There is increasing evidence for involvement of reactive oxygen species (ROS) in many neurodegenerative diseases. A particularly vulnerable target within cells for both endogenous and exogenous ROS insults is mitochondrial DNA(mtDNA). Mutations in mtDNA whether inherited or acquired, lead to impaired electron transport chain functioning and cellular dysfunction. The present application is designed to address questions relating to the hypothesis that elevations in the lesion equilibrium in mtDNA in neurons resulting from the processing of damage caused by reactive oxygen species is an early key factor that sets in motion the cascade of events that ultimately leads to the phenotypes associated with certain neurodegenerative diseases. Specifically, we will focus on factors that are involved in disruption of the normal lesion equilibrium in mtDNA, which exists between the formation of oxidative damage and its subsequent removal by repair processes. This will be accomplished through the pursuit of two specific aims. The first specific aim is to assess how steps involved in mtDNA repair in neurons affect the re-establishment of the normal lesion equilibrium in mtDNA. These studies will use a new application for ligation-mediated PCR to determine the rate limiting step(s) for repair of mtDNA damage caused by ROS in cultured neurons. The second specific aim is to evaluate the effects of recombinant DNA repair proteins targeted to the mitochondria. These studies will use primary cultures of neurons that are transfected with genes that encode mtDNA repair enzymes to modulate mtDNA repair capacity and directly alter the lesion equilibrium following stress caused by ROS. When successfully completed, these studies will provide a more thorough understanding of the role that mtDNA damage and its subsequent repair can play in the pathogenesis of various neurodegenerative diseases and the normal process of aging. Also, they will provide an enhanced comprehension of the basic biology of neurons in general, and the mitochondrion in particular.