The long-term goal of this proposal is to elucidate the mechanism by which mitochondrial oxidative stress produces dopaminergic neuronal death in Parkinson's Disease (PD). The precise mechanism by which mitochondrial oxidative stress, bioenergetic decline and iron overload arise and collaborate to produce age-related neuronal death in Parkinson's disease remains unclear. It is hypothesized that neuronal damage in Parkinson's disease results, in part from direct superoxide radical toxicity due to oxidative inactivation of mitochondrial aconitase. The hypothesis predicts that superoxide production, arising from Complex I inhibition or abnormal dopamine metabolism, inactivates [4Fe-4S]Z+-containing mitochondrial aconitase, resulting in loss of aconitase activity and release Fe z+ and H202. Posttranslational modification of this key TCA cycle enzyme can therefore result in an increased iron load, oxidant burden and bioenergetic decline. The presence of an iron responsive element (IRE) in the 5' untranslated region of the mitochondrial aconitase Mrna provides an additional mechanism for iron dysregulation in Parkinson's disease. The proposal will utilize human PD samples, animal models of PD (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and 6-hydroxydopamine) and dopaminergic cell culture models in conjunction with a diversity of tools and techniques that include biochemical analyses, confocal microscopy, molecular biology and transgenic/knockout/aging mice.
Specific Aim 1 will determine whether mitochondrial aconitase is inactivated in human and experimental Parkinson's disease. The influence of aging and chronic mitochondrial oxidative stress will be determined using mice deficient in MnSOD, a critical mitochondrial antioxidant.
Specific Aim 2 will determine whether mitochondrial aconitase inactivation contributes to impaired iron homeostasis.
Specific Aim 3 will determine whether scavenging mitochondrial superoxide using native or synthetic antioxidants (e.g. MnSOD transgenic mice or metalloporphyrins) protect against mitochondrial aconitase inactivation in a manner that correlates with decreased iron overload and neuronal death in experimental Parkinson's disease.
Specific Aim 4 will determine the downstream consequences of mitochondrial aconitase inactivation in experimental Parkinson's disease. Specifically, regulation of brain mitochondrial aconitase synthesis by the 5' IRE in its mRNA, impact on the TCA cycle capacity and direct neurotoxicity of aconitase gene silencing will be examined. These studies can advance our understanding of the oxidative mechanisms of neuronal death in Parkinson's disease and suggest novel therapeutic strategies for rescuing neurons from age-related neurodegeneration. Additionally, this line of investigation may explain Parkinson's disease arising from genetic factors uncovered by aging as well as environmental factors. ? ?
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