7926 Nucleic Acids Research, 2012, Vol. 40, No. 16 mtDNA copy number decreases with age. This indicates that degradation of mtDNA is occurring and the resultant decline in copy number is not compensated for by increased biogenesis. This decrease in copy number likely relates to the decreased metabolic rate and ATP production shown in aging nematodes (70). We therefore used developing rather than adult C. elegans to determine the signiﬁcance of removal of damaged mtDNAs in recovery from UVC exposure. During the L3 and L4 tran- sition, nematodes switch to OXPHOS to produce energy; this is accompanied by a signiﬁcant increase in energy demand and mtDNA copy number (52). Inhibition of mitochondrial replication or translation or mutations in mitochondrial electron transport chain components all result in arrest at or before the L3 stage (51–53). Using a novel protocol designed to permit accumulation of a high level of mtDNA damage early in L1 (Figure 4a), we showed that UVC-induced mtDNA damage caused dose-dependent increases in mitochondrial dysfunction as indicated by L3 arrest, reduced steady state ATP level and reduced oxygen consumption. Treatment with low levels of ethidium bromide nearly abolished recovery from UVC-induced L3 arrest suggesting that recovery of mitochondrial function is facilitated by dilution of damaged mtDNA via mtDNA replication. unc-51 mutants displayed a signiﬁcant increase in L3 arrest as did nematodes in which autophagy was inhibited with 3-MA. These data indicate that autophagy (and presumably removal of damaged mtDNA via autophagy) is involved in functional recovery from persistent mtDNA damage. Autophagy provides protection against mitochondrial dysfunction, apoptosis and cell death following toxicant exposure or cell stress (40,41,96,97), and abnormal mitochondrial autophagy coupled with mitochondrial dysfunction occurs in a variety of pathologies including mitochondrial diseases (40), lysosomal storage disorders (98) and neurodegenerative disorders including Parkinson’s, Alzheimer’s and Huntington’s disease (99–101). It is not unlikely that autophagy protects against mitochondrial stressors, in part, by removing mitochondria that would otherwise generate reactive oxygen species and trigger apoptosis or necrosis (48,49). Thus, the reduction in autophagy with age particularly in post-mitotic cells may result in persistence of unstable mitochondria potentially leading to development of age-related disorders (102). Surprisingly, pink-1 mutants did not exhibit increased larval arrest despite the role of this protein in removal of damaged mtDNA in adult C. elegans. This may indicate that removal of damage in replicating cells is not PINK-1 dependent. On the other hand, PINK1 knockdown in C. elegans increases mitochondrial fusion (Unpublished observations made by Maxwell C.K. Leung, Duke University) and in other model organisms is associated with increased mitochondrial fusion (103,104) which is critical for recovery from L3 arrest as discussed below. Mutations in the fusion genes fzo-1 and eat-3 resulted in complete arrest of larval development following serial UVC, suggesting that mitochondrial fusion is critical in recovery of mitochondrial function following UVC-induced mtDNA damage. Although it is likely that the complete larval arrest observed in these mutants is partially because of inhibition of damage removal, the de- gree of exacerbation suggests failure of other recovery mechanisms. Fusion is critical for recovery of mitochon- drial function following toxicant exposure and cell stress. Loss of fusion capacity increases susceptibility to loss of MP (105), respiratory deﬁciency (105,106), oxidative stress (61,107), apoptosis and cell death (61,108,109). Additionally, enhanced mitochondrial fusion has been shown to be protective against UV irradiation-induced de- pletion of ATP (94,95) and blockage of mtDNA replication by mtDNA intercalators (110). Fusion provides protection against the effects of mtDNA mutations by facilitating ‘functional complemen- tation’ (111,112), the mixing of lipids, proteins and mtDNA to compensate for mutated DNA (80) and this mechanism may explain the lack of phenotypic expression of mtDNA mutations until the occurrence is high (>40–90%) (113). This function of fusion also equally dis- tributes proteins involved in mtDNA replication and repair (80). Mice harboring mutations in fusion proteins exhibit severe depletion of mtDNA (114) because of defects in mtDNA replication (80), and polg-1–mutant C. elegans exhibit increased mitochondrial fusion in response to mtDNA depletion (57). As described earlier in the text, mtDNA replication is a particularly important mechanism of recovery from L3 arrest. Therefore, mitochondrial fusion may aid in recovery from UVC-induced larval arrest by promoting functional complementation, mtDNA replication and removal of damaged mtDNAs. Consistent with this idea, mutation in the ﬁssion gene drp-1 did not increase L3 arrest. Although this could suggest that mtDNA damage removal is DRP-1 independ- ent in developing C. elegans, we hypothesize that enhanced fusion resulting from the drp-1 mutation may compensate for lack of mtDNA damage removal. Decreased mitochondrial fragmentation has been shown to be protective against toxicant-induced mitochondrial dysfunction, ATP depletion and apoptosis (89,90,94). Conversely, Yang et al. found that drp-1 nematodes are more sensitive to heat stress and paraquat (115) and, in mammalian cells, knockdown of drp1 results in mitochon- drial dysfunction, loss and disorganization of mtDNA and decreased membrane ﬂuidity (92,116). We were unable to directly test in developing C. elegans if mutations in fusion, ﬁssion and autophagy/mitophagy genes inhibit removal of UVC-induced mtDNA damage or if lack of damage removal correlates with the frequency and duration of L3 arrest because of developmental changes (i.e. somatic and germ cell proliferation and sub- stantial increases in mtDNA copy number) that would have confounded those measurements. Assuming that larval C. elegans employ the same mechanism as adults to remove UVC-induced mtDNA damage, our genetic and pharmacologic data suggest that removal of mtDNA damage aids in recovery from UVC-induced mitochondrial dysfunction facilitated by autophagy and mitochondrial fusion.