Fig 1.
Schematic chart of experimental design.
Diagram outlining the experimental groups employed to study aging in the mouse and definition of mtDNA mutations into germline, embryonic and somatic origin. MtDNA samples were collected from bulk tissues or individual skin fibroblast-derived iPSC clones or clonally expanded skin fibroblasts from wt mice (2–34 months) and Polg mutator mice (2–9 months). All mtDNA samples were subjected to whole mtDNA sequencing by Miseq (Illumina).
Fig 2.
Characterization of mtDNA mutations in wild type mice with aging.
(A) Quantification of mtDNA mutations (mean ± SEM; asterisk, P < 0.05, Student’s t-test) for different mutation types in young wt mice at age 2–13 months (green bars; n = 31) and old mice at age 18–34 months (orange bars; n = 44). Somatic mutations were undetectable in wt mice. Asterisk represents a significant increase in number of germline mutations with age. (B) Mean heteroplasmy levels of non-synonymous germline and early embryonic mutations as a function of age. Error bars, mean ± SEM. Asterisk represents a significant increase in the mean heteroplasmy levels of non-synonymous germline mutations in old wt mice compared to the young group (P < 0.05, Student’s t-test). (C) Pie chart showing gene distribution of non-synonymous germline mutations in protein-coding and RNA coding genes in old wt mice. (D) Bar graphs showing mean heteroplasmy levels for non-synonymous germline mutations in protein-coding and RNA-coding genes in old wt mice. (E) Heteroplasmy of non-synonymous mutations in protein-coding and RNA-coding genes of early embryonic origin. (F) Pie chart showing relative proportion of mutation types in young and old wt mice. (G) Mitochondrial OXPHOS complex I activity in iPS cells carrying non-synonymous mutations in protein-coding genes and in age-matched control ESCs or iPS cells. The complex I activity was measured in cell homogenates (n = 12 per cell line, technical replicates) and was expressed as “% rotenone inhibition”. 10m-iPS7, 34m-iPS9 and 34m-iPS10 cells displayed reduced activities compared to controls. (H) Mitochondrial OXPHOS complex IV activity in iPS cells carrying non-synonymous mutations in protein-coding genes and in age-matched control ES or iPS cells. The complex IV activity was measured in cell homogenates (n = 12 per cell line, technical replicates) and was expressed as “nmol / min / mg protein”. 18m-iPS2 cells showed decreased activity compared to controls. In (G and H), ns denotes p ≥ 0.05.
Table 1.
Summary of mtDNA mutations detected in wild type and Polg mice.
Table 2.
Dynamics of mtDNA mutations during mouse aging.
Fig 3.
Characterization of mtDNA mutations in homozygous Polg mice with aging.
(A). Comparison of mean number of germline mutations in wt and Polg mice at young and old age (n = 31 for young wt, n = 8 for young Polg, n = 44 for old wt and n = 14 for old Polg). Error bars, mean ± SEM. Asterisk indicates a significant increase in the number of mutations per tissue in old Polg compared to the old wt (P < 0.05, Student’s t-test). (B). Mean heteroplasmy levels of non-synonymous germline mutations with ≥2% heteroplasmy in Polg mice (mean ± SEM; asterisk, P < 0.05, Student’s t-test). (C) Pie charts showing gene distributions of non-synonymous germline mutations in young Polg mice (2 months, left) and old Polg mice (9 months, right). (D) Bar graphs representing the mean heteroplasmy levels of non-synonymous germline mutations in protein-coding and RNA-coding genes in Polg mice (asterisks, P < 0.05, Student’s t-test). (E) Pie charts showing the distribution of shared and unique mtDNA mutations detected in single skin fibroblast (SF) clones in young and old Polg mice. (F) Mean heteroplasmy changes for non-synonymous somatic mutations with ≥15% heteroplasmy in Polg mice. Error bars, mean ± SEM. Asterisk, P < 0.05, Student’s t-test. (G) Changes in number of non-synonymous somatic mutations with heteroplasmy levels ≥ 15% among different gene types with Polg mice aging. Error bars, mean ± SEM. Asterisks, P < 0.05, Student’s t-test.
Fig 4.
Early embryonic and NCR mtDNA mutations in Polg mice.
(A) Bar graphs representing changes in mean heteroplasmy of early embryonic mutations with Polg mice aging. Error Bars, mean ± SEM. (B) Distribution of non-synonymous early embryonic mtDNA mutations among different genes. Error bars, mean ± SEM. (C) Quantification of mtDNA mutations in the non-coding region (NCR) in Polg mice (n = 12 for 2 months; n = 24 for 9 months). Error bars, mean ± SEM. (D) Bar graphs representing mean heteroplasmy levels of NCR mtDNA mutations in Polg mice with aging. Error bars, mean ± SEM. (E) Summary of mtDNA mutations found in the NCR region (mtDNA15423-16299) in Polg mice. Dots represent mtDNA mutations and numbers under dots represent the heteroplasmy levels. ETAS indicates the extended termination associated sequence and CSB indicates the conserved sequence block.