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

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

Reverse your mtDNA control region and you’ll reverse your mutation bias.

The Inversion of the Control Region in Three Mitogenomes Provides Further Evidence for an Asymmetric Model of Vertebrate mtDNA Replication, PlosOne 2014

As many of you are probably aware of there is an ongoing debate regarding the mechanism of mitochondrial DNA (mtDNA) replication (see for instance Holt and Reyes, 2012). The models were summarized well in a recent paper from Fonseca et al. in PlosOne 2014.

 

Fonseca MM et al. PlosOne 2014

They analyzed the base composition of the mtDNA protein-coding genes (2500 mtDNAs) and found a clear bias, which correlated with the direction of the control region. Based on this they concluded that the strand-displacement model (SDM) or the ribonucleotides-incorporation throughout the lagging strand model (RITOLS) is likely to be the replication mechanism instead of the strand-coupled model (SCM).

Of course one can always hypothesize that the SCM is specific for cell cultures (as in the original paper describing this model) or is tissue specific. However, from the evolutionary point of view the strand-displacement model seems to be the more important one. Whether the single-strand DNA is covered by mtSSB or RNA remains to be seen.