Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.     

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.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O^{⋅ –}), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

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.     

Mitochondrial complex III ROS regulate adipocyte differentiation

Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation.     

Yeast Colony Survival Depends on Metabolic Adaptation and Cell Differentiation Rather Than on Stress Defense

Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Δ and ctt1Δ colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling.     

Oxidative stress and mitochondrial impairment can be separated from lipofuscin accumulation in aged human skeletal muscle

According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain. However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high-resolution respirometry, and ROS production was analyzed by in situ staining with the redox-sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional mitochondria and that the level of ROS production is higher in young compared to aged muscle. Accordingly, we could not find any increase in oxidative modification of proteins in muscle from elderly donors. However, the accumulation of lipofuscin was identified as a robust marker of human muscle aging. The data support a model, where ROS-induced molecular damage is continuously removed, preventing the accumulation of dysfunctional mitochondria despite ongoing ROS production.     

Mitochondria and aging: A role for the mitochondrial transition pore?

The cellular mechanisms responsible for aging are poorly understood. Aging is considered as a degenerative process induced by the accumulation of cellular lesions leading progressively to organ dysfunction and death. The free radical theory of aging has long been considered the most relevant to explain the mechanisms of aging. As the mitochondrion is an important source of reactive oxygen species (ROS), this organelle is regarded as a key intracellular player in this process and a large amount of data supports the role of mitochondrial ROS production during aging. Thus, mitochondrial ROS, oxidative damage, aging, and aging‐dependent diseases are strongly connected. However, other features of mitochondrial physiology and dysfunction have been recently implicated in the development of the aging process. Here, we examine the potential role of the mitochondrial permeability transition pore (mPTP) in normal aging and in aging‐associated diseases.     

Protein-mediated energy-dissipating pathways in mitochondria

Mitochondrial production of reactive oxygen species (ROS) is a well-established fact of fundamental importance to aging and etiology of many pathologies with serious public health implications. The ROS production is an innate property of mitochondrial biochemistry inseparable from the oxidative metabolism. Recent discoveries indicate that in addition to several ROS-detoxifying enzyme systems, which remove ROS, mitochondria may also be able to limit their ROS production by the mechanism comprising several protein-mediated energy-dissipating ("uncoupling") pathways. Although the physiological significance and in vivo modus operandi of these pathways remain to be elucidated, several proteins potentially capable of energy dissipation are known. This mini-review addresses the identity of mitochondrial protein-mediated energy-dissipating pathways and the experimental evidence to their role in controlling ROS production.     

Protein-mediated energy-dissipating pathways in mitochondria

Mitochondrial production of reactive oxygen species (ROS) is a well-established fact of fundamental importance to aging and etiology of many pathologies with serious public health implications. The ROS production is an innate property of mitochondrial biochemistry inseparable from the oxidative metabolism. Recent discoveries indicate that in addition to several ROS-detoxifying enzyme systems, which remove ROS, mitochondria may also be able to limit their ROS production by the mechanism comprising several protein-mediated energy-dissipating ("uncoupling") pathways. Although the physiological significance and in vivo modus operandi of these pathways remain to be elucidated, several proteins potentially capable of energy dissipation are known. This mini-review addresses the identity of mitochondrial protein-mediated energy-dissipating pathways and the experimental evidence to their role in controlling ROS production.     

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background

Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria.

Methodology/Principal Findings

We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells.

Conclusion/Significance

Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.     

Characterization of the effects of a polyunsaturated fatty acid (PUFA) on mitochondrial bioenergetics of chronologically aged yeast

Increased membrane unsaturation has been associated with shorter longevity due to higher sensitivity to lipid peroxidation (LP) leading to enhanced mitochondrial dysfunction and ROS overproduction. However, the role of LP during aging has been put in doubt along with the participation of electron leak at the electron transport chain (ETC) in ROS generation in aged organisms. Thus, to test these hypothesis and gain further information about how minimizing LP preserves ETC function during aging, we studied the effects of α-linolenic acid (C18:3) on in situ mitochondrial ETC function, ROS production and viability of chronologically aged cells of S. cerevisiae, whose membranes are intrinsically resistant to LP due to the lack of PUFA. Increased sensitivity to LP was observed in cells cultured with C18:3 at 6 days of aging. This was associated with higher viability loss, dissipated membrane potential, impaired respiration and increased ROS generation, being these effects more evident at 28 days. However, at this point, lower sensitivity to LP was observed without changes in the membrane content of C18:3, suggesting the activation of a mechanism counteracting LP. The cells without C18:3 display better viability and mitochondrial functionality with lower ROS generation even at 28 days of aging and this was attributed to full preservation of complex III activity. These results indicate that the presence of PUFA in membranes enhances ETC dysfunction and electron leak and suggest that complex III is crucial to preserve membrane potential and to maintain a low rate of ROS production during aging.     

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C .  elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C .  elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C .  elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C .  elegans lifespan.     

Specificity of coenzyme Q10 for a balanced function of respiratory chain and endogenous ubiquinone biosynthesis in human cells

Coenzyme Q (Q) is an obligatory component of both respiratory chain and uncoupling proteins. Also, Q acts as an antioxidant in cellular membranes. Several neurodegenerative diseases are associated with modifications of Q10 levels. For these reasons, therapies based on Q supplementation in the diet are currently studied in order to mitigate the symptoms of these diseases. However, the incorporation of exogenous Q also affects aging process in nematodes probably affecting reactive oxygen species (ROS) production. The aim of the present work is to clarify if supplementation with both Q10 and Q6 isoforms affects mitochondrial Q10 content, respiratory chain activity and ROS levels in human cells. Cells incorporated exogenously added Q10 and Q6 isoforms into mitochondria that produced changes in mitochondrial activity depending on the side chain length. Supplementation with Q10, but not with Q6, increased mitochondrial Q-dependent activities. However, Q6 affected the mitochondrial membrane potential, ROS production, and increased the protein levels of both catalase and Mn-superoxide dismutase (Mn-SOD). Also, Q6 induced a transient decrease in endogenous mitochondrial Q10 levels by increasing its catabolism. These results show that human cells supplemented with Q6 undergo a mitochondrial impairment, which is not observed with Q10 supplementation.     

Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging.