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. 2009 Feb;5(2):e1000361.
doi: 10.1371/journal.pgen.1000361. Epub 2009 Feb 6.

Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans

Jeremy M Van Raamsdonk  1 , Siegfried Hekimi
Affiliations

Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans

Jeremy M Van Raamsdonk et al. PLoS Genet. 2009 Feb.

Abstract

The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants-including slow development, low brood size, and slow defecation-this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Deletion of Individual sod Genes Does Not Decrease Lifespan Despite Increasing Sensitivity to Oxidative Stress.
A–E) sod-1(A), sod-3(C), sod-4(D) and sod-5(E) mutant worms live as long as wild-type N2 worms while sod-2 mutant worms (B) live significantly longer than wild-type worms. F–J) sod-1 (F), sod-2 (G) and sod-3 (H) mutant worms showed decreased survival on 4 mM paraquat plates compared to N2 worms, while sod-4 (I) and sod-5 (J) mutant worms survived as long as N2 worms. K–O) Similarly, sod-1 (K) and sod-2 (L) mutant worms showed decreased survival on 240 µM juglone plates compared to N2 worms, while the survival of sod-3 (M), sod-4 (N) and sod-5 (O) mutant worms was not significantly different than N2 worms. Overall, sod deletion increases sensitivity to oxidative stress but does not decrease lifespan. * p<0.05, ** p<0.01, ***p<0.001.
Figure 2
Figure 2. Deletion of Individual sod Genes Results in Mild Compensatory Upregulation of sod mRNA.
A) sod mRNA levels for each of the five sod genes was examined in the sod deletion mutants by quantitative real-time RT-PCR. While compensatory upregulation was observed in sod-2, sod-3 and sod-5 mutant worms, the magnitude was small, generally 2-fold or less. No sod mRNA upregulation in sod-1 or sod-4 mutant worms. B) Examination of SOD-1 and SOD-2 protein levels by Western blotting reveals no SOD-1 expression in sod-1 mutant worms and no SOD-2 expression in sod-2 mutant worms. C) Quantification of SOD protein levels reveals no significant upregulation of SOD-1 or SOD-2 proteins in any of the sod deletion mutants. * p<0.05.
Figure 3
Figure 3. sod-sod Double Deletion Mutants Do Not Exhibit Decreased Lifespan Despite Increased Sensitivity to Oxidative Stress.
A) None of the sod-1 double deletion mutants showed a decreased lifespan compared to wild-type or sod-1 mutant worms. sod-1;sod-2 mutant worms show extended longevity. B) All of the sod-2 double deletion mutants exhibited extended lifespan compared to wild-type worms. C) Despite exhibiting a normal or extended lifespan, all of the sod-1 double deletion mutants as well as sod-2;sod-3 mutant worms were very sensitive to paraquat-induced oxidative stress. D) As with the paraquat assay, sod-1 double deletion mutants and sod-2;sod-3 mutant worms showed the greatest sensitivity to juglone-induced oxidative stress. Overall, sod-sod double deletion mutants show increased sensitivity to oxidative stress but exhibit a normal or extended lifespan.
Figure 4
Figure 4. Worms Lacking Combinations of Three sod Genes Show Normal or Extended Lifespan.
To model sod-1 and sod-2 knockouts in species which have only three sod genes, sod-1;sod-3;sod-5 (A) and sod-2;sod-3;sod-5 (B) mutant worms were generated respectively. sod-1;sod-2;sod-4 (C) mutant worms which lack the primary cytoplasmic, mitochondrial and extracellular sod genes were also generated. Surprisingly, none of the sod triple deletion mutants showed decreased lifespan compared to wild-type worms and the two triple mutants bearing the sod-2 allele lived significantly longer than wild-type. Thus, C. elegans is able to compensate for the loss of multiple sod genes to achieve a normal or extended lifespan.
Figure 5
Figure 5. sod-2 Deletion Alters the Lifespan of Extended Longevity Mitochondrial Mutants.
To gain insight into the mechanism of lifespan extension in sod-2 mutant worms, we examined the effect of sod-2 deletion on other genes known to increase longevity. A) sod-2 deletion did not affect lifespan in daf-2 worms (insulin/IGF1 signaling). B) sod-2 deletion markedly increases lifespan in clk-1 worms (decreased mitochondrial function). C) sod-2 deletion shortens lifespan in isp-1 worms (decreased mitochondrial function) such that isp-1;sod-2 worms do not survive as long as wild-type worms. D) sod-2 deletion moderately extends mean and maximum lifespan in eat-2 worms (dietary restriction). E) sod-2 deletion extends mean but not maximum lifespan of glp-1 worms (germ-line ablation). Thus, sod-2 deletion has the greatest impact on the lifespan of clk-1 and isp-1 worms, which extend lifespan by decreasing mitochondrial function.
Figure 6
Figure 6. sod-2 Mutant Worms Exhibit the Hallmark Features of Extended Longevity Mitochondrial Mutants in C. elegans.
Similar to extended longevity mitochondrial mutants such as clk-1 and isp-1, sod-2 mutant worms show slow post-embryonic development (A), slow defecation rate (B), decreased self-brood size (C) and long life (D). In clk-1 worms, the deletion of sod-2 slows development, slows defecation rate, decreases brood size and increases lifespan. In isp-1 worms, sod-2 deletion slows development, increases defecation rate, decreases brood size and decreases lifespan. *p<0.05, **p<0.01, ***p<0.001.
Figure 7
Figure 7. sod-2 Mutant Worms Show Decreased Oxygen Consumption and Increased Oxidative Damage But Normal Tolerance to Heat and Osmotic Stress.
A) In order to assess mitochondrial function in sod-2 mutant worms, whole worm oxygen consumption was measured and compared to wild-type N2 controls. Oxygen consumption was significantly decreased in sod-2 mutant worms compared to wild-type worms. B) Examination of carbonylated proteins by Oxyblot reveals increased oxidative damage in sod-2 mutant worms compared to wild-type worms (N = 10 N2, 8 sod-2). Nonetheless, sod-2 mutant worms survive both heat stress (C) and osmotic stress (D) as well as wild-type worms (stress results represent three independent trials).
Figure 8
Figure 8. Proposed Model Relating Mitochondrial Function and Lifespan in sod-2 Mutant Worms and sod-2 Double Mutants.
N2 exhibits normal mitochondrial function and a normal lifespan. In sod-2 and clk-1 worms, mitochondrial function is decreased leading to compensatory changes which increase lifespan. Deleting sod-2 in clk-1 worms further decreases mitochondrial function towards that of isp-1 worms with a coincident increase in lifespan. Deleting sod-2 in isp-1 worms decreases mitochondrial function past a threshold after which the organism is no longer able to compensate for the degree of mitochondrial impairment and lifespan decreases.
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