GENETIC ANALYSIS OF HYPOXIC CELL DEATH IN C. ELEGANS

描述（由申请人提供）：在美国，缺氧细胞死亡导致的死亡人数超过任何其他原因；中风是导致残疾的主要原因。然而，没有任何疗法显示出对缺氧细胞死亡的益处。秀丽隐杆线虫的多种正向遗传筛选表明，蛋白质稳态对缺氧后的生存至关重要。利用线虫和小鼠海马神经元的互补方法，我们提出确定可以保护神经元免受缺氧细胞损伤的蛋白质平衡机制。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，

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.