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Keywords:

  • aging;
  • Caenorhabditis elegans;
  • dauer;
  • genetics;
  • lifespan;
  • stress resistance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The great majority of lifespan-augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin-like signaling extends longevity 1.5- to 3-fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age-1 gene, which encodes the class-I phosphatidylinositol 3-kinase catalytic subunit (PI3KCS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long-lived adults with near-normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10-fold extension of both median and maximum adult lifespan relative to N2DRM, a long-lived wild-type stock into which the null mutant was outcrossed. PI3K-null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long-lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3KCS or its products, and consequent modification of kinase signaling cascades.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Reduction-of-function mutations, usually identified in mutagenesis screens, have been found to extend the lifespan of invertebrates, with reported effects ranging from less than 1.1-fold to 3-fold. The first-characterized longevity mutation was discovered by Michael Klass in a sib-screen for long-lived progeny arising from chemical mutagenesis (Klass, 1983). All long-lived isolates in this screen were shown to derive from a single mutant allele, age-1(hx546), increasing survival by 40% over controls at 20 °C, and by 65% at 25 °C (Friedman & Johnson, 1988b). Another longevity gene, daf-2, was initially identified in 1980 through temperature-sensitive mutants that, at ≥ 25 °C, undergo constitutive dauer larva formation (developmental arrest or diapause) (Riddle et al., 1981). Over a decade passed before survivals were conducted on daf-2 mutant adults that had matured at 20 °C, a permissive temperature allowing worms to bypass larval arrest; only then were daf-2 mutations discovered to confer dramatic increases in adult lifespan (Kenyon et al., 1993). A survey of 15 daf-2 alleles showed 1.1- to 2.5-fold life extension at either 15 °C or 22.5 °C (Gems et al., 1998). Specific allelic combinations of mutated daf-2 and daf-12 (encoding a nuclear hormone receptor) can increase longevity at 25.5 °C as much as 4.4-fold (Larsen et al., 1995; Gems et al., 1998), or 5-fold with the addition of germ-line ablation (Rottiers & Antebi, 2006). Daf-2 encodes a cell membrane receptor for insulin-like ligands, while age-1 encodes a catalytic (p110α) subunit of phosphatidylinositol-3-kinase (PI3K); these are two of the first components in the insulin/insulin-like growth factor 1 (IGF-1) response pathway of nematodes, regulating dauer formation, fecundity, stress resistance and longevity. Addition of a second mutation inactivating the daf-16 gene, which encodes a FOXO ‘forkhead’ transcription factor phosphorylated via the DAF-2/AGE-1/PDK-1/AKT kinase cascade, abrogates all reported phenotypes of both age-1 and daf-2 mutations (Larsen et al., 1995; Tissenbaum & Ruvkun, 1998; Guarente & Kenyon, 2000).

To date, the greatest extensions of longevity by single mutations have been in the vicinity of 3-fold for Caenorhabditis elegans hermaphrodites (Larsen et al., 1995; Lin et al., 1997; Gems et al., 1998; Tissenbaum & Ruvkun, 1998) although somewhat higher in males (Partridge & Gems, 2002), nearly 2-fold in Drosophila (Rogina et al., 2000; Tatar et al., 2001), but only 1.3- to 1.5-fold in mice (Bartke et al., 2001; Clancy et al., 2001; Tatar et al., 2001; Bluher et al., 2003; Holzenberger et al., 2003; Tirosh et al., 2004; Kurosu et al., 2005). It is clear, however, that these mutants do not define the upper limit for life extension, as combinations of two or three interventions, for example, augmenting such mutations with germ-cell ablation or dietary restriction, can result in a total life extension of 4- to 6-fold in the nematode (Arantes-Oliveira et al., 2003; Houthoofd et al., 2003). Similarly, the greatest life extension obtained thus far in mice, 1.7-fold, required the combined effects of a life-extending mutation and caloric restriction (Bartke et al., 2001). Many long-lived mutations also confer resistance to a variety of stresses (Lithgow et al., 1995; Lin et al., 1998; Murakami, 2006), whereas naturally occurring genetic variants known as longevity quantitative trait loci display selective resistance to some stresses and not others (Shmookler Reis et al., 2007).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Greatly increased longevity of second-generation age-1-null worms with intact DAF-16

We obtained three alleles of age-1 from the Caenorhabditis Genetics Center (CGC), as strains TJ1052 [age-1(hx546)], GR1168 [age-1(mg44)/mnC1], and DR722 [age-1(m333)/mnC1]. Each allele was outcrossed six generations into N2DRM (CGC ‘N2 Male Strain’), a direct continuation of the Bristol-N2 DRM stock from the Riddle laboratory, to eliminate allelic differences at other loci (background effects). N2DRM is the wild-type stock reported to be the longest lived of six Bristol-N2 stocks tested (Gems & Riddle, 2000). Age-1(hx546) is a reduction-of-function mutation (Friedman & Johnson, 1988a), for which the sequence alteration had not previously been identified. We found age-1(hx546) to differ from the N2DRM allele by a C[RIGHTWARDS ARROW]T transition at position 2416, converting a proline to a serine in the AGE-1 peptide sequence (788 KLRDELRSISHKMENMDSPLDPVYKLGEM 816) just C-terminal to the α-helical domain (Fig. 1). The other two alleles are nonsense mutations in which stop codons truncate the PI3K catalytic subunit (PI3KCS) protein upstream of its kinase domain (Morris et al., 1996) (Fig. 1). By determining the DNA sequence of age-1 exons, we confirmed the TGA/amber stop codon replacing TGG (Trp) at amino acid 387 in age-1(mg44) worms (strain SR808 after outcrossing into N2DRM), corresponding to the mutation described previously at position 405. Similarly, the age-1(m333) mutation (strain SR809 after N2DRM outcrossing) was confirmed as a Trp to TAG/opal change at position 641, corresponding to residue 659 in the sequence reported previously (Morris et al., 1996).

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Figure 1. Wild-type and mutant alleles of age-1, encoding the catalytic subunit of a class Ia phosphatidylinositol 3-kinase. The nonsense mutations responsible for the mg44 and m333 alleles were reported previously and confirmed here; the missense mutation underlying age-1(hx546) is reported here for the first time.

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The two kinase-null daf-c mutant strains, producing truncated AGE-1 proteins (data not shown), are maintained as heterozygotes with the mnC1 balancer chromosome. Of the progeny arising from self-fertilization of these heterozygotes, one-fourth are homozygous for the age-1 mutation. The essentially normal development of such age-1/age-1 homozyotes is attributed to maternal protection due to the oocyte complement of wild-type age-1 mRNA or AGE-1 protein encoded by the normal age-1 allele on mnC1 (Gottlieb & Ruvkun, 1994; Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998). Their progeny, devoid of maternally contributed AGE-1 or its PIP3 product, constitutively form dauer larvae at 25.5 °C as reported previously (Gottlieb & Ruvkun, 1994; Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998). At 15 or 20 °C, however, 95% of mg44/mg44 progeny of mg44/mg44 hermaphrodites matured to adults within 8–10 days (Fig. 2; Table 1). Development is even more protracted for progeny of maternally protected m333 homozygotes, with only 2–3% forming nearly full-sized adults within 16–19 days, and another 5–10% becoming very small adults or ‘runts’ (Table 1).

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Figure 2. Caenorhabditis elegans larvae of age-1(mg44) genotype, born of homozygous age-1(mg44) hermaphrodites, mature at 20 °C into adults of near-normal appearance and extraordinary longevity. (A) Larval arrest of worms maintained at 25.5 °C for 6 days after hatching; their morphology does not change over periods of at least 14 days at this temperature. (B) Adults matured in 8 days at 20 °C, here imaged 9 days after hatching.

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Table 1.  Developmental and adult survival times for wild-type and age-1 mutant Caenorhabditis elegans
Strain (outcross generations)Development time, 15 ± 0.1 °C (% adult formation, n)Development time, 20 ± 0.1 °C (% adult formation, n)Development time, 25.5 ± 0.1 °C (% adult formation, n)Mean survival time ± SEM (adult days) at 20 ± 1 °CMean of survivals (± SEM), for two to three biological replicates normalized to N2DRM, at the 25th, 50th, and 75th percentiles*
# 1# 2
  • Development times (days after eclosion) and adult survival times (days after L4/adult moult) are given. N2DRM and age-1(hx546) worms were propagated as homozygous lines. Other age-1 homozygotes arose by self-fertilization of age-1(m333)/mnC1 or age-1(mg44)/mnC1 heterozygotes, and their progeny (‘second-generation’age-1 homozygotes) were tested as indicated. Developmental time is taken as the age at 90% of maximal adult formation; percent adult formation (in parentheses, followed by the number of worms counted) was scored 1–2 days later as the number of full-size adults.

  • ND, not determined.

  • *

    Means ± SEM are shown for quartile survivals of two or three independent biological replicates, each divided by the mean N2DRM quartile value; the SEM of a ratio A/B is derived as (inline image+inline image)0.5.

  • Different from N2DRM, by one-tailed Behrens–Fisher t-test (for unequal or unknown variances), n = 2, 3, P < 0.025.

  • P < 0.015.

  • §

    P < 0.005.

  • Different from the longest-lived N2DRM group by one-tailed Fisher t-test, P < 10−6.

  • ††

    P < 10−15. Each P-value is calculated for a single comparison between the groups indicated, without correction for multiple comparisons.

N2DRM (control)3.5 days (100%, 200)2.5 days (99%, 200)2.0 days (98%, 200)15.1 ± 0.617.2 ± 0.71.00 ± 0.05; 1.00 ± 0.04; 1.00 ± 0.06
age-1(hx546) × 64.5 days (99.5%, 203)     3 days (98.5%, 198)2.5 days (94%, 207)29.2 ± 2.030.3 ± 1.62.1 ± 0.1; 1.9 ± 0.1; 2.1 ± 0.01§
age-1(m333) × 619 days (3%, 207)     16 days (2%, 207)Indefinite (0%, 189)NDNDND
age-1(mg44) × 610 days (95%, 207)      8 days (95 ± 2%, 621)Indefinite (0%, 540)136 ± 16††170 ± 12††8.6 ± 0.3; 10.3 ± 0.7; 11.0 ± 0.5
age-1(mg44) × 010 days (ND)  8 days (ND)Indefinite (ND)130 ± 18††; 184 ± 7††;163 ± 10††10.6 ± 1.9; 10.1 ± 1.0§; 10.2 ± 0.8§

Second-generation homozygous age-1(mg44) adults appear normal apart from a slight protrusion of the vulva (Fig. 2B, arrows), but are totally infertile, as are second-generation m333 adults (Fig. 3A). Their pharyngeal pumping rates (Fig. 3B) are indistinguishable from wild-type N2DRM or age-1(hx546) adults, and similar to those reported by others (Tanaka et al., 2007) for Bristol-N2 worms on plates seeded with Escherichia coli OP50. In contrast, age-1(m333) adults are smaller than normal and their pharyngeal pumping is a little slower (83% of the N2DRM rate, P < 0.01). Spontaneous movement in the absence of bacterial food is slightly reduced for second-generation age-1(mg44) day-10 adults (Fig. 3C), and declines by 82 days of adulthood to 69% of the movement seen in day-4 N2DRM (P < 10−9), similar to N2DRM worms on adult day 11 (data not shown). For both strong and weak age-1 alleles, tropism to distal bacteria is more severely impaired (reduced to 43–54% of young N2DRM levels), largely reversed by daf-16 mutation (Fig. 3C, striped bars). Like wild-type adults, but unlike dauer larvae, age-1(mg44) adults are fully susceptible to disruption by 1% sodium dodecyl sulfate (10/10 killed; data not shown).

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Figure 3. Reproductive and activity phenotypes of age-1-mutant and wild-type Caenorhabditis elegans adults. (A) Fertility of first- and second-generation age-1 homozygotes (F1 and F2, respectively) for null alleles m333 and mg44, compared to isogenic wild-type controls. Worms were placed on OP50-seeded 60-mm agar plates and viable progeny were counted when most had reached adulthood (3 days for N2DRM, 18 days for age-1(m333), and 9 days for age-1(mg44)). For age-1, F2 worms, n > 1000 (≥ 50 worms per plate); for others, n = 5 (one worm per plate). (B) Pharyngeal pumping rates, in pumps per min, of extreme-longevity worms (second-generation age-1 adults, m333 and mg44 alleles), compared to wild-type and age-1(hx546) adults. Adult worms were picked from uncrowded, unstarved plates onto seeded 60-mm plates (one worm per plate), and monitored 2 h later; for each group, n = 12. (C) Motility index, defined as the fraction of worms (n = 69–160) more than 2 or 4 cm from their starting position, at the times indicated. Age-1(mg44) and (m333) worms are second-generation homozygotes without maternal protection (F2). Statistics: error bars are SEMs. Numbers above bars indicate significance (P-values) of differences from N2DRM, by one-tailed t-tests (A, B), or χ2-tests (C), each corrected for multiple assessments.

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Remarkably, however, F2-homozygous adult worms bearing either m333 or mg44 nonsense mutations (and born to age-1/age-1 hermaphrodites, hence lacking any maternal protection) are much longer lived than any mutant worms previously reported – averaging more than 10 times the median or 75th percentile adult lifespan of the congenic N2DRM controls, and 9–10 times the mean adult survival of controls (Fig. 4A and Table 1). This was not due to background gene effects, that is, stock-specific genetic interactions with other loci, as the original mutant stocks survived just as long as those outcrossed six generations into the N2DRM background (Fig. 4A and Table 1). Most F2-homozygous, age-1-null worms that survived past the population median lifespan (145–189 adult days) remained remarkably active, even at more than eight times the N2DRM median lifespan (see Supplementary material, videos). Both developmental and adult lifespan effects of the age-1(mg44) nonsense mutation (Table 1) were largely or entirely reversed by a second mutation, daf-16(m26), inactivating the DAF-16 (FOXO family, or forkhead) transcription factor downstream of the AGE-1 PI 3-kinase (Fig. 4B).

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Figure 4. Second-generation age-1(mg44) homozygous-null mutant adults are much longer lived than wild-type or age-1(hx546) worms, dependent on functional DAF-16/FOXO, but not on genetic background. (A) Survivals, on agar medium, are shown for N2DRM control worms (results were combined for two independent cultures, each comprising two plates of 25 worms), GR1168 (second-generation age-1(mg44) homozygotes, combining data from three independent cultures of 50 worms each), and SR808 [derived from GR1168 by six generations of outcrossing to N2DRM; second-generation age-1(mg44) homozygotes, combining two independent cultures of 50 worms each]. Each age-1(mg44) group (final n = 42–50 uncensored deaths) differed from either N2DRM control group, by log-rank test, at P < 10−9. (B) Survivals on solid-agar medium seeded with Escherichia coli strain OP50. Age-1 alleles hx546 and mg44 were crossed with daf-16(m26), all in N2DRM background; double-mutant homozygous progeny were selected and propagated. Each symbol represents 100 worms (four plates, 25 each). Adult age (following the L4/adult molt) is displayed on the axis, and excludes the duration of larval development at 20 °C: 8 days for age-1(mg44), 3 days for age-1(hx546), and 2.5 days for all other strains, including double mutants with daf-16.

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Exceptional stress resistance of age-1 null alleles depends largely on DAF-16/FOXO

We asked whether these extreme-longevity strains were also unusually resistant to stresses administered to day-2 adults. We first assessed survival of hyperthermia, upon shifting the temperature from 20 to 35.5 °C. The longest lived age-1 alleles were slightly less tolerant of thermal stress than worms bearing the weaker age-1(hx546) allele (Fig. 5A). This was rather surprising in view of the strong correlation (r ≈ 0.9) observed previously between thermotolerance and longevity among naturally occurring genetic variants of C. elegans (Shmookler Reis et al., 2007). In contrast, resistances to two oxidative stresses and to an electrophilic stress associated with lipid peroxidation were strikingly correlated with longevity of age-1 alleles. Short-term survivals in the presence of hydrogen peroxide are shown in Fig. 5B. The two very long lived age-1 alleles confer roughly 9-fold longer survival in 3-mm H2O2 (i.e. mortality at 30 h for those alleles was similar to that of wild-type at 3.3 h). Survival in 150-mm paraquat, which generates superoxide, is also extended by 3.3- to 5.2-fold (Fig. 5C). Electrophilic toxicity of 4-hydroxynonenal (4-HNE, at 10 mm), a lipoperoxidation end-product, is postponed 4.4-fold (Fig. 5D). In each case, stress resistance was markedly greater for age-1(mg44) and age-1(m333) worms than those bearing the weaker age-1 allele, hx546. Almost identical results to those shown in Fig. 5 were obtained in one or more independent repeats (not shown). This reproducibility extends in particular to the nearly identical survival curves of age-1(mg44) and (m333) worms exposed to hydrogen peroxide, paraquat or 4-HNE (Fig. 5B–D), despite consistent differences in other phenotypes (e.g. Table 1 and Fig. 3B) and in thermotolerance (Fig. 5A). We infer that these survival traits reflect primarily the absence of functional AGE-1.

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Figure 5. Second-generation homozygous age-1(mg44) adults are less thermotolerant, but more resistant to oxidative and electrophilic stresses, than age-1(hx546) homozygotes. (A) Thermotolerance assay. Worms, on agar plates, were shifted from 20° to 35.5 °C and maintained at the higher temperature for the duration of the assay. (B) Stress survivals in 3-mm hydrogen peroxide (H2O2). (C) Survivals in 150-mm paraquat. (D) Survivals in 10-mm 4-hydroxynonenal (4-HNE). Survival cultures were set up at adult day 2; with the noted exception of (A), all were maintained at 20 ± 0.3 °C in liquid culture medium without bacteria. Results of two or three independent experiments were very similar. Runts were excluded from all age-1(m333) assays. Strains: all age-1 lines indicated had been outcrossed six generations into N2DRM; age-1(mg44) worms were F2-homozygous adults, the second-generation progeny of heterozygous carriers. Double mutants were constructed as described in Fig. 4. Statistics (by log-rank test, each n = 40–50): (A) age-1(mg44) or (m333) vs. either N2DRM or age-1(hx546): P < 10−7; (B) age-1(hx546) vs. N2DRM or daf-16;age-1(hx546): P < 0.004; daf-16;age-1(hx546) vs. N2DRM: P < 10−3; age-1(mg44) or (m333) vs. N2DRM or daf-16;age-1(hx546): P < 10−4; (C) age-1(hx546) vs. N2DRM or daf-16;age-1(hx546): P < 0.005; age-1(mg44) or (m333) vs. daf-16;age-1(hx546) or N2DRM: P < 10−9.

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We also assessed the ability of a second mutation, daf-16(m26), disabling the forkhead transcription factor believed to mediate insulin-like signaling in C. elegans, to epistatically reverse phenotypes of resistance to 4-HNE and paraquat. The daf-16(m26) mutation fully reversed the stress-resistance trait of the weaker age-1 allele (hx546), but only partially reversed protection due to the stronger mg44 and m333 alleles: 80% for 4-HNE, but just 40% for paraquat (Fig. 5C,D). This contrasts with essentially full reversion, by the same daf-16(m26) mutation, of lifespan effects arising from strong or weak mutant alleles of age-1 (Fig. 4B).

Oogenesis requires PI3K

Homozygous null mutants of age-1 are fertile when maternally protected, yielding 150 ± 14 (SEM) progeny per SR808 (mg44) hermaphrodite, and 131 ± 13 progeny per SR809 (m333) parent (Fig. 3), at the low end of the range (100–340) reported (Friedman & Johnson, 1988a) for wild-type N2 worms. From > 1000 of those offspring, born of age-1-null parents, no progeny were produced (differing from their parents, each P < 10−6), and neither embryos nor unfertilized oocytes were laid. 4′,6-Diamidino-2-phenylindole-stained nuclei of day-1 adults (Fig. 6) demonstrate germ-cell syncytia in second-generation mg44-homozygous nematodes (arrows, Fig. 6C), containing fewer nuclei than their wild-type (Fig. 6A) or age-1(hx546) (Fig. 6B) counterparts. In the total absence of full-length PI3KCS, these syncytial nuclei fail to complete oogenesis, producing no mature oocytes or embryos (Fig. 6, compare C to A and B). Because spermatozoa also appear to be deficient, we tested whether any mature oocytes are formed in age-1(mg44) hermaphrodites by mating them to N2DRM males. Again, no progeny were observed, confirming that oocyte development is fully blocked in worms without endogenous or maternally provided PI3KCS, and consistent with previous reports that daf-2 signaling in germ cells is required for fertility (Apfeld & Kenyon, 1998; Arantes-Oliveira et al., 2002).

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Figure 6. DNA fluorescence images of control and age-1 mutant worms. Day 1 adult worms were permeabilized and stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged by fluorescence microscopy (350-nm excitation, 450-nm emission) to highlight nuclei. (A) N2DRM control worm; (B) age-1(hx546) homozygous worm; (C) age-1(mg44) second-generation (F2) homozygote. The approximate position of the vulva is indicated by an asterisk.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The age-1(mg44) and (m333) mutations, in homozygous form, were reported initially to extend lifespan by just 2.2- to 2.6-fold (Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998). These effects were similar in magnitude to those reported for other age-1 and daf-2 mutations, but at least fourfold less than we now observe. How did the unprecedented phenotypes of these null-mutant alleles escape detection for over a decade? Most other long-lived mutations in the insulin-like pathway are temperature sensitive, allowing their propagation at a permissive temperature as homozygous strains. Age-1(mg44) and (m333), in contrast, are unconditional null mutations that when homozygous form dauer larvae or infertile adults, and therefore need to be propagated as heterozygotes. Self-fertilization of those carrier stocks produces one-fourth homozygous-null progeny, which develop at a near-normal pace and are themselves fertile. Those first-generation homozygotes were the focus of most previous studies, and in particular of all lifespan survivals conducted on these strains. When such first-generation homozygotes are maintained at 25.5 °C, 100% of their progeny arrest development as dauer (alternative stage 3) larvae (Gottlieb & Ruvkun, 1994; Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998). At lower temperatures, it had been noticed that the progeny appear to mature very slowly into adults (Larsen et al., 1995; Morris et al., 1996), but it was not realized that the absence of maternal protection would produce a remarkably distinctive adult phenotype in these second-generation homozygotes. We confirmed the inability of those progeny to develop into adults under growth conditions most commonly employed (Gottlieb & Ruvkun, 1994; Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998), whereas at 15 or 20 °C we observed formation of 95%mg44 adults and 2–3%m333 adults (Table 1). The two age-1-null alleles, mg44 and m333, are almost identical with respect to longevity and resistance to most stresses. However, age-1(m333) adults are smaller, slower to develop and in feeding, and modestly but consistently less thermotolerant than age-1(mg44). The interpretation of their properties is also complicated by the possibility of selection for the small percentage of worms which are capable of forming adults – in contrast to age-1(mg44) worms, of which 95% form adults.

The necessity to maintain unconditional null age-1 mutants as heterozygotes may also explain why no other, comparably long-lived mutants have been observed in the same pathway. If extreme longevity arises, directly or indirectly, from elimination of the last vestiges of PI3KCS activity, which itself causes sterility and hence failure to propagate, such mutants will not easily be recovered from mutagenesis screens. They can only be carried as heterozygotes with a wild-type allele on a balancer chromosome (Edgley & Riddle, 2001).

The AGE-1 protein is the catalytic subunit of PI3K, which converts phosphatidylinositol 4,5-phosphate to phosphatidylinositol 3,4,5-phosphate, PI(3,4,5)P3. PI3KCS also has protein kinase activity, as evidenced by phosphorylation of its own regulatory subunit. However, PI(3,4,5)P3 is regarded as the principal effector through which PI3K modulates other kinases, including AKT-1, AKT-2 and PDK-1; it is a docking factor for many membrane-associated kinases (Gami et al., 2006; Heo et al., 2006), as well as an allosteric activator of AKT-1 (Gami et al., 2006; Remenyi et al., 2006). Both modes of regulation are presumed to be involved in insulin-like signaling (Gami et al., 2006).

The finding that extremely long-lived age-1 mutant worms are also exceptionally resistant to oxidative and electrophilic stresses lends support to the hypothesis that such stresses comprise key contributors to the process of aging (Ayyadevara et al., 2005a,b, 2007), and that longevity depends (at least in part) on the balance between generation of such stresses and resistance to them (Gems & McElwee, 2005; Shmookler Reis et al., 2007). The consequences of AGE-1/PI3K deficiency are complex, impacting the canonical insulin/IGF-1 kinase cascade, transactivation and suppression of transcription via DAF-16, and cross-talk to other signaling pathways via PIP3 tethering, protein kinases, and/or transcription factors. The greatest enhancement of longevity is attained only for kinase-null mutants, and only when maternal carry-over of PI3KCS activity has been eliminated.

To what extent might defective oogenesis contribute to the extraordinary longevity of worms bearing prematurely terminating age-1 alleles? Life extension by 1.4- to 1.6-fold has been reported for germ-line-defective mes-1, daz-1 or glp-1 mutants, and also for worms in which germ-line progenitor cells were laser ablated (Arantes-Oliveira et al., 2002). When combined with two other interventions, laser ablation of germ-line gonadal precursor cells extended lifespan to a total of four times (Hsin & Kenyon, 1999; Lin et al., 2001) or in one experiment, six times (Arantes-Oliveira et al., 2003) that of normal controls. However, the signal restricting lifespan emanates from germ-line stem cells (Hsin & Kenyon, 1999; Lin et al., 2001; Arantes-Oliveira et al., 2002); in view of the presence of syncytial germ nuclei (Fig. 6), these stem cells clearly are not absent from second-generation age-1(mg44) homozygotes.

It appears likely that the more modest life extensions seen previously were due to maternal protection, known to attenuate other age-1 phenotypes for the first generation of homozygous-null mutants (Larsen et al., 1995; Morris et al., 1996; Tissenbaum & Ruvkun, 1998). Second-generation homozygotes were not assessed for longevity in earlier studies, in part because they did not mature into adults under the conditions employed. What mechanisms could account for the extraordinary enhancement of lifespan and stress resistance, requiring an age-1 kinase-null allele, intact DAF-16, and the absence of maternal protection?

The canonical kinase-mediated insulin/IGF signaling pathway leads from the DAF-2 receptor via PI3K, PDK-1, SGK-1 and AKT-1/2 to phosphorylate and block transcriptional effects of DAF-16; this pathway is augmented by cross-talk with other signaling pathways that employ some of the same kinases or phosphatases (Singh et al., 1993; Kondo et al., 2005; Gami et al., 2006; Troemel et al., 2006; Matsumoto et al., 2006). These signals serve to maintain normal development in a benign environment; during starvation, overcrowding or stress, abatement of insulin/IGF signaling triggers the dauer program of developmental arrest and thus postponed reproduction. Most of the previously studied insulin/IGF signaling mutations were conditional (temperature sensitive) dauer-constitutive mutations, allowing maturation of larvae at permissive temperatures into long-lived adults. However, the age-1(mg44) and (m333) alleles are unconditional dauer-constitutive mutations, which under many culture conditions yield no adults. We have shown that at sufficiently low temperature, they slowly mature into infertile adults, incapable of producing progeny. In studies to be reported elsewhere, we find these adults to have quite distinctive profiles with respect to transcripts, proteins, lipids and small metabolites. At all of these levels, they are clearly distinct from dauer larvae, and also from any other insulin/IGF signaling-mutant adults. They thus have the potential to reveal novel mechanisms of life extension.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Strains

Nematode strains used in the present study were either provided by the Caenorhabditis Genetics Center (CGC), Minneapolis, MN, USA, or were derived in our laboratory from CGC-supplied strains. Working stocks were initiated as needed (typically every 6 months) from aliquots stored frozen at –80 °C or under liquid nitrogen. Unless noted otherwise, worm strains were maintained at 20 °C on nematode growth medium–agar plates supplemented to 1% with Bacto-Peptone and spread with a lawn of E. coli strain OP50, as described previously (Sulston & Hodgkin, 1988; Ebert et al., 1993; Ayyadevara et al., 2003).

Determination of lifespan

Worms were grown on nematode growth medium–agar plates with 0.6% peptone (Sulston & Hodgkin, 1988; Ebert et al., 1993; Ayyadevara et al., 2003) and harvested by rinsing with S buffer (0.1 m NaCl, 0.05 m potassium phosphate, pH 6.0) (Sulston & Hodgkin, 1988; Ebert et al., 1993; Ayyadevara et al., 2003). Adults, > 99.6% hermaphrodites, were allowed to settle and then were suspended in alkaline hypochlorite (5 min at 20 °C in 0.5 N NaOH, 1.05% hypochlorite). Uneclosed larvae (eggs) were recovered, rinsed with S buffer, and transferred to fresh agar plates containing E. coli OP50. Survival cultures (in 60-mm dishes) were set up 1 day after the L4/adult molt by transferring 25–50 adults to 60-mm dishes containing nematode growth agar seeded with a lawn of E. coli OP50 bacteria. Worms were incubated at 20 °C unless otherwise noted; live worms were counted and transferred daily to fresh dishes prepared as above. Those failing to move spontaneously or in response to touch were counted as dead. Worms lost, stranded (e.g. on dish walls or beneath the agar) or ‘bagged’ (endotokia matricida) were scored as ‘censored’ at the mid-point of the measurement interval in which this occurred; those inadvertently killed were censored at the time of the event.

Stress survivals

Young adult worms were transferred to wells of a 24-well plate (25 worms/well) at 9 days posthatch for second-generation age-1(mg44) homozygotes derived from strains SR808 and GR1168; at 19 days for second-generation age-1(m333) homozygotes derived from strains SR809 and DR722; and at 5 days for all other strains. Worms were maintained at 20 °C, in S medium (S suffer plus 0.5% cholesterol) also containing 3 mm hydrogen peroxide (Sigma, St. Louis, MO), 10 mm 4-HNE (synthesized as previously described; Ayyadevara et al., 2005b), or 150 mm paraquat (Sigma), as noted. Worms were scored for survival, as described above, at regular intervals – typically once per hour, until none remained alive.

Staining nuclear DNA

Worms were washed and fixed according to the protocol of Finney and Ruvkun (1990). DAPI (4’,6-diamidino-2-phenylindole) was added to phosphate-buffered saline (PBS, pH 7.0) to a final concentration of 0.1 µg mL−1, and incubated for 30 min at room temperature. Worms were then washed three times with PBS and GIF images captured on a Nikon (Melville, NY) Cool-Pix digital camera through a Nikon Eclipse E1000 fluorescence microscope.

Statistical analyses

Survivals (Figs 4 and 5) compared by the Gehans–Wilcoxon log-rank test, indicated many highly significant differences between age-1 strains and their controls (N2DRM or daf-16 double-mutants), as indicated in the figure legends. Table 1 also presents t-test comparisons of median survivals across biological replicates (independently generated cohorts), addressing purely intergroup variation. Such t-tests are single tailed where the direction of expected change is established, two-tailed otherwise; actual P-values are given in each case.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank Vasudha Kondopally and Rajani Ayyadevara for expert technical assistance; Eric Siegel for statistical consultation; and Heidi Tissenbaum (University Massachusetts Medical School, Boston) and Khaled Machaca (University of Arkansas for Medical Sciences) for helpful suggestions. This work was supported by a grant (to Robert J. Shmookler Reis) from the US National Institutes of Health, and by a Research Career Scientist Award (to Robert J. Shmookler Reis) from the US Department of Veterans Affairs.

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  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Video of an age-1(mg44) worm at 82 days of adult age (90 days post-eclosion).

Video of an age-1(mg44) worm at 140 days of adult age (148 days post-eclosion).

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