Neuroprotection from ETOH-mediated apoptosis: Nrf2/ARE control of GSH homeostasis

描述（由申请人提供）：拟议的研究将阐明响应乙醇(E)的神经元谷胱甘肽（GSH）稳态调节的基本机制，并将增强这些系统以增强神经保护。调节谷胱甘肽稳态的关键转录因子是Nrf2，它通过与抗氧化反应元件（ARE）结合来驱动必要的转录。假设。核心假设是3-谷氨酰循环的成分，包括神经元内细胞GSH稳态成分，可以上调以保护神经元免受e相关的凋亡性死亡。实验将确定这些成分依赖于e的调节的基本机制，并将其扩展到将减轻e介导的神经元凋亡的干预措施。方法。体内研究将利用大鼠和小鼠暴食模型。体外模型将是胎儿大脑皮质神经元的原代培养。方法将包括转染WT和显性阴性对照，RNA沉默，RT-PCR，荧光素酶测定和凝胶移位。具体目标1：在Nrf2/ARE水平上，通过优化神经元GSH稳态机制的特定成分，可以缓解乙醇相关神经元损伤的假设。目的是利用Nrf2/ARE细胞保护系统在两个水平上防止e介导的凋亡神经元死亡；首先通过增加控制谷胱甘肽合成及其还原状态的酶（3 -谷氨酰半胱氨酸连接酶、谷胱甘肽还原酶）的表达，其次通过增强对谷胱甘肽清除系统（-谷氨酰转肽酶（3GT）和氨基肽酶n）的调节，实验将确定乙醇对控制这些蛋白质表达的转录和转录后事件的影响机制。他们将通过调控神经元谷胱甘肽稳态机制关键控制点上Nrf2/ARE驱动的转录，开发减轻e诱导的凋亡死亡的方法。具体目标2。确定乙醇影响神经元中Nrf2表达的机制。初步研究表明，E诱导神经元中Nrf2蛋白的上调，可能发生在转录和/或转录后水平。然而，这不足以最佳地保护细胞免受乙醇介导的凋亡死亡。因此，我们将阐明乙醇是如何影响Nrf2表达的，目的是最终增强神经保护潜力。Nrf2的调控将首先通过翻译后修饰、Keap1依赖的氧化还原开关和泛素化来解决，其次通过启动子水平的调控来解决。具体目标3。为了将这些概念扩展到活体动物，使用了一个有充分证据的大鼠模型和一个转基因小鼠模型（Nrf2-/-）。实验将验证Nrf2在发育中的大脑中的激活可以增强对e介导的神经元损伤的保护。这些研究将确定GSH稳态控制成分的发育特征，它们对E的反应，Nrf2和3GT在细胞保护中的作用，以及增强这种神经保护的方法。

DESCRIPTION (provided by applicant): The proposed studies will elucidate fundamental mechanisms underlying regulation of neuron glutathione (GSH) homeostasis in response to ethanol (E) and will enable augmentation of these systems to enhance neuroprotection. A key transcription factor regulating GSH homeostasis is Nrf2 which drives requisite transcriptions via binding to the antioxidant response element (ARE). Hypothesis. The central hypothesis is that components of the 3-glutamyl cycle, including internal cellular GSH homeostasis components in neurons, can be up-regulated to protect neurons from E-related apoptotic death. Experiments will define essential mechanisms of ARE-dependent regulation of these components and extend this to interventions that will mitigate E-mediated apoptotic death of neurons. Methods. In vivo studies will utilize rat and mouse binge models. In vitro models will be primary cultures of fetal cerebral cortical neurons. Approaches will include transfections with WT and dominant negative controls, RNA silencing, RT-PCR, luciferase assays, and gel shifts. Specific Aim 1: To address the hypothesis that alleviation of ethanol-related damage to neurons can be achieved by optimizing specific components of neuron GSH homeostasis machinery, at the Nrf2/ARE level. The intent is to utilize the Nrf2/ARE cytoprotective system to prevent E-mediated apoptotic neuron death at two levels; first by increased expression of enzymes controlling GSH synthesis and its reduced state ( 3-glutamyl cysteine ligase, glutathione reductase) and second by enhancing regulation of a GSH scavenging system (;-glutamyl transpeptidase (3GT), and aminopeptidase N. Experiments will define mechanisms of ethanol effects on transcriptional and posttranscriptional events controlling expression of these proteins. They will develop means to mitigate E-induced apoptotic death by manipulating Nrf2/ARE driven transcription at key control points of neuron GSH homeostasis machinery. Specific Aim 2. To determine mechanisms by which ethanol impacts on Nrf2 expression in the neuron. Preliminary studies show that E elicits the up-regulation of Nrf2 protein in neurons which could occur at transcriptional and/or posttranscriptional levels. However, this is insufficient to optimally protect the cells from ethanol-mediated apoptotic death. Thus, we will elucidate how ethanol impacts on Nrf2 expression, with the intent of ultimately enhancing neuroprotective potential. Regulation of Nrf2 will be addressed first by post-translational modifications, Keap1 dependent redox switching, and ubiquitination, and second by regulation at the promoter level. Specific Aim 3. To extend these concepts to the live animal using a well-documented rat model and a transgenic mouse model (Nrf2-/-). Experiments will test the hypothesis that Nrf2 activation in the developing brain enhances protection against E-mediated neuron damage. These studies will determine developmental profiles of controlling components of GSH homeostasis, their responses to E, the role of Nrf2 and 3GT in cytoprotection from E, and means to enhance this neuroprotection.