Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes

Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats.

Mitochondrial dysfunction appears to contribute to some of the loss of function accompanying ageing. Mitochondria from aged tissue use oxygen inefficiently impairing ATP synthesis and results in increased oxidant production. A high flux of oxidants not only damages mitochondria, but other important cell biomolecules as well. In the present investigation, the levels of lipid peroxidation, oxidized glutathione, non-enzymatic antioxidants and the activities of mitochondrial enzymes were measured in liver and kidney mitochondria of young and aged rats before and after lipoic acid supplementation. In both liver and kidney increase in the levels of mitochondrial lipid peroxidation and oxidized glutathione and decrease in the levels of antioxidants and the activities of mitochondrial enzymes were observed in aged rats. DL-alpha-lipoic acid supplemented aged rats showed a decrease in the levels of lipid peroxidation and oxidized glutathione and increase in the levels of reduced glutathione, vitamins C and E and the activities of mitochondrial enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Thus, lipoic acid reverses the age-associated decline in endogenous low molecular weight antioxidants and mitochondrial enzymes and, therefore, may lower the increased risk of oxidative damage that occurs during ageing. From our results it can be concluded that lipoic acid supplementation enhances the activities of mitochondrial enzymes and antioxidant status and thereby protects mitochondria from ageing.     

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.     

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.     

Progressive accumulation of mitochondrial DNA mutations and decline in mitochondrial function lead to beta-cell failure

A key adaptation enabling the fetus to survive in a limited energy environment may be the reprogramming of mitochondrial function, which can have deleterious effects. Critical questions are whether mitochondrial dysfunction progressively declines after birth, and if so, what mechanism might underlie this process. To address this, we developed a model of intrauterine growth retardation (IUGR) in the rat that leads to diabetes in adulthood. Reactive oxygen species (ROS) production and oxidative stress gradually increased in IUGR islets. ATP production was impaired and continued to deteriorate with age. The activities of complex I and III of the electron transport chain progressively declined in IUGR islets. Mitochondrial DNA point mutations accumulated with age and were associated with decreased mitochondrial DNA content and reduced expression of mitochondria-encoded genes in IUGR islets. Mitochondrial dysfunction resulted in impaired insulin secretion. These results demonstrate that IUGR induces mitochondrial dysfunction in the fetal beta-cell, leading to increased production of ROS, which in turn damage mitochondrial DNA. A self-reinforcing cycle of progressive deterioration in mitochondrial function leads to a corresponding decline in beta-cell function. Finally, a threshold in mitochondrial dysfunction and ROS production is reached, and diabetes ensues.     

The Effects and Mechanisms of Mitochondrial Nutrient α-Lipoic Acid on Improving Age-Associated Mitochondrial and Cognitive Dysfunction: An Overview

We have identified a group of nutrients that can directly or indirectly protect mitochondria from oxidative damage and improve mitochondrial function and named them “mitochondrial nutrients”. The direct protection includes preventing the generation of oxidants, scavenging free radicals or inhibiting oxidant reactivity, and elevating cofactors of defective mitochondrial enzymes with increased Michaelis–Menten constant to stimulate enzyme activity, and also protect enzymes from further oxidation, and the indirect protection includes repairing oxidative damage by enhancing antioxidant defense systems either through activation of phase 2 enzymes or through increase in mitochondrial biogenesis. In this review, we take α-lipoic acid (LA) as an example of mitochondrial nutrients by summarizing the protective effects and possible mechanisms of LA and its derivatives on age-associated cognitive and mitochondrial dysfunction of the brain. LA and its derivatives improve the age-associated decline of memory, improve mitochondrial structure and function, inhibit the age-associated increase of oxidative damage, elevate the levels of antioxidants, and restore the activity of key enzymes. In addition, co-administration of LA with other mitochondrial nutrients, such as acetyl-l-carnitine and coenzyme Q10, appears more effective in improving cognitive dysfunction and reducing oxidative mitochondrial dysfunction. Therefore, administrating mitochondrial nutrients, such as LA and its derivatives in combination with other mitochondrial nutrients to aged people and patients suffering from neurodegenerative diseases, may be an effective strategy for improving mitochondrial and cognitive dysfunction.     

Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.     

Hippocampal mitochondrial dysfunction in rat aging

Hippocampus mitochondrial dysfunction with impaired electron transfer and increased oxidative damage was observed upon rat aging. Hippocampal mitochondria of aged (12 mo) and senescent (20 mo) rats showed, compared with young (4 mo) rats, marked decreases in the rate of state 3 respiration with NAD-dependent substrates (32-51%) and in the activities of mitochondrial complexes I (57-73%) and IV (33-54%). The activity of mitochondrial nitric oxide synthase was also decreased, 53-66%, with age. These losses in enzymatic activity were more marked in the hippocampus than in brain cortex or in whole brain. The histochemical assay of mitochondrial complex IV in the hippocampus showed decreased staining upon aging. Oxidative damage, determined as the mitochondrial content of thiobarbituric-acid reactive substances (TBARS) and protein carbonyls, increased in aged and senescent hippocampus (66-74% in TBARS and 48-96% in carbonyls). A significant statistical correlation was observed between mitochondrial oxidative damage and enzymatic activity. Mitochondrial dysfunction with shortage of energy supply is considered a likely cause of dysfunction in aged hippocampus.     

Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Mitochondrial respiration is an important site of ROS production and hence a potential contributor to brain functional changes with aging. In this study we examined the effect of aging on complex I activity, oxygen consumption, ROS production and phospholipid composition in rat brain mitochondria. The activity of complex I was reduced by 30% in brain mitochondria from 24 months aged rats relative to young animals. These changes in complex I activity were associated with parallel changes in state 3 respiration. H(2)O(2) generation was significantly increased in mitochondria isolated from aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 31% as function of aging, while there was a significant increase in the level of peroxidized cardiolipin. The age-related decrease in complex I activity in brain mitochondria could be reversed by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids. It is proposed that aging causes brain mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with brain aging.     

Mitochondrial Decay in the Brains of Old Rats: Ameliorating Effect of Alpha-Lipoic Acid and Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine

To investigate the mitochondrial decay and oxidative damage resulting from aging, the activities/kinetics of the mitochondrial complexes were examined in the brains of young and old rats as well as in old rats fed R-α-lipoic acid plus acetyl-l-carnitine (LA/ALC). The brain mitochondria of old rats, compared with young rats, had significantly decreased endogenous antioxidants and superoxide dismutase activity; more oxidative damage to lipids and proteins; and decreased activities of complex I, IV and V. Complex I showed a decrease in binding affinity (increase in K_m) for substrates. Feeding LA/ALC to old rats partially restored age-associated mitochondrial dysfunction to the levels of the young rats. These results indicate that oxidative mitochondrial decay plays an important role in brain aging and that a combination of nutrients targeting mitochondria, such as LA/ALC, could ameliorate mitochondrial decay through preventing mitochondrial oxidative damage. Special issue article in honor of Dr. Akitane Mori.     

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.     

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.     

Mitochondrial DNA, base excision repair and neurodegeneration

Neurodegeneration is a growing public health concern because of the rapid increase in median and maximum life expectancy in the developed world. Mitochondrial dysfunction seems to play a critical role in neurodegeneration, likely owing to the high energy demand of the central nervous system and its sole reliance on oxidative metabolism for energy production. Loss of mitochondrial function has been clearly demonstrated in several neuropathologies, most notably those associated with age, like Alzheimer's, Parkinson's and Huntington's diseases. Among the common features observed in such conditions is the accumulation of oxidative DNA damage, in particular in the mitochondrial DNA, suggesting that mitochondrial DNA instability may play a causative role in the development of these diseases. In this review we examine the evidence for the accumulation of oxidative DNA damage in mitochondria, and its relationship with loss of mitochondrial function and cell death in neural tissues. Oxidative DNA damage is repaired mainly by the base excision repair pathway. Thus, we review the molecular events and enzymes involved in base excision repair in mitochondria, and explore the possible role of alterations in mitochondrial base excision repair activities in premature aging and age-associated neurodegenerative diseases.     

Effects of aging on activities of mitochondrial electron transport chain complexes and oxidative damage in rat heart

Mitochondrial dysfunction and accumulation of oxidative damage have been implicated to be the major factors of aging. However, data on age-related changes in activities of mitochondrial electron transport chain (ETC) complexes remain controversial and molecular mechanisms responsible for ETC dysfunction are still largely unknown. In this study, we examined the effect of aging on activities of ETC complexes and oxidative damage to proteins and lipids in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. ETC complexes I-IV displayed different extent of inhibition with age. The most significant decline occurred in complex IV activity, whereas complex II activity was unchanged in old rats and was only slightly reduced in senescent rats. Compared to adult, old and senescent rat hearts had significantly higher levels of malondialdehyde, 4-hydroxynonenal (HNE) and dityrosine, while thiol group content was reduced. Despite marked increase in HNE content with age (25 and 76 % for 15- and 26-month-old rats, respectively) Western blot analysis revealed only few HNE-protein adducts. The present study suggests that non-uniform decline in activities of ETC complexes is due, at least in part, to mitochondrial oxidative damage; however, lipid peroxidation products appear to have a limited impact on enzyme functions.     

Sex-dependent differences in aged rat brain mitochondrial function and oxidative stress

Females show lower incidences of several neurodegenerative diseases related to oxidative stress and mitochondrial dysfunction than males. In addition, female rats show more differentiated mitochondria than males in several tissues. The aim of this work was to investigate the existence of sex-dependent differences in brain mitochondrial bioenergetics and oxidative balance in aged rats. Results showed that aged female rat brain had a lower mitochondria content than aged male brain but with a greater differentiation degree given the higher mitochondrial protein content and mitochondrial complex activities in females. Female rat brain also showed a better oxidative balance than that of males, reflected by the fact that higher mitochondrial respiratory chain function is accompanied by a similar ROS production and greater antioxidant enzyme activities, which could be responsible for the lesser oxidative damage observed in proteins and lipids in this sex. Interestingly, levels of UCP4 and UCP5--proteins related to a decrease in ROS production--were also higher in females. In conclusion, aged female rat brain had more differentiated mitochondria than male brain and showed a better control of oxidative stress balance, which could be due, in part, to the neuroprotective effect of UCPs.     

Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal

Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders.     

Carbonylation of ER chaperone proteins in aged mouse liver

Progressive accumulation of oxidative damage to macromolecules in aged tissues is thought to contribute to the decline in tissue function characteristic of the aged phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants, which can damage macromolecules. We, therefore, sought to examine the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, we have fractionated young and aged liver homogenates, resolved the proteins by 2D gel electrophoresis, assayed for oxidative damage as indicated by protein carbonylation, and identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting an age-associated increase in oxidative damage. Increased carbonylation of these key proteins in aged liver suggests an age-associated impairment in protein folding, disulfide crosslinking, and glycosylation in the aged mouse liver.