The purpose of this renewal application is to continue studies designed to provide information related to mechanisms that underlie alcohol- related birth defects (ARBDs). The proposal is based on the supposition that given the wide spectrum of ARBDS that have been identified, and the variety of conditions under which they can be produced, it is extremely unlikely that a single mechanism underlies all of the damage. Based on data gathered from the preceding funding period, the proposed studies focus on a single, reliably produced quantitative deficit, cerebellar Purkinje cell death, as a consequence of alcohol exposure during part of the third trimester equivalent. The proposal is designed around two Specific Aims.
SPECIFIC AIM 1 has three objectives: 1) to characterize the time course for blood alcohol concentration (BAC)-dependent Purkinje cell death; 2) to identify the primary mode of cell death that occurs following a short episode of alcohol exposure; and 3) test the hypothesis that moderate BACs will induce Purkinje cell death with the majority of cells dying via apoptosis, but at higher BACs the predominant mode of cell death will sift from apoptosis to necrosis.
SPECIFIC AIM 2 will investigate mechanisms underlying Purkinje cell death by testing the following four hypotheses. Hypothesis 1: Alcohol exposure disrupts critical ratios of key apoptotic regulator proteins, that are involved in Purkinje cell death. Hypothesis 2: Alcohol reduces neurotrophic factors associated with Purkinje cell survival. Hypothesis 3: Replacing neurotrophic factors reduced by alcohol can rescue a portion of Purkinje cells from alcohol-induced death. Hypothesis 4: Replacing neurotrophic factors will suppress the cell death cascade by readjusting the balance of apoptotic regulator proteins. The proposal incorporates an in vivo model system (the artificial rearing of neonatal rat pups to evaluate cerebellar damage) and a complementary in vitro system (organotypic cerebellar explants). A variety of techniques will be used in the proposal, including light and electron microscopy, 3-D stereology, cell sorting and counting by flow cytometry, ELISAs, Western blots, and immunocytochemistry. The proposed studies will generate new data related to how alcohol kills postmitotic neurons during development. Understanding the regulation of alcohol-induced cell death is necessary for developing strategies to reduce the deleterious effects associated with fetal alcohol syndrome.
