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

 描述(由申请人提供)：母亲摄入乙醇(E)对胎儿的毒性效应在动物模型和人类中都有很好的记录。对体内E暴露的主要反应是神经发育缺陷。其中一个来源是与E相关的神经元死亡，这与胎儿大脑中GSH的减少有因果关系。体内和体外的支持性研究表明，E对胎儿神经元Cys的两个主要来源产生显著的抑制作用，Cys是GSH合成的控制点。这些半胱氨酸的来源是EAAC1半胱氨酸转运体和在体内合成半胱氨酸的跨硫途径(TSP)。E-抑制TSP中EAAC1和胱硫醚-γ-裂解酶(CSE)的蛋白和基因表达。体内和体外数据支持这一机制是钙调神经磷酸酶/NFAT信号通路的中断，该信号通路与唐氏症和胎儿酒精综合征的头面部和神经学表型有关。假设：Nrf2/ARE神经保护通路完全阻止E诱导的胎儿大脑皮层神经元死亡的能力受到与E相关的细胞半胱氨酸的干扰。这是由于兴奋性氨基酸转运蛋白(EAAC1)(Aim 1)和CSE(Aim 2)对E的抑制作用。这种对EAAC1和CSE蛋白的E抑制是由于NFAT信号通路的中断导致基因表达减少(AIM 3)。具体目的1将验证一种假设，即Nrf2/ARE相互作用无法提供完全保护，以对抗E诱导的胎儿神经元GSH稳态破坏的机制是通过EAAC1转运蛋白损害Cys的内化。实验将阐明E对EAAC1的调控及其维持胎儿神经元GSH的潜在机制。实验将开发增强神经元Cys/GSH动态平衡作为一种救援策略。具体目标2将验证这样的假设，即神经元GSH稳态E损伤的第二个和功能相关的成分是通过跨硫途径减少半胱氨酸的合成。其机制是E相关的胱硫氨酸γ裂解酶表达和功能的降低。这些实验将讨论E对TSP调控点的潜在机制，由此产生的后果，E抑制CSE在TSP功能中的作用，它在维持胎儿神经元GSH动态平衡中的作用，以及EAAC1半胱氨酸运输和半胱氨酸合成途径(TSP)之间的代偿串扰。特异靶3将验证这一假设，即EAAC1和CSE蛋白的E抑制是由于NFAT信号通路中断导致的基因表达减少。实验将阐明NFAT信号在胎儿酒精综合征中的作用，NFAT信号与颅面和神经表型有关。重点是NFAT3在E诱导的EAAC1和CSE转录下调中的作用。尽管EAAC1和CSE是由E激活的Nrf2/ARE信号的靶标，但仍会发生这种情况。实验将开发NFAT3的增强作为一种救援策略。

 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.