Timo E. S. Kauppila, Ana Bratic, Martin Borch Jensen, Francesca Baggio, Linda Partridge, Heinrich Jasper, Sebastian Grönke, and Nils-Göran Larsson

PNAS October 9, 2018 115 (41) E9620-E9629

Significance

Mutations of mtDNA accumulate in aging humans and other mammals to cause mitochondrial dysfunction in a subset of cells in various tissues. Furthermore, experimental induction of mtDNA mutations causes a premature aging syndrome in the mouse. To study if mitochondrial dysfunction is universally involved in shortening life span in metazoans, we generated a series of fruit fly lines with varying levels of mtDNA mutations. Unexpectedly, we report that fruit flies are remarkably tolerant to mtDNA mutations, as exemplified by their lack of effect on physiology and lifespan. Only an artificially induced, very drastic increase of the mtDNA mutation load will lead to reduced lifespan, showing that mtDNA mutations are unlikely to limit lifespan in natural fruit fly populations.

Abstract

Mammals develop age-associated clonal expansion of somatic mtDNA mutations resulting in severe respiratory chain deficiency in a subset of cells in a variety of tissues. Both mathematical modeling based on descriptive data from humans and experimental data from mtDNA mutator mice suggest that the somatic mutations are formed early in life and then undergo mitotic segregation during adult life to reach very high levels in certain cells. To address whether mtDNA mutations have a universal effect on aging metazoans, we investigated their role in physiology and aging of fruit flies. To this end, we utilized genetically engineered flies expressing mutant versions of the catalytic subunit of mitochondrial DNA polymerase (DmPOLγA) as a means to introduce mtDNA mutations. We report here that lifespan and health in fruit flies are remarkably tolerant to mtDNA mutations. Our results show that the short lifespan and wide genetic bottleneck of fruit flies are limiting the extent of clonal expansion of mtDNA mutations both in individuals and between generations. However, an increase of mtDNA mutations to very high levels caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan under standard conditions. In addition, the effects of dietary restriction, widely considered beneficial for organismal health, were attenuated in flies with very high levels of mtDNA mutations.

See http://www.pnas.org/content/115/41/E9620

Figure 1: Accumulation of mtDNA mutations with age, mtDNA turnover, and lifespan effects of DmPOLγA alleles. (A) Pulse-chase experiments with BrdU to detect mtDNA turnover in adult fruit flies. MtDNA was extracted from flies after pulse labeling and after 3, 6, 9, and 13 d of chase. MtDNA was digested using SacI, producing three fragments: 10.3, 5.2, and 4 kb (SI Appendix, Fig. S1A). The two shorter fragments were used for Southwestern blotting to detect mtDNA incorporated BrdU and the 10.3-kb fragment was detected using P³² -12S probe and used as a loading control. (B) Quantification of unique (white bar) and total (black bar) mtDNA mutation loads in thorax from young (1–3 d) and old (48 d) heterozygous mtDNA mutator flies with only somatic (+/D263A) or both inherited and somatic mutations (D263A/+). Error bars represent SD. Student’s two-tailed t test. (C) Quantification of unique (white bar) and total (black bar) mtDNA mutation loads from the WT progeny of young (1 d) and old (20 d) WT and heterozygous mtDNA mutator female flies. Error bars represent SD. Student’s two-tailed t test. *P < 0.05. (D) Lifespan analyses of flies inheriting DmPOLγA alleles paternally and therefore carrying only somatic mtDNA mutations. +/Rescue F₀ (red), +/D263A F₀ (blue), +/H1038A F₀ (green), and +/Q1009A F₀ (orange). P < 0.0001, log-rank test. (E and F) Lifespan analyses of flies originating from either (E) young (1 d) or (F) old (20 d) female flies. Only the progeny with WT nuclear background was used for experiments. Progeny from WT mother (black), +/Rescue F₀ mother (red), and +/D263A F₀ mother (blue). P < 0.0001, log-rank test. Flies labeled as +/D263A inherited the D263A allele paternally (no inherited mtDNA mutations), whereas D263A/+ flies inherited the D263A allele maternally (inherited and somatic mtDNA mutations). In this way, fly genotypes are always presented as follows: DmPOLγA allele from mother/DmPOLγA allele from father.