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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.

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

“…The cell lines were described earlier by Michikawa et al (10). In particular, the AL4.8 and AL4.27 are homoplasmic wild-type at position 414 of the human mitochondrial genome, whereas the AL4.3 and AL4.5 carried the age-associated T414G transversion at a homoplasmic level.…”

“…The AL4.3, AL4.5, 1AC1.2 human cell lines, carrying in homoplasmic form the T414G transversion in the mtDNA and the AL4.8, AL4.27 and 1AC1.48 cell lines, carrying in homoplasmic form the wild-type version (T) of the same position (414) of the mtDNA, were previously isolated by transfer into human mtDNA-less 143B.TK − ρ 0 206 cells of mitochondria from fibroblasts of the individuals 100-3y (for the AL series of transformants) and 98y (for the 1AC series of transformants) (6,10). All cell lines were grown in DMEM, supplemented with 10% fetal bovine serum (FBS).…”

A mitochondrial etiology of Alzheimer and Parkinson disease

“…We have found that AD brains, but not control brains, harbor a mtDNA control region mutation, T414G. This mutation alters the mitochondrial transcription factor (Tfam) binding site associated with the L-strand promoter, which transcribes the ND6 gene [87, 88]. This mutation was originally observed to accumulate with age in human skin fibroblasts [89].…”

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

“…Most strikingly, a T414G transversion was found in a high proportion (up to 50%) of mtDNA molecules from individuals above 65 years of age. However, when several fibroblasts populations carrying the heteroplasmic T414G mutation were grown in vitro , outgrowth of the mutant cells by wild-type cells was observed ( 101 ). This led to the suggestion that accumulation of mtDNA point mutations is a phenomenon occurring exclusively in vivo.…”