Mark Prendergast - US grants

Long-term ethanol abuse and withdrawal in humans is associated with deleterious effects on neuronal function, including neurodegeneration that may be consequences of dysregulated Ca2+ homeostasis. This is reflected in elevated function of neuronal NMDA receptors and voltage-operated Ca2+ channels (VOCC). During ethanol withdrawal, increased function of these channels likely contribute to the accumulation of toxic levels of Ca2+ in neurons. The proposed studies are designed to elucidate mechanisms of ethanol-associated neuronal damage as they related to changes in neuronal Ca2+ buffeting. Radioligand binding and functional assays of radiolabeled Ca2+ entry into neurons will examine changes in neuronal density and function of NMDAr and VOCC in response to ethanol and correlated these changes with neuronal damage in cultured neurons of the rat hippocampus. Further, ethanol-induced reductions in glucose utilization, ATP content, and activity of neuronal Ca2+-ATPases will also be examined using autoradiography and functional assays, as these may represent effects of ethanol which reduce the efflux of Ca2+ following neuronal accumulation. Finally, recent evidence suggests that exposure to the nicotinic acetylcholine receptor agonist, (-)-nicotine, may be of benefit in reducing neuronal damage produced by ethanol abuse and/or withdrawal. It is hypothesized that this benefit is provided, in part, by preventing adaptive changes to ethanol which may increase intracellular accumulation of Ca2+ during periods of ethanol withdrawal. Indeed, this may be one reason underlying the close association between ethanol abuse and cigarette smoking. Thus, the ability of (-)-nicotine to prevent the effects of ethanol described above will be evaluated. It is intended that by conducting these studies, a better understanding of the mechanisms associated with ethanol abuse and withdrawal associated neurodegeneration will be provided and that the study of (-)-nicotine will aid in suggesting novel means of attenuating these effects. In particular, exposure to agents which interact with nicotinic receptors may be useful in this regard and, possibly, may be useful in reducing the incidence of cigarette smoking during chronic ethanol abuse.

DESCRIPTION (provided by applicant): Alcohol dependence or abuse is often observed in some populations of patients infected with human immunodeficiency virus type-1 (HIV-1). However, the consequences of alcohol intake in those who are HIV- 1 infected, with regard to function of the brain, have been little studied. This is significant in that HIV-1 infection often results in motor and cognitive impairments, collectively termed HIV-associated dementia (HAD). Long-term ethanol intake produces increased expression or sensitivity of N-methyl-D-aspartate type glutamate receptors (NMDAr). This effect may sensitize the brain to insult caused by excitotoxins such as the HIV-1 protein Tat, a viral transcription factor thought to contribute to the development of HAD. In vitro and in vivo studies using Tat have shown that it causes NMDAr-mediated excitation, Ca 2+ influx into neurons, and neurotoxicity. Thus, it was hypothesized that chronic ethanol exposure would sensitize the brain to the neurotoxic effects of Tat. The aims of these studies are to: 1. Determine if chronic ethanol ingestion sensitizes the NMDAr system to the detrimental effects of intra-hippocampal Tat injection on behavior and neuronal viability in rats; 2. Determine if Tat (1-72 and 1-86 amino acid forms) may directly interact with specific sites on the NMDAr using radioligand binding studies of rat hippocampus; 3. Determine the role of NMDAR polyamine-site over-activity and Ca 2v influx into neurons in the ability of chronic ethanol exposure to sensitize the hippocampus to Tat toxicity. These studies will employ fluorescent microscopy and immunohistoehemical methods in an organotypic slice-culture model derived from male rats; and 4. Evaluate the ability of novel, synthetic NMDAr polyamine-site antagonists to attenuate the neurodegenerative and behavioral effects of ethanol and Tat exposure. In sum, these studies are designed to elucidate the means by which ethanol intake sensitizes the brain to Tat toxicity and to identify potential therapeutic strategies that may be useful in reducing the incidence of neurological problems caused by HIV-1 infection in those who abuse alcohol or are alcohol-dependent.

Alcohol dependence and abuse likely cost more than $200 billion in lost productivity and healthcare annually in the United States. Brain injury is among the most prominent effects of prolonged alcohol use and appears to contribute to the development of neurological abnormalities. Thus, examination of biochemical pathways involved in this form of brain injury may be of value in identifying therapeutic targets to be exploited in treating alcohol-related brain injury. Excess activity of N-methyl-D-asparate (NMDA)-type glutamate receptors has been implicated in both the behavioral and neurotoxic effects of alcoholism, particularly during alcohol withdrawal, when excess release of glutamte and polyamines occurs. Polyamines are endogenous substances that, among other things, act as allosteric activators of NMDA receptors at NR 2 subunits. Substances that further promote activity of NMDA receptor systems, such as glucocorticoids, are hypothesized to exacerbate alcohol withdrawal effects. Indeed, prolonged alcohol intake is associated with hypercortisolemia likely resulting from alcohol effects on hypothalamic corticotropin releasing hormone (CRH) receptors. However, the consequences of elevated stress hormone release with regard to alcoholrelated neuronal injury have been little studied. The proposed in vitro and in vivo rodent studies will test the hypothesis that alcohol-induced activation of the hypothalamic pituitary adrenal axis promotes NMDA receptor-mediated seizure and neurotoxicity during alcohol withdrawal by: (1) stimulating the synthesis and release of polyamines via upregulation of the synthetic enzyme ornithine decarboxylase, an effect dependent upon glucocorticoid receptor (GR) activation; and (2) upregulating the expression of polyamine-sensitive NR2B subunits, thus, promoting NMDA channel opening and neuronal excitation and/or neurotoxicity in a GR-dependent manner. These studies will employ biochemical, immunohistochemical, confocal imaging, and behavioral studies using an in vivo "moderate" binge-alcohol exposure paradigm that produces peak B.A.L.s of ~160 mg/dl and organotypic hippocampal slice cultures, in parallel studies. It is the aim of these studies to elucidate the means by which glucocorticoids may exacerbate alcohol withdrawal behavioral and physiological effects, as these relevant biochemical pathways may represent therapeutic targets to be exploited in the treatment of alcohol withdrawal effects.

DESCRIPTION (provided by applicant): Brain injury is among the most prominent effects of prolonged alcohol use or abuse and evidence suggests that females may be more sensitive than males to the neurotoxic effect of prolonged alcohol intake. Thus, examination of biochemical pathways involved in this form of brain injury may be of value in identifying therapeutic targets to be exploited in treating alcohol-related brain injury. Excess activity of N-methyl-D-asparate (NMDA)- type glutamate receptors has been implicated in both the behavioral and neurotoxic effects of alcoholism, particularly during alcohol withdrawal, when excess release of glutamate and polyamines occurs. Preliminary findings suggest that the rat female brain may be more sensitive to the toxic effects of polyamine exposure during ethanol withdrawal. Polyamines are endogenous substances that act as allosteric activators of NMDA receptors at NR2 subunits. Substances that further promote activity of NMDA receptor systems, such as glucocorticoids, are hypothesized to exacerbate alcohol withdrawal effects. Indeed, prolonged alcohol intake is associated with hypercortisolemia resulting from alcohol effects the hypothalamic pituitary adrenal (HPA) axis. However, the consequences of elevated stress hormone release with regard to alcohol-related neuronal injury have been little studied, particularly with regard to potential sex differences that may exist. The proposed in vitro and in vivo rodent studies will test the hypothesis that alcohol-induced activation of the HPA axis promotes NMDA receptor- mediated seizure and/or neurotoxicity during alcohol withdrawal in a sex-dependent manner by: (1) stimulating the synthesis of polyamines via upregulation of the synthetic enzyme ornithine decarboxylase, an effect dependent upon glucocorticoid receptor (GR) activation;and (2) upregulating the expression of polyamine-sensitive NR2B subunits, thus, promoting NMDA channel opening and neuronal excitation and/or neurotoxicity in a GR-dependent manner. These studies will employ biochemical, immunohistochemical, confocal imaging, and behavioral studies using an in vivo "moderate" binge-alcohol exposure paradigm that produces peak BALs of ~160 mg/dl and organotypic hippocampal slice cultures, in parallel studies. The aim of these studies is to elucidate the means by which glucocorticoids may exacerbate alcohol withdrawal effects in males and females, as these relevant biochemical pathways may represent therapeutic targets to be exploited in the treatment of alcohol withdrawal effects. PUBLIC HEALTH RELEVANCE Brain injury is among the most common consequences of alcohol abuse or dependence and limited evidence suggests that the female brain may be more sensitive than the male brain to injurious effects of alcohol use. These studies will examine effects of alcohol on the stress hormone response and will test the hypothesis that stress hormones stimulate biochemical cascades that produce alcohol withdrawal excitability and brain injury, particularly in the female brain. It is the goal of these studies to identify a potential novel therapeutic target for the treatment of alcohol-related brain injury.

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.

ABSTRACT/SUMMARY The aim of this training program is to provide graduate students with the research skills to identify alcohol-related risk factors by investigating their expression in response to acute and chronic exposure to alcohol at the cellular level, behaving laboratory animal, and the human subject. The training program will emphasize training in cellular and behavioral pharmacology of alcohol with the broad objective to understand how such acute and chronic reactions combine or interact with personal and environmental factors to confer risk for alcohol use disorders. The training is designed to achieve three objectives: 1) deliver intensive training in alcohol research; 2) foster interdisciplinary and translational perspectives on alcohol research; and 3) develop professional skills to support career development in alcohol research fields. The proposal requests support for five pre-doctoral appointments. The appointments will be for a two-year period. Training will be primarily delivered within the research programs of the faculty and the core facilities at UK. This includes a training faculty team of 20 researchers drawn from six academic and research units of the University of Kentucky. The rich environment provides opportunities for translational and multidisciplinary bio-behavioral research training in facilities such as the: 1) Center on Drug and Alcohol Research; 2) Center for Clinical and Translational Science; and 3) The Substance-abuse Prevention in Emerging Adults Research Center (SPEAR). The alcohol-specific expertise and broad research foci of our faculty, coupled with their interdisciplinary research provides a solid foundation by which implementation of the proposed T32 training program will bolster the research skills of our students for the ultimate goal of producing bright, productive scientists capable of significant developments and discovery in the etiology, prevention, and treatment of alcohol use disorders.