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.     

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.     

Manipulation of mitochondrial DNA gene expression in the mouse

Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre-loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.     

Mitochondrial energy metabolism and ageing

Ageing can be defined as “a progressive, generalized impairment of function, resulting in an increased vulnerability to environmental challenge and a growing risk of disease and death”. Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. During the last 20 years, gerontological studies have revealed different molecular pathways involved in the ageing process and pointed out mitochondria as one of the key regulators of longevity. Increasing age in mammals correlates with increased levels of mitochondrial DNA (mtDNA) mutations and a deteriorating respiratory chain function. Experimental evidence in the mouse has linked increased levels of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, graying of the hair, weight reduction and decreased fertility. A mosaic respiratory chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and an increased cell loss is therefore likely of importance in the age-associated mitochondrial dysfunction. In this review, we would like to point out the link between the mitochondrial energy balance and ageing, as well as a possible connection between the mitochondrial metabolism and molecular pathways important for the lifespan extension.     

Human stem cell aging: do mitochondrial DNA mutations have a causal role?

A decline in the replicative and regenerative capacity of adult stem cell populations is a major contributor to the aging process. Mitochondrial DNA (mtDNA) mutations clonally expand with age in human stem cell compartments including the colon, small intestine, and stomach, and result in respiratory chain deficiency. Studies in a mouse model with high levels of mtDNA mutations due to a defect in the proofreading domain of the mtDNA polymerase γ (mtDNA mutator mice) have established causal relationships between the accumulation of mtDNA point mutations, stem cell dysfunction, and premature aging. These mtDNA mutator mice have also highlighted that the consequences of mtDNA mutations upon stem cells vary depending on the tissue. In this review, we present evidence that these studies in mice are relevant to normal human stem cell aging and we explore different hypotheses to explain the tissue‐specific consequences of mtDNA mutations. In addition, we emphasize the need for a comprehensive analysis of mtDNA mutations and their effects on cellular function in different aging human stem cell populations.     

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.     

Mitochondrial DNA mutations regulate metastasis of human breast cancer cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.     

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.     

Generation and bioenergetic analysis of cybrids containing mitochondrial DNA from mouse skeletal muscle during aging

Mitochondrial respiratory chain defects have been associated with various diseases and normal aging, particularly in tissues with high energy demands including skeletal muscle. Muscle-specific mitochondrial DNA (mtDNA) mutations have also been reported to accumulate with aging. Our understanding of the molecular processes mediating altered mitochondrial gene expression to dysfunction associated with mtDNA mutations in muscle would be greatly enhanced by our ability to transfer muscle mtDNA to established cell lines. Here, we report the successful generation of mouse cybrids carrying skeletal muscle mtDNA. Using this novel approach, we performed bioenergetic analysis of cells bearing mtDNA derived from young and old mouse skeletal muscles. A significant decrease in oxidative phosphorylation coupling and regulation capacity has been observed with cybrids carrying mtDNA from skeletal muscle of old mice. Our results also revealed decrease growth capacity and cell viability associated with the mtDNA derived from muscle of old mice. These findings indicate that a decline in mitochondrial function associated with compromised mtDNA quality during aging leads to a decrease in both the capacity and regulation of oxidative phosphorylation.     

Mitochondrial DNA maintenance and bioenergetics

Oxidative phosphorylation requires assembly of the protein products of both mitochondrial and of nuclear genomes into functional respiratory complexes. Cellular respiration can be compromised when mitochondrial DNA (mtDNA) sequences are corrupted. Oxidative damage resulting from reactive oxygen species (ROS) produced during respiration is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction. Here, we review mechanisms of mitochondrial ROS production, mtDNA damage and its relationship to mitochondrial dysfunction. We focus particular attention on the roles of mtDNA repair enzymes and processes by which the integrity of the mitochondrial genome is maintained and dysfunction prevented.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

Mitochondrial DNA maintenance and bioenergetics

Oxidative phosphorylation requires assembly of the protein products of both mitochondrial and of nuclear genomes into functional respiratory complexes. Cellular respiration can be compromised when mitochondrial DNA (mtDNA) sequences are corrupted. Oxidative damage resulting from reactive oxygen species (ROS) produced during respiration is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction. Here, we review mechanisms of mitochondrial ROS production, mtDNA damage and its relationship to mitochondrial dysfunction. We focus particular attention on the roles of mtDNA repair enzymes and processes by which the integrity of the mitochondrial genome is maintained and dysfunction prevented.     

The mitochondrial genome is a "genetic sanctuary" during the oncogenic process

Since Otto Warburg linked mitochondrial physiology and oncogenesis in the 1930s, a number of studies have focused on the analysis of the genetic basis for the presence of aerobic glycolysis in cancer cells. However, little or no evidence exists today to indicate that mtDNA mutations are directly responsible for the initiation of tumor onset. Based on a model of gliomagenesis in the mouse, we aimed to explore whether or not mtDNA mutations are associated with the initiation of tumor formation, maintenance and aggressiveness. We reproduced the different molecular events that lead from tumor initiation to progression in the mouse glioma. In human gliomas, most of the genetic alterations that have been previously identified result in the aberrant activation of different signaling pathways and deregulation of the cell cycle. Our data indicates that mitochondrial dysfunction is associated with reactive oxygen species (ROS) generation, leading to increased nuclear DNA (nDNA) mutagenesis, but maintaining the integrity of the mitochondrial genome. In addition, mutational stability has been observed in entire mtDNA of human gliomas; this is in full agreement with the results obtained in the cancer mouse model. We use this model as a paradigm of oncogenic transformation due to the fact that mutations commonly found in gliomas appear to be the most common molecular alterations leading to tumor development in most types of human cancer. Our results indicate that the mtDNA genome is kept by the cell as a "genetic sanctuary" during tumor development in the mouse and humans. This is compatible with the hypothesis that the mtDNA molecule plays an essential role in the control of the cellular adaptive survival response to tumor-induced oxidative stress. The integrity of mtDNA seems to be a necessary element for responding to the increased ROS production associated with the oncogenic process.     

Rethinking the Mitochondrial Theory of Aging: The Role of Mitochondrial Gene Expression in Lifespan Determination

The Mitochondrial Theory of Aging postulates that accumulation of mtDNA mutations and mitochondrial dysfunction are responsible for generating aging phenotypes and limiting lifespan. Although widely accepted, this theory remains unproven because the evidence supporting it, while substantial, is largely correlative. Furthermore, recent experimental results in mice with accelerated rates of mtDNA mutagenesis have challenged the traditional formulation of the Mitochondrial Theory, perhaps warranting a reevaluation of some of its core principles. In this perspective, we summarize recent work suggesting that both the quantity and the quality of mitochondrial gene expression play a much greater role in the aging process than previously appreciated. We speculate that this form of mitochondrial dysfunction may operate independently or in concert with mtDNA mutations to promote age-related pathology and limit lifespan.     

Reduced Adolescent-Age Spatial Learning Ability Associated with Elevated Juvenile-Age Superoxide Levels in Complex I Mouse Mutants

Large-scale, heteroplasmic and generally pathogenic mtDNA defects (as induced by defective mitochondrial DNA polymerase, clonal mutations or DNA deletions) are known to negatively impact on life span and can result in apoptosis and tissue loss in, e.g., skeletal muscle or reduce learning abilities. The functional impact of homoplasmic specific mtDNA point mutations, e.g., in genes coding for the electron transport chain, however, remains a matter of debate. The present study contributes to this discussion and provides evidence that a single point mutation in complex I of the respiratory chain is associated with impairment of spatial navigation in adolescent (6-month-old) mice, i.e., reduced performance in the Morris Water Maze, which goes along with increased production of reactive oxygen species (ROS) in juvenile mice (3 months) but not at the age of phenotype expression. A point mutation in complex III goes along with only a mild and non-significant negative effect on cognitive performance and no significant changes in ROS production. These findings suggest to also consider the ontogenetic development of phenotypes when studying mtDNA mutations and highlights a possible impact of complex I dysfunction on the emergence of neurological deficits.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

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.