article: Evolutionarily conserved mechanisms that control aging are predicted to have prereproductive functions in order to be subject to natural selection. Genes that are essential for growth and development are highly conserved in evolution, but their role in longevity has not previously been assessed. We screened 2,700 genes essential for Caenorhabditis elegans development and identified 64 genes that extend lifespan when inactivated postdevelopmentally. These candidate lifespan regulators are highly conserved from yeast to humans. Classification of the candidate lifespan regulators into functional groups identified the expected insulin and metabolic pathways but also revealed enrichment for translation, RNA, and chromatin factors. Many of these essential gene inactivations extend lifespan as much as the strongest known regulators of aging. Early gene inactivations of these essential genes caused growth arrest at larval stages, and some of these arrested animals live much longer than wild-type adults. daf-16 is required for the enhanced survival of arrested larvae, suggesting that the increased longevity is a physiological response to the essential gene inactivation. These results suggest that insulin-signaling pathways play a role in regulation of aging at any stage in life.
  The lifespan of an organism is regulated by both genetic and environmental influences in many species [1]. Recent work has identified specific components from a variety of cellular processes that regulate lifespan. In C. elegans loss-of-function mutations in the insulin/insulin-like growth factor-1/daf-2 signaling pathway can more than double the lifespan of an animal [2–6]. The regulation of lifespan by DAF-2 occurs during adulthood [7]. The insulin-signaling pathway negatively regulates the forkhead (FOXO) transcription factor DAF-16, which ultimately functions to both positively and negatively regulate transcription of metabolic, chaperone, cellular defense, and other genes [8–11]. The regulation of lifespan through an insulin-like signaling cascade is an evolutionarily conserved mechanism and has been demonstrated in flies and mice [12–15]. The regulation of DAF-16 activity is also modulated by the JNK signaling pathway, the SIR-2. 1 deacetylase, and HSF-1, LIN-14, and SMK-1 in the nucleus [16–20]. In many organisms the rate of aging is tied to reproduction. In C. elegans germline proliferation produces a DAF-16 and KRI-1 mediated signal that negatively regulates lifespan while the somatic gonad promotes lifespan extension [21–23]. Caloric restriction (CR) also extends lifespan across species including yeast, worms, flies, and mice [24–29]. The sir-2. 1 and let-363 genes in C. elegans regulate lifespan via CR [30,31]. Unlike insulin signaling, in flies, imposing CR at anytime can increase lifespan [32]. Finally, perturbations in mitochondrial function have been shown to increase lifespan [33–36]. Diminished function of the mitochondrial electron transport system can further extend mutations in the insulin-like signaling pathway, however mutations in the ubiquinone biosynthesis gene clk-1 do not further increase the longevity phenotype from CR. The mechanism of longevity induced by defective mitochondria is thought to occur during development, as previous attempts to use RNA interference (RNAi) to inhibit mitochondrial function in adults has not been shown to increase lifespan [34]. Recent genome-wide RNAi screens for increased longevity have identified ∼100 potential regulators of lifespan in C. elegans from diverse cellular pathways, many of which are evolutionarily conserved [37,38], but genes essential for viability are underrepresented in genome-wide RNAi screens for postdevelopmental phenotypes such as aging. These RNAi screens for adult longevity preclude the identification of gene inactivations that cause lethality, larval arrest, sterility, and/or other developmental pleiotropies (i. e. , essential genes), unless these genes are inactivated after their required developmental roles. Here we report 64 genes that, when inactivated postdevelopmentally by RNAi, increase adult lifespan. We also report the enhanced survival phenotype of the animals arrested during development by these essential gene inactivations.
  To identify essential genes that function in adulthood to regulate lifespan, we selected 2,700 RNAi clones that, if fed from the previous generation or from the L1 larval stage, cause arrest at embryonic or larval stages, and screened them for increased lifespan after initiating RNAi at the L4 larval/young adult stage (Figure 1). We found three observations that validate this approach: First, C. elegans lifespan can be extended when fed dsRNA targeting the insulin receptor/daf-2 at any developmental larval stage through adulthood [7]; second, conditional daf-2 alleles cause dauer arrest if the gene is inactivated at the L1 stage but increased longevity if inactivated in adults [39]; and third, null alleles of daf-2 are lethal [40] demonstrating the necessity of uncoupling the developmental and aging phenotypes of essential genes as well as the utility of producing non-null phenotypes by RNAi. We performed the RNAi screen utilizing the eri-1 (mg366) [41] strain to improve RNAi in all cells types including neurons, which are normally refractory to RNA interference. In fact, many of the longevity genes we identify are expressed in the nervous system, which has been implicated in insulin regulation of longevity (Figure S1; Table S1) [42]. Gene inactivations that caused increases in mean lifespan of at least 10% were scored as positive in our screen. Endocrine signaling, stress adaptation, metabolism, and reproduction are potent and evolutionarily conserved regulators of aging [43]. Our screen identified genes from these canonical longevity-promoting pathways in addition to pathways not previously implicated in aging (Figures S2 and S3; Table 1). Essential genes are more conserved in phylogeny than genes with no obvious developmental phenotype; more than 90% of the genes we identify are conserved from yeast to humans. The theory of antagonistic pleiotropy suggests that any genes that function in postreproductive longevity control should be under natural selection at prereproductive stages. Our data support this notion: our yield of major lifespan regulators is 64 gene inactivations out of 2,700 tested (∼2. 4%), a 4-fold increased yield compared to the previous 89 gene inactivations out of 16,000 screened (∼0. 6%), and a higher proportion of the gene inactivations cause large increases in longevity (percent increase compared to vector control denoted in parentheses for each RNAi clone), although the use of an enhanced RNAi strain may also contribute to the increased sensitivity. One established method of validating genes that emerge from RNAi screens is to test gene knockouts for the same phenotype. For these essential genes, it may prove possible to study the longevity of the arrested animals. However, to study adult longevity conditional alleles will be required. Such alleles tend to emerge from detailed genetic analysis rather than genomic knockout projects and are not currently available. The aging research community has characterized several central mechanisms that mediate lifespan regulation including insulin/insulin-like growth factor signaling, CR, and mitochondrial function. To classify the pathways represented by these new genes, we performed secondary assays: DAF-16 localization, sod-3 expression, arrested larval survival, suppression of polyglutamine aggregation, and aberrant fat metabolism and clustered the genes by the phenotypes observed (Figure 2; Figure S4; Table 2; Tables S1 and S2). In this study, we have screened a large portion of the genome previously underrepresented in genome-wide based RNAi screens for aging phenotypes and identified 64 genes that normally function to shorten lifespan. Characterization of these gene knockdowns by epistasis with the insulin-signaling pathway and use of biomarkers of aging places them into distinct classes. Because these molecules are predicted to function in complex cellular pathways (Figure S5), future work will focus to dissect the mechanisms employed by these essential processes to regulate lifespan.
  Strains were maintained and cultured using standard techniques [65]. We used the following C. elegans strains and mutant alleles: wild-type N2 Bristol, eri-1 (mg366) IV, daf-16 (mgDf47) I; eri-1 (mg366) IV; zIs356, (daf-16p: : daf-16: : gfp; rol-6[su1006]); muIs84, (sod-3p: : gfp); huIs33, (sod-3p: : gfp; rol-6[su1006]); and rmIs133: (unc-54p: : Q40yfp). Eggs were isolated from gravid eri-1 (mg366) worms and synchronized by hatching overnight in the absence of food. The synchronized L1 larvae were then placed on OP50-containing agar plates and allowed to develop to L4-stage larvae at 20 °C. The L4-stage larvae were washed thoroughly, cleaned by sucrose flotation, and placed on 12-well plates with Escherichia coli expressing double-stranded RNA (dsRNA) (described below). We carried out a large-scale RNAi screen using the enhanced RNAi strain eri-1 (mg366) (Figure S1). Briefly, each RNAi colony was grown overnight in LB with 50 μg/ml ampicillin and then seeded onto 12-well RNAi agar plates containing 5 mM isopropylthiogalactoside (IPTG). The RNAi bacteria were induced overnight at room temperature for dsRNA expression. We then added ∼30 synchronized L4-stage animals to each well, allowed worms to develop to adults, and then added 5-fluorodexoyuridine (FUdR) solution to a final concentration of 0. 1 mg/ml. Worms were kept at 20 °C, and their lifespan was monitored. Worms feeding on bacteria carrying the empty vector or targeting eri-1 were used as negative controls. At least 96 wells of the empty vector control were included. At the time when all of the control worms were dead, each well containing the different RNAi bacteria was scored for live worms. RNAi wells in which live worms were observed were scored as positives. RNAi clones that were scored as positive in the first and second passes of screening (Figure S1) were retested in duplicate during a third pass using a conventional longitudinal RNAi lifespan assays (see below). RNAi bacteria were prepared as described above. daf-2, eri-1, and daf-16 RNAi clones were included as positive and negative controls in blind fashion in the conventional lifespan assays (daf-2 RNAi construct kindly provided by M. Vidal, Harvard Medical School, Boston, Massachusetts, United States). Synchronous L4/young adult stage eri-1 (mg366) animals treated with FUdR were prepared as above and placed on 6-well plates containing the positive RNAi clones and controls in duplicate. Approximately 40–60 adult animals were scored in each well (two wells for each RNAi clone, in two biological replicates). The animals were kept at 20 °C and scored every two days by gentle prodding with a platinum wire to test for viability. To ensure the continued efficacy of RNAi knockdown, animals were fed freshly induced RNAi bacteria every five to seven days. Lifespan is defined as the first day of adulthood (adult lifespan = 0) to when they were scored as dead. Worms that died of protruding/bursting vulva, bagging, or crawling off the agar were censored from the analysis. For epistasis analysis, RNAi lifespan assays were performed as described above, except that daf-16 (mgDf47); eri-1 (mg366) worms were used. Statistical analyses were performed using SPSS software (http: //www. spss. com). The survival experience of each RNAi-treated population is compared with that of the population treated with control RNAi using the log rank test. A p-value <0. 05 was considered as significantly different from control. Synchronized L1-stage eri-1 (mg366) or eri-1 (mg366); daf-16 (mgDf47) animals were placed on RNAi clones that induce larval arrest phenotypes at 20 °C. To ensure that any animals bypassing lethality did not reproduce the plates were moved to 25 °C to exploit the temperature-sensitive sterility associated with eri-1 (mg366). The wells were qualitatively monitored every two days for arrested larvae survival. Synchronized L1-stage animals or freshly egg-prepped embryos carrying the integrated transgenes sod-3p: : gfp, daf-16p: : daf-16-gfp, and myo-3p: : Q40yfp were each placed onto RNAi bacteria as described above. The fluorescence intensity of each population was monitored two and three days following RNAi treatment. Nile Red experiments were performed as previously described [66] except that L4-stage eri-1 (mg366) animals were fed RNAi clones induced on plates containing 5 mM IPTG. Promoter elements were amplified by PCR from wild-type genomic DNA and fused to RFP [67]. The transcriptional reporters were then injected into the gonads of wild-type adult hermaphrodites. Transgenic animals harboring the extrachromosomal arrays were then imaged. The primer pairs used to amplify promoter elements are as follows: c56g2. 1: F: 5′-CAGACAGGTGAAGCTGAGCGTGGC-3′, R: 5′-AAACGCAGAAAACGTCGGTGACGGAATG-3′; egl-45: F: 5′-CCAGCCAGGAAAAATCGATTATATTAAG, R: 5′-AGTTGTGCTCGGATTACCGCTGAATTG-3′; unc-62: F: 5′-CCCTGAAATTGTTGCGAAAGTTTCTG, R: 5′-GTTCCTGCAAGAGAGAAATATTAAATTTTG-3′; htp-3: F: 5′-CTCCCGAAGATTCCGCATTTGCTC, R: 5′-TTTGACACTTAAAATATTTTAAAACATTTTTTTTAA-3′; f26a3. 4: F: 5′-GACCGGAACAGGTGGGCAATGTCGAC, R: 5′-TTTGTAGTGTATCTGTAATCATATTAAATTTGATTC-3′; inf-1: F: 5′-GTATGTGTTTATGGTGTGTGCACAAG, R: 5′-GACAGGTGGGTTGAAAAGTTAAAAATTAAC-3′; f08b4. 1: F: 5′-CTAACCGATTCCTCAAGCCACGTGGG, R: 5′-TTTTGATTATTGATATTTCATTCGAATTTGCCAG-3′; y54e10br. 4: F: 5′-CTCTCCCGATTCCGCCATAATGCCCG, R: 5′-TCTCTGAAATATCGAAAAGAAATGAGATAATTG-3′; sem-5: F: 5′-GGGTTAGAGCACTCTTAATGAGTCATG, R: 5′-CGTCTCGCTACCTGAAATATACTCTT-3′; zk686. 2: F: 5′-GGTTCCGGAGATAACCAAGCAGTATTGG, R: 5′-TATCTGGAGAAATTAAAATATGAACCAAAAAATGCG-3′.
summary: The lifespan of an animal is determined by both environmental and genetic factors, and many of the mechanisms identified to increase lifespan are evolutionarily conserved across organisms. Previous longevity screens in C. elegans have identified over 100 genes, but ∼2,700 essential for normal development were excluded from analysis. Paradoxically, these essential genes are five times more likely to be highly conserved in phylogeny than genes with no obvious developmental phenotypes. We screened these 2,700 essential genes for increased adult lifespan by initiating the gene knockdown once the animal had reached adulthood, thus bypassing earlier developmental roles. We identified 64 genes that can extend lifespan when inactivated postdevelopmentally. More than 90% of the genes we identified are conserved from yeast to humans. Many of the newly identified longevity genes extend lifespan as robustly as the most well-characterized longevity mutants. It is possible that the homologues of these genes may also regulate lifespan in other organisms as well. Genetic analysis places some of these genes in known pathways regulated by insulin-like signaling, although many of these gene inactivations function independently of this mechanism of lifespan extension. Surprisingly, a subset of these gene inactivations that induce potent developmental arrest also facilitate enhanced survival in the arrested state, suggesting that aging at any stage may be subject to regulatory control.
keywords: animals
  developmental biology
  cell biology
  caenorhabditis
  eukaryotes
  evolutionary biology
  molecular biology
  genetics and genomics
  nematodes
title: Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability