To investigate the role that aging-dependent accumulation of mitochondrial DNA (mtDNA) mutations plays in the senescence processes, mitochondria from fibroblasts of 21 normal human individuals between 20 weeks (fetal) and 103 years of age were introduced into human mtDNA-less ( o ) 206 cells by cytoplast ؋ o cell fusion, and 7-31 transformant clones were isolated from each fusion. A slight cell donor age-dependent decrease in growth rate was detected in the transformants. Using an O 2 consumption rate of 1 fmol/min/cell, which was not observed in any transformant among 158 derived from individuals 20 weeks (fetal) to 37 years of age, as a cutoff to identify respiratory-deficient clones, 11 such clones were found among 198 transformants derived from individuals 39 -103 years of age. Furthermore, conventional and nonparametric analysis of the respiratory rates of 356 clones revealed a very significant decrease with donor age. In other analyses, a very significant age-dependent decline in the mtDNA content of the clones was observed, without, however, any significant correlation with the decrease in O 2 consumption rate in the defective transformants. These observations clearly indicate the occurrence in the fibroblast-derived transformants of two independent, age-related functional alterations of mtDNA, presumably resulting from structural damage to this genome.Since the free radical theory of aging was first proposed (1), a voluminous amount of data has accumulated indicating that free radicals contribute to the degeneration of biological systems (2-4). Over 90% of the oxygen consumed by mammalian cells is utilized in mitochondria, and up to 4% of this oxygen is transformed into excited oxygen species (5). It is assumed that oxygen-free radicals are produced in mitochondria at a rate proportional to cellular metabolism, and that reaction with these free radicals may cause progressive damage to mitochondrial macromolecules during the life of the organism, contributing to the phenotypic effects of aging (3, 6 -8). In particular, it has been hypothesized by Linnane and collaborators (6) that accumulation of mtDNA 1 mutations is a major contributor to aging and degenerative diseases, due to the high mutation rate of mtDNA, which is 10 times higher than that of single-copy nuclear genes (9), to the size and compactness of the mitochondrial genome, to the lack of histones, to the lack or inefficiency of repair mechanisms, and to the somatic segregation of mitochondrial genomes during cell division (6). Substantial support for this suggestion has come from biochemical, histochemical, and immunohistochemical evidence of a progressive deterioration with aging of the respiratory capacity of different tissues, which exhibits a characteristic intercellular mosaicism of mitochondrial dysfunction (10 -16), as well as from the demonstration of aging-related mtDNA damage in the form of large deletions (17-23), small deletions and insertions (24), and oxidative adducts of DNA (25-28). However, it has been impossible thus far to d...

No abstract

No abstract

Previous work had shown a large accumulation (up to 50% of mtDNA) of a noninherited T414G transversion at a critical control site for mtDNA replication in skin fibroblasts from the majority of human subjects above 65 years old, and its absence in younger individuals. In the present studies, long-term in vitro culture of several fibroblasts populations carrying the heteroplasmic T414G mutation revealed an outgrowth of the mutant cells by wild-type cells. This observation supported the previous conclusion that the mutation accumulation is an in vivo phenomenon, while, at the same time, indicating intrinsic physiological differences between mutant and wild-type cells. Furthermore, subcloning experiments revealed a striking mosaic distribution of the mutation in the original fibroblasts populations, as shown by its presence, in heteroplasmic or homoplasmic form, in a fraction (18-32%) of the fibroblasts, and its absence in the others. In other investigations, transfer of mitochondria from mutation-carrying fibroblasts into mtDNA-less 143B.TK- rho0 206 cells revealed the persistence of the mosaic distribution of the mutation, however, with a near-complete shift to homoplasmy. The generality of the latter phenomenon would exclude a founder effect by one or few mitochondria in the transformation experiments, and would rather point to the important role of the nuclear background in the in vitro behavior of the T414G mutation. The stability of the homoplasmic mutation in rho0 cell transformants provides a powerful tool for analyzing its biochemical effects.