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Keywords:

  • insulin;
  • long-lived mutant;
  • oxidative stress;
  • redox regulation;
  • superoxide dismutase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

Lifespan can be lengthened by genetic and environmental modifications. Study of these might provide valuable insights into the mechanism of aging. Low doses of radiation and short-term exposure to heat and high concentrations of oxygen prolong the lifespan of the nematode Caenorhabditis elegans. These might be caused by adaptive responses to harmful environmental conditions. Single-gene mutations have been found to extend lifespan in C. elegans, Drosophila and mice. So far, the best-characterized system is the C. elegans mutant in the daf-2, insulin/IGF-I receptor gene that is the component of the insulin/IGF-I signaling pathway. The mutant animals live twice as long as the wild type. The insulin/IGF-I signaling pathway regulates the activity of DAF-16, a FOXO transcription factor. However, the unified explanation for the function of DAF-16 transcription targets in the lifespan extension is not yet fully established. As both of the Mn superoxide dismutase (MnSOD) isoforms (sod-2 and sod-3) are found to be targets of DAF-16, we attempted to assess their functions in regulating lifespan and oxidative stress responsivity. We show that the double deletions of sod-2 and sod-3 genes induced oxidative-stress sensitivity but do not shorten lifespan in the daf-2 mutant background, indicating that oxidative stress is not necessarily a limiting factor for longevity. Furthermore, the deletion in the sod-3 gene lengthens lifespan in the daf-2 mutant. We conclude that the MnSOD systems in C. elegans fine-tune the insulin/IGF-I-signaling based regulation of longevity by acting not as anti-oxidants but as physiological-redox-signaling modulators. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S59–S69.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

The lifespan of metazoans is determined by genetic and environmental factors. A variety of environmental conditions are known to extend lifespan: caloric restriction in a wide range of organisms,1 low temperature in some poikilothermic animals2 and low oxygen concentrations in the nematode Caenorhabditis elegans.3 The mechanisms by which each condition slows the aging rate have not yet been fully elucidated. Function of caloric restriction has been postulated as hormonal changes, altered gene expression, lowered metabolic rate and a reduced generation rate of mitochondrial reactive oxygen species (ROS). Lifespan could also be lengthened by environmental perturbations. Hormesis is a phenomenon occurring when agents that are harmful in high doses or over long periods actually produce beneficial effects, such as lifespan extension, when used at low doses or over short periods. C. elegans shows lifespan-extension hormesis when exposed to low doses of radiation,4 or short-term heat,5 hyperoxia6 or hyperbaric oxygen.7 These treatments are associated with adaptive resistance to lethal thermal or oxidative stress, and the gene expression of stress-defense proteins.5,6,8

Recently, lifespan-extension mutants of the nematode C. elegans have been extensively isolated, and the gene network responsible for its longevity has been unraveled.9 Two main classes of lifespan-extension mutants have been reported; one class is related to the activity of the mitochondrial electron transport chains, such as clk-110 and isp-1,11 and the other is related to hormonal mechanisms, especially an insulin/IGF-I signaling pathway, such as daf-212,13 and age-1.14,15

Hormesis and oxygen

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

Lifespan hormesis

Hormesis is a phenomenon that occurs when agents that are harmful in high doses or over long periods, actually produce beneficial effects, such as lifespan extension, when used in low doses or over short periods. C. elegans shows lifespan-extension hormesis when exposed to low doses of radiation4 or short-term heat exposure.5 We showed that long-term hyperoxia shortened the lifespan of C. elegans.16 During the course of this investigation, we noticed slight extension of lifespan by normoxic incubation after hyperoxia.6,16 Darr and Fridovich showed an adaptive induction of anti-oxidant defense in C. elegans.8

Lifespan extension by short-term exposure to hyperoxia

The wild-type strain that was reared under normoxic conditions was exposed to 90% oxygen for 2 days from a 6-day adult age. The lifespan was measured after it was returned to normoxic conditions until the end of life. Figure 1 shows that a two-day exposure to 90% oxygen slightly, but nevertheless significantly, increases in mean and maximum lifespan.6 It is well known that one of the best criteria of the aging process is an exponential acceleration of mortality rate with chronological age, which is formulated by the Gompertz equation. The acceleration rate, that is the Gompertz component, is a parameter of aging rate and has been estimated in C. elegans.17 The Gompertz analysis of the survival data of control and hyperoxia-exposed animals was carried out in three different ways. In any analyses, the mortality rate of the hyperoxia-exposed animals was found to be smaller than that of the control animals, indicating that short-term exposure to hyperoxia slowed the aging rate.6

image

Figure 1. The wild-type C. elegans was exposed to 90% oxygen for 2 days from a 6-day adult age. The oxygen-exposed animals lived longer than the unexposed animals.

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Adaptive response to oxidative stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

To test whether hyperoxia exposure can increase lethal oxidative stress resistance, the wild-type strain was pre-exposed to 90% oxygen for 2 days, and subsequently treated with 50 mmol paraquat (PQ) under 98% oxygen, compared with animals that were treated with 50 mmol PQ under 98% oxygen without pre-exposure. Exposure to 90% oxygen increased oxidative stress resistance.6 This indicated that exposure to 90% oxygen induced an adaptive response for protection against oxidative stress, supporting the observation of Darr and Fridovich.8 To test whether hyperoxia can induce the gene expression of anti-oxidant enzymes, we measured the gene expression of three types of SOD and catalase after exposure to 90% oxygen and found that 90% oxygen exposure induces the gene expression of sod-1, sod-2, sod-3 and catalase. Exposure of C. elegans to hyperoxia adaptably increases resistance to subsequent lethal oxidative stress and increases expression of anti-oxidant defense enzymes, such as SOD and catalase. The most plausible explanation is that an increase in the ability to withdraw ROS induced by hyperoxia could reduce normally occurring oxidative stress that could be the cause of aging.

Oxygen concentrations and lifespan

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

Lifespan is known to be affected by environmental conditions: food concentrations,1 temperature in some poikilothermic animals2 and oxygen concentrations in the nematode C. elegans.3 We showed that the lifespan of C. elegans was decreased with increasing environmental oxygen concentrations.3,6 In contrast, low oxygen concentrations lengthened the lifespan.3 The Gompertz component, a parameter of aging rate, increased with an increase in oxygen concentrations and decreased under 1% oxygen.3,6 These results suggest that the environmental oxygen concentration is a lifespan determinant. Because ROS are thought to arise in organisms with a dependence on the oxygen concentration,18 these findings suggest that ROS-mediated oxidative stress is involved in the determination of lifespan.

When the animals were exposed to a high concentration of oxygen at the earlier phase of the adult lifespan, oxygen-induced reduced lifespan shortening was not observed.16 Furthermore, a split of exposure to the high concentration of oxygen reduced the oxygen-induced reduced lifespan. These results suggest that there exists a mechanism that repairs the oxygen-induced damage leading to reduced lifespan.

Lifespan extension mutants

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

Single-gene mutations have been found to extend lifespan in C. elegans,9Drosophila and mice.4 So far, the best-characterized system is an insulin/IGF-1 signaling pathway that regulates the lifespan of C. elegans. Insulin/IGF-I signaling is mediated by the DAF-2 insulin/IGF-I receptor. The C. elegans daf-2 mutants that reduce the activity of DAF-2 remain youthful and active much longer than the wild-type animals and live more than twice as long.12 The lifespan-extension phenotype of the daf-2 is suppressed by mutations in daf-16, indicating that daf-16 is negatively regulated by DAF-2 signaling and is the major downstream effector. The daf-16 encodes a FOXO transcription factor.19,20

Binding of insulin/IGF-I-like ligands to the DAF-2 insulin/IGF-I receptor controls insulin/IGF-I signaling. There are at least 38 genes (ins) encoding insulin/IGF-I-like peptides in C. elegans.21,22 Many of these genes are divergent insulin superfamily members, and as the specific ligand has not yet been identified, it is possible that these members might have complex and redundant roles. The daf-28 gene encodes insulin-like peptide. A dominant-negative allele of the daf-28 mutant lives 10% longer than wild-type animals. A phenotype of the daf-28 mutant is rescued by the ins-4 or ins-6 transgene, suggesting a redundant nature.22 Some ins genes are expressed in sensory neurons.22,23 Environmental cues such as food, pheromones and temperature might affect insulin/IGF signaling through different expression and the secretion of various INS peptides. The mutation of age-1, which encodes the phosphoinositide-3-OH (PI3) kinase catalytic subunit, doubles the lifespan in C. elegans.14,15

The current model of insulin/IGF-I signaling is as follows (Fig. 2): the DAF-2 insulin/IGF-I receptor transduces signals by activating AGE-1. AGE-1 PI3 kinase phosphorylates PIP2 to generate the second messenger PIP3. In contrast, DAF-18 PTEN dephosphorylates PIP3 and thus antagonizes the action of AGE-1. Thus, the PIP3 level is determined by a balance between generation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN. PIP3 activates 3-phosphoinositide-dependent kinase-1 (PDK-1), which in turn phosphorylates and activates AKT-1/AKT-2 Ser/Thr kinase. AKT-1/AKT-2 phosphorylates and inactivates the DAF-16 transcription factor to be sequestered from the nucleus to the cytoplasm. In this state, adults age rapidly. On the contrary, when DAF-2 signaling is reduced, DAF-16 is eventually translocated to the nucleus to promote transcription of target genes. In fact, disrupting AKT-consensus phosphorylation sites in DAF-16 causes nuclear accumulation, although the nuclear accumulation is not sufficient for lifespan extension.24

image

Figure 2. The insulin/IGF-1 signaling regulates stress resistance and longevity in C. elegans and mammals.

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C. elegans worms grow through four larval stages (L1-L4) before reaching maturity. However, when the food supply is limited and the population density is high at the L1 stage, animals become dauer larvae after the L2 stage. The dauer larva is a developmentally arrested dispersal stage and lives up to several months, greatly exceeding the normal adult lifespan of about 3 weeks under stressful environmental conditions.25 It seems that the dauer stage is non-aging, because the post-dauer lifespan is not affected by a prolonged dauer stage of up to 2 months.26 The dauer larva is more resistant to a variety of environmental stresses, including hypoxia, heat, desiccation and oxidative stress, and has increased levels of SOD and catalase.27 We showed that the expression of the Mn superoxide dismutase (MnSOD) gene (sod-3) is higher in the dauer larvae than in the adults.28 As dauer larvae live much longer than adults, some genes expressing altered levels in dauer state might be the key to longevity. By using serial analysis of gene expression (SAGE), Jones et al. found that the expression of tts-1 (transcribed telomere-like sequence), a variant histone H1 and a nucleosome assembly protein possibly relating to the structure or stability of chromatin is high in dauer larvae. These results suggest that the chromatin structure might change to be more stable in the dauer state than in the growing state.29 Holt and Riddle examined gene expression profiles of carbohydrate metabolism in dauer larvae by using SAGE. A high gene expression of pyruvate kinase, alcohol dehydrogenase, a putative cytosolic fumarate reductase, two pyruvate dehydrogenase components and succinyl CoA synthetase α subunit implies that anaerobic metabolism is prominent in dauer larvae.30

The formation of the dauer larva in C. elegans is normally triggered by food limitation and/or pheromones.25,31,32 The switch between development and dauer diapause is regulated by both the nervous and endocrine systems through the recognition and transduction of environmental cues through the TGF-β,33 cGMP,34 serotonin35 and insulin/IGF-113 signaling pathways. In this regard, the TGF-β and insulin/IGF-1 signaling pathways converge on a steroid hormone pathway.36 By genetic analysis of mutants showing “dauer larva formation abnormal” Daf phenotype, a number of genes that regulate dauer formation have been identified.25 Among the mutants that show the constitutive formation of dauer larvae, those that downregulate insulin/IGF-1 signaling, such as the daf-2 mutants but not a TGF-β signaling such as the daf-7 (TGF-β) mutants, also manifest an adult-lifespan extension phenotype. This suggests that the insulin/IGF-1 signaling pathway initiates the program for long survival in the dauer state and that the ectopic activation of this program in the adult stage by its mutations might extend adult lifespan.12 We discovered that the crude pheromone extract of C. elegans culture medium extends the adult lifespan in C. elegans.32 This extension does not occur in the daf-16 mutant animal that suppresses the expansion of lifespan caused by reducing the insulin/IGF-1 signaling indicating relevancy of the insulin/IGF-1 signaling in the lifespan extension caused by the extract.

Insulin/IGF-I signaling pathway and stress resistance

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

To investigate the relationship between lifespan extension and oxidative-stress resistance, we screened the oxidative-stress resistance phenotype in various mutants.6,28 We examined the survival period of each mutant under experimentally induced, acute oxidative stress. We used paraquat, an intracellular superoxide inline image generator, under hyperoxia for acute oxidative stress. The daf-2 mutants with an extended lifespan survived for a longer period of time than wild-type animals in the presence of paraquat under hyperoxic or normoxic conditions. The daf-2 mutant is also more resistant to menadione, another intracellular inline image generator, under hyperoxia than wild-type animals. The mutants in the TGF-β pathway and cGMP pathway do not show oxidative-stress resistance. The oxidative-stress resistance seen in the daf-2 mutants is suppressed by mutations in daf-18 or daf-16 indicating that daf-16 and daf-18 act downstream of daf-2 to confer oxidative-stress resistance, as well as extended lifespan. Vanfleteren37 and Larsen38 showed that the lifespan extension mutant age-1 is more resistant to oxidative stress in old age than wild-type animals at the same age. We showed that the age-1 mutants of young adults also display the oxidative-stress resistance.6 The oxidative-stress resistance in age-1 mutants is suppressed by daf-16 mutation, indicating that daf-16 is located downstream of age-1 in the pathway for regulating oxidative-stress resistance.

In contrast, oxidative-stress resistance in two alleles of age-1 mutants (m333 and mg44) is not fully suppressed by daf-18 mutation, indicating that daf-18 does not act downstream of age-1.6daf-16 and daf-18 act downstream of daf-2 in the insulin/IGF-I signaling pathway for oxidative-stress resistance.28 Taken together, we postulate the following pathway for oxidative-stress resistance:

  • image

This pathway is essentially identical to the pathway regulating longevity,39 suggesting a strong association between lifespan extension and oxidative-stress resistance. However, Dorman and Canyon showed that the daf-18 mutation suppressed the lifespan-extension phenotype of another allele of age-1 (hx546) indicating that daf-18 acts downstream of age-1.40 Such differences could be attributed to the differences in severity of the age-1 alleles used.

Two alleles of age-1 mutants (m333 and mg44) display the Daf phenotype, which is completely suppressed by daf-18 or daf-16 mutations, indicating the following pathway for dauer formation:

  • image

Thus, oxidative-stress resistance is closely associated with longevity but not with dauer formation. The PIP3 level is maintained under a balance between generation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN, which could determine the impact of this pathway. The loss or reduction of function mutations in age-1 could reduce PI3 kinase activity to drop this second messenger level. When DAF-18 is reduced, only the pre-existing pool of the second messenger might be insufficient to inhibit longevity and oxidative-stress resistance, but sufficient to inhibit dauer formation. Dillin et al. found that the inactivation of daf-2 during adulthood by RNAi extends lifespan and increases oxidative-stress resistance.41 Because dauer formation is switched in the early larval stage, the insulin/IGF-I pathway controls the dauer switch and oxidative-stress resistance/longevity independently.

There are several genes in C. elegans that encode SOD enzymes: sod-1 encodes cytosolic CuZnSOD,38sod-2 and sod-3 each encodes mitochondrial MnSOD,42–44 and sod-4 encodes extracellular CuZnSOD.45 We showed that the level of sod-3 mRNA in daf-2 mutants is higher than that in wild-type animals.28 The levels of mRNA transcripts of sod-1, sod-2, and catalase in the daf-2 are similar to those in wild-type animals.28 The level of sod-3 mRNA in daf-2 mutants increases as it develops from the egg to the L2 larval stage coinciding with increases in oxidative-stress resistance. The elevated level of sod-3 mRNA in the daf-2 mutants is suppressed by daf-16 and daf-18 mutation.28Figure 3 shows that the level of sod-3 mRNA in the age-1 mutants is higher than that in wild-type animals.6 The elevated level of sod-3 mRNA in age-1 mutants is suppressed by daf-16 mutation but is not fully suppressed by daf-18 mutation (Fig. 3). These results provide further evidence that the insulin/IGF-I signaling pathway regulates extended lifespan, oxidative-stress resistance and sod-3 expression in a similar way. These results suggest that the extended lifespan is correlated with the efficient withdrawal of ROS generated in mitochondria during normal metabolism.

image

Figure 3. The gene expression of superoxide dismutases and catalase in C. elegans longevity mutants. RT–PCR analysis of the mRNA level of sod-1, sod-2, sod-3 and ctl-1 was carried out in age-1 and/or daf-16 and daf-18 mutants.

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Another class of the lifespan-extension mutations, clk-1, does not show oxidative-stress resistance or increased expression of sod-3. However, clk-1 mutation largely promotes oxidative-stress resistance and an increased expression of sod-3 in the daf-2 mutant background,28 as well as extended lifespan.10 In contrast, mutation in isp-1 cannot promote an extended lifespan of daf-2 and itself causes oxidative-stress resistance and increased expression of sod-3.11 Insulin/IGF-I signaling seems to regulate the expression of sod-3 in concert with mitochondrial energy metabolites.

Redox signaling in lifespan regulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

With respect to the role of MnSOD in organismal aging, the seemingly conflicting observations so far reported suggest that MnSOD overexpression extends lifespan in Drosophila46 and rodents,47 shortens lifespan when catalase is co-overexpressed48 and has little effect on lifespan49 in Drosophila. In the mouse, heterozygous MnSOD knockouts that show about half of the wild-type MnSOD activity indicate no changes in their lifespan despite an increase in nuclear and mitochondrial oxidative-DNA-damage accumulation and cancer incidence.50

To further clarify the roles of MnSOD in C. elegans aging, we explored the effects of the deletion mutations for the individual MnSOD genes, sod-2 and sod-3, on oxidative-stress responsivity and lifespan in the wild-type and long-lived daf-2 mutant.28 We assessed oxidative-stress responsivity by using hyperoxia and PQ under hyperoxia. The sod-2/sod-3 double deletion mutant animals at adult stage all died within 2 days under hyperoxia (98% oxygen), whereas the wild type, sod-2 and sod-3 single deletion mutant adults all survived longer than 10 days under hyperoxia. The sod-2 deletion mutant was found to be more sensitive to PQ-hyperoxia than the wild type and the sod-3 deletion mutant, but less sensitive to PQ-hyperoxia than the sod-2; sod-3 double mutant (Fig. 4a). These results indicate that SOD-2 enzyme plays a more important role for anti-oxidant defense than SOD-3, and that SOD-2 and SOD-3 cooperatively exert an anti-oxidant-defense function.

image

Figure 4. Oxidative-stress responsivity of sod-2/sod-3 deletion mutants. (a) Oxidative-stress responsivity of sod-2 and/or sod-3 deletion mutants in the background of wild-type C. elegans. (b) Oxidative-stress responsivity of sod-2 and/or sod-3 deletion mutants in the background of daf-2 mutation of C. elegans. Survival of adult animals is plotted as a function of treatment period with paraquat.

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In order to elucidate a role of MnSOD in resistance to oxidative stress (Oxr phenotype) in the daf-2 mutants,28 we measured the PQ-hyperoxia sensitivities of the sod-2 and/or sod-3 deletions in the daf-2 mutant.51 Neither the sod-2 nor sod-3 deletion mutation alone suppressed Oxr in the daf-2 mutant (Fig. 4b). However, the sod-2; daf-2; sod-3 triple mutant showed oxidative-stress sensitivity and, therefore, did not show Oxr (Fig. 4b). These results show that Oxr in the daf-2 mutant requires at least one functioning MnSOD isoform and is, therefore, totally ameliorated by the two MnSOD double deletions.

Next, we examined the lifespan of the MnSOD-deletion mutants.51 The sod-2 and sod-3 single and double deletions did not appear to significantly affect the lifespan. These data indicate that the MnSOD do not participate in lifespan determination and suggest that the sensitivity to oxidative stress does not necessarily correlate with lifespan in C. elegans. To explore the possible involvement of the MnSOD in the lifespan extension phenotype (Age) of the daf-2 mutants, we examined the lifespan of the MnSOD-deletions in the daf-2 mutant. The sod-2 and sod-3 single and double deletions did not abolish Age of the daf-2 mutant. Because the sod-2/sod-3 double deletions eliminated Oxr in daf-2, as mentioned above (Fig. 4b), these results indicate that Oxr is not causally linked to Age in the daf-2 mutant. Interestingly, however, the deletions of the sod-2 and/or sod-3 deletions appeared to cause fluctuations in the degree of the Age phenotype in the daf-2 mutant (Fig. 5a,b). Specifically, the sod-2; daf-2 double mutant showed a shorter lifespan than that of the daf-2 mutant counterparts (Fig. 5a). In contrast, the daf-2; sod-3 double mutant had a slightly but significantly longer lifespan than that of the daf-2 mutant (Fig. 5b). The introduction of a wild-type sod-2 transgene extended the lifespan of the sod-2; daf-2 mutant, (Fig. 5a) whereas a wild-type sod-3 transgene shortened the lifespan of the daf-2; sod-3 mutant (Fig. 5b), indicating that SOD-2 and SOD-3 actually function as lifespan modulators in the daf-2 mutant. In addition, the sod-2; daf-2; sod-3 triple mutant lived longer than the daf-2 mutant, similarly to the daf-2; sod-3 double mutant, showing that sod-3 is epistatic to sod-2 in Age phenotype.

image

Figure 5. Lifespan of sod-2/sod-3 deletion mutants. (a) Lifespan of sod-2 deletion mutants in the background of daf-2 mutation of C. elegans. (b) Lifespan of sod-3 deletion mutants in the background of daf-2 mutation of C. elegans.

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Similar effects of sod-2 and sod-3 deletions on Daf-c: abnormal dauer formation, the other daf-2 phenotype, were observed in the daf-2 mutants.51 Because the Age did not correlate with Oxr in the sod-2 and/or sod-3 deletion mutants under the daf-2 mutant background, it is likely that the MnSOD in C. elegans function in longevity, by acting not as anti-oxidant-defense enzymes but as regulatory-signaling modulators. Taken together, the MnSOD are likely to constitute a redox regulation pathway to modulate the longevity and o developmental switching processes similarly under the control of the insulin-like signaling.

From our observations that sod-3 is epistatic to sod-2 in the Age and Daf-c in the daf-2 mutants, we propose a working model whereby SOD-2 regulates the function of SOD-3, and whereby SOD-3 more directly modulates both longevity and development under insulin-like signaling control. Hu et al. reported that superoxide is a mediator of signal transduction during homeostasis and in developmental processes in multicellular systems.47 SOD-2 and SOD-3 express in different cells in the head of C. elegans,51 suggesting that two MnSOD isoforms expressed in different cells are involved in signal transduction between these cells regulating longevity and development.

In contrast to C. elegans, MnSOD has been shown to have only a single form in mammals, but it is noteworthy that this gene is induced in a number of mammalian cell types in response to a variety of stimuli, including TNF-α,52 NGF,53 VEGF,54 IL-1,55 interferon-γ,56 insulin,57 thyroid hormone,58 endotoxin-like LPS59 and hyperoxia,55 possibly resulting in heterogeneities of its cellular expressions. Thus, we hypothesize that these heterogeneities might participate in formation of a signaling network to regulate diverse complex biological processes, including longevity regulation (Fig. 6).

image

Figure 6. Mn superoxide dismutase (MnSOD) induced in various cells by diverse stimuli such as cytokines, hormones and physical agents might be involved in signal transduction between these cells, resulting in the formation of a signaling network to regulate longevity.

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Uncoupling between aging and oxidative stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

As described above, the sensitivity to oxidative stress does not necessarily correlate with lifespan in C. elegans mutants. In order to further examine the relationship between oxidative stress sensitivity and aging, we used a mutation that enhances oxidative-stress sensitivity other than the MnSOD deletions.51 A mutant in the mev-1 gene, which encodes the cytochrome b subunit of the succinate dehydrogenase (complex II) of the electron-transport chain shows an elevated generation of ROS, an increased sensitivity to oxidative stress and a reduced lifespan.60 We analyzed the effects of the mev-1 mutation on oxidative-stress responsivity and lifespan under the daf-2 mutant background. The daf-2; mev-1 double mutant animals showed severe oxidative-stress sensitivity similar to the mev-1 mutant counterparts.51 (Fig. 7a), indicating that the mev-1 mutation also ameliorated Oxr in daf-2. In contrast, the daf-2; mev-1 double mutant animals still lived as long as the daf-2 mutant (Fig. 7b).51 These results indicate the uncoupling between aging and oxidative stress.

image

Figure 7. Oxidative-stress responsivity and lifespan of mev-1 and/or daf-2 mutants. (a) The mev-1 mutant is sensitive to oxidative-stress, even in the daf-2 mutant background. (b) The mev-1 mutant did not have a short lifespan in the daf-2 mutant background.

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The free radical hypothesis of aging postulates that senescence is a result of an accumulation of molecular oxidative damage caused by ROS that are produced as by-products of normal metabolic processes.61,62 A logical prediction based on this hypothesis is that the oxidative-stress sensitivity should inversely correlate with longevity. The present results that the sod-2 deletion and sod-2/sod-3 double deletions caused oxidative-stress sensitivity but had no effect on lifespan in C. elegans conflict with this prediction. Furthermore, it has been implicated that oxidative-stress resistance induced by reduction in the insulin-like signaling is causally linked to lifespan extension in C. elegans,6,28,37,38Drosophila63 and mice.64 The present findings that the sod-2/sod-3 double deletions and mev-1 mutation ablated Oxr but did not abolish Age in the daf-2 mutant do not support this implication. The SOD mimetics has been reported to induce oxidative-stress resistance but does not lengthen the lifespan of C. elegans65 and Drosophila,66 also arguing against the free radical hypothesis of aging. Alternatively, the ROS-induced damaging processes that are not affected by these mutations or mimetics might lead to senescence.

Cellular aging and oxidative stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

Cultured normal cells generally have a finite replicative lifespan as originally shown by Hayflick and Moorhead.67 Human diploid fibroblasts lose the capacity to replicate after vigorous proliferation and enter a viable but non-proliferative state of senescence. Senescent cells are characterized by the upregulation of cyclin-dependent kinase inhibitors, p21SDI1/WAF1/CIP1,68 p16INK4a69 and hypophosphorylated Rb.70 Human fibroblasts grown in hypoxia have an extended replicative lifespan.71

Mouse embryonic fibroblasts (MEF) senesce after vigorous proliferation, and then grow into immortal cells in normoxia (20% oxygen), whereas human normal fibroblasts never achieve immortality. Parrinello et al. found that MEF do not senesce in hypoxia (3% oxygen) and that MEF accumulate more DNA damage in normoxia than hypoxia, and more damage than human fibroblasts in normoxia.72 These results suggest that human cells have a superior ability to prevent or repair oxidative DNA damage compared with murine cells.

Stress-induced premature senescence (SIPS) occurs when cells are grown in hyperoxia,73 and treated with hyperbaric oxygen74 and H2O2,75 with the appearance of several biomarkers of replicative senescence. Hyperoxia (40% oxygen) increases the rate of telomere shortening from 90 bp per population doubling (normoxia) to more than 500 bp per population doubling (hyperoxia),76 suggesting that telomere shortening causes premature senescence. In contrast, Gorbunova et al. indicated that the overexpression of the catalytic subunit of telomerase cannot prevent SIPS, but can protect from stress-induced apoptosis and necrosis. This finding suggests that SIPS might not be a result of telomere shortening.77

As shown in Figure 8, the addition of N-acetylcysteine, which increases the level of cellular reduced glutathione (GSH) known as ROS scavenger, into culture medium extends the replicative lifespan of human embryonic fibroblasts.78 In contrast, the addition of l-buthionine-(R,S)-sulfoximine, a specific inhibitor of GSH synthetase, shortens the replicative lifespan.78 These results suggest that cellular ROS levels are a determinant of replicative lifespan.

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Figure 8. Human diploid fibroblasts at 25 population doubling level (PDL) were serially cultivated in a medium containing 2 mmol or 5 mmol N-acetylcysteine (NAC) or 2 mmol or 5 mmol l-buthionine-(R,S)-sulfoximine (BSO). The cumulative PDL is shown.

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The inactivation of cytosolic CuZnSOD (SOD1) by RNAi induces premature senescence in human fibroblasts depending on p53 induction.79 In human fibroblasts with inactivated p53, the SOD1 RNAi is without effect, suggesting that oxidative DNA damage mediates premature senescence. The overexpression of extracellular CuZnSOD decreases the intracellular peroxide content, slows the telomere-shortening rate, and extends the replicative lifespan under normoxia and hyperoxia.80

The overexpression of an activated V12Ras induces premature senescence in human fibroblasts and induces increased mitochondrial ROS generation.81 In hypoxia, the overexpression of the V12Ras cannot induce premature senescence or increase the level of p21 that is related to the senescent phenotype. These findings suggest that ROS play a role in the regulation of the replicative lifespan.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References

In the past decades, gene-manipulation studies have shown that gene networks exist for the determination of lifespan in diverse species. There appear to be common and different features among species. ROS appear at various points in the aging processes, and play a variety of roles, including the manifestation of oxidative stress and the mediation of signal transduction. The precise role of ROS in the aging process is not yet clear. DNA microarray technology allows us to show global gene expression profiles governing the lifespan and aging rate, although these studies are just beginning. Using the data from DNA microarray analysis, painstaking studies are needed to clarify the aging mechanism to analyze how various gene products work in concert to regulate the aging rate.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Hormesis and oxygen
  5. Adaptive response to oxidative stress
  6. Oxygen concentrations and lifespan
  7. Lifespan extension mutants
  8. Insulin/IGF-I signaling pathway and stress resistance
  9. Redox signaling in lifespan regulation
  10. Uncoupling between aging and oxidative stress
  11. Cellular aging and oxidative stress
  12. Conclusion
  13. Conflicts of interest
  14. References