Regulation of defective mitochondrial DNA accumulation and transmission in C. elegans by the programmed cell death and aging pathways
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This manuscript suggests a novel mechanism of purifying selection by which programmed cell death contributes to the selective removal of mtDNA deletion mutations in C. elegans. The evidence for this mechanism of removal is strong although questions remain regarding the underlying mechanism and the role of canonical ageing pathways. Because of the likely central role of mtDNA deletions in ageing and age-dependent diseases, this work will be of interest to scientists in the field of mitochondrial biology as well as ageing.
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Abstract
The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). ‘Purifying selection’ mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5 , which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNA uaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNA uaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.
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eLife assessment
This manuscript suggests a novel mechanism of purifying selection by which programmed cell death contributes to the selective removal of mtDNA deletion mutations in C. elegans. The evidence for this mechanism of removal is strong although questions remain regarding the underlying mechanism and the role of canonical ageing pathways. Because of the likely central role of mtDNA deletions in ageing and age-dependent diseases, this work will be of interest to scientists in the field of mitochondrial biology as well as ageing.
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Reviewer #1 (Public Review):
Flowers et al. studied requirements for the persistence and clearance of mutant mtDNA in C. elegans using the uaDf5 deletion in mtDNA. This mutant mtDNA persists at relatively constant levels, despite clearly having detrimental effects. Surprisingly, no mutations were found in the existing wt copies, which would have otherwise explained the persistence of mutant DNA by complementation. The authors then investigated the potential role of programmed cell death in the removal of mutant mtDNA from the germline using crosses with existing cell death mutants. They observed increased amounts of uaDf5 DNA in 1 day old progeny in strains with mutations in the caspases ced-3 and csp-1 and in several other cell death genes, showing that a significant amount of uaDF5 is removed by PCD in the germline. The authors also …
Reviewer #1 (Public Review):
Flowers et al. studied requirements for the persistence and clearance of mutant mtDNA in C. elegans using the uaDf5 deletion in mtDNA. This mutant mtDNA persists at relatively constant levels, despite clearly having detrimental effects. Surprisingly, no mutations were found in the existing wt copies, which would have otherwise explained the persistence of mutant DNA by complementation. The authors then investigated the potential role of programmed cell death in the removal of mutant mtDNA from the germline using crosses with existing cell death mutants. They observed increased amounts of uaDf5 DNA in 1 day old progeny in strains with mutations in the caspases ced-3 and csp-1 and in several other cell death genes, showing that a significant amount of uaDF5 is removed by PCD in the germline. The authors also observed increased uaDf5 over time in the germline, and effects lifespan mutations on the mount of uaDf5. This was true both for the insulin signaling pathway and the clk-1 pathway, suggesting that both pathways regulate uaDf5 levels consistent with the connection between longevity and mitochondrial homeostasis. Finally, the authors discuss results showing that PCD mutants with high amounts of uaDf5 in the germline, have surprisingly low amounts of uaDf5 in their progeny, which would suggest that PCD can be replaced by another clearance method.
This manuscript is of general interest because it demonstrates the importance of PCD for clearance of mutant mtDNA. The evidence for this mechanism of removal is strong. The effects of the aging mutants are more difficult to understand and the discussion of these effects is therefore somewhat speculative.
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Reviewer #2 (Public Review):
In this study, the authors sought to elucidate regulators of mitochondrial DNA (mtDNA) quality control in the germline. To this end, the authors used Caenorhabditis elegans as a model organism and 3.1kb mtDNA deletion mutation uaDf5 that is stably transmitted across generations. The key data presented were the heteroplasmy level of mtDNA, specifically the molar ratio of mutant vs. wildtype (WT) mtDNA molecules, at different ages. The authors specifically focused on the role of programmed cell death (PCD) signaling and a few well-known aging pathways in C. elegans. The data showed that attenuation of PCD has the general effect of increasing the steady-state mutant-to-WT ratio, while increasing PCD does not reduce this ratio. The data also showed that this mutant-to-WT ratio increases with age, an effect that …
Reviewer #2 (Public Review):
In this study, the authors sought to elucidate regulators of mitochondrial DNA (mtDNA) quality control in the germline. To this end, the authors used Caenorhabditis elegans as a model organism and 3.1kb mtDNA deletion mutation uaDf5 that is stably transmitted across generations. The key data presented were the heteroplasmy level of mtDNA, specifically the molar ratio of mutant vs. wildtype (WT) mtDNA molecules, at different ages. The authors specifically focused on the role of programmed cell death (PCD) signaling and a few well-known aging pathways in C. elegans. The data showed that attenuation of PCD has the general effect of increasing the steady-state mutant-to-WT ratio, while increasing PCD does not reduce this ratio. The data also showed that this mutant-to-WT ratio increases with age, an effect that is transmitted to progenies, and that perturbations to well-known insulin signaling and CLK-1 aging pathways affect the rate of this increase, where a longer lifespan is correlated with a slower increase. Finally, the data demonstrated an intergenerational reduction in mutant-to-WT ratio and that the degree of this reduction has a nonlinear ultrasensitive-like dependence on the ratio.
A strength of the study is the comprehensive exploration of the role of key molecules of the PCD machinery in mtDNA quality control in the germline. Also, the data on the effects of age and aging pathways on the maintenance of mtDNA quality in the germline, as well as on intergenerational mtDNA quality control, are extremely interesting and have the potential to trigger transformative studies that connect mtDNA purifying selection and aging.
A major weakness of the study is that the key findings are predominantly based on data of the mutant-to-WT mtDNA ratio. But, a higher mutant-to-WT ratio does not necessarily equate to an increase/accumulation of mutant mtDNA in the cell population, since the same increase can also be caused by a decrease in WT mtDNA. No data for copy numbers of WT and mutant mtDNA or their proxies were analyzed. As a consequence, some of the major findings, such as the non-canonical/non-apoptotic role of PCD machinery in mediating mitochondrial purifying selection and the accumulation of mutant mtDNA with age, cannot be uniquely concluded from the data. Alternative explanations could be given to explain the observed trends of mutant-to-WT ratios.
Another weakness is that the connection between the two pathways in this study: PCD and aging, in regulating mtDNA quality control was not more deeply explored. The study did not delve into how the interplay of aging and PCD if any, affects mtDNA quality control in the germline.
Finally, as the authors noted, the important role of stochasticity in purifying selection against pathogenic mtDNA is established. Yet, this aspect of purifying selection is not explored in this study (e.g., how such stochasticity is working with PCD in mtDNA quality control in the germline), nor it is accounted for in the analysis of the data and the discussion of the observation.
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