Senior Scholar Award in Aging
California Institute of Technology
Aging-dependent Large Accumulation of Mutations at Specific Sites in Human Mitochondrial DNA Control Region
Recently, the use of a novel approach for specific detection of heteroplasmic mutations in mitochondrial DNA (mtDNA) has surprisingly revealed high copy point mutations (present in up to 50% of mtDNA) at specific positions in the control region of human fibroblast mtDNA from normal old, but not young individuals. Furthermore, longitudinal studies showed the appearance of the mutations in a given individual only at advanced age. Most strikingly, some mutations appeared in more than one individual. The occurrence of these mutations at sites critical for mtDNA replication and their abundance strongly argue for their having important functional effects.
An exciting very recent development of this work has been the discovery that an aging-dependent accumulation of specific mutations also occurs in the mtDNA control region of skeletal muscle, a post-mitotic tissue. Most significantly, there is a tissue specificity of such mutations, with the muscle exhibiting two aging-dependent mutations that were not previously found in fibroblasts of older subjects, and, similarly, with the site-specific point mutations discovered in fibroblasts being absent in muscle mtDNA. Such an unexpected tissue specificity of aging-related mtDNA mutations in a functionally critical mtDNA region points to tissue-specific factors controlling the appearance and/or expansion of these mutations.
The aims of the proposed project are:
1. To investigate the functional consequences of the aging-dependent mutations, with the initial attention being given to the most abundant one found in fibroblasts, i.e., a T to G transversion located at position 414 of mtDNA, in the middle of the promoter for the synthesis of the RNA primer of mtDNA heavy (H)-strand synthesis and for light (L)-strand transcription, and to the two mutations found in muscle. This aim will be pursued by both in vivo and in vitro approaches. The in vivo approach will take advantage of a powerful tool developed in our laboratory, involving the construction of transmitochondrial cell lines by mitochondria-mediated transfer of mutation-carrying mtDNA into human mtDNA-less cells. By this approach, cell lines carrying 100% mtDNA molecules with the fibroblast T414G mutation or 100% wild-type mtDNA molecules from the same individuals have already been constructed. The in vitro approach will use a transcription system developed in our laboratory to investigate the effects of the mutations on the synthesis of potential primers for H-strand synthesis and/or on L-strand transcription.
2. To search for the possible occurrence of proteins which bind to the mtDNA segment carrying the fibroblast-specific T414G mutation or to the mtDNA segments carrying the two muscle-specific mutations, and subsequently to clone their cDNAs and to investigate their role in mtDNA H-strand synthesis and/or L-strand transcription.
3. To analyze the mechanisms underlying the appearance of the T414G mutation at that particular position in fibroblast mtDNA and that of the two muscle-specific mutations at different positions, and their dramatic expansion, with particular attention being given to oxygen radical damage to mtDNA and to possible aging-related damage to the mtDNA-specific g-DNA polymerase.
4. To investigate the generality of the phenomenon, i.e., the aging-dependent occurrence in other tissues from normal individuals and from individuals affected by degenerative diseases and in cancer cells of the high copy specific mtDNA mutations found in fibroblast and muscle mtDNA or of other specific point mutations.