Alcohol Impairs Neonatal Astrocyte GSH Homeostasis

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Prior studies in our laboratory and others have documented ethanol- related oxidative stress in the developing brain and there is evidence suggesting that it may cause at least some of the neurotoxic effects of ethanol (E). The mechanism(s) by which E causes this oxidative stress remain to be determined, but it is likely related to relatively low anti- oxidant status. There is a compelling literature illustrating that astrocytes play a vital neuroprotective role by producing and exporting reduced glutathione (GSH) and we include, as preliminary data, evidence that astrocyte GSH homeostasis may be perturbed by E in the developing brain. Hypothesis. We hypothesize that one mechanism underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH. Hypothesis: We hypothesize that one mechanisms underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH. Specific Aims. The following specific aims are extensions of our preliminary studies and those of others which document an E-mediated reduction of GSH in cultured neonatal cortical astrocytes. They will determine the means by which E causes this effect. They focus on E effects on well established control points in GSH synthesis and on its efflux. Additionally, experiments will determine the effect of E on viability of astrocytes with respect to mitochondrial functions key to survival and generation of oxygen species which may deplete astrocyte GSH. Specific Aim One will determine the effect(s) of E on astrocyte uptake of precursors required for the synthesis of GSH. We will focus on the effects of E on inward directed transport of cystine and cysteine, which are known determinants of GSH synthesis, but we will also determine effects of E on cellular content of other relevant amino acids e.g. glutamate glycine. These studies will also determine effects of E on GSH uptake and efflux by astrocytes. Specific Aim Two will determine effects of E on the enzymatic synthesis of GSH by astrocytes with a focus on the impact on activity of gamma- glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. Additionally, effects of E on astrocyte-bound gamma-glutamyl transpeptidas4e as a source on CysGly for neuronal GSH synthesis. will be determined. Specific Aim Three will determine the effects of E on astrocyte variability at the mitochondrial . The emphasis on the mitochondrion is due to it being a target for E toxicity, the source of ATP for GSH synthesis, and a primary source of oxidative stress. These experiments will determine E-related depletion of mitochondrial GSH, production of oxidative products which deplete mitochondria of GSH, and induction of mitochondrially-mediated cell death which we document in the appendix.