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

ETOH 介导的细胞凋亡的神经保护：Nrf2/ARE 控制 GSH 稳态

基本信息

批准号：
8713885
负责人：
GEORGE I HENDERSON
金额：
$ 31.15万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2016-05-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8713885
关键词：
Address Adenoviruses Animals Antioxidants Apoptosis Apoptotic Astrocytes Binding Brain Cells Cerebrum Cessation of life Cysteine Cytoprotection Data Development Dominant-Negative Mutation Electrophoretic Mobility Shift Assay Enzymes Ethanol Event Gamma-glutamyl transferase Gene Expression Genes Genetic Transcription Glutathione Glutathione Reductase Homeostasis In Vitro Intervention Life Ligase Luciferases Mediating Methods Modeling Molecular Mus NF-E2-related factor 2 Neurons Oxidation-Reduction Oxidative Stress Pathway interactions Post-Translational Protein Processing Proteins RNA Interference Rattus Regulation Response Elements Reverse Transcriptase Polymerase Chain Reaction Role System Testing Transcriptional Regulation Transfection Transgenic Mice Translating Ubiquitination Up-Regulation alanine aminopeptidase alcohol effect alcohol response fetal in vitro Model in vivo mouse model neuron apoptosis neuron component neuron loss neuroprotection overexpression oxidative damage prevent promoter protein expression public health relevance research study response stressor therapy development transcription factor

项目摘要

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.

描述（由申请人提供）：拟议的研究将阐明乙醇（E）响应神经元谷胱甘肽（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 GSH 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-谷氨酰循环的组成部分，包括神经元中的内部细胞 GSH 稳态组成部分，可以上调以保护神经元免受 E 相关的细胞凋亡死亡。实验将定义这些成分的 ARE 依赖性调节的基本机制，并将其扩展到减轻 E 介导的神经元凋亡死亡的干预措施。方法。体内研究将利用大鼠和小鼠暴饮暴食模型。体外模型将是胎儿大脑皮层神经元的原代培养物。方法包括用 WT 和显性阴性对照转染、RNA 沉默、RT-PCR、荧光素酶测定和凝胶迁移。具体目标 1：提出这样的假设：可以通过在 Nrf2/ARE 水平上优化神经元 GSH 稳态机制的特定组件来减轻乙醇相关的神经元损伤。目的是利用 Nrf2/ARE 细胞保护系统在两个层面上预防 E 介导的细胞凋亡神经元死亡；首先通过增加控制 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，其次通过增强 GSH 清除系统（;-谷氨酰转肽酶 (3GT) 和氨肽酶 N）的调节。实验将确定机制他们将开发通过操纵E诱导的细胞凋亡的方法来减轻乙醇对转录和转录后事件的影响。 Nrf2/ARE 在神经元 GSH 稳态机制的关键控制点驱动转录。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 会引起神经元中 Nrf2 蛋白的上调。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，我们将阐明乙醇如何影响。 Nrf2 表达，最终增强神经保护潜力。 Nrf2 的调节首先通过翻译后修饰、Keap1 依赖性氧化还原开关和泛素化来解决，其次通过启动子水平的调节来解决。具体目标 3. 使用有据可查的大鼠模型和转基因小鼠模型 (Nrf2-/-) 将这些概念扩展到活体动物。实验将检验这样的假设：发育中的大脑中 Nrf2 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概况、它们对 E 的反应、Nrf2 和 3GT 在 E 细胞保护中的作用，以及增强这种神经保护的方法。