Jeffrey A. Stuart

A midlife crisis for the mitochondrial free radical theory of aging

Since its inception more than four decades ago, the Mitochondrial Free Radical Theory of Aging (MFRTA) has served as a touchstone for research into the biology of aging. The MFRTA suggests that oxidative damage to cellular macromolecules caused by reactive oxygen species (ROS) originating from mitochondria accumulates in cells over an animal’s lifespan and eventually leads to the dysfunction and failure that characterizes aging. A central prediction of the theory is that the ability to ameliorate or slow this process should be associated with a slowed rate of aging and thus increased lifespan. A vast pool of data bearing on this idea has now been published. ROS production, ROS neutralization and macromolecule repair have all been extensively studied in the context of longevity. We review experimental evidence from comparisons between naturally long- or short-lived animal species, from calorie restricted animals, and from genetically modified animals and weigh the strength of results supporting the MFRTA. Viewed as a whole, the data accumulated from these studies have too often failed to support the theory. Excellent, well controlled studies from the past decade in particular have isolated ROS as an experimental variable and have shown no relationship between its production or neutralization and aging or longevity. Instead, a role for mitochondrial ROS as intracellular messengers involved in the regulation of some basic cellular processes, such as proliferation, differentiation and death, has emerged. If mitochondrial ROS are involved in the aging process, it seems very likely it will be via highly specific and regulated cellular processes and not through indiscriminate oxidative damage to macromolecules.

Higher levels of heat shock proteins in longer-lived mammals and birds.

Cellular stress resistance is generally associated with longevity, but the mechanisms underlying this phenotype are not clear. In invertebrate models there is a clear role for heat shock proteins (Hsps) and organelle-specific unfolded protein responses (UPR) in longevity. However, this has not been demonstrated in vertebrates. Some Hsp amino acid sequences are highly conserved amongst mammals and birds. We used antibodies recognizing conserved regions of Hsp60 (primarily mitochondrial), Hsp70 (primarily cytosolic), GRP78 (Bip) and GRP94 (endoplasmic reticulum) to measure constitutive levels of these proteins in brain, heart and liver of 13 mammalian and avian species ranging in maximum lifespan from 3 to 30 years. In all three tissues, the expression of these proteins was highly correlated with MLSP, indicating higher basal levels of Hsp expression are characteristic of longer-lived species. We also quantified the levels of Hsp60, Hsp70 and GRP78 in brain and heart tissue of young adult (6-7 month old) Snell dwarf mice and normal littermates. Snell dwarf mice are characterized by a single gene mutation that is associated with an ∼50% increase in lifespan. However, neither Hsp60, nor Hsp70, nor GRP78 levels were elevated in brain or heart tissue from Snell dwarf mice compared to normal littermates.

Mitochondrial redox metabolism: aging, longevity and dietary effects.

Mitochondrial redox metabolism has long been considered to play important roles in mammalian aging and the development of age-related pathologies in the major oxidative organs. Both genetic and dietary manipulations of mitochondrial redox metabolism have been associated with the extension of lifespan. Here we provide a broad overview of the circumstantial evidence showing associations between mitochondrial reactive oxygen species (ROS) metabolism, aging and longevity. We address most aspects of mitochondrial ROS metabolism, from superoxide production, to ROS detoxification and the repair/removal of ROS-mediated macromolecular damage. Finally, we discuss the effects of dietary manipulations (e.g. caloric restriction, methionine restriction), dietary deficiencies (e.g. folate) and dietary supplementation (e.g. resveratrol) on mitochondrial ROS metabolism and lifespan.

Resveratrol interacts with estrogen receptor-β to inhibit cell replicative growth and enhance stress resistance by upregulating mitochondrial superoxide dismutase

trans-Resveratrol (RES) is one of a number of dietary polyphenols that have been reported to beneficially affect human physiology. Although numerous studies have attributed this to direct interactions between RES and histone deacetylases, recently the reliability of these results has been questioned. We have shown that the mitochondrial superoxide dismutase (MnSOD) is substantially upregulated in RES-treated cells. Here we explore the mechanisms underlying this, showing that two of RESs more interesting effects, inhibition of replication and enhancement of stress resistance, are mediated by MnSOD upregulation in three cell lines: MRC5 human lung fibroblasts, C2C12 mouse myoblasts, and SHSY5Y human neuroblastoma cells. When small interfering RNA was used to prevent induction of MnSOD expression, the effects of RES on population doubling time of cells in culture, and resistance to cell death after exposure to hydrogen peroxide or paraquat, were abolished. Interestingly, the RES-induced upregulation of MnSOD levels could be prevented by the estrogen receptor antagonist ICI 182780. RESs effects also could be reproduced using estradiol or the estrogen receptor-β agonist diarylpropionitrile, but not using the estrogen receptor-α agonist propylpyrazole triol. Thus, we suggest that RES interacts with estrogen receptor-β to induce the upregulation of MnSOD, which affects cell cycle progression and stress resistance. These results have important implications for our understanding of RESs biological activities and potential applications to human health.

Antioxidant enzyme activities are not broadly correlated with longevity in 14 vertebrate endotherm species.

The free radical theory of ageing posits that accrual of oxidative damage underlies the increased cellular, tissue and organ dysfunction and failure associated with advanced age. In support of this theory, cellular resistance to oxidative stress is highly correlated with life span, suggesting that prevention or repair of oxidative damage might indeed be essential for longevity. To test the hypothesis that the prevention of oxidative damage underlies longevity, we measured the activities of the five major intracellular antioxidant enzymes in brain, heart and liver tissue of 14 mammalian and avian species with maximum life spans (MLSPs) ranging from 3xa0years to over 100xa0years. Our data set included Snell dwarf mice in which life span is increased by ∼50% compared to their normal littermates. We found that CuZn superoxide dismutase, the major cytosolic superoxide dismutase, showed no correlation with MLSP in any of the three organs. Similarly, neither glutathione peroxidase nor glutathione reductase activities correlated with MLSP. MnSOD, the sole mitochondrial superoxide dismutase in mammals and birds, was positively correlated with MLSP only for brain tissue. This same trend was observed for catalase. For all correlational data, effects of body mass and phylogenetic relatedness were removed using residual analysis and Felsenstein’s phylogenetically independent contrasts. Our results are not consistent with a causal role for intracellular antioxidant enzymes in longevity, similar to recent reports from studies utilising genetic modifications of mice (Pérez et al., Biochim Biophys Acta 1790:1005–1014, 2009). However, our results indicate a specific augmentation of reactive oxygen species neutralising activities in brain associated with longevity.

trans-Resveratrol as A Neuroprotectant

Epidemiological evidence indicates that nutritionally-derived polyphenols such as resveratrol (RES) have neuroprotective properties. Administration of RES to culture media protects a wide variety of neuronal cell types from stress-induced death. Dietary supplementation of RES can ameliorate neuronal damage and death resulting from both acute and chronic stresses in rodents. The specific molecular mechanisms by which RES acts at the cellular level remain incompletely understood. However, many experimental data indicate that RES reduces or prevents the occurrence of oxidative damage. Here we discuss possible mechanisms by which RES might exert protection against oxidative damage and cell death. Evidence suggesting that RES’s chemical antioxidant potential is not sufficient explanation for its effects is discussed. Putative biological activities, including interactions with estrogen receptors and sirtuins are critically discussed. We provide a synthesis of how RES’s phytoestrogenic properties might mediate the neuronal stress resistance underlying its observed neuroprotective properties.

Plasma IGF-1 is negatively correlated with body mass in a comparison of 36 mammalian species

In mammals, insulin-like growth factor-1 (IGF-1) is positively correlated with adult body mass, in comparisons made within a given species. In mice, IGF-1 deficiency is associated with dwarfism, whereas IGF-1 overproduction in transgenic animals causes gigantism. Surprisingly, the opposite is true in an inter-species context. We collected published plasma total IGF-1 data for adults of 36 mammalian species and analyzed it with respect to body mass. In contrast to the intra-species observation, this analysis revealed a significant negative correlation of plasma IGF-1 with body mass. Interestingly, IGF-1 is negatively correlated with longevity, and suppression of IGF-1 signalling in worms, flies and mice increases lifespan. Smaller mouse strains, for example, tend to have lower plasma IGF-1 levels and to be longer-lived. However, when plasma total IGF-1 was analyzed relative to the maximum lifespans of the 36 species examined here, there was no statistically significant correlation. Low plasma IGF-1 levels in larger mammalian species may be physiologically significant, considering the roles of this hormone in metabolism, tissue regeneration, and cancer incidence.

Enhanced protein repair and recycling are not correlated with longevity in 15 vertebrate endotherm species

Previous studies have shown that longevity is associated with enhanced cellular stress resistance. This observation supports the disposable soma theory of aging, which suggests that the investment made in cellular maintenance will be proportional to selective pressures to extend lifespan. Maintenance of protein homeostasis is a critical component of cellular maintenance and stress resistance. To test the hypothesis that enhanced protein repair and recycling activities underlie longevity, we measured the activities of the 20S/26S proteasome and two protein repair enzymes in liver, heart and brain tissues of 15 different mammalian and avian species with maximum lifespans (MLSP) ranging from 3 to 30xa0years. The data set included Snell dwarf mice, in which lifespan is increased by ∼50% compared to their normal littermates. None of these activities in any of the three tissues correlated positively with MLSP. In liver, 20S/26S proteasome and thioredoxin reductase (TrxR) activities correlated negatively with body mass. In brain tissue, TrxR was also negatively correlated with body mass. Glutaredoxin (Grx) activity in brain was negatively correlated with MLSP and this correlation remained after residual analysis to remove the effects of body mass, but was lost when the data were analysed using Felsenstein’s independent contrasts. Snell dwarf mice had marginally lower 20S proteasome, TrxR and Grx activities than normal controls in brain, but not heart tissue. Thus, increased longevity is not associated with increased protein repair or proteasomal degradation capacities in vertebrate endotherms.

Mitochondria, Cellular Stress Resistance, Somatic Cell Depletion and Lifespan

The causes of aging and determinants of maximum lifespan in animal species are multifaceted and complex. However, a wealth of experimental data suggests that mitochondria are involved both in the aging process and in regulating lifespan. Here we outline a somatic cell depletion (SCD) model to account for correlations between: (1) mitochondrial reactive oxygen species and lifespan; (2) mitochondrial antioxidant enzymes and lifespan; (3) mitochondrial DNA mutation and lifespan and (4) cellular stress resistance and lifespan. We examine the available data from within the framework of the SCD model, in which mitochondrial dysfunction leading to cell death and gradual loss of essential somatic cells eventually contributes to the decline in physiological performance that limits lifespan. This model is useful in explaining many of the mitochondrial manipulations that alter maximum lifespan in a variety of animal species; however, there are a number of caveats and critical experiments outstanding, and these are outlined in this review.

Correlation of mitochondrial superoxide dismutase and DNA polymerase β in mammalian dermal fibroblasts with species maximal lifespan

Eukaryotic cells have evolved elaborate mechanisms to preserve the fidelity of their genomic material in the face of chronic attack by reactive byproducts of aerobic metabolism. These mechanisms include antioxidant and DNA repair enzymes. Skin fibroblasts of long-lived mammalian species are more resistant to oxidative stress than those of shorter-lived species [Kapahi, P., Boulton, M.E., Kirkwood, T.B., 1999. Positive correlation between mammalian life span and cellular resistance to stress. Free Radic. Biol. Med. 26, 495-500], and we speculated that this is due to greater antioxidant and/or DNA repair capacities in longer-lived species. We tested this hypothesis using dermal fibroblasts from mammalian species with maximum lifespans between 5 and 122 years. The fibroblasts were cultured at either 18 or 3% O(2). Of the antioxidant enzymes only manganese superoxide dismutase was found to positively correlate with maximum lifespan (p<0.01). Oxidative damage to DNA is primary repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation (apurinic/apyrimidinic endonuclease), or correlated negatively (p<0.01) with donor species MLS (polymerase beta). Standard culture conditions (18% O(2)) induced both antioxidant and BER enzymes activities, suggesting that the normal cell culture conditions widely employed are inappropriately hyperoxic, which likely confounds the interpretation of studies of cellular oxidative stress responses in culture.