Drosophila Longevity Assurance Conferred by Reduced Insulin Receptor Substrate <i>Chico</i> Partially Requires <i>d4eBP</i>

<div><p>Mutations of the insulin/IGF signaling (IIS) pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit, suggesting there are additional outputs of IIS to mediate aging. One candidate is target of rapamycin complex 1 (TORC1). Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP) is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. Here we use genetic epistasis analyses to test whether longevity assurance mutants of <i>chico</i>, the Drosophila insulin receptor substrate homolog, require Drosophila <i>d4eBP</i> to slow aging. In <i>chico</i> heterozygotes, which are robustly long-lived, <i>d4eBP</i> is required but not sufficient to slow aging. Remarkably, <i>d4eBP</i> is not required or sufficient for <i>chico</i> homozygotes to extend longevity. Likewise, <i>chico</i> heterozygote females partially require <i>d4eBP</i> to preserve age-dependent locomotion, and both <i>chico</i> genotypes require <i>d4eBP</i> to improve stress-resistance. Reproduction and most measures of growth affected by either <i>chico</i> genotype are always independent of <i>d4eBP</i>. In females, <i>chico</i> heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP). Altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP.</p></div

Proportional hazard analysis for survival during paraquat exposure and starvation.

<p>Proportional hazard modeled for <i>ch</i>^<i>+/-</i> and <i>d4eBP</i> as single and double mutants relative to wildtype, and <i>ch</i>^<i>-/-</i> and <i>d4eBP</i> as single and double mutants, with likelihood ratio test. Coefficient <i>β</i> for single loci: when significantly less than zero indicates reduction in mortality, estimates greater than zero indicate elevated mortality. Double mutants: coefficient <i>β</i> when significantly different from zero indicates gene interaction where the effect of the double mutant differs from expectation from product of single mutants. Epistasis inferred when significant gene interaction increases mortality (positive <i>β</i>) relative to expected product of <i>chico</i> and <i>d4eBP</i>; synergy inferred when gene interaction reduces mortality (negative <i>β</i>) relative to product of <i>chico</i> and <i>d4eBP</i> genotypes. Female and male survivorship when exposed to paraquat. Female and male survivorship during fasting.</p

Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in <i>Drosophila</i>

<div><p>Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in <i>Drosophila</i>, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of <i>Autophagy-specific gene 8a (Atg8a)</i> within muscle, a factor controlling the rate of autophagy. Expression of <i>Atg8a</i> within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.</p></div

Abundance of AKT, pAKT, 4E-BP and p4E-BP in whole adults of each <i>chico</i> genotype.

<p><b>A)</b> and <b>C)</b> Western blots with actin loading control. <b>B)</b> and <b>D)</b> Means (s.e.) from quantified replicate blots (n = 3) relative to actin within each matched sample. Significant differences (ANOVA, post hoc test) indicated by brace-lines: p < 0.05.</p

Survivorship and mortality of male and female adult Drosophila with single and combined mutations of <i>chico</i> and <i>d4eBP</i>.

<p>Cohorts of all genotypes were aged concurrently in two independent trials. Deaths in Trial 2 were recorded beginning at 10 days of age. Mortality rate is plotted as ln(<i>μ</i>_<i>x</i>), estimated as ln(-ln(1-<i>q</i>_<i>x</i>)) where <i>q</i>_<i>x</i> is age-specific mortality. Panels <b>A</b>, <b>C</b>, <b>E</b> and <b>G</b> plot <i>chico</i> heterozygotes <i>ch</i>^<i>+/-</i> relative to wildtype, <i>d4eBP</i> null mutant and the double mutant <i>ch</i>^<i>+/-</i><i>d4eBP</i>. Panels <b>B</b>, <b>D</b>, <b>F</b> and <b>H</b> plot <i>chico</i> homozygotes <i>ch</i>^<i>-/-</i> relative to wildtype, <i>d4eBP</i> null mutant and the double mutant <i>ch</i>^<i>-/-</i><i>d4eBP</i>.</p

Life table and proportional hazard survival analysis statistics of adult Drosophila wildtype, <i>chico</i>, <i>d4eBP</i> and <i>chico d4eBP</i> genotypes.

<p>Independent replicate trials, sexes (males & once mated females) maintained as separate cohorts. Number: adults for combined cages of synchronous cohorts. Upper (UL) and lower (LL) 95% confidence intervals for median lifespan. Relative risk estimated from Cox proportional hazard analyses for each genotype (row) relative to <i>chico</i> genotype (column); probability > χ ² based on log-likelihood (***) for p < 0.0001. Relative risk indicates fold change of the row genotype relative to the column <i>chico</i> genotype. Relative risk less than one (significant estimates in italics) indicate reduced mortality relative to the column <i>chico</i> genotype. Relative risk greater than one (significant estimates underlined) indicates increased mortality relative to the column <i>chico</i> genotype.</p

ChIP-Seq to identify dFOXO direct target genes and lifespan screen for 23 selected candidates.

<p>(A) Venn diagram to show dFOXO target genes identified in ChIP-Seq analysis. 15-day-old female insulin mutants (<i>chico ^−/+</i> and IPC ablation) were used in ChIP-Seq experiments. dFOXO was enriched at promoters of 273 genes common to these genotypes. (B) Pathway analysis for 273 dFOXO targets, determined by DAVID functional classification. (C–E) Expression analysis of 23 selected dFOXO target genes indicates dFOXO acts as both activator and repressor. Asterisk indicates significant difference between <i>chico^−/−</i> and wildtype (p<0.05); three biological replicates per genotype. (F–H) Lifespan analysis for three dFOXO target genes (<i>daw</i>, <i>Glyp</i> and <i>Tsp42Ef</i>) (Log-rank test, <i>p</i><0.0001). Ubiquitous GeneSwitch (GS)-Gal4 drivers, Tub-GS-Gal4 or Tub-GS-dicer2-Gal4 (with UAS-dicer2 to enhance the knockdown) were used in lifespan screen (lifetable statistics summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen.1003941.s009" target="_blank">Table S1</a>).</p

Muscle Activin signaling regulates longevity through <i>Atg8a</i> and remotely controls brain insulin secretion.

<p>(A) Lifespan analysis of muscle-specific <i>Atg8a</i> overexpression. (B) Genetic epistasis between <i>daw</i> and <i>Atg8a</i> in muscle (MHC-Gal4). Simultaneous expression of RNAi for <i>daw</i> and <i>Atg8a</i> blocks the longevity benefit of <i>daw</i> RNAi alone (while <i>Atg8a</i> RNAi alone does not affect survival). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen.1003941.s013" target="_blank">Table S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen.1003941.s014" target="_blank">S6</a> for survival analysis. (C, D) Muscle-specific <i>daw</i> RNAi reduces circulating DILP2 level, but has no effects on <i>dilp2</i> mRNA expression in the head. (E) <i>4ebp</i> mRNA expression in fat body is regulated by muscle Activin signaling. <i>4ebp</i> mRNA is elevated in fat body when <i>daw</i> is reduced in muscle, while it is repressed when muscle <i>babo</i> is induced. (F) Female fecundity is not affected by reducing muscle Activin signaling. Asterisk indicates significant difference between treatment and control (<i>p</i><0.05).</p

Activin signaling regulates muscle aging and proteostasis.

<p>(A–C) Decline of flight with age is delayed in <i>daw</i>, <i>Smox</i> and <i>babo</i> RNAi flies. 40 females were scored for each genotype at each time point. Flying ability was measured at one week, three weeks and five weeks. (D) Poly-Ubiquitin-positive protein aggregates are reduced at old age in <i>daw</i>, <i>Smox</i> and <i>babo</i> RNAi flies. Aggregates were visualized with Poly-Ubiquitin FK2 antibody at one week, three weeks and five weeks. Scale bar: 20 µm. (E) RNAi for <i>daw</i>, <i>Smox</i> and <i>babo</i> preserves the decline of lysotracker-positive organelles (lysosomes). Scale bar: 20 µm. (F) Quantification of the cumulative area of protein aggregates for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen-1003941-g004" target="_blank">Figure 4D</a> (n = 20). (G) Quantification of the number of lysotracker-positive stain for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen-1003941-g004" target="_blank">Figure 4E</a> (n = 10). Asterisk indicates significant difference between treatment and control (<i>p</i><0.05).</p

Inactivation of genes in Activin signaling (<i>daw</i>, <i>Smox</i> and <i>babo</i>) in muscle, but not in fat body extended lifespan.

<p>(A) Tissue-specific gene expression pattern of <i>daw</i>. (B) Tissue-specific distribution of transcription factor Smox using 7-day-old <i>Oregon R</i> females. (C–E) Lifespan analysis of Activin signaling using muscle-specific Gal4 driver (MHC-Gal4). Lifespan was extended by inactivating Activin genes (<i>daw</i>, <i>Smox</i> and <i>babo</i>) in muscle (Log-rank test, <i>p</i><0.0001). (F–H) Lifespan analysis of Activin signaling using adult fat body-specific Gal4 driver (S106-GS-Gal4). Fat body-specific inactivation of Activin genes (<i>daw</i> and <i>Smox</i>) shortens lifespan (Log-rank test, <i>p</i><0.0001). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003941#pgen.1003941.s012" target="_blank">Table S4</a> for survival analysis. (I, J) mRNA expression of <i>daw</i> and phosphorylation of Smox are down-regulated by <i>chico</i> mutation and rescued by mutation of dFOXO. Muscle and fat body were dissected from 7-day-old female wildtype, <i>chico^−/−</i> and <i>chico;foxo</i> double mutants. Band intensity was quantified using Bio-Rad Image Lab software. The average band intensity from four independent experiments is shown. Asterisk indicates significant difference between treatment and control (<i>p</i><0.05).</p