application): Harman originally proposed that cellular damage by oxygen-free radicals may be the principle driving force for aging and substantial evidence has accumulated that suggests a major contribution of reactive oxygen species (ROS) to the age-dependent deteriorization of physiological functions. Because mitochondria constitute the main site of free radical generation (and other reactive oxygen forms) in the cell, they represent a significant and highly vulnerable target of oxidative stress. Oxidant stress is also a well-known inducer of apoptosis, causing programmed cell death by compromising mitochondrial structure and function. The findings that mitochondria: a) generate reactive oxygen species; b) are targets of oxidative stress; c) initiate oxidant-induced apoptosis; and d) exhibit increased oxidant-induced damage with age, suggest that oxidant-induced alterations in mitochondrial function may underlie in part the aging phenotype. Unfortunately, the mechanisms by which oxidant stress damages mitochondria and leads to dysfunction remain unknown. Using a variety of transgenic mouse models coupled with new and novel optical imaging approaches, the investigators in this proposal will test the hypothesis that oxidative stress contributes to aging by alterations in mitochondrial structure and function. One theory advanced to explain aging is failure of normal apoptotic regulation, leading to accumulation of damaged cells and/or loss of differentiated ceils. In Project 1, the hypothesis that that oxidative stress over the lifespan of an organism, leads to failure of normal apoptotic regulation, resulting in accelerated apoptosis contributing to aging will be examined. Age associated increases in mtDNA deletions and other mutations, increased oxidative damage to mtDNA and increased levels of aberrant forms of mtDNA have all been observed. Project 2 will examine the hypothesis that increased oxidative base damage in mtDNA leads to increased mitochondrial dysfunction and aging. If free radical reactions are the major cause of aging, over expression of cellular/mitochondrial antioxidants should in principle retard aging with a concomitant increase in maximum lifespan. Project 3 will test the hypothesis that altered steady state accumulation of oxidative damage in mitochondria (through transgenic manipulation of mitochondrial antioxidant levels), results in altered mitochondrial function and aging. Oxidant-induced mitochondrial damage has been implicated in a number of age-dependent neuronal diseases, and may involve oxidant-induced alterations in mitochondrial Ca2+ metabolism. Studies in Project 4 will examine the hypothesis that age-dependent oxidant stress alters astrocyte mitochondrial Ca2+ signaling leading to increased neuronal excitotoxicity and apoptosis. Lastly, two of the most important lipid peroxides formed during oxidant stress are 4-hydroxynonenol and malondialdehyde. Project 5 will test the hypothesis that age-related oxidative stress damages key components of the mitochondrial respiratory chain by oxidation of cardiolipin and by direct inhibition by 4-hydroxynonenal, a toxic product of fatty acid oxidation.
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