The main objective of this application is to establish the functional significance of a novel type of aging-related fibroblast- or muscle-specific mtDNA point mutations, which accumulate with extraordinary nucleotide selectivity in the majority of old individuals above a certain age at critical control sites for mtDNA replication. In particular, it is planned to use cellular models of these mutations, constructed by mitochondriamediated transfer of the mutations into mtDNA-less (px) cells, in order 1) to determine in a definitive way whether these mutations have a positive or negative effect on mitochondrial function, by comparing, at the biochemical and bioenergetic level, cell lines carrying a given mutation in 100% or 0% of mtDNA in the same mitochondrial genome and in the same nuclear background; 2) to determine whether these mutations increase or decrease the rate of mtDNA synthesis and affect its initiation at different heavy-strand synthesis origins; 3) to establish whether any identified replicative advantage of the mtDNA harboring these mutations involves preferentially molecules structurally intact and functionally normal or molecules structurally damaged and functionally deteriorated. Furthermore, we will determine whether the muscle-specific mutations arise and/or expand in the differentiated fibers or in undifferentiated muscle precursor cells (satellite cells). It is also planned to investigate the role of the nuclear background and mtDNA haplotype in the observed effects, by analyzing mitochondrial transformants constructed with genetically different px cell lines and different mitochondria donors, to determine the role of the in vivo environment in the appearance and/or expansion of the fibroblast-specific T414G mutation, and to correlate the effects of the mutations in cellular model systems with their effects in ex vivo tissue or cells. The achievements of the above aims will have important implications for understanding the role of these novel mtDNA mutations in aging, as well as their possible involvement in aging-related degenerative diseases.
