GENETIC ANALYSIS OF HYPOXIC CELL DEATH IN C. ELEGANS

线虫缺氧细胞死亡的遗传分析

基本信息

批准号：
8714068
负责人：
C. Michael Crowder
金额：
$ 49.52万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-12-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8714068
关键词：
Address Applications Grants Behavioral Brain Ischemia Caenorhabditis elegans Calcium Channel Categories Cell Death Cell Survival Cells Cessation of life Chemicals Consumption Coupled Cytoprotection Defect Disease Endoplasmic Reticulum Event Foundations Genes Genetic Genetic Determinism Genetic Screening Glucose HSF1 Hippocampus (Brain)Histone Deacetylase Inhibitor Homeostasis Homologous Gene Human Hypoxia Injury Invertebrates Lead Measures Mitochondria Modeling Mus Muscle Cells Mutant Strains Mice Mutate Mutation Myocardial Infarction Nematoda Neurons Oxygen Oxygen Consumption Pathology Pathway interactions Pharmaceutical Preparations Phenotype Proteasome Inhibitor Proteins RNA Interference Reagent Recovery Regulation Research Resistance Rodent Role Stress Stroke Surveys Testing Therapeutic Time Transgenic Organisms Translations Ursidae Family Work base cell injury cell type deprivation disability effective therapy efficacy testing follow-up genetic analysis improved inhibitor/antagonist killings mortality multicatalytic endopeptidase complex mutant neuron loss neuroprotection novel therapeutics permanent cell line phosphatase inhibitor research study response selective expression therapy development trait translation factor

项目摘要

DESCRIPTION (provided by applicant): Hypoxic cell death kills more people in the USA than any other cause; stroke is the leading cause of disability. However, no therapy has shown benefit against hypoxic cell death. A variety of forward genetic screens in C. elegans have implicated protein homeostasis as critical to survival after hypoxia. Using complementary approaches in C. elegans and mouse hippocampal neurons, we propose to define proteostasis mechanisms that can protect neurons from hypoxic cellular injury. Our specific aims are: 1) Determine the role of protein homeostasis in cell autonomous and non-autonomous, early and delayed, neuronal cell death. Utilizing a mutant where all cells are protected from hypoxic injury, we will selectively express a wild type copy of this gene in neurons and myocytes. We will utilize these unique transgenic strains and others that we will generate along with cell-specific RNAi to examine the role of protein homeostasis in cell autonomous, non-autonomous, early, and delayed, neuronal death. 2) Define the mechanisms whereby translation factor knockdown increase survival from hypoxic injury. Translational suppression has been associated with hypoxia resistance in a variety of experimental paradigms. The mechanism whereby translational suppression protects from hypoxic injury has been nearly universally attributed to a decrease in oxygen consumption. We have performed a survey of the effect of knockdown of various translation factors on C. elegans organismal survival after hypoxia and correlated the level of hypoxia resistances with oxygen consumption, resistance to perturbation of protein homeostasis, and other traits. The correlation of hypoxia resistance with oxygen consumption was weak and correlated strongly only with resistance to perturbations in protein homeostasis. This argues that translational suppression protects from hypoxic injury by improving protein homeostasis. Focusing on established proteostasis pathways, we propose to utilize a variety of C. elegans genetic reagents to define the mechanisms whereby translational suppression protects from hypoxia. 3) Examine the ability of protein homeostasis compounds to protect from immediate and delayed hypoxic injury of mouse hippocampal and C. elegans neurons. We have strong evidence from RNAi knockdown experiments that modulation of proteostasis before oxygen/glucose deprivation is an important determinant of survival of mouse hippocampal neurons. We now propose to determine whether and, if so, when proteostasis compounds are neuroprotective. We will test various categories of chemical proteostasis regulators. We will add the drugs before or after hypoxia and measure if and when these compounds can provide neuroprotection in primary mouse hippocampal neuronal cultures and in our C. elegans neuronal cell death models generated in specific aim 1.

描述（由申请人提供）：在美国，缺氧细胞死亡导致的死亡人数比任何其他原因都要多；中风是导致残疾的主要原因。然而，没有任何疗法显示出对抗缺氧细胞死亡的益处。秀丽隐杆线虫的各种正向遗传筛选表明蛋白质稳态对于缺氧后的生存至关重要。使用线虫和小鼠海马神经元的互补方法，我们建议定义可以保护神经元免受缺氧细胞损伤的蛋白质稳态机制。我们的具体目标是： 1) 确定蛋白质稳态在细胞自主和非自主、早期和延迟神经细胞死亡中的作用。利用所有细胞都受到保护免受缺氧损伤的突变体，我们将在神经元和肌细胞中选择性表达该基因的野生型副本。我们将利用这些独特的转基因菌株和其他与细胞特异性 RNAi 一起产生的菌株来检查蛋白质稳态在细胞自主、非自主、早期和延迟神经元死亡中的作用。 2) 定义翻译因子敲低可提高缺氧损伤存活率的机制。在各种实验范式中，翻译抑制与耐缺氧性有关。翻译抑制防止缺氧损伤的机制几乎普遍归因于耗氧量的减少。我们对各种翻译因子的敲低对缺氧后秀丽隐杆线虫有机体存活的影响进行了调查，并将缺氧抵抗水平与耗氧量、对蛋白质稳态扰动的抵抗力和其他性状相关联。耐缺氧性与耗氧量的相关性较弱，仅与对蛋白质稳态扰动的抵抗力强相关。这表明翻译抑制通过改善蛋白质稳态来防止缺氧损伤。着眼于已建立的蛋白质稳态途径，我们建议利用各种秀丽隐杆线虫遗传试剂来定义翻译抑制防止缺氧的机制。 3) 检查蛋白质稳态化合物保护小鼠海马和秀丽隐杆线虫神经元免受立即和延迟缺氧损伤的能力。我们从 RNAi 敲低实验中获得强有力的证据，表明缺氧/葡萄糖剥夺之前蛋白质稳态的调节是小鼠海马神经元存活的重要决定因素。我们现在建议确定蛋白质稳态化合物是否具有神经保护作用，如果是的话，何时具有神经保护作用。我们将测试各种类别的化学蛋白质稳态调节剂。我们将在缺氧之前或之后添加药物，并测量这些化合物是否以及何时可以在原代小鼠海马神经元培养物和特定目标 1 中生成的线虫神经元细胞死亡模型中提供神经保护。