- Email: [email protected]
Evolving insight into the role of mitochondrial DNA mutations in aging
Available online at www.sciencedirect.com
Experimental Gerontology 43 (2008) 20–23 www.elsevier.com/locate/expgero
Mini Review
Evolving insight into the role of mitochondrial DNA mutations in aging Gregory C. Kujoth *, Tomas A. Prolla Department of Genetics and Medical Genetics, University of Wisconsin, 425-G Henry Mall, Madison, WI 53706, USA Received 22 September 2007; accepted 28 September 2007 Available online 5 October 2007
Abstract Mitochondria have occupied a central place in theories on the underlying cellular mechanisms of eukaryotic aging for several decades and much debate has ensued regarding the role of oxidative stress and mitochondrial genomic damage in these processes. Mouse models with greatly enhanced mitochondrial mutagenesis have produced dramatic aging-like phenotypes but recent results have led some to reassess whether such models are relevant to naturally occurring aging mechanisms. Here, we discuss the evolving insight that may be gained from these models regarding the contribution of mitochondrial DNA mutations to aging. 2007 Elsevier Inc. All rights reserved. Keywords: mtDNA; Mutations; Deletions; Polg; Mice; Random mutation capture
1. Mitochondrial mutator mice: testing the mitochondrial theory of aging The decline of cellular functioning with age has long been postulated to be linked to the mitochondrial production of reactive oxygen species (ROS) and the resultant damage to multiple classes of macromolecules, including carbohydrates, lipids and nucleic acids (Harman, 1972). In contrast to oxidized biomolecules that undergo rapid replacement, damage to the mitochondrial DNA (mtDNA) may be particularly harmful in that DNA repair processes in the mitochondria are less robust than those in the nucleus and the majority of the 16.5 kb circular mtDNA is coding in nature. Oxidative damage to mtDNA has been proposed to lead to a ‘‘vicious cycle’’ of further ROS production due to impaired electron transport that could result from mutations in the gene-dense mitochondrial genome (Harman, 1972). One approach to testing this idea has been the generation of mouse models of increased mtDNA mutagenesis. Ubiquitous, homozygous expression of a proofreadingdeficient isoform (D257A) of the nucleus-encoded mitochondrial DNA polymerase c (Polg) results in mice with
functional deficits in many tissues and some of these phenotypes resemble an accelerated aging syndrome (Kujoth et al., 2005; Trifunovic et al., 2004). For example, PolgD257A/D257A mice display age-related declines in adiposity, bone density, fertility, hearing and muscle mass, as well as accelerated thymic atrophy, hair graying and loss (alopecia), dilated cardiac hypertrophy and reduced survival. Impaired mitochondrial respiratory activity in these mice does not lead to increased oxidative stress but is associated with activation of apoptosis (Kujoth et al., 2005; Trifunovic et al., 2005). Eventual depletion of differentiated and regenerative cells may lead to decline of tissue functioning. Mutations accumulate in mtDNA beginning during development in these mice. It was originally thought that levels of mtDNA point mutations were 3–11-fold higher in PolgD257A/D257A mice than in heterozygous or wild type mice when assessed using a conventional PCR amplification and cloning approach. Recently developed methodology, however, has generated new insight into the complexities of the effects of mitochondrial mutagenesis.
2. The random mutation capture assay *
Corresponding author. Tel.: +1 608 265 5205; fax: +1 608 262 2976. E-mail address: [email protected] (G.C. Kujoth).
0531-5565/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2007.09.010
Critical to these new developments was the recent application of the so-called random mutation capture (RMC)
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23
during PCR). Using this approach, Vermulst et al. (2007a) have found that the levels of point mutations in wild type mice were several orders of magnitude lower than previously reported. Young (1–3 month) mice had an average base substitution mutation frequency of 6–8 · 10 7 per bp in the mitochondrial 12S rRNA subunit gene (in brain and heart). By 24–33 months, this climbed to 1 · 10 5 per bp, with an exponential increase occurring after 16 months of age. Determination of mutation frequencies in the mitochondrial cytochrome b gene gave similar results. The frequency of base substitutions in tissues from young PolgD257A/D257A mice was as high as 1.5 · 10 3 per bp, an increase of 2500-fold over wild
assay (Bielas and Loeb, 2005) to the study of mitochondrial mutations by Loeb and colleagues (Vermulst et al., 2007a). In this assay, mtDNA is isolated and subjected to restriction digestion before being amplified by PCR. Primers for PCR are chosen to flank the restriction site such that the only mtDNA molecules that can be amplified are those that are uncut, due to a mutation in the recognition site of the restriction enzyme (Fig. 1A). The presence of a selection event prior to PCR avoids the de novo introduction of mutations during amplification (this includes not only intrinsic Taq polymerase fidelity errors, which had been subtracted in previous studies, but also fixation of in vivo- or ex vivo-generated base adducts into mutations TaqI
21
TaqI
TaqI TaqI TaqI
3 CYTB
4
1
2 TaqI TaqI
12S rRNA 16S rRNA
TaqI ND6
TaqI ND1
6 ND5
TaqI
ND2
TaqI
ND4
TaqI ND4L ND3
TaqI
COX1
COX3 ATP6
COX2
5 TaqI TaqI
ATP8 TaqI TaqI TaqI
Base pairs screened: = 10,000 copies per well x 4bp (TaqI recognition site) x 84 wells = 3.36x106 bp Mutation frequency: = 9 positives / 3.36x106 bp =2.6x10-6 per bp
Fig. 1. Detection of mtDNA point mutations and large deletions by the random mutation capture assay. (A) The random mutation capture (RMC) assay principle. Mitochondrial DNA is isolated and digested with TaqI endonuclease before dilution to 10,000 copies per well in a microtiter plate. PCR amplification using primers (arrowheads) flanking a restriction site (primers 1/2) will produce a product (black wells) only when a mutation has inactivated the restriction site, leaving the mtDNA template uncut in that region. Neighboring primers (3/4) are used in a control PCR reaction to determine copy number per well in conjunction with a dilution series (light gray wells, row A). Mutation frequency is calculated as the number of positive wells per total bp screened. Cloning and sequencing of amplified products provides mutational spectra. The assay can be adapted to detect large deletions by selecting a primer set that spans multiple restriction sites (e.g., primers 5/6). The probability of inactivation by base substitution of multiple sites is rare, so that for practical purposes, loss of these sites can be deemed to be due to large deletion events. For example, assuming a conservative mutation frequency in wild type cells of 1 · 10 6 per bp, the probability of inactivating three restriction sites would be 1 · 10 18. PCR products are cloned and sequenced for verification and to map deletion endpoints. For additional technical description of the assay, see Vermulst et al. (2007a). (B) Frequency of base substitution and small insertion/deletions in aging wild type and mitochondrial mutator mice. The mean mutation frequency (±SEM) as determined at the TaqI site 634 (12S rRNA gene) in mtDNA isolated from brain. Young mice are 1–3 months, old mice are 24–33 months old. RMC data are taken from Vermulst et al. (2007a).
22
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23
type. Surprisingly, young Polg+/D257A mice also had a strikingly high mutational frequency of 3.3 · 10 4 per bp. This level is 500-fold higher than that of young wild type mice and 29-fold greater than that seen in even old wild type mice. To date, no significant pathology has been observed in Polg+/D257A mice, as they lack the accelerated aging features of homozygous mice. These data suggest that the mutational threshold at which tissue dysfunction and aging phenotypes develop must be very high and that it is unlikely that point mutations in naturally aging wild type mice will accumulate to such an extent. This argues against a causal role for point mutations in the aging process. The RMC assay described above detects base substitutions and small insertions or deletions only so these studies have not addressed the potential contribution of large mtDNA deletions. Such deletions are commonly present in aged tissues (for example, see Bender et al., 2006; Kraytsberg et al., 2006; Melov et al., 1995, 1997) and deleted mitochondrial genomes may have a replicative advantage which may contribute to their attainment of homoplasmy (where most or all of the mtDNA molecules in a mitochondrion or cell are identical), although data on this latter point are mixed (Diaz et al., 2002; Tang et al., 2000). The RMC assay can be adapted to quantify the levels of large mtDNA deletions in the mitochondrial mutator mice (Vermulst and Loeb, 2007b) by positioning primers in such a way that multiple restriction sites must all be inactivated in order for the mtDNA template to be amplified. Because the mutational frequency at each individual site is very low, removal of all the sites by a large deletion is the primary means of generating a detectable product (see Fig. 1). Similarly to the pattern observed with point mutations, the frequency of large mtDNA deletions in brain and heart tissues of wild type mice would be expected to increase with age. Homozygous mitochondrial mutator mice do show elevated levels of large deletions compared to wild type controls when detected by Southern blot analysis (Trifunovic et al., 2004). The key question is how the levels of deletions will compare quantitatively between wild type, Polg+/D257A and PolgD257A/D257A mice with age. In other words, will large mtDNA deletion mutations correlate with the presence of accelerated aging phenotypes and how similar will the levels in prematurely aged mice be to those in naturally aged mice? Data to answer these important questions should be available soon. Interestingly, modulation of mtDNA deletion levels in transgenic mice carrying a mutated allele of the mitochondrial helicase, Twinkle, has not produced accelerated aging phenotypes despite increased mtDNA deletions in several post-mitotic tissues (Tyynismaa et al., 2005). Currently, it is not clear how the levels of mtDNA deletions compare quantitatively between the Twinkle, PolgD257A/D257A and naturally aged mouse models. Additional models of increased mtDNA deletion frequencies with ubiquitous tissue distribution (as generated by gene targeting) would aid in such comparisons. Ultimately, decreasing the level of
large mtDNA deletions would be the most interesting test of a causal role in aging. 3. Conclusions The seemingly simple approach of engineering mice with elevated mtDNA mutation levels as a test of the mitochondrial theory of aging is not as straight-forward as it first seems. Aside from the complexities of temporal onset of mutations (developmental versus adult-onset) and quantitative comparisons between mutant and naturally aged wild type individuals, the qualitative class of mtDNA mutation (point mutation versus large deletion) may also be relevant. The development of the innovative RMC assay should enable a much better understanding of the relative importance of mutation type to the development of tissue dysfunction and aging phenotypes. Acknowledgement This work was supported by NIH Grant AG021905 to T.A.P. References Bender, A., Krishnan, K.J., Morris, C.M., Taylor, G.A., Reeve, A.K., Perry, R.H., Jaros, E., Hersheson, J.S., Betts, J., Klopstock, T., Taylor, R.W., Turnbull, D.M., 2006. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat. Genet. 38, 515–517. Bielas, J.H., Loeb, L.A., 2005. Quantification of random genomic mutations. Nat. Methods 2, 285–290. Diaz, F., Bayona-Bafaluy, M.P., Rana, M., Mora, M., Hao, H., Moraes, C.T., 2002. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 30, 4626–4633. Harman, D., 1972. The biologic clock: the mitochondria? J. Am. Geriatr. Soc. 20, 145–147. Kraytsberg, Y., Kudryavtseva, E., McKee, A.C., Geula, C., Kowall, N.W., Khrapko, K., 2006. Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat. Genet. 38, 518–520. Kujoth, G.C., Hiona, A., Pugh, T.D., Someya, S., Panzer, K., Wohlgemuth, S.E., Hofer, T., Seo, A.Y., Sullivan, R., Jobling, W.A., Morrow, J.D., Van Remmen, H., Sedivy, J.M., Yamasoba, T., Tanokura, M., Weindruch, R., Leeuwenburgh, C., Prolla, T.A., 2005. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309, 481–484. Melov, S., Hinerfeld, D., Esposito, L., Wallace, D.C., 1997. Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age. Nucleic Acids Res. 25, 974–982. Melov, S., Shoffner, J.M., Kaufman, A., Wallace, D.C., 1995. Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle. Nucleic Acids Res. 23, 4122–4126. Tang, Y., Manfredi, G., Hirano, M., Schon, E.A., 2000. Maintenance of human rearranged mitochondrial DNAs in long-term cultured transmitochondrial cell lines. Mol. Biol. Cell 11, 2349–2358. Trifunovic, A., Hansson, A., Wredenberg, A., Rovio, A.T., Dufour, E., Khvorostov, I., Spelbrink, J.N., Wibom, R., Jacobs, H.T., Larsson, N.G., 2005. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc. Natl. Acad. Sci. USA 429, 357–359.
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23 Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J.N., Rovio, A.T., Bruder, C.E., Bohlooly, Y.M., Gidlof, S., Oldfors, A., Wibom, R., Tornell, J., Jacobs, H.T., Larsson, N.G., 2004. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423. Tyynismaa, H., Mjosund, K.P., Wanrooij, S., Lappalainen, I., Ylikallio, E., Jalanko, A., Spelbrink, J.N., Paetau, A., Suomalainen, A., 2005.
23
Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc. Natl. Acad. Sci. USA 102, 17687–17692. Vermulst, M., Bielas, J.H., Kujoth, G.C., Ladiges, W.C., Rabinovitch, P.S., Prolla, T.A., Loeb, L.A., 2007a. Mitochondrial point mutations do not limit the natural lifespan of mice. Nat. Genet. 39, 540–543. Vermulst, M., Loeb, L.A., 2007b. Personal communication.
Experimental Gerontology 43 (2008) 20–23 www.elsevier.com/locate/expgero
Mini Review
Evolving insight into the role of mitochondrial DNA mutations in aging Gregory C. Kujoth *, Tomas A. Prolla Department of Genetics and Medical Genetics, University of Wisconsin, 425-G Henry Mall, Madison, WI 53706, USA Received 22 September 2007; accepted 28 September 2007 Available online 5 October 2007
Abstract Mitochondria have occupied a central place in theories on the underlying cellular mechanisms of eukaryotic aging for several decades and much debate has ensued regarding the role of oxidative stress and mitochondrial genomic damage in these processes. Mouse models with greatly enhanced mitochondrial mutagenesis have produced dramatic aging-like phenotypes but recent results have led some to reassess whether such models are relevant to naturally occurring aging mechanisms. Here, we discuss the evolving insight that may be gained from these models regarding the contribution of mitochondrial DNA mutations to aging. 2007 Elsevier Inc. All rights reserved. Keywords: mtDNA; Mutations; Deletions; Polg; Mice; Random mutation capture
1. Mitochondrial mutator mice: testing the mitochondrial theory of aging The decline of cellular functioning with age has long been postulated to be linked to the mitochondrial production of reactive oxygen species (ROS) and the resultant damage to multiple classes of macromolecules, including carbohydrates, lipids and nucleic acids (Harman, 1972). In contrast to oxidized biomolecules that undergo rapid replacement, damage to the mitochondrial DNA (mtDNA) may be particularly harmful in that DNA repair processes in the mitochondria are less robust than those in the nucleus and the majority of the 16.5 kb circular mtDNA is coding in nature. Oxidative damage to mtDNA has been proposed to lead to a ‘‘vicious cycle’’ of further ROS production due to impaired electron transport that could result from mutations in the gene-dense mitochondrial genome (Harman, 1972). One approach to testing this idea has been the generation of mouse models of increased mtDNA mutagenesis. Ubiquitous, homozygous expression of a proofreadingdeficient isoform (D257A) of the nucleus-encoded mitochondrial DNA polymerase c (Polg) results in mice with
functional deficits in many tissues and some of these phenotypes resemble an accelerated aging syndrome (Kujoth et al., 2005; Trifunovic et al., 2004). For example, PolgD257A/D257A mice display age-related declines in adiposity, bone density, fertility, hearing and muscle mass, as well as accelerated thymic atrophy, hair graying and loss (alopecia), dilated cardiac hypertrophy and reduced survival. Impaired mitochondrial respiratory activity in these mice does not lead to increased oxidative stress but is associated with activation of apoptosis (Kujoth et al., 2005; Trifunovic et al., 2005). Eventual depletion of differentiated and regenerative cells may lead to decline of tissue functioning. Mutations accumulate in mtDNA beginning during development in these mice. It was originally thought that levels of mtDNA point mutations were 3–11-fold higher in PolgD257A/D257A mice than in heterozygous or wild type mice when assessed using a conventional PCR amplification and cloning approach. Recently developed methodology, however, has generated new insight into the complexities of the effects of mitochondrial mutagenesis.
2. The random mutation capture assay *
Corresponding author. Tel.: +1 608 265 5205; fax: +1 608 262 2976. E-mail address: [email protected] (G.C. Kujoth).
0531-5565/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2007.09.010
Critical to these new developments was the recent application of the so-called random mutation capture (RMC)
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23
during PCR). Using this approach, Vermulst et al. (2007a) have found that the levels of point mutations in wild type mice were several orders of magnitude lower than previously reported. Young (1–3 month) mice had an average base substitution mutation frequency of 6–8 · 10 7 per bp in the mitochondrial 12S rRNA subunit gene (in brain and heart). By 24–33 months, this climbed to 1 · 10 5 per bp, with an exponential increase occurring after 16 months of age. Determination of mutation frequencies in the mitochondrial cytochrome b gene gave similar results. The frequency of base substitutions in tissues from young PolgD257A/D257A mice was as high as 1.5 · 10 3 per bp, an increase of 2500-fold over wild
assay (Bielas and Loeb, 2005) to the study of mitochondrial mutations by Loeb and colleagues (Vermulst et al., 2007a). In this assay, mtDNA is isolated and subjected to restriction digestion before being amplified by PCR. Primers for PCR are chosen to flank the restriction site such that the only mtDNA molecules that can be amplified are those that are uncut, due to a mutation in the recognition site of the restriction enzyme (Fig. 1A). The presence of a selection event prior to PCR avoids the de novo introduction of mutations during amplification (this includes not only intrinsic Taq polymerase fidelity errors, which had been subtracted in previous studies, but also fixation of in vivo- or ex vivo-generated base adducts into mutations TaqI
21
TaqI
TaqI TaqI TaqI
3 CYTB
4
1
2 TaqI TaqI
12S rRNA 16S rRNA
TaqI ND6
TaqI ND1
6 ND5
TaqI
ND2
TaqI
ND4
TaqI ND4L ND3
TaqI
COX1
COX3 ATP6
COX2
5 TaqI TaqI
ATP8 TaqI TaqI TaqI
Base pairs screened: = 10,000 copies per well x 4bp (TaqI recognition site) x 84 wells = 3.36x106 bp Mutation frequency: = 9 positives / 3.36x106 bp =2.6x10-6 per bp
Fig. 1. Detection of mtDNA point mutations and large deletions by the random mutation capture assay. (A) The random mutation capture (RMC) assay principle. Mitochondrial DNA is isolated and digested with TaqI endonuclease before dilution to 10,000 copies per well in a microtiter plate. PCR amplification using primers (arrowheads) flanking a restriction site (primers 1/2) will produce a product (black wells) only when a mutation has inactivated the restriction site, leaving the mtDNA template uncut in that region. Neighboring primers (3/4) are used in a control PCR reaction to determine copy number per well in conjunction with a dilution series (light gray wells, row A). Mutation frequency is calculated as the number of positive wells per total bp screened. Cloning and sequencing of amplified products provides mutational spectra. The assay can be adapted to detect large deletions by selecting a primer set that spans multiple restriction sites (e.g., primers 5/6). The probability of inactivation by base substitution of multiple sites is rare, so that for practical purposes, loss of these sites can be deemed to be due to large deletion events. For example, assuming a conservative mutation frequency in wild type cells of 1 · 10 6 per bp, the probability of inactivating three restriction sites would be 1 · 10 18. PCR products are cloned and sequenced for verification and to map deletion endpoints. For additional technical description of the assay, see Vermulst et al. (2007a). (B) Frequency of base substitution and small insertion/deletions in aging wild type and mitochondrial mutator mice. The mean mutation frequency (±SEM) as determined at the TaqI site 634 (12S rRNA gene) in mtDNA isolated from brain. Young mice are 1–3 months, old mice are 24–33 months old. RMC data are taken from Vermulst et al. (2007a).
22
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23
type. Surprisingly, young Polg+/D257A mice also had a strikingly high mutational frequency of 3.3 · 10 4 per bp. This level is 500-fold higher than that of young wild type mice and 29-fold greater than that seen in even old wild type mice. To date, no significant pathology has been observed in Polg+/D257A mice, as they lack the accelerated aging features of homozygous mice. These data suggest that the mutational threshold at which tissue dysfunction and aging phenotypes develop must be very high and that it is unlikely that point mutations in naturally aging wild type mice will accumulate to such an extent. This argues against a causal role for point mutations in the aging process. The RMC assay described above detects base substitutions and small insertions or deletions only so these studies have not addressed the potential contribution of large mtDNA deletions. Such deletions are commonly present in aged tissues (for example, see Bender et al., 2006; Kraytsberg et al., 2006; Melov et al., 1995, 1997) and deleted mitochondrial genomes may have a replicative advantage which may contribute to their attainment of homoplasmy (where most or all of the mtDNA molecules in a mitochondrion or cell are identical), although data on this latter point are mixed (Diaz et al., 2002; Tang et al., 2000). The RMC assay can be adapted to quantify the levels of large mtDNA deletions in the mitochondrial mutator mice (Vermulst and Loeb, 2007b) by positioning primers in such a way that multiple restriction sites must all be inactivated in order for the mtDNA template to be amplified. Because the mutational frequency at each individual site is very low, removal of all the sites by a large deletion is the primary means of generating a detectable product (see Fig. 1). Similarly to the pattern observed with point mutations, the frequency of large mtDNA deletions in brain and heart tissues of wild type mice would be expected to increase with age. Homozygous mitochondrial mutator mice do show elevated levels of large deletions compared to wild type controls when detected by Southern blot analysis (Trifunovic et al., 2004). The key question is how the levels of deletions will compare quantitatively between wild type, Polg+/D257A and PolgD257A/D257A mice with age. In other words, will large mtDNA deletion mutations correlate with the presence of accelerated aging phenotypes and how similar will the levels in prematurely aged mice be to those in naturally aged mice? Data to answer these important questions should be available soon. Interestingly, modulation of mtDNA deletion levels in transgenic mice carrying a mutated allele of the mitochondrial helicase, Twinkle, has not produced accelerated aging phenotypes despite increased mtDNA deletions in several post-mitotic tissues (Tyynismaa et al., 2005). Currently, it is not clear how the levels of mtDNA deletions compare quantitatively between the Twinkle, PolgD257A/D257A and naturally aged mouse models. Additional models of increased mtDNA deletion frequencies with ubiquitous tissue distribution (as generated by gene targeting) would aid in such comparisons. Ultimately, decreasing the level of
large mtDNA deletions would be the most interesting test of a causal role in aging. 3. Conclusions The seemingly simple approach of engineering mice with elevated mtDNA mutation levels as a test of the mitochondrial theory of aging is not as straight-forward as it first seems. Aside from the complexities of temporal onset of mutations (developmental versus adult-onset) and quantitative comparisons between mutant and naturally aged wild type individuals, the qualitative class of mtDNA mutation (point mutation versus large deletion) may also be relevant. The development of the innovative RMC assay should enable a much better understanding of the relative importance of mutation type to the development of tissue dysfunction and aging phenotypes. Acknowledgement This work was supported by NIH Grant AG021905 to T.A.P. References Bender, A., Krishnan, K.J., Morris, C.M., Taylor, G.A., Reeve, A.K., Perry, R.H., Jaros, E., Hersheson, J.S., Betts, J., Klopstock, T., Taylor, R.W., Turnbull, D.M., 2006. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat. Genet. 38, 515–517. Bielas, J.H., Loeb, L.A., 2005. Quantification of random genomic mutations. Nat. Methods 2, 285–290. Diaz, F., Bayona-Bafaluy, M.P., Rana, M., Mora, M., Hao, H., Moraes, C.T., 2002. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 30, 4626–4633. Harman, D., 1972. The biologic clock: the mitochondria? J. Am. Geriatr. Soc. 20, 145–147. Kraytsberg, Y., Kudryavtseva, E., McKee, A.C., Geula, C., Kowall, N.W., Khrapko, K., 2006. Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat. Genet. 38, 518–520. Kujoth, G.C., Hiona, A., Pugh, T.D., Someya, S., Panzer, K., Wohlgemuth, S.E., Hofer, T., Seo, A.Y., Sullivan, R., Jobling, W.A., Morrow, J.D., Van Remmen, H., Sedivy, J.M., Yamasoba, T., Tanokura, M., Weindruch, R., Leeuwenburgh, C., Prolla, T.A., 2005. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309, 481–484. Melov, S., Hinerfeld, D., Esposito, L., Wallace, D.C., 1997. Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age. Nucleic Acids Res. 25, 974–982. Melov, S., Shoffner, J.M., Kaufman, A., Wallace, D.C., 1995. Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle. Nucleic Acids Res. 23, 4122–4126. Tang, Y., Manfredi, G., Hirano, M., Schon, E.A., 2000. Maintenance of human rearranged mitochondrial DNAs in long-term cultured transmitochondrial cell lines. Mol. Biol. Cell 11, 2349–2358. Trifunovic, A., Hansson, A., Wredenberg, A., Rovio, A.T., Dufour, E., Khvorostov, I., Spelbrink, J.N., Wibom, R., Jacobs, H.T., Larsson, N.G., 2005. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc. Natl. Acad. Sci. USA 429, 357–359.
G.C. Kujoth, T.A. Prolla / Experimental Gerontology 43 (2008) 20–23 Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J.N., Rovio, A.T., Bruder, C.E., Bohlooly, Y.M., Gidlof, S., Oldfors, A., Wibom, R., Tornell, J., Jacobs, H.T., Larsson, N.G., 2004. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423. Tyynismaa, H., Mjosund, K.P., Wanrooij, S., Lappalainen, I., Ylikallio, E., Jalanko, A., Spelbrink, J.N., Paetau, A., Suomalainen, A., 2005.
23
Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc. Natl. Acad. Sci. USA 102, 17687–17692. Vermulst, M., Bielas, J.H., Kujoth, G.C., Ladiges, W.C., Rabinovitch, P.S., Prolla, T.A., Loeb, L.A., 2007a. Mitochondrial point mutations do not limit the natural lifespan of mice. Nat. Genet. 39, 540–543. Vermulst, M., Loeb, L.A., 2007b. Personal communication.