A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells.

Journal Article Unusual pattern of mitochondrial DNA deletions in skeletal muscle of an adult human with chronic fatigue syndrome Get access Chunfang Zhang, Chunfang Zhang * Department of Biochemistry and Centre for Molecular Biology and Medicine, Monash UniversityClayton, Victoria 3168, Australia *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Alessandra Baumer, Alessandra Baumer Department of Biochemistry and Centre for Molecular Biology and Medicine, Monash UniversityClayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Ian R. Mackay, Ian R. Mackay Department of Biochemistry and Centre for Molecular Biology and Medicine, Monash UniversityClayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Anthony W. Linnane, Anthony W. Linnane Department of Biochemistry and Centre for Molecular Biology and Medicine, Monash UniversityClayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Phillip Nagley Phillip Nagley Department of Biochemistry and Centre for Molecular Biology and Medicine, Monash UniversityClayton, Victoria 3168, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 4, Issue 4, April 1995, Pages 751–754, https://doi.org/10.1093/hmg/4.4.751 Published: 01 April 1995 Article history Received: 16 November 1994 Revision received: 04 January 1995 Accepted: 04 January 1995 Published: 01 April 1995

A comprehensive hypothesis concerning the contribution of mitochondrial DNA (mtDNA) mutations to the human ageing process is reviewed and the implications for cellular bioenergy loss and pharmacological therapy are considered. The central idea is that random mutations in the population of mtDNA molecules of each cell occur throughout life, and that this is a major contributor to the gradual loss of cellular bioenergy capacity within tissues and organs, associated with general senescence and diseases of ageing. An elaboration of four major aspects of the general proposition, together with relevant supporting data, is presented. (1) An extensive array of deletions in mtDNA of many tissues of humans and other mammals has been observed to occur in an age-related manner. (2) The preservation and selection of fully functional mtDNA molecules in the female germ line cells is proposed to occur via a human mtDNA cycle, in which selective amplification of a limited number of mtDNA templates occurs during oocyte development. This proposal explains the endowment of normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA molecules. The phenomena of maternal inheritance and rapid fixation of sequence variants of mtDNA in mammals, as well as selection of cells based on mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics result from accumulated mtDNA damage during ageing, representing different rates of cellular bioenergy loss within individual cells of a tissue. The random segregation of mtDNA during cell division will also further contribute to the tissue energy mosaic. Cells unable to meet their particular bioenergy demand will become non-functional, leading to cell death; the bioenergy threshold is different for the various cell types in the tissues of the body. (4) In order to bioenergetically resuscitate cells and tissues suffering from impaired mitochondrial functions as a result of the ageing process, we propose that redox compounds may be used therapeutically in the pharmacological configurations of a by-pass strategy or as a redox sink therapy. The role of these compounds is to maintain at least part of the mitochondrial respiratory chain function (by-pass) as well as to maintain adequate levels of cellular NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic pathway, with some contribution from mitochondrial oxidative phosphorylation.