Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.     

Isolation of long-lived mutants in Caenorhabditis elegans using selection for resistance to juglone

The accumulation of molecular damage from attack by reactive oxygen species is one cause of aging. Therefore, some mutant organisms showing increased resistance to reactive oxygen species should live longer. We show that selection for Caenorhabditis elegans mutants that are resistant to juglone, a reactive oxygen species-generating compound, leads to the identification of long-lived mutants. Indeed, four of six resistant mutants isolated were also long-lived. This study illustrates once more the strong relationship between oxidative stress and the aging processes.     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.     

pkc-1 regulates daf-2 insulin/IGF signalling-dependent control of dauer formation in Caenorhabditis elegans

In Caenorhabditis elegans, the insulin/IGF pathway participates in the decision to initiate dauer development. Dauer is a diapause stage that is triggered by environmental stresses, such as a lack of nutrients. Insulin/IGF receptor mutants arrest constitutively in dauer, an effect that can be suppressed by mutations in other elements of the insulin/IGF pathway or by a reduction in the activity of the nuclear hormone receptor daf‐12. We have isolated a pkc‐1 mutant that acts as a novel suppressor of the dauer phenotypes caused by insulin/IGF receptor mutations. Interactions between insulin/IGF mutants and the pkc‐1 suppressor mutant are similar to those described for daf‐12 or the DAF‐12 coregulator din‐1. Moreover, we show that the expression of the DAF‐12 target daf‐9, which is normally elevated upon a reduction in insulin/IGF receptor activity, is suppressed in a pkc‐1 mutant background, suggesting that pkc‐1 could link the daf‐12 and insulin/IGF pathways. pkc‐1 has been implicated in the regulation of peptide neurosecretion in C. elegans. Although we demonstrate that pkc‐1 expression in the nervous system regulates dauer formation, our results suggest that the requirement for pkc‐1 in neurosecretion is independent of its role in modulating insulin/IGF signalling. pkc‐1 belongs to the novel protein kinase C (nPKC) family, members of which have been implicated in insulin resistance and diabetes in mammals, suggesting a conserved role for pkc‐1 in the regulation of the insulin/IGF pathway.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Genome-Wide RNAi Longevity Screens in Caenorhabditis elegans

Progress in aging research has identified genetic and environmental factors that regulate longevity across species. The nematode worm Caenorhabditis elegans is a genetically tractable model system that has been widely used to investigate the molecular mechanisms of aging, and the development of RNA interference (RNAi) technology has provided a powerful tool for performing large-scale genetic screens in this organism. Genome-wide screens have identified hundreds of genes that influence lifespan, many of which fall into distinct functional classes and pathways. The purpose of this review is to summarize the results of large-scale RNAi longevity screens in C. elegans, and to provide an in-depth comparison and analysis of their methodology and most significant findings.     

Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans.

Acquired resistance of mammalian cells to multiple chemotherapeutic drugs can result from enhanced expression of the multidrug resistance-associated protein (MRP), which belongs to the ABC transporter superfamily. ABC transporters play a role in the protection of organisms against exogenous toxins by cellular detoxification processes. We have identified four MRP homologues in the soil nematode Caenorhabditis elegans, and we have studied one member, mrp-1, in detail. Using an mrp::lacZ gene fusion, mrp-l expression was found in cells of the pharynx, the pharynx-intestinal valve and the anterior intestinal cells, the rectum-intestinal valve and the epithelial cells of the vulva. Targeted inactivation of mrp-l resulted in increased sensitivity to the heavy metal ions cadmium and arsenite, to which wild-type worms are highly tolerant. The most pronounced effect of the mrp-1 mutation is on the ability of animals to recover from temporary exposure to high concentrations of heavy metals. Nematodes were found to be hypersensitive to heavy metals when both the MRP homologue, mrp-1, and a member of the P-glycoprotein (Pgp) gene family, pgp-1, were deleted. We conclude that nematodes have multiple proteins, homologues of mammalian proteins involved in the cellular resistance to chemotherapeutic drugs, that protect them against heavy metals.     

Uncoupling of longevity and paraquat resistance in mutants of the nematode Caenorhabditis elegans

To analyze the relationship between resistance to oxidative stress and longevity, we isolated three novel paraquat-resistant mutants, mev-5, mev-6, and mev-7, from the nematode Caenorhabditis elegans. They all showed the Dyf (defective in dye filling) phenotype, but not always resistance to heat or UV. Life-span extension was observed only in the mev-5 mutant at 26 degrees C. These results indicate that longevity is uncoupled with the phenotype of paraquat resistance.     

Positive selection of Caenorhabditis elegans mutants with increased stress resistance and longevity

We developed selective conditions for long-lived mutants of the nematode Caenorhabditis elegans by subjecting the first larval stage (L1) to thermal stress at 30 degrees for 7 days. The surviving larvae developed to fertile adults after the temperature was shifted to 15 degrees. A total of one million F(2) progeny and a half million F(3) progeny of ethyl-methanesulfonate-mutagenized animals were treated in three separate experiments. Among the 81 putative mutants that recovered and matured to the reproductive adult, 63 retested as thermotolerant and 49 (80%) exhibited a >15% increase in mean life span. All the known classes of dauer formation (Daf) mutant that affect longevity were found, including six new alleles of daf-2, and a unique temperature-sensitive, dauer-constitutive allele of age-1. Alleles of dyf-2 and unc-13 were isolated, and mutants of unc-18, a gene that interacts with unc-13, were also found to be long lived. Thirteen additional mutations define at least four new genes.     

daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.

Evolutionary models of aging propose that a trade-off exists between the resources an organism devotes to reproduction and growth and those devoted to cellular maintenance and repair, such that an optimal life history always entails an imperfect ability to resist stress. Yet, since environmental stressors, such as caloric restriction or exposure to mild stress, can increase stress resistance and life span, it is possible that a common genetic mechanism could regulate the allocation of resources in response to a changing environment (for overview, see ). Consistent with predictions of evolutionary trade-off models, we show that nematodes carrying an integrated DAF-16::GFP transgene grow and reproduce more slowly yet are more stress resistant and longer lived than controls carrying the integration marker alone. We also show that the nuclear localization of the DAF-16::GFP fusion protein responds to environmental inputs as well as genetic. Environmental stresses, such as starvation, heat, and oxidative stress, cause rapid nuclear localization of DAF-16. In conditions rich in food, we find that DAF-16::GFP is inhibited from entry into the nucleus by daf-2 and akt-1/akt-2, both components of insulin-like signaling in nematodes. We suggest that changes in the subcellular localization of DAF-16 by environmental cues allows for rapid reallocation of resources in response to a changing environment at all stages of life.     

Temperature-sensitive developmental mutants of Caenorhabditis elegans

About 200 temperature-sensitive mutants of the nematode Caenorhabditis elegans have been isolated. At restrictive temperature, the mutants are blocked in the reproductive life cycle. They have been placed into six broad categories based on their defective phenotypes. The six categories are: (1) mutants blocked in embryogenesis; (2) mutants defective in gonadogenesis; (3) mutants defective in spermatogenesis; (4) mutants that accumulate at an intermediate growth stage; (5) mutants that produce sterile adult progeny; (6) mutants that have a temperature-sensitive morphological defect that interrupts the reproductive life cycle. The critical times of temperature sensitivity have been measured using temperature-shift experiments. Most of the gonadogenesis and spermatogenesis mutants are temperature sensitive during the period of cellular differentiation rather than proliferation. The temperature responses of the gonadogenesis and zygote-defective mutants indicate a common association between functions in gonadogenesis and early embryogenesis. Many of the mutants placed in different categories share other temperature-sensitive phenotypes upon close examination. This implies that many of the functions required for development are general metabolic reactions under increased demand during differentiation and embryogenesis.     

Longevity determined by developmental arrest genes in Caenorhabditis elegans

The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.     

Two pleiotropic classes of daf-2 mutation affect larval arrest,adult behavior,reproduction and longevity in Caenorhabditis elegans.

The nematode Caenorhabditis elegans responds to overcrowding and scarcity of food by arresting development as a dauer larva, a nonfeeding, long-lived, stress-resistant, alternative third-larval stage. Previous work has shown that mutations in the genes daf-2 (encoding a member of the insulin receptor family) and age-1 (encoding a PI 3-kinase) result in constitutive formation of dauer larvae (Daf-c), increased adult longevity (Age), and increased intrinsic thermotolerance (Itt). Some daf-2 mutants have additional developmental, behavioral, and reproductive defects. We have characterized in detail 15 temperature-sensitive and 1 nonconditional daf-2 allele to investigate the extent of daf-2 mutant defects and to examine whether specific mutant traits correlate with each other. The greatest longevity seen in daf-2 mutant adults was approximately three times that of wild type. The temperature-sensitive daf-2 mutants fell into two overlapping classes, including eight class 1 mutants, which are Daf-c, Age, and Itt, and exhibit low levels of L1 arrest at 25.5 degrees. Seven class 2 mutants also exhibit the class 1 defects as well as some or all of the following: reduced adult motility, abnormal adult body and gonad morphology, high levels of embryonic and L1 arrest, production of progeny late in life, and reduced brood size. The strengths of the Daf-c, Age, and Itt phenotypes largely correlated with each other but not with the strength of class 2-specific defects. This suggests that the DAF-2 receptor is bifunctional. Examination of the null phenotype revealed a maternally rescued egg, L1 lethal component, and a nonconditional Daf-c component. With respect to the Daf-c phenotype, the dauer-defective (Daf-d) mutation daf-12(m20) was epistatic to daf-2 class 1 alleles but not the severe class 2 alleles tested. All daf-2 mutant defects were suppressed by the daf-d mutation daf-16(m26). Our findings suggest a new model for daf-2, age-1, daf-12, and daf-16 interactions.