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Aging, The Molecular Concepts

2.5. MITOCHONDRIAL DECLINE AND mtDNA MUTATIONS IN AGING

Mitochondrial OXPHOS has been shown to decline in a variety of post-mitotic tissues including skeletal muscle (60-62), liver (63), and brain (64). This is correlated with the accumulation of the wide spectrum of somatic mtDNA mutations, including rearrangements and base substitutions. Using long extension-polymerase chain (LX-PCR) to amplify full-length mtDNAs (16.3 kb), the relative level and size distribution of mtDNA rearrangements has been assessed in tissue DNA samples. Applying this technique to DNA samples isolated from skeletal muscle of normal subjects of different ages revealed that DNA from donors under age 40 contained primarily full length mtDNAs, while DNA from donors over age 50 harbored a wide array of mtDNA molecules, indicating high levels of deleted mtDNAs. Since every subject had a different array of deletions, the deletions are likely to be the product of individual spontaneous somatic mutations. Hence, after age 40, humans accumulate a wide range of de novo mtDNA rearrangements in skeletal muscle (65).

The DNAs or two CPEO patients harboring the common 5 kb deletion were run as positive controls. In addition to expected full length linear, supercoiled and nicked circular molecules, the Southern blots of the CPEO patients had prominent linear and supercoiled deletion bands at 11.5 kb. Muscle DNA samples from individuals under age 40 gave only the three normal full length mtDNA bands. However, samples from individuals 54-77 years of age gave an additional diffuse band that ran at the same position in the gel as the supercoiled deletion molecules of the CPEO patients. The intensity of this additional band in the older subjects was comparable to that of the CPEO patients with 39 and 52% detected mtDNAs (65). Hence, it would appear that skeletal muscle of older individuals accumulates high levels of rearranged molecules.

One could observe that in normal hearts, the 5 kb deletion was essentially undetectable up to age 40. However, after age 40, the level rose steadily with age (66, 67). In the brain, it was found that the 5 kb deletion remained low in the cerebellum throughout life. However, the 5kb deletion was present in the cerebral cortex and basal ganglia and its level increased rapidly in those tissues after age 70. The cortical deletion levels reached a maximum of about 2-3%, while the basal ganglia level rose to 10-12% by age 80 (68). Since this assay only quantitates one of many deletions, it seems likely that the basal ganglia of elderly individuals must contain a large percentage of deleted mtDNAs. This is interesting because it is the basal ganglia cells that are preferentially lost in Leigh's patients with the np 8993 mutation and dystonia patients with the np 14459 mutation.

Quantitation of the 5 kb deletion by other groups has also shown the age-related rise in mtDNA damage in skeletal muscle (69, 70), heart (71), and brain (72). All of these observations are consistent with the age-related accumulation of deleted mtDNA.

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