GENETIC ANALYSIS OF HYPOXIC CELL DEATH IN C. ELEGANS

线虫缺氧细胞死亡的遗传分析

• 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
• 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

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，低分化细胞死亡比任何其他原因都要多;中风是残疾的主要原因。然而，没有治疗显示出对缺氧细胞死亡的益处。在C.秀丽隐杆线虫暗示蛋白质稳态对于缺氧后的存活至关重要。在C. elegans和小鼠海马神经元，我们建议定义蛋白质稳定机制，可以保护神经元免受缺氧细胞损伤。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，神经元细胞死亡中的作用。利用所有细胞都能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地表达该基因的野生型拷贝。我们将利用这些独特的转基因菌株和其他我们将产生的沿着细胞特异性RNAi来研究蛋白质稳态在细胞自主性、非自主性、早期和延迟性神经元死亡中的作用。2)明确翻译因子敲低增加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相关。翻译抑制保护免受缺氧损伤的机制几乎普遍归因于氧消耗的减少。我们已经进行了一个调查的影响敲低各种翻译因素对C。缺氧后线虫生物体的生存和相关的耐缺氧水平与耗氧量，抗蛋白质稳态的扰动，和其他性状。耐缺氧性与耗氧量的相关性很弱，仅与蛋白质稳态的抗干扰性密切相关。这表明，翻译抑制通过改善蛋白质稳态来保护缺氧损伤。针对已建立的蛋白抑制途径，我们建议利用多种C。elegans基因试剂来定义翻译抑制保护免受缺氧的机制。3)检测蛋白质稳态化合物对小鼠海马和C.线虫神经元我们从RNAi敲除实验中获得了强有力的证据，即在氧/葡萄糖剥夺之前调节蛋白质稳态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抑制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。我们将测试各种类型的化学蛋白质稳定调节剂。我们将在缺氧前或缺氧后加入药物，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我们的C。elegans神经元细胞死亡模型在特定目标1中产生。