Research Article: Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Date Published: July 24, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Lakshmi Narayanan Lakshmanan, Zhuangli Yee, Li Fang Ng, Rudiyanto Gunawan, Barry Halliwell, Jan Gruber.


Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.

Partial Text

Eukaryotic cells typically contain connected networks of mitochondria, each containing multiple redundant copies of mitochondrial DNA (mtDNA). The copy number of mtDNA per cell is highly dependent on cell type and energy requirements but usually cells contain 1,000–10,000 s of mtDNA (Miller, Rosenfeldt, Zhang, Linnane, & Nagley, 2003). The nematode Caenorhabditis elegans(C. elegans), a widely used model organism in developmental biology, neurobiology, and aging, comprises 959 cells, together typically containing between 200,000 and 300,000 mtDNA molecules (Gruber et al., 2011; Tsang & Lemire, 2002). The copy number of mtDNA per animal declines with age, with aged nematodes containing almost 30% fewer mtDNA compared to young adults (Gruber et al., 2011). Errors during mtDNA replication or during repair of DNA damage can result in mtDNA mutations, including large deletions spanning multiple mtDNA genes (Alexeyev, Shokolenko, Wilson, & LeDoux, 2013; Krishnan et al., 2008). Mutant mtDNA molecules can be amplified during normal mtDNA maintenance, creating additional copies of the mutant sequence within the same cell (Stewart & Chinnery, 2015). During clonal expansion, mutant mtDNA increases in abundance relative to the wild‐type (WT) sequence (Stewart & Chinnery, 2015). When the fraction of mutant mtDNA exceeds ~60%, the excess mutant mtDNA can cause mitochondrial dysfunction, cell atrophy, or death (Rossignol et al., 2003; Wanagat, Cao, Pathare, & Aiken, 2001).

Mitochondrial decline and dysfunction are common features of aging and of many age‐dependent diseases in mammals and in model organisms including nematodes (Braeckman et al., 2002; Gruber et al., 2011; Herndon et al., 2002; López‐Otín, Blasco, Partridge, Serrano, & Kroemer, 2013; Yasuda et al., 2006). In humans, one of the mechanisms that is believed to be involved in mitochondrial aging is loss of mtDNA integrity and accumulation of mutant mtDNA (Stewart & Chinnery, 2015). The accumulation and clonal expansion of mutant mtDNA are processes that have been causally linked to neurodegeneration and sarcopenia (Bender et al., 2006; Herbst et al., 2016). Metabolic decline, mitochondrial dysfunction, and sarcopenia are all conserved features of aging in C. elegans(Braeckman et al., 2002; Fisher, 2004; Gruber et al., 2011; Herndon et al., 2002; Yasuda et al., 2006). While some evidence for an age‐dependent increase in mtDNA deletion probability has been reported in C. elegans over 20 years ago, the question if these deletions are functionally linked to metabolic and mitochondrial aging has remained unanswered (Melov et al., 1994, 1995). Using cohorts of aging nematodes and 236 distinct primer pairs, we comprehensively determined the mtDNA deletion spectrum and mutation burden in aging C. elegans,identifying 266 novel mtDNA deletions (see Supporting information Appendix S2). Partially duplicated mtDNA (pd‐mtDNA), which are large circular mtDNA molecules containing one deleted molecule and one wild‐type molecule, has been reported in mitochondria (Holt, Dunbar, & Jacobs, 1997). Differentiating a pd‐mtDNA from a mtDNA deletion is challenging and can require a large DNA sample, single cell data, and specific PCR design (Bodyak, Nekhaeva, Wei, & Khrapko, 2001). Our mutation capture method is not designed to identify pd‐mtDNA. The abundance of any specific type of pd‐mtDNA is suggested to be low (Bodyak et al., 2001) and hence, for simplicity, we assume that all the identified breakpoint junctions are deletions and not duplications. However, the presence of pd‐mtDNA within our dataset, if any, would only reduce the mtDNA deletion fraction further and would therefore not affect the key conclusions from this study. On average, we detected a slightly larger number of deletions per experimental replicate in aged nematode cohorts compared with young cohorts (Table 1), a finding that is consistent with the only previous report (Melov et al., 1995). For quantification, we employed Monte Carlo simulations, taking into account the experimental design and sampling statistics. We find that the apparent age‐dependent increase in mutation burden is not statistically significant and that overall burden remains low over the course of life, with on average 82 ± 9 (mean ± 1 SD over all three age groups) mutant mtDNA molecules per worm irrespective of age. This mutation burden is well below the 60% threshold where mtDNA deletions would become physiologically limiting in terms of mitochondrial energy production (Rossignol et al., 2003). Even under the extremely unlikely assumption that all mutant molecules in each worm were concentrated in a single cell, the levels of mtDNA deletion in even that single cell remain under this 60% threshold. Under the much more plausible assumption that these ~80 mutations are relatively equally distributed among the 959 cells of an adult nematode, these data suggest that most cells in C. elegans harbors no mutant mtDNA and that, therefore, mtDNA mutations at this level are unlikely to be sufficient to cause significant physiological detriments. We determined deletion burden up to Day 10 of life because at a temperature of 25°C, this is an age where rapidly rising mortality, functional, and metabolic decline and loss of mitochondrial integrity suggest significant age‐dependent failure (Gruber et al., 2011).

None declared.

LNL, ZL, and LF devised, designed, and performed experiments and analyzed results. JG, RG, and BH participated in the experimental design and in the interpretation, conceived the project, and coordinated its execution. All authors commented and approved the manuscript.