Ageing, age-related diseases and oxidative stress: What to do next?

There is still ongoing controversy about the oxidative stress theory of aging, particularly in mammals, after a significant number of studies have been conducted to test this theory. Results generated from the studies strongly indicate that accumulation of oxidative damage alone does not play a significant role as an underlying mechanism of aging, which calls into question that significant modifications to the theory are required to understand the relationship between oxidative stress and aging. To examine the exact role of oxidative stress in aging and age-related diseases, our laboratory has been conducting studies with unique animal models: 1) mice overexpressing or down-regulating thioredoxin (Trx) in the cytosol (Trx1) or in mitochondria (Trx2); and 2) rats overexpressing Cu/Zn superoxide dismutase (SOD). Results generated from these studies strongly indicate that: 1) changes in oxidative stress and redox state could play more important roles in age-related pathological changes, e.g., cancer and metabolic disorders; 2) redox regulation of signaling pathways could play more important roles in aging than accumulation of oxidative damages; 3) the potential benefit of changes in oxidative stress and redox state could be organ/tissue specific; 4) the roles of oxidative stress could vary in different stages of life (i.e., young versus old); and 5) synergetic effects of changes in oxidative stress in multiple cellular compartments may be required to have a significant impact on aging. Therefore, the studies with more careful approach would uncover the exact roles and pathophysiological consequences of oxidative stress during aging. Keywords: Oxidative stress, aging, age-related diseases, cancer, obesity, healthspan

Generation of reactive oxygen species (ROS) constantly occurs in healthy cells and is inevitable for aerobic organisms. Controlled ROS production provides the optimal redox state for the maintaining proper cellular function. When large amounts of ROS accumulated, oxidative cellular stress occurs. Under conditions of oxidative stress, overproduction of ROS production can lead to damage to membrane lipids, proteins, and nucleic acids. Oxidative damage of these biomolecules is associated with a range of pathophysiological processes, including aging, carcinogenesis, ischemic reperfusion injury, and neuro-degenerative diseases. The pathogeneses of oxidative stress-mediated diseases are complex in nature. Through the use of our newly synthesized fluorescent probes, the role of oxidative damage in the pathogenesis of ischemia/reperfusion injury has been updated. However, there is a need of adequately powered trials to confirm the validity of these fluorescent probes for monitoring cellular response to oxidative damage, initiation of treatment, and reliably assessing therapy efficacy. Towards this, a correlation between oxidative stress and mitochondrial dysfunction was further examined in in vitro and in vivo models of ischemia/reperfusion injury (Chapter 1). Moreover, these newly validated organelle-targetable fluorescent probes and their relevance in the detection of diseases and assessing of treatment efficacy were further investigated in the experimental models of ischemia/ reperfusion injury (Chapter 1) and cancer (Chapter 2). The overproduction of ROS can cause severe damage to cellular macromolecules, especially the DNA. It is important to develop new molecular probes for sensing DNA oxidative damage. Finally, a series of novel nucleic acid-based fluorescent probes are developed (Chapter 3) for biosensing of DNA oxidative damage to alterations in the cellular redox state during hypoxia or oxidative stress. Identification of oxidative DNA damage allows us to have better understanding of how it is implicated in a number of diseases. For my future research direction, I am interested in developing new strategy to improve a cell’s ability to withstand oxidative damage thereby protecting its DNA. I am also interested in finding new ways to increase the activity of DNA repair genes, which may enable the genes to better handle oxidative damage.

Markers of oxidant stress that are clinically relevant in aging and age-related disease

Aging is characterized by a progressive decline in cellular function, organismal fitness and increased risk of age-associated diseases and death. One potential cause of aging is the progressive accumulation of dysfunctional mitochondria and oxidative damage with age. Considerable efforts have been made in our understanding of the role of mitochondrial dysfunction and oxidative stress in aging and age-associated diseases. This chapter outlines the interplay between oxidative stress and mitochondrial dysfunction, and discusses their impact on senescence, cell death, stem cell function, age-associated diseases and longevity.

In vivo studies reveal that mitochondrial Complex I deficiencies induce iron misregulation and liver iron overload that may contribute to neurodegeneration in mitochondrial disease mice, and that iron restriction is effective in reducing disease progression.

In vivo studies reveal that mitochondrial Complex I deficiencies induce iron misregulation and liver iron overload that may contribute to neurodegeneration in mitochondrial disease mice, and that iron restriction is effective in reducing disease progression.

In vivo studies reveal that mitochondrial Complex I deficiencies induce iron misregulation and liver iron overload that may contribute to neurodegeneration in mitochondrial disease mice, and that iron restriction is effective in reducing disease progression.

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

Oxidative stress plays a causative role in the development of age-related degenerative diseases and may be an underlying determinant of the aging process itself. In this review, we summarize the evidence from a variety of experimental systems that supports this hypothesis. We discuss the molecular basis for the control of gene expression by oxidants, with particular focus on recent studies demonstrating the activation of mitogen-activated protein kinase signal transduction by oxidative stress. How alterations in such pathways may contribute to aging is addressed, and specific examples of transcription factors whose activities are altered both by oxidants and with aging are given. We provide a model illustrating the multiple sites in signal transduction pathways sensitive to oxidative stress, which may indicate targets for strategies aiming to retard or reverse age-related phenotypic changes. OXIDATIVE STRESS IN AGING: CURRENT VIEWS AND HYPOTHESES Exposure to reactive oxygen intermediates (ROI), including molecular oxygen, superoxide, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals, occurs ubiquitously in an aerobic environment. Accordingly, aerobic organisms have widely adapted oxidation-reduction reactions to function in key metabolic and regulatory pathways necessary for normal cell growth. In addition, a number of defense mechanisms to control the level of ROI have evolved as an accessory to these processes. However, when ROI and other oxidizing species exceed the cellular antioxidant capacity, oxidative stress results and oxidative damage to lipids, protein, and DNA ensues. The accumulation of oxidative modifications as a causative factor in aging and degenerative processes was originally proposed by Harman 40 years ago (Harman...

Oxidative stress and vascular inflammation in aging

Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation

The role of oxidative stress in aging proposed by free radical theory has been focus of investigations for more than fifty years. The results of a large number of these investigations provide support for this theory. However, numerous recent findings point to an unexpected complexity in relationships between oxidative stress and aging. This complexity is highlighted by discovery by Timmermann et al. described in this issue of Aging [1] that a mutation in Tsa1p, a key element of oxidative stress defenses in model organism budding yeast, shortens lifespan in concert with enhanced resistance to oxidative stress. In addition to implications of this finding for understanding aging, identification of this mutation by massively parallel sequencing of whole genomes emphasizes enormous utility of next-generation sequencing technologies as investigative tools that will likely revolutionize genetics. The starting point for Timmermann et al. study was random mutagenesis of yeast cells that were subsequently screened for resistance to cumene hydroperoxide (CHP), an oxidizing agent employed in industrial processes and in studies of oxidative stress responses in yeast and other organisms. The authors isolated a mutant strain resistant to CHP and another oxidizing agent, (tert-butyl hydroperoxide). Classical approaches determined that this phenotype was inherited as a dominant allele in a monogenic fashion independently of genetic background. Even the awesome power of yeast genetics is not sufficiently powerful to tackle some experimental challenges. For a variety of reasons, dominant mutations conferring resistance to stresses are difficult to identify using classic yeast approaches, including those that employ strategies based on complementation using clone libraries. Timmermann et al. approached this task instead by sequencing genomes of mutant strain and its wild type parent using Roche 454 massively parallel sequencing platform. Whole-genome sequencing using next-generation sequence technology was also recently employed to identify mutations underlying Mendelian diseases in humans [2]. The Timmermann et al. study is first to apply this technology to map mutations in randomly mutagenized yeast, including strains with phenotypes that are not easily identified by classic functional approaches. Subtraction of surprisingly large number of sequence variants found in both wild type and mutant genomes, but not in a previously sequenced reference genome (as well as identification of non-uniform sequences and alignment artifacts) allowed Timmermann et al. to quickly narrow their search to four candidate mutations predicted to cause amino acid changes in proteins. Phenotypic analysis of cloned sequences containing each of these mutations revealed that TSA1-B7, a dominant allele of TSA1 encoding a peroxiredoxin, was responsible for increased resistance to CHP. Tsa1p catalyzes H2O2 reduction and acts as a molecular chaperone, and disruption of Tsa1p shortens replicative lifespan (RLS) and confers sensitivity to a variety of oxidants, including hydrogen peroxide and CHP [3]. The increased resistance to CHP conferred by B7 allele thus establishes it as a gain-of-function mutation. Although resistance to oxidative stress is often associated with lifespan extension (as predicted by free radical theory), surprisingly, strains harboring B7 mutation shortened, rather than lengthened RLS. This is reminiscent of findings of several recent studies that were similarly discordant in connections they established between oxidative stress and predictions of free radical theory. For example, under glucose-restricted conditions C. elegans lifespan correlates with increased oxidative stress [4]. Although catalase inactivation shortens RLS of budding yeast [5], it lengthens chronological lifespan of this organism in a different model of yeast aging in concert with elevated levels of hydrogen peroxide and increased oxidative damage. Similar enhancement of chronological lifespan by hydrogen peroxide is detected in calorie-restricted cells [6]. Perhaps most dramatic is approximately 10-fold increase in lifespan of naked mole rats compared to mice accompanied by high levels of oxidative damage [7]. In some cases, apparent disconnect between experimental results and predictions of free radical theory is related to hormesis effects that elevate oxidative and other stress defenses in response to low levels of oxidative stress [8]. Interestingly, although B7 mutation in TSA1 confers resistance to CHP, Timmermann et al. also show that cells harboring this mutation are sensitive to hydrogen peroxide. This finding is more in line with shorter RLS of these cells and with free radical theory. Perhaps hydrogen peroxide accumulates in B7 mutant and shortens RLS, similar to effects on RLS of inactivating catalases, but triggers stress responses that protect against CHP. The more transparently clear lesson here is that not all forms of oxidative stress are equivalent in their effects on aging. This isn't surprising in context of multitude of pathways that respond to different forms of oxidative stress [9] and numerous mechanisms by which oxidants can modify different macromolecular targets. Whatever explanation, findings by Timmermann et al. emphasize enormous complexity of relationships between oxidative stress and aging. They also illustrate awesome potential of next-generation sequencing technology combined with classical yeast to make sense of complexity revealed by these and other findings.

Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1(+/-) mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-dependent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2(+/-) mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1(+/-) mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging.

Positive oxidative stress in aging and aging-related disease tolerance

The telomere length (TL) has increasingly been used as a biomarker of human aging because it has been shown to predict the chances of survival and longevity. Oxidative stress is presumed to be a major cause of telomere shortening, but the importance of oxidative stress as a determinant of telomere shortening remains less clear and has recently been questioned. We analyzed 105 healthy subjects of both sexes between the ages of 20-77 years. The TL and biomarkers of oxidative stress were estimated as per standard protocols. A significant (p < 0.001) age-dependent decline in TL was observed. TL was positively correlated with the ferric reducing ability of plasma value (r = 0.8811) and reduced glutathione (r = 0.8209), whereas negatively correlated with malondialdehyde (r = -0.7191). Our findings supported the idea of a possible correlation between the TL and biomarkers of oxidative stress in aging. The study has remarkable scope in medical science as the findings on correlation of TL with biomarkers of oxidative stress in aging are novel and they will help in further research against oxidative stress.