Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.     

Oxidative stress, mitochondria and mtDNA-mutator mice

The oxidative stress theory of aging, an expansion of the mitochondrial theory of aging, is based around the idea of a vicious cycle, in which somatic mutations of mitochondrial DNA (mtDNA) provoke respiratory chain dysfunction leading to enhanced ROS production and in turn to the accumulation of further mtDNA mutations. Mitochondrial dysfunction and mtDNA mutations are amplified during the course of aging. Recently, results obtained from mtDNA-mutator mice further strengthen the role of mitochondria in the aging process. However, lack of increased oxidative stress in the mtDNA-mutator mice raises doubts in the direct connection of mtDNA mutations with increased ROS production, challenging the oxidative stress theory of aging. The purpose of this short review is to highlight several studies that provide direct evidence that accelerated aging is linked to mtDNA mutations, without an increase in oxidative damage.     

Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys

While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.     

Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells

Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.     

Das mitochondriale Genom und Altern

There is a lot of evidence that age-associated alterations of the mitochondrial genome occur, especially in postmitotic tissues such as brain, heart and skeletal muscle. These alterations are supposed to be a result of an attack of free radicals generated as normal byproducts of oxidative phosphorylation and lead to damage of proteins, lipids, and DNA. The alterations of mtDNA include oxidative damage of base pairs, point mutations, large-scale deletions or duplications. The 4977 bp deletion or "common deletion" reveals an age-dependent accumulation in postmitotic tissues, but not in fast-dividing tissues such as blood cells. In addition, it is observed that a tissue-specific accumulation occurs with the highest abundance in the basal ganglia, followed by skeletal muscle, heart, and lowest in cerebellar tissue. Third, pathological alterations of specific tissue, like ischemia/reperfusion events, display a pronounced accumulation of the deletion compared to age-matched controls. Because there are many mtDNA mutations, further analysis of all alterations of mtDNA will elucidate its role in the phenomenon of aging. Despite some criticisms of this free radical theory of aging, there is a lot of experimental evidence to support the important role of mitochondria in organismal aging.     

Mitochondrial dysfunction as a cause of ageing

Mitochondrial dysfunction is heavily implicated in the ageing process. Increasing age in mammals correlates with accumulation of somatic mitochondrial DNA (mtDNA) mutations and decline in respiratory chain function. The age-associated respiratory chain deficiency is typically unevenly distributed and affects only a subset of cells in various human tissues, such as heart, skeletal muscle, colonic crypts and neurons. Studies of mtDNA mutator mice has shown that increased levels of somatic mtDNA mutations directly can cause a variety of ageing phenotypes, such as osteoporosis, hair loss, greying of the hair, weight reduction and decreased fertility. Respiratory-chain-deficient cells are apoptosis prone and increased cell loss is therefore likely an important consequence of age-associated mitochondrial dysfunction. There is a tendency to automatically link mitochondrial dysfunction to increased generation of reactive oxygen species (ROS), however, the experimental support for this concept is rather weak. In fact, respiratory-chain-deficient mice with tissue-specific mtDNA depletion or massive increase of point mutations in mtDNA typically have minor or no increase of oxidative stress. Mitochondrial dysfunction is clearly involved in the human ageing process, but its relative importance for mammalian ageing remains to be established.     

Mitochondrial-nuclear Cross-talk in the Aging and Failing Heart

Hypothesis Damage to heart mitochondrial structure and function occur with aging, and in heart failure (HF). However, the extent of mitochondrial dysfunction, the expression of mitochondrial and nuclear genes, and their cross-talk is not known. Observations Several observations have suggested that somatic mutations in mitochondrial DNA (mtDNA), induced by reactive oxygen species (ROS), appear to be the primary cause of energy decline, and that the generation of ROS is mainly the product of the mitochondrial respiratory chain. The free radical theory of aging, that could also be applied to HF, and in particular the targeting of mtDNA is supported by a plurality of observations from both animal and clinical studies showing decreased mitochondrial function, increased ROS levels and mtDNA mutations in the aging heart. Discussion Aging and HF with their increased ROS-induced defects in mtDNA, including base modifications and frequency of mtDNA deletions, might be expected to cause increased errors or mutations in mtDNA-encoded enzyme subunits, resulting in impaired oxidative phosphorylation and defective electron transport chain (ETC) activity which in turn creates more ROS. These events in both the aging and failing heart involve substantial nuclear–mitochondrial interaction, which is further illustrated in the progression of myocardial apoptosis. In this review the cross-talk between the nucleus and the mitochondrial organelle will be examined based on a number of animal and clinical studies, including our own.     

The role of mitochondrial DNA mutations in aging and sarcopenia: implications for the mitochondrial vicious cycle theory of aging

Aging is associated with a progressive loss of skeletal muscle mass and strength and the mechanisms mediating these effects likely involve mitochondrial DNA (mtDNA) mutations, mitochondrial dysfunction and the activation of mitochondrial-mediated apoptosis. Because the mitochondrial genome is densely packed and close to the main generator of reactive oxygen species (ROS) in the cell, the electron transport chain (ETC), an important role for mtDNA mutations in aging has been proposed. Point mutations and deletions in mtDNA accumulate with age in a wide variety of tissues in mammals, including humans, and often coincide with significant tissue dysfunction. Here, we examine the evidence supporting a causative role for mtDNA mutations in aging and sarcopenia. We review experimental outcomes showing that mtDNA mutations, leading to mitochondrial dysfunction and possibly apoptosis, are causal to the process of sarcopenia. Moreover, we critically discuss and dispute an important part of the mitochondrial 'vicious cycle' theory of aging which proposes that accumulation of mtDNA mutations may lead to an enhanced mitochondrial ROS production and ever increasing oxidative stress which ultimately leads to tissue deterioration and aging. Potential mechanism(s) by which mtDNA mutations may mediate their pathological consequences in skeletal muscle are also discussed.     

Mitochondrial protein oxidation and degradation in response to oxidative stress and aging

Mitochondria are a major source of intracellular reactive oxygen species (ROS), the production of which increases with age. These organelles are also targets of oxidative damage. The deleterious effects of ROS may be responsible for impairment of mitochondrial function observed during various pathophysiological states associated with oxidative stress and aging. An important factor for protein maintenance in the presence of oxidative stress is enzymatic reversal of oxidative modifications and/or protein degradation. Failure of these protein maintenance systems is likely a critical component of the aging process. Mitochondrial matrix proteins are sensitive to oxidative inactivation and oxidized proteins are known to accumulate during aging. The ATP-stimulated mitochondrial Lon protease is a highly conserved protease found in prokaryotes and the mitochondrial compartment of eukaryotes and is believed to play an important role in the degradation of oxidized mitochondrial matrix proteins. Age-dependent declines in the activity and regulation of this proteolytic system may underlie accumulation of oxidatively modified and dysfunctional protein and loss in mitochondrial viability.     

Oxidative stress and protein aggregation during biological aging

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation.Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.     

The role of mitochondrial DNA mutations and free radicals in disease and ageing

Considerable efforts have been made to understand the role of oxidative stress in age‐related diseases and ageing. The mitochondrial free radical theory of ageing, which proposes that damage to mitochondrial DNA (mtDNA) and other macromolecules caused by the production of reactive oxygen species (ROS) during cellular respiration drives ageing, has for a long time been the central hypothesis in the field. However, in contrast with this theory, evidence from an increasing number of experimental studies has suggested that mtDNA mutations may be generated by replication errors rather than by accumulated oxidative damage. Furthermore, interventions to modulate ROS levels in humans and animal models have not produced consistent results in terms of delaying disease progression and extending lifespan. A number of recent experimental findings strongly question the mitochondrial free radical theory of ageing, leading to the emergence of new theories of how age‐associated mitochondrial dysfunction may lead to ageing. These new hypotheses are mainly based on the underlying notion that, despite their deleterious role, ROS are essential signalling molecules that mediate stress responses in general and the stress response to age‐dependent damage in particular. This novel view of ROS roles has a clear impact on the interpretation of studies in which antioxidants have been used to treat human age‐related diseases commonly linked to oxidative stress.     

The flux of free radical attack through mitochondrial DNA is related to aging rate

Aging is a progressive and universal process originated endogenously which manifests best in post-mitotic cells. Available data indicate that the relation between oxidative stress and aging is due to the presence of low rates of mitochondrial free radical production and low degrees of fatty acid unsaturation of cellular membranes in the post-mitotic tissues of long-lived animals in relation to those of short-lived ones. Recent research shows that long-lived animals also have lower steady-state levels of oxidative damage in the mitochondrial DNA (mtDNA) of post-mitotic cells than short-lived species. This study shows that the flux of free radical attack to mtDNA is higher in short- than in long-lived animals, and proposes that this is a main determinant of the rate of accumulation of mtDNA mutations, and thus the rate of aging. This implies that aging has been slowed evolutionarily by mechanisms that decrease the generation of endogenous damage rather than try to intercept damaging agents, or to repair the damage already inflicted. The first kind of mechanisms are more efficient and less energetically expensive. Free radicals of mitochondrial origin, oxidative damage to DNA, evolution of aging rate, and possibilities and consequences of their future modification are also discussed.     

Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress

A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment flanking the β-globin gene. Simian virus 40-transformed fibroblasts treated with 200 μM hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROS-associated diseases.     

Decline in skeletal muscle mitochondrial function with aging in humans

Cumulative mtDNA damage occurs in aging animals, and mtDNA mutations are reported to accelerate aging in mice. We determined whether aging results in increased DNA oxidative damage and reduced mtDNA abundance and mitochondrial function in skeletal muscle of human subjects. Studies performed in 146 healthy men and women aged 18-89 yr demonstrated that mtDNA and mRNA abundance and mitochondrial ATP production all declined with advancing age. Abundance of mtDNA was positively related to mitochondrial ATP production rate, which in turn, was closely associated with aerobic capacity and glucose tolerance. The content of several mitochondrial proteins was reduced in older muscles, whereas the level of the oxidative DNA lesion, 8-oxo-deoxyguanosine, was increased, supporting the oxidative damage theory of aging. These results demonstrate that age-related muscle mitochondrial dysfunction is related to reduced mtDNA and muscle functional changes that are common in the elderly.     

增龄大鼠心肌线粒体DNA损伤及DNA修复酶γ表达的变化

目的:探讨大鼠增龄过程中心肌组织DNA损伤和DNA修复酶表达的变化。方法:从不同月龄的大鼠心肌组织中提取DNA、RNA及蛋白。采用定量多聚酶链反应(Q-PCR)检测心肌DNA损伤;用RT-PCR和Western blot检测DNA修复酶γ(Polγ)表达的变化。用ELISA法检测DNA内8羟基脱氧鸟苷(8-OHdG)的水平。结果:随着鼠龄的增长,大鼠心肌组织中核DNA(nDNA)损伤不明显,线粒体DNA(mtDNA)损伤严重,DNA内8-OHdG的水平增加,Polγ的表达下降。结论:随着鼠龄的增长,大鼠心肌组织中DNA修复酶Polγ的表达下降,mtDNA损伤增加。DNA损伤与DNA修复能力下降间会引起恶性循环,最终可导致DNA损伤的增加加快心脏衰老。     

Testing the rate-of-living/oxidative damage theory of aging in the nematode model Caenorhabditis elegans

The integration of the rate-of-living and oxidative damage theory of aging predicts that lifespan extension is linked to low energy metabolism, low ROS production rates, low molecular damage and a slow aging rate. In the long-lived Caenorhabditis elegans Ins/IGF-1 mutant daf-2(e1370), low carbonylation levels and postponed morphological decline comply with the latter two of these predictions. However, metabolic rates in daf-2(e1370) refute the rate-of-living theory. The apparent contradiction between increased ROS generation and long lifespan in daf-2(e1370) is reconciled by an enhanced stress defense, acknowledging oxidative damage as a probable cause of aging.