Mitigation of age-dependent accumulation of defective mitochondrial genomes - PubMed (original) (raw)

Mitigation of age-dependent accumulation of defective mitochondrial genomes

Pei-I Tsai et al. Proc Natl Acad Sci U S A. 2022.

Abstract

Unknown processes promote the accumulation of mitochondrial DNA (mtDNA) mutations during aging. Accumulation of defective mitochondrial genomes is thought to promote the progression of heteroplasmic mitochondrial diseases and degenerative changes with natural aging. We used a heteroplasmic Drosophila model to test 1) whether purifying selection acts to limit the abundance of deleterious mutations during development and aging, 2) whether quality control pathways contribute to purifying selection, 3) whether activation of quality control can mitigate accumulation of deleterious mutations, and 4) whether improved quality control improves health span. We show that purifying selection operates during development and growth but is ineffective during aging. Genetic manipulations suggest that a quality control process known to enforce purifying selection during oogenesis also suppresses accumulation of a deleterious mutation during growth and development. Flies with nuclear genotypes that enhance purifying selection sustained higher genome quality, retained more vigorous climbing activity, and lost fewer dopaminergic neurons. A pharmacological agent thought to enhance quality control produced similar benefits. Importantly, similar pharmacological treatment of aged mice reversed age-associated accumulation of a deleterious mtDNA mutation. Our findings reveal dynamic maintenance of mitochondrial genome fitness and reduction in the effectiveness of purifying selection during life. Importantly, we describe interventions that mitigate and even reverse age-associated genome degeneration in flies and in mice. Furthermore, mitigation of genome degeneration improved well-being in a Drosophila model of heteroplasmic mitochondrial disease.

Keywords: aging; heteroplasmy; mitochondria; mtDNA; mutations.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Fig. 1.

Quality control modulates the ratio of heteroplasmic mitochondrial genomes during development. (A) Heteroplasmy for schematized _Yak-_mt and _Mel-_mtts genomes was established by transferring cytoplasm of D. yakuba embryos into D. melanogaster embryos carrying the doubly mutant genome mt:ND2del + mt:ColT300I (_Mel-mtt_s). (B) The proportion of _Yak-_mt (_Yak-_mt/total-mt) following development from egg to adult shows action of quality control. Eggs (2-h collection at 29 °C) were assayed (gray bar) or allowed to develop to 5 d after eclosion at 22 °C (blue) or 29 °C (amber). Adult females have a large contribution from oocyte mtDNA. (C) Quality control operates in multiple tissues with different effectiveness. The blue and amber bars (22 °C or 29 °C, respectively) show the proportion of Yak-mt in different tissues of late third instar larvae. (D) The impact of purifying selection declined with age in the CNS. (E) The decline in relative abundance of wild-type, indicated by the dashed regression lines (orange for 29°C and blue for 22°C), _Yak_-mt following eclosion was fast. (F) The signature of quality control is absent during maturation of adults. _Yak_-mt/total-mt ratios from gut and brain taken from 5-d (gray bars) or 20-d adults aged at 22 °C or 29 °C (blue and amber bars, respectively). Here and below, *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA/Tukey’s multiple comparison test. Data represent eight independent biological repeats, with each repeat being an average of ratios assessed in three samples of eggs or adults. For tissues, data represent tissues dissected from eight individuals. Error bars represent standard error. In E, slopes differ (*P < 0.05) by linear regression. Cyto, cytoplasm; AED, after egg deposition; ns, not significant.

Fig. 2.

Fig. 2.

The nuclear genotype impacts the maintenance of mitochondrial genome quality in adult brains. (A) Loss-of-function of Pink1 leads to decline in Yak-mt/total-mt, indicating that PINK1 helps sustain high levels of the functional genome. Data show genome ratios for 5-d-old flies of the indicated genotype raised at 29 °C. The Pink1B9/Y flies lack Pink1 function, while the Pink1B9/Y; Dp(1:3)DC026/+ flies carry a rescuing autosomal duplication of the normally X chromosomal PINK1 gene. The PINK1-deficient flies and rescued flies are sibs from the same cross and therefore have the same pool of maternally contributed mitochondrial genomes. (B) The influence of nuclear genotype on mitochondrial genome quality of adult brains. Bars show the ratio of _Yak-_mt/total-mt in eggs (gray) and in brains of newly eclosed (0 d: yellow) or 5-d-old (red) adults when the indicated genotypes are raised at 29 °C. Data reflect the consequences of zygotic dose-reduction of the tested gene. *P < 0.05; and ***P < 0.001 by one-way ANOVA/Tukey’s multiple comparisons test. Data represent eight independent biological repeats, with each repeat being an average of ratios assessed in three samples of eggs or adults. For tissues, data represent tissues dissected from eight individuals. Error bars represent standard error. ns, not significant.

Fig. 3.

Fig. 3.

Genetically induced improvements in mitochondrial genome quality and vigor during aging. (A) A time course during adulthood shows the consequence of removing one of the two alleles of the gene encoding the mtDNA polymerase (tamKO/+) on _Yak-_mt/total-mt (red) compared to two controls (black is wild type, and green is a revertant tam allele). Flies were held at 29 °C and sampled at the indicated times. Data represent tissues dissected from eight individuals. (B) Removing one allele of mtDNA polymerase gene suppresses age-associated decline in climbing activity in heteroplasmic flies (tamKO/+ in red, wild-type in gray, and revertant in green). Ten flies collected from an independent cross were grouped into a single task; 15∼25 tasks (150∼250 flies from 15 to 25 independent crosses) were tested for each genotype. (C) Example images of DA neurons (marked by anti-Tyrosine hydroxylase) in the PPL1 clusters of adult brains 20 d after eclosion, in heteroplasmic flies with the three nuclear genotypes indicated to the right of vertical bar. Data represent 20 to 30 brains collected from three independent experiments. (D) Quantification of DA neuron number in PPL1 clusters. *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA/Tukey’s multiple comparisons test. The error bars in A,B and D represent standard error. TH, tryosine hydroxylase; ns, not significant.

Fig. 4.

Fig. 4.

Pharmacological enhancement of PINK1 improves mitochondrial genome quality and vigor. (A) Kinetin treatment increases _Yak-_mt/total-mt in adulthood. Wild-type adult male heteroplasmic flies at 29 °C were fed (Materials and Methods) with solvent control (DMSO), 100 μM kinetin, or 100 μM adenine from day 3, and _Yak-_mt/total-mt was measured in brains at the times indicated. Data represent tissues dissected from eight individuals. (B) Kinetin-induced increase in _Yak-_mt/total-mt required Pink1. _Yak-_mt/total-mt increase in the CNS of control late third instar larvae (29 °C) that were fed kinetin, but not larvae lacking Pink1, Pink1B9/Y. Data represent tissues dissected from eight individuals. (C) Kinetin sustains climbing activity in heteroplasmic flies with age. Heteroplasmic males at 29 °C were fed with control (DMSO, gray), 100 μM adenine (green), or 100 μM kinetin (red) from day 3 and tested for climbing activity at the indicated times. Ten flies collected from an independent cross were grouped into a single task; 15∼25 tasks (150∼250 flies from 15 to 25 independent crosses) were tested for each genotype. (D) Kinetin treatment prevents neuronal loss in PPL1 clusters of heteroplasmic males. Quantification of neuron number of PPL1 clusters in each hemisphere in heteroplasmic male adult flies at 29 °C fed with DMSO, 100 μM adenine, or 100 μM kinetin from day 3 and assayed at day 20. Data represent 20 to 30 brains collected from three independent experiments. (E) Illustration of deletion (gray) located between two 9-bp repeat sequences (orange “R”) within minor arc. (F) Age dependency of deletion frequency in liver of C57BL6/J mice. Two samples from each of five mice were measured by ddPCR for each age group. (G) Kinetin effect on minor arc deletion levels in aged mouse liver. For 6 wk, 16.5-mo-old mice were fed chow supplemented with kinetin. This treatment reduced minor arc deletion levels 10-fold compared to control chow–fed mice of the same age. **P < 0.01; and ***P < 0.001 by one-way ANOVA/Tukey’s multiple comparisons test. The error bars represent standard error. sol., solution; OH and OL, origin for heavy and light strand replication, respectively; ns, not significant.

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