Oxygen in mitochondrial disease: can there be too much of a good thing?

Vamsi Mootha and Patrick Chinnery wrote a short letter to the editors discussing whether or not mitochondrial disease patients should be given high-flow oxygen in medical care (Mootha & Chinnery 2018). A recommended read but I wanted to add few references describing also models where mild hypoxia has been shown to have negative effects.

Indeed, a genome-wide Cas9 screen done in Mootha’s lab identified hypoxia response to be protective against oxidative phosphorylation system (OXPHOS) defects (Jain et al. 2016) and even extend the lifespan of mice lacking a OXPHOS complex I subunit Ndusf4 (Ferrari et al. 2017). They also mentioned in the text that fruit flies carrying a mutation in OXPHOS complex II (succinate dehydrogenase, SDH) are sensitive to high oxygen (Walker et al. 2006) as are the Ndusf4 knockout mice (Jain et al. 2016).

On the other hand, the mitochondrial DNA (mtDNA) mutator mouse suffers from an anemia (Ahlqvist et al. 2015) as do many POLG disease patients (Hikmat et al. 2017) so decreasing oxygen levels is unlikely to be helpful. Also in fruit flies, hypoxia worsens many of the observed phenotypes of mitochondrial mutants (Burman et al. 2014, Whelan et al. 2010).

Clearly, as Mootha and Chinnery advocate, we need more randomized trials to better understand whether some specific mitochondrial disease patients would benefit for not having high-flow oxygen given to them.

References:

Ahlqvist KJ, Leoncini S, Pecorelli A, Wortmann SB, Ahola S, Forsström S, Guerranti R, De Felice C, Smeitink J, Ciccoli L, Hämäläinen RH, Suomalainen A. MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction. Nat Commun. 2015. PMID: 25751021

Burman JL, Itsara LS, Kayser EB, Suthammarak W, Wang AM, Kaeberlein M, Sedensky MM, Morgan PG, Pallanck LJ. A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer. Dis Model Mech. 2014. PMID: 25085991

Ferrari M, Jain IH, Goldberger O, Rezoagli E, Thoonen R, Cheng KH, Sosnovik DE, Scherrer-Crosbie M, Mootha VK, Zapol WM. Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome. Proc Natl Acad Sci U S A. 2017. PMID: 28483998

Hikmat O, Charalampos T, Klingenberg C, Rasmussen M, Tallaksen CME, Brodtkorb E, Fiskerstrand T, McFarland R, Rahman S, Bindoff LA. The presence of anaemia negatively influences survival in patients with POLG disease. J Inherit Metab Dis. 2017. PMID: 28865037

Jain IH, Zazzeron L, Goli R, Alexa K, Schatzman-Bone S, Dhillon H, Goldberger O, Peng J, Shalem O, Sanjana NE, Zhang F, Goessling W, Zapol WM, Mootha VK. Hypoxia as a therapy for mitochondrial disease. Science. 2016. PMID: 26917594

Mootha VK, Chinnery PF. Oxygen in mitochondrial disease: can there be too much of a good thing? J Inherit Metab Dis. 2018. PMID: 29948481

Walker DW, Hájek P, Muffat J, Knoepfle D, Cornelison S, Attardi G, Benzer S. Hypersensitivity to oxygen and shortened lifespan in a Drosophila mitochondrial complex II mutant. Proc Natl Acad Sci U S A. 2006. PMID: 17056719

Whelan J, Burke B, Rice A, Tong M, Kuebler D. Sensitivity to seizure-like activity in Drosophila following acute hypoxia and hypercapnia. Brain Res. 2010. PMID: 20034480

Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse.

Mitochondrial DNA (mtDNA) mutator mouse carries a proofreading-deficient mitochondrial DNA polymerase leading to accumulation of mtDNA mutations. This mouse was originally engineered in two independent labs (Trifunovic et al. 2004, Kujoth et al. 2005) and has since been used to study various aspects of mitochondrial dysfunction.

Several studies have tried to find ways to improve the various phenotypes of the mtDNA mutator mouse, which include sarcopenia, hearing loss, osteoporosis, alopecia, weight loss, testicular atrophy, enlarged heart etc. For instance, our group showed recently that increasing mtDNA copy number of mtDNA mutator mice can partially rescue the testicular atrophy phenotype (Jiang et al. 2017)

It was published already seven years ago that exercise might improve the phenotype of the mitochondrial DNA (mtDNA) mutator mouse (Safdar et al. 2011). Like most papers using mtDNA mutator mouse as the model organism, Safdar et al. has the shortcoming that their WT control mice also inherited mtDNA mutations from a heterozygous mtDNA mutator mother, a.k.a. they made a “dirty” breeding (Kauppila et al. 2017). Therefore, this study had no “true” WT mouse model as a control. Also more recently, two papers from these authors have now Editorial Expression of Concerns (JBC study, PNAS study) so let’s see what happens with these findings.

Now a recent paper from a different group suggests that exercising mtDNA mutator mice might affect oocyte quality (Faraci et al. 2018). It was not reported how these mice have been bred, so one should be very cautious when interpreting the results. For instance, it would be important to know how many generations the heterozygous mtDNA mutator females have been bred and whether the WT control mice are a WT littermate from a heterozygote X heterozygote cross.

All in all, exercise has almost no effect on the oocytes of mtDNA mutator mice. For some of the differences seen it is also impossible to tell whether they are caused by changes in oocytes themselves of some systemic changes.

References:

Editorial Expression of Concern: Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice. Proc Natl Acad Sci U S A. 2018. PMID: 29891666

Expression of concern for: Exercise increases mitochondrial PGC-1 α content and promotes nuclear-mitochondrial cross-talk to coordinate mitochondrial biogenesis. J Biol Chem. 2018. PMID: 29602880

Faraci C, Annis S, Jin J, Li H, Khrapko K, Woods D. Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse. Reproduction. 2018. PMID: 29875308

Jiang M, Kauppila TES, Motori E, Li X, Atanassov I, Folz-Donahue K, Bonekamp NA, Albarran-Gutierrez S, Stewart JB, Larsson NG. Increased Total mtDNA Copy Number Cures Male Infertility Despite Unaltered mtDNA Mutation Load. Cell Metab. 2017. PMID: 28768180

Kauppila TES, Kauppila JHK1, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, Morrow JD, Van Remmen H, Sedivy JM, Yamasoba T, Tanokura M, Weindruch R, Leeuwenburgh C, Prolla TA. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005. PMID: 16020738

Safdar A, Bourgeois JM, Ogborn DI, Little JP, Hettinga BP, Akhtar M, Thompson JE, Melov S, Mocellin NJ, Kujoth GC, Prolla TA, Tarnopolsky MA. Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice. Proc Natl Acad Sci U S A. 2011. PMID: 21368114

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064

Replication machinery degrading linear mitochondrial DNA

Linear mitochondrial DNA (mtDNA) molecules with deletions are a curious case. To my knowledge, they are only present at high levels in a few genetic models, including the mtDNA mutator mouse(Trifunovic et al. 2004, Bailey et al. 2009), mtDNA mutator flies (Bratic et al. 2015), patients carrying mutations in mitochondrial genome maintenance exonuclease 1 (MGME1)(Nicholls et al. 2014) and MGME1 knockout mouse (Matic et al. 2018).

In the case of mtDNA mutator mouse, the level of this linear mtDNA with deletion remains stable with time (Kukat et al. 2009). Additionally, it has been shown using mitochondrially targeted restriction enzymes that linear mtDNA is rapidly degraded (Bayona-Bafaluy et al. 2005). Therefore the question remains, who is degrading these mtDNA fragments?

Moretton et al. tried to answer this question by linearizing mtDNA with mitochondrial targeted restriction enzyme followed by systematic knockdown of exonucleases known/suggested to be in mitochondria (Moretton et al. 2017). These exonucleases included ExoG, EndoG, MGME1, DNA2 and FEN1. EXD2 was not tested, but that was later shown to be localized on the mitochondrial outer membrane (Hensen et al. 2018). Perhaps surprisingly, none of the exonucleases studied by Moretton et al. seemed to participate in degradation of linear mtDNA fragments.

There is still at least one exonuclease that Moretton et al. did not study, which is the exonuclease activity of mitochondrial DNA polymerase (POLG). Indeed, in the absence of nucleotides POLG can engage in so called exonuclease mode where it starts degrading the primer in 3’-5’ direction (Bratic et al. 2015). A recent study in fruit flies tried to answer the question what enzyme degrades mtDNA in fly sperm (Yu et al. 2017). Yu et al. concluded that DmPOLG is involved in this degradation but it takes place independent of its exonuclease activity. I was not convinced by this paper because they saw mtDNA replication in punctae without DmPOLG-GFP and more importantly all the models were based on knocking down various proteins of the minimal mitochondrial replisome with variable efficiencies. Also, when I read this paper I was aware of some in-house work taking place in the group of Martin Graef.

Medeiros et al. showed in yeast that under starvation there is a strong mtDNA depletion if autophagy is inhibited and that this depletion can be rescued by increasing nucleoside pools through supplementation or genetically (Medeiros et al. 2018). Therefore, the authors hypothesized that under starvation the autophagy-deficient cells might have changes in nucleotide pools causing MIP1 (yeast POLG) to enter the above mentioned exonuclease mode and degrade mtDNA. Using an exonuclease-deficient POLG the authors showed that indeed it seems to be POLG which is degrading mtDNA.

The most recent paper on the subject comes from Peeva et al. (Peeva et al. 2018). Peeva et al. tested multiple exonucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, RBBP8, MGME1 and POLG) to see whether the knockout or knockdown of these enzymes would affect the degradation of linear mtDNA molecules. Somewhat similar to Medeiros et al., Peeva et al. also suggest that POLG together with MGME1 and TWINKLE would be degrading linear mtDNA.

So there it is. Both in yeast and human cells mtDNA seems to be degraded by the exonuclease activity of POLG. It would be interesting to see how the yeast mtDNA actually looks like at the molecular level when degraded as yeasts do not possess MGME1. One could therefore predict that these degraded molecules would not have blunt ends similarly to the results from humans where POLG is suggested to degrade mtDNA to the 3’-5’ direction and MGME1 to the 5’-3’ direction. It is still open question what controls this degradation, if it is controlled to begin with. Both Medeiros et al. and Peeva et al. hypothesize that low nucleotide pools might be a controlling factor. Although this hypothesis would make sense in the case of yeast under starvation and inhibited autophagy, I don’t see how this would work with these human cells under normal culture conditions. Clearly just linearizing mtDNA is sufficient to promote mtDNA degradation without manipulation of the dNTP pools.

Some open questions for the future:

• Is this kind of degradation present also in more in vivo conditions or whether it is only present in somewhat artificial conditions where most of mtDNA is linearized by restriction enzymes.
• What, if any, is controlling the balance between mtDNA synthesis and degradation?
• Is there a specific endonuclease cleaving the yeast mtDNA before POLG can start degrading mtDNA?
• How is mtDNA degraded in organisms without MGME1?

Perhaps this is a stretch but in Peeva et al. study the authors showed how in WT cells linear mtDNA is degraded piece by piece. Where mitophagy selective for damaged mtDNA to exist, which I think it does not (Kauppila et al. 2017), one would not expect to see these slowly degrading molecules but instead a bulk disappearance of linear mtDNA fragments.

References:

Bailey LJ, Cluett TJ, Reyes A, Prolla TA, Poulton J, Leeuwenburgh C, Holt IJ. Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA. Nucleic Acids Res. 2009. PMID: 19244310

Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A. 2005. PMID: 16179392

Bratic A, Kauppila TE, Macao B, Grönke S, Siibak T, Stewart JB, Baggio F, Dols J, Partridge L, Falkenberg M, Wredenberg A, Larsson NG. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat Commun. 2015. PMID: 26554610

Hensen F, Moretton A, van Esveld S, Farge G, Spelbrink JN. The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair. Sci Rep. 2018. PMID: 29599527

Kauppila TES, Kauppila JHK, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kukat A, Trifunovic A. Somatic mtDNA mutations and aging--facts and fancies. Exp Gerontol. 2009. PMID: 18585880

Matic S, Jiang M, Nicholls TJ, Uhler JP, Dirksen-Schwanenland C, Polosa PL, Simard ML, Li X, Atanassov I, Rackham O, Filipovska A, Stewart JB, Falkenberg M, Larsson NG, Milenkovic D. Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria. Nat Commun. 2018. PMID: 29572490

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Moretton A, Morel F, Macao B, Lachaume P, Ishak L, Lefebvre M, Garreau-Balandier I, Vernet P, Falkenberg M, Farge G. Selective mitochondrial DNA degradation following double-strand breaks. PLoS One. 2017. PMID: 28453550

Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D, Reyes A, Kornblum C, Sciacco M, Moggio M, Dziembowski A, Kunz WS, Minczuk M. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet. 2014. PMID: 24986917

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064

Yu Z, O'Farrell PH, Yakubovich N, DeLuca SZ. The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes. Curr Biol. 2017. PMID: 28318978

Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans.

Mitochondrial DNA (mtDNA) mutagenesis can be increased by inactivating the proofreading activity of the main (or only, but let’s not go there today) mitochondrial DNA-dependent DNA polymerase (POLG). During my PhD I have been working with these mtDNA mutators in flies (Bratic et al. 2015) and mice (Jiang et al. 2017). Therefore, it piqued my interest when I saw that a C. elegans mtDNA mutator was published (Haroon et al. 2018). Having a nematode, fly and a mouse carrying the analogous proofreading-deficient variant of POLG allows one to do some interesting comparisons.

To get it out of the system I just want to express first my annoyance. This nematode paper wanted specifically to establish a simpler mtDNA mutator organism which could be used to generate animals carrying mtDNA mutations and to perform suppressor screens. However, they failed to mention our mtDNA mutator fruit fly paper published 2 years ago (Bratic et al. 2015). On another note, they should have referenced the POLG knockout paper from 9 years ago (Bratic et al. 2009). Now to the actual paper.

The authors of this paper showed that the homozygous mutator nematodes have a 50% decrease in mtDNA copy number at stage L4 (Fig. 1B). Similar mtDNA depletion have been described in the mtDNA mutator fly and mouse models (Bratic et al. 2015,Jiang et al. 2017) but it should be also pointed out that the POLG knockout nematode has similar decrease (~41%) in mtDNA levels (Bratic et al. 2009). This makes one wonder whether the mutator POLG is replicating any mtDNA in nematodes. It is likely that most of this mtDNA depletion in contributed by the decrease in mtDNA levels in the gonads similar to the knockout (Bratic et al. 2009) and would also explain the fertility problems of the mtDNA mutator nematode as mentioned by Haroon et al. in the methods. One reason for this mtDNA depletion in mtDNA mutator nematodes could stem from our fly work where we showed that the fly POLG mutator variant should be considered dominant negative, because the hemizygous flies developed further in comparison with the homozygous (Bratic et al. 2015). Indeed, the exonuclease-deficient POLG is known to have increased strand-displacement activity (Farge et al. 2007) and even be insensitive to altered dNTP/NTP ratios (Forslund et al. 2018).

Interestingly, there aren’t any high heteroplasmy level mutations even after five generations of consecutive intercrosses of mtDNA mutator nematodes. It seems that like in our mtDNA mutator flies (Bratic et al. 2015), also in mtDNA mutator nematodes mtDNA mutations accumulate slowly across generations.

It has been now documented both in the mtDNA mutator mice (Trifunovic et al. 2004) and mutator flies (Bratic et al. 2015) that abolishing proofreading activity of POLG result in the formation of linear mtDNA with deletions. A model how these deletions are formed has also been put forward (Macao et al. 2015). Based on studies done using two-dimensional neutral agarose gel electrophoresis (2DNAGE), mtDNA replication in nematodes seems to be quite different to the ones in other metazoans (Lewis et al. 2015). I would have loved to see using Southern what happens to the mtDNA integrity of these mtDNA mutator nematodes but unfortunately the authors did not carry out this analysis.

They authors also tried to use this mtDNA mutator nematode to establish nematode lines carrying pathogenic mtDNA mutations. I feel they came a bit short on this one as they only established a nematode line, which mtDNA is littered with mutations. Some more careful breeding scheme, as we have used in the mouse (Kauppila et al. 2016), would have avoided this issue. The key is to carry the mtDNA mutator allele for only a one or two generations, so that each mtDNA molecule has only a few mutations. These lines should then be bred further to isolate lines with high heteroplasmy levels of certain mutations.

In the end the authors performed targeted RNAi screen against multiple cellular pathways to find suppressors of the neuromuscular defects of these mtDNA mutator nematodes. It has been shown in fruit flies that RNAi based screens have to be well controlled (Alic et al. 2012) and in this study the authors did not mention whether they included any negative controls, such as RNAi against GFP or LacZ. The reason I mention this is because nearly every pathway tested was able to suppress the neuromuscular defects, which feels a bit too good to be true (Cell Reports seem to have linked a wrong file to the Supplementary materials so I wasn’t able to see all the data). Also, they should have taken into account that the RNAi alone might be sufficient to affect the neuromuscular function of these nematodes. Some of these RNAi targets were verified further using hypomorphic and knockout mutant nematodes. In all cases the rescue of mtDNA copy number was modest so it is unclear to me how these mutants are rescuing the phenotype.

It should be also pointed out that in Fig. 2C-E and Fig. 4 the authors performed multiple compaisons using t-tests whereas ANOVA would have been more appropriate instead.

In the end the authors didn’t really use this model to its full capacity to establish nematode lines carrying pathogenic mtDNA mutations but perhaps that will happen in the future. There is always the question though whether POLG and/or mtDNA disease nematode has scientific value, because the POLG knockout nematode is viable.

References:

Alic N, Hoddinott MP, Foley A, Slack C, Piper MD, Partridge L. Detrimental effects of RNAi: a cautionary note on its use in Drosophila ageing studies. PLoS One. 2012

Bratic A, Kauppila TE, Macao B, Grönke S, Siibak T, Stewart JB, Baggio F, Dols J, Partridge L, Falkenberg M, Wredenberg A, Larsson NG. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat Commun. 2015.

Bratic I, Hench J, Henriksson J, Antebi A, Bürglin TR, Trifunovic A. Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development. Nucleic Acids Res. 2009

Farge G, Pham XH, Holmlund T, Khorostov I, Falkenberg M. The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function. Nucleic Acids Res. 2007. PMID: 17251196

Forslund JME, Pfeiffer A, Stojkovič G, Wanrooij PH, Wanrooij S. The presence of rNTPs decreases the speed of mitochondrial DNA replication. PLoS Genet. 2018 PMID: 29601571

Haroon S, Li A, Weinert JL, Fritsch C, Ericson NG, Alexander-Floyd J, Braeckman BP, Haynes CM, Bielas JH, Gidalevitz T, Vermulst M. Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans. Cell Rep. 2018

Jiang M, Kauppila TES, Motori E, Li X, Atanassov I, Folz-Donahue K, Bonekamp NA, Albarran-Gutierrez S, Stewart JB, Larsson NG. Increased Total mtDNA Copy Number Cures Male Infertility Despite Unaltered mtDNA Mutation Load. Cell Metab. 2017

Kauppila JHK, Baines HL, Bratic A, Simard ML, Freyer C, Mourier A, Stamp C, Filograna R, Larsson NG, Greaves LC, Stewart JB. A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease. Cell Rep. 2016

Lewis SC, Joers P, Willcox S, Griffith JD, Jacobs HT, Hyman BC. A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans. PLoS Genet. 2015

Macao B, Uhler JP, Siibak T, Zhu X, Shi Y, Sheng W, Olsson M, Stewart JB, Gustafsson CM, Falkenberg M. The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication. Nat Commun. 2015

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004