The long term objective of the proposed study is to test the validity of the oxidative stress hypothesis of aging, which postulates that deleterious changes caused by reactive oxygen species (ROS) are the primary cause of senescence. The historical approach to establish causality between oxidative stress and aging have been the manipulation of the latter by overexpression of antioxidant enzyme defenses, however, results of such studies have been quite equivocal. In the prospective study, an alternative approach that alters the rate of mitochondrial production of ROS will be used to manipulate oxidative stress. Mitochondrial superoxide generation is known to be controlled by the activity of cytochrome c oxidase (COX), the terminal component of the mitochondrial electron transport chain. In Drosophila melanogaster, COX activity and the abundance of some COX subunits, encoded in both nuclear and mitochondrial DNA, decline while mitochondrial ROS generation increases during aging. Experimental decrease in COX activity causes an increase in mitochondrial ROS production. Accordingly, the specific hypothesis to be tested is that enhancement of COX activity will decrease the rate of mitochondrial superoxide and hydrogen peroxide generation, lower the level of oxidative stress, and extend the life span of D. melanogaster. The three specific aims are: (I) Determine the effect of over- and underexpression of nuclear DNA-encoded COX subunits, exhibiting age-associated decline in abundance on COX activity, mitochondrial production of superoxide/hydrogen, life span, markers of oxidative stress and aging in transgenic D. melanogaster. (II) Determine the effects of overexpression of mitochondrial DNA- encoded COX subunits, showing decline with age, on COX activity, life span and indicators of oxidative stress and aging. (Ill) Determine the effects of co-overexpression of selected COX subunits on COX activity and various indicators of aging. A novel feature of this study is that results should indicate whether attenuation of the rate of production of ROS rather than their elimination after they have been generated would lower the level of oxidative stress, slow the age-associated changes and extend the life span of flies. More broadly, results should provide a direct test of the validity of oxidative stress hypothesis.
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