C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins

Abstract

Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work.

Keywords: C. elegans; FOXO; FUdR; Hormesis; Hypertonic stress; Sirtuin.

Figure 1. Hypertonic stress extends lifespan in the presence of FUdR

A. Hypertonic stress can extend lifespan at some NaCl concentrations. Restricted mean lifespans for survival curves shown in panel A. Maximal extension was observed at 250 or 350 mM NaCl in the presence of FUdR. Restricted mean lifespans for each group are shown, with n = 48 to 69 animals per condition. B. Lifespan extension is independent of osmolyte. Osmotic equivalents of NaCl, KCl, glycerol, or sorbitol extend lifespan equivalently in the presence of FUdR. n = 60 to 106 animals per condition. All experiments were performed at 25°C with 400 µM FUdR. Error bars indicate S.E.M., * indicates p<0.0001 in log rank test vs. 50 mM NaCl control.

Figure 2. Hypertonic stress lifespan extension is dependent on the concentration of FUdR

A. Increasing hypertonicity decreased lifespan, but the addition of FUdR in combination with moderate hypertonic stress increased lifespan. Pooled analysis of two independent replicates at 20° C with 0 or 400 µM FUdR, for a total of n ≥ 178 animals per condition. B. Lifespan extension is dependent on FUdR concentration. Little impact of FUdR was observed at 50 mM NaCl. Under moderate hypertonic stress, lifespan was increased by 40 µM or 400 µM FUdR. Pooled analysis of three independent replicates at 20° C, for a total of n ≥ 226 animals per condition. C. Loss of Notch co-ligands OSM-7 or OSM-11 increased lifespan on 50 mM NaCl. Three independent replicate experiments were pooled for analysis, for a total of n ≥ 273 animals per condition. Experiments were performed at 25°C with 400 µM FUdR. D. Lifespan extension due to hypertonic stress or loss of Notch co-ligands is dependent on the presence of FUdR. Hypertonic stress alone decreased wild type (WT) lifespan, while hypertonic stress combined with FUdR increased. Loss of Notch co-ligands OSM-7 and OSM-11 decreased lifespan in the absence of FUdR, but increased in the presence of FUdR, regardless of hypertonic stress. Pooled analysis of two independent replicates at 20° C with 0 or 400 µM FUdR, for a total of n ≥ 140 animals per condition. Error bars indicate SEM, * indicates p<0.01 in log rank test vs. 50 mM NaCl control or between indicated columns, n.s. indicates p > 0.05.

Figure 3. Lifespan extension by thymidylate synthase inhibition and osmotic stress is partially dependent on DNA base excision repair

A. FUdR inhibition of thymidylate synthase increases lifespan under hypertonic stress. Lifespan was determined for transgenic F1 animals carrying ubiquitously expressed control or tyms-1(RNAi) transgenes (dpy-30p::empty or dpy-30p::tyms-1(RNAi)). Hypertonic stress increased dpy-30p::tyms-1(RNAi) lifespan, but not control. FUdR slightly extended lifespan when combined with hypertonic stress in dpy-30p::tyms-1(RNAi) animals. Pooled analysis of 3 independent replicate experiments, for a total of n ≥ 148 animals per condition. B. Loss of uracil-DNA glycosylase NTH-1 partially abrogated FUdR-mediated lifespan extension under hypertonic stress, but did not completely eliminate the beneficial impact of FUdR under hypertonic stress. Pooled analysis of 3 independent replicate experiments, for a total of n ≥ 313 animals per condition. Experiments were performed at 20 °C with 0 or 400 µM FUdR. Error bars indicate SEM, * indicates p<0.01 in log rank test vs. 50 mM NaCl, no FUdR or between indicated columns, n.s. indicates p>0.05.

Figure 4. Thymidylate synthase inhibition induces broad stress resistance

A. FUdR or tyms-1(RNAi) increases resistance to acute hypertonic stress. Fraction of motile animals was measured 10 minutes after transfer to 500 mM NaCl plates. n = 3 independent replicate experiments, with 30 animals per replicate. B. FUdR increases resistance to anoxia. Fraction surviving anoxia in BioBag was assessed at 24 hours. n = 3 independent replicate experiments, with at least 20 animals per replicate. C. FUdR confers resistance to thermal stress. Fraction surviving was assessed after 12 hours at 35°C. n = 4 independent replicate experiments, with at least 28 animals per replicate. D. Either FUdR treatment or hypertonic stress induced expression of gpdh-1p::GFP. Average percent change in GFP fluorescence for gpdh-1::GFP at rear of intestine after 48 hours in 3 replicate experiments, with 10 animals per replicate. Dotted lines show outline of animal (top panels). E. nth-1 loss partially abrogates FUdR-mediated resistance to acute hypertonic stress. n = 4 independent replicate experiments, with 30 animals per replicate. Young adult animals were pre-treated with 400 µM FUdR or bacteria expressing tyms-1(RNAi) for 48 hours prior to stress assays. Error bars indicate SEM, * indicates p < 0.05, n.s. indicates p > 0.05 in Student’s T-test vs. control or between indicated groups.

Figure 5. Sirtuin function is required for FUdR to induce stress resistance or increase lifespan

A. sir-2.1 plays a role in FUdR-induced hypertonic stress resistance. Animals lacking sir-2.1 were hypersensitive to acute hypertonic stress; the effect of FUdR pre-treatment was reduced compared to wild type. Hypertonic stress resistance and FUdR response of animals lacking other sirtuin genes was normal. n = 3 independent replicate experiments, with 45 animals per replicate. B. Simultaneous loss of three sirtuin genes eliminated the ability of FUdR to induce acute hypertonic stress resistance. For either triple mutant strain, FUdR had no impact on hypertonic stress resistance. n = 5 independent replicate experiments, with at least 34 animals per replicate. C. Loss of sir-2.1, sir-2.2, sir-2.3, or sir-2.4 did not prevent lifespan extension by FUdR and hypertonic stress. Pooled analysis of three independent replicates at 25° with 400 µM FUdR, for a total of n ≥ 253 animals per condition. D. Sirtuin function is required for FUdR-induced lifespan extension under hypertonic stress. Loss of three sirtuins partially abrogated lifespan extension induced by FUdR and hypertonic stress. Pooled analysis of three independent replicates at 25° with 400 µM FUdR, for a total of n ≥ 193 animals per condition. For stress resistance experiments, young adult animals were pre-treated for 48 hours with 400 µM FUdR prior to stress assays. Error bars indicate SEM, * indicates p<0.05 vs. control or between designated columns using Student’s T-test for stress assays and the log-rank test for lifespan.

Figure 6. DAF-16 is required for lifespan extension, but not hypertonic stress resistance induced by FUdR

A. FUdR treatment, but not hypertonic stress, induced expression of daf-16p::DAF-16::GFP. FUdR treatment for 48 hours increased DAF-16::GFP levels by one third, regardless of external osmolarity. Average percent change in GFP fluorescence at rear of intestine after 48 hours in 3 replicate experiments, with 10 animals per replicate. B. Animals lacking daf-16 were hypersensitive to acute hypertonic stress, but FUdR pre-treatment conferred resistance. n = 5 independent replicate experiments, with at least 21 animals per replicate. C. DAF-16 FOXO is required for FUdR to extend lifespan under hypertonic conditions. daf-16 loss modestly decreased lifespan under standard culture conditions, regardless of FUdR treatment. In the absence of FUdR, daf-16 loss had no impact on lifespan under hypertonic stress, but loss of daf-16 partially abrogated lifespan extension induced by hypertonic stress with FUdR. Pooled analysis of two independent replicates at 20° with 0 or 400 µM FUdR, for a total of n ≥ 161 animals per condition. Error bars indicate SEM, * indicates p<0.05 vs. control or between designated columns using Student’s T-test for stress assays and the log-rank test for lifespan.