Mitochondrial dynamics and autophagy aid in removal of persistent mitochondrial DNA damage in Caenorhabditis elegans

Abstract

Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mitochondrial DNA (mtDNA) by important environmental genotoxins and endogenous metabolites. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. We report that removal of such mtDNA damage is detectable by 48 h in Caenorhabditis elegans, and requires mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.

Figure 1.

mtDNA lesion frequency slowly decreases after a single dose of UVC in post-mitotic adult C. elegans (glp-1). Nematodes were exposed to UVC and analyzed for DNA damage immediately (0 h), or after 24, 48 or 72 h via QPCR. (a) mtDNA damage is removed by ∼40% over 72 h at 50 and 100 J/m². Two-way ANOVA indicated a significant effect of recovery (P < 0.0001) and a recovery × treatment interaction (P < 0.05). Asterisks denote a significant difference compared with 0 h lesion frequency within each UVC treatment (Fisher’s PLSD, P < 0.05). (b) nDNA damage is repaired following a single dose of UVC. Two-way ANOVA indicated a significant effect of treatment (P < 0.0001) and a recovery × treatment interaction (P < 0.0001). Asterisks denote a significant difference compared with undosed control lesion frequency at each recovery timepoint (Fisher’s PLSD, P < 0.05). (c) No significant increase in mtDNA copy number was observed during the recovery period. (d) mtDNA copy number significantly decreased during the recovery period irrespective of UVC exposure. Two-way ANOVA indicated a significant effect of time (P < 0.0001) but no significant treatment effect (P = 0.4714) or treatment × time interaction (P = 0.3246). Asterisks denote a significant difference compared with 0 h mtDNA copy number (Fisher’s PLSD, P < 0.05). Bars ± SEM.

Figure 2.

Fusion, fission and autophagy gene knockdown inhibits mtDNA damage removal. mtDNA lesions are removed in empty vector control (L4440) by 30–40% 120 h post a single UVC exposure in post-mitotic adult C. elegans (glp-1). RNAi knockdown of eat-3, fzo-1, drp-1, bec-1, unc-51 and pink-1 inhibited mtDNA damage removal (inhibition of removal was determined by a lack of a significant difference between 0 and 120 h lesion frequency within each RNAi treatment). Two-way ANOVA indicated a significant interaction between RNAi and recovery (P < 0.05). Asterisks denote a significant difference between 0 h and 120 h mtDNA lesions within RNAi treatment (Fisher’s PLSD, P < 0.05). Percent mtDNA lesions remaining after 120 h was calculated based on 0 h lesion frequency within each RNAi treatment. Bars ± SEM.

Figure 3.

UVC exposure induces autophagy but no detectable changes in mitochondrial morphology in adult C. elegans. Mitochondrial morphology was assessed using form factor as a measure of elongation (perfect circles = 1) and mean area/perimeter ratio as a measure of interconnectivity. No detectable differences were observed at (a) 24 h or (b) 48 h following UVC exposure. Each data point represents the mean area/perimeter ratio and form factor of mitochondria within a single muscle cell. Mitochondrial morphology was highly variable within each treatment group. Representative images show a (c) tubular, (d) intermediate and (e) fragmented mitochondrial morphology in muscle cells of untreated controls 48 h after UVC exposure. (f) The number of LGG-1::GFP foci per seam cell increased 24 h following UVC exposure compared with untreated controls (Fisher’s PLSD, P = 0.004). Inhibition of autophagy with 3-MA exposure reduced the formation of LGG-1::GFP foci compared with untreated controls (Fisher’s PLSD, P < 0.0001). An overall effect of treatment was determined by ANOVA (P < 0.0001). Bars ± SEM.

Figure 4.

Serial UVC exposure results in mtDNA damage accumulation. (a) Schematic of serial UVC protocol. L1 nematodes on unseeded plates are dosed with UVC at 254 nm every 24 h for a total of three doses. Following the third dose, nematodes are provided with food and developmental stage recorded at 48, 72 and 96 h post UVC exposure. (b) mtDNA damage accumulates over serial UVC exposure and persists 24 h after the last UVC exposure and addition of food. Following 24 h recovery periods, nDNA damage was repaired. Bars ± SEM.

Figure 5.

Serial UVC exposure results in dose-dependent L3 arrest, lower steady state ATP level and reduced O₂ consumption. (a) Larval arrest increased in a dose-dependent manner following serial UVC exposure. (b) Steady state ATP levels were significantly lower by 24 h after UVC exposure and addition of food. Asterisks denote a significant treatment effect compared with untreated control (Fisher’s PLSD, P < 0.05). (c) Nematodes exposed to serial UVC 10 J/m² had significantly lower O₂ consumption compared with untreated nematodes at 0, 24 and 48 h post exposure and addition of food. At 48 h, O₂ consumption was further decreased in arrested nematodes compared with non-arrested nematodes and both were decreased compared with the untreated group. Two-way ANOVA indicated a significant treatment × recovery interaction (P < 0.0001) and asterisks denote significant differences at each time point (Fisher’s PLSD, P < 0.05). Bars ± SEM.

Figure 6.

Co-exposure to UVC and ethidium bromide exacerbates L3 arrest. Co-exposure to UVC (10 J/m²) and ethidium bromide (5 µg/ml) further exacerbates L3 arrest compared with UVC (asterisks) or chemical (hash) exposure alone at every time point (Fisher’s PLSD, P < 0.05). Bars ± SEM.

Figure 7.

Mutations in fusion and autophagy genes exacerbate L3 arrest following serial UVC exposure. Mutations in fusion genes fzo-1 and eat-3 and autophagy gene unc-51, as well as inhibition of autophagy with 3-MA, exacerbated L3 arrest compared with wild-type at 72 and 96 h. Asterisks denote a significantly different effect of treatment on mutant compared with wild-type strain (two-way ANOVA, P < 0.05). Bars ± SEM.

Figure 8.

Proposed model of the effect of mitochondrial fusion, fission and autophagy processes on persistent mtDNA damage-induced mitochondrial dysfunction and removal of mtDNA damage. Persistent mtDNA damage leads to mitochondrial dysfunction (e.g. reduced ATP levels and larval arrest). Mitochondrial fusion protects against dysfunction, as does autophagy, presumably by promoting functional complementation, mtDNA replication and removal of dysfunctional mitochondria (red solid lines). Mitochondrial fission and autophagy promote mtDNA damage removal (green dashed lines), whereas mitochondrial fusion is indirectly required for mtDNA damage removal because it is required to preserve basic mitochondrial function and mtDNA replication (dashed green line denotes the indirect effect of fusion on mtDNA damage removal).