The miR-58 microRNA family is regulated by insulin signaling and contributes to lifespan regulation in Caenorhabditis elegans

microRNAs regulate diverse biological processes such as development and aging by promoting degradation or inhibiting translation of their target mRNAs. In this study, we have found that the miR-58 family microRNAs regulate lifespan in C.elegans. Intriguingly, members of the miR-58 family affect lifespan differently, sometimes in opposite directions, and have complex genetic interactions. The abundances of the miR-58 family miRNAs are up-regulated in the long-lived daf-2 mutant in a daf-16-dependent manner, indicating that these miRNAs are effectors of insulin signaling in C. elegans. We also found that miR-58 is regulated by insulin signaling and partially required for the lifespan extension mediated by reduced insulin signaling,germline ablation, dietary restriction, and mild mitochondrial dysfunction. We further identified the daf-21, ins-1, and isw-1 mRNAs as endogenous targets of miR-58. Our study shows that miRNAs function in multiple lifespan extension mechanisms,and that the seed sequence is not the dominant factor defining the role of a miRNA in lifespan regulation.     

The anticonvulsant ethosuximide disrupts sensory function to extend C. elegans lifespan

Ethosuximide is a medication used to treat seizure disorders in humans, and we previously demonstrated that ethosuximide can delay age-related changes and extend the lifespan of the nematode Caenorhabditis elegans. The mechanism of action of ethosuximide in lifespan extension is unknown, and elucidating how ethosuximide functions is important for defining endogenous processes that influence lifespan and for exploring the potential of ethosuximide as a therapeutic for age-related diseases. To identify genes that mediate the activity of ethosuximide, we conducted a genetic screen and identified mutations in two genes, che-3 and osm-3, that cause resistance to ethosuximide-mediated toxicity. Mutations in che-3 and osm-3 cause defects in overlapping sets of chemosensory neurons, resulting in defective chemosensation and an extended lifespan. These findings suggest that ethosuximide extends lifespan by inhibiting the function of specific chemosensory neurons. This model is supported by the observation that ethosuximide-treated animals displayed numerous phenotypic similarities with mutants that have chemosensory defects, indicating that ethosuximide inhibits chemosensory function. Furthermore, ethosuximide extends lifespan by inhibiting chemosensation, since the long-lived osm-3 mutants were resistant to the lifespan extension caused by ethosuximide. These studies demonstrate a novel mechanism of action for a lifespan-extending drug and indicate that sensory perception has a critical role in controlling lifespan. Sensory perception also influences the lifespan of Drosophila, suggesting that sensory perception has an evolutionarily conserved role in lifespan control. These studies highlight the potential of ethosuximide and related drugs that modulate sensory perception to extend lifespan in diverse animals.     

Dietary deprivation extends lifespan in Caenorhabditis elegans

Dietary restriction (DR) is well known as a nongenetic intervention that robustly extends lifespan in a variety of species; however, its underlying mechanisms remain unclear. We have found in Caenorhabditis elegans that dietary deprivation (DD) during adulthood, defined as removal of their food source Escherichia coli after the completion of larval development, increased lifespan and enhanced thermotolerance and resistance to oxidative stress. DD-induced longevity was independent of one C. elegans SIRTUIN, sir-2.1, which is required for the effects of DR, and was independent of the daf-2/insulin-like signaling pathway that independently regulates longevity and larval diapause in C. elegans. DD did not significantly alter lifespan of fem-1(hc17); eat-2(ad465) worms, a genetic model of DR. These findings suggest that DD and DR share some downstream effectors. In addition, DD was detrimental for longevity when imposed on reproductively active young adults, suggesting that DD may only be beneficial in the absence of competing metabolic demands, such as fertility. Adult-onset DD offers a new paradigm for investigating dietary regulation of longevity in C. elegans. This study presents the first evidence that long-term DD, instead of being detrimental, can extend lifespan of a multicellular adult organism.     

5'-AMP-activated protein kinase signaling in Caenorhabditis elegans

5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.     

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C .  elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C .  elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C .  elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C .  elegans lifespan.     

An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.

Mutations in daf-2 and age-1 cause a dramatic increase in longevity as well as developmental arrest at the dauer diapause stage in Caenorhabditis elegans. daf-2 and age-1 encode components of an insulin-like signaling pathway. Both daf-2 and age-1 act at a similar point in the genetic epistasis pathway for dauer arrest and longevity and regulate the activity of the daf-16 gene. Mutations in daf-16 cause a dauer-defective phenotype and are epistatic to the diapause arrest and life span extension phenotypes of daf-2 and age-1 mutants. Here we show that mutations in this pathway also affect fertility and embryonic development. Weak daf-2 alleles, and maternally rescued age-1 alleles that cause life span extension but do not arrest at the dauer stage, also reduce fertility and viability. We find that age-1(hx546) has reduced both maternal and zygotic age-1 activity. daf-16 mutations suppress all of the daf-2 and age-1 phenotypes, including dauer arrest, life span extension, reduced fertility, and viability defects. These data show that insulin signaling, mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates reproduction and embryonic development, as well as dauer diapause and life span, and that DAF-16 transduces these signals. The regulation of fertility, life span, and metabolism by an insulin-like signaling pathway is similar to the endocrine regulation of metabolism and fertility by mammalian insulin signaling.     

Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blueberry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.     

Variable pathogenicity determines individual lifespan in Caenorhabditis elegans

A common property of aging in all animals is that chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for Caenorhabditis elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with the superoxide dismutase gene sod-3 expression being the best single predictor of remaining lifespan. Correlation with remaining lifespan became stronger if expression from two aging markers was monitored simultaneously, accounting for up to 49% of the variation in individual lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability is different pathogenicity from individual to individual and that the mechanism involves variable activation of the insulin-signaling pathway.     

Fitness cost of extended lifespan in Caenorhabditis elegans

An insulin/IGF-I-like signalling pathway determines the rate of aging of the adult nematode, Caenorhabditis elegans. Mutations in genes encoding this pathway can result in a doubling of lifespan. While such mutations may appear to have little effect on development or fertility, evolutionary theory predicts that large increases in lifespan will not be optimal for fitness. We demonstrate by laboratory natural selection that partial loss of function of the insulin receptor-like protein DAF-2 results in dramatically reduced fitness even under laboratory conditions. Despite long-lived mutants appearing healthy, they exhibit a heavy fitness cost consistent with an evolutionary theory of aging.     

Distribution of an L-isoaspartyl protein methyltransferase in eubacteria.

A protein carboxyl methyltransferase (EC 2.1.1.77) that recognizes age-damaged proteins for potential repair or degradation reactions has been found in all vertebrate tissues and cells examined to date. This enzyme catalyzes the transfer of methyl groups from S-adenosylmethionine to the carboxyl groups of D-aspartyl or L-isoaspartyl residues that are formed spontaneously from normal L-aspartyl and L-asparaginyl residues. A similar methyltransferase has been found in two bacterial species, Escherichia coli and Salmonella typhimurium, suggesting that this enzyme performs an essential function in all cells. In this study, we show that this enzyme is present in cytosolic extracts of six additional members of the alpha and gamma subdivisions of the purple bacteria: Pseudomonas aeruginosa (gamma), Rhodobacter sphaeroides (alpha), and the gamma enteric species Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens. DNA probes from the E. coli methyltransferase gene hybridized only to the chromosomal DNA of the enteric species. Interestingly, no activity was found in the plant pathogen Erwinia chrysanthemi, a member of the enteric family, nor in Rhizobium meliloti or Rhodopseudomonas palustris, two members of the alpha subdivision. Additionally, we could not detect activity in the four gram-positive species Bacillus subtilis, B. stearothermophilus, Lactobacillus casei, and Streptomyces griseus. The absence of enzyme activity was not due to the presence of inhibitors in the extracts. These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology.     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

A pharmacological network for lifespan extension in Caenorhabditis elegans

One goal of aging research is to find drugs that delay the onset of age‐associated disease. Studies in invertebrates, particularly Caenorhabditis elegans, have uncovered numerous genes involved in aging, many conserved in mammals. However, which of these encode proteins suitable for drug targeting is unknown. To investigate this question, we screened a library of compounds with known mammalian pharmacology for compounds that increase C. elegans lifespan. We identified 60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. Enhanced resistance to oxidative stress was associated primarily with compounds that target receptors for biogenic amines, such as dopamine or serotonin. A pharmacological network constructed with these data reveal that lifespan extension and increased stress resistance cluster together in a few pharmacological classes, most involved in intercellular signaling. These studies identify compounds that can now be explored for beneficial effects on aging in mammals, as well as tools that can be used to further investigate the mechanisms underlying aging in C. elegans.     

Caenorhabditis elegans integrates food and reproductive signals in lifespan determination

Dietary restriction extends lifespan and inhibits reproduction in many species. In Caenorhabditis elegans, inhibiting reproduction by germline removal extends lifespan. Therefore, we asked whether the effect of dietary restriction on lifespan might proceed via changes in the activity of the germline. We found that dietary restriction could increase the lifespan of animals lacking the entire reproductive system. Thus, dietary restriction can extend lifespan independently of any reproductive input. However, dietary restriction produced little or no increase in the long lifespan of animals that lack germ cells. Thus, germline removal and dietary restriction may potentially activate lifespan-extending pathways that ultimately converge on the same downstream longevity mechanisms. In well-fed animals, the somatic reproductive tissues are generally completely required for germline removal to extend lifespan. We found that this was not the case in animals subjected to dietary restriction. In addition, in these animals, loss of the germline could either further lengthen lifespan or shorten lifespan, depending on the genetic background. Thus, nutrient levels play an important role in determining how the reproductive system influences longevity.     

Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans

Background

Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan.

Results

Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension.

Conclusions

Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0167-2) contains supplementary material, which is available to authorized users.     

Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap-binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg-1, the worm homologue of eIF4G, which is a scaffold protein in the cap-binding complex; and rsks-1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg-1 or rsks-1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg-1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.