A substantial amount of evidence has indicated that a progressive accumulation of damage, including oxidation and alkylation derivatives of nucleotides and the products of their mutagenic activity, large deletions and small deletions/insertions, occurs during aging in mitochondrial DNA (mtDNA) of skeletal muscle, heart, liver and other tissues of man and other mammals. Furthermore, an aging-dependent decline in oxidative phosphorylation capacity has been demonstrated in several tissues of man. In view of the dual genetic control, nuclear and mitochondrial, of the assembly and function of the oxidative phosphorylation apparatus, a critical test of the role that the progressive structural damage of mtDNA plays in the observed aging-dependent decline in oxidative phosphorylation capacity requires the availability of an approach which would distinguish between effects of nuclear gene mutations or activity changes and effects of mtDNA mutations. We have recently developed a generalized method for mitochondria-mediated transformation of human cells, which utilizes mtDNA- less cells as recipients, and applied successfully this method for the construction of cellular models of encephalomyopathies caused by mtDNA mutations. We intend to use this approach to investigate the role that the progressive accumulation of mutations that occurs with aging in mtDNA plays in the senescence processes. For this purpose, we plan to transfer by cytoplast fusion mitochondria of several proliferating, specialized cell types (fibroblasts, myoblasts, lymphoblasts) from human individuals of different ages, from fetuses to centenarians, into human mtDNA-less cells. The cybrids thus produced will be examined for 1) overall respiratory capacity; 2) mitochondrial protein synthesis; 3) assembly and activity of individual respiratory complexes; and 4) mtDNA content and structural alterations. In another part of the proposal, we plan to test the aging-dependent structural and functional alterations occurring in mtDNA of blood platelets, taken as an accessible representative of a post- mitotic, fully differentiated cell type, by analyzing the cybrids produced by mtDNA-less cell x platelet fusion.
