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Faculty List
Giuseppe Attardi
attardi@caltech.edu
M.D. 1947, University of Padua, Italy
Genetics of Human Mitochondrial Function
Recently, a considerable part of our efforts has been aimed at understanding the phenomenon that we recently discovered of an aging-dependent large accumulation of specific point mutations in the control region of human mtDNA, in particular at establishing the generality of the phenomenon, its mechanism and its functional relevance. Experiments of subcloning of fibroblast cultures carrying a high proportion of the most common of these point mutations, i.e., the T to G transversion at position 414 within the promoter for the primer of heavy mtDNA strand synthesis, and mitochondrial-mediated transformation of mtDNA-less cells have revealed the clonal nature of this phenomenon. Thus, a significant fraction of the original fibroblasts and of the cells in the derived transmitochondrial cell lines carry the mutation in a very high proportion of their mtDNA, up to a homoplasmic form. These findings have pointed to an intracellular replicative advantage of the mutant molecules, and to either multiple independent initiating events or a growth advantage of the mutant cells. Furthermore, long-term in vitro culture of fibroblasts carrying the mutation from three different individuals showed a clear tendency of the cultures to lose the mutation within two or three months, although at different rates. Fibroblast cultures from three individuals lacking the mutation failed to acquire it spontaneously during the same period. These experiments indicated that the observed dramatic expansion of the mutations requires an in vivo specific environment to occur.
As concerns the mechanism(s) leading to the appearance of these aging-dependent mutations, a start has been made towards identifying by gel-shift techniques, UV-crosslinking and affinity chromatography the proteins which interact with the mtDNA control region. Experiments are also in progress to analyze, by in vitro transcription experiments using as a template mtDNA carrying or lacking the 414G transversion, quantitative or qualitative changes in the pattern of synthesis of the primer for heavy mtDNA strand synthesis.
The search for an aging-dependent accumulation of specific point mutations in the mtDNA control region of other cell types, besides fibroblasts, in particular, of post-mitotic cells, has yielded positive results, revealing, however, an unexpected tissue specificity of such mutations. Thus, in muscle from older human individuals, mutations in the mtDNA control region have been identified that were not previously found in the corresponding mtDNA region from fibroblasts of older subjects. Conversely, the aging-dependent site-specific point mutations which have been discovered in fibroblast mtDNA control region have so far not been found in either muscle or blood cell mtDNA. Such a striking tissue specificity of aging-related mtDNA mutations in a functionally critical mtDNA region may be related to a tissue-specific susceptibility to oxidative damage or replication errors.
Considerable progress has also been made in the past year in the analysis of the early involvement of mitochondria in the apoptotic process. In Jurkat cells undergoing Fas-mediated apoptosis, the use of double-labeling confocal immunofluorescence microscopy has revealed a massive release of cytochrome c from mitochondria into the cytosol, which occurred in an increasing fraction of the cell population after the apoptotic stimulus. This release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and, with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Moreover, sorting on paramagnetic beads of fully apoptotic cells and apparently normal cells and an in-depth analysis of the respiratory changes have led to the surprising discovery of a rate-limiting step in cells already primed for apoptosis, but still respiring and with normal cytochrome c localization. Furthermore, most striking was the demonstration that controlled digitonin treatment can specifically bypass or accelerate this rate-limiting step, causing a rapid and massive cytochrome c release and complete loss of respiration. In another cellular system, osteosarcoma derived 143B cells induced to apoptosis by staurosporine, a surprisingly different sequence of events was detected, with cytochrome c release into the cytosol following a decrease in overall endogenous respiration rate and preceding a reduction in COX respiratory activity. The decrease in endogenous respiration was insensitive to the anti-apoptotic protein Bcl-2 and to the caspase inhibitor z-VADfmk. In still another system, PC12 phenochromocytoma cells induced to apoptosis by staurosporine, a fairly fast decrease in both endogenous and COX-dependent respiration rates in the first two to three hours after the apoptotic stimulus, and a much slower decrease of both types of respiration thereafter have been detected. The variability of the respiratory changes accompanying cytochrome c release in the different systems that we have analyzed reflects the great heterogeneity of the pathways leading to apoptosis in different cell types.
Analysis of disease-causing mtDNA mutations has continued to be actively pursued in our laboratory in the past year. Thus, definitive evidence has been obtained, by mitochondria-mediated transformation, that the nuclear background plays a determinant role in the manifestation of the biochemical phenotype underlying the non-syndromic deafness associated with a mitochondrial 12S rRNA mutation. Furthermore, a biochemical basis has been identified for the maternally-inherited susceptibility to aminoglycoside ototoxicity associated with the same mtDNA mutation. In another area, the role of post-transcriptional modifications of the human mitochondrial tRNAs in the expression of their structural and functional features, as well as in the pathogenesis of some tRNA-related diseases, like the MERRF and MELAS encephalomyopathies and maternally-inherited diabetes, has continued to be investigated. The efforts are presently being concentrated on the isolation and characterization of the methyltransferases responsible for methylation of N1A9 and N2G10 in mitochondrial tRNAs. Methylation at the first site has previously been shown to be very important for the folding of the tRNALys, while undermethylation at the second site has been demonstrated in this laboratory in the mutant tRNALeu(UUR) associated with the MELAS encephalomyopathy and maternally-inherited diabetes.
Mitochondria transfer into mtDNA-less ( r0) cells by fusion of enucleated r+ cells (enr+) with r0 cells in the presence of polyethylene glycol, and selection of the fusion products in the absence of uridine or pyruvate.
