RNase L IN EXCITOTOXIN MITOCHONDRIAL mRNA DEGRADATION

描述（由申请人提供）：调节线粒体基因表达的机制尚不清楚，尽管基因表达调节的改变可能有助于急性缺血性损伤的病理生理学。我们已经观察到，另外的Na+离子载体或兴奋性神经递质谷氨酸引起的线粒体DNA编码的mRNA（MT-mRNA）在培养的神经元的水平显着下降。这一发现是出乎意料的，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RNases负责线粒体基因产物的减少。一种特异性的RNase，RNase L最近被报道降低干扰素处理的细胞中mt-mRNA的稳定性。我们推测，细胞内钠离子升高（由Na+离子载体或兴奋性毒性引起）激活线粒体中的RNase L通路，导致线粒体mRNA加速降解，并导致神经元对额外代谢损伤引起的死亡的脆弱性增加。将在以下具体目标中检验这一假设：目标1。确定RNase L是否介导细胞内钠升高时mt-mRNA的降解。方法：将在RNA酶L缺陷的细胞（RNA酶L -/-）中存在或不存在Na+离子载体（莫能菌素）的情况下，与野生型细胞的mt-mRNA的半衰期进行比较。用对照载体或编码RNA酶L抑制剂或反义RNA酶L的载体转染PC 12 S嗜铬细胞瘤细胞。将比较这些细胞系中莫能菌素诱导的mt-mRNA降解速率。目的#2：确定RNase L是否介导原代神经元兴奋性毒性损伤后线粒体mRNA的降解。方法：将在由RNase L敲除（RNase L -/-）和野生型小鼠制备的原代神经元培养物中比较谷氨酸诱导的mtmRNA降低和神经元死亡的程度。目的#3：确定mtmRNA中RNase L依赖性降低如何影响细胞对代谢损伤引起的死亡的脆弱性。方法：具有正常或降低的线粒体基因表达和蛋白质水平的细胞对代谢损伤如一氧化氮（NO.）- 将研究诱导的细胞死亡。重要性：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的鉴定可能导致针对这两种情况的治疗干预的发展。

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.