The overall goal of the Principal Investigator's laboratory has been to elucidate the biochemical mechanisms underlying several models of neurodegeneration. A common theme in these studies has been the role of metabolic inhibition in mediating cell death. The applicant proposes to continue with studies on the intimate interaction between inhibition of energy metabolism and activation of excitotoxic processes initially involving the NMDA subtype of glutamate receptor. The studies will utilize two model systems: the well characterized ex vivo chick retina and the rat mesencephalic culture system. All of the proposed experiments are a direct outgrowth of studies done during previous grant periods. Metabolic stress will be induced by various paradigms which will decrease the ATP formed by glycolysis or mitochondrial oxidative phosphorylation.
Aim A will continue the PI's studies on acute excitotoxicity. These will expand previous work showing protection against metabolic stress and excitotoxicity by hypothermia, and include studies of temperature range effects on protection, measurement of ATP and phosphocreatine levels, and the possible role of Na+,K+-ATPase in this protection. Free fatty acid, arachidonate release and the role of reactive oxygen species during this acute toxicity will also be examined.
Aim B will carefully assess the mechanisms operative in delayed excitotoxicity including whether prior acute toxicity play any role. Using reversible glycolytic or mitochondrial inhibition, optimal temporal conditions will be determined, as will the role of NMDA and non-NMDA receptors, and the involvement of free radical and reactive oxygen species. The putative role of extracellular and intracellular calcium will also be determined.
In aim C the hypotheses concerning the role of the various intracellular messenger systems to mediate the toxicity will be examined. These will include the role of protein kinases A and C, interactions of hypothermia and protein kinase activation, and whether macromolecular synthesis is necessary for the delayed toxicity to occur.
Aim D will utilize the rat mesencephalic culture system to test the hypothesis that excitotoxicity is potentiated by energy depletion, especially in the dopaminergic cells of these cultures. Overall these studies will expand on previous findings from work funded by this grant to give a more detailed knowledge of the role of excitotoxicity in neurodegenerative processes and the interaction of this toxicity with metabolic stress leading to energy depletion in the CNS.
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