Mechanism of Alcohol Induced Neurotoxicity

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DESCRIPTION (provided by applicant): Excessive intake of alcohol increases blood ethanol concentration and induces brain developmental disorders manifested as fetal alcohol syndrome (FAS). Children born with FAS have abnormal facial features with mild to sever mental retardation. Cerebellum is one of the most sensitive areas in the brain that is affected by ethanol. However, mechanisms underlying the deleterious effects of ethanol in cerebellum are largely unknown. Our preliminary studies demonstrated that moderate dose of ethanol (blood ethanol concentration of 40 mM) inhibits differentiation, and high dose of ethanol (blood ethanol concentration 80 mM) induces apoptosis in cerebellar granular neurons (CGNs) under in vivo conditions. But under in vitro conditions, higher concentration of ethanol is required for producing similar results, suggesting that some other factor(s) might also be involved. It was further observed that under in vitro conditions ethanol (40 mM and 80 mM) in the presence of physiological concentration of retinoic acid (100 nM) had similar deleterious effects as observed under in vivo conditions. Based on these studies, it is our working hypothesis that harmful effects of ethanol on differentiation and survival of CGNs are mediated by retinoic acid. To test this hypothesis we will determine whether (1) ethanol exposure in vivo affects activation of retinoic acid receptors and (2) whether administration of retinoic acid receptor antagonists protect against harmful effects of ethanol on differentiation and survival of CGNs. To address the issue, rat pups (postnatal day 7) which are widely used as a rodent model of fetal alcohol syndrome, will be exposed to ethanol. Cerebellar granular neurons will be isolated and used for the proposed studies. The transcriptional activities of retinoic acid receptors will be studied by electromobility shift assay. If ethanol interferes in transcriptional activities, it will suggest that retinoic acid receptors might be mediating ethanol effects. Our studies demonstrated that Rho GTPases play a crucial role in differentiation of CGNs; and, exposure of ethanol in vivo affected the activation of these GTPases. It is plausible that impaired activation of receptors affects Rho GTPase signaling. To test this possibility retinoic acid receptor antagonists, known to prevent the activation, will be used. Studies will determine whether antagonists prevent the effects of ethanol on activation of Rho GTPases and protect CGNs against harmful effects of ethanol. To study the role of Rho GTPases we will use in vitro cell culture model. In this cell culture model CGNs will be exposed to ethanol (40 mM, 80 mM) in the presence of retinoic acid (100 nM). Whether, expression of constitutively active or dominant negative forms of Rho GTPases prevent toxic effects of ethanol on differentiation and survival of CGNs will be investigated.