RNase L IN EXCITOTOXIN MITOCHONDRIAL mRNA DEGRADATION

描述（由申请人提供）：线粒体基因表达的调控机制尚不清楚，尽管基因表达的调控改变可能与急性缺血性损伤的病理生理有关。我们观察到，添加Na+离子载体或兴奋性神经递质谷氨酸导致培养神经元线粒体dna编码mRNA （mt-mRNA）水平显著降低。这一发现出乎意料，因为将离子（Na+和Ca+）泵出细胞会消耗能量，而能量消耗通常会上调mt-mRNA的表达。我们的初步结果表明，降解mt-mRNA的rna酶是导致线粒体基因产物减少的原因。一种特殊的RNase， RNase L最近被报道在干扰素处理的细胞中降低mt-mRNA的稳定性。我们假设细胞内钠的升高（由Na+离子载体或兴奋性毒性引起）激活线粒体中的RNase L途径，导致mt-mRNA的加速降解，并导致神经元因额外的代谢损伤而死亡的易感性增加。这一假设将在以下具体目标中进行检验：确定RNase L是否介导细胞内钠升高时mt-mRNA的降解。方法：在缺乏RNase L （RNase L -/-）的细胞中，在Na+离子载体（莫能菌素）存在或不存在的情况下，mt-mRNA的半衰期将与野生型细胞进行比较。将编码RNase L抑制剂或反义RNase L的对照载体或载体转染PC12S嗜铬细胞瘤细胞，比较莫能菌素诱导的mt-mRNA降解率。目的2：确定RNase L是否介导兴奋毒性损伤的原代神经元培养物中mt-mRNA的降解。方法：比较RNase L敲除（RNase L -/-）和野生型小鼠制备的原代神经元培养物中谷氨酸诱导的mtmRNA减少和神经元死亡的程度。目的3：确定RNase l依赖性mtmRNA的减少如何影响细胞对代谢损伤引起的死亡的脆弱性。方法：线粒体基因表达和蛋白水平正常或降低的细胞对代谢损伤（如一氧化氮（NO.））的脆弱性。-诱导的细胞死亡将被研究。意义：确定代谢应激和炎症诱导的细胞损伤之间共同的潜在基本机制可能导致针对这两种情况的治疗干预措施的发展。

DESCRIPTION (provided by applicant): Mechanisms regulating mitochondrial gene expression are not well understood, although altered regulation of gene expression probably contributes to the pathophysiology of acute ischemic injury. We have observed that addition of a Na+ ionophore or the excitatory neurotransmitter glutamate causes a marked decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) in cultured neurons. This finding was unexpected, since pumping ions (Na+ and Ca+) out of the cell consumes energy, and energy consumption normally upregulates mt-mRNA expression. Our preliminary results suggest that the RNases that degrade mt-mRNA are responsible for the decrease in mitochondrial gene products. A specific RNase, RNase L has recently been reported to decrease the stability of mt-mRNA in interferon-treated cells. We hypothesize that that elevated intracellular sodium (caused by the Na+ ionophore or excitotoxicity) activates the RNase L pathway in mitochondria, causing accelerated degradation of mt-mRNA and resulting in increased vulnerability of neurons to death caused by additional metabolic insults. This hypothesis will be tested in the following specific aims: Aim # 1. To determine if RNase L mediates the degradation of mt-mRNA in cells subjected to elevated intracellular sodium. Approach: The half-life of mt-mRNA will be compared in the presence or absence of the Na+ ionophore (monensin) in cells that are deficient in RNase L (RNase L -/-) with that, of wild type cells. PC12S pheochromocytoma cells will be transfected with control vector or vector coding for either RNase L inhibitor or for antisense RNase L. The rates of monensin-induced mt-mRNA degradation in these cell lines will be compared. Aim # 2: To determine if RNase L mediates the degradation of mt-mRNA in primary neuronal cultures subjected to excitotoxic injury. Approach: The extent of the glutamate-induced mtmRNA decrease and neuronal death will be compared in primary neuronal cultures prepared from RNase L knock out (RNase L -/-) and wild type mice. Aim #3: To determine how RNase L-dependent decreases in mtmRNA affect vulnerability of cells to death caused by metabolic insults. Approach: The vulnerability of cells with normal or decreased mitochondrial gene expression and protein levels to metabolic insults such as nitric oxide (NO.) - induced cell death will be investigated. Significance: Identification of a common underlying basic mechanism between metabolic stress- and inflammation-induced cell injury could lead to development of therapeutic interventions to target both conditions.