RNase L IN EXCITOTOXIN MITOCHONDRIAL mRNA DEGRADATION

兴奋毒素线粒体 mRNA 降解中的 RNase L

• 批准号: 6561761
• 负责人: KRISH CHANDRASEKARAN
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-15 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561761
• 关键词: PC12 cells; apoptosis; cerebral ischemia /hypoxia; endoribonucleases; fibroblasts; gene expression; gene targeting; genetic regulation; genetically modified animals; glutamates; granule cell; laboratory mouse; laboratory rat; messenger RNA; mitochondria; monensin; neurotoxins; nitric oxide; nucleic acid metabolism; pheochromocytoma; sodium ion; transfection /expression vector

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.

描述（由申请人提供）：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，但调节线粒体基因表达的机制尚不清楚。我们观察到，添加 Na+ 离子载体或兴奋性神经递质谷氨酸会导致培养神经元中线粒体 DNA 编码的 mRNA (mt-mRNA) 水平显着降低。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表达。我们的初步结果表明，降解 mt-mRNA 的 RNase 是线粒体基因产物减少的原因。最近有报道称，一种特定的 RNase，RNase L 会降低干扰素处理细胞中 mt-mRNA 的稳定性。我们假设细胞内钠含量升高（由 Na+ 离子载体或兴奋性毒性引起）会激活线粒体中的 RNase L 通路，导致 mt-mRNA 加速降解，并导致神经元更容易因额外的代谢损伤而死亡。该假设将在以下具体目标中进行检验： 目标#1. 确定 RNase L 是否介导细胞内钠浓度升高的细胞中 mt-mRNA 的降解。方法：在缺乏 RNase L (RNase L -/-) 的细胞中，将在存在或不存在 Na+ 离子载体（莫能菌素）的情况下，将 mt-mRNA 的半衰期与野生型细胞进行比较。 PC12S 嗜铬细胞瘤细胞将用对照载体或编码 RNase L 抑制剂或反义 RNase L 的载体转染。将比较这些细胞系中莫能菌素诱导的 mt-mRNA 降解率。目标#2：确定 RNase L 是否介导遭受兴奋性毒性损伤的原代神经元培养物中 mt-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 减少和神经元死亡的程度。目标#3：确定 RNase L 依赖性 mtmRNA 减少如何影响细胞因代谢损伤而死亡的脆弱性。方法：将研究线粒体基因表达和蛋白质水平正常或降低的细胞对代谢损伤（如一氧化氮 (NO.)）诱导的细胞死亡的脆弱性。意义：识别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会导致针对这两种情况的治疗干预措施的开发。