Scientists have long struggled to explain the evolutionary basis of aging. In particular, how can there be a genetic program of aging, if aging manifests itself long after the reproductive period has passed and, therefore, after all the forces of natural selection have long since abated? Potential explanations have come from Medawar’s mutation accumulation hypothesis, Kirkwood’s disposable soma theory, and the concept of antagonistic pleiotropy (). The latter hypothesis revolves around the notion that genes regulating aging in the old organism actually have a different, antagonistic function when the animal is young (). In this scenario, those genes positively regulating growth and fertility in the young animal might serve to accelerate senescence and aging in the older animal. While, from an evolutionary viewpoint, this concept has been exclusively applied to our genetic inheritance, the notion of antagonistic pleiotropy actually provides a useful framework to understand the role of mitochondria in aging. Perhaps, no structure is so intimately and simultaneously connected to both the energy of youth and the decline of the old. The revelation of these complex and antagonistic functions of mitochondria has slowly transformed how we view this subcellular organelle. Mitochondria can no longer be viewed as simple bioenergetics factories but rather as platforms for intracellular signaling, regulators of innate immunity, and modulators of stem cell activity. In turn, each of these properties provides clues as to how mitochondria might regulate aging and age-related diseases. Here, we review how mitochondria participate in aging and how these insights may usher in a new era of mitochondrial-targeted therapies to potentially slow or reverse the aging process.

Regardless of mtDNA, in humans, the link between mitochondrial function and aging has been, perhaps, best studied by analyzing skeletal muscle. While all studies are not in complete concordance, the majority of reports have found that aging is generally accompanied by a decline in activity of mitochondrial enzymes (e.g., citrate synthase), a decrease in respiratory capacity per mitochondria (e.g., substrate-dependent oxygen consumption), an increase in reactive oxygen species (ROS) production, and a reduced phosphocreatine (PCr) recovery time (an in vivo measurement of mitochondrial respiratory capacity). Nonetheless, the literature is also filled with many counterexamples that may reflect differences in how the specific assays were performed or differences in the muscle fiber type studied (). Most studies have also noted that aging is accompanied by an accelerated rate of muscle loss, in terms of both mass and activity (e.g., strength). Although muscle strength over a lifetime declines at an average rate of roughly 1% per year, for patients in their 70s, that rate of decline can increase 2- to 4-fold (). At present, perhaps the best intervention to counteract this age-dependent decline in muscle function, termed sarcopenia, is physical exercise. Indeed, accumulating evidence from epidemiological studies and randomized clinical trials illustrates that regular physical activity and endurance exercise benefits a range of human age-related pathologies, including sarcopenia, as well as the age-dependent decline in cardiac and cognitive functions (). Interestingly, endurance exercise also conferred phenotypic protection and prevented the premature mortality observed in the mitochondrial mutator mice mentioned earlier (). The therapeutic effects of endurance exercise are accompanied by a number of physiological adaptations; however, one of the most beneficial effects appears to be stimulation of mitochondrial biogenesis in a wide variety of tissues, including the brain (). Mitochondrial biogenesis is largely coordinated by the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) (). PGC-1α, in turn, regulates the activity of several transcription factors involved in creating new mitochondria, including nuclear respiratory factors NRF1 and NRF2 and mitochondrial transcription factor A (TFAM) (). Increasing PGC-1α levels in mouse skeletal muscle is sufficient to forestall the development of age-dependent sarcopenia, again emphasizing the potential importance of this pathway for aging biology (). The development of sarcopenia is not, however, solely an issue of impaired mitochondrial biogenesis (). Recently, it was also shown that phosphorylation—and, hence, activity of ATP citrate lyase (ACL), a key regulator of acetyl-coenzyme A (CoA) levels—was markedly reduced in sarcopenic muscle (). In this study, ACL phosphorylation was stimulated by insulin-like growth factor 1 (IGF-1), a growth factor known to increase muscle mass () and also known to decline in the serum of aging men and woman (). Increasing ACL levels in mice resulted in improved mitochondrial function, suggesting that this might be a complementary approach to combat the deleterious effects of skeletal muscle aging ().

Serum levels of insulin-like growth factor-I are related to age and not to body composition in healthy women and men.

It has been long appreciated that aging in model organisms is accompanied by a decline in mitochondrial function and that this decline might, in turn, contribute to the observed age-dependent decline in organ function (). Similarly, a decline in mitochondrial function in humans has also been observed; again, this decrement may pre-dispose humans to certain age-related diseases (). It is also known that mitochondrial mutations increase in frequency with age in both animal models and in humans (), although the levels and kind of mutations appear to differ between tissues and even within tissues (). While some have speculated that the increased levels of mitochondrial mutations contribute to aging and age-related diseases (), others have questioned whether these mutations ever reach a significant enough level to contribute to the aging process (). Indeed, since mtDNA exists in hundreds to thousands of copies per cell, the detection of mutant mtDNA does not, in itself, imply dysfunction, as it is generally believed that mutational load must exceed a threshold value (perhaps exceeding 60% of all mitochondria within a given tissue) for there to be a significant phenotype (). Perhaps the strongest evidence for a potential causative role for mtDNA mutations in mammalian aging comes from analyzing the “mitochondrial mutator mice,” which are knockin mice containing a mutated (D257A), proofreading-deficient form of the mtDNA polymerase POLGγ. This nuclear-encoded gene is the sole mtDNA polymerase, and the mutation at amino acid position 257 results in an enzyme that retains normal polymerase function but has impaired proofreading activity. Mice containing one or two copies of this proofreading-deficient POLG accumulate a significant level of mitochondrial mutations, and homozygous knockin mice exhibit an accelerated aging phenotype (). Nonetheless, while this model clearly links mitochondrial mutations to aging, it should be noted that the type and magnitude of mitochondrial mutations do not appear to faithfully replicate what is seen during normal aging (). Thus, while the levels of mitochondrial mutations increase with age, it remains unclear whether this increase plays a fundamental role in the aging process.

Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging.

The unique properties of stem cells suggest that these cells might have mechanisms to ensure that these critical cells do not accumulate old and dysfunctional mitochondria. Preliminary evidence suggests that, in the brain, areas enriched for NSCs appear to have augmented rates of mitophagy (). Another potential mechanism appears to be a unique capacity of adult stem cells to exclude older mitochondria. Indeed, a recent report studying the stem-like cells within immortalized human mammary epithelial cell cultures noted that there was an uneven distribution of mitochondria after cell division (). This asymmetry was not a difference in mitochondrial number between the two daughter cells but, rather, a difference in the segregation of young and old mitochondria ( Figure 1 ). Stem-like cells getting young mitochondria maintained their stem cell properties much more robustly then those cells receiving older mitochondria. This unequal distribution of mitochondria based on the age of the mitochondria was only seen within the stem-like cells in the culture, not the more differentiated mammary epithelial cells. In addition, this property was only seen with mitochondrial segregation and not with other organelles, such as lysosomes or ribosomes, or with cellular components such as chromatin. Currently, it is unclear whether this property is present in vivo and, if so, whether it is present in all, or just some, types of stem cells. However, it should be noted that, in yeast, where there is asymmetric division between the mother cell and the bud, there is also evidence of a corresponding asymmetric inheritance of both mitochondria () and misfolded proteins ().

Analysis of stem-like cells within immortalized, transformed epithelial cultures revealed that young mitochondria (shown in green) and old mitochondria (depicted in orange) are not symmetrically distributed after the stem-like cell divides. Moreover, the daughter cell inheriting the younger mitochondria also exhibits higher stem-like activity. The molecular basis for this asymmetric mitochondrial distribution is not clear, nor is it known whether similar mechanisms exist in vivo.

Another mechanism by which mitochondria might contribute to stem cell maintenance is through regulation of specific metabolites. Increasingly, there is evidence that metabolic intermediates play an important role in regulating the transcriptional and epigenetic states of cells. It is no presumed accident that chromatin modifications are largely dependent on the same carbon intermediates (e.g., methyl, acetyl, etc.) that are generated during normal mitochondrial metabolism. For example, one recent study demonstrated that, in mouse embryonic stem (ES) cells, the intracellular ratio of α-ketoglutarate (αKG) to succinate was important in maintaining pluripotency (). Both of these metabolites are generated as a result of tricarboxylic acid (TCA) metabolism in the mitochondrial matrix. In turn, it was shown that levels of αKG modulated distinct chromatin modifications. This modulation was mediated, at least in part, by the activity of αKG-dependent demethylases, including Jumonji C (JmjC)-domain-containing enzymes and the ten-eleven translocation (Tet)-dependent DNA demethylases (). Another important set of metabolites that connect stem cells to the mitochondria is the NAD/NADH ratio. Levels of NADappears to decline in tissues as they age (). Analysis of neural stem cells (NSCs) has shown that reducing NADlevels recapitulates at least some of the phenotypes of stem cell aging, while NADsupplementation can restore function to old NSCs (). These effects appear to be mediated, in part, by the sirtuin family of NAD-dependent enzymes. This connection has also been observed in HSC biology. SIRT3 is one of seven mammalian sirtuin family members and is found within the mitochondria, where it regulates the mitochondrial acetylome in an NAD-dependent fashion (). Interestingly, SIRT3 is highly enriched in HSCs, although its expression declines with age. Augmenting SIRT3 levels in old HSCs results in improved regenerative capacity in these aging stem cells (). Similar results have been recently observed with overexpression of SIRT7 ().

One clear connection between mitochondria and stem cell function has come from the analysis of the previously described mtDNA mutator mice (). Several reports have analyzed the stem cell function of the POLG knockin mice and found a range of defects. These include the development of a severe and often fatal anemia in the mice, as well as abnormalities in B cells (). A similar impairment was observed in neural stem cell populations derived from POLG knockin mice (). Several features of these analyses deserve mentioning. First, the stem cell defects could, at least, be partially ameliorated by the administration of the antioxidant N-acetylcysteine (). Indeed, follow-up studies have demonstrated that POLG knockin cells also have markedly impaired capacity to be reprogrammed into pluripotent stem cells, a defect again related to an increase in mitochondrial ROS production (). The second point to emphasize is that the observed stem cell defects appear to arise because of cell-autonomous mitochondrial defects. This mutator mouse model affects a multitude of cell types, including the stem cell and their progeny, as well as the niche. Nonetheless, transplantation of POLG knockin HSCs into a normal host recapitulates the observed defect (), and other mouse models that have large-scale mitochondrial deletions only within post-mitotic tissues do not exhibit any stem cell defects (). Thus, even though stem cells do not seem to rely on oxidative phosphorylation for their energetics, mitochondria are clearly required for the long-term function of these cells and their progenitors in a cell-autonomous capacity. Finally, as mentioned previously, it is important to note that these stem cell defects do not appear to accurately recapitulate aging (). Indeed, from a histological viewpoint, the anemia observed in these animals looks less like the anemia of aging and more like the pre-leukemic abnormality known as myelodysplastic syndrome (). It should also be noted, that the level of mitochondrial mutation seen in these models is also dramatically higher than that seen during the normal aging process, which may account for why the observed stem cell defects do not faithfully recapitulate what is seen during normal aging.

While aging is accompanied by a general decline in mitochondrial function in all tissues, the effects of mitochondrial dysfunction might be particularly important within certain specialized cell types. Since a decline in adult stem cell function is thought to contribute to various aspects of aging (), the role of mitochondrial dysfunction in stem cell biology has become a subject of increasing interest. In the case of hematopoietic stem cells (HSCs), perhaps the best studied stem cell population, mitochondria are thought to play a relatively minor role in the resting bioenergetics profile of these cells (). Quiescent HSCs are generally thought to, instead, rely on glycolytic metabolism as the major source of their ATP, presumably in keeping with the low oxygen environment of the HSC niche, and as a mechanism to minimize the long-term deleterious effects of mitochondrial ROS production (). Indeed, a number of links suggest that a rise in ROS might be harmful for stem cell function (), although there are also increasing examples in which ROS appear to play a positive and necessary signaling role in stem cell biology ().

There is also a strong link between mitochondrial metabolism, ROS generation, and the senescent state. Almost 4 decades ago, it was noted that the lifespan of human cells in culture could be significantly extended by culturing the cells in a low-oxygen environment (). A similar effect was also observed in mouse cells (). Similarly, OIS triggered by Ras expression results in an increase in ROS levels, and OIS can be prevented by growing these cells in a low-oxygen state or supplementing the media with an antioxidant (). Similar relationships have been observed between other regulators of senescence and ROS, including the p53 target and cell-cycle regulator p21, which also appears to regulate senescence in a redox-dependent fashion (). All of these observations fit well with the long-standing notions of the free-radical theory of aging that postulated a causal role for ROS in the aging process (). Nonetheless, there are a number of observations that suggest that the cellular effects of ROS, with regard to inducing senescence, do not unequivocally transfer to organismal aging. For instance, while in some animal models, scavenging mitochondrial oxidants appears to extend lifespan (), in other cases, a consistent relationship between ROS levels and lifespan was seemingly absent (). Moreover, in some cases, a rise in ROS appears to actually increase, rather than reduce, overall lifespan ().

As noted in the discussion of stem cell biology, mitochondria can regulate cellular aging through the modulation of the metabolic profile of the cell. Cellular senescence is accompanied by profound changes in the metabolome, and although different triggers of senescence all have a similar morphological appearance, the metabolic profiles of oncogene-induced senescence and replicative senescence appear distinct (). There is increasing evidence that these metabolic changes are casually related to the senescent state. For instance, p53 plays an important role in senescence, and evidence suggests that it can also repress expression of mitochondrial malic enzyme (ME2), which converts the TCA metabolite malate to pyruvate via oxidative decarboxylation (). Moreover, knockdown of ME2 results in the induction of senescence, while forced expression allows cells to escape from senescence. This argues that the ability of p53 to mediate senescence may partially be through its ability to modulate TCA metabolism. Interestingly, previous observations have established that overexpression of malate dehydrogenase also results in lifespan extension in yeast (). The link between mitochondrial metabolism and senescence is also observed in oncogene-induced senescence (OIS). Analysis of cells undergoing OIS precipitated by expression of the BRAF oncogene demonstrated an increase in pyruvate oxidation that contributed to the generation of increased mitochondrial ROS and entry into the senescent state (). This increase in pyruvate utilization was due to alteration in the phosphorylation state—and, hence, the activity—of the mitochondrial pyruvate dehydrogenase (PDH) complex. Again, gain- and loss-of-function studies suggest that these metabolic changes appear to be necessary for BRAF-induced senescence. Interestingly, the PDH complex is also regulated by the mitochondrial sirtuins, particularly SIRT3 and SIRT4 (). Similarly, from an organismal context, a recent large-scale screen of yeast single-gene deletion mutants uncovered a number of enzymes involved in the TCA cycle as potent regulators of lifespan (). Together, these argue that mitochondrial-induced metabolic changes might be necessary—and, in some cases, sufficient—to trigger cellular senescence and, potentially, to regulate overall longevity.

The Mitochondrial Unfolded Protein Response and Longevity

mt is regulated, in part, by a unique transcription factor termed Activating Transcription Factor associated with Stress-1 (ATFS-1). ATFS-1 was identified initially in a screen for factors that mediate the UPRmt in C. elegans ( Haynes et al., 2010 Haynes C.M.

Yang Y.

Blais S.P.

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Ron D. The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Nargund et al., 2012 Nargund A.M.

Pellegrino M.W.

Fiorese C.J.

Baker B.M.

Haynes C.M. Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. mt appears to require a number of other factors, including the homeobox transcription factor DVE-1, the ubiquitin-like protein UBL-5, the mitochondrial protease ClpP, and the inner mitochondrial membrane transporter HAF-1 ( Jensen and Jasper, 2014 Jensen M.B.

Jasper H. Mitochondrial proteostasis in the control of aging and longevity. It is now known that, in worms, the UPRis regulated, in part, by a unique transcription factor termed Activating Transcription Factor associated with Stress-1 (ATFS-1). ATFS-1 was identified initially in a screen for factors that mediate the UPRin C. elegans (). It was subsequently demonstrated that ATFS-1 has both a nuclear localization targeting sequence and a mitochondrial targeting sequence (). While a mitochondrial localization predominates under basal conditions, mitochondrial stress results in reduced importation of ATFS-1, leading to nuclear accumulation and the transcriptional response delineated earlier. In addition to ATFS-1, in worms, the UPRappears to require a number of other factors, including the homeobox transcription factor DVE-1, the ubiquitin-like protein UBL-5, the mitochondrial protease ClpP, and the inner mitochondrial membrane transporter HAF-1 ().

mt and lifespan was made initially in the setting of attempting to explain why mutant worms, such as those with knockdown of cco-1, live longer ( Durieux et al., 2011 Durieux J.

Wolff S.

Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. mt for their lifespan extension. In contrast, other long-lived mutants, such as those involved in insulin/IGF signaling, appeared to extend lifespan independent of UPRmt activation ( Durieux et al., 2011 Durieux J.

Wolff S.

Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. mt in other, distal tissues (e.g., intestine). This suggested the existence of a circulating factor that signals, and perhaps coordinates, metabolism between tissues. The authors called this factor a mitokine ( Durieux et al., 2011 Durieux J.

Wolff S.

Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. Conboy et al., 2013 Conboy M.J.

Conboy I.M.

Rando T.A. Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity. Owusu-Ansah et al., 2013 Owusu-Ansah E.

Song W.

Perrimon N. Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. mt through what appeared to involve a redox-sensitive pathway. Indeed, overexpression of hydrogen-peroxide-scavenging enzymes, such as catalase or glutathione peroxidase, suppressed the induction of the UPRmt and also abrogated the increased longevity seen with Complex I inhibition ( Owusu-Ansah et al., 2013 Owusu-Ansah E.

Song W.

Perrimon N. Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. Ristow et al., 2009 Ristow M.

Zarse K.

Oberbach A.

Klöting N.

Birringer M.

Kiehntopf M.

Stumvoll M.

Kahn C.R.

Blüher M. Antioxidants prevent health-promoting effects of physical exercise in humans. mt in muscle, the authors also observed a systemic effect on insulin signaling mediated by changes in the level of a particular circulating IGF-binding partner ( Owusu-Ansah et al., 2013 Owusu-Ansah E.

Song W.

Perrimon N. Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. The link between the UPRand lifespan was made initially in the setting of attempting to explain why mutant worms, such as those with knockdown of cco-1, live longer (). These results demonstrated that this mutant, as well as other long-lived mitochondrial mutants, all appeared to require activation of the UPRfor their lifespan extension. In contrast, other long-lived mutants, such as those involved in insulin/IGF signaling, appeared to extend lifespan independent of UPRactivation (). Remarkably, when cco-1 was knocked down in one tissue (e.g., neurons), it appeared to activate induction of the UPRin other, distal tissues (e.g., intestine). This suggested the existence of a circulating factor that signals, and perhaps coordinates, metabolism between tissues. The authors called this factor a mitokine (), although, to date, its molecular makeup remains undefined. Whether such factors exist in higher organisms is unclear, but there is clearly a growing interest in circulating factors that regulate aging, as evidenced by the renewed interest in parabiosis studies (). This notion of mitochondrial dysfunction in one tissue acting as a signal for other tissues has also been observed in Drosophila. In a recent example, muscle-specific impairment of a component of Complex I resulted in an increase in the overall lifespan of the fly (). This mitochondrial stress resulted in the activation for at least two separate pathways that appeared to contribute to the observed longevity effects. In the muscle itself, disruption of Complex I resulted in the induction of the UPRthrough what appeared to involve a redox-sensitive pathway. Indeed, overexpression of hydrogen-peroxide-scavenging enzymes, such as catalase or glutathione peroxidase, suppressed the induction of the UPRand also abrogated the increased longevity seen with Complex I inhibition (). These negative effects of redox scavengers are reminiscent of similar observations in humans where, for instance, the beneficial effects of exercise appear to be abrogated by antioxidant supplementation (). In addition to the induction of the UPRin muscle, the authors also observed a systemic effect on insulin signaling mediated by changes in the level of a particular circulating IGF-binding partner (). Again, these results argue that mitochondrial dysfunction in one tissue can signal through secreted factors in the circulation to alter the function of distal tissues. This inter-organ communication appears to be ultimately required for the observed increase in lifespan.