Mitochondrial DNA Damage Does Not Determine C. elegans Lifespan

Abstract

The mitochondrial free radical theory of aging (mFRTA) proposes that accumulation of oxidative damage to macromolecules in mitochondria is a causative mechanism for aging. Accumulation of mitochondrial DNA (mtDNA) damage may be of particular interest in this context. While there is evidence for age-dependent accumulation of mtDNA damage, there have been only a limited number of investigations into mtDNA damage as a determinant of longevity. This lack of quantitative data regarding mtDNA damage is predominantly due to a lack of reliable assays to measure mtDNA damage. Here, we report adaptation of a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the detection of sequence-specific mtDNA damage in C. elegans and apply this method to investigate the role of mtDNA damage in the aging of nematodes. We compare damage levels in old and young animals and also between wild-type animals and long-lived mutant strains or strains with modifications in ROS detoxification or production rates. We confirm an age-dependent increase in mtDNA damage levels in C. elegans but found that there is no simple relationship between mtDNA damage and lifespan. MtDNA damage levels were high in some mutants with long lifespan (and vice versa). We next investigated mtDNA damage, lifespan and healthspan effects in nematode subjected to exogenously elevated damage (UV- or γ-radiation induced). We, again, observed a complex relationship between damage and lifespan in such animals. Despite causing a significant elevation in mtDNA damage, γ-radiation did not shorten the lifespan of nematodes at any of the doses tested. When mtDNA damage levels were elevated significantly using UV-radiation, nematodes did suffer from shorter lifespan at the higher end of exposure tested. However, surprisingly, we also found hormetic lifespan and healthspan benefits in nematodes treated with intermediate doses of UV-radiation, despite the fact that mtDNA damage in these animals was also significantly elevated. Our results suggest that within a wide physiological range, the level of mtDNA damage does not control lifespan in C. elegans.

Keywords: DNA damage; aging; healthspan; hormesis; lifespan; mitochondrial DNA; quantitative PCR; radiation.

Diagram 1

Schematic diagram of region amplified by short and long target using the S-XL-qRT-PCR DNA damage assay within C. elegans mitochondrial genome. The short target (71 bp) is represented by a green arrow and the long amplicon (6300 bp) is represented by the red arrow. The short target is too short and is much less affected by DNA damage, therefore can be used as an approximation for the undamaged DNA while the long target is used for DNA quantification as the probability of encountering DNA lesions increases with template size and this long amplicon amplifies the majority of the C. elegans mitochondrial genome.

Figure 1

The effect of dose-dependent UV- and γ-irradiation on induction of DNA damage in mitochondria of Day 4 young glp-1 C. elegans. (A) UV-radiation dose values given to nematodes ranging from 0 to 1000 J/m². Overall, there was a significant dose-dependent increase in the damaged mtDNA in animals exposed to UV-radiation (p < 0.0001, One-way ANOVA, n = minimum 3 independent experiments). 100 and 200 J/m² had no significant effect on the level of damaged mtDNA relative to untreated control animals (p > 0.05, One-way ANOVA with Bonferroni’s post-test). Exposure of nematodes to 400–1000 J/m² UV-radiation significantly elevated the DNA lesions in the nematodes (P < 0.0001 for all other conditions, One-way ANOVA with Bonferroni’s post-test). (B) Nematodes were exposed to increasing doses of γ-radiation ranging from 0 to 40 kRad. There was a statistically significant elevation in the extent of mtDNA damage (p < 0.0001, One-way ANOVA, n = minimum 3 independent experiments). The level of damaged mtDNA in nematodes exposed to 20 kRad γ-radiation was at 0.5 lesions per 10 kbp from a baseline level of 0.4 lesions per 10 kbp in non-irradiated control animals, the change in the mtDNA damage level was not statistically significant (p > 0.05, One-way ANOVA with Bonferroni’s post-test). Relative to control nematodes, 40 kRad γ-radiation significantly elevated the mtDNA damage levels as evaluated by our S-XL-qRT-PCR DNA damage assay (p < 0.01, One-way ANOVA with Bonferroni’s post-test).

Figure 2

Pilot experiments of various doses of γ-radiation. (A) Egg-laying study comparing number of eggs laid by C. elegans exposed to 0 (control), 20 and 40 kRad γ-radiation doses. Animals exposed to γ-radiation laid less eggs than non-irradiated control animals. (B) Distance traveled by C. elegans exposed to 0 (control), 20 and 40 kRad γ-radiation doses. Non-irradiated animals were healthy and active. Animals exposed 40 kRad of γ-radiation explored less than 20 kRad and control.

Figure 3

DNA strand breaks induced in γ-irradiated wild type N2 C. elegans measured using the comet assay. (A) Typical comet images of wild type N2 C. elegans embryonic cells obtained from (i) untreated control animals (undamaged DNA sample). (ii) C. elegans exposed to 20 kRad γ-radiation (damaged DNA sample) and (iii) C. elegans exposed to 40 kRad γ-radiation (damaged DNA sample). (i) In undamaged DNA samples, the DNA remains intact within the highly organized structure and is confined to the nucleus, resulting a halo-like structure. (ii,iii) When DNA is damaged, the relaxed DNA expands out from the nucleoid during electrophoresis, resulting in a structure that resembles a comet with a head composed of intact undamaged DNA and a tail that consists of damaged/broken fragments of DNA. (B) Effects of γ-radiation on DNA damage on nematodes exposed to 0, 20, 40 kRad γ-radiation, determined using the comet assay as the percentage of DNA in the tail. There were 36, 41, and 45% of DNA in tail of the comets in the control, 20 and 40 kRad γ-irradiated animals, respectively. There was a linear relationship between radiation dosage and DNA in tail (R-squared = 1.00). n = minimum 55 comets analyzed per condition.

Figure 4

High correlation between the DNA damage level from both comet assay and S-XL-qRT-PCR assay. Both comet assay and S-XL-qRT-PCR DNA damage assay are able to detect significant dose-dependent increase in DNA damage in animals irradiated with 0, 20, and 40 kRad γ-radiation. Each point is a mean of minimum 55 comets (for data plotted in Y-axis) or minimum of 12 replicates (for data plotted on X-axis).

Figure 5

Age-dependent changes in the mtDNA damage level in nematodes. Using our S-XL-qRT-PCR DNA damage assay, there were a statistically significantly more mtDNA lesions in old nematodes compared to young nematodes. mtDNA lesions in old animals were 2.3-fold more than young animals (p = 0.04, Student’s t-test), n = minimum 9 independent experiments.

Figure 6

MtDNA damage in different mutant strains relative to young wild type N2 animals as measured using S-XL-qRT-PCR DNA damage assay. Relative to wild type animals, mpst-1, sod-2/-3, and daf-2 mutant strains had 3.4-, 2.7-, and 4.0-fold higher mtDNA damage, respectively (p < 0.01 for both mpst-1 and sod-2/-3 mutants and p < 0.0001 for daf-2 mutant, Student’s t-test). mev-1 mutant C. elegans strain had no significant difference in mtDNA damage level compared to wild type N2 C. elegans (p > 0.05, Student’s t-test), n = minimum 3 independent experiments.

Figure 7

Dose-response study of UV-irradiated young glp-1 nematodes. (A) Survival curves from each condition were compared to that of the non-irradiated control animals and analyzed using Log-rank (Mantel-Cox) test. The survival of nematodes irradiated with 100 J/m² was not significantly different from the control. Lifespan of nematodes irradiated with 200 J/m² was significantly extended, while exposure to 400 J/m² significantly shortened the overall nematodes lifespan (p < 0.001). n = 200 worms per conditions. (B) Comparison of the percentage of lifespan difference and the mtDNA damage level of nematodes irradiated with 0, 100, 200, and 400 J/m² UV-radiation. There was a trend toward dose dependent increase in damaged mtDNA in nematodes exposed to UV-radiation, interestingly, there was a hormetic lifespan extension effect in nematodes irradiated with 200 J/m² UV-radiation compared to control animals (p < 0.05, survival curve comparison Log-rank (Mantel-Cox) test) (C) Overall, 200 J/m² UV-irradiated animals were healthier (p < 0.0001, Log-rank (Mantel-Cox) test) (D) Relative to non-irradiated control animals, 200 J/m² UV-radiation extended the mean healthspan by 12% but 400 J/m² UV-radiation significantly shortened the mean healthspan of the nematodes by 25% (p < 0.05 and p < 0.001, respectively, Log-rank test with Bonferroni multiple comparisons test, OASIS 2), n = 200 worms per conditions (E) After UV-radiation, 100 and 200 J/m² UV-irradiated animals traveled significantly more distance than control animals (p < 0.05 and p < 0.0001, respectively, One-way ANOVA with Bonferroni post-test). n = minimum 10 animals per condition.

Figure 8

Dose-response study of young glp-1 nematodes exposed to γ-radiation. The survival of day 4 young glp-1 nematodes exposed to 40 kRad γ-radiation. (A) Survival curves from nematodes irradiated with 40 kRad γ-radiation are compared to that of the non-irradiated control animals. The lifespan of the 40 kRad γ-irradiated animals was not significantly shortened relative to the non-irradiated control animals [p > 0.05, Log-rank (Mantel-Cox) test]. (B) γ-radiation had no significant effect on the mean lifespan of the nematodes, both control and γ-irradiated animals have mean lifespan of 13 days (p > 0.05, Log-rank test with Bonferroni multiple comparisons test, OASIS 2). n = 200 worms per condition. Healthspan of the surviving day 4 glp-1 nematodes exposed to 40 kRad γ-radiation. (C) As shown in the healthspan curve, the overall health status of the γ-irradiated animals was similar to control animals (p > 0.05, Log-rank (Mantel-Cox) Test for survival curve). (D) Both non-irradiated control nematodes and 40 kRad γ-irradiated nematodes had similar mean healthspan of 13.8 days (p > 0.05, Log-rank test with Bonferroni multiple comparisons test, OASIS 2). n = 200 worms per condition. (E) Average distance traveled by nematodes exposed to 40 kRad γ-radiation. γ-radiation significantly shortened the distance traveled by the nematodes compared to control animals. γ- irradiated nematodes traveled about 63% less than non-irradiated control animals (p < 0.0001, Student’s t-test). n = minimum 10 animals per condition.