Kimberly Nixon, Ph.D. - US grants

ADHD, a widely diagnosed disorder in the school-aged population, is correlated with a variety of causes, but its etiology remains a mystery. Alcohol exposure during pregnancy has been correlated with the occurrence of ADHD in childhood. As ADHD may have several subtypes, one of which is the result of hippocampal dysfunction, and alcohol acts primarily on the hippocampus, alcohol-induced damage to the hippocampus may describe a subtype of ADHD. The project is also a further extension of the investigation of the effect of alcohol on the NMDA receptor, a primary receptor in the hippocampus. The project determines the composition of NMDA receptor protein subunits in rats exposed to alcohol during the third trimester equivalent and second and third trimesters combined. The secondary subunits of the NMDA modulate its function by altering Mg2+ affinity to the attached calcium channel, which in specific configurations may promote either excitotoxicity or apoptosis. Differential NMDA receptor function is observed after exposure to EtOH, as well as cell death, but the means by which cells die has not been described. This project seeks to answer whether altered NMDA receptor subunit composition is responsible for the change in NMDA receptor function, whether it causes apoptosis or excitotoxicity, and its subsequent effects on behavior.

Alcohol use disorders remain as one of the nation's major public health problems with over 17 million Americans meeting the diagnostic criteria for alcohol abuse or dependence. Chronic alcoholics demonstrate cognitive impairments that are related to a loss of brain mass or neurodegeneration, effects that may recover with abstinence. Many assumed that the mechanism of this recovery was due to glial regeneration, however recent discoveries from our laboratory show that alcohol-induced regulation of neural stem cells (NSCs) parallels the changes in brain mass and cognition during active alcoholism (decrease) versus abstinence (increase) in the hippocampus. The regulation of NSCs relies on the milieu of the local environment, or neurogenic niche. Microglial, one of three types of glia, contribute to this niche. Though microglial events historically were synonymous with cytotoxicity, a new role in neurogenesis is emerging. Some activated microglia secrete growth factors and anti-inflammatory cytokines, an effect that is consistent with recent data that certain types of microglia promote NSC proliferation and adult neurogenesis. Thus, when we observed a microglial response that precedes the neurogenic response in our model of chronic alcoholism, we suspected a causal link between microglial events and the promotion of neurogenesis. Therefore, this proposal will test the hypothesis that binge alcohol exposure produces a graded microglial response that drives the recruitment of quiescent NSCs into proliferation and neurogenesis in abstinence. Three specific aims address this hypothesis by asking: (1) whether binge alcohol exposure recruits additional NSCs, (2) whether microglia show a graded, nonphagocytic phenotype predictive of a proneurogenic microenvironment and (3) whether can we modulate this phenotype to alter neurogenesis in a model of chronic alcoholism. Multiple approaches will be used, namely immunohistochemistry to assess the recruitment and proliferative dynamics of NSCs, the morphology of microglia, as well as in situ hybridization, receptor autoradiography and Enzyme-Linked ImmunoSorbant Assaysto determine microglia phenotype and cytokine expression. And finally, neuroanatomical and behavioral work will confirm the role of microglia phenotype in neurodegneration and regeneration following binge alcoholexposure. Relevance to public health: This proposal will uncover a mechanism of brain regrowth in abstinence from alcohol by investigating the role of activated microglia on neural stem cells and the neurogenic environment. The results will lead to a novel approach in our long term goal of treating brain damage associated with chronic alcoholism: Identifying agents or behaviorsthat promote protective actions of microglia in recruiting NSCs to repair sites of damage with the hope of reversing or preventing cognitive deficits associated with alcoholic neurodegeneration.

[unreadable] DESCRIPTION (provided by applicant): Adolescence is a time when many individuals begin to experiment with alcohol. Alcohol abuse and alcohol dependence, collectively termed alcohol use disorders, are diagnosed in ~6% of adolescents and result in significant neuropsychological and/or cognitive deficits. As many as half of all high school students may currently drink alcohol and up to 70% of those students are binge drinkers. Considering that binge drinking is one of the factors that predicts brain damage from alcohol, understanding the impact of binge drinking on adolescent brain structure is an important public health concern. The neuroanatomical consequences of adolescent drinking are not well described, though two studies have shown hippocampal volume reductions in adolescents with alcohol use disorders. This finding confirmed behavioral and neurophysiological work in animal models that the adolescent hippocampus is targeted by alcohol. However, no one has examined whether alcohol directly produces neurodegeneration in the adolescent rat or the basic mechanism of cell or neuron reduction. The recent discovery that neural stem cells contribute to ongoing neurogenesis and to hippocampal structure suggests a novel potential mechanism of neurodegeneration alcohol-induced [unreadable] neurodegeneration. Thus, this application will test the overall hypothesis that binge alcohol exposure in the adolescent rat alters neural stem cells and neurogenesis to produce neurodegeneration. Three specific aims will address this hypothesis in an in vivo rat model of an adolescent alcohol use disorder by (1) investigating the effects of adolescent binge alcohol administration on the components of neurogenesis, (2) determining the mechanism by which alcohol intoxication inhibits neural stem cell proliferation and (3) evaluating whether changes in neurogenesis are associated with neurodegeneration (net reduction of cells) in the hippocampal dentate gyrus. Within each aim, important questions regarding the contribution of alcohol dose or duration necessary to alter the components of neurogenesis and the contribution of cell death will be investigated. Understanding the mechanism of alcohol-induced effects on neural stem cells and dentate gyrus granule cell loss forms a solid foundation to investigate the differential sensitivity of the adolescent brain to alcohol effects and the dynamic role of both cell death and cell birth mechanisms in neurodegeneration. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The University of Kentucky Summer Training in Alcohol Research (STAR) program provides 12 weeks of intensive, hands-on alcohol research for 11 outstanding undergraduate students. A broad range of research opportunities exist that are truly bench to bedside, because of the breadth of empirical approaches employed by our alcohol research faculty. Research topics available to the trainees range from the cell biology of alcohol neurodegeneration to applied survey research on the role that alcohol may have in moderating interpersonal domestic violence. This program will mesh well with the long-standing existing infrastructure of the Department of Pharmaceutical Science's summer undergraduate research program. Trainees will perform independent research under the supervision of alcohol research faculty, participate in weekly educational and professional development seminars and be trained in responsible conduct of research. Hard work, discovery and great science will culminate in a research presentation to faculty, laboratory personnel and peers at our annual Summer Alcohol Research Symposium. Aggressive advertising of the program through scientific organizations, networking websites, and directly to regional liberal arts colleges, undergraduate institutions, and especially those schools serving underrepresented groups in STEM fields will allow for the recruitment of a diverse pool of summer undergraduate trainees. In concert with the main goals of providing hands on experience in basic and applied alcohol research, we will be promoting the biomedical and behavioral sciences as career choices for these students. Evaluations of short-term and long-term successes of the STAR program on our primary objective of increasing the pool of diverse applicants to graduate programs and biomedical research careers will be conducted and followed throughout the years of the program.

Alcohol use and abuse often begins in adolescence where many factors collide to promote excessive intake. Most striking though is that those who drink during adolescence - specifically before age 15 - are four times more likely to develop an alcohol use disorder in adulthood. This suggests that alcohol impacts the adolescent brain in such a way to make it more susceptible to developing an alcohol use disorder. Indeed, the adolescent brain is more susceptible to brain damage due to alcohol, which has led several groups to examine subsequent neuroinflammatory signaling in alcohol use disorders. A hallmark of neuroinflammation is microglial activation. Microglia are one of the three types of non-neuronal, glia cells in the brain that act as the brain?s immune system, but their role in alcohol use disorders is poorly understood. Microglia display a full spectrum of phenotypes from beneficial to cytotoxic and the phenotype of these microglia after alcohol exposure has not been defined. Further, microglia may become ?primed? by an event, then upon subsequent challenge they aggressively over-respond, a phenomenon intertwined with their phenotype. Microglia priming appears to be more evident in development, where early life exposure to immune insult has long-term consequences on a variety of adult outcomes. Thus, the priming or activation of microglia by alcohol during adolescent development may result in long term consequences. Therefore, the overarching hypothesis of this proposal is that young adolescents are more to susceptible to microglia priming by alcohol versus adults and that alcohol priming produces long term effects on alcohol-induced neuropathology and addiction-relevant behavior. We will test this hypothesis through three specific aims that (1) determine the adolescent?s susceptibility to alcohol-induced effects on microglia, (2) examine whether adolescents have a greater susceptibility to alcohol priming microglia and (3) elucidate the role of microglia priming in addiction-relevant behavior. By understanding the events that prime the adolescent brain to be susceptible to developing an alcohol use disorders, better interventions and treatments can be developed so that we can reduce the incidence of alcohol use disorders.

Abstract Excessive alcohol consumption, the defining characteristic of an alcohol use disorder (AUD), damages the nervous system, producing the cognitive impairments observed in those afflicted with an AUD. Preclinical and clinical evidence from our lab and others suggest that microglia play a key role in alcohol-induced neuroinflammation and neurotoxicity. Various types of microglia have been categorized based on their function: classically activated (M1) proinflammatory microglia and alternatively activated (M2) growth-promoting microglia. However, the mechanisms responsible for alcohol activating microglia and polarizing them to either phenotype are poorly understood. MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation of microglia polarization. Although miRNAs act mainly in a cell- autonomous manner, accumulating evidence suggests that miRNAs can be packaged into exosomes for intercellular signaling. Exosomes are small, 50-150 nm vesicles that are secreted into the extracellular space, fuse to neighboring cells, and release their contents (proteins, mRNAs, and miRNAs), thus serving as a novel intercellular genetic regulatory mechanism. Many cells in the nervous systems have the capacity to release exosomes, including neurons. Our preliminary data demonstrated that neuronal exosomes contain miRNAs that can alter microglia activation. Therefore, we hypothesize that miRNA-containing exosomes derived from alcohol-treated neurons transport messages, namely miRNA, to microglia and modulate their activation and function, and thus potentially contribute to neuroinflammation in AUDs. We will test this hypothesis with the following specific aims: Aim 1: Identify the miRNA elements that regulate microglia activation and function in alcohol-treated neuronal exosomes. Aim 2. Determine the role of brain exosomes derived from 4-day binge alcohol-exposed rat brains in mediating brain inflammation and microglia activation. This proposal is novel in exploring the role of exosomes in cellular communication between neurons and microglia in AUDs. This unique and novel regulatory mechanism in neuron-to-microglia communication will provide new insight into miRNA- mediated microglia activation in pathological conditions such as AUDs.

PROJECT SUMMARY/ABSTRACT This is a proposal for the renewal of a training grant, the primary purpose of which is to provide stipends for five predoctoral and three postdoctoral fellows who will be broadly and intensively trained to conduct research on the neurobiological and behavioral effects of alcohol. This program has a long history of training predoctoral and postdoctoral students in alcoholism research to help the national effort of producing the next generation of independent scientists with a focus on alcohol research. Training opportunities span the breadth of state-of- the-art approaches including molecular biology and genetics, electrophysiology, cellular imaging, neurochemistry, and behavior. Research models include both animal and human. The training program will promote and support collaborative research across multiple departmental units and Ph.D. programs including Clinical Psychology, Behavioral Neuroscience, Pharmacology, Neuroscience, and Cellular and Molecular Biology. Molecular biology training will encompass 1) studies of the function of excitatory and inhibitory ion channels in cellular expression systems (Mihic, Harris), 2) development of new transgenic animal models (Messing, Harris, Mihic, Pierce-Shimomura, Atkinson), and 3) identification of ethanol responsive genes (Atkinson, Harris, Mihic, Pierce-Shimomura). Genetics training will involve genotyping of human and animal subjects (Fromme, Harris, Mayfield, Ponomarev). Electrophysiological and microscopic imaging training will comprise experiments with whole cell and intracellular methods in single cells and in brain slices (Morrisett, Mihic, Harris, Atkinson, Pierce-Shimomura, Messing, Marinelli). Training in neurochemistry will focus on 1) intracellular and extracellular signaling mechanisms with an emphasis on phosphorylation (Morrisett, Mayfield), 2) release and transport of glutamate, dopamine, and other neurotransmitters (Gonzales, Harris, Dominguez), and 3) expression of proteins (Harris, Mayfield, Morrisett, Messing). Research on the behavioral effects of ethanol will include its influence on motor skills, reinforcement, anxiety, and withdrawal (Harris, Gonzales, Dominguez, Morrisett). Training in psychosocial aspects of alcohol research (Fromme) and the interaction of genotype and effects of alcohol drinking in humans (Fromme, Harris) will also be done. The training program will continue to emphasize professional development including improvement of oral and written communication skills and grant writing. Predoctoral students will be required to complete a series of core course requirements, scientific ethics, experimental design, and statistical analysis, and will end up with a Ph.D. degree. Postdoctoral training will be for three years and consist of focused alcohol research guided by a faculty mentor. We will continue to focus on minority recruitment. The training faculty has an excellent history of collaboration with a primary focus on alcohol research. The research laboratories are well equipped with the latest instrumentation for neurochemical and behavioral testing.