Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age‐related diseases, but forestalling these changes has been a major challenge. One of the best‐studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock‐in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase‐γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In‐depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear–mitochondrial crosstalk. There was also a specific increase in the nuclear‐encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial‐encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue‐specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.     

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3‐isoprostane measurement

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3‐isoprostanes. F3‐isoprostanes are prostaglandin‐like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev‐1 and clk‐1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro‐oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress‐resistant and long‐lived daf‐2 insulin/IGF‐1 receptor mutant involved distinct daf‐16‐dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS.     

Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury

Mouse models that accumulate high levels of mitochondrial DNA (mtDNA) mutations owing to impairments in mitochondrial polymerase γ (PolG) proofreading function have been shown to develop phenotypes consistent with accelerated aging. As increase in mtDNA mutations and aging are risk factors for neurodegenerative diseases, we sought to determine whether increase in mtDNA mutations renders neurons more vulnerable to injury. We therefore examined the in vivo functional activity of retinal neurons and their ability to cope with stress in transgenic mice harboring a neural‐targeted mutant PolG gene with an impaired proofreading capability (Kasahara, et al. (2006) Mol Psychiatry 11 (6):577–93, 523). We confirmed that the retina of these transgenic mice have increased mtDNA deletions and point mutations and decreased expression of mitochondrial oxidative phosphorylation enzymes. Associated with these changes, the PolG transgenic mice demonstrated accelerated age‐related loss in retinal function as measured by dark‐adapted electroretinogram, particularly in the inner and middle retina. Furthermore, the retinal ganglion cell–dominant inner retinal function in PolG transgenic mice showed greater vulnerability to injury induced by raised intraocular pressure, an insult known to produce mechanical, metabolic, and oxidative stress in the retina. These findings indicate that an accumulation of mtDNA mutations is associated with impairment in neural function and reduced capacity of neurons to resist external stress in vivo, suggesting a potential mechanism whereby aging central nervous system can become more vulnerable to neurodegeneration.     

Premature aging is associated with higher levels of 8‐oxoguanine and increased DNA damage in the Polg mutator mouse

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg ^D257A/D257A mutator mouse (Polg ^mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg ^mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg ^mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.     

Defects in mitochondrial DNA replication and oxidative damage in muscle of mtDNA mutator mice

A causal role for mitochondrial dysfunction in mammalian aging is supported by recent studies of the mtDNA mutator mouse (“PolG” mouse), which harbors a defect in the proofreading-exonuclease activity of mitochondrial DNA polymerase gamma. These mice exhibit accelerated aging phenotypes characteristic of human aging, including systemic mitochondrial dysfunction, exercise intolerance, alopecia and graying of hair, curvature of the spine, and premature mortality. While mitochondrial dysfunction has been shown to cause increased oxidative stress in many systems, several groups have suggested that PolG mutator mice show no markers of oxidative damage. These mice have been presented as proof that mitochondrial dysfunction is sufficient to accelerate aging without oxidative stress. In this study, by normalizing to mitochondrial content in enriched fractions we detected increased oxidative modification of protein and DNA in PolG skeletal muscle mitochondria. We separately developed novel methods that allow simultaneous direct measurement of mtDNA replication defects and oxidative damage. Using this approach, we find evidence that suggests PolG muscle mtDNA is indeed oxidatively damaged. We also observed a significant decrease in antioxidants and expression of mitochondrial biogenesis pathway components and DNA repair enzymes in these mice, indicating an association of maladaptive gene expression with the phenotypes observed in PolG mice. Together, these findings demonstrate the presence of oxidative damage associated with the premature aging-like phenotypes induced by mitochondrial dysfunction.     

Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging

A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging has been observed in animals and humans. These include (i) decline in mitochondrial respiratory function; (ii) increase in mitochondrial production of reactive oxygen species (ROS) and the extent of oxidative damage to DNA, proteins, and lipids; (iii) accumulation of point mutations and large-scale deletions of mtDNA; and (iv) enhanced apoptosis. Recent studies have provided abundant evidence to substantiate the importance of mitochondrial production of ROS in aging. On the other hand, somatic mtDNA mutations can cause premature aging without increasing ROS production. In this review, we focus on the roles that ROS play in the aging-associated decline of mitochondrial respiratory function, accumulation of mtDNA mutations, apoptosis, and alteration of gene expression profiles. Taking these findings together, we suggest that mitochondrial dysfunction, enhanced oxidative stress, subsequent accumulation of mtDNA mutations, altered expression of a few clusters of genes, and apoptosis are important contributors to human aging.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

Naked mole‐rats (NMRs) are mouse‐sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging‐related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long‐lived species produce less ROS than do mitochondria of short‐lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long‐lived species from short‐lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H₂O₂; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H₂O₂ generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.     

Age‐dependent cardiomyopathy in mitochondrial mutator mice is attenuated by overexpression of catalase targeted to mitochondria

Mitochondrial defects have been found in aging and several age‐related diseases. Mice with a homozygous mutation in the exonuclease encoding domain of mitochondrial DNA polymerase gamma (Polg^m/m) are prone to age‐dependent accumulation of mitochondrial DNA mutations and have shown a broad spectrum of aging‐like phenotypes. However, the mechanism of cardiac phenotypes in relation to the role of mitochondrial DNA mutations and oxidative stress in this mouse model has not been fully addressed. We demonstrate age‐dependent cardiomyopathy in Polg^m/m mice, which by 13–14 months of age displays marked cardiac hypertrophy and dilatation, impairment of systolic and diastolic function, and increased cardiac fibrosis. This age‐dependent cardiomyopathy is associated with increases in mitochondrial DNA (mtDNA) deletions and protein oxidative damage, increased expression of apoptotic and senescence markers, as well as a decline in signaling for mitochondrial biogenesis. The relationship of these changes to mitochondrial reactive oxygen species (ROS) was tested by crossing Polg^m/m mice with mice that overexpress mitochondrial targeted catalase (mCAT). All of the above phenotypes were partially rescued in Polg^m/m/mCAT mice. These data indicate that accumulation of mitochondrial DNA damage with age can lead to cardiomyopathy and that this phenotype is partly mediated by mitochondrial oxidative stress.     

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.     

Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins

Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice) have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho^–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho^–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.     

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.     

Mitochondria,oxidative stress and aging

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver.

Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.     

Oxidative stress,mitochondrial DNA mutation,and impairment of antioxidant enzymes in aging

Mitochondria do not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism in the aging tissues of the human and animals. It is now generally accepted that aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the elevation of oxidative stress in aging tissues. Within a certain concentration range, ROS may induce stress response of the cells by altering expression of respiratory genes to uphold the energy metabolism to rescue the cell. However, beyond the threshold, ROS may cause a wide spectrum of oxidative damage to various cellular components to result in cell death or elicit apoptosis by induction of mitochondrial membrane permeability transition and release of apoptogenic factors such as cytochrome c. Moreover, oxidative damage and large-scale deletion and duplication of mitochondrial DNA (mtDNA) have been found to increase with age in various tissues of the human. Mitochondria act like a biosensor of oxidative stress and they enable cell to undergo changes in aging and age-related diseases. On the other hand, it has recently been demonstrated that impairment in mitochondrial respiration and oxidative phosphorylation elicits an increase in oxidative stress and causes a host of mtDNA rearrangements and deletions. Here, we review work done in the past few years to support our view that oxidative stress and oxidative damage are a result of concurrent accumulation of mtDNA mutations and defective antioxidant enzymes in human aging.