To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer's disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less ( 0 ) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with 0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in 0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

We addressed the question of whether both mitochondrial and cytoplasmic translation activities decreased simultaneously in human skin fibroblasts with the age of the donors and found that the age-related reduction was limited to mitochondrial translation. Then, to determine which genome, mitochondrial or nuclear, was responsible for this age-related, mitochondria-specific reduction, pure nuclear transfer was carried out from mitochondrial DNA (mtDNA)-less HeLa cells to four fibroblast lines, two from aged subjects, one from a fetus, and one from a patient with cardiomyopathy, and their nuclear hybrid clones were isolated. A normal fibroblast line from the fetus and a respiration-deficient fibroblast line from the patient were used as a positive and a negative control, respectively. Subsequently, the mitochondrial translation and respiration properties of the nuclear hybrid clones were compared. A negative control experiment showed that this procedure could be used to isolate even nuclear hybrids expressing overall mitochondrial respiration deficiency, whereas no respiration deficiencies were observed in any nuclear hybrids irrespective of whether their mtDNAs were exclusively derived from aged or fetal donors. These observations suggest that nuclear-recessive mutations of factors involved in mitochondrial translation but not mtDNA mutations are responsible for age-related respiration deficiency of human fibroblasts.It has been presumed that somatic mutations accumulate in mitochondrial DNA (mtDNA) much faster than in nuclear DNA because mitochondria are highly oxygenic organelles due to their function in producing energy, mtDNA lacks histones protecting it from mutagenic damage, and its repair systems are limited (1). Therefore, it has been proposed that the accumulation of various somatic mutations in mtDNA and the resultant decrease in mitochondrial respiratory function could be involved in aging processes in mammals (2-4). There have been many reports that the respiration capacity of mitochondria in highly oxidative tissues decreases during aging (4). Moreover, the accumulation of somatic and pathogenic mtDNA mutations, which have been shown to cause various kinds of mitochondrial encephalomyopathies (5-8), was also shown to increase with age in normal subjects (9, 10). However, as the nuclear genome encodes most mitochondrial proteins including factors necessary for replication and expression of the mitochondrial genome, it is possible that only mutations in the nuclear genome contribute to the age-related decline of mitochondrial respiratory function. In fact, there is no convincing evidence that mtDNA somatic mutations are responsible for this age-related phenotype.Previously, we observed age-related reduction of cytochrome c oxidase (COX) 1 activity and mitochondrial translation in cultured human skin fibroblasts isolated from donors of various ages (0 -97 years), and in studies on their mtDNA transfer to mtDNA-less ( 0 ) HeLa cells, we showed that mtDNA mutations were not responsible for the observed age...

For isolation of mouse mtDNA-less ( 0 ) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting 0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the 0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (⌬mtDNA 5823 ) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.

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