Experimental Control Of Oxidative Stress Pathways

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A. Genetic Manipulations

Work with Drosophila, yeast cells, Neurospora (a type of fungus), and the nematode Caenorhabditis elegans has established a genetic link between stress responsiveness and lifespan. For example, when exposed to a low-energy environment, C. elegans converts to its dauer state in which reproductive function is arrested. During this state, this organism is more resistant to stress. C. elegans mutations have been reported to extend life expectancy by 40% to more than 100%. The first of these mutations to be discovered involved the age-1 gene; mutations in this gene have been shown to increase longevity by about 100% but do not affect reproduction or movement. Biochemical studies have revealed that strains carrying age-1 alleles have enhanced oxidative defenses. For example, when the wild-type and age-1 strains were examined for resistance to H202 exposure, the 50% effective lethal dose (LD50) of the wild type remained constant over the lifespan whereas the LD50 of the age-1 strain increased with aging [75]. Moreover, the increased resistance to oxidative stress was associated with elevated antioxidant activity, as shown by an increase in the activity of SOD and catalase [78]. A variety of other life-extending mutations that are correlated with enhanced stress tolerance have been described.

There are a number of mutated genes that regulate the insulin/insulin growth factor 1 (IGF-1) signaling pathway. Age-1, daf-2, and daf-16 genes in C. elegans are associated with an insulin-like signaling pathway. Age-1 and daf-2 suppress the activity of the downstream target daf-16, a transcription factor that belongs to the Forkhead family of proteases [79]. Hence, loss of function of either of these upstream regulators enhances daf-16 function and leads to increased lifespan. Importantly, loss-of-function mutations in daf-16 not only prevents longevity conferred by the age-1 and daf-2 mutations, but also abolishes stress resistance [80], thereby strengthening the intimate link between longevity and the stress responsiveness associated pathway. These animals are smaller in size, and have a decreased body temperature and a modest increase in antioxidant capacity. Recent studies have shown that knockout mice for the IGF receptor live longer and display greater resistance to oxidative stress [81, 82].

One additional long-lived mutant strain of C. elegans, which provides an important link between metabolism, oxidants, and aging, is clk-1. Clk-1 lacks an enzyme required for the synthesis of ubiquinone, or coenzyme Q [83]. Coenzyme Q is an important electron acceptor for both complex I- and complex Il-dependent respiration. Overexpression of clk-1 leads to a reduction in lifespan [84], probably by increasing the rate of metabolism, which in turn might lead to a faster accumulation of damage resulting from metabolic by-products such as ROS. Another mutant with reduced longevity is mev-1. Mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, a component of complex II of the mitochondrial electron transport chain. These animals show hypersensitivity to hyperoxia, and have compromised mitochondrial function and increased ROS generation [85]. SOD activity is about half that found in the wild type, and the average lifespan is reduced by approximately 35% [86]. These worms also were shown to exhibit increased levels of nuclear DNA damage [87]. Similarly, mice heterozygous for SOD2 have an increased incidence of nDNA as well as a significant increase in tumor formation. Another interesting mutation affecting longevity involves the p66shc gene. The p66shc protein belongs to a family of adaptor proteins that regulate protein-protein interaction for several cell surface receptors. These mice live 30% longer than control mice and also have an increased resistance to oxidative stress [88].

Links between longevity and stress resistance, similar to those demonstrated in C. elegans, also exist in Drosophila melanogaster. Various strains of flies selected for extended lifespan display increased resistance to oxidative stress that in some cases is correlated with enhanced activity of antioxidant enzymes. Methuselah (mth), a long-lived mutant, encodes a G protein-coupled receptor that is thought to play a role in signal transduction [89]. This mutant not only enhances longevity, but also increases resistance to heat stress and paraquat (an intracellular ROS generator). Mth exhibits a 35% increase in average lifespan and is resistant to several stressors, such as oxidants, starvation, and heat [89]. Another Drosophilamutant, Indy, belongs to a family of proteins involved in the Krebs cycle. This mutant shows a 50% increase in lifespan [90].

Several groups have been developing animal models with mitochondria deficiencies [91-93]. These models include the adenine nucleotide translocator (ANT-1), mitochondria superoxide dismutase- (SOD2-) deficient mice, Tfam-deficient mice, and the PolgA. ANT-1- deficient mice are a model for chronic ATP deficiency. These mice have increased production of ROS and hydrogen peroxide and a parallel increase in mtDNA mutations consistent with levels seen in much older mice [94]. SOD2-deficient mice die in the neonatal period from dilated cardiomyopathy or neonatal degeneration in the brain stem [92, 93]. The Tfam-deficient mice exhibit cytochrome c oxidase deficiency and die at around 3 weeks of age [95]. PolgA is a more recent mouse model, independently developed by two groups [29, 96], that expresses a deficient version of the nucleus-encoded catalytic subunit of mtDNA polymerase. These mice develop a mtDNA mutator phenotype with a three- to fivefold increase in levels of mtDNA point mutations, as well as an increase in the amount of deleted DNA. This increase in mtDNA is associated with a premature onset of aging-related phenotypes, including osteoporosis, alopecia, kuphosis, and a median survival of 48 weeks of age and a maximum survival of 61 weeks. Interestingly, these mice do not show an increase in oxidative damage markers [96, 97]. One possible explanation for the decreased longevity in these mice is an increase in markers of apoptosis, suggesting a possible decline in regenerative capacity of tissues in these mice [96, 97].

B. Caloric Restriction

Caloric restriction is the only reproducible experimental manipulation for extending lifespan in many species. Restriction of food intake by 30 to 50% below ad libitum levels during the early growth phase of life has been shown to produce significant increases in the mean lifespan of several species, including insects, mice, fish, and rats [98]. Moreover, calorically restricted rhesus monkeys showed physiological changes similar to those observed in rodents on a calorie-restricted diet. Both calorically restricted monkeys and rats are smaller, mature later, have lower blood glucose and insulin levels, lower body temperature, and increased daytime activity [99]. Although several theories have been proposed to explain the anti-aging effects of caloric restriction, one hypothesis proposes that it acts by decreasing oxidative stress. In support of this hypothesis, it has been shown that caloric restriction can stabilize mitochondrial function and reduce oxidative stress in brain cells [100]. In the same study, the authors showed that the reduction in ROS production is not due to reduced mitochondria oxygen consumption, but rather to a lower percentage release of ROS per total flow in the respiratory chain [100]. It has been shown that caloric restriction decreases H2O2 in rat heart mitochondria. On the other hand, contradictory results have been obtained on the effect of caloric restriction on the expression of the antioxidant enzymes SOD, catalase, and GSH-peroxidase. In particular, some reports have shown that caloric restriction did not increase antiox-idant defenses, while other studies demonstrated that the activity of SOD, catalase, and GSH was increased in older ages [101, 102]. Caloric restriction also prevents many of the changes in gene expression and transcription-factor activity that normally occur with aging, including basal elevations in expression of heat-shock proteins. Caloric restriction can also induce the expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). Increased levels of BDNF have been found in neurons in the hippocampus and other brain regions of rodents maintained on caloric restriction [103]. Given all the positive effects of caloric restriction, one could assume that this translates to an improvement in cognition in animals with caloric restriction; however, the data are mixed on this aspect of behavior; although some reports have found positive benefits [104-106], others have found either only small benefits or no benefits at all [107, 108]. Mixed benefits have also been reported on motor behaviors; improvements were noted on some motor learning tasks and complex locomotor behaviors [107, 109] but negative effects of caloric restriction were observed when testing some aspects of drug-induced rotational behavior and stereotopy [110].

Although caloric restriction is a reproducible way to increase the functional and maximal lifespan, it is questionable whether humans will choose to adopt this lifestyle change and it is still controversial whether caloric restriction will increase lifespan in nonhuman and human primates [111]. There is now accumulating evidence that selection of appropriate whole foods or the addition of antioxidants into the diet is beneficial to increasing the functional lifespan, if not the maximal lifespan (for a review, see [112]). One could then argue that caloric selection may be as important as caloric restriction.

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