RNase L IN EXCITOTOXIN MITOCHONDRIAL mRNA DEGRADATION

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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.