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Species-specific lifespans: Can it be a lottery based on the mode of mitochondrial DNA replication?
Mechanisms of Ageing and Development 155 (2016) 1–6
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Original article
Species-specific lifespans: Can it be a lottery based on the mode of mitochondrial DNA replication? Magomed Khaidakov MD, PhD ∗ Central Arkansas Healthcare System and the University of Arkansas for Medical Sciences, Little Rock, AR, USA
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 28 January 2016 Accepted 25 February 2016 Available online 27 February 2016
a b s t r a c t Accumulating evidence suggests that the aging process is, in part, driven by accumulation of large deletions in mitochondrial DNA (mtDNA). Here, I present a hypothesis that significant variations in lifespans can be explained by species-specific mtDNA sequence features that cause a shift in the mode of mtDNA replication and thus preclude the formation of large deletions. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: mtDNA Direct repeats Deletions Alternative origins of replication Lifespan
1. Introduction There are in excess of three hundred theories of why and how we age (Medvedev, 1990). The majority of these theories, while highlighting specific aspects of this multidimensional process, are secondary to relatively few basic molecular mechanisms that include DNA damage/repair followed by mutations, epigenetic alterations, limitations of recycling machinery and telomere shortening (Kirkwood and Austad, 2000; López-Otín et al., 2013; Terman and Brunk, 2004). A combination of mutations, epigenetic changes and accumulation of extra- and intracellular residual waste likely drives senescence and is responsible for gradual destabilization, functional decline and global aging phenotype. The role of telomeres is less certain but, intuitively, the depletion of telomeres may contribute to cell attrition followed by system/organ-specific failure or the phenomenon of compressed morbidity observed in the “escapers” sub-group of supercentenarians (Andersen et al., 2012). Animal lifespans have been shown to correlate with various parameters including DNA damage/repair, metabolic rate, mitochondrial ROS production, length of telomeres, membrane fatty acid composition, etc (Barja, 2004; Bernardes de Jesus and Blasco, 2013; Pamplona et al., 1998; Promislow, 1994; Speakman, 2005). However, there is little experimental evidence that would permit an evaluation of the actual contribution of suspected mechanisms
∗ Correspondence to: 4301 West Markham Street, #567, Shorey Building, Little Rock, AR 72205, USA. E-mail address: [email protected] http://dx.doi.org/10.1016/j.mad.2016.02.012 0047-6374/© 2016 Elsevier Ireland Ltd. All rights reserved.
to species-specific physiological aging rate and lifespan. Although the basic mechanisms of aging should be operational in all living things, it is not at all certain that they maintain the same relative weight across species. On the contrary, several lines of evidence suggest that different animals likely have unique sets of leading determinants. For example, oxidative stress may be one of the important contributors in fruit flies since antioxidant treatments are highly efficient in significantly extending their lifespans (Parkes et al., 1998; Sun and Tower, 1999). When similar approaches are utilized in mammals, they largely fail (Barja, 2004; Pérez et al., 2009). This suggests that mammals have developed highly efficient mechanisms for reduction of ROS production and/or antioxidant defense and, therefore, oxidative stress is no longer at the forefront of factors responsible for their lifespan. Similarly, telomere attrition does not appear to be a significant factor in inbred mice with long telomeres, as telomerase deficient animals do not experience reduction in lifespan or have any detrimental consequences for several generations (Blasco et al., 1997). Yet, when telomerase is inactivated in mice with much shorter chromosomes (Cast/EiJ), degenerative effects appear in the first generation (Armanios et al., 2009). Clearly, the contribution of telomeres to senescence and lifespan may be context-dependent. The available data indicate that many proposed determinants of lifespan can be bypassed, offset or ignored in long-lived animals. It also remains possible that the contribution of suspected mechanisms of aging is relatively minor or derivative in relation to yet to be discovered and much more potent determinant(s).
2
M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
Fig. 1. (a)—Map of detected homology-based deletions in human mtDNA. Insert—an illustration of strand-displacement mechanism of mtDNA replication; (b) and (c)—graphic representation of direct repeats originating from major arc (“active repeats”) and minor arc (“dormant repeats”).
2. Materials and methods 2.1. Screening for direct repeats and OL-like stem-loop secondary structures Direct repeats were identified using the REPuter program (http://bibiserv.techfak.uni-bielefeld.de/reputer/, Kurtz et al., 2001). Selection criteria for OL-like secondary structures were based on the results of the recent OL mutagenesis study (Wanrooij et al., 2012) and included the following requirements: (1) loop size
should be no less than 10 nucleotides; (2) the loop is required to contain stretches of at least 3 thymines (H-strand) or adenines (Lstrand) and/or (3) the second position at the base of the loop relative to the 3 component of the stem should be occupied by thymine (Adenine for L-strand)
2.2. Screening for potential alternative OLs It was conducted in following steps: (1) All reverse-complement repeats equal or longer than 8 nucleotides without mismatches
M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
3
and equal or longer than 10 nucleotides with 2 allowed mismatches were identified using the REPuter program; (2) We limited our analysis to repeats with projected loop size not exceeding 50 nucleotides; (3) Of these, we selected sequences with at least 30% GC content in reverse complement repeats (stems). (4) The thermodynamic stability of secondary structures formed by candidate sequences were determined using Mfold software (Zuker, 2003).
3. Results and discussion Accumulating evidence suggests that the aging process may, in part, be driven by mutations in mitochondrial DNA (mtDNA) that have a particularly strong impact on non- or slowly-renewable cell populations such as neurons or myocytes (Bua et al., 2006; Fayet et al., 2002; Itoh et al., 1996; Kraytsberg et al., 2006). The proportion of cytochrome c oxidase (COX)-negative neurons has been reported to reach around 30% in the substantia nigra of elderly subjects (Itoh et al., 1996), with all respiration-deficient cells carrying clonally expanded large deletions in mtDNA (Kraytsberg et al., 2006). Similarly, analysis of skeletal muscles revealed significant aging-associated accumulation (up to 31%) of compromised cells with clonally expanded deletions found in all tested COX-negative fibers (Bua et al., 2006). Presence of mutations in a majority of cellular mtDNA copies results in mitochondrial dysfunction with far ranging detrimental consequences including respiratory deficiency, enhanced susceptibility to apoptosis, increased burden of recycling and accelerated accumulation of residual waste. Of all classes of mutations, deletions are reported to be a primary factor implicated in accelerated aging phenotype and reduced lifespan of the mtDNA mutator (Polgmut/mut ) mouse (Vermulst et al., 2008). The importance of deletions is likely based on predominantly homology-driven mechanisms of their formation in mtDNA, as the vast majority of detected deletions are flanked by direct repeats (Ruiz-Pesini et al., 2007). This ensures multiple occurrences of the same deletion and, therefore, a much higher likelihood for the development of homoplasmy with the attending functional consequences. It is of great interest that the distribution of deletions within mtDNA sequence is far from random (Fig. 1a). In humans, the vast majority of deletions have been detected within the major arc between replication origins for heavy and light strands (OH and OL, respectively). This does not mean, however, that the minor arc is devoid of direct repeats (Fig. 1b and c). On the contrary, the frequency of direct repeats originating from the minor arc is almost double compared to that of the major arc (31.3/kb vs. 16.9/kb for ≥10nt DRs, Fig. 1B). The obvious over-representation of detected deletions within the major arc is consistent with the strand displacement model (Clayton, 1982) according to which mtDNA replication proceeds unidirectionally from OH until about 2/3 (about 11 kb) of the heavy strand is synthesized, OL is then exposed and the replication of the light strand commences (Fig. 1A insert). This is in stark contrast with the replication of nuclear DNA (Fig. 2) where the lagging strand is synthesized simultaneously with the leading strand in a series of small 200–300 nt fragments − a mechanism that practically excludes the possibility of interaction between widely separated repeats. The relative excess of direct repeats in the minor arc also strongly suggests that the existing positioning of OL protects mtDNA from even more accelerated homology-driven mutagenesis. After a period of controversial reports regarding the particulars of mammalian mtDNA replication (Brown et al., 2005; Holt et al., 2000), it has been re-confirmed that, at least in the mouse, the strand-displacement model is by far the most dominant (Bogenhagen and Clayton, 2003). Equally important, it has been shown that a parallel minor streams of more symmetrical repli-
Fig. 2. Comparison in replicational modes of nuclear and mitochondrial DNA. The mode of mtDNA replication (synthesis of lagging strand of major arc in one large fragment) facilitates interaction between distantly spaced direct repeats (rectangles).
cation originating from yet unknown alternative OLs also exist (Bogenhagen and Clayton, 2003). By the virtue of secondary structure similarities, some tRNAs have been suggested to act as such alternative OLs (Seligmann, 2010), although it is possible that OLlike structures can appear anywhere in the mitochondrial genome. This also raises several possibilities. Birds and several reptiles show that it is not essential to have OL as the dedicated origin of replication and that its absence can be offset by alternative OLs. Indeed, the analysis of avian mtDNA has demonstrated that its replication occurs from multiple positions, albeit with different intensity (Reyes et al., 2005). Let’s also note that, with few exceptions, avians have considerably greater lifespans compared to similarly sized mammals (Tacutu et al., 2013). In the context of a connection between the position of the OL and the frequency of deletions in mtDNA, it is reasonable to assume that escape from rigidly asymmetric replication would largely solve the problem of deletion events based on widely separated repeats. Species-specific variations in the ratio of non-symmetrical versus symmetrical replication modes may, therefore, have profound effects on the rate of deletion formation and, by extension, on mitochondrial function, cell attrition, aging rate and lifespan. In principle, there are several possible strategies to eliminate the mtDNA mutational load associated with direct repeats. For example, a reduction of deletion incidence could be achieved by either lowering number and length of direct repeats, or creating several
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Fig. 3. Candidate alternative OLs in M. musculus and H. glaber mitochondrial genomes (OL-like stem-loop structures that reached greater than 40% level (G ≥ −5.4 kcal/mol) of thermodynamic stability of canonical OL). A—candidate OLs found in M. musculus mtDNA; B—candidate OLs found in H. glaber mtDNA. C—Potential effect of additional OLs on interactions between widely separated repeats (rectangles).
Fig. 4. Positioning of additional OL within HSL tRNA cluster is expected to disrupt interactions between most repeats involved in formation of deletions in human mtDNA.
additional OLs within the major arc of mtDNA. The removal of direct repeats implies multiple point mutations throughout the mitochondrial genome and is constrained by a necessity to preserve the coding sequence and avoid generation of new repeats. Although the existence of a trend toward reducing the frequency of direct
repeats in the mtDNA of long-lived species has been reported by us and others (Khaidakov et al., 2006; Samuels, 2004), it has rather limited potential. In comparison, generating thermodynamically stable OL-like stem-loop secondary structures is mechanistically
M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
much simpler and may require only minor changes of mtDNA sequence. To tentatively test our hypothesis, we searched for OL-like secondary structures (see Supplemental Table 1) in mitochondrial genomes of Mus musculus (mouse, MM) and Heterocephalus glaber (naked mole-rat, NMR) as comparably sized rodents with dramatically (10-fold) different maximum lifespans (MLSs). NMR is a unique outlier within the mammalian community, as numerous studies have failed to identify any compelling reason that would account for its extraordinary longevity (Buffenstein, 2005). In fact, most parameters currently thought to be implicated in aging, including oxidative stress, antioxidant defense, DNA damage and telomere length suggest that the NMR should age faster than the mouse. Mitochondrial genomes from both species did not lack potential candidates for alternative OLs with close to perfect loop sequences (see Supplemental Table 1). The GC content of MM mtDNA, however, is almost 14% (35.4% vs. 41.2%) lower than in NMR mtDNA. A strong correlation between mtDNA GC content and lifespan has been recently reported (Lehmann et al., 2008) and it likely increases probability for the formation of thermodynamically stable secondary structures. The mouse genome yielded 11 potential candidates that roughly conformed to the criteria for functional OL identified in a recent mutagenesis study (Wanrooij et al., 2012), whereas NMR mtDNA contained 17 OL-like secondary structures. The mouse mtDNA coding sequence contained only three OL-like stem-loop structures that reached greater than 40% (G ≥ −5.4 kcal/mol) of the thermodynamic stability of canonical OL (Fig. 3) with only two of them located within major arc (ND3 and tRNA-Pro). Both candidate OLs were positioned in the vicinity of OH. Assuming that they are functional, mtDNA replication would still remain severely asymmetrical and proceed through synthesis of continuous ∼10 Kb fragment enclosing all active repeats. In contrast, the NMR genome contained five OL-like structures all of which were located within the major arc in Cox2, tRNA-Lys, Cox3, ND4L and CytB at different distances from each other and OL ranging from 4 to 6 Kb. If these alternative OLs were functional, a majority of direct repeats would be synthesized on different fragments and, therefore, would not be able to facilitate formation of large deletions. It should be noted that the presence of 5 secondary structures similar to OL in the major arc of NMR mtDNA is merely suggestive since there is no evidence that they can function as bona fide origins of replication. Based on this hypothesis, however, it can be expected that, compared to mice, replication of NMR mtDNA is initiated at multiple sites and that the spectrum of mtDNA deletions significantly differs in relation to expected based on distribution of direct repeats. The stochastic nature of the cumulative effects of alternative OLs on deletion formation caused by species-specific differences in their functionality, number, relative strength and location creates a peculiar scenario where species may have vastly different lifespans without any significant disparities in other “key” parameters. This “lottery” mechanism does not imply any physiological footprint and may work in both directions. For example, if a functional and stable alternative OL forms in close proximity to canonical OL or more distally in relation to OH, their impact on deletion formation and, therefore, lifespan is expected to range from negligible to even detrimental. Alternatively, even one strategically placed functional alternative OL can result in a dramatic reduction in deletion accumulation. For example, positioning of an additional OL in another tRNA cluster HSL should theoretically disrupt interactions between the vast majority of repeats flanking known large deletions (Fig. 4). Elimination of large deletions, in turn, is likely to change a dynamic of clonal expansion of mutations in mtDNA and, therefore, decrease the incidence of respiration-deficient cells.
5
Our analysis suggests that, compared to the mouse, the NMR may have a greater portion of mtDNA replication channeled through a more symmetrical mode, which discourages interactions between widely spaced direct repeats and should, in principle, result in diminished accumulation of large deletions and slower aging. On the other hand, mouse mitochondrial DNA replicates almost exclusively via strand displacement mechanism and, therefore, is expected to accumulate deletions at relatively accelerated rate. Following the same logic, birds – whose mtDNAs are lacking canonical OL by default – appear to employ an exclusively symmetrical, albeit with species-specific differences, replication mechanism and, therefore, stand to benefit most (as a group) in terms of extended lifespan. 4. Conclusions In summary, we present a novel possible determinant of species lifespan based on sequence features of mtDNA that can change its mode of replication and dramatically decrease its susceptibility to deletion formation. If such mechanism exists, it may have profound effect on the rate of senescence and global aging. In this context, one can make a number of predictions awaiting experimental validation: • Comparably sized mammals with significantly different lifespans will display species-specific deviations from the strand displacement mode of mtDNA replication with a number of additional functional OLs found in longer-lived animals; • Compared to their similarly sized short-lived counterparts, longer lived mammals will exhibit lower rate of deletion accumulation in mtDNA and a significantly lower incidence of large deletions; • Due to decelerated mitochondrial aging and, therefore, diminished need for recycling, there will be less accumulation of intracellular waste. • The combination of aforementioned factors will result in delayed senescence, fewer respiration deficient/dysfunctional cell and reduced cell attrition in slowly renewable cell populations such as neurons and myocytes. Acknowledgements I am very grateful to Dr. M. Dobretsov for helping with analysis of mtDNA sequences for presence of OL-like secondary structures. I am also grateful to Drs. M. Dobretsov and R. Shmookler Reis for their constructive comments and critique. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2016.02. 012. References Andersen, S.L., Sebastiani, P., Dworkis, D.A., Feldman, L., Perls, T.T., 2012. J. Gerontol. A. Biol. Sci. Med. Sci. 67, 395–405. Armanios, M., Alder, J.K., Parry, E.M., Karim, B., Strong, M.A., Greider, C.W., 2009. Short telomeres are sufficient to cause the degenerative defects associated with aging. Am. J. Hum. Genet. 85, 823–832. Barja, G., 2004. Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism? Biol. Rev. Camb. Philos. Soc. 79, 235–251. Bernardes de Jesus, B., Blasco, M.A., 2013. Telomerase at the intersection of cancer and aging. Trends Genet. 29, 513–520. Blasco, M.A., Lee, H.W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A., Greider, C.W., 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34.
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Bogenhagen, D.F., Clayton, D.A., 2003. The mitochondrial replication bubble has not burst. Trends Biochem. Sci. 28, 357–360. Brown, T.A., Cecconi, C., Tkachuk, A.N., Bustamante, C., Clayton, D.A., 2005. Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev. 19, 2466–2476. Bua, E., Johnson, J., Herbst, A., Delong, B., McKenzie, D., Salamat, S., Aiken, J.M., 2006. Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am. J. Hum. Genet. 79, 469–480. Buffenstein, R., 2005. The naked mole-rat: a new long-living model for human aging. J. Gerontol. A. Biol. Sci. Med. Sci. 60, 1369–1377. Clayton, D.A., 1982. Replication of animal mitochondrial DNA. Cell 28, 693–705. Fayet, G., Jansson, M., Sternberg, D., Moslemi, A.R., Blondy, P., Lombès, A., Fardeau, M., Oldfors, A., 2002. Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function. Neuromuscul. Disord. 12, 484–493. Holt, I.J., Lorimer, H.E., Jacobs, H.T., 2000. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524. Itoh, K., Weis, S., Mehraein, P., Müller-Höcker, J., 1996. Cytochrome c oxidase defects of the human substantia nigra in normal aging. Neurobiol. Aging 17, 843–848. Khaidakov, M., Siegel, E.R., Shmookler Reis, R.J., 2006. Direct repeats in mitochondrial DNA and mammalian lifespan. Mech. Ageing Dev. 127, 808–812. Kirkwood, T.B.L., Austad, S.N., 2000. Why do we age? Nature 408, 233–238. 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. Kurtz, S., Choudhuri, J.V., Ohlebusch, E., Schleiermacher, S., Stoye, J., Giegerich, R., 2001. The Manifold Applications of Repeat Analysis on a Genomic Scale. Nucleic Acids Res. 29, 4633–4642. López-Otín, C., Blasco, M.A., Partridge, L., Serrano, M., Kroemer, G., 2013. The hallmarks of aging. Cell 153, 1194–1217. Lehmann, G., Segal, E., Muradian, K.K., Fraifeld, V.E., 2008. Do mitochondrial DNA and metabolic rate complement each other in determination of the mammalian maximum longevity? Rejuvenation Res. 11, 409–417. Medvedev, Z.A., 1990. An attempt at a rational classification of theories of ageing. Biol. Rev. Camb. Philos. Soc. 65, 375–398. Pérez, V.I., Van Remmen, H., Bokov, A., Epstein, C.J., Vijg, J., Richardson, A., 2009. The overexpression of major antioxidant enzymes does not extend the lifespan of mice. Aging Cell 8, 73–75.
Pamplona, R., Portero-Otin, M., Riba, D., Ruiz, C., Prat, J., Bellmunt, M.J., Barja, G., 1998. Mitochondrial membrane peroxidizability index is inversely related to maximum lifespan in mammals. J. Lipid Res. 39, 1989–1994. Parkes, T.L., Elia, A.J., Dickinson, D., Hilliker, A.J., Phillips, J.P., Boulianne, G.L., 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19, 171–174. Promislow, D.E., 1994. DNA repair and the evolution of longevity: a critical analysis. J. Theor. Biol. 170, 291–300. Reyes, A., Yang, M.Y., Bowmaker, M., Holt, I.J., 2005. Bidirectional replication initiates at sites throughout the mitochondrial genome of birds. J. Biol. Chem. 280, 3242–3250. Ruiz-Pesini, E., Lott, M.T., Procaccio, V., Poole, J.C., Brandon, M.C., Mishmar, D., Yi, C., Kreuziger, J., Baldi, P., Wallace, D.C., 2007. An enhanced MITOMAP with a global mtDNA mutational phylogeny. Nucleic Acid Res. 35, D823–D828. Samuels, D.C., 2004. Mitochondrial DNA repeats constrain the life span of mammals. TRIGS 20, 226–229. Seligmann, H., 2010. Mitochondrial tRNAs as light strand replication origins: similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication. Biosystems 99, 85–93. Speakman, J.R., 2005. Body size, energy metabolism and lifespan. J. Exp. Biol. 208, 1717–1730. Sun, J., Tower, J., 1999. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies. Mol. Cell. Biol. 19, 216–228. Tacutu, R., Craig, T., Budovsky, A., Wuttke, D., Lehmann, G., Taranukha, D., Costa, J., Fraifeld, V.E., de Magalhães, J.P., 2013. Human Ageing Genomic Resources: Integrated databases and tools for the biology and genetics of ageing. Nucleic Acids Res. 41, D1027–D1033. Terman, A., Brunk, U.T., 2004. Aging as a catabolic malfunction. Int. J. Biochem. Cell. Biol. 36, 2365–2375. Vermulst, M., Wanagat, J., Kujoth, G.C., Bielas, J.H., Rabinovitch, P.S., Prolla, T.A., Loeb, L.A., 2008. DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat. Genet. 40, 392–394. Wanrooij, S., Miralles Fusté, J., Stewart, J.B., Wanrooij, P.H., Samuelsson, T., Larsson, N.G., Gustafsson, C.M., Falkenberg, M., 2012. In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication. EMBO Rep. 13, 1130–1137. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Original article
Species-specific lifespans: Can it be a lottery based on the mode of mitochondrial DNA replication? Magomed Khaidakov MD, PhD ∗ Central Arkansas Healthcare System and the University of Arkansas for Medical Sciences, Little Rock, AR, USA
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 28 January 2016 Accepted 25 February 2016 Available online 27 February 2016
a b s t r a c t Accumulating evidence suggests that the aging process is, in part, driven by accumulation of large deletions in mitochondrial DNA (mtDNA). Here, I present a hypothesis that significant variations in lifespans can be explained by species-specific mtDNA sequence features that cause a shift in the mode of mtDNA replication and thus preclude the formation of large deletions. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: mtDNA Direct repeats Deletions Alternative origins of replication Lifespan
1. Introduction There are in excess of three hundred theories of why and how we age (Medvedev, 1990). The majority of these theories, while highlighting specific aspects of this multidimensional process, are secondary to relatively few basic molecular mechanisms that include DNA damage/repair followed by mutations, epigenetic alterations, limitations of recycling machinery and telomere shortening (Kirkwood and Austad, 2000; López-Otín et al., 2013; Terman and Brunk, 2004). A combination of mutations, epigenetic changes and accumulation of extra- and intracellular residual waste likely drives senescence and is responsible for gradual destabilization, functional decline and global aging phenotype. The role of telomeres is less certain but, intuitively, the depletion of telomeres may contribute to cell attrition followed by system/organ-specific failure or the phenomenon of compressed morbidity observed in the “escapers” sub-group of supercentenarians (Andersen et al., 2012). Animal lifespans have been shown to correlate with various parameters including DNA damage/repair, metabolic rate, mitochondrial ROS production, length of telomeres, membrane fatty acid composition, etc (Barja, 2004; Bernardes de Jesus and Blasco, 2013; Pamplona et al., 1998; Promislow, 1994; Speakman, 2005). However, there is little experimental evidence that would permit an evaluation of the actual contribution of suspected mechanisms
∗ Correspondence to: 4301 West Markham Street, #567, Shorey Building, Little Rock, AR 72205, USA. E-mail address: [email protected] http://dx.doi.org/10.1016/j.mad.2016.02.012 0047-6374/© 2016 Elsevier Ireland Ltd. All rights reserved.
to species-specific physiological aging rate and lifespan. Although the basic mechanisms of aging should be operational in all living things, it is not at all certain that they maintain the same relative weight across species. On the contrary, several lines of evidence suggest that different animals likely have unique sets of leading determinants. For example, oxidative stress may be one of the important contributors in fruit flies since antioxidant treatments are highly efficient in significantly extending their lifespans (Parkes et al., 1998; Sun and Tower, 1999). When similar approaches are utilized in mammals, they largely fail (Barja, 2004; Pérez et al., 2009). This suggests that mammals have developed highly efficient mechanisms for reduction of ROS production and/or antioxidant defense and, therefore, oxidative stress is no longer at the forefront of factors responsible for their lifespan. Similarly, telomere attrition does not appear to be a significant factor in inbred mice with long telomeres, as telomerase deficient animals do not experience reduction in lifespan or have any detrimental consequences for several generations (Blasco et al., 1997). Yet, when telomerase is inactivated in mice with much shorter chromosomes (Cast/EiJ), degenerative effects appear in the first generation (Armanios et al., 2009). Clearly, the contribution of telomeres to senescence and lifespan may be context-dependent. The available data indicate that many proposed determinants of lifespan can be bypassed, offset or ignored in long-lived animals. It also remains possible that the contribution of suspected mechanisms of aging is relatively minor or derivative in relation to yet to be discovered and much more potent determinant(s).
2
M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
Fig. 1. (a)—Map of detected homology-based deletions in human mtDNA. Insert—an illustration of strand-displacement mechanism of mtDNA replication; (b) and (c)—graphic representation of direct repeats originating from major arc (“active repeats”) and minor arc (“dormant repeats”).
2. Materials and methods 2.1. Screening for direct repeats and OL-like stem-loop secondary structures Direct repeats were identified using the REPuter program (http://bibiserv.techfak.uni-bielefeld.de/reputer/, Kurtz et al., 2001). Selection criteria for OL-like secondary structures were based on the results of the recent OL mutagenesis study (Wanrooij et al., 2012) and included the following requirements: (1) loop size
should be no less than 10 nucleotides; (2) the loop is required to contain stretches of at least 3 thymines (H-strand) or adenines (Lstrand) and/or (3) the second position at the base of the loop relative to the 3 component of the stem should be occupied by thymine (Adenine for L-strand)
2.2. Screening for potential alternative OLs It was conducted in following steps: (1) All reverse-complement repeats equal or longer than 8 nucleotides without mismatches
M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
3
and equal or longer than 10 nucleotides with 2 allowed mismatches were identified using the REPuter program; (2) We limited our analysis to repeats with projected loop size not exceeding 50 nucleotides; (3) Of these, we selected sequences with at least 30% GC content in reverse complement repeats (stems). (4) The thermodynamic stability of secondary structures formed by candidate sequences were determined using Mfold software (Zuker, 2003).
3. Results and discussion Accumulating evidence suggests that the aging process may, in part, be driven by mutations in mitochondrial DNA (mtDNA) that have a particularly strong impact on non- or slowly-renewable cell populations such as neurons or myocytes (Bua et al., 2006; Fayet et al., 2002; Itoh et al., 1996; Kraytsberg et al., 2006). The proportion of cytochrome c oxidase (COX)-negative neurons has been reported to reach around 30% in the substantia nigra of elderly subjects (Itoh et al., 1996), with all respiration-deficient cells carrying clonally expanded large deletions in mtDNA (Kraytsberg et al., 2006). Similarly, analysis of skeletal muscles revealed significant aging-associated accumulation (up to 31%) of compromised cells with clonally expanded deletions found in all tested COX-negative fibers (Bua et al., 2006). Presence of mutations in a majority of cellular mtDNA copies results in mitochondrial dysfunction with far ranging detrimental consequences including respiratory deficiency, enhanced susceptibility to apoptosis, increased burden of recycling and accelerated accumulation of residual waste. Of all classes of mutations, deletions are reported to be a primary factor implicated in accelerated aging phenotype and reduced lifespan of the mtDNA mutator (Polgmut/mut ) mouse (Vermulst et al., 2008). The importance of deletions is likely based on predominantly homology-driven mechanisms of their formation in mtDNA, as the vast majority of detected deletions are flanked by direct repeats (Ruiz-Pesini et al., 2007). This ensures multiple occurrences of the same deletion and, therefore, a much higher likelihood for the development of homoplasmy with the attending functional consequences. It is of great interest that the distribution of deletions within mtDNA sequence is far from random (Fig. 1a). In humans, the vast majority of deletions have been detected within the major arc between replication origins for heavy and light strands (OH and OL, respectively). This does not mean, however, that the minor arc is devoid of direct repeats (Fig. 1b and c). On the contrary, the frequency of direct repeats originating from the minor arc is almost double compared to that of the major arc (31.3/kb vs. 16.9/kb for ≥10nt DRs, Fig. 1B). The obvious over-representation of detected deletions within the major arc is consistent with the strand displacement model (Clayton, 1982) according to which mtDNA replication proceeds unidirectionally from OH until about 2/3 (about 11 kb) of the heavy strand is synthesized, OL is then exposed and the replication of the light strand commences (Fig. 1A insert). This is in stark contrast with the replication of nuclear DNA (Fig. 2) where the lagging strand is synthesized simultaneously with the leading strand in a series of small 200–300 nt fragments − a mechanism that practically excludes the possibility of interaction between widely separated repeats. The relative excess of direct repeats in the minor arc also strongly suggests that the existing positioning of OL protects mtDNA from even more accelerated homology-driven mutagenesis. After a period of controversial reports regarding the particulars of mammalian mtDNA replication (Brown et al., 2005; Holt et al., 2000), it has been re-confirmed that, at least in the mouse, the strand-displacement model is by far the most dominant (Bogenhagen and Clayton, 2003). Equally important, it has been shown that a parallel minor streams of more symmetrical repli-
Fig. 2. Comparison in replicational modes of nuclear and mitochondrial DNA. The mode of mtDNA replication (synthesis of lagging strand of major arc in one large fragment) facilitates interaction between distantly spaced direct repeats (rectangles).
cation originating from yet unknown alternative OLs also exist (Bogenhagen and Clayton, 2003). By the virtue of secondary structure similarities, some tRNAs have been suggested to act as such alternative OLs (Seligmann, 2010), although it is possible that OLlike structures can appear anywhere in the mitochondrial genome. This also raises several possibilities. Birds and several reptiles show that it is not essential to have OL as the dedicated origin of replication and that its absence can be offset by alternative OLs. Indeed, the analysis of avian mtDNA has demonstrated that its replication occurs from multiple positions, albeit with different intensity (Reyes et al., 2005). Let’s also note that, with few exceptions, avians have considerably greater lifespans compared to similarly sized mammals (Tacutu et al., 2013). In the context of a connection between the position of the OL and the frequency of deletions in mtDNA, it is reasonable to assume that escape from rigidly asymmetric replication would largely solve the problem of deletion events based on widely separated repeats. Species-specific variations in the ratio of non-symmetrical versus symmetrical replication modes may, therefore, have profound effects on the rate of deletion formation and, by extension, on mitochondrial function, cell attrition, aging rate and lifespan. In principle, there are several possible strategies to eliminate the mtDNA mutational load associated with direct repeats. For example, a reduction of deletion incidence could be achieved by either lowering number and length of direct repeats, or creating several
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Fig. 3. Candidate alternative OLs in M. musculus and H. glaber mitochondrial genomes (OL-like stem-loop structures that reached greater than 40% level (G ≥ −5.4 kcal/mol) of thermodynamic stability of canonical OL). A—candidate OLs found in M. musculus mtDNA; B—candidate OLs found in H. glaber mtDNA. C—Potential effect of additional OLs on interactions between widely separated repeats (rectangles).
Fig. 4. Positioning of additional OL within HSL tRNA cluster is expected to disrupt interactions between most repeats involved in formation of deletions in human mtDNA.
additional OLs within the major arc of mtDNA. The removal of direct repeats implies multiple point mutations throughout the mitochondrial genome and is constrained by a necessity to preserve the coding sequence and avoid generation of new repeats. Although the existence of a trend toward reducing the frequency of direct
repeats in the mtDNA of long-lived species has been reported by us and others (Khaidakov et al., 2006; Samuels, 2004), it has rather limited potential. In comparison, generating thermodynamically stable OL-like stem-loop secondary structures is mechanistically
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much simpler and may require only minor changes of mtDNA sequence. To tentatively test our hypothesis, we searched for OL-like secondary structures (see Supplemental Table 1) in mitochondrial genomes of Mus musculus (mouse, MM) and Heterocephalus glaber (naked mole-rat, NMR) as comparably sized rodents with dramatically (10-fold) different maximum lifespans (MLSs). NMR is a unique outlier within the mammalian community, as numerous studies have failed to identify any compelling reason that would account for its extraordinary longevity (Buffenstein, 2005). In fact, most parameters currently thought to be implicated in aging, including oxidative stress, antioxidant defense, DNA damage and telomere length suggest that the NMR should age faster than the mouse. Mitochondrial genomes from both species did not lack potential candidates for alternative OLs with close to perfect loop sequences (see Supplemental Table 1). The GC content of MM mtDNA, however, is almost 14% (35.4% vs. 41.2%) lower than in NMR mtDNA. A strong correlation between mtDNA GC content and lifespan has been recently reported (Lehmann et al., 2008) and it likely increases probability for the formation of thermodynamically stable secondary structures. The mouse genome yielded 11 potential candidates that roughly conformed to the criteria for functional OL identified in a recent mutagenesis study (Wanrooij et al., 2012), whereas NMR mtDNA contained 17 OL-like secondary structures. The mouse mtDNA coding sequence contained only three OL-like stem-loop structures that reached greater than 40% (G ≥ −5.4 kcal/mol) of the thermodynamic stability of canonical OL (Fig. 3) with only two of them located within major arc (ND3 and tRNA-Pro). Both candidate OLs were positioned in the vicinity of OH. Assuming that they are functional, mtDNA replication would still remain severely asymmetrical and proceed through synthesis of continuous ∼10 Kb fragment enclosing all active repeats. In contrast, the NMR genome contained five OL-like structures all of which were located within the major arc in Cox2, tRNA-Lys, Cox3, ND4L and CytB at different distances from each other and OL ranging from 4 to 6 Kb. If these alternative OLs were functional, a majority of direct repeats would be synthesized on different fragments and, therefore, would not be able to facilitate formation of large deletions. It should be noted that the presence of 5 secondary structures similar to OL in the major arc of NMR mtDNA is merely suggestive since there is no evidence that they can function as bona fide origins of replication. Based on this hypothesis, however, it can be expected that, compared to mice, replication of NMR mtDNA is initiated at multiple sites and that the spectrum of mtDNA deletions significantly differs in relation to expected based on distribution of direct repeats. The stochastic nature of the cumulative effects of alternative OLs on deletion formation caused by species-specific differences in their functionality, number, relative strength and location creates a peculiar scenario where species may have vastly different lifespans without any significant disparities in other “key” parameters. This “lottery” mechanism does not imply any physiological footprint and may work in both directions. For example, if a functional and stable alternative OL forms in close proximity to canonical OL or more distally in relation to OH, their impact on deletion formation and, therefore, lifespan is expected to range from negligible to even detrimental. Alternatively, even one strategically placed functional alternative OL can result in a dramatic reduction in deletion accumulation. For example, positioning of an additional OL in another tRNA cluster HSL should theoretically disrupt interactions between the vast majority of repeats flanking known large deletions (Fig. 4). Elimination of large deletions, in turn, is likely to change a dynamic of clonal expansion of mutations in mtDNA and, therefore, decrease the incidence of respiration-deficient cells.
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Our analysis suggests that, compared to the mouse, the NMR may have a greater portion of mtDNA replication channeled through a more symmetrical mode, which discourages interactions between widely spaced direct repeats and should, in principle, result in diminished accumulation of large deletions and slower aging. On the other hand, mouse mitochondrial DNA replicates almost exclusively via strand displacement mechanism and, therefore, is expected to accumulate deletions at relatively accelerated rate. Following the same logic, birds – whose mtDNAs are lacking canonical OL by default – appear to employ an exclusively symmetrical, albeit with species-specific differences, replication mechanism and, therefore, stand to benefit most (as a group) in terms of extended lifespan. 4. Conclusions In summary, we present a novel possible determinant of species lifespan based on sequence features of mtDNA that can change its mode of replication and dramatically decrease its susceptibility to deletion formation. If such mechanism exists, it may have profound effect on the rate of senescence and global aging. In this context, one can make a number of predictions awaiting experimental validation: • Comparably sized mammals with significantly different lifespans will display species-specific deviations from the strand displacement mode of mtDNA replication with a number of additional functional OLs found in longer-lived animals; • Compared to their similarly sized short-lived counterparts, longer lived mammals will exhibit lower rate of deletion accumulation in mtDNA and a significantly lower incidence of large deletions; • Due to decelerated mitochondrial aging and, therefore, diminished need for recycling, there will be less accumulation of intracellular waste. • The combination of aforementioned factors will result in delayed senescence, fewer respiration deficient/dysfunctional cell and reduced cell attrition in slowly renewable cell populations such as neurons and myocytes. Acknowledgements I am very grateful to Dr. M. Dobretsov for helping with analysis of mtDNA sequences for presence of OL-like secondary structures. I am also grateful to Drs. M. Dobretsov and R. Shmookler Reis for their constructive comments and critique. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2016.02. 012. References Andersen, S.L., Sebastiani, P., Dworkis, D.A., Feldman, L., Perls, T.T., 2012. J. Gerontol. A. Biol. Sci. Med. Sci. 67, 395–405. Armanios, M., Alder, J.K., Parry, E.M., Karim, B., Strong, M.A., Greider, C.W., 2009. Short telomeres are sufficient to cause the degenerative defects associated with aging. Am. J. Hum. Genet. 85, 823–832. Barja, G., 2004. Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism? Biol. Rev. Camb. Philos. Soc. 79, 235–251. Bernardes de Jesus, B., Blasco, M.A., 2013. Telomerase at the intersection of cancer and aging. Trends Genet. 29, 513–520. Blasco, M.A., Lee, H.W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A., Greider, C.W., 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34.
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M.K. MD, PhD / Mechanisms of Ageing and Development 155 (2016) 1–6
Bogenhagen, D.F., Clayton, D.A., 2003. The mitochondrial replication bubble has not burst. Trends Biochem. Sci. 28, 357–360. Brown, T.A., Cecconi, C., Tkachuk, A.N., Bustamante, C., Clayton, D.A., 2005. Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev. 19, 2466–2476. Bua, E., Johnson, J., Herbst, A., Delong, B., McKenzie, D., Salamat, S., Aiken, J.M., 2006. Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am. J. Hum. Genet. 79, 469–480. Buffenstein, R., 2005. The naked mole-rat: a new long-living model for human aging. J. Gerontol. A. Biol. Sci. Med. Sci. 60, 1369–1377. Clayton, D.A., 1982. Replication of animal mitochondrial DNA. Cell 28, 693–705. Fayet, G., Jansson, M., Sternberg, D., Moslemi, A.R., Blondy, P., Lombès, A., Fardeau, M., Oldfors, A., 2002. Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function. Neuromuscul. Disord. 12, 484–493. Holt, I.J., Lorimer, H.E., Jacobs, H.T., 2000. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524. Itoh, K., Weis, S., Mehraein, P., Müller-Höcker, J., 1996. Cytochrome c oxidase defects of the human substantia nigra in normal aging. Neurobiol. Aging 17, 843–848. Khaidakov, M., Siegel, E.R., Shmookler Reis, R.J., 2006. Direct repeats in mitochondrial DNA and mammalian lifespan. Mech. Ageing Dev. 127, 808–812. Kirkwood, T.B.L., Austad, S.N., 2000. Why do we age? Nature 408, 233–238. 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. Kurtz, S., Choudhuri, J.V., Ohlebusch, E., Schleiermacher, S., Stoye, J., Giegerich, R., 2001. The Manifold Applications of Repeat Analysis on a Genomic Scale. Nucleic Acids Res. 29, 4633–4642. López-Otín, C., Blasco, M.A., Partridge, L., Serrano, M., Kroemer, G., 2013. The hallmarks of aging. Cell 153, 1194–1217. Lehmann, G., Segal, E., Muradian, K.K., Fraifeld, V.E., 2008. Do mitochondrial DNA and metabolic rate complement each other in determination of the mammalian maximum longevity? Rejuvenation Res. 11, 409–417. Medvedev, Z.A., 1990. An attempt at a rational classification of theories of ageing. Biol. Rev. Camb. Philos. Soc. 65, 375–398. Pérez, V.I., Van Remmen, H., Bokov, A., Epstein, C.J., Vijg, J., Richardson, A., 2009. The overexpression of major antioxidant enzymes does not extend the lifespan of mice. Aging Cell 8, 73–75.
Pamplona, R., Portero-Otin, M., Riba, D., Ruiz, C., Prat, J., Bellmunt, M.J., Barja, G., 1998. Mitochondrial membrane peroxidizability index is inversely related to maximum lifespan in mammals. J. Lipid Res. 39, 1989–1994. Parkes, T.L., Elia, A.J., Dickinson, D., Hilliker, A.J., Phillips, J.P., Boulianne, G.L., 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19, 171–174. Promislow, D.E., 1994. DNA repair and the evolution of longevity: a critical analysis. J. Theor. Biol. 170, 291–300. Reyes, A., Yang, M.Y., Bowmaker, M., Holt, I.J., 2005. Bidirectional replication initiates at sites throughout the mitochondrial genome of birds. J. Biol. Chem. 280, 3242–3250. Ruiz-Pesini, E., Lott, M.T., Procaccio, V., Poole, J.C., Brandon, M.C., Mishmar, D., Yi, C., Kreuziger, J., Baldi, P., Wallace, D.C., 2007. An enhanced MITOMAP with a global mtDNA mutational phylogeny. Nucleic Acid Res. 35, D823–D828. Samuels, D.C., 2004. Mitochondrial DNA repeats constrain the life span of mammals. TRIGS 20, 226–229. Seligmann, H., 2010. Mitochondrial tRNAs as light strand replication origins: similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication. Biosystems 99, 85–93. Speakman, J.R., 2005. Body size, energy metabolism and lifespan. J. Exp. Biol. 208, 1717–1730. Sun, J., Tower, J., 1999. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies. Mol. Cell. Biol. 19, 216–228. Tacutu, R., Craig, T., Budovsky, A., Wuttke, D., Lehmann, G., Taranukha, D., Costa, J., Fraifeld, V.E., de Magalhães, J.P., 2013. Human Ageing Genomic Resources: Integrated databases and tools for the biology and genetics of ageing. Nucleic Acids Res. 41, D1027–D1033. Terman, A., Brunk, U.T., 2004. Aging as a catabolic malfunction. Int. J. Biochem. Cell. Biol. 36, 2365–2375. Vermulst, M., Wanagat, J., Kujoth, G.C., Bielas, J.H., Rabinovitch, P.S., Prolla, T.A., Loeb, L.A., 2008. DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat. Genet. 40, 392–394. Wanrooij, S., Miralles Fusté, J., Stewart, J.B., Wanrooij, P.H., Samuelsson, T., Larsson, N.G., Gustafsson, C.M., Falkenberg, M., 2012. In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication. EMBO Rep. 13, 1130–1137. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.