Aging and Cellular Stress Resistance

Stressors for cells include the nonenzymatic glycation of proteins and DNA, as well as oxidative damage to diverse molecules that could be caused by free radicals generated mainly as a by-product of metabolism, by solar radiation, by a variety of stressors that elicit the heat shock response, and by numerous other toxic factors in the environment. Normal function, and even survival, is dependent on the ability of cells to sustain homoeostasis by being able to adapt to and/or resist stressors, and to repair or replace damaged molecules and organelles.

Oxidative Stress. Oxygen radicals are reactive molecular species produced by the one-electron reduction of oxygen. It is estimated that approximately 4% of the oxygen consumed by mitochondria is converted to the reactive oxygen species (ROS) hydrogen peroxide and superoxide during oxidative phosphorylation.

These ROS can be converted by a number of distinct processes to highly reactive hydroxyl radicals, which can damage proteins, lipids, and DNA. Not surprisingly, organisms have important defenses against oxidative stress, including the small molecules uric acid, glutathione, ascorbic acid, and vitamin E. Oxidative stress induces the synthesis of several antioxidant enzymes including superoxide dismutase (SOD), catalase, and glutathione peroxidase. These enzymes protect cells and tissues against oxidative damage by converting ROS to nonreactive species.

What is the importance of defenses against oxidative damage during aging? The continued effectiveness of inducible responses to environmental damage are certainly thought to be major factors in resistance to diseases and even in the rate of aging. Indeed, it has been proposed that a capacity for increased life span is latent within most (if not all) species and might be uncovered by treatments that enhance cellular stress resistance.

This is based on the observation that all known laboratory-based strategies that extend organism life span, whether by genetic intervention or environmental manipulation, are associated with an increased ability to respond to environmental stressors and decreased susceptibility to stress-induced damage.

Systemic Stress Responses and Aging. One of the most exciting concepts relating stress and aging is that the rate of aging is governed by the rate of accumulation of macromolecular damage and, therefore, that processes determining this rate also define life span. Damage accumulates when repair or degradation of damaged molecules cannot keep pace with the rate of damage, which itself is determined by the production of damaging agents (e.g., ROS) and their subsequent detoxification (e.g., by antioxidant enzymes).

This idea is well developed in the free-radical theory of aging and its subsequent modifications The modern oxygen radical theory states that the decline in function and increasing mortality rate associated with aging are due to ROS production and their subsequent reaction with cellular components, causing irreparable damage.

Despite considerable interest in this theory, there is virtually no evidence that it explains the natural aging rate. However, we are now developing a more general picture of how cellular stress resistance and aging rates might be related, mainly from studies of model genetic systems (Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster).

Fungal Systems and Aging. S. cerevisiae divides by asymmetric budding, leaving a mother cell and a smaller daughter cell. The mother cell exhibits limited division potential and consequently, a limited life span. At the end of the yeast life span, the cell becomes granulated and dies. Yeasts provide excellent systems for the dissection of complex biological problems, and aging is no exception.

Many genes are now known to influence yeast life span. The first mutations isolated that conferred an increased life span came from a genetic screen for mutations that also conferred stress resistance, in this case to starvation. These mutations clustered in four genes, UTH1, UTH3, UTH4, and SIR4.

An important biomarker of aging, observed in species as diverse as primates and nematodes, is the accumulation of damaged mitochondrial DNA (mtDNA). It is thought that the cause of these altered forms is oxygen radical damage. The filamentous fungi Podospora anserina and Neurospora crassa have provided interesting information on at least one form of aging-related mtDNA mutation. In Podospora, aging is associated with the release of a circular DNA molecule (asenDNA) from the cytochrome oxidase subunit I gene (COI) in the mitochondrial genome. Mutations in the nuclear genes gr (greisea) or i viv result in the repression of the production of this asenDNA.

Combinations of these mutations result in indefinite life spans. The gr gene has been cloned and encodes a protein homologous to the angiotensin-converting enzyme (ACE)1 transcription factor, known to regulate the superoxide dismutase (SOD1) and metallothionein (CUP1) genes in budding yeast. Therefore, the alterations in stress-associated transcription factors cause dramatic alterations in the rate of accumulation of aging-associated damage, which correlates with life span.

Caenorhabditis elegans. C. elegans is a small (1.2-mm) soil-dwelling roundworm. It has a 3-day life cycle with four larval stages (L1-L4) before the final molt to the adult form. Most worms are hermaphrodites (XX), although males (X0) do exist at a ratio of approximately 1:500. The hermaphrodites reproduce by self-fertilization and live for approximately 20 days. This organism has proved to be very useful in aging research because single-gene mutations have arisen that both extend life span (age mutations) and increase resistance to environmental stressors.

The age mutations can bring about an up to threefold increases in life span, attributable to a smaller acceleration of mortality rate with advancing age. Further, the mutations also induce increased resistance to several stressors: heat, ultraviolet (UV) radiation, oxidation, and heavy metals. Many, but not all, mutations that enhance resistance to such stressors are associated with increased life span, which suggests a direct relationship between longevity and stress resistance.

The epistasis analysis of age mutations has established two genetic pathways that determine life span. The cloning of genes in the first pathway, age-1, daf-2, and daf-16 has shown that an insulin-like signaling pathway governs both aging and responses to stressors in this species. The age-1 gene encodes a homolog of the mammalian phosphatidyl inositol- 3-kinase catalytic subunit (PI3K). The daf-2 gene encodes a protein that is 35% identical to human insulin receptor and 34% identical to the insulinlike growth factor 1 receptor (IGF-1R). The daf-16 gene is a Fork head transcription factor.

With the free-radical theory in mind, worms carrying either age-1 (hx546) or daf-2 (e1370) have been assessed for antioxidant enzyme activities, Cu/Zn SOD, and catalase. The net effect of these two enzymes is to prevent the production of the highly reactive hydroxyl radical, which is thought to be responsible for age-associated damage to proteins, lipids, and nucleic acids. The activities of both of these enzymes are elevated in age-1 and daf-2 mutant worms in mid- and late life.

This is consistent with the idea that age mutations result in resistance to intrinsic oxidative stressors. Worms mutant in age-1 are certainly resistant to extrinsic oxidative stress, as measured by survival during exposure to hydrogen peroxide (H₂O₂) and the O₂ generator paraquat. These results suggest that the insulin signaling pathway negatively regulates antioxidant enzyme activity and that extended life span results from enhanced antioxidant defenses. This strongly suggests that over expression of antioxidant enzymes is sufficient to confer extended life span.

Oxidative stress is also thought to be one mechanism by which insulin action may be impaired. For example, it has been shown that whole-body glucose uptake (reflecting metabolic clearance rate, MCR) during a euglycemic glucose clamp declines with age in humans. On the basis of parallel increases in plasma superoxide production, red blood cell membrane microviscosity, and oxidation state of glutathione, it was postulated that insulin action is impaired by free-radical production. However, any such hypothesis based purely on correlation studies must be regarded as very tentative.

Drosophila melanogaster. D. melanogaster is an insect has been widely used in both stress and aging studies. Transgenic Drosophila lines containing additional copies of the Cu/ZnSOD and catalase genes have been generated, and most lines that have elevated antioxidant-enzyme levels are also long-lived. This demonstrates the importance of these two enzymes in determining life span. More recent experiments suggest that the expression of MnSOD in neurons alone is sufficient to extend life span.

The transgenic Drosophila experimental system has also been used to test the role of molecular chaperones in determining aging rates. In these experiments, it was shown that additional copies of heat shock protein (HSP)-70 could reduce age-specific mortality rates in Drosophila. Just how HSP-70 retards aging is unknown, but these findings are consistent with other experiments in both C. elegans and Drosophila in which animals given a mild thermal stress and allowed to recover go on to develop acquired thermotolerance and exhibit extended life span. In these experiments, HSPs are induced and may be the principal cause of slowed aging.

Drosophila has also been used for studying the effects of multiple genes on stress responses and aging. Selection has been undertaken for Drosophila lines that differ in their levels of stress tolerance, and at least one laboratory has demonstrated that selection for stress-resistance produces lines with increased longevity. In addition, some long-lived Drosophila lines, selected for late reproduction, are stress resistant.