The proposed research will investigate the relationship between developmental ethanol neurotoxicity and the expression of apoptosis effector and repressor molecules of the Bcl-2 family. Members of this family can inhibit apoptosis (e.g., Bcl-2, Bcl- xl) or promote it (e.g., Bax, Bad, Bcl-xs). These relationships will be explored in the developing cerebellum, which exhibits a differential temporal susceptibility to ethanol during the early postnatal period, with a brief period of sensitivity on postnatal days 45 (P45), which results in loss of Purkinje and granule cells, followed by a period during which this region is relatively refractory to ethanol effects (P7-8). We hypothesize that alterations in the expression of Bcl-2-related molecules contribute significantly to this population's relative temporal vulnerability to ethanol. Neonatal rats will be exposed to ethanol via artificial rearing, which we have found to produce increases in bax and bcl-xs mRNA on P4, but not on P7. In specific experiments we will use quantitative Western blot procedures to characterize the dynamics of expression of Bcl-2- related proteins following ethanol exposure at P4 and P7. We will also examine the influence of ethanol on certain post- translational modifications of Bcl-2-related proteins (e.g., dimerization, phosphorylation, and altered molecular integrity). Such processes affect the capacity of these molecules to implement or inhibit cell death. We will determine the regional distribution of Bcl-2-related proteins within the developing cerebellum following ethanol exposure at a vulnerable time (P4) and at a """"""""protected"""""""" time (P7), using immunohistochemical procedures. Finally, we will establish whether a causal relationship exists between expression of Bcl-2-related proteins and ethanol-induced neurotoxicity. For this study we will use genetically engineered animals lacking the pro-apoptotic bax gene. Homozygous, heterozygous and wild-type animals will be exposed to ethanol on P45 and Purkinje and granule cell counts subsequently made. We hypothesize that loss of this apoptosis promoter will eliminate or significantly mitigate ethanol-induced cerebellar neuronal death. These studies will be important in producing the first characterization of the role of cell death effector and repressor molecules in developmental ethanol neurotoxicity, and will provide new information concerning a critical mechanism underlying the devastating central nervous system damage seen in the Fetal Alcohol Syndrome.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project (R01)
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Alcohol and Toxicology Subcommittee 4 (ALTX)
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Velazquez, Jose M
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University of Florida
Schools of Medicine
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Heaton, Marieta B; Paiva, Michael; Kubovec, Stacey (2015) Differential effects of ethanol on bid, tBid, and Bax:tBid interactions in postnatal day 4 and postnatal day 7 rat cerebellum. Alcohol Clin Exp Res 39:55-63
Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael et al. (2013) Ethanol influences on Bax associations with mitochondrial membrane proteins in neonatal rat cerebellum. Dev Neurobiol 73:127-41
Heaton, Marieta Barrow; Paiva, Michael; Kubovic, Stacey et al. (2012) Differential effects of ethanol on c-jun N-terminal kinase, 14-3-3 proteins, and Bax in postnatal day 4 and postnatal day 7 rat cerebellum. Brain Res 1432:15-27
Heaton, Marieta B; Paiva, Michael; Siler-Marsiglio, Kendra (2011) Ethanol influences on Bax translocation, mitochondrial membrane potential, and reactive oxygen species generation are modulated by vitamin E and brain-derived neurotrophic factor. Alcohol Clin Exp Res 35:1122-33
Siler-Marsiglio, Kendra I; Madorsky, Irina; Pan, Qun et al. (2006) Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods. Alcohol Clin Exp Res 30:1031-8
Heaton, Marieta Barrow; Paiva, Michael; Madorsky, Irina et al. (2006) Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum. J Neurobiol 66:95-101
Siler-Marsiglio, Kendra I; Paiva, Michael; Madorsky, Irina et al. (2005) Functional mechanisms of apoptosis-related proteins in neonatal rat cerebellum are differentially influenced by ethanol at postnatal days 4 and 7. J Neurosci Res 81:632-43
Siler-Marsiglio, Kendra I; Pan, Qun; Paiva, Michael et al. (2005) Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems. Brain Res 1052:202-11
Moore, D Blaine; Madorsky, Irina; Paiva, Michael et al. (2004) Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of neonatal exposure. J Neurobiol 60:114-26
Moore, D Blaine; Madorsky, Irina; Paiva, Michael et al. (2004) Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of prenatal exposure. J Neurobiol 60:101-13

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