Superoxide dismutase is dispensable for normal animal lifespan

Abstract

Reactive oxygen species (ROS) are toxic oxygen-containing molecules that can damage multiple components of the cell and have been proposed to be the primary cause of aging. The antioxidant enzyme superoxide dismutase (SOD) is the only eukaryotic enzyme capable of detoxifying superoxide, one type of ROS. The fact that SOD is present in all aerobic organisms raises the question as to whether SOD is absolutely required for animal life and whether the loss of SOD activity will result in decreased lifespan. Here we use the genetic model organism Caenorhabditis elegans to generate an animal that completely lacks SOD activity (sod-12345 worms). We show that sod-12345 worms are viable and exhibit a normal lifespan, despite markedly increased sensitivity to multiple stresses. This is in stark contrast to what is observed in other genetic model organisms where the loss of a single sod gene can result in severely decreased survival. Investigating the mechanism underlying the normal lifespan of sod-12345 worms reveals that their longevity results from a balance between the prosurvival signaling and the toxicity of superoxide. Overall, our results demonstrate that SOD activity is dispensable for normal animal lifespan but is required to survive acute stresses. Moreover, our findings indicate that maintaining normal stress resistance is not crucial to the rate of aging.

Fig. 1.

sod quintuple mutant worms have no SOD activity. (A) PCR genotyping reveals that sod-12345 worms have deletions in all five sod genes. (B) Quantitative real-time RT-PCR shows that sod-12345 worms have little or no sod mRNA expression. (C) Western blotting using polyclonal antibodies against SOD-1 and SOD-2 reveals that sod-12345 worms have no SOD-1 or SOD-2 protein expression. (D) sod-12345 worms have no detectable SOD activity. The generation of a bona fide sod quintuple mutant was thus confirmed by DNA, mRNA, protein, and activity. Error bars indicate SEM. M, sod-12345 worms.

Fig. 2.

Loss of SOD activity results in increased sensitivity to multiple stresses. sod-12345 worms have markedly increased sensitivity to superoxide-mediated oxidative stress induced by exposure to low concentrations of paraquat during development (A), by acute exposure to juglone during adulthood (B), and by chronic exposure to 4 mM paraquat beginning on day 1 of adulthood (C). In contrast, sod-12345 worms exhibit normal sensitivity to H₂O₂-mediated oxidative stress (D). sod-12345 worms are also sensitive to osmotic stress (E), cold stress (F), and heat stress (G). Results from each stress assay represent the average of at least three independent trials with 20 worms or 40 eggs per trial. Whereas there was a trend toward increased resistance to H₂O₂-mediated oxidative stress at 2 mM H₂O₂, the difference was not significant. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001. FA, fertile adult.

Fig. 3.

Worms lacking SOD activity live as long as wild-type worms. (A) Examination of worm lifespan revealed no difference in overall survival between sod-12345 and wild-type worms. This survival plot is the average of 17 independent trials with a total of 1,571 deaths recorded. (B) Scatterplot of the mean lifespan from 17 independent lifespan assays shows no difference between sod-12345 and wild-type lifespan. (C) Maximum lifespan of sod-12345 is significantly greater than wild-type worms.

Fig. 4.

Genotype determines the effect of superoxide on lifespan. (A) Increasing superoxide levels through addition of paraquat has a biphasic effect on the lifespan of wild-type worms. (B) This pattern can be explained by a model for the effect of superoxide on lifespan in which superoxide has two opposing effects on lifespan: prosurvival signaling and toxicity. Initially, the effect of the prosurvival signaling is greater than the toxic effect, resulting in a dose-dependent increase in lifespan. At higher superoxide levels, the toxic effect of superoxide overwhelms the prosurvival signaling effect leading to decreased lifespan. (C) Increasing superoxide levels with paraquat resulted in a dose-dependent decrease in sod-12345 worm lifespan but a dose-dependent increase in wild-type lifespan. (D) Paraquat did not increase the lifespan of sod-12345 worms at any concentration. (E) Decreasing ROS with 10 mM vitamin C or 0.1 mM manganese increased the lifespan of sod-12345 worms with little or no effect on wild-type lifespan. (F) Superoxide levels in sod-12345 worms are past their optimum superoxide concentration. (G) gst-4 promoter shows increased expression levels with increasing concentrations of paraquat. (H) isp-1 and sod-12345 worms show increased gst-4 expression, suggesting elevated levels of superoxide. Results from the paraquat and antioxidant survival studies are the average of at least three independent trials of at least 20 worms per strain per trial. Error bars indicate SEM. ***P < 0.001.