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）：拟议的研究将阐明神经元谷胱甘肽（GSH）稳态的基本机制，以响应乙醇（E），并将增强这些系统以增强神经保护作用。调节GSH稳态的关键转录因子是NRF2，它通过与抗氧化剂响应元件结合（AS）来驱动必要的转录。假设。中心假设是，可以上调3-谷氨酰基循环的成分，包括神经元中的内部细胞GSH稳态成分，以保护神经元免受电子相关的凋亡死亡的影响。实验将定义对这些组件的依赖性调节的基本机制，并将其扩展到可以减轻神经元的E介导的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。体外模型将是胎儿脑皮质神经元的原发性培养物。方法将包括具有WT和显性阴性对照的转染，RNA沉默，RT-PCR，荧光素酶测定和凝胶移位。具体目的1：解决以下假设：可以通过优化NRF2/是NARF2/是水平的神经元GSH稳态机械的特定组件来减轻与神经元的损害。目的是利用NRF2/是细胞保护系统来防止在两个层次上进行电子介导的凋亡神经元死亡。首先，通过增加控制GSH合成的酶的表达及其降低（3-谷氨酰基半胱氨酸连接酶，谷胱甘肽还原酶），然后通过增强对GSH清除系统的调节（; -glutamylylymyl thrutamyl transeptidase（3GGT）和氨基肽N.的实验性效应机械效应的透明度效应，从这些蛋白质将发展为通过操纵NRF2/的凋亡死亡，这是在神经元GSH的关键控制点上驱动的转录。但是，/或转录后水平。 NRF2的调节将首先通过翻译后修改，KEAP1依赖性氧化还原切换和泛素化来解决，其次是在启动子级别进行调节。特定目的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动物。实验将检验以下假设：发育中的大脑中的NRF2激活增强了对电子介导的神经元损伤的保护。这些研究将确定控制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中的细胞保护中的作用，以及增强这种神经保护作用的方法。