Over the past decade numerous diseases affecting highly active metabolic tissues including the brain and skeletal musculature have been shown to be associated with mutations in the genome of the sub-cellular organelle, the mitochondrion. This organelle is of importance because of its critical role in aerobic metabolism. A number of diseases have been linked to mutations to the genome of this organelle, which is inherited in a maternal or non-Mendelain manner. Mendelian, or nuclear, inherited genetic variations and diseases have also been found to affect mitochondrial function, and structure. Previous research in this Laboratory, employing PCR assays to examine brain tissue from 43 age-comparable individuals, demonstrated 12- and 5 fold higher levels or the 4977-bp “common” mitochondrial DNA (mtDNA4977) deletion mutation in the putamen and the superior frontal gyrus of the cortex respectively in conditions associated with chronic hypoxia. Further examination of these tissue demonstrated a correlation between a decrease in the levels of genomic DNA adducts, 8-hydroxydeoxyguanine, (8-OHdG), and levels of mtDNA4977 deletions. Previous studies, monitoring translational activity and the efficiency of antibiotic resistance mutations, suggested effects of deletions could not be detected until the deletion levels approached 60% to 10% respectively. However, significant effects could be observed at mtDNA4977 deletion levels as low as 0.01%, by measuring 8-OHdG levels in genomic DNA. Recently, the Laboratory developed a highly efficient, reproducible in situ, and in vitro, PCR-based system to detect the mtDNA4977 deletions so that the deletions can now be studied in single cells. The development of these methods has resulted in a number of new collaborations with both intramural and extramural Agencies. Cytological identification and localization of the deletions can be useful in the investigation of the nature and genesis of various human diseases, and in radiation epidemiology studies. Indeed, the mtDNA4977 deletion mutation has been associated with different diseases including Pearsons syndrome, Kearns-Sayre syndrome, ophtalmoplegia, cardiomyopathy, as well as exposure to ionizing radiation. Chronic exposure to free radicals created by electron-transport-chain activity, absence of sophisticated repair mechanisms, and frequent mtDNA replication can contribute to accumulation of mutations in the mitochondrial genome. We had previously shown, in the mouse brain, the presence of two mtDNA deletions that are located in an analogous position to the human mtDNA4977 deletion. We are currently refining detection of these deletions in collaborative projects to develop in vivo detection of radiodosimetry in an animal model, and to investigate oxidative stress in animals transformed with the human APP protein.The human mtDNA4977 deletion is associated with apoptotic changes in the cultured lymphocytes from a mother, a son with Pearsons syndrome (containing the deletion at 50% levels), and an asymptomatic daughter. The distribution of the mtDNA4977 deletions in the cells ranged from a few deletions in isolated regions of cells, to cells that were saturated with mtDNA4977 deletions. These observations are consistent with progression of the deletion from a single locus to deletions throughout the cell. Maximal rate of oxygen consumption were measured in these cells by monitoring the electron transport chain (ETC) through complex I, or through complex II. The sons lymphocytes gave the lowest values and the mothers lymphocytes always gave the highest. The daughters lymphocytes gave values comparable to the sons through complex II and intermediate between the mothers and sons (although closer to the sons) through complex I. The results clearly show that sons and daughters lymphocytes have diminished capacities for oxygen consumption compared to the mothers, with the deficit worse in the sons. The cells from this family displayed the same susceptibility to apoptosis-inducing agents as control lymphocyte lines. Although the deletions affect oxygen consumption, they appear to have no effect on the induction of apoptosis when compared to control lymphocyte lines, suggesting further experiments to determine the role, if any, of the deletions in apoptosis.The apparent inheritance of the mtDNA deletion occurrence in the afore-mentioned family suggests that the deletion could result from an active enzymatic process. Thus, we began to search for potential mtDNA deletion enzymes. The mtDNA polymerase γ is one such enzyme. The Laboratory utilized cross-species hybridization (yeast to human and mouse) to identify the genomic sequences of the human (POLG) and mouse (Polg) genes. We are currently comparing the intron-exon configuration of the respective genomic sequences.The Laboratory is collaborating in experiments on the nature of oxidative damage in hypertension, Alzheimer’s disease, and aging. This collaboration derives from the fact that persons with hypertension are at high risk for cerebral infarcts and ischaemic subcortical lesions. Hypertension may also disrupt the blood-brain barrier, which has been suggested to be involved in the aetiology and pathogenesis of Alzheimers disease. Psychological stress, and the formation of free oxygen radicals have also been postulated to be involved. Thus, our understanding of mitochondria and oxidative damage in hypertension may provide clues to understanding the pathology of Alzheimers disease in a hypertension.Our recent gene therapy experiments suggest it may be possible to ameliorate both the neuropathological effects of somatic mitochondrial DNA mutations, and the effects of similar age-related mitochondrial mutations that have been observed in the brain. We used the mtDNA-encoded ATPase6 mutant gene of a chinese hamster ovary (CHO) cell line that confers oligomycin resistance by converting the mtDNA code of this gene to the nuclear DNA universal code and by attaching a mitochondrial targeting sequence to the synthetic gene. This construct was introduced into CHO cells sensitive to oligomycin. Transformed CHO cell lines have been isolated that are capable of growing in up to 0.1 ug/mL oligomycin while untransformed sensitive CHO cells are eliminated at only 0.001 ug/mL oligomycin. We also recently transformed human cybrids containing a mutation, the T8993G mutation, in the human mtDNA-encoded ATPase6 gene in 100% of the mitochondrial genomes with the CHO oli-r ATPase6 construct described above. The ATPase6 T8993G mutation results in a condition known as Leigh Syndrome, Subacute Necrotizing Encephalopathy (SNE), or NARP (neuropathy, ataxia, retinitis pigmentosa). Cybrids transformed with UOATP6 are growing in 0.1ug/mL oligomycin, while the untransformed cybrids are eliminated in 0.001ug/mL oligomycin.The Laboratory has established and is maintaining the MitoDat database (http://www-lecb.ncifcrf.gov/mitoDat/). The database consolidates information from various biological databases, e.g., GenBank and Online Mendelian Inheritance in Man. Because the mitochondrion has a central role in cellular metabolism, it is involved in most human diseases. This database should help us in studying these diseases.The Laboratory has taken the initiative to establish the Mitochondria Interest Group (MIG), and is involved in the formation of the Mitochondria Research Society (MRS) and the new journal “Mitochondria”, published by Elsevier. The Laboratory is currently adapting fluorescence in situ hybridization technologies for use in comparative genomic hybridization to study nuclear and mtDNA genomes in CNS regions in which increased levels of mtDNA deletions have been reported.
