Neuroprotection from alcohol: Control of fetal neuron cysteine and GSH homeostasis by Cys transport/synthesis

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 DESCRIPTION (provided by applicant): Toxic effects of maternal ethanol (E) intake on the fetus are well documented both in animal models and humans. Central among the responses to in vivo E exposure are neuro-developmental deficits. A source of this is E-related neuron death that is causally connected to decreased GSH in fetal brain. In vivo and in vitro supporting studies illustrate that E generates striking inhibition of the two primary sources of fetal neuron Cys, the control point for GSH synthesis. These Cys sources are the EAAC1 Cys transporter and the transsulfuration pathway (TSP), which internally synthesizes Cys. E-inhibits both protein and mRNA expression of EAAC1 and cystathionine-γ-lyase (CSE), a rate-limiting enzyme in TSP. In vivo and in vitro data support the mechanism underlying this being disruption of the calcineurin/NFAT signaling pathway which is linked to craniofacial and neurological phenotypes in Down's and Fetal Alcohol Syndrome. Hypothesis: The ability of Nrf2/ARE neuroprotective pathways to fully prevent E-induced death of fetal cerebral cortical neurons is impaired by an E-related perturbation of cellular Cys. This is due to E inhibiting activities of the excitatory amin acid transporter (EAAC1) (Aim 1) and CSE (Aim 2). This E-inhibition of EAAC1 and CSE protein is due to decreased gene expressions caused by disruption of the NFAT signaling pathway (Aim 3). Specific Aim 1 will test the hypothesis that a mechanism underlying the inability of Nrf2/ARE interactions to provide complete protection from E-induced damage of fetal neuron GSH homeostasis is impaired Cys internalization by the EAAC1 transporter. Experiments will elucidate mechanisms underlying E effects on regulation and control of EAAC1 and its maintenance of fetal neuron GSH. Experiments will develop augmentation of neuronal Cys/GSH homeostasis as a rescue strategy. Specific Aim 2 will test the hypothesis that the second and functionally connected component of E impairment of neuron GSH homeostasis is reduced synthesis of Cys by the transsulfuration pathway. The mechanism underlying this is an E-related decrease in cystathionine-γ-lyase expression and function. These experiments will address mechanisms underlying E effects on regulation and control points of the TSP, consequences of this, the role of E inhibition of CSE in TSP function, its role in maintenance of fetal neuron GSH homeostasis, and compensatory crosstalk between EAAC1 Cys transport and the Cys synthesis pathway, TSP. Specific Aim 3 will test the hypothesis that E-inhibition of EAAC1 and CSE protein is due to decreased gene expressions caused by disruption of the NFAT signaling pathway. Experiments will elucidate the role of NFAT signaling which has been linked to craniofacial and neurological phenotypes in Fetal Alcohol Syndrome. Focus is on NFAT3 in E induced transcriptional down regulation of EAAC1 and CSE. This occurs despite EAAC1 and CSE being targets of Nrf2/ARE signaling which is activated by E. Experiments will develop the enhancement of NFAT3 as a rescue strategy.