Fulton T. Crews - US grants

Chronic ethanol consumption and senescence are both known to cause memory deficits and to cause similar morphdogical changes in the hippocampus, a brain region associated with memory. In Addition, both ethanol and senescence alter cholinergic neurotransmission, a process implicated in the cognitive processes which decline during normal aging and in chronic alcoholics. Recently, we have shown that chronic ethanol administration increases the cholesterol to phospholipid molar ratio in synaptosomal membranes. In a separate series of experiments we found that the cholesterol to phospholipid ratio of synaptosomal membranes increases as rats approach senescence. This change in membrane composition alters membrane properties and neurotransmission. Acetylcholine is known to be a potent stimulant of hippocampal pyramidal cell firing and to increase phospholipid metabolism. We have recently developed a technique to reversibly increase the relative cholesterol content of hippocampal brain slices. An increased in the relative cholesterol content markedly reduces the response to acetylcholine. Furthermore, iontophoretic as well as hippocampal slice studies have shown that the hippocampal response to acetylcholine declines in senesent rats. Thus, we hypothesize that changes in membrane composition which occur during chronic ethanol consumption and aging can reduce the response of hippocampal pyramidal cells to acetylcholine and thereby may cause memory dysfunction. Using hippocampal slices prepared from young and old Fisher 344 rats we propose to determine the relationship between the alterations in membranes which occur during chronic ethanol consumption and the electrophysiological and phospholipid metabolism responses to acetylcholine. The changes which occur during chronic ethanol treatment of both young and old rats will be studied as well as the effects of in-vitro modification of membrane lipids on the responses to acetylcholine and ethanol). In addition, the effects of in-vitro ethanol on membrane properties will be compared to the effects of in-vitro ethanol on the responses to acethylcholine in hippocampal slices. These studies will provide insight into the pathological mechanisms of chronic ethanol consumption; the interaction between ethanol and aging, as well as the susceptability of old animals to the pathological actions of ethanol.

Cholinergic and adrenergic neurotransmission in brain is mediated in part through muscarinic and alpha-1-adrenergic receptor stimulated phosphoinositide hydrolysis. A variety of evidence suggests that cholinergic and adrenergic neurotransmission is disrupted during aging. The loss of cognitive function which occurs during normal aging, senile dementia and Alzheimer's disease is likely to be related to change in cholinergic neurotransmission during aging. The administration of muscarinic cholinergic antagonists produces memory deficits in young animals similar to those found to occur naturally in aged animals. An understanding of muscarinic cholinergic and alpha 1- adrenergic receptor stimulated phosphoinositide hydrolysis is essential for a complete understanding of the mechanisms of cholinergic and adrenergic transmission in brain. The long-term objective of this proposal is to determine the properties and mechanisms of adrenergic and cholinergic receptor coupling to phosphoinositide hydrolysis in brain and how these processes are altered during senescence.

The broad, long-term, objectives of this proposal are to sensitivity to excitotoxicity. Stimulation of excitatory amino acid receptors, particularly those of the N-methyl-D-aspartic acid (NMDA) and kainate subtype of glutamate receptor, can trigger delayed neuronal death that occurs over a period of hours to days. This process, commonly referred to as excitotoxicity, is thought to involve excessive and prolonged increases of intracellular calcium that ultimately lead to calcium-induced neuronal death. Recent studies have shown that ethanol has potent inhibitory actions on NMDA and kainate receptor mediated calcium influx. Preliminary data contained in this proposal shows that in primary neuronal cultures, acute in vitro ethanol can potently inhibit NMDA receptor mediated excitotoxicity. The first portion of this proposal (specific aims 1 and 2) consist of in vitro studies using primary rat neuronal cultures that will fully characterize the acute and chronic effects of ethanol on excitotoxicity. Acute studies will examine the effects of ethanol on calcium influx, the role of voltage-dependent calcium channels in excitotoxicity and their interaction with ethanol in this process, and the differential sensitivity of certain neurons to excitotoxicity and to excitoprotection by ethanol. The effects of chronic in vitro ethanol treatment on neuronal cultures, including the ability of chronic ethanol to sensitize neurons to excitotoxicity via upregulation of both excitatory amino acid-operated and voltage-operated calcium channels, will also be examined. The in vitro studies will provide a foundation for the in vivo studies (specific aims 3 and 4) contained in the second portion of this proposal. A rat model of ishemic brain damage is proposed that will allow investigations of the interactions of both acute and chronic ethanol consumption with ischemia induced delayed neuronal death. Excitatory amino acid receptors, ion channels and neuronal cell death will be quantitated by histopathological and autoradiographic analysis of brain sections. The interaction of ethanol with ischemic brain damage could yield important and clinically significant information on the neurotoxic effects of ethanol and the neuropathology of chronic alcohol abuse.

The Bowles Center for Alcohol Studies (BCAS) training program at the University of North Carolina at Chapel Hill is designed to promote the development of promising postdoctoral research fellows as independent investigators and future University faculty members who will investigate the pathogenesis of alcoholism and alcohol abuse using modern molecular, genetic, biochemical and imaging techniques. Training of the postdoctoral fellows will be individualized with the most important component being the research conducted by the trainee in the faculty mentor's laboratory. In addition to hands-on alcohol research, training will include seminars and conferences, activities on responsible conduct of research, professional development, didactic courses, and other training as needed to prepare fellows for independent research. The training faculty will consist of 14 funded investigators from multiple departments at the University of North Carolina at Chapel Hill. The faculty has a documented history of close interaction and collaboration. The trainees will benefit from the unique strengths of alcohol research at the University of North Carolina BCAS, which include the NIAAA-funded Alcohol Research Center with its research cores, the UNC Neuroscience Center, and the North Carolina Translational and Clinical Sciences Institute (NIH-funded Clinical and Translational Science Award). The training program will be led by Co-Directors, Drs. Fulton Crews, Donita Robinson and Thomas Kash, with the assistance of two senior alcohol researchers, Drs. Clyde Hodge and Leslie Morrow, who will constitute the Training Program Advisory Committee. The External Advisory Committee provides another level of oversight. The program proposes seven post-doctoral fellow slots. Trainees will receive two years of research training with the possibility of a third year and with external support sought for later years. This institutional training grant has a strong track record and will promote intensive training in molecular, biochemical and imaging techniques and basic pathophysiology in a stimulating environment, leading to broadly trained independent investigators capable of adapting to the rapid advances in research in the 21st century.

Gene delivery can be used to test hypothesis on brain mechanisms of alcoholism as well as providing a potential new therapeutic approach to drug abuse. Both human and animal data have implicated decreased dopamine (DA)and/or serotonin (5-hydroxytryptamine-5HT) in the nucleus accumbens (NAc) and other forebrain areas with high alcohol drinking behaviors. The hypothesis to be tested in this proposal is that viral vectors enhancing levels of the biogenic amines, DA and/or 5HT in specific brain regions will alter responses to ethanol, particularly high alcohol drinking behavior. Levels of DA and 5HT will be selectively increased in specific brain regions by constructing vectors containing synthetic enzymes using the adeno-associated virus (AAV) vector system. This approach prepares a therapeutic agent-drug that contains less than 5% of the original viral genome and the enzymes tyrosine hydroxylase (TH), aromatic amino acid decarboxylase( AADc) and tryptophan decarboxylase.( TRYPH). Preliminary studies have shown that the AAV vector is not neurotoxic and can express TH-AADC in brain to levels that raise the concentration of brain DA sufficient to partially restore motor abnormalities in models of Parkinson's disease. Expression is known to occur for at least several months in many brain regions. These preliminary studies indicate the AAV- Th-AADC vector can be used to increase DA levels in brain. The aims of this proposal are: 1. To investigate the expression of AAV-tyrosine hydroxylase-aromatic amino acid decarboxylase (AAV-TH-AADC) vectors in various brain regions including the ventral tegmental area (VTA) , NAc, and forebrain. Variables will include vector load and brain region(s) of expression. Preliminary studies have indicated that a AAV-lacZ vector is expressed in NAc. The AAV-lacZ vector will be used to investigate specific cell sites of expression and to serve as a "vehicle" control for AAV vectors containing the TH-AADC.Th-AADC expression will be determined using a FLAG epitope included in the vector for immunohistochemical distinction from endogenous enzyme, total TH immunohistochemistry, total TH activity, the content of DA and DA metabolites as well as PCR of viral DNA. 2. To determine the effects of AAV-Th-AADC vector induced increased dopamine in nucleus accumbens and/or VTA on ethanol behaviors, particularly high ethanol drinking behaviors in P and NP rats. 3. Develop an AAV-Tryptophan hydroxylase-aromatic amino acid decarboxylase vector (AAV-TRYPH-AADC) to enhance 5HT levels in NAc, VTA, Raphe, forebrain and other areas of interest. Variables will include vector load and brain region(s) of expression. 4. Determine the effects of increased 5HT synthesis in NAc, forebrain and/or Raphe on behaviors as in Specific Aim 2.

DESCRIPTION (provided by applicant): Overview: Molecular and Cellular Pathogenesis in Alcoholism. The UNC NIAAA Alcohol Research Center (ARC) fosters interdisciplinary collaborative research on alcoholism, alcohol abuse and the impact of alcohol on health and disease - exactly the goal of an NIAAA ARC. The UNC Bowles Center for Alcohol Studies (CAS) provides a foundation of administrative support and dedicated space for alcohol research. Across ARC components molecular signaling, synaptic networks, neurocircuitry and psychopathology are investigated in models of binge drinking. The scope of these studies requires the Center mechanism to integrate the multiple signaling systems and neurocircuits that underlie complex addiction-like behaviors. The UNC ARC is a catalyst of discovery that promotes collaboration, expands use of new methods and scientific knowledge through regular research meetings, scientific seminars, core research services, annual clinical conferences, pilot projects and through stimulation of interest in the effects of alcohol on health across the University and State. Training and mentoring of students and junior faculty contribute to scholarly interactions and successful laboratory research programs through the ARC. Health professional and youth curricula promote interest in science and knowledge that contribute to improved health. The ARC synergizes with existing investigator funding to promote interactions among multidisciplinary investigators focused on molecular mechanisms of ethanol-related behavioral and tissue pathology. The ARC Specific Aims are to investigate mechanisms of alcohol-induced behavioral, molecular, cellular and neural circuit pathogenesis and to disseminate information on alcohol to health professionals and youth. Research components investigate hypotheses on ethanol-induced regulation of signaling kinases, receptor expression and trafficking, cytokine and peptide gene induction, changes in brain networks and circuitry as well as alcohol induced mood, behavior and drug taking as addiction-like behavioral pathologies. By conducting focused investigations that integrate molecular signaling mechanisms across neural networks and neurocircuitry, the ARC creates synergies that promote and catalyze discoveries. This ARC proposal continues a research focus on molecular and cellular mechanisms with a new emphasis on dysfunctional brain networks and neurocircuitry, a theme at the cutting edge of neuroscience. The ARC will conduct, promote, support, and mentor research on alcoholic pathology and educate broad groups of health professionals and youth in North Carolina.

This FIRCA proposal will investigate changes in acetylcholine (ACh) and dopamine (DA) release from the nucleus accumbens shell (NA) following chronic ethanol treatment. Chronic ethanol is known to cause damage to cholinergic neurons (Arendt, 1993) and to sensitize DA neurons to glutamate excitotoxicity (Crews et a1. 1998). Recent studies have indicated that endogenous opiates are involved in alcohol addiction, leading to the approval of naltrexone, an opiate antagonist, for treatment of alcohol dependence. Studies of morphine and ethanol dependence have indicated changes in the interaction of DA and ACh during dependence and physical withdrawal and sustained changes during prolonged abstinence. Fiserova et.al.,1998 found that acute morphine decreases ACh release whereas a morphine challenge weeks after chronic morphine during prolonged abstinence results in an increase in ACh response to morphine. Endogenous opiates in the NA regulate DA release which regulates release from ACh interneurons in the NA. The reversal of the opiate response is consistent with studies of the effects of chronic ethanol on ACh release in hippocampus. These studies will be extended to NA with additional investigations of the interaction of DA and opiates on DA and ACh release. The Specific aims are: 1. It is hypothesized that physical withdrawal from chronic ethanol treatment will alter ACh release in the NA. A chronic binge ethanol treatment protocol well known to cause substantial physical dependence will be used. Levels of microdialysate ACh will be determined in diet controls or ethanol-treated groups just after the last dose and throughout the physical withdrawal syndrome (0-96hrs) as well as during prolonged abstinence, e.g., 15 and 35 days after the last dose of ethanol. It is expected that the hyperexcitability of withdrawal will damage ACh neurons, reducing basal ACh release levels during prolonged abstinence and altering neuronal architecture and/or receptor density. 2. Aim 2 will test the hypothesis that DA microdialysate levels decrease during withdrawal from chronic ethanol and remain decreased throughout prolonged abstinence due to changes in DA neuronal architecture and/or receptor density. 3. Aim 3 will test the hypothesis that chronic ethanol treatment followed by prolonged abstinence results in changes in the interaction of DA and ACh. In these studies challenges with various receptor specific drugs will be used to investigate mechanisms of changes in neurotransmission. Post mortem studies will determine if changes in the density or distribution of receptors occur that could underlie changes in neurotransmission.

This is a request for partial funding for an international symposium sponsored by the UNC Bowles Center for Alcohol Studies entitled "Genes and Gene Delivery for Diseases of Alcoholism Symposium" to be held April 18-21, 2001 at the Carolina Inn, adjacent to the UNC-CH campus. Recent technological advances in gene delivery and DNA microarrays have enhanced our abilities to understand pathological processes at the molecular level and prompt a meeting to review recent advances in our understanding of the role of genes and gene expression in alcohol related pathologies. The goal of this meeting is to bring together scientists that have made significant contributions in these areas and provide a forum for discussing the state of the art studies on genes; gene delivery and pathological processes to application in a variety of alcohol related pathologies. This proposal requests travel support for speakers and travel awards for meeting participants as partial support of the meeting costs. Alcohol related morbidity is among the most common worldwide health problems. As many as 10-15% of Americans will suffer from alcohol dependence within their lifetime and is related to changes in brain function secondary to gene expression and genetic risk factors. Alcoholic cirrhosis is the fourth leading cause of death among urban males. Fetal Alcohol syndrome is the largest known cause of mental retardation and more individuals suffer from the less pronounced Fetal Alcohol Effects syndrome. Thus, directly or indirectly, alcoholism causes a spectrum of pathologies all of which involve changes in gene expression. Gene cascades involved in pathological processes often are similar among different cell types. Studies detailing gene identification and gene delivery techniques will have broad application to other pathologies as well as studies in other tissues. The topics that will be covered in this meeting include 1) CNS Genes and Pathology 2) Genes, Cells and Hepatotoxicity and 3) Stem Cells, Development and Teratology. The format will include invited presentations from prominent investigators and poster sessions. Researchers with interest in the field of neurobiology of alcohol addiction, molecular neuropharmacology, gene therapy, hepatic and fetal pathologies and toxicology will attend this meeting as participants.

DESCRIPTION (provided by applicant): Alcoholics suffer from neuroticdeneration and loss of CNS function, which could occur due to hinge drinking. Studies in vivo using a 4-day animal binge model found neurodegeneration in corticolimbic areas, i.e. Binge-Induced Brain Damage (BIBD). BIBD is characterized by significant neuronal damage, long-lasting expression of markers of inflammation, and changes in behavior. Although in vitro studies have found glutamate-NMDA excitotoxicity following chronic ethanol treatment, BIBD is not blocked by glutamate-NMDA antagonists, hut BIBD is blocked by antioxidants. This proposal will investigate the role of oxidative stress in BIBD and how it alters signaling pathways involved in neuronal survival. We will also determine the contribution of multiple ethanol exposures and ethanol-induced plasma toxins to alcoholic neurodegeneration as both of these pathological states can lead to oxidative stress. Ethanol-induced plasma toxins may contribute to oxidative stress and multiple ethanol exposures may lead to cumulative stress prompting experiments to determine the contributions of these factors to alcoholic neurodegeneration. The following hypotheses will be tested. Aim 1. BIBD is related to oxidative stress. This hypothesis will be tested by measuring nitro-tyrosine, 4-hydroxynenenol and 8-hydroxyguanosine, indicators of protein, lipid and nucleic acid oxidation respectively. In addition, genes related to oxidative stress, e.g., NADPH oxidase, cytochrome P(450)2E1,TNFalpha and NFkappaB, will be studied as potential components of BIBD. Oxidative stress and TNFalpha can activate NFkappaB, which is known to induce COX-2, iNOS and MnSOD expression. We will also investigate prosurvival signaling molecules such as MAPK/ERK and CREB, which provide resistance to neurotoxic events such as oxidative stress. Preliminary studies comparing the P and NP rat strains indicate that ethanol increases MAPK/ERK activation, atrophic signaling pathway that causes prosurvival gene induction, likely as a protective response to ethanol-induced oxidative stress. MAPK/ERK activation is inversely related to BIBD, that is, P rats have greater BIBD and reduced MAPK/ERK activation compared to NP rats. Thus, a second component of this aim will be to investigate aspects of P-MAPK/ERK and P-CREB. The antioxidant butylated hydroxytoluene (BHT) pre-vents BIBD and these studies will be extended using other antioxidants. The effect of antioxidants on induction of oxidative stress markers, enzymes, and the signaling cascades mentioned above will also be determined using immunohistochemistry, EMSA, western and blot analyses. Experiments using transgenic mice strains including NFkappaB reporter gene mice, TNFalpha knockouts, CYP2E1 knockouts and PKC knockouts will test the role of these specific genes in B1BD. Aim 2. Peripheral tissue damage is involved in BIBD. Our 4-day binge elevates plasma ammonia and enzyme levels. Further, intragastric models of liver fibrosis induce CNS inflammation. Thus, models that are known to induce hepatotoxicity will be investigated for brain gene activation and neuropathology as described above. Aim 3. Repeated binge exposure will increase BIBD. Clinical studies have shown that multiple withdrawals lead to a higher incidence of seizures and correlates with memory and learning impairments. Thus, the effects of multiple episodes of alcohol exposure on BIBD, gene induction and oxidative stress will be determined. These in vivo studies will greatly increase our understanding of the factors that cause neurodegeneration in alcoholics.

DESCRIPTION (provided by applicant): The core of this alcohol research center: Molecular and Cellular Pathogenesis in Alcoholism is organized to promote research and education regarding the effects of ethanol at the molecular and cellular level, including effects on gene regulation, transcription factor activation, oxidative stress, and the use of transgenic animals and gene delivery to investigate and/or modify the effects of ethanol. The specific aims of the Core are to provide scientific and administrative leadership for the Alcohol Research Center, monitor and enhance interaction among the research cores and research components, optimize synergy and sharing of resources and services among the research cores, organize seminars, conferences, and workshops to stimulate exchange of scientific information among the Center faculty and staff. This core will also be responsible for administration of the pilot project program. Further, it will work to stimulate new alcohol research programs at UNC, disseminate new research knowledge to academic, student, health professional and lay communities particularly in its role as administrator of the educational component. The Administrative core in the current funded period has been involved in community outreach activities designed to improve awareness, prevention, and treatment of alcoholism and support regional and national research initiatives aimed at improving knowledge, prevention and treatment of alcoholism. These activities complement the aims of the Education Component of this comprehensive Alcohol Research Center. The Education Component will organize annual Research to Practice symposia that educate health professional students and practicing health professionals. It will translate research to practice through annual one-day conferences, targeting specific "cutting-edge" topics on addiction and the brain, on alcohol and birth defects and on alcohol and liver disease, current areas of expertise of Center researchers. Highlights of research to practice symposia will be disseminated through the preparation of interactive CD-ROM teaching modules for continuing medical education (CME) credit for the broader scientific, academic, clinical and treatment communities. The administrative core will be responsible for the activities of the educational component, therefore these components are combined. The Administrative Core of this Comprehensive Alcohol Research Center will continue to provide scientific and administrative leadership and expand the outreach activities in close coordination with the Education Component.

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The administrative core of this alcohol research center: MOLECULAR AND CELLULAR PATHOGENESIS IN ALCOHOLISM is organized to promote research and education regarding the pathological effects of repeated ethanol exposure at the molecular, cellular and circuit levels. In this renewal, we build upon the strengths of our Center by extending our understanding of ethanol pathology mediated by cytokine, GABA, glutamate, corticotropin releasing factor and dopamine signaling and delineating the circuitry involved in these adaptations. The specific aims of the Administrative Core are to provide scientific inspiration and administrative leadership for the Alcohol Research Center, monitor and enhance interaction among the research components, optimize synergy and sharing of resources and services among the components, organize seminars, conferences, and workshops to stimulate exchange of scientific information among the Center faculty and staff. This core is also responsible for administration of the pilot project program. Further, it will work to stimulate new alcohol research programs at UNC, disseminate new research knowledge to academic, student, health professional and lay communities. The Administrative core in the current funded period has been involved in community outreach activities designed to improve awareness, prevention, and treatment of alcoholism and support regional and national research initiatives aimed at improving knowledge, prevention and treatment of alcoholism. These activities complement the aims of the Research Translation Information Dissemination Component of this comprehensive Alcohol Research Center. In addition, the Administrative Core oversees all budgetary issues, appropriate allocation of funds as well as preparation of progress reports and renewal of the ARC.

2,6-Bis(1,1-dimethylethyl)-4-methylphenol; 2,6-Di-t-butyl-4-methylphenol; 2,6-Di-tert-butyl-p-cresol; 4-Methyl-2,6-ditertbutylphenol; Absolute ethanol; Acquired brain injury; Acute; Adolescent; Adolescent Youth; Affect; Agonist; Alcohol, Ethyl; Alcohol-Induced Neurotoxicity; Alcoholic; Alcohols; Ammon Horn; Antibodies; Antioxidants; BHT; Behavior; Behavioral; Binding; Binding (Molecular Function); Blood Serum; Boozer; Brain; Brain Injuries; Brain region; Butylated Hydroxytoluene; Butylhydroxytoluene; Cell Communication and Signaling; Cell Count; Cell Number; Cell Signaling; Cell/Tissue, Immunohistochemistry; Cells; Chemical Class, Alcohol; Chronic; Cornu Ammonis; Cytokine Gene; DNA Binding; DNA Binding Interaction; Dependent drinker; Dose; Dysfunction; EC 2.7; ELISA; EMSA; ETOH; Elements; Encephalitis; Encephalon; Encephalons; Endotoxins; Entorhinal Area; Entorhinal Cortex; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Enzymes; Ethanol; Fiber; Functional disorder; Gene Expression; Gene Transcription; Genes; Genetic; Genetic Transcription; Glia; Glial Cells; Grain Alcohol; Grant; Health; Hippocampus; Hippocampus (Brain); Histocytochemistry; IHC; INFLM; Immunohistochemistry; Immunohistochemistry Staining Method; In Vitro; Inflammation; Inflammation, Brain; Inflammatory; Inflammatory Response; Intracellular Communication and Signaling; Kinases; Kolliker's reticulum; Kupffer Cells; Lead; Link; Liver; Localized; Mammals, Mice; Mediator; Mediator of Activation; Mediator of activation protein; Messenger RNA; Methylcarbinol; Mice; Molecular; Molecular Interaction; Murine; Mus; Myelin; Nerve Cells; Nerve Degeneration; Nerve Impulse Transmission; Nerve Transmission; Nerve Unit; Nervous System, Brain; Neural Cell; Neural Growth; Neurocyte; Neuroglia; Neuroglial Cells; Neuron Degeneration; Neuronal Growth; Neuronal Transmission; Neurons; Non-neuronal cell; Oxidative Stress; Pathogenesis; Pathology; Pb element; Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-; Phosphotransferases; Physiopathology; Predisposition; Proteins; RNA Expression; RNA, Messenger; RNA, Small Interfering; RT-PCR; RTPCR; Reporter; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Serum; Signal Transduction; Signal Transduction Systems; Signaling; Silver Staining; Slice; Small Interfering RNA; Stellate Sinusoidal Macrophage; Structure of entorhinal cortex; Susceptibility; Testing; Time; Toxic effect; Toxicities; Transcription; Transcription Activation; Transcription, Genetic; Transcriptional Activation; Transgenic Animals; Transphosphorylases; Up-Regulation; alcohol effect; alcohol exposed; alcohol exposure; anti-oxidant; biological signal transduction; body system, hepatic; brain damage; brain lesion (from injury); cell type; craving; cytokine; density; entorhinal cortex; ethanol effect; ethanol exposed; ethanol exposure; ethanol-induced neurotoxicity; experiment; experimental research; experimental study; exposed to alcohol; exposure to alcohol; gene induction; gene product; heavy metal Pb; heavy metal lead; hippocampal; histochemistry; histochemistry/cytochemistry; in vivo; in vivo Model; insight; juvenile; juvenile human; liver macrophage; mRNA; molecular pathology; nerve cement; neural degeneration; neurodegeneration; neurogenesis; neuroinflammation; neuron toxicity; neuronal; neuronal degeneration; neuronal toxicity; neuropathology; neurotoxic; neurotoxicity; neurotransmission; organ system, hepatic; oxidation; pathophysiology; problem drinker; regional difference; research study; response; reverse transcriptase PCR; siRNA; silver impregnation; social role; time use; transcription factor

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DESCRIPTION (provided by applicant): The primary objective of this NADIA core is to integrate and expand the Consortium research projects. The hypothesis of the Scientific Core is that adolescent intermittent ethanol (AIE) impacts brain development and induces specific neuro-adaptations that persist into adulthood as a specific neurochemical and morphological phenotype. Thus, the Scientific Core will serve and link NADIA components by providing neurochemical and morphological brain atlases that will define the phenotype of the brain exposed to alcohol during adolescence. Each component will send brains to the core for immunohistochemical determination of pivotal neurotransmitter gene expression in brain regions related to the physiology and behaviors being studied. Additionally, Magnetic Resonance Imaging (MRI) and Diffusion Tensor Imaging (DTI) data will be collected on select data sets in order to provide global structural information on the impact of different AIE models on adult brain. Finally, all the resulting data will be shared and unused brain tissue will be archived to allow for future analysis. Together, these approaches will provide critical information on neurobiological mechanisms that underlie AlE-induced changes in adult behavior and physiology. The Scientific Core will use an animal model of adolescence alcohol drinking to investigate how alcohol changes brain structure and neurochemistry. This endeavor, along with the resulting data repository, serves to integrate the research components of the NADIA Consortium and will provide a foundation for understanding differences in adolescent life trajectories and maturation of important behavioral repertoires. PUBLIC HEALTH RELEVANCE: The Scientific Core will use an animal model of adolescence alcohol drinking to investigate how alcohol changes brain structure and neurochemistry. This endeavor, along with the resulting data repository, serves to integrate the research components of the NADIA Consortium and will provide a foundation for understanding differences in adolescent life trajectories and maturation of important behavioral repertoires.

DESCRIPTION (provided by applicant): The purpose of this application is to develop a Consortium for the initiative Neurobiology of Adolescent Drinking in Adulthood (NADIA). The NADIA will coordinate a diverse group of basic neuroscientists in a multidisciplinary research project to clearly define the persistent effects of adolescent alcohol exposure on adults, and to begin to explore the neurobiological mechanisms. The overarching hypothesis of this consortium is that models of human underage drinking will impact brain maturation resulting in persistent changes in adult brain function and structure that relate to changes in behavior. This NADIA will use adolescent intermittent ethanol (AIE) rat models that mimic episodic human adolescent underage drinking. Multiple research components will integrate molecular, cellular, physiological, endocrine, genetic neuroanatomical and behavioral studies utilizing cutting edge and novel approaches to investigate potential long term consequences of human underage drinking. An abundance of evidence suggests that during adolescence, cognition, affect, and reward driven behavioral repertoires are uniquely plastic and responsive to environmental influences. Maturation of brain circuitry that underlies motivation, affect and decision making are expected to be sensitive to ethanol disruption, resulting in increased adult psychopathology. This consortium will integrate investigators that share common hypotheses and overlapping protocols. Each will contribute to an improved understanding of the consequences of adolescent alcohol exposure on brain physiology, structure, chemistry, maturation and behavioral induces of affect, motivation, social functioning, decision-making cognitive assessments, impulsivity, circadian rhythms, and alcohol drinking behaviors using AIE. Components cover broad inter-related investigations of the neurocircuitry between frontal-cortical, striatal, hippocampal, extended amygdala and hypothalamic nuclei as well as hormonal maturation of hypothalamic-adrenal interactions across gender, providing a broad global investigation of the development of neural networks that underlie maturation of complex behaviors. The scientific core will provide components with brain MRI-DTI (brain volume - structure), brain regional histology-immunohistochemistry and establish a data repository for future brain network analysis. Understanding the impact of underage drinking on adult neurobiology is important to guide public health initiatives. PUBLIC HEALTH RELEVANCE: Drinking in adolescence is common but the consequences of excessive drinking during adolescence are unknown. This proposal will establish a consortium of investigators to determine the consequences of adolescent alcohol exposure on brain physiology, structure, chemistry, maturation and behavioral indices of affect, motivation and/or cognition. This information will be important to guide public health policy and develop better tools for the prevention of risky behavior in adolescents.

DESCRIPTION (provided by applicant): This is an application for a cooperative agreement (U54), "Mechanisms of Alcoholic Pathology" (MAP), between faculties of the Biomedical/Biotechnology Research Institute (BBRI) at North Carolina Central University (NCCU), a Minority-Serving Institution, and the Bowles Center for Alcohol Studies at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program. Within this proposal, an NCCU Administrative component and three Research Components will integrate with the UNC-ARC Administrative, Education, 5 Research Components, and Cores, as well as other activities at the Bowles Center for Alcohol Studies. The UNC-ARC investigates mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that easily integrate into the ongoing overall theme of the UNC-ARC. Thus, both the UNC-ARC research components and this U54 proposal are integrated around the central theme that alcohol-induced pathology involves molecular and cellular changes that occur with alcohol abuse and alcoholism. The objectives of this U54 partnership are: 1) To investigate molecular mechanisms of alcohol-induced cellular pathology. By conducting an integrated and focused investigation into the molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create an active and successful research program on alcohol pathology at NCCU;2) To provide scholarly education on Alcohol Pathology. The educational efforts in this proposal will educate NCCU students on alcohol pharmacology and alcohol related pathologies and health disparities through a combined Annual Alcohol Research Day, new alcohol course curricula developed by NCCU faculty, an alcohol seminar series hosted by the BBRI, the UNC Bowles Center for Alcohol Studies seminar series, and NCCU fellowships to students on alcohol pathology and minorities. Ultimately, this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an acfive and successful alcohol research program within the NCCU-BBRI that synergizes with the UNC-ARC to advance education and discoveries.

DESCRIPTION (provided by applicant): Maturation of reward, affect and behavioral control coincides with morphological changes in frontal and limbic brain regions during adolescence and the transition to adulthood. Previous studies from our lab have shown that adolescent rats are far more sensitive to ethanol-induced forebrain neurodegeneration and to inhibition of hippocampal neurogenesis. We have also observed in adult mice that adolescent ethanol exposure reduced the volume of the forebrain and several other regions as determined by MRI. In addition to a reduction in MRI volume, histological measurements indicated corresponding reduction of forebrain area and decreased cholinergic neuron density. These pathological changes were associated with functional deficits in reversal learning. This Research Component-7 of the NADIA consortium will extend these studies to determine the long-term consequences of adolescent alcohol exposure. This NADIA Research Component- 7 hypothesizes that adolescent intermittent ethanol (AIE) treatment will alter adult brain regional cellular structure, neurogenesis, microglia, astrocyte phenotypes and myelin, as well as brain regional MRI volumes and histologic areas. Additional hypotheses are that higher alcohol doses, younger individuals and longer periods of abuse will cause more pathology and will be reflected in diffuse neurocircuitry responses to stress and ethanol challenges. It is further hypothesized that AIE will alter stress and alcohol induction of neuronal activation markers in adults. The following 4 Aims test hypotheses: Aim 1 will test the hypothesis that AIE alters adult brain neurogenesis and neuroprogenitor-stem cells (NPC). Aim 2 will test the hypothesis that AIE alters adult brain white matter. Aim 3 will determine if AIE alters adult brain microglia and astrocytes. Aim 4 will determine if AIE alters adult brain neuronal responses to stress or ethanol. The proposed studies will be conducted using different AIE protocols that vary age of ethanol exposure, e.g. youth-adolescent-young adult (P25-55), and dose;i.e. binge, heavy and moderate self-administration. Adults following AIE, are assessed for cognition (Barnes maze, learning and reversal learning), affect-anxiety behaviors (elevated plus maze), open field test (center time anxiety-overall locomotor) and social interaction (anxiety). Histochemistry on adult brain cellular structure and composition will be related to MRI regional volumes, DTI structure and behavior. In addition, studies will challenge adult rats with ethanol or stress to investigate neurocircuitry through IHC for neuronal activation markers (e.g. fos, zif268, pERK1/2, pDARPP). It is expected that AIE will have brain region specific effects with younger ages of AIE treatment inducing larger changes in adult brain cell structure. These studies will determine if models of underage drinking result in persistent structural, cellular, and neuronal activation responses that impact adult brain networks and behavior.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Magnetic resonance histology has become an important tool for quantitative volume changes in neurologic studies. We are particularly interested in changes in specific regions of the brain of animals (Wistar rats) exposed to alcohol. We are currently performing longitudinal studies at the BRIC at UNC to follow such changes. Part of our protocol involves end point ex vivo (MR Histology) to determine the relative accuracy and reproducibility (in vivo vs. ex vivo). Reviewers have raised questions over the validity of inferring volumes measured in ex vivo to the in vivo case. Our expectation is that the ex vivo studies will be far more accurate measures of volumes. MR Histology has not been as fully developed at the BRIC as it has at the CIVM. We would seek the CIVM's help in translating the perfusion and scanning protocols. At the same time we would like to understand the relevant scanning methods. Animals supplied by: Perfusion-fixed rat heads will be prepared at UNC from animals that have been scanned (both in vivo and ex vivo) at the BRIC. CIVM will provide high-resolution MR Histology to match the studies performed at UNC.

DESCRIPTION (provided by applicant): This is an application for a cooperative agreement (U54), Mechanisms of Alcoholic Pathology (MAP), between faculties of the Biomedical/Biotechnology Research Institute (BBRI) at North Carolina Central University (NCCU), a Minority-Serving Institution, and the Bowles Center for Alcohol Studies at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program. Within this proposal, an NCCU Administrative component and three Research Components will integrate with the UNC-ARC Administrative, Education, 5 Research Components, and Cores, as well as other activities at the Bowles Center for Alcohol Studies. The UNC-ARC investigates mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that easily integrate into the ongoing overall theme of the UNC-ARC. Thus, both the UNC-ARC research components and this U54 proposal are integrated around the central theme that alcohol-induced pathology involves molecular and cellular changes that occur with alcohol abuse and alcoholism. The objectives of this U54 partnership are: 1) To investigate molecular mechanisms of alcohol-induced cellular pathology. By conducting an integrated and focused investigation into the molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create an active and successful research program on alcohol pathology at NCCU; 2) To provide scholarly education on Alcohol Pathology. The educational efforts in this proposal will educate NCCU students on alcohol pharmacology and alcohol related pathologies and health disparities through a combined Annual Alcohol Research Day, new alcohol course curricula developed by NCCU faculty, an alcohol seminar series hosted by the BBRI, the UNC Bowles Center for Alcohol Studies seminar series, and NCCU fellowships to students on alcohol pathology and minorities. Ultimately, this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an acfive and successful alcohol research program within the NCCU-BBRI that synergizes with the UNC-ARC to advance education and discoveries.

This ARC proposal tests the hypothesis that repeated ethanol binges (REB) persistently change neuroimmune signaling that alters neuronal activation in frontal cortical (FC), amygdala (Amyg), ventral striatum (VS, nucleus accumbens) and hippocampus (Hip) that contributes to the psychopathology of alcohol dependence. Progress in the previous funding cycle discovered that neuroimmune signals are activated by REB altering behavior consistent with addiction. Breese progress linked ethanol cycles, stressors and/or brain injection of neuroimmune agonists into Amyg with decreased social interaction, an index of negative affect. In parallel. Crews discovered REB induces neuroimmune genes through glial NFKB transcription of chemokines, cytokines, toll-like receptors (TLR) and the TLR agonist HMGB1 that persist for long periods of abstinence and contribute to neurodegeneratlon and reversal learning cognitive deficits. Increased neuroimmune protein expression was also discovered in post-mortem human alcoholic brain. These labs partner within this renewal component. REB induced changes in neuronal activation using cfos and Zif268 markers (Aim 1) will be related to increases in neuroimmune signals (Aim 2) within FC, Amyg, VS and Hip during abstinenece following REB. These brain regions are networked and associated with the persistent alcohol induced arousal that occurs in alcohol dependence. Optogenetics will investigate changes in FC circuits. Naltrexone, minocycline and knock out mice will test causal relationships between neuroimmune induction and altered neuronal activation (Aim 3) as well as addiction related behaviors (Aim 4). These experiments will enhance understanding of the molecular and cellular mechanisms of alcohol induced brain pathology. Ethanol induced neuroimmune activation could become central to the neurobiology of addiction and translate to new treatments. The ARC broadens and strengthens the proposed experiments with additional related studies on binge induced alterations in neurocircuits, addiction-like behaviors and signaling mechanisms.

The Pilot Project program is designed to promote alcohol research across the UNC campus. We provide funds to new investigators without current or past research support and to established investigators who propose projects that represent a significant departure from their ongoing research. The goal of the program is to provide sufficient funds to generate pilot data on alcohol research projects that can be used to support applications for research grants. A detailed review process includes Internal solicitation for proposals, involvement and advice from appropriate Center faculty, as well as internal and external review to select studies of the highest quality. The pilot program also takes advantage of funds from the NC Clinical and Translational Research Consortium (NIH Clinical Translation, Science Award) that will match funding by the ARC to outstanding proposals. Previous pilot projects funded by our Center grant have led to new NIH funding for UNC researchers. This process brings new people and new ideas into the center, renewing Its vibrance and innovation and helping to keep us on the forefront of the field. This ARC includes two Pilot Projects (Project 16 and 17) as part of this proposal. Both projects integrate nicely with our theme of ALCOHOL PATHOGENESIS. Project 16 (Ramon Bataller, PI) will test whether p90RSK mediates liver injury and inflammation in alcoholic hepatitis (AH). His group will conduct a translational study with liver samples from patients, where p90RSK expression and activation will be determined and correlated with disease severity. Next, they will investigate whether p90RSK inhibition in mice with experimental AH attenuates liver Injury and inflammation. Project 17 (Donald Lysle, PI) will examine the role of interteukin-1|3 (IL-1 (3) In ethanol binge drinking. His group will test the hypotheses that repeated binge-like drinking increases IL-1 (3 expression in brain and that blocking 1L-1(3 using small interfering RNA (siRNA) or IL-13 receptor antagonist will protect against binge-like ethanol drinking. These Pilot Projects will provide the resources for new investigators to develop preliminary findings important for success In larger research grants. This effort supports the long term growth and success of our ARC.

DESCRIPTION (provided by applicant): Overview: Molecular and Cellular Pathogenesis in Alcoholism. The UNC NIAAA Alcohol Research Center (ARC) fosters interdisciplinary collaborative research on alcoholism, alcohol abuse and the impact of alcohol on health and disease - exactly the goal of an NIAAA ARC. The UNC Bowles Center for Alcohol Studies (CAS) provides a foundation of administrative support and dedicated space for alcohol research. Across ARC components molecular signaling, synaptic networks, neurocircuitry and psychopathology are investigated in models of binge drinking. The scope of these studies requires the Center mechanism to integrate the multiple signaling systems and neurocircuits that underlie complex addiction-like behaviors. The UNC ARC is a catalyst of discovery that promotes collaboration, expands use of new methods and scientific knowledge through regular research meetings, scientific seminars, core research services, annual clinical conferences, pilot projects and through stimulation of interest in the effects of alcohol on health across the University and State. Training and mentoring of students and junior faculty contribute to scholarly interactions and successful laboratory research programs through the ARC. Health professional and youth curricula promote interest in science and knowledge that contribute to improved health. The ARC synergizes with existing investigator funding to promote interactions among multidisciplinary investigators focused on molecular mechanisms of ethanol-related behavioral and tissue pathology. The ARC Specific Aims are to investigate mechanisms of alcohol-induced behavioral, molecular, cellular and neural circuit pathogenesis and to disseminate information on alcohol to health professionals and youth. Research components investigate hypotheses on ethanol-induced regulation of signaling kinases, receptor expression and trafficking, cytokine and peptide gene induction, changes in brain networks and circuitry as well as alcohol induced mood, behavior and drug taking as addiction-like behavioral pathologies. By conducting focused investigations that integrate molecular signaling mechanisms across neural networks and neurocircuitry, the ARC creates synergies that promote and catalyze discoveries. This ARC proposal continues a research focus on molecular and cellular mechanisms with a new emphasis on dysfunctional brain networks and neurocircuitry, a theme at the cutting edge of neuroscience. The ARC will conduct, promote, support, and mentor research on alcoholic pathology and educate broad groups of health professionals and youth in North Carolina.

DESCRIPTION (provided by applicant): The Bowles Center for Alcohol Studies (CAS) training program at the University of North Carolina at Chapel Hill is designed to promote the development of promising postdoctoral research fellows as independent investigators and future University faculty members who will investigate the pathogenesis of alcoholism and alcohol abuse using modern molecular medicine, biochemical and imaging techniques. Training of the postdoctoral fellows will be individualized with the most important component being the research conducted by the trainee in the faculty mentor's laboratory. In addition to hand's-on alcohol research, additional training will include didactic courses, seminars and conferences, activities on responsible conduct of research and other training as needed to prepare fellows for independent research. The training faculty will consist of 14 funded investigators from multiple departments at the University of North Carolina at Chapel Hill. The faculty has a documented history of close interaction and collaboration. The trainees will benefit from the unique strengths of alcohol research at the University of North Carolina, which include the CAS with its research cores, UNC-Neuroscience Center, a research-oriented Mental Health Research Center with its research cores in Psychiatry, a CTSA and the Program in Molecular Biology and Biotechnology. The training program will be directed by Dr. Fulton T. Crews with the assistance of three senior alcohol researchers, Drs. Clyde Hodge, Kathy Sulik and Leslie Morrow, who will constitute the Training Program Advisory Committee. The program proposes six post-doctoral fellow slots. Trainees will receive two years of research training with the possibility of a third year and with external support sought for later years. This institutional training grant will promote intensive training in molecular, biochemical and imaging techniques and basic pathophysiology in a stimulating environment leading to broadly trained independent investigators capable of adapting to the rapid advances in research in the 21st century.

Abstract: Excessive alcohol use damages many organs, but most commonly results in alcoholic liver disease (ALD). The molecular mechanisms by which ethanol (EtOH) exerts its damaging effects are extensively studied, but not fully understood, thus hampering the development of therapeutic options for the treatment of ALD. Furthermore, to date, there are no experimental animal models that recapitulate the full spectrum of ALD symptoms. The pregnane X receptor (PXR), expressed primarily in the liver and intestine, is a xenobiotic nuclear receptor that defends against toxic agents. PXR activation in humans and rodents leads to hepatic triglyceride (TG) accumulation, one feature of ALD, suggesting that the PXR may be involved in ALD pathogenesis. However, the role of mouse and human PXRs in EtOH hepatotoxicity has not been characterized. The earliest sign of liver injury observed in alcoholics is steatosis. While alcoholic steatosis progresses to alcoholic steatohepatitis during the development of ALD, the mechanisms that contribute to this deterioration are unclear. However, we have intriguing preliminary data suggesting the possibility that PXR is involved in the progression of alcoholic steatosis to more severe liver disease. Basal hepatic TG levels were significantly higher in male mice that lacked the PXR (PXR-KO) than in wild type (WT) or PXR-humanized (hPXR) transgenic mice; conversely WT and hPXR mice accumulated higher levels of TG when treated with EtOH and EtOH-treated hPXR mice were most prone to liver injury. The objectives of this R01 proposal are: 1) to explore the underlying molecular mechanisms by which PXR signaling regulates EtOH-induced TG accumulation and related lipid metabolic pathways and 2) to identify the molecular features of hPXR vs. WT mice that contribute to their increased susceptibility to EtOH-induced liver injury. Based upon our compelling preliminary data, our central hypothesis is that the nuclear receptor PXR is required for both ethanol-induced steatosis and progressive hepatotoxicity. We will carry out molecular and cellular studies using the three mouse models (WT, PXR-KO, and hPXR) and hepatocytes isolated from these animals to achieve the following aims. Specific Aim 1 will monitor i) the effect of PXR signaling on lipid synthesis, oxidation and export in hepatic steatosis and ii) corresponding changes in lipid composition in plasma and liver after ethanol ingestion. Specific Aim 2 will determine whether PXR regulates the main enzyme that metabolizes EtOH, ADH gene transcription and how it affects ethanol and acetaldehyde elimination rates. Specific Aim 3 will investigate the role of PXR signaling in hepatocyte proliferation following acute and chronic EtOH exposure. Our proposed study is novel in its use of humanized mice to examine the function of PXR in ALD, providing data that can be more directly translated into clinical research. This study is also significant in a) providing valuable insights into the role of mouse and human PXRs in ethanol hepatotoxicity via their regulation of alcohol metabolism and liver regeneration, and b) offering new therapeutic targets for the treatment of ALD.

This U54 Mentoring/Development Core: Mechanisms of alcohol pathology proposal will continue and expand mentoring and collaborative partnerships between the UNC-Bowles Center for Alcohol Studies (UNC-BCAS) and NCCU faculty and students that over the past 4 years has involved 8 NCCU Biomedical faculty, as many as 15 students each year, 12 manuscripts, and multiple alcohol NIH grant applications, none of which occurred prior to this partnership. This partnership has been extremely productive in initiating alcohol research at NCCU, a Historically Black College and University (HBCU) where 81% of students are underrepresented minorities (African-American or Hispanic). It continues to expose large numbers of underrepresented minority students to alcohol research to inform them regarding alcohol related pathology. The Mentoring and Research Development Core will support UNC faculty mentors providing research expertise to educate, involve and develop faculty and student researchers from NCCU through productive research collaboration and mentoring. The Mentoring/Development Core will provide funds for two years to maintain NCCU essential research staff in the 3 active laboratories with the goal of obtaining independent research support within this period. UNC mentors are supported for active NCCU programs as well as future new programs. By conducting an integrated and focused investigation into the genetic and molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create successful research programs exposing large numbers of NCCU students to alcohol research and alcohol pathology. Blacks are at high risk for fetal alcohol syndrome (FAS), alcoholic liver disease, and mortality from cancer of the oral cavity, pharynx, larynx, and esophagus. Undergraduate research internships and alcohol curriculum development will help identify and involve individuals in learning about and reducing alcohol-related health issues. Active NCCU alcohol research laboratories are training students from diverse backgrounds and are uniquely positioned to engage in research and in the translation of research advances into culturally appropriate education and health care.

Abstract: The objective of the proposed research is to identify genetic factors that modify sensitivity to prenatal alcohol exposure-induced birth defects, with the long-term goals of informing clinical practice, and ultimately reducing the incidence and severity of Fetal Alcohol Spectrum Disorders (FASD). This objective will be met by applying an innovative approach that includes utilization of high-throughput transcriptome sequencing (RNA-Seq), genetically modified mice, and the developing mammalian limb as a readily examined target tissue whose molecular signaling pathways and vulnerability to alcohol teratogenesis are well documented. This research proposal is founded on 1) strong evidence that at early developmental stages, alcohol-induced postaxial limb deficiencies occur as a result of a primary interference with developmental events that are upstream of antero-posterior (AP) limb patterning, 2) recognition that AP patterning is dependent upon signaling through the Sonic hedgehog (Shh) pathway, 3) illustration that genetically-based reduction in Shh signaling results in increased vulnerability to alcohol-induced birth defects, and 4) the hypothesis that haploinsufficiency of additional genes, both up-and downstream of Shh, will impart enhanced sensitivity to alcohol teratogenesis. The latter will be tested with experiments that employ a well-established FASD mouse model and will address the following Specific Aims: Aim 1 is to define gene expression differences between control and alcohol-exposed C57Bl/6J mouse embryos at the time of initial forelimb bud outgrowth and concurrent with peak sensitivity to alcohol teratogenesis; Aim 2 is to define gene expression differences between control and alcohol-exposed C57Bl/6J mouse forelimb buds at times concurrent with a later developmental epoch, the establishment of AP patterning; and the Aim 3 studies will identify the significance of specific genes that are modified following alcohol exposure by comparing pertinent genetically engineered mice relative to their sensitivity to alcohol?s teratogenicity. The proposed studies will be conducted by NCCU faculty and trainees under the mentorship of UNC?s Bowles Center for Alcohol Studies researchers. In addition to expanding our understanding of genetic susceptibilities to alcohol?s teratogenicity, this research project will foster state of the art research by budding minority scientists and will provide the foundation for important FASD studies to be addressed in future grant applications/research.

This Administrative Core (U54), ?Mechanisms of Alcohol Pathology? (MAP), is a partnership between the Biomedical/Biotechnology Research Institute (BBRI) and Biomanufacturing Research Institute and Technology Enterprise (BRITE) institutes at North Carolina Central University (NCCU), a Historically Black College and University, and The University of North Carolina School of Medicine Bowles Center for Alcohol Studies (UNC-BCAS). Collaborative partnerships between these groups initated alcohol research at NCCU during the previous funded period with 3 active laboratories that have produced about a dozen publications, have involved 15-20 NCCU students per year in alcohol research with faculty submitting several NIH grants. This proposal continues to expand alcohol research to 8 active faculty alcohol research laboratories. This proposal includes an Administrative core, Mentoring and Development Core, and five Research Components, two full projects and 3 pilot projects, all with preliminary data and structured collaborative partnerships between NCCU and UNC-BCAS faculty. This U54 Mechanisms of Alcohol Pathology has research projects focused on fetal alcohol spectrum disorders, alcoholic liver disease, alcohol induced oral and breast cancers and alcohol-cannabinoid neurotoxicity. Health disparities in African-Americans is related to increased alcohol pathology, and not increased alcohol use disorder. These projects address alcohol health disparities by involving NCCU students in alcohol research that educates them about mechanisms of alcohol pathology associated with health disparities as well as building on the expertise of NCCU faculty. UNC collaborators chosen for appropriate expertise in the scientific area and include senior mentors who provide leadership, experimental design and scientific expertise as well as junior mentors who can help with hands on methods mentoring. Thus, UNC and NCCU this are integrated around the central theme of alcohol-induced pathology promoting molecular and cellular research involving NCCU faculty and students. The objectives of this U54 Administrative Core are: 1) Provide scientific and administrative leadership by advocating with leadership for staff support of research as well as supporting specific staff for alcohol research. By assuring UNC mentors collaborate and meet regularly with NCCU faculty and by organizing seminars, and annual steering committee meeting to review progress with NIAAA staff. NCCU faculty and students will do experiments in UNC laboratories, and gain access to UNC research cores and libraries. Research laboratory experience, access to full text journals and key research core facilities will improve It will also mentor NCCU undergraduate and graduate students training future alcohol researchers. Finally, the U54 Administrative Core will define expectations and metrics for assessment of progress. The MAP Administrative core will provide support that will allow NCCU faculty to develop independent self-supporting research programs at NCCU that will involve many students in AHDRC.

? DESCRIPTION (provided by applicant): Alcohol abuse is common in adolescence, a time of marked neurocircuitry development. Recently, we discovered that NADIA adolescent intermittent ethanol (AIE) treatment of rats increased frontal cortical neuroimmune molecules, blunted frontal cortical cFos responses to ethanol, reduced hippocampal neurogenesis, reduced choline acetyltransferase (ChAT, the enzyme synthesizing acetylcholine), and caused deficits in reversal learning in adulthood. Reduced hippocampal neurogenesis and ChAT are unique to AIE and did not occur after similar ethanol treatment in adulthood. In an important parallel with human studies, we found increased expression of neuroimmune genes and reduced expression of ChAT and other cholinergic neuron markers in the post-mortem brains of alcoholic patients. This is consistent with adolescent alcohol exposure contributing to adult alcoholic neuropathology. This proposal will test multiple hypotheses: 1) that inhibiting AIE-induced neuroimmune signaling will protect against loss of adult neurogenesis and ChAT+ neurons, and reduce reversal learning deficits. 2) That reduced cholinergic signaling contributes to AIE-induced adult neuroimmune gene induction, reduced neurogenesis, reduced adult prefrontal cortical neuronal responses to ethanol, and reversal learning deficits. As well as investigating adolescent rat brain connectivity using resting-state functional Magnetic Resonance Imaging (rsfMRI) for direct comparisons to human studies testing hypothesis 3, i.e. that adolescent alcohol exposure alters neural connectivity in the adult brain through persistent increases in neuroimmune signaling and deficits in ChAT. It is expected that AIE will alter adult functional connectivity through pro-inflammatory gene induction and decreased cholinergic signaling. These studies will link key signaling mechanisms to AIE-induced adult neuropathology and lead to therapies.

This U54 component, Research Project 3-NCCU-UNC Small Projects, will support the research development of three NCCU investigators new to alcohol research. These three small projects will receive funding to obtain pilot data that will allow the submission of grant applications to transition to independent funded alcohol research programs, or the transition to full research projects in later years of this U54 program. Through active mentoring received from the Mentoring and Development Core these NCCU alcohol researchers will further the understanding of the mechanisms of alcohol pathology as related to the role of alcohol exposure during development on microglia function, the role of alcohol on mammary tumor development, and the role of alcohol in pulmonary inflammation. It is expected that these pilot projects will introduce underrepresented students to alcohol research while enhancing the alcohol research expertise of the NCCU faculty. Ultimately these three pilot projects will continue the expansion of alcohol research at an IUCP, NCCU, and will advance our understanding of the contribution of alcohol abuse to cancer, lung inflammation, and the role of microglia in FASD.

? DESCRIPTION (provided by applicant): This is a renewal application for a cooperative agreement (U54), Mechanisms of Alcohol Pathology (MAP), between faculties of North Carolina Central University (NCCU), a Historically Black College and University, and the Bowles Center for Alcohol Studies (BCAS) at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this Alcohol and Health Disparity Research Center (AHDRC) Program. Within this proposal, an NCCU Administrative Core, Mentoring and Development Core, and five Research Components will integrate with the UNC-BCAS Administrative, Mentoring and Development Cores and Research Components, as well as other activities at the Bowles Center for Alcohol Studies. The UNC NIAAA Alcohol Research Center (ARC) and BCAS have many faculty studying mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that allow collaborative research partnerships on common areas of interest between UNC and NCCU faculty. These partnerships open UNC research laboratories, core facilities and libraries to NCCU faculty improving opportunities for developing productive research laboratories at NCCU. In addition, large numbers of NCCU students will continue to become involved in research on mechanisms of alcohol pathologies providing education on African-American alcohol related health disparities which are due to increased morbidity to alcohol related pathologies and not increased alcohol use disorder. The objectives of this U54 partnership are to investigate molecular mechanisms of alcohol- induced cellular pathology. This proposal includes eight NCCU research projects that will investigate the molecular mechanisms of fetal alcohol pathology, alcohol induced liver disease, alcohol induced oral and breast cancers as well as alcohol-cannabinoid neurotoxicity. A second objective is to provide scholarly education on Alcohol Pathology for NCCU students. The educational efforts in this proposal will involve NCCU undergraduate and graduate students through the BCAS seminar series, through training in UNC laboratories and research development and mentor meetings, attendance and presentation of data at the annual RSA meeting, an alcohol seminar series hosted by NCCU, a new course curricula on alcohol pathology developed with a focus on health disparities, as well as specific undergraduate student summer internships for NCCU students to study and learn about alcohol pathology and health disparities within laboratories in both institutions. Ultimately this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an active and successful alcohol research program within NCCU that synergizes with UNC to advance education and discoveries.

DESCRIPTION (provided by applicant): This is the renewal of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Administrative Core (NADIA Administrative Core-U24, RFA-AA-15-005). The Administrative Core provides administrative and scientific leadership to achieve the goal of our Consortium: to elucidate persistent changes in complex brain function-behavior relationships following adolescent alcohol exposure. In the previous funding period, the NADIA discovered that adolescent intermittent ethanol (AIE) led to multiple pathologies in adulthood, including altered anxiety, social behavior, cognitive flexibility, conflict behavior, sleep, impulsivity, decision making, incentive salience, increased adult alcohol drinking and decreased response to alcohol, as well as altered neuroimmune gene expression, neurogenesis, epigenetic histone-methylation changes, and decreased choline acetyltransferase expression. To promote and facilitate this progress, the NADIA Administrative Core is the main organizational unit of the Consortium and serves as the liaison between the eight Research Components, the two Scientific Cores, the NIAAA, the NADIA Steering Committee, and the External Advisory Board. This Core organizes all Consortium activities including retreats, progress reports and External Advisory Board evaluations of cores and components. The Administrative Core ensures consistent and forward progress by facilitating communication and promoting integration of data among the components and cores. The Core develops scientific and conceptual themes as well as standard operating procedures that cross components. The Core provides a repository for all NADIA publications (at the time of submission, 119 publications were published or in press from the previous funded period) and data generated by the Consortium. Finally, the Core facilitates dissemination and translation of NADIA-generated data. Understanding the impact of underage drinking on adult neurobiology is important to guide public health initiatives, and the NADIA Administrative Core creates synergies for such discoveries across the Consortium.

Abstract: Excessive alcohol use damages many organs, but most commonly results in alcoholic liver disease (ALD). The molecular mechanisms by which ethanol (EtOH) exerts its damaging effects are extensively studied, but not fully understood, thus hampering the development of therapeutic options for the treatment of ALD. Furthermore, to date, there are no experimental animal models that recapitulate the full spectrum of ALD symptoms. The pregnane X receptor (PXR), expressed primarily in the liver and intestine, is a xenobiotic nuclear receptor that defends against toxic agents. PXR activation in humans and rodents leads to hepatic triglyceride (TG) accumulation, one feature of ALD, suggesting that the PXR may be involved in ALD pathogenesis. However, the role of mouse and human PXRs in EtOH hepatotoxicity has not been characterized. The earliest sign of liver injury observed in alcoholics is steatosis. While alcoholic steatosis progresses to alcoholic steatohepatitis during the development of ALD, the mechanisms that contribute to this deterioration are unclear. However, we have intriguing preliminary data suggesting the possibility that PXR is involved in the progression of alcoholic steatosis to more severe liver disease. Basal hepatic TG levels were significantly higher in male mice that lacked the PXR (PXR-KO) than in wild type (WT) or PXR-humanized (hPXR) transgenic mice; conversely WT and hPXR mice accumulated higher levels of TG when treated with EtOH and EtOH-treated hPXR mice were most prone to liver injury. The objectives of this R01 proposal are: 1) to explore the underlying molecular mechanisms by which PXR signaling regulates EtOH-induced TG accumulation and related lipid metabolic pathways and 2) to identify the molecular features of hPXR vs. WT mice that contribute to their increased susceptibility to EtOH-induced liver injury. Based upon our compelling preliminary data, our central hypothesis is that the nuclear receptor PXR is required for both ethanol-induced steatosis and progressive hepatotoxicity. We will carry out molecular and cellular studies using the three mouse models (WT, PXR-KO, and hPXR) and hepatocytes isolated from these animals to achieve the following aims. Specific Aim 1 will monitor i) the effect of PXR signaling on lipid synthesis, oxidation and export in hepatic steatosis and ii) corresponding changes in lipid composition in plasma and liver after ethanol ingestion. Specific Aim 2 will determine whether PXR regulates the main enzyme that metabolizes EtOH, ADH gene transcription and how it affects ethanol and acetaldehyde elimination rates. Specific Aim 3 will investigate the role of PXR signaling in hepatocyte proliferation following acute and chronic EtOH exposure. Our proposed study is novel in its use of humanized mice to examine the function of PXR in ALD, providing data that can be more directly translated into clinical research. This study is also significant in a) providing valuable insights into the role of mouse and human PXRs in ethanol hepatotoxicity via their regulation of alcohol metabolism and liver regeneration, and b) offering new therapeutic targets for the treatment of ALD.

This NIAAA Alcohol Research Center (ARC) Grant is the catalytic element that integrates a large group of investigators across the University of North Carolina at Chapel Hill (UNC). The UNC School of Medicine Bowles Center for Alcohol Studies (BCAS), provides a foundation of administrative support and dedicated Bowles building space for alcohol research. The UNC ARC fosters interdisciplinary collaborative research on alcohol use disorders, alcohol abuse and the impact of alcohol on health and disease - exactly the goal of an NIAAA ARC. The ARC has facilitated the growth and development of UNC into an outstanding alcohol research University, among the best in the world. Research and education have always centered on a theme of molecular and cellular pathology in alcohol use disorders. This renewal focuses on the molecular mechanisms that underlie alcohol-induced circuit pathology across the stages of addiction. Ultimately, our guiding hypothesis is that alcohol-induced dysregulation of neural circuitry drives pathological behaviors and is thus the key cause of all alcohol-related pathology. This 4th renewal of the UNC ARC continues an emphasis on alcohol use disorder pathology, integrating existing and new faculty to investigate changes in neural circuits and molecular signaling in models of drinking across the proposed stages of addiction. The scope of these studies addresses the critical neurobiological changes leading to all alcoholic pathologies, i.e. the mechanisms leading to heavy chronic drinking. The ARC integrates multiple signaling systems and neurocircuits that each focus on specific mechanisms within and across brain regions. The research components also include the translational endpoint, functional magnetic resonance imaging (fMRI) connectivity of each component?s pathological circuit model in the Scientific Resource Core. This approach is expected to increase discovery, improve models and gain strength from common assessments across preclinical models to the ARC human studies. The ARC through the Information Translation Core informs practicing health professionals, health professional and college students as well as youth through specific alcohol curricula for each group to have the greatest impact on health. This proposal connects principle investigators of involving 15 independent funded faculty. By design, each research component of this ARC will focus on specific models that capture distinct endophenotypes associated with alcohol abuse. A range of molecular mechanisms that drive these circuit alterations will be explored, including kinases, cytokines and neuropeptides. This ARC renewal continues to be the catalytic element that integrates a broad group of investigators, pairing senior and junior faculty within ARC components that promote discovery across the BCAS and UNC as well as educating many within NC. This ARC proposal continues a research focus on pathogenesis of alcohol addiction with emphasis on molecular and circuit mechanisms that lead to dysfunctional brain networks, a theme at the cutting edge of neuroscience. The ARC will conduct, promote, support, and mentor research on the pathology of alcohol use disorders and educate broad groups of the public, including health professionals, families, college students and youth in North Carolina on the causes and prevention of these disorders.

Administrative Core Project Summary The administrative core of the UNC Alcohol Research Center entitled ?MOLECULAR AND CIRCUIT PATHOGENESIS IN ALCOHOL ADDICTION? is organized to foster and conduct interdisciplinary and collaborative research on alcohol use disorders with a specific emphasis on molecular and circuit pathogenesis underlying these disorders. In this renewal, we build upon the strengths of our Center by extending our understanding of ethanol pathology mediated by dysregulation of neural circuits involving cytokine, dopamine, dynorphin, GABA, glutamate, histone deacetylase and kinase signaling. The specific aims of the Administrative Core are to provide scientific inspiration and administrative leadership for the Alcohol Research Center, monitor and enhance interaction among the research components, optimize synergy and sharing of resources and services, organize seminars and workshops to stimulate exchange of scientific information among the Center faculty and staff. Further, we will work to stimulate new alcohol research programs at UNC, disseminate new research knowledge to health professionals, academics, college students, families and youth in NC. The Administrative core is involved in community outreach activities designed to improve awareness, prevention, and treatment of alcohol use disorders. In addition, the Administrative Core oversees all budgetary issues, appropriate allocation of funds as well as preparation of progress reports and the renewal of the ARC. In summary, the administrative core is designed to maximize research rigor, significance, innovation, collaboration and productivity to advance the research aims and to use scientific knowledge to improve health by educating health professionals, parents, college students and youth.

Project Summary. Neuroimmune Sensitization and Circuit Pathogenesis: An Alzheimer's supplement to P60-AA011605 in response to (PA-18-591). Emerging data suggest that Toll-like receptors (TLRs) and neuroimmune signaling are involved in Alzheimer's disease (AD). Neuroimmune signaling and ethanol drinking induce neuroimmune and microglial activation. Additionally, microglial priming of proinflammatory responses increases over time. Our studies find that ethanol and stress increase brain microglial, astrocyte and neuronal neuroimmune gene expression with progressive increases over time. Microglia are unique brain neuroimmune cells that are sensitized to proinflammatory responses by environmental and genetic factors that induce Toll-like receptors. We discovered that cycles of ethanol exposure, similar to stress cycles, lead to long-lasting changes in expression of neuroimmune genes, including neuronal Toll-like receptor 3 (TLR3). We have found that TLR3 expression is increased in post-mortem human alcoholic brains, and is increased by ethanol exposure in both cortex and striatum in rats. These are highly relevant findings given that TLR3 expression, both protein immunoreactivity and gene mRNA expression, are also increased in post-mortem Alzheimer's disease (AD) brain tissue. Neuroimmune gene induction of oxidative stress and neurodegeneration are well known to contribute to AD. Importantly, our TLR3 finding is consistent with increases in TLR3 signaling persisting for long periods that could sensitize and contribute to neurodegeneration and the development of AD. This result emphasizes TLR3 as a potential therapeutic target for AD. Additionally, we find that treatment with TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) results in escalated neuroimmune gene induction and markers of activated astrocytes and neuronal synapse markers. Moreover, we observe parallel poly(I:C)-induced increases in TLR3 gene expression, Gi-coupled Group II metabotropic glutamate receptors (mGluR2 and 3) consistent with increased glutamate signaling. We propose to expand our analyses to include AD markers (APP, Tau, beta-secretase [BACE1], GSK3?, A?42), and mRNA determination of TLR3 and other TLR signaling genes (MyD88, IRF, STAT, NFkB), as well as neuroimmune signaling genes (TLR3, HMGB1, MCP1, IL6, TNF?). We propose to extend our studies of glutamatergic synapse markers (PSD95, mGluR2, mGluR3, GLT1, AMPA, NMDA subunits) to include the brain regions involved in AD pathology (prefrontal cortex, entorhinal cortex and hippocampus). In addition, we propose to assess microglial markers (Iba1, CD11b), as well as microglia synaptic regulatory proteins (C1q, CR3, CD200, CD200R), astrocyte markers (Aq4, GFAP, GLT1, ALDH), and astrocyte synaptic proteins (i.e., thrombospondins). In this supplement, we will expand our investigation of TLR3 neuroimmune signaling synaptic changes to test the hypothesis that poly(I:C)-TLR3 activation induces neuroadaptations in glutamatergic synapses and glia that contribute to AD pathology. As such, experiments in Aim 1 will examine changes in neuroimmune signaling and markers of AD-related pathology. Experiments in Aim 2 will focus on neuroadaptations in glutamatergic synapses and glia that may contribute to AD pathology. Together these studies will determine the impact of TLR3 sensitization on markers of AD pathology across multiple brain regions and brain cell types for neuroimmune, synaptic and AD degeneration genes.

ABSTRACT 1/1 U24 NADIA ADMINISTRATIVE CORE - SUMMARY This is the second renewal of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Administrative Core (NADIA Administrative Core-U24, RFA-AA-20-004). The Administrative Core provides organizational and scientific leadership to achieve the goal of our Consortium: to elucidate persistent changes in complex brain function-behavior relationships following adolescent alcohol exposure. Adolescent binge and extreme drinking are common, and while many factors contribute to human brain development and alcohol use during adoles- cence, animal models are critical for understanding the specific consequences of alcohol exposure on the brain and behavior during this crucial developmental period. In the previous funding periods, the NADIA discovered that adolescent intermittent ethanol (AIE) led to multiple behavioral, neurochemical and physiological patholo-gies in adulthood. Mechanistic studies revealed that these AIE-induced changes could be prevented or re-versed by targeting specific neurotransmission, neuroinflammation, or epigenetic regulation of genes. The Consortium has been very productive, as evidenced by our recent literature reviews and over 200 publications. To promote and facilitate continued progress, the NADIA Administrative Core is the main organizational unit of the Consortium and serves as the liaison between the Scientific Core, the eight Research Components, the NADIA Steering Committee, the External Advisory Board, and the NIAAA. The Administrative Core organizes all Consortium activities including retreats, progress reports and External Advisory Board evaluations of cores and components. The Administrative Core ensures consistent and forward progress by facilitating communica-tion and promoting integration of data among the components and cores. The Core develops scientific and conceptual themes as well as recommended operating procedures that cross components. The Core provides a repository for all publications generated by the Consortium. Finally, the Core facilitates dissemination and translation of NADIA-generated data, in part by targeted interactions with the National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA) and other alcohol research groups. This NADIA renewal exactly fulfills the molecular mechanism and translation goals of this RFA leading to development of therapeu-tic interventions of adult psychopathologies of adolescent alcohol consumption. Understanding the impact of underage drinking on adult neurobiology is important to guide public health initiatives, and the NADIA Adminis-trative Core creates synergies for such discoveries across the Consortium.

5/8 NADIA UO1. Effects of Adolescent Alcohol on Adult Brain Neurocircuitry. Abstract Our studies find adolescent intermittent ethanol (AIE) exposure increases adult risky decisions, disinhibition, behavioral inflexibility, and reduces sensitivity to adult ethanol challenge as well as increasing alcohol drinking, all risk factors for human AUD. Acetylcholine is reduced by AIE and plays a critical role in cortical activation and integration as well as maturation of adolescent neurocircuits that could impact ethanol sensitivity. Although AIE does not change adult ethanol elimination, it does reduce adult ethanol motor-sedative, anxiolytic, PFC immediate early gene (IEG) c-fos, and cortical functional connectivity magnetic resonance imaging (fcMRI) responses. We discovered AIE increases adult HMGB1 and its receptors (i.e., Toll-like receptor 4 [TLR4] and receptor for advanced glycation end products [RAGE]) on neurons, and these signals are linked to epigenetic silencing of cholinergic phenotype genes (i.e., ChAT, VAChT, TrkA) and loss of hippocampal neurogenesis, which are examples of AIE-altered neurocircuitry. Low response to ethanol is associated with AUD, and these changes are found in post-mortem human adult AUD brain. Basal forebrain cholinergic neurons integrate neurocircuits, and reductions of these neurons may contribute to AIE pathology. We hypothesize AIE HMGB1?TLR4/RAGE epigenetic silencing of cholinergic neurons alters basal forebrain-cortical- hippocampal neurocircuitry, leading to reduced ethanol sensitivity and increased alcohol drinking in adulthood. Reductions of adult ChAT+ neurons caused by AIE were initially interpreted as neurodegeneration; however, the surprising discovery that AIE reductions of ChAT+ neurons are reversible by exercise and indomethacin supports HMGB1?TLR4/RAGE?NF?B epigenetic silencing of ChAT within neurons persisting into adulthood. Reversal of neuroimmune ChAT silencing suggests AIE pathology is persistent, but not permanent. However, it is not known if AIE-mediated adolescent-like low adult ethanol sensitivity can be prevented or reversed. This proposal therefore has the following Aims. AIM 1. Test the hypothesis that AIE decreases adult ethanol sensitivity to brain fcMRI changes, c-fos expression, ethanol response behaviors (ERBs), and increases adult alcohol drinking. AIM 2 will extend studies testing the hypothesis that HMGB1?TLR4/RAGE?NF?B signaling reduces basal forebrain ChAT+ cholinergic neurons by reversible epigenetic mechanisms. AIM 3 will test the hypothesis that adolescent ChAT silencing mimics adult AIE low- ethanol response and other pathology. AIM 4 will test the hypothesis that anti-inflammatory treatment known to reverse AIE-altered ChAT, neurogenesis, and epigenetic changes, will also recover AIE-altered c-fos, fcMRI, ethanol low behavioral response sensitivity, and adult ethanol drinking. Identifying molecular mechanisms and key neurocircuits regulating low response to alcohol will contribute to our understanding of adolescent brain development and the mechanisms determining low response to ethanol challenge, a known risk factor for AUD, as well as possibly identifying treatment targets.

Abstract Chronic heavy or binge alcohol use is associated with dementia and increased risk for Alzheimer's disease (AD), though mechanisms connecting the disease pathologies have not been identified. AD pathology follows a temporal progression of proinflammatory signaling in brain with A? deposition, tau fibril formation, synapse loss, neurodegeneration, and cognitive decline. We find increased AD pathology proteins A?1-42 and phosphorylated (p)-tau-181 in human alcohol use disorder (AUD) brain and after chronic binge ethanol during early life in the 3x- Tg-AD mouse model. Toll-like Receptor (TLR) proinflammatory signaling is thought to precede gross pathology in AD, and is a prominent feature of AUD. We find AUD in young individuals is associated with AD protein accumulation, and chronic binge ethanol treatment in 3x-Tg-AD mice causes persistent upregulation of proinflammatory genes that correlated strongly with levels of neurotoxic A?1-42 and p-tau-181 protein. Thus, we hypothesize ethanol promotes AD neurotoxic protein pathology by enhancing proinflammatory signaling. Microglia have emerged as mediators of AD pathology, contributing to A? plaque formation, tau propagation, synapse loss and possibly neurodegeneration. Microglial change phenotype (e.g. resting, phagocytic, proinflammatory, pruning, or neurotoxic) and microglial activation is thought to precede and promote AD pathology. Human GWAS as well as human and AD mouse single nuclei RNA sequencing (snRNA-seq) have identified AD microglia as altered, having increased C1q (pro-synaptic pruning) and reduced resting state markers (e.g. Tmem119). In human AUD brain, we find reduced resting state microglia (reduced Tmem119) with proinflammatory microglia. Further, we find chronic binge ethanol in vivo persistently increases C1q and proinflammatory cytokines mirroring AD. Thus, we hypothesize proinflammatory microglial activation by chronic heavy/binge ethanol promotes AD A? and tau pathology, synapse loss and neurodegeneration. This hypothesis will be tested in human AD post-mortem brain (Aim 1), and the 3x-Tg-AD mouse model (Aim 2) that features progressive A? and tau pathology. Apoptotic neuronal cell death is a feature of AD pathology thought to contribute to irreversible cognitive decline. We find binge ethanol increases neurodegeneration in brain regions known to have neuronal loss in AD such as prefrontal cortex (PFC) and hippocampus. Our studies implicate proinflammatory Toll-like Receptor (TLR) activation and microglial to neuronal interactions that lead to apoptotic neuronal cell death. We find chronic binge ethanol increases HMGB1-TLR4 signaling to activate microglia. Proinflammatory microglia secrete the miRNA let-7b that we found activates TLR7 in neurons. Let-7b-TLR7 signaling in neurons induces apoptotic neuronal cell death via the TNF-receptor superfamily apoptosis-inducing ligand (TRAIL). TLR4, HMGB1, let-7 and TRAIL have each been implicated in AD pathology. However, a link in AD neurodegeneration has not been identified. We hypothesize chronic binge ethanol promotes AD neurodegeneration by enhancing TRAIL- mediated apoptotic neuronal cell death. This hypothesis will be tested in human AD post-mortem brain (Aim 1), and the 5xFAD AD mouse model (Aim 3) that features A?-induced apoptotic neuronal death.