GENETIC ANALYSIS OF HYPOXIC DEATH IN C ELEGANS

以中风和心肌梗塞形式的低分化细胞死亡是老年人死亡的最大单一原因。 美国的缺氧细胞死亡的基本分子机制尚未完全了解。 在遗传上易处理的模式生物如秀丽隐杆线虫和果蝇中的研究已经取得了进展， 在细胞死亡、缺氧感知和适应领域的重大进展。拟议的工作将利用 强大的遗传工具在C。探索蛋白质合成、运输和降解的操纵 作为控制缺氧细胞死亡的手段。我们的具体目标是：（1）确定机制， 氨酰-tRNA合成酶（阿尔斯）和其它翻译机器蛋白控制缺氧损伤。中 C中的屏幕。为了寻找产生耐缺氧性的突变，我们在rrt中分离出了一个错义突变， 1基因，该基因编码一种翻译所需的基本蛋白质-乙酰tRNA合成酶。这 specific aim提出了一个系统的研究翻译因子作为低氧敏感性的调节因子。通过 通过这些实验，我们应该了解哪些翻译因子是缺氧靶向的最佳候选因子。 保护我们还将了解什么水平的翻译抑制是耐缺氧所必需的。最后， 我们将了解是否广泛持有的假设，翻译抑制产生耐缺氧 通过减少能源消耗是正确的，或者如果保护机制更复杂。(2)检查 未折叠蛋白和未折叠蛋白反应（UPR）在缺氧损伤中的作用。多项研究主要 研究人员发现，缺氧导致未折叠蛋白质的增加， 死亡我们有证据表明，翻译抑制通过降低水平或毒性来保护缺氧 未折叠的蛋白质。利用C.线虫遗传工具，我们将进行更广泛的研究如何 翻译抑制、未折叠蛋白和细胞对未折叠蛋白的反应相互作用， 急性缺氧损伤(3)确定是否有直系同源的牵连C。elegans基因类似地控制 小鼠原代神经元缺氧性死亡。与病理学系的Jeff Milbrandt合作， 华盛顿大学，我们将确定是否减少基因的表达牵连在特定的目标 1和2保护小鼠神经元免受创伤和缺氧损伤。这些实验将实现两个 目的：首先，确定小鼠C.线虫的基因在 第二，这些基因是否可以靶向而没有基线神经毒性。 完成这些目标将提供一个更完整的理解机制， 抑制和未折叠蛋白反应控制缺氧敏感性，并将决定 这些途径的遗传操作对哺乳动物神经元的缺氧保护。

Hypoxic cell death in the form of stroke and myocardial infarction is the largest single cause of death in the United States. The fundamental molecular mechanisms of hypoxic cell death are incompletely understood. Studies in genetically tractable model organisms such as Caenorhabditis elegans and Drosophila have made major advances in the fields of cell death, hypoxia sensing, and adaptation. The proposed work will utilize the powerful genetic tools in C. elegans to explore manipulation of protein synthesis, trafficking, and degradation as a means to control hypoxic cell death. Our specific aims are: (1) Define the mechanisms whereby aminoacyl-tRNA synthetases (aaRS's) and other translational machinery proteins control hypoxic injury. In a screen in C. elegans for mutations that produce hypoxia resistance, we isolated a missense mutation in the rrt- 1 gene, which encodes an arginyl tRNA synthetase, a fundamental protein required for translation. This specific aim proposes a systematic study of translation factors as regulators of hypoxic sensitivity. Through these experiments, we should learn which translation factors are the best candidates to target for hypoxic protection. We will also learn what level of translational suppression is required for hypoxia resistance. Finally, we will learn whether the widely held assumption that translational suppression produces hypoxia resistance by reducing energy consumption is correct or if the mechanism of protection is more complex. (2) Examine the role of unfolded proteins and the unfolded protein response (UPR) in hypoxic injury. Multiple studies primarily with cancer cells have found that hypoxia results in an increase in unfolded proteins that may promote cell death. We have evidence that translational suppression protects from hypoxia by reducing the level or toxicity of unfolded proteins. Using C. elegans genetic tools, we will perform a more extensive examination of how translational suppression, unfolded proteins, and the cellular response to unfolded proteins interact to control acute hypoxic injury. (3) Determine whether the orthologs of the implicated C. elegans genes similarly control hypoxic death of mouse primary neurons. In collaboration with Jeff Milbrandt in the Department of Pathology at Washington University, we will determine whether reducing the expression of genes implicated in specific aims 1 and 2 protect mouse neurons from traumatic and hypoxic injury. These experiments will accomplish two goals: First, determine whether the mouse orthologs of the C. elegans genes play similarly important roles in hypoxic neuronal injury and second, whether these genes can be targeted without baseline neural toxicity. Completion of these aims will provide a more complete understanding of the mechanism whereby translational suppression and the unfolded protein response control hypoxic sensitivity and will determine the feasibility of genetic manipulation of these pathways for hypoxic protection of mammalian neurons.