Related Experiment Videos
mtDNA replicative potential remains constant during ageing: polymerase gamma activity does not correlate with age
R M Kapsa1, A F Quigley, T F Han
1Melbourne Neuromuscular Research Centre and Department of Clinical CSIRO Division of Molecular Science, Parkville Laboratory, Parkville, Victoria 3052, Australia. rkapsa@ariel.unimelb.edu.au
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.
Insights
Mitochondrial DNA polymerase gamma (Polgamma) activity remains stable during aging, suggesting it does not drive age-related bioenergetic decline. This new assay aids in studying mitochondrial diseases.
Area of Science:
- Biochemistry
- Cell Biology
- Aging Research
Background:
- Age-related decline in oxidative phosphorylation (OxPhos) is a known phenomenon in human tissues.
- Mitochondrial DNA (mtDNA) depletion and replication defects are linked to OxPhos dysfunction in diseases and aging.
- Previous research indicates that certain AIDS treatments can cause OxPhos defects via mtDNA depletion.
Purpose of the Study:
- To investigate the potential link between mtDNA replication and age-related bioenergetic decline.
- To develop and validate an in vitro assay for mitochondrial DNA polymerase gamma (Polgamma) function in small tissue samples.
Main Methods:
- Developed an in vitro assay to measure Polgamma activity in platelet homogenates.
- Characterized Polgamma activity kinetics (Km, Vmax) and inhibition by ethidium bromide.
- Assessed Polgamma activity in 45 subjects and compared it with age-related changes in cytochrome oxidase (CytOx) and citrate synthase activities.
Main Results:
- Platelet Polgamma exhibited Michaelis-Menten kinetics and was inhibited by ethidium bromide.
- Polgamma activity showed no significant age-related decline.
- A significant age-related decrease in cytochrome oxidase (CytOx) activity was observed in elderly individuals (>50 years).
Conclusions:
- Cellular mtDNA Polgamma levels appear to be maintained during aging, independent of bioenergetic status or mitochondrial density.
- The processive activity of Polgamma is unlikely to be a major contributor to age-related bioenergetic decline.
- The developed assay is valuable for studying Polgamma activity in conditions involving mtDNA depletion or rearrangement.