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Keywords:

  • insulin signaling;
  • insulin receptor substrate;
  • chico;
  • foxo;
  • epistasis;
  • proportional hazard analysis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

Although extensively studied in Caenorhabditis elegans, no work has yet demonstrated for Drosophila melanogaster whether reduced insulin/IGF signaling (IIS) requires the FOXO transcription factor (foxo) to extend lifespan. Here, we conduct genetic epistasis analysis to determine whether foxo is required for chico mutants (insulin receptor substrate) to reduce age-specific mortality and thus extend lifespan. The mutant chico1 allele strongly extends lifespan relative to wild-type sibs. A mutant of foxo eliminates most of this chico survival benefit. In addition, we used a factorial proportional hazard analysis to formally study the main effects of chico and of foxo and to determine how these genes interact to influence mortality. We document that foxo indeed contributes to how chico increases lifespan, but part of the convergence in survival between chico genotypes in the foxo-mutant background may occur because chico mutation exacerbates the negative effects of foxo mutation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

The benefits of reduced insulin/IGF signaling (IIS) upon aging are thought to be mediated by FOXO class transcription factors. In studies with Caenorhabditis elegans, hypomorphic mutants of daf-2, which encode an insulin-like receptor, extend adult lifespan (Kenyon et al., 1993; Lin et al., 1997; Ogg et al., 1997). DAF-2 negatively regulates a FOXO transcription factor encoded by daf-16 (Lee et al., 2001). Based on data from genetic epistasis analysis, the beneficial effects of daf-2 upon survival are mediated by the downstream factor FOXO (daf-16) because the double mutant of daf-2; daf-16 is not long lived relative to wild-type (Kenyon et al., 1993; Dorman et al., 1995; Larsen et al., 1995).

Beyond C. elegans, there is only indirect evidence to suggest that insulin signaling extends lifespan through FOXO. Mutants of the Drosophila insulin-like receptor (InR) or of the insulin receptor substrate (encoded by chico) increase fly lifespan (Clancy et al., 2001; Tatar et al., 2001; Tu et al., 2002), while overexpression of foxo in adult fat body likewise increases longevity (Hwangbo et al., 2004; Giannakou et al., 2005). Although such foxo overexpression is tissue limited, FOXO may still act throughout the animal to confer longevity assurance: foxo overexpression in fat body represses insulin-like peptide mRNA produced in the brain and appears to reduce the level of circulating insulin peptide, as inferred from the observation that FOXO concentrates into nuclei in unmanipulated peripheral tissues (Hwangbo et al., 2004). Furthermore, transgene expression of foxo in cardiac cells alone is sufficient to slow heart functional aging (Wessells et al., 2004). Yet to date, we lack direct evidence in Drosophila to determine whether mutants of InR or chico require foxo to assure longevity. (Note: in the review of this current work, Slack et al. (2011) reported that IIS-dependent lifespan extension requires FOXO).

Here, we report an explicit epistasis analysis between chico and foxo to determine how these genes interact to affect Drosophila mortality rate and survivorship. We produce life tables for mutants of each gene alone and together and determine whether the longevity benefits of chico mutation require downstream FOXO.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

We began with the stock cn1, chico1/cn1; ry506 where all chico genotypes segregated as uniquely identifiable sibs with coisogenic backgrounds (Tu et al., 2002). Note that chico1 (also annotated ch1 and chico[1]) is a p-element insert marked with rosy (ry+). Thus, cn1/cn1; ry506 has apricot eyes, cn1, chico1/cn1; ry506 has cinnabar (cn) eyes and normal size, and cn1, chico1/chico1cn1; ry506 has cinnabar eyes and small size. With these genotypes, Tu et al (2002) reported that males and females carrying one or two copies of chico1 were 36–57% longer lived than wild-type sibs. Here, we backcrossed this stock into y1; cn1; ry506 to introduce a yellow marker and establish the new chico segregation stock y1; cn1chico1/cn1; ry506 (see Data S1). In parallel, we established the stock y1; cn1chico1/cn1; dfoxo21ry506, which was also built from strains backcrossed into y1; cn1; ry506. While the allele dfoxo21 (Junger et al., 2003) produces no detectable protein (Min et al., 2008), the emerging paper by Slack et al. (2011) suggests that this allele is hypomorphic; we shall therefore refer it as foxo-mutant rather than as foxo-null. In these genotypes, chico wild-type are distinguished by apricot eyes, chico heterozygotes by cinnabar eyes and normal body size, and chico homozygotes by cinnabar eyes but only slightly reduced body size as expected, as foxo mutation can suppress the chico size phenotype (Junger et al., 2003; Kramer et al., 2003; Puig et al., 2003; although see Slack et al., 2011). Because the foxo mutation increased the size of chico homozygotes, we balanced second chromosomes from the y1; cn1chico1/cn1; dfoxo21ry506 stock with CyO so that we could subsequently generate unambiguous chico heterozygotes and homozygotes in different foxo backgrounds from mating with the appropriate parental stock (see Data S1). This approach produced large cohorts of chico wild-type (ch+/ch+) and chico heterozygote (ch1/ch+) in foxo wild-type and foxo21/foxo21 backgrounds appropriate for demographic analysis. We attempted to generate ch1/ch1; foxo21/foxo21 but this genotype exhibited synthetic lethality: we recovered almost no viable males, and adult females were developmentally delayed and had excessive early adult mortality.

For simplicity, we refer to these genotypes only by their alleles at chico and foxo. As expected with a foxo wild-type background, chico heterozygotes (ch1/ch+) lived considerably longer (median lifespan: males 70 d, females 72 d) than wild-type (ch+/ch+) (males 48 d, females 44 d) (Fig. 1A,B) (Table S1). As seen in previous reports (Giannakou et al., 2008; Min et al., 2008), the foxo-mutant on its own was somewhat shorter lived than coisogenic wild-type (males 36 d, females 36 d). The survival of (ch1/ch+); foxo21/foxo21 (males 42 d, females 44 d) was similar to that of ch+/ch+ wild-type, and this contrasts to the 22- to 28-day benefit produced by chico heterozygotes relative to ch+/ch+ in the foxo wild-type background. The mortality plots of these genotypes (Fig. 1C,D) demonstrate that differences in chico survival and suppression of this benefit in the foxo-mutant occurred across ages where mortality progressively increased. These data together suggest that chico extends Drosophila longevity by acting through FOXO.

image

Figure 1.  Life table plots for chico, foxo and chico, foxo genotypes. Survivorship: (A) females, (B) males. Mortality rate: (C) females, (D) males. In each plot, wild-type chico is compared with heterozygote (chico[1]/ch+) in a wild-type foxo and foxo-null background.

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Because foxo mutation alone is somewhat deleterious, some attention is required to understand whether foxo mutation acts only to block the longevity benefits of chico mutation or whether chico mutation itself exacerbates the deleterious effects of foxo mutation, which would be the evidence for partial synthetic lethality. Note that synthetic lethality was strong in males with foxo mutation in the ch1/ch1 homozyogote background.

To solve this problem, we apply proportional hazard analysis to determine the simultaneous contribution of two variables upon mortality and estimate their main and interactive effects (Parmer & Machin, 1995). We estimate how chico mutation affects mortality with and without foxo and how foxo itself affects mortality with or without chico. All coefficients were significant including both main effects of chico and foxo and their interaction (Table S2). chico mutation significantly decreases mortality [hazard ratio: males, 0.56 ± 0.03 (SE); females 0.54 ± 0.02 (SE)] while foxo mutation independently increases mortality [hazard ratio: males, 2.05 ± 0.03 (SE); females 2.8 ± 0.03 (SE)]. The significant interaction between these factors verifies that chico and foxo function together to affect mortality and thus survivorship. At the same time, the interaction suggests that foxo mutation blocks the benefits of chico heterozygote and the chico heterozygote increases the deleterious effects of foxo mutation. A potential implication of this interaction is that chico mutation may extend longevity to some extent independent of FOXO. The reduced survival observed in the chico/foxo double mutant could arise in part from losing the partial role FOXO plays in the benefits conferred by chico coupled with the partial synthetic deleterious interaction of these mutants. Because foxo mutation alone reduces survival, in genetic epistasis analysis we cannot fully determine whether all or just part of the survival benefit of chico mutation is FOXO-dependent.

Many investigations aim to understand the mechanisms by which reduced insulin/IGF signaling (IIS) increases lifespan and slows aging. Work from C. elegans indicates that signaling through the FOXO/DAF-16 transcription factor is a step in this process. Mutant alleles of daf-2 and of age-1 extend lifespan of adult worms but not so when combined with mutants of daf-16 (Kenyon et al., 1993; Dorman et al., 1995; Larsen et al., 1995). Multiple isoforms of daf-16 exist, and epistasis analysis shows that these cooperate to modulate longevity conferred by daf-2 mutation (Kwon et al., 2010). Accordingly, discovering the direct targets of DAF-16/FOXO when reduced IIS extends lifespan may provide insights into how insulin/IGF confers longevity assurance (Murphy, 2006). Manipulation of genes downstream of daf-16 has been able to improve survival in some cases, but there are few epistasis analyses to determine whether these downstream targets are required for mutant daf-2 to extend lifespan (McElwee et al., 2003; Oh et al., 2006; Honda et al., 2008).

Much less is known about how reduced IIS slows aging in animals beyond C. elegans. Drosophila FOXO is thought to play a role because foxo overexpression is sufficient to extend lifespan (Hwangbo et al., 2004; Giannakou et al., 2005). Here, we present an epistasis test to determine whether FOXO is required for reduced insulin/IGF signaling to extend Drosophila lifespan. We document a beneficial effect of chico mutation, a deleterious effect of foxo mutation, and a significant interaction between these main effects. This result is also observed in both sexes and in a preliminary trial (Data S1). We find that the poor survivorship of foxo-mutants is made worse in chico-mutants, but also that the benefit of chico upon survival acts through FOXO. Understanding the targets of FOXO through which reduced IIS extends longevity in Drosophila will be productive.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

This work was supported by The National Institute of Aging (R01 AG024360), The Ellison Medical Foundation, and the The Glenn Foundation for Medical Research.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgments
  6. References
  7. Supporting Information

Data S1 Methods.

Fig. S1 Life table plots for chico, foxo and chico;foxo genotypes, females.

Table S1 Sample size and median lifespan of chico, foxo and chico; foxo genotypes.

Table S2 Cox proportional hazard statistics for main and interaction effects.

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