The objectives of the proposed research will be to define cellular mechanisms critical to the devastating consequences produced by exposure to ethanol during the development of the nervous system. Such exposure can lead to the fetal alcohol syndrome (FAS) or alcohol-related birth defects (ARBD). Improved understanding of these mechanisms will make it possible to eventually devise therapeutic strategies for preventing or mitigating ethanol neurotoxicity. Of particular interest in these studies is the effect of ethanol on proteins of the Bcl-2 survival-regulatory gene family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl-xl) or promote it (e.g., Bax, Bad, Bid). Proposed experiments will focus on the Bax protein, an apoptosis agonist strongly linked to ethanol-induced cell death. These relationships are being explored in developing cerebellum, which is highly susceptible to ethanol during the early postnatal period. This region is maximally vulnerable to ethanol on postnatal days 4-5 (P4-5), but is resistant to these effects by P7-9. For these studies, we will use a two-pronged in vivo <>in vitro approach. P4 and P7 neonatal rats will be exposed to ethanol via vapor inhalation, and cultured cerebellar granular cells will be used for parallel manipulative analyses. Techniques to be used include Western blot protein analyses for characterizing Bax activation and subcellular localization, and the ELISA procedure to assess protein-protein interactions. Cell survival assays will be made in the cultured cells, via the MTT assay. In addition, protein purification and circular dichroism (CD) methodologies will be used to examine protein structure. Specific experiments will define (1) ethanol influences on activation of the JNK kinase;JNK phosphorylation of the 14-3-3 Bax anchoring protein;subsequent Bax activation and insertion into the mitochondrial membrane;(2) ethanol effects on cleavage of the Bid protein, and subsequent tBid:Bax dimerization;and (3) the pathway of Bax disruption of the mitochondrial membrane, i.e., via the mitochondrial permeability transition pore or by Bax formation of membrane channels. In each of these series of studies, the granule cell model system will enable us to perform manipulative assessments, and to measure cell death in order to confirm the importance of the events of interest. In addition, purified P4 and P7 Bax will be subjected to CD analyses of protein conformation at the two ages, and ethanol effects on this conformation.
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