GENETIC ANALYSIS OF HYPOXIC CELL DEATH IN C. ELEGANS

描述(由申请人提供)：在美国，缺氧性细胞死亡比其他任何原因导致的死亡人数都多；中风是导致残疾的主要原因。然而，没有任何治疗方法显示出对抗缺氧性细胞死亡的益处。线虫中的各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，我们建议确定可以保护神经元免受缺氧性细胞损伤的蛋白稳定机制。我们的具体目标是：1)确定蛋白质稳态在细胞自主性和非自主性、早期和延迟性神经细胞死亡中的作用。利用一种突变体，其中所有细胞都受到保护，免受缺氧损伤， 我们将在神经元和心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细胞特异性RNAi来检测蛋白质稳态在细胞自主、非自主、早期和延迟的神经元死亡中的作用。2)明确翻译因子基因敲除增加缺氧损伤存活率的机制。在各种实验范例中，翻译抑制与耐缺氧有关。翻译抑制保护缺氧损伤的机制几乎被普遍归因于氧气消耗的减少。我们已经研究了不同翻译因子的敲除对线虫在低氧后存活的影响，并将耐低氧水平与耗氧量、对蛋白质动态平衡的破坏的抗性等特性进行了关联。低氧抗性与耗氧量的相关性很弱，只与对蛋白质动态平衡的扰动的抗性有很强的相关性。这认为，翻译抑制通过改善蛋白质的动态平衡来保护机体免受缺氧性损伤。着眼于已建立的蛋白平衡途径，我们建议利用各种线虫遗传试剂来定义翻译抑制保护缺氧的机制。3)检测蛋白质稳态化合物对小鼠海马神经元和线虫神经元即刻和延迟性缺氧损伤的保护作用。我们从RNAi敲除实验中得到了强有力的证据，即在缺氧/葡萄糖剥夺之前，蛋白平衡的调节是小鼠海马神经元存活的一个重要决定因素。我们现在建议确定蛋白抑制剂化合物是否具有神经保护作用，如果是，则确定何时具有神经保护作用。我们将测试各种类型的化学蛋白抑制剂调节剂。我们将在缺氧前或缺氧后加入药物，并在原代小鼠海马神经元培养中以及在我们的线虫神经细胞死亡模型中测量这些化合物是否以及何时可以提供神经保护。

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.