MtDNA replicative potential remains constant during ageing: polymerase gamma activity does not correlate with age related cytochrome oxidase activity decline in platelets.

Abstract

Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.