulation of cocaine conditioned reward (conditioned place preference) and self-administration 2) NPAS2Q mediated circadian transcription on cocaine-induced dendritic plasticity in the NAcR and 3) elucidating the cell type specific molecular mechanisms of NPAS2 regulation of cocaine reward using confocal microscopy, FACs, RNA seq, and integrative analyses with preliminary ChIPseq data. These studies will further clarify the role of the molecular clock in the transition to addiction, and importantly, provide the broader scientific community with novel transgenic mice to further investigate the molecular mechanisms of 'direct' and 'indirect' pathway regulation of drug reward and addiction phenotypes. These studies will leverage CRISPR/Cas9 technologies and advanced molecular and behavioral approaches to study a novel mechanism of circadian regulation of addiction behaviors.

PROJECT SUMMARY Sleep and circadian disruption are highly prevalent in Alzheimer?s disease, emerging decades prior to cognitive decline. Evidence from animals and humans suggests these disruptions directly lead to Alzheimer?s disease pathology that further exacerbate sleep and circadian dysfunctions. An important breakthrough for studying these connections was the recent development of a genetically diverse mouse panel that incorporates high-risk familial Alzheimer?s disease mutations (termed AD-BXD) that recapitulate key aspects of human Alzheimer?s pathophysiology, including aging-related neurodegeneration, progressive cognitive deficits, and sleep disruption. Using these mice, identified a new marker of genetic vulnerability to cognitive decline and sleep disruption in Alzheimer?s disease: the transient-receptor potential nonselective cation channel type 3 (TRPC3). TRPC3 has already been implicated as a target for modifying the development of normal cognitive aging and Alzheimer?s disease. We found that viral-mediated knockdown of TRPC3 diminished amyloid load and enhanced cognition in susceptible AD-BXD mice, providing the preclinical basis for investigating the mechanistic links that connect sleep disruption and cognitive decline in Alzheimer?s disease. However, these methods do not offer the biology by which TRPC3 moderate disease-related pathology. We propose to comprehensively map the spatial location and expression of TRPC3 to identify whether this localization changes in key brain regions related to sleep and cognition due to Alzheimer?s pathology. We developed a new approach to rapidly image multiple cell-types and markers of Alzheimer?s neurodegeneration within a whole brain in three-dimensional (3D) space at single-cell resolution. Our ultra-fast high-resolution confocal ribbon-scanning approach reaches diffraction limited resolution (~200nm) and collects 3D rendered whole brain maps in less than 24-hours. The goals of the supplement are to take advantage of the discovery of TRPC3 as a new target for Alzheimer?s-related changes in cognition and sleep and use our innovative tools to answer fundamental questions: In mice, 1) Is TRPC3 located in disease-related brain areas linked to cognition and sleep?; and 2) Does TRPC3 interact with biological hallmarks of Alzheimer?s disease brain pathology, including amyloid beta and hyperphosphorylated Tau? We will use human postmortem brains from Alzheimer?s disease patients to ask, 3) Is brain TRPC3 expression altered in Alzheimer?s disease? We predict TRPC3 localizes to subregions of the hypothalamus associated with sleep and hippocampal and cortical regions associated with cognition, and this distribution will be differentially impacted by sleep deprivation in susceptible vs. resilient mice. In human brains, we expect that TRPC3 expression will be higher in advanced Alzheimer?s disease patient brains and display altered rhythmicity. Ultimately, using our novel suite of genetic, imaging, and computational tools, we will we will answer longstanding questions about how changes in sleep and cognitive decline are linked to Alzheimer?s disease pathology.

PROJECT SUMMARY Sleep and circadian disruption are highly prevalent in Alzheimer?s disease, emerging decades prior to cognitive decline. Evidence from animals and humans suggests these disruptions directly lead to Alzheimer?s disease pathology that further exacerbate sleep and circadian dysfunctions. An important breakthrough for studying these connections was the recent development of a genetically diverse mouse panel that incorporates high-risk familial Alzheimer?s disease mutations (termed AD-BXD) that recapitulate key aspects of human Alzheimer?s pathophysiology, including aging-related neurodegeneration, progressive cognitive deficits, and sleep disruption. Using these mice, identified a new marker of genetic vulnerability to cognitive decline and sleep disruption in Alzheimer?s disease: the transient-receptor potential nonselective cation channel type 3 (TRPC3). TRPC3 has already been implicated as a target for modifying the development of normal cognitive aging and Alzheimer?s disease. We found that viral-mediated knockdown of TRPC3 diminished amyloid load and enhanced cognition in susceptible AD-BXD mice, providing the preclinical basis for investigating the mechanistic links that connect sleep disruption and cognitive decline in Alzheimer?s disease. However, these methods do not offer the biology by which TRPC3 moderate disease-related pathology. We propose to comprehensively map the spatial location and expression of TRPC3 to identify whether this localization changes in key brain regions related to sleep and cognition due to Alzheimer?s pathology. We developed a new approach to rapidly image multiple cell-types and markers of Alzheimer?s neurodegeneration within a whole brain in three-dimensional (3D) space at single-cell resolution. Our ultra-fast high-resolution confocal ribbon-scanning approach reaches diffraction limited resolution (~200nm) and collects 3D rendered whole brain maps in less than 24-hours. The goals of the supplement are to take advantage of the discovery of TRPC3 as a new target for Alzheimer?s-related changes in cognition and sleep and use our innovative tools to answer fundamental questions: In mice, 1) Is TRPC3 located in disease-related brain areas linked to cognition and sleep?; and 2) Does TRPC3 interact with biological hallmarks of Alzheimer?s disease brain pathology, including amyloid beta and hyperphosphorylated Tau? We will use human postmortem brains from Alzheimer?s disease patients to ask, 3) Is brain TRPC3 expression altered in Alzheimer?s disease? We predict TRPC3 localizes to subregions of the hypothalamus associated with sleep and hippocampal and cortical regions associated with cognition, and this distribution will be differentially impacted by sleep deprivation in susceptible vs. resilient mice. In human brains, we expect that TRPC3 expression will be higher in advanced Alzheimer?s disease patient brains and display altered rhythmicity. Ultimately, using our novel suite of genetic, imaging, and computational tools, we will we will answer longstanding questions about how changes in sleep and cognitive decline are linked to Alzheimer?s disease pathology.

PROJECT SUMMARY Opioid use and dependence prevalence have skyrocketed in the United States. A majority of patients with opioid use disorder (OUD) relapse within months despite treatment. Recent human neuroimaging and postmortem brain studies in OUD reveal the degree of dysfunction within cortical and striatal brain circuits, particularly within dorsolateral prefrontal cortical (DLPFC) and nucleus accumbens (NAc) regions, strongly relates to the opioid use and dependence risk. The PFC provides top-down inhibitory cognitive and emotional control to the NAc, which mediates goal-directed and reward behaviors. Relapse vulnerability in OUD is strongly associated with the severity and persistency of disruptions to sleep and circadian rhythms, raising the possibility that therapeutic interventions which mitigate these disruptions during abstinence may be effective for reducing opioid craving and relapse. However, our understanding of the biological mechanisms underlying the relationships between circadian rhythms and OUD is limited, especially at the molecular level in the brains of people with OUD. We and others have developed novel, innovative approaches using time of death (TOD) to measure molecular rhythms in the human postmortem brain to investigate the mechanistic links between substance use and molecular brain rhythms. Using TOD approaches, we recently found a marked loss of molecular rhythms in the prefrontal cortex associated with normal aging and psychiatric disorders. Notably, we also discovered a gain of rhythmicity in genes within disease-specific molecular pathways, providing novel insights into the biology of brain aging and psychiatric pathology. Preliminary TOD analyses on large-scale gene expression in human subjects with OUD revealed enrichment for pathways related to circadian rhythms in the PFC and NAc. In our proposal, we will directly investigate the relationship between molecular rhythm disruption and opioid use and relapse using both human postmortem brains from subjects with OUD and mouse models of circuit-specific targeting and opioid self-administration. Specifically, we will investigate molecular rhythms in postmortem DLPFC and NAc using RNA-sequencing from a large cohort of subjects with OUD (Aim 1A). We will also examine the impact of specific clinical features (e.g., toxicology reports and overdoses, comorbid psychiatric disorders, history of use, polysubstance use, illness duration) on molecular rhythms in OUD (Aim 1B). We will then directly test the functional relevance of molecular rhythm disruptions in specific brain regions (PFC and NAc; Aim 2A) and circuits (PFC projections to NAc; Aim 2B) during opioid self-administration behavior in mice. Our studies will identify molecular rhythm abnormalities in the brains of subjects with OUD and begin to determine the mechanisms linking circadian rhythms and addiction, which will provide important insight into disease-related pathways and also potential treatment strategies.

PROJECT SUMMARY Opioid and cocaine abuse prevalence has skyrocketed in the United States, fueling the current epidemic of overdose deaths. Despite the public health impact of opioids and cocaine, we still lack a fundamental understanding of the mechanisms by which these drugs work, particularly across cellular and circuit levels. Further understanding of the neuroanatomy of the neural circuitry underlying opioid and cocaine reward is a critical initial step in targeting and elucidating their mechanisms. However, comprehensively visualizing relevant circuits in drug reward has been limited by approaches to contextualize these circuits and their response to drugs of abuse in the whole brain. We developed an approach to rapidly image the whole brain in three-dimensional (3D) space using ultra-fast high-resolution ribbon-scanning confocal microscopy. Our ribbon-scanning confocal imaging approach can image and visualize an entire rodent brain in less than 24 hours, where more conventional approaches (e.g., light-sheet) currently require days or even weeks. Furthermore, our ribbon-scanning confocal approach reaches diffraction-limited resolutions (~200-300nm), enabling us to visualize individual cells in the brain and their ultrastructure. We can apply these unique tools to begin solving the fundamental questions: 1) What is the precise circuitry that defines drug reward? And 2) What are the differential effects of cocaine and opioids on this circuitry? Like many drugs of abuse, cocaine and opioids rely on neurotransmission from dopamine (DA) neurons in the ventral tegmental area (VTA). However, until recently, parsing the connectivity of unique subpopulations of DA neurons and their potential roles in drug reward has been difficult. We developed a suite of intersectional genetic tools to definitively dissect the anatomical and functional properties of these different subpopulations within the same brain. We will integrate our 3D ribbon-scanning confocal imaging of DA neuron subpopulations with immunolabeling of neuronal activity markers to visualize precisely which DA neurons are activated in response to cocaine and opioids. Using whole brain immunolabeling and imaging, we will also visualize and map drug-dependent neuronal activity changes in the whole brain with the potential to reveal new populations of neurons differentially response to cocaine and opioids. Our overall objectives are to: Comprehensively map the distribution of DA neuron subpopulations including DA/glutamate co-transmitting cells relative to the overall DA system within whole brain (Aim 1); and to determine how cocaine and opioids differentially affect the activity of these DA neuron subpopulations (Aim 2). We will generate a comprehensive 3D brain atlas to identify the roles of unique subpopulations of DA neurons highly relevant to cocaine and opioids, which will serve as a proof of principle for the implementation of our ultra-fast high-resolution 3D ribbon-scanning confocal microscopy. Our proposal will foster future development of the first 3D high-resolution comprehensive maps of neurotransmission within in whole brain to study addiction.