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Aging Cell (2006) 5, pp279–282 Doi: 10.1111/j.1474-9726.2006.00209.x Blackwell Publishing Ltd COMMENTARY Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging? Konstantin Khrapko,1 Yevgenya Kraytsberg,1 Aubrey DNJ de Grey,2 Jan Vijg3 and Eric A. Schon4 1 Gerontology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA 2 Department of Genetics, University of Cambridge, Cambridge, UK 3 University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 4 Departments of Neurology and of Genetics and Development, Columbia University, New York, NY, USA Summary Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice. Key words: aging; human; mouse; mtDNA mutation. Correspondence Konstantin Khrapko, Gerontology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA. e-mail: [email protected] Accepted for publication 8 February 2006 Two recent studies reported the creation of transgenic mice with proofreading-deficient mitochondrial DNA polymerase gamma (Trifunovic et al., 2004; Kujoth et al., 2005). In these mice, high levels of mutations (i.e. over 10 mutations per 10 000 bp or 15 per coding region of mitochondrial genome) accumulate by the age of 25 weeks in all tissues studied (summarized in Fig. 1, black bars). Intriguingly, both mouse lines present similar premature aging phenotypes including osteoporosis, hair loss, cardiomyopathy, anemia, sarcopenia, fertility problems, and shortened lifespan. The authors of the reports concluded that their data provided a ‘causative link between mtDNA mutations and aging phenotypes in mammals’ (Trifunovic et al., 2004) and that ‘mtDNA mutations … may be a central mechanism driving mammalian aging’ (Kujoth et al., 2005). Intact mitochondrial function is vital for the normal functioning of any cell. The presence of more than 15 random point mutations per 15-kb-long coding region of the mitochondrial genome (Fig. 1) almost entirely constituted of essential coding sequences is likely to severely affect mitochondrial physiology. This expectation is confirmed by a substantial drop of mitochondrial enzyme activities observed in heart tissue of the mutator mouse (Trifunovic et al., 2004). High levels of mutations are particularly toxic to dividing cells: mtDNA mutator cell lines have been shown to enter crisis at about 10 mutations per genome (Spelbrink et al., 2000). Thus the severe multisystem phenotype of the mutator mouse is not surprising. To further explore whether premature aging in mutator mice corroborates the hypothesis that mtDNA mutations are involved in mammalian aging in general and in human aging in particular, we surveyed the data regarding levels of mtDNA mutations in older humans (Fig. 1, white bars). We limited our search to studies which used, similar to Trifunovic et al. (2004) and Kujoth et al. (2005), the clone-by-clone sequencing approach to measure the mutations (see online supplementary material for details of the data analysis and validation). As shown in Fig. 1, levels of mtDNA mutations in human tissues are more than an order of magnitude lower than in mutator mice. In our opinion, this huge gap in mtDNA mutant fractions makes it difficult to conclude that the same types of mutations are causally related to human aging. One potential caveat in our argument is that mtDNA mutations may be so infrequent in old humans because they have already fulfilled their role in the aging process by killing the cells that had carried them, and thus had become ‘self-eliminated’. The data, however, do not support this scenario. Most likely only © 2006 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006 279 280 Mutator mouse: a model of human aging? K. Khrapko et al. Fig. 1 Fractions of somatic point mutations in the coding region of mutator mice mtDNA as compared to human. The mutator mice data were measured at mid-life (5–6 months) in duodenum and heart (right bar) (Kujoth et al., 2005), and brain, heart (left bar), and liver (Trifunovic et al., 2004). The data for adult and old humans (over 30 years old, mostly over 70 years old) have been adapted from published reports and our unpublished data: leukocytes (Monnat & Loeb, 1985a), colon (Taylor et al., 2003), skeletal muscle (Del Bo et al., 2003a; Wanrooij et al., 2004), retina (Bodenteich et al., 1991), brain (Simon et al., 2004; Smigrodzki et al., 2004), and heart (Kraytsberg & Khrapko, unpublished); 95% confidence intervals are shown where available. Note that the low levels of mtDNA mutations in humans cannot be explained by preferential apoptosis of mutant cells (see text). a small fraction of mtDNA mutations are capable of causing cell death. Hence, their self-elimination should not have significantly affected the total mutant fraction. If, on the other hand, the set of deadly mutations were broad enough to cause a significant reduction in the overall mutant fraction, then this should have resulted in an equally significant enrichment of silent mutations (i.e. mutations that do not cause an amino acid change and thus are neutral). Normally silent mutations constitute about 25% of all mutations in a protein-coding sequence. Elimination of a large number of non-silent mutations sufficient for a severalfold reduction in overall mutant fraction should have increased the fraction of silent mutations considerably, but this is not observed in human tissues. For example, in colonic crypts (Taylor et al., 2003), all five mutations in the protein-coding sequences that were found in normal crypts change highly conserved amino acids. Although more data would be desirable, those available are clearly incompatible with a significant elimination of mutations by apoptosis. Note that colon is a tissue with rapid cellular turn- over, which is expected to be particularly susceptible to mtDNA mutation-driven apoptosis. Similarly, no bias in favor of silent mutations was observed in the much larger database of brain mtDNA mutations (Smigrodzki et al., 2004): 127 silent mutations (24%) and 411 amino acid changing mutations (76%). Analysis of the data in Fig. 1 could potentially be taken one step further. The levels of mtDNA mutations associated with premature aging in mice are so much higher than the levels of point mutations present in aged human tissues that it seems reasonable to ask whether mtDNA mutator mice actually demonstrate that mtDNA mutations might not be involved in human aging! Such an idea would be consistent with an observation that non-transgenic littermates of mutator mice accumulate five 4 mutations per 10 bp at 40 weeks (i.e. over five times more than aged humans) and they still show no signs of aging (Trifunovic et al., 2005) (see online supplementary material for discussion of conflicting estimates of mutant fractions in wild-type mice). This conclusion, however, would probably be incorrect, in part because the types and distribution of mtDNA mutations in aging humans may be very different from those in mutator mice, which may allow them to play a role in aging despite their overall low abundance, as discussed below. In the mutator mouse, the major type of mtDNA mutations (i.e. polymerase proofreading errors) start to accumulate rapidly early in embryogenesis [presumably immediately following blastocyst implantation, when organellar division and mtDNA replication resume (Smith & Alcivar, 1993)], and thus distribute at high levels among most if not all tissues (Trifunovic et al., 2004; Kujoth et al., 2005). In contrast, in humans the major sources and the types of mutations (e.g. large-scale deletions vs. point mutations, or proofreading errors vs. errors at chemically modified nucleotides), and thus the age- and tissue-specificity of mutation accumulation, are probably different from those in the mutator mouse. In accord with this, the mutational spectra of mtDNA mutator human cells are different from the spectra of human polymorphisms (Spelbrink et al., 2000). The spectra of affected tissues and the relative timing of the onset and mechanisms of pathology may be very different, too. For example, in humans, the highest proportions of cells with defects of mitochondrial function (about 20%) were found in the substantia nigra in the brain (Itoh et al., 1996). Moreover, these defects were caused not by point mutations, but by large-scale mtDNA deletions (Kraytsberg et al., 2006), which also account for a majority of the mitochondrial defects in skeletal muscle (Gokey et al., 2004). Deletions, unlike point mutations in the mutator mouse, accumulate much later in life, and are extremely tissue-specific (Soong et al., 1992). Finally, the intracellular distribution on mtDNA mutations may differ between mutator mice and aged humans. Each potentially detrimental mtDNA mutation alone is benign below the threshold of about 90% per cell, and several different mutations, when present in the same cell, may complement each other’s defects. mtDNA mutations are known to have a tendency to expand clonally within cells, but the rate of this process is not known. Admittedly, mutations may have had a better chance © 2006 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006 Mutator mouse: a model of human aging? K. Khrapko et al. 281 to expand clonally above the threshold within about 70 years in an aged person compared to 6 months in mutator mouse. Thus, we believe that the rapid generation of mtDNA mutations in a relatively small number of progenitor cells beginning at the earliest stages of mouse embryonic development has a disproportionately large effect on the mutant load in postnatal life, and probably does not mimic the slow and tissue-specific accumulation of mutations seen in normal human aging. As an example, the presence of anemia in almost all of the mutator mice stands in contrast to the lack of anemia in most (although not all) patients with authentic mitochondrial disease harboring high levels of mutant mtDNA in blood [e.g. the A8344G mutation in MERRF, myoclonus epilepsy with ragged-red fibers (Silvestri et al., 1993)]. Perhaps a mouse with a conditional postnatal expression of an error-prone mtDNA polymerase, or one expressing a less aggressively error-prone enzyme variant, or a collection of mice with tissue-specific elevations of mtDNA mutation rates, similar to that reported in cardiac tissue (Zhang et al., 2000; Mott et al., 2005), might provide a model of natural aging in mammals. The remarkably low abundance of mtDNA mutations reported by the latter group in association with severe cardiac dysfunction reinforces our view that there is much more to be learned about the species- and tissuespecificity of pathologies associated with mitochondrial mutations (see also online supplementary material for discussion of mutant fraction estimates in mice). Another very recent example of a mild mutator is the ‘deletor’ transgenic mouse expressing a defective human Twinkle gene (Tyynismaa et al., 2005). The above arguments are supported further by the independent observation that osteoporosis, curvature of the spine, hair and weight loss, reduced fertility, anemia, and enhanced cell death – i.e. those phenotypes that collectively make aging of the mtDNA mutator mice particularly similar to human aging – are not specific to these mice, as they are also present in other premature aging mouse models, for example in those with impaired nuclear (but not mitochondrial) DNA metabolism (Hasty et al., 2003; Chang et al., 2004; Lombard et al., 2005). It follows that while the mutator mouse does show that large amounts of mtDNA mutations can cause certain aging-like phenotypes, it does not follow that mtDNA mutations actually cause similar phenotypes in natural aging. Particular caution should be exercised in using mutator mice ‘to test compounds intended for treating or staving off osteoporosis, sarcopenia, and other age-related conditions’, as has been suggested in the media (Travis, 2004). It is worth emphasizing that mutator mice, although they are not, in our opinion, an appropriate test of the involvement of mtDNA mutations in natural aging, are certainly an indispensable instrument for in vivo studies of the effects of somatic point mutations in mtDNA. This is illustrated by the demonstration that somatic mtDNA mutations may not cause oxidative stress in mutator mice (Kujoth et al., 2005; Trifunovic et al., 2005), which is a powerful argument against the influential ‘vicious cycle’ hypothesis. In conclusion, prematurely aging mtDNA mutator mice, however important they are for the studies of somatic mtDNA mutations, do not prove that mtDNA mutations are involved in natural aging, although these mice do not exclude this possibility either. We hope that this discussion will stimulate new experiments to test the relationship between mtDNA mutations and the aging process. Acknowledgments This work was supported in part by NIH grants ES 11343 and AG 19787 to K.K. 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Supplementary material The following supplementary material is available for this article online from http://www.blackwellsynergy.com Appendix S1 Comments regarding Fig. 1, and Mutant fractions in non-mutator mice. © 2006 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006