The effects of complex I function and oxidative damage on lifespan and anesthetic sensitivity in Caenorhabditis elegans
Introduction
A missense mutation in the 49 kDa subunit of complex I of the mitochondrial electron transport chain causes Caenorhabditis elegans to be hypersensitive to all volatile anesthetics, to have fewer offspring, and to have a shortened lifespan (Morgan and Sedensky, 1994, Kayser et al., 1999). This mutation, gas-1(fc21), leads to a decrease in the maximal rate of oxidative phosphorylation via complex I and an increase in the complex II-dependent rate (Kayser et al., 2001). Measurement of electron transport chain activity also indicated a complex I specific defect in gas-1 mitochondria (Kayser et al., 2001). Increased ambient oxygen concentration decreases survival and fecundity of both gas-1 and wild type animals but increases their anesthetic sensitivity (Hartman et al., 2001, Kayser et al., in press–a). Growth under low oxygen tension reverses the phenotypes of gas-1 toward those of N2. Growth of N2 under 60% oxygen caused adults to be hypersensitive to volatile anesthetics as well as to have a shortened lifespan (Hartman et al., 2001). These data strongly suggest that both anesthetic sensitivity and lifespan are affected by aspects of mitochondrial function. We hypothesized that specific facets of mitochondrial function cause changes in both of these phenotypes, and that the study of other key mutants may sort out their relative contribution to anesthetics sensitivity and to longevity.
Several other mutations that may decrease mitochondrial function have been noted to alter lifespan. Mutations in the gene clk-1 have been shown to extend average lifespan in C. elegans (Wong et al., 1995). clk-1 affects the synthesis of ubiquinone, an electron acceptor from both complex I and II in the electron transport chain. Indirect measurements of mitochondrial function indicate that clk-1 mutants have oxidative rates similar to those of wildtype nematodes (Felkai et al., 1999, Miyadera et al., 2001). However, oxygen consumption in intact worms was noted to be lower in clk-1 animals than in N2 (VanFleteren and De Vreese, 1996). Our studies of mitochondria from clk-1 showed that complex I-dependent oxidative phosphorylation is dramatically decreased in these animals (Kayser et al., in press-b). A mutation in the gene mev-1, which codes for a component of complex II, shortens lifespan in air similarly to gas-1 but does not alter anesthetic sensitivity (Hartman et al., 2001). Measurements of complex II function indicate that it is decreased in mev-1 animals (Senoo-Matsuda et al., 2001, Ishii et al., 1998). Thus, decreased mitochondrial function may be associated with increases or decreases in lifespan. Several other recent studies have also indicated that decreased mitochondrial function may be correlated with increases in lifespan. Using RNAi to decrease components of the respiratory chain Dillin et al. (2002) showed that lifespan was increased. Lowering electron transport chain activity with antimycin A, a complex III inhibitor, also lengthened lifespan. In a systematic screen of 5690 genes for lengthened lifespan using RNAi, Lee et al. (2003) found 52 genes which caused reproducible increases in lifespan. Of these, 11 of the genes were specific for mitochondrial function. Thus, that mitochondrial function affect lifespan seems established.
However, there is some question as to whether free radical damage or rates of respiration are more important in affecting lifespan (Braeckman et al., 1999, Van Voorhies and Ward, 1999, Lee et al., 2003). Since gas-1, clk-1 and mev-1 present different combinations of lifespan and mitochondrial dysfunction, they could be used to separate the effects of rates of oxidative phosphorylation from free radical damage in affecting lifespan. In order to further dissect which aspects of mitochondrial function contribute most to lifespan in C. elegans, we studied free radical damage, oxidative phosphorylation and lifespan in gas-1, clk-1 and mev-1 mutants. In addition, we isolated two dominant suppressors of gas-1 and studied their effects on lifespan, oxidative phosphorylation and oxidative damage. Finally, we constructed two double mutants that we predicted to have virtually no complex I function, hypothesizing that the doubles would be very short-lived. Surprisingly, the lifespans of these mutants were extremely long, contrary to our predictions.
We noted earlier that complex I function correlates with anesthetic sensitivity. gas-1 animals have decreased complex I activity and an increase in anesthetic sensitivity while mev-1 animals have decreased complex II activity and no change in anesthetic sensitivity (Morgan and Sedensky, 1994, Hartman et al., 2001). Our observations in children have fully corroborated this correlation, as children with mitochondrial myopathies affecting complex I are hypersensitive to the volatile anesthetic sevoflurane (Morgan et al., 2002). Since we found clk-1 complex I activity to be decreased, we studied the anesthetic sensitivity of these animals. We also studied the responses of the suppressors of gas-1 to volatile anesthetics. We find that aging is related to oxidative damage in the nematode, but that sensitivity to volatile anesthetics is related to both complex I function and to oxidative damage.
Section snippets
Nematode techniques
The conventions for C. elegans nomenclature are followed throughout (Horvitz et al., 1979). Standard techniques were used for growing and maintaining cultures of C. elegans (Brenner, 1974). Genetic mapping of mutations was as described by Brenner (1974) and by use of single nucleotide polymorphisms (snp) between the Bristol strain and Hawaiian strain, CB4856 (Wicks et al., 2001).
The wild-type C. elegans, N2, was obtained from the Caenorhabditis Genetics Center (Minneapolis, MN). gas-1(fc21) was
Results
The lifespans of wildtype C. elegans (N2), gas-1(fc21), mev-1(kn1) and clk-1(e2519) are presented in Table 1. In addition, the oxidative phosphorylation rates of each of these strains for complex I and II are listed in Table 2. Some of these results have been reported elsewhere (N2, gas-1, clk-1), but they are presented here as current controls for the studies of other mutations.
Discussion
We previously studied the gas-1 mutant to determine the relationship of mitochondrial function to the behavior of C. elegans in volatile anesthetics. However, other aspects of the gas-1 phenotype, including changes in lifespan, are shared by other mutants that also affect mitochondrial function. Therefore, we have extended our analysis to determine which aspect of mitochondrial function contributes to each phenotype (i.e. longevity and anesthetic sensitivity).
It is clear that rates of oxidative
Acknowledgements
The authors deeply appreciate the efforts of Luke and Pam Szweda for their generous sharing of reagents, protocols and their discussions and of Ms. Judy Preston for her invaluable technical expertise. They also thank Phil Hartman for his reading of and suggestions concerning the manuscript. Finally, they thank Charles Hoppel for his advice, insight and sharing of his laboratory for mitochondrial studies.
Support: M.M.S. and P.G.M. were supported in part by NIH grants GM51881 and GM45402. E.B.K.
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