Genetically controlled mtDNA editing prevents ROS damage by arresting oxidative phosphorylation

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Deletion of mitochondrial DNA in eukaryotes is mainly attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that the regulatory circuitry underlying this editing critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. Our results may therefore be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.

One-Sentence Summary

Genetically controlled editing of mitochondrial DNA is an integral part of the yeast’s defenses against oxidative damage.

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  1. Evaluation Summary:

    The authors report that upon exposure of yeast cells to paraquat-induced superoxide production, specific mitochondrial DNA genes encoding electron transport chain proteins are deleted to minimize the generation of endogenous superoxide. Reversible loss of mitochondrial DNA as an adaptive response to paraquat stress is an interesting idea. The data presented appear to support the proposed model, but could be further strengthened as alternative interpretations of the described observations are possible.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):


    In this paper, authors used S. cerevisiae to explore the mechanism of tolerance to paraquat, a drug that produces the superoxide anion with electrons from the respiratory chain or other NADH dehydrogenases. They find that yeast adapt to PQ after a few generation, recovering growth by 50 % from the 2.5-3 fold growth inhibition caused by PQ. Adaptation is paralleled by mitochondrial fragmentation, but is not the result of a mitophagic response, the loss of part of mtDNA consistently in similar regions, and the inability to respire. Growth adaptation, the loss of mtDNA and the ability to respire are recovered after a few generations after stress release, but long, chronic PQ exposure causes irreversible mtDNA total loss. Adaptation appears to depend on the presence of SOD2 and is absent in the Rtg1 and Rtg3 mutants of the retrograde response. Sequence data analysis also indicate the duplication of chromosomes II, III and V, the duplication of Chr II and V having an additive effect on the adaptation to paraquat.

    General comments:

    The reversible loss of mtDNA as an adaptive response to PQ stress is novel and interesting, and the data provided support this conclusion. The evaluation of PQ stress tolerance by real time DNA sequencing represents a huge work task and is a reflection of the rigor used throughout the experiments. The paper however suffers from its style, very elliptic, the use of complicated, long sentences, the use of terms such as growth cycles, generations etc. that have not been clearly defined at start, which makes it difficult to read and to understand the protocols used, for instance when PQ is added and removed. For instance, if cells adapt to PQ stress by decreasing their doubling time, the severity of growth rate reduction should be initially quantified. The authors are encouraged to take this criticism seriously. Below are a few specific points that do not detract from the general very positive impression of this paper.

    Specific comments:

    1. page 4, and S1 legends says that " we see PQ causes doubling time to increase", but where do we have to see that? It is not clear how the growth rate is calculated? How are experiments performed on solid, liquid medium? The figure only shows gene expression data? What is a growth cycle and what is its length? What do you mean by 240 generations? What is the length of one generation in hours? What is a population, and what is the difference with "clonally reproducing cell populations? At best a picture of the plates used to monitor growth should be shown for one to understand how it is done. How do you calculate that 106 min doubling time reduction equals 49.3 % of the maximum possible reduction? And what is this maximum? Fig. 1B is confusing: it is understandable that cells are exposed to PQ enough to adapt, then grown without PQ, and then again with PQ, but over the generations shown in the picture, do one not expect to see adaptation after a few "cycles"?

    2. Page 7. The experiment described in S6A cannot be used to rule out signaling by H2O2: adding 3 mM H2O2 to cells that have already adapted to PQ, whether or not by use of an H2O2 signal, amounts to a severe H2O2 stress, exacerbated by the lack of a functional respiratory chain (petite cells are more sensitive to H2O2, relative to WT). One way of tackling this question would be to see whether adaptive doses of H2O2 (100-300 microM) prior to exposure to paraquat would speed up growth adaptation or not (cross adaptations have been described in the past). Similarly, the WT PQ adaptative response of cells lacking Yap1 or other antioxidants does not prove anything: signaling by H2O2 is mostly localized in confined areas, and this should persist even in a Yap1 mutant.

    3. Page 8. The need of SOD2 for PQ adaptation to occur is not really convincing because of the sickness of SOD mutants in general. Further, it shows that there is no adaptation at 12 microG/mL PQ, but then adaptation occurs at a higher dose, but slower, relative to WT. What is the point authors want to make? That SOD by dismutation of the superoxide anion produces H2O2 needed for signaling? But, authors already ruled out the need for H2O2 to signal adaptation? Please don't be too peremptory in your conclusion on this experiment. In addition, it is hard to follow the writer: "in four populations, the copy number..." then "two of these fail to adapt" then "the remaining four populations", but which ones? Lastly the text of Fig 4c indicates 12.5 mG/mL, but the figure 50?

  3. Reviewer #2 (Public Review):

    The authors used extended yeast cell culture to determine how cells adapt to paraquat-induced oxidative stress by following the deletion pattern of mtDNA and the role of nuclear genes such as SOD2, MIP1, RTG2 and RTG3 in the process. The idea that specific deletions of mtDNA genes may reduce superoxide production endogenously which may help cells to grow is novel. However, there are several major concerns that severely diminish the validity of the conclusion. First, the manuscript suffers from overinterpretation of the experimental data. The key observation in the manuscript is that growth adaptation to paraquat correlates with mtDNA deletions in the COX1-VAR1 region. There is no data showing that loss of these genes actually reduces superoxide production and is causative for adaptive growth. In fact, yeast mitochondria produce superoxide mainly in the bc1 complex. Loss of Cox1 would be expected to increase electron leak at the bc1 complex. Secondly, the authors overlooked the dynamic nature of mtDNA mutations in yeast and the data on mtDNA deletions are incorrectly interpreted in many places. Thirdly, the budding yeast is anaerobic and the physiological implication of mtDNA mutations would be different from mammalian cells. The authors made many unfunded claims for the implication of the work to mitophagy, cancer therapy and aging-related diseases. Finally, describing paraquat-induced mtDNA mutation as a regulatory "gene editing" program is inappropriate.