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. 2010 Dec 7;8(12):e1000556.
doi: 10.1371/journal.pbio.1000556.

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

Wen Yang  1 , Siegfried Hekimi
Affiliations

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

Wen Yang et al. PLoS Biol. .

Abstract

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O(⋅) (-)), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reactive oxygen species (ROS) in isolated mitochondria of long-lived mutants and in response to paraquat (PQ) and N-acetyl-cysteine (NAC) treatment.
Global ROS levels were measured by quantifying the fluorescence of the reporter dye H2DCFDA, and superoxide with the dye MitoSox, in FACS-sorted mitochondria. Values are normalized to the value of the untreated sample or the wild type. PQ and NAC, respectively, increase and decrease the levels of both global ROS (A) and superoxide (B). Mitochondria isolated from isp-1(qm150) and nuo-6(qm200) mutants show slightly decreased global ROS generation (C) but significantly increased superoxide generation (D). Mitochondria from clk-1 mutants, but not from daf-2(e1370), eat-2(ad1116), and sod-2(ok1030) mutants, show significantly increased global ROS levels (E). Mitochondria from daf-2 mutants, but not from clk-1, eat-2, and sod-2 mutants, show increased superoxide levels (F). * p<0.05, ** p<0.001, *** p<0.0001, by the paired t test.
Figure 2
Figure 2. Lifespans of wild-type animals and mutants treated with 10 mM NAC.
The treatment has no effect on wild type (A) but dramatically suppresses the lifespan extension of the respiratory chain subunit mutants nuo-6 (B) and isp-1 (C). In contrast, it has no lifespan-shortening effects on the long-lived clk-1 mutants (D). NAC treatment has only a moderate effect on the very long-lived daf-2 mutants (E) but also completely abolished the extended longevity of sod-2 mutants (F). See Tables 1 and S1 for details of genotype, sample size, and statistical analysis.
Figure 3
Figure 3. Treatment with 0.1 mM of paraquat (PQ) increases protein oxidative damage, superoxide dismutase expression, and lifespan.
(A) Young wild type adults treated with 0.1 mM PQ have higher protein oxidative damage compared to untreated control. (B) Young wild type adults treated with 0.1 mM PQ since hatching express significantly more SOD-1 protein than untreated animals. (C) Young wild type adults treated with 0.1 mM paraquat since hatching express significantly more SOD-2 protein compared to untreated wild type worms. (D) Treatment with 0.05, 0.1, or 0.2 mM PQ increases mean and maximum lifespan significantly (see also Tables 1 and S1). *<0.05, ** p<0.001.
Figure 4
Figure 4. Lifespans of wild-type animals and mutants treated with 0.1 mM paraquat (PQ).
The treatment had a dramatic lifespan-lengthening effect on the wild type (A) but no effect on the nuo-6 (B) and isp-1 (C) respiratory chain subunit mutants. In contrast, the treatment had a dramatic additive effect on the long-lived clk-1 (D) and eat-2 (E) mutants. The treatment has only a very moderate effect on daf-2 mutants (F) but a strong effect on sod-2 mutants (G). See Tables 1 and S1 for details of genotype, sample size, and statistical analysis.
Figure 5
Figure 5. Treatment with 0.1 mM PQ does not affect mitochondrial abundance.
Worms were treated with Mitotracker Red at 50 nM (final concentration) in M9 buffer for 20 min. All pictures were taken by confocal microscopy at 400×. A scale bar of 20 µm is shown in the upper right corner of the figure. For each genotype/treatment three tissues (hypodermis, muscle, and germline) were selected, and for each tissue at least five pictures from different worms were taken. An equal section of each picture was enlarged for quantitative comparisons. The percentage of surface occupied by mitochondria as stained by Mitotracker Red was measured and related to the total area of the selected region. A representative example for each tissue and condition is shown in the figure. The quantification for each sample is also shown in the figure below the enlarged areas. The sample size for the hypodermis was >15, and it was 5 for muscles and the germline. In muscles and in the hypodermis, the difference between the ETC mutants and the wild type was significant (p<0.001), while the difference between PQ treatment and untreated wild type worms was not. Thus, the nuo-6 and isp-1 mutations, but not treatment with 0.1 mM PQ, affect mitochondrial abundance.
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