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. 2013 Dec 26;5(6):1600-10.
doi: 10.1016/j.celrep.2013.11.018. Epub 2013 Dec 12.

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Di Chen  1 , Patrick Wai-Lun Li  2 , Benjamin A Goldstein  3 , Waijiao Cai  4 , Emma Lynn Thomas  2 , Fen Chen  5 , Alan E Hubbard  3 , Simon Melov  2 , Pankaj Kapahi  6
Affiliations

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Di Chen et al. Cell Rep. .

Abstract

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. Here, we demonstrate that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, we identify germline as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, our findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms.

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Figures

Figure 1
Figure 1. Double mutations in daf-2 and rsks-1 lead to synergistically prolonged longevity that requires DAF-16
(A) The daf-2 rsks-1 double mutant showed synergistically prolonged longevity (454% extension compared to N2) that is dependent on DAF-16. (B) Inhibition of TOR by rapamycin led to increased lifespan extension in daf-2 compared to N2. Rapamycin (100 μM) extended N2 and daf-2 lifespan by 26% and 45%, respectively (log-rank, p < 0.0001). Animals treated with the vehicle (DMSO) alone did not show significantly affected lifespan (log-rank, p > 0.05). Quantitative data and statistical analyses are included in Table S1. (C) daf-2 rsks-1 animals showed significantly increased DAF-16 transcriptional activity. mRNA levels of DAF-16 targets that are either activated (sod-3 and hsp-12.3) or inhibited (sams-1) by DAF-16 were quantified using qRT-PCR. Asterisks indicate statistical differences using two-tailed t tests: ***, p < 0.001.
Figure 2
Figure 2. Effects of daf-2 rsks-1 on development, reproduction, stress resistance and dietary restriction
(A) rsks-1 did not affect daf-2 dauer formation at 22.5°C. ns, not significant. (B) daf-2 rsks-1 animals showed delayed, prolonged and overall reduced reproduction. (C) daf-2 rsks-1 animals were more sensitive to heat stress (35°C) than daf-2 (log-rank, p < 0.0001). (D) daf-2 rsks-1 animals were more resistant to oxidative stress by paraquat compared to daf-2 (**, p < 0.01, t - test). (E) daf-2 rsks-1 animals were more resistant to UV stress (2,000 J/m2) than daf-2 (log-rank, p < 0.0001). (F) daf-2 rsks-1 animals showed significantly increased survival under DR by bacterial food deprivation compared to daf-2 (log-rank, p < 0.0001).
Figure 3
Figure 3. The synergistic longevity by daf-2 rsks-1 is mediated by positive feedback regulation of DAF-16 via AMPK
(A) Genes that are differentially expressed in daf-2 rsks-1 were identified by microarrays analyses. (B) Identification of aakg-4 as a strong suppressor of daf-2 rsks-1. rsks-1-mediated lifespan extension in daf-2 (daf-2 vs. daf-2 rsks-1): 89% (control RNAi), 18% (aakg-4 RNAi). (C) A deletion in aak-2 suppressed the synergistic longevity by daf-2 rsks-1. rsks-1-mediated lifespan extension in daf-2 (daf-2 vs. daf-2 rsks-1): 85% (with aak-2), 18% (without aak-2). Quantitative data and statistical analyses are included in Table S1. (D) The daf-2 rsks-1 double mutant showed further increased phosphorylation of AAK-2. Inhibition of aakg-4 significantly decreased AAK-2 phosphorylation in daf-2 rsks-1. (E) The aak-2 deletion suppressed the significantly increased DAF-16 transcriptional activity in daf-2 rsks-1. ***, p < 0.001. (F) Inhibition of aakg-4 in daf-2 rsks-1 reduced DAF-16 transcriptional activity. ***, p < 0.001. (G) A model depicting the synergistic lifespan extension through positive feedback regulation of DAF-16 via AMPK.
Figure 4
Figure 4. Tissue-specific regulation of the synergistically prolonged longevity by daf-2 rsks-1
(A) Mean lifespan extension by rsks-1 RNAi knocking-down in different tissues of daf-2 animals. (B) Changes in mean lifespan relative to the control RNAi-treated animals by tissue-specific RNAi knocking-down of daf-16, hsf-1 and aak-2 in daf-2 rsks-1. Data from three independent experiments are shown. Survival curves are included in Figure S4. Quantitative data and statistical analyses are included in Table S1.
Figure 5
Figure 5. Germline signaling modulates the daf-2 rsks-1-mediated synergistic lifespan extension through AMPK and DAF-16
(A) The glp-1(gf) mutation suppressed the synergistic longevity by daf-2 rsks-1. rsks-1-mediated lifespan extension in daf-2 (daf-2 vs. daf-2 rsks-1): 86% [without glp-1(gf)], 14% [with glp-1(gf)]. (B) The glp-1(gf) mutation decreased phosphorylation of AAK-2 in daf-2 rsks-1. (C) The glp-1(gf) mutation suppressed the significantly increased DAF-16 transcriptional activity in daf-2 rsks-1. ***, p < 0.001. (D) Inhibition of DAF-12 or KRI-1, essential mediators of the germline signaling, significantly suppressed the synergistic longevity by daf-2 rsks-1 (log-rank, p < 0.0001). Quantitative data and statistical analyses are included in Table S1.
Figure 6
Figure 6. Cell-non-autonomous activation of DAF-16 by rsks-1 RNAi knocking-down in the germ line
(A) Activation of the DAF-16 target stdh-1 by rsks-1 RNAi in daf-2 and daf- 2; rrf-1. rsks-1 RNAi knocking-down in the germ line of daf-2 animals (daf-2; rrf-1) significantly increased the intestinal expression of GFP driven by the stdh-1 promoter. Arrows indicate the anterior and posterior intestine. (B) Quantification of GFP expression driven by the stdh-1 promoter. Thirty animals were examined for each treatment. (C) Measurement of Pstdh-1::gfp expression by Western blots using anti-GFP antibodies. (D) Quantification of GFP expression from Western blots. Relative GFP levels were calculated by normalizing the intensities of GFP bands to Actin. Fold increase in Pstdh-1::gfp expression induced by rsks-1 RNAi was calculated through dividing the relative GFP levels in rsks-1 RNAi-treated animals by those in control RNAi-treated animals. **, p < 0.01. The quantification was performed with four biological replicates.
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