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

描述(申请人提供)：拟议的研究将阐明乙醇(E)对神经元谷胱甘肽(GSH)稳态调节的基本机制，并将使这些系统得以增强以增强神经保护。调节GSH动态平衡的一个关键转录因子是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。中心假设是，3-谷氨酰循环的组成部分，包括神经元内细胞内GSH稳态成分，可以上调，以保护神经元免受E相关的凋亡死亡。实验将确定这些成分依赖ARE调节的基本机制，并将其扩展到将减轻E介导的神经元凋亡死亡的干预措施。方法：研究方法。体内研究将利用大鼠和小鼠的暴饮暴食模型。体外模型将是胎儿大脑皮层神经元的原代培养。方法将包括WT和显性阴性对照、RNA沉默、RT-PCR、荧光素酶检测和凝胶移位。具体目标1：解决这一假设，即可以通过在Nrf2/ARE水平上优化神经元GSH动态平衡机制的特定组件来减轻乙醇对神经元的损伤。目的是利用Nrf2/ARE细胞保护系统在两个水平上防止E介导的神经元凋亡：第一，通过增加控制GSH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，第二，通过加强对GSH清除系统的调节(；-谷氨酰转肽酶(3GT)，和氨基肽酶N)，实验将确定乙醇对控制这些蛋白表达的转录和转录后事件的影响的机制。他们将开发通过操纵Nrf2/Are在神经元GSH稳态机制的关键控制点驱动转录来减轻E诱导的细胞凋亡的方法。具体目的2.确定乙醇影响神经元Nrf2表达的机制。初步研究表明，E可诱导神经元中Nrf2蛋白的上调，这种上调可能发生在转录和/或转录后水平。然而，这并不足以最好地保护细胞免受乙醇介导的细胞凋亡的影响。因此，我们将阐明乙醇如何影响Nrf2的表达，以期最终增强神经保护潜力。对Nrf2的调控将首先通过翻译后修饰、Keap1依赖的氧化还原转换和泛素化来解决，其次通过启动子水平的调控来解决。具体目的3.使用有充分证据的大鼠模型和转基因小鼠模型(Nrf2-/-)将这些概念扩展到活动物。实验将检验这一假设，即Nrf2在发育中的大脑中的激活增强了对E介导的神经元损伤的保护。这些研究将确定GSH稳态的控制成分的发育概况，它们对E的反应，Nrf2和3GT在E的细胞保护中的作用，以及加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.