Roles for ROS and hydrogen sulfide in the longevity response to germline loss in Caenorhabditis elegans Yuehua Weia and Cynthia Kenyona,1,2 a

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158

Edited by Gary Ruvkun, Massachusetts General Hospital, Boston, MA, and approved April 5, 2016 (received for review December 15, 2015)

In Caenorhabditis elegans, removing germ cells slows aging and extends life. Here we show that transcription factors that extend life and confer protection to age-related protein-aggregation toxicity are activated early in adulthood in response to a burst of reactive oxygen species (ROS) and a shift in sulfur metabolism. Germline loss triggers H2S production, mitochondrial biogenesis, and a dynamic pattern of ROS in specific somatic tissues. A cytoskeletal protein, KRI-1, plays a key role in the generation of H2S and ROS. These kri-1– dependent redox species, in turn, promote life extension by activating SKN-1/Nrf2 and the mitochondrial unfolded-protein response, respectively. Both H2S and, remarkably, kri-1–dependent ROS are required for the life extension produced by low levels of the superoxide-generator paraquat and by a mutation that inhibits respiration. Together our findings link reproductive signaling to mitochondria and define an inducible, kri-1–dependent redox-signaling module that can be invoked in different contexts to extend life and counteract proteotoxicity. Nrf2

| SKN-1 | hormesis | KRIT1 | Aβ


he concept that low levels of harmful substances can induce protective responses is an old one, exemplified by Evelyn Witkin’s ground-breaking discovery of the bacterial SOS system (1). Mild exposure to environmental stress, as well as mutations that activate stress-response pathways, can also extend the lifespan of many animal species. In some cases, cell-protective pathways are activated by the generation of reactive oxygen species (ROS) within the animal. For example, low levels of the toxin paraquat or inhibition of mitochondrial superoxide dismutase, both of which increase mitochondrial superoxide levels, extend the lifespan of Caenorhabditis elegans (2, 3). Lifespan is also extended in response to elevated ROS levels when C. elegans is fed the glucose analog 2-deoxyglucose (4). Likewise, ROS is part of the mechanism by which TOR inhibition extends lifespan in yeast (5), and acute inhibition of the daf-2 insulin/insulin-like growth factor 1 (IGF-1) receptor extends life in C. elegans (6). ROS also plays an important role in the extension of lifespan caused by a mild inhibition of respiration in worms and flies (2, 3, 7). Whether ROS can promote life extension in mammals is not yet clear, but ROS does enhance immune function in mice carrying respiration mutations (8), and it enhances glucose uptake in response to exercise in humans (9). Recently, low levels of another toxic substance, hydrogen sulfide (H2S), have been linked to the life extension produced by caloric restriction in yeast, worms, and flies (10, 11). H2S is a product of the transsulfuration pathway, a pathway activated in response to caloric restriction, specifically by limitation of sulfur-containing amino acids. The transsulfuration pathway generates sulfur-containing metabolites, including cysteine, taurine, glutathione, and hydrogen sulfide. The pathway is required for caloric restriction to extend lifespan in many species, and several lines of evidence, particularly the finding that low levels of exogenous H2S extend C. elegans’ lifespan dramatically (10), suggest that this effect involves H2S (10, 11). H2S production also may be responsible for the beneficial effect of food restriction on recovery from ischemic injury, potentially through an effect on respiration (11). Thus, like ROS, H2S appears to be a signal generated in response to stress that can induce processes that extend life. E2832–E2841 | PNAS | Published online May 2, 2016

In this study, we investigate the roles of ROS, the transsulfuration pathway, and H2S in a particularly interesting lifeextending system in C. elegans, one that is activated when the germ line is compromised. Because it slows aging, this system could couple the rate of aging to the timing of reproduction, thereby ensuring that reproduction can proceed before aging decreases the animal’s fitness. Reproductive signals influence lifespan in many species. For example, as in worms (12, 13), removing germline stem cells extends life in flies (14), and removing flowers can extend the lifespan of plants (15). In mice, lifespan can be extended by transplanting young ovaries into old females (16) and possibly by increasing levels of certain reproductive hormones (17). The reproductive system may affect lifespan in humans as well, because castration has been reported to increase longevity in men (18). Little is known about the underlying mechanisms in most species, but in C. elegans, germ-cell loss is known to activate several pro-longevity transcription factors in somatic tissues, including the DAF-16/FOXO transcription factor, which in turn influence downstream cellular processes (12, 13, 19–26), including chaperone and proteasome activities and autophagy, that contribute more directly to life extension. One protein required for germline loss to extend life, KRI-1 (27), is a cytoskeletal adaptor protein whose human homolog KRIT1/CCM1 is implicated in cerebral cavernous malformation. In this study, we show that KRI-1 enables the production of two distinct redox species, one an unidentified ROS and the other H2S, both of which appear to lie in the causal pathway for life extension and also for the increased resistance to Aβ toxicity produced by germline loss. Upon loss of germ cells, ROS and H2S are generated cell nonautonomously in somatic endodermal tissues during early adulthood, where they activate two distinct transcriptional programs. Our findings argue that this ROS species activates the mitochondrial unfolded-protein response Significance Signals from reproductive tissues and germ cells influence the lifespans of many organisms, including mammals. How germ cells, which give rise to the next generation, control the aging of the animal in which they reside is poorly understood. Counter-intuitively, we found that removing germ cells in Caenorhabditis elegans triggers the generation of two potentially toxic substances, reactive oxygen species and hydrogen sulfide, in nonreproductive somatic tissues. These substances, in turn, induce protective responses that slow aging. Author contributions: Y.W. and C.K. designed research; Y.W. performed research; Y.W. contributed new reagents/analytic tools; Y.W. and C.K. analyzed data; and Y.W. and C.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1

Present address: Calico Life Sciences, South San Francisco, CA 94080.


To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at 1073/pnas.1524727113/-/DCSupplemental.

Results The Longevity of Germline-Deficient Animals Is Redox Regulated. The germ line of C. elegans can be removed by laser-ablating the two germline precursor cells, Z2 and Z3, at hatching or by blocking germline stem-cell proliferation with the temperature-sensitive (ts) glp-1(e2144ts) mutation (13, 27). To ask whether the life extension produced by germ-cell loss might involve ROS, we treated germline-deficient glp-1(ts) animals with various concentrations of the antioxidant N-acetylcysteine (NAC) and measured life-

ROS Promotes Life Extension in Germline-Deficient Animals. To test more specifically whether ROS might play a role in this pathway, we treated the animals with vitamin C, an antioxidant that does not affect transsulfuration. Vitamin C decreases levels of ROS but not H2S in mice (11), and, we found, in C. elegans as well (Fig. 1 E and SI Appendix, Fig. S1B). Quenching ROS with vitamin C shortened the extended lifespan of germline-defective worms but had a relatively minor effect on wild-type worms (Fig. 1F). To test further whether ROS might be part of this pathway, we asked whether low levels of the superoxide generator paraquat,

Fig. 1. Loss of germ cells generates redox signaling to extend lifespan. (A) The antioxidant NAC shortened the mean lifespan of germline-less animals. Wildtype and germline-deficient glp-1(ts) animals were raised on plates containing different concentrations of NAC and were transferred every 4 d to fresh NAC plates. Statistical analyses are shown in SI Appendix, Fig. S1A. (B) ROS was generated in the intestine by germ-cell removal. The germline precursors Z2 and Z3 were removed through microsurgery by laser ablation at the L1 stage, and day-1 adults were stained with DHE. (C) ROS production was attenuated by the antioxidant NAC. Animals were treated with 8 mM NAC from hatching and were stained with DHE at day 1 of adulthood. Worms were grouped as having high, medium, or low levels of DHE signal (see SI Appendix for details). n, number of worms pooled from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups) using GraphPad Prism software: *P < 0.05; **P < 0.001. (D) Germline loss triggered production of H2S, and H2S levels were reduced by NAC. Young-adult worms were lysed, and various concentrations of protein lysate were spotted on lead acetate paper. (E) ROS production in germline-mutant (glp-1) worms was reduced by the antioxidant vitamin C. Animals were treated with 100 μg/mL vitamin C (VC) from the time of hatching and were stained with DHE at day 1 of adulthood. n, number of worms pooled from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups): *P < 0.05; **P < 0.001. (F) The antioxidant vitamin C attenuated the extended lifespan of germline mutant (glp-1) worms. Animals were treated with vitamin C from the time of hatching throughout life. The percentage alive was recorded every 2 or 3 d. Changes in mean lifespan are shown in parentheses. n >60 for each line. P values were determined using the log-rank test in Stata 10 software: *P < 0.05; **P < 0.001. (G) Further increasing ROS levels did not further extend the lifespan of germline-less animals. Animals were treated with various concentrations of the ROS generator paraquat from the L4 stage. Error estimates in percentage lifespan extensions were computed from mean lifespans and errors using standard propagation of error formulas. P values compared the treated vs. nontreated controls and were determined using the logrank test. Statistical tests of overall effects on wild-type vs. mutant backgrounds were performed by using a two-tailed t test (see SI Appendix, SI Experimental Procedures for details): ns, not significant; *P < 0.05; **P < 0.001; ***P < 0.0001.

Wei and Kenyon


span. NAC reduced the mean lifespan of germline-deficient glp-1(ts) animals substantially but had no effect on wild-type animals (Fig. 1A and SI Appendix, Fig. S1A). Both ROS and H2S production are sensitive to NAC treatment, ROS because of the reducing activity of NAC, and H2S because of NAC’s suppression of the transsulfuration pathway (11). To investigate which type of redox species might be involved in this case, we first asked whether ROS levels increased in response to germline removal, using the ROS indicator dihydroethidium (DHE). We found that DHE levels were higher in germline-deficient mutants than in wild-type animals during early (day-1) adulthood and that the signal was sensitive to NAC treatment (Fig. 1 B and C). To test for the presence of H2S, we used the H2S sensor lead acetate (11). H2S levels were higher in germlinedeficient mutants than in wild-type animals, and the H2S signal also was sensitive to NAC treatment (Fig. 1D). Thus, either ROS or H2S, or both, could potentially act in this pathway.

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(mito-UPR) and that H2S up-regulates SKN-1/Nrf2–dependent gene expression to extend life and restore redox homeostasis. The activation of this kri-1–dependent signaling axis may be triggered in response to germline loss by an enhancement of mitochondrial biogenesis and an increase in mitochondrial superoxide levels during development. Our findings suggest that this same signaling system can be activated in other ways to extend lifespan. We find that it is activated by low, life-extending levels of paraquat; not directly by the superoxide produced by paraquat, but apparently by a secondary form of ROS that requires KRI-1. In addition, the pathway is activated, again in a kri-1–dependent fashion, by a mild inhibition of respiration, another condition that generates mitochondrial ROS (2, 3). Thus, this study identifies a dynamic redox-regulatory cassette that can be induced in response to multiple signals, including endogenous signals alerting the animal to germline damage, to activate protective mechanisms that extend life.

which increases C. elegans’ lifespan (2, 3), would further increase the longevity of germline-deficient animals. If germline-deficient animals were intrinsically experiencing the same ROS benefit as that conferred by low levels of paraquat, then additional ROS might not extend their lifespan further. Wild-type and germlinedeficient animals differ in their resistance to paraquat (13), so we tested a range of paraquat concentrations (0.05–2.5 mM). We found that optimal paraquat treatment produced only a small increase in the lifespan of germline-deficient glp-1 mutants (Fig. 1G). These findings (and others described below) confirmed that ROS is part of the mechanism by which germline loss extends life. H2S Promotes the Life Extension of Germline-Deficient Animals. To determine whether the transsulfuration pathway and H2S also might contribute to life extension, we RNAi-inhibited the transsulfuration-pathway gene cbs-1, which encodes cystathionine beta-synthase (11). Reducing cbs-1 expression decreased H2S levels (SI Appendix, Fig. S1C) and also shortened the lifespan of germlinedeficient mutants to a greater extent than it shortened the lifespan of wild type (SI Appendix, Fig. S1D). Because cbs-1 RNAi did not change the levels of ROS in germline-deficient worms (SI Appendix, Fig. S1E), we concluded that the reduction in lifespan was likely caused by reduced transsulfuration-pathway activity and H2S levels. ROS Extends Life by Activating the Mito-UPR. How might ROS and the transsulfuration pathway influence lifespan? We asked whether they might be required for DAF-16 nuclear localization or for expression of the DAF-16 target gene sod-3, but neither

was affected by NAC treatment. ROS, produced for example by paraquat treatment, can activate the mito-UPR, which in turn can promote longevity (28–31). Therefore, we asked whether ROS might activate the mito-UPR in animals lacking the germ line. The nuclear-encoded mitochondrial Hsp70 chaperone gene hsp-6 is a transcriptional target of the mito-UPR, and we found that germline loss activated a Phsp-6::GFP reporter (Fig. 2A). This induction was specific, because the endoplasmic reticulumUPR was not induced (SI Appendix, Fig. S1F). Consistent with a role for ROS in mito-UPR induction, Phsp-6::GFP expression was attenuated by the antioxidant NAC (Fig. 2B) and by vitamin C treatment (Fig. 2C). We asked whether this reporter also might respond to the transsulfuration pathway, but its expression in germline-deficient animals was not affected by loss of the transsulfuration-pathway gene cbs-1 (SI Appendix, Fig. S1G). In C. elegans, activation of the mito-UPR is mediated by the transcription factor DVE-1 and its coactivator, the UBL-5 ubiquitin ligase (29). We found that both DVE-1 and UBL-5 were required for germline loss to activate Phsp-6::GFP expression (Fig. 2D). In addition, expression of ubl-5::GFP rose, specifically in the intestine, when the germ-cell precursors Z2 and Z3 were ablated (Fig. 2E and SI Appendix, Fig. S1H). Neither dve-1 nor ubl-5 was required for the DHE-reactive ROS signal (SI Appendix, Fig. S1I); thus, these genes likely act downstream of ROS to activate the mito-UPR. To investigate whether the mito-UPR contributed to the longevity of germline-deficient animals, we examined the lifespans of animals subjected to dve-1 or ubl-5 RNAi. ubl-5 was

Fig. 2. ROS activates the mito-UPR upon germ-cell loss. (A) Germline loss activates the mito-UPR. Animals expressing the mito-UPR marker Phsp-6::GFP were examined at day 1 of adulthood. (B) Mito-UPR induction by germline loss was reduced by the antioxidant NAC. Animals were treated with 8 mM NAC from the time of hatching and were examined at day 1 of adulthood. Worms were grouped as exhibiting high, medium, or low levels of GFP (see SI Appendix, SI Experimental Procedures). n, number of worms pooled from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups): *P < 0.05; **P < 0.001. (C) Mito-UPR induction by germline loss was reduced by vitamin C. Animals were treated with 100 μg/mL vitamin C from the time of hatching and were examined at day 1 of adulthood. *P < 0.05; **P < 0.001. (D) The transcription factor DVE-1 and its cofactor UBL-5, but not the mitochondrial protease CLPP-1, were required for mito-UPR induction in germline-less worms. RNAi was initiated at the time of hatching, and Phsp-6::GFP expression was examined at day 1 of adulthood. **P < 0.001. clpp-1 knockdown was confirmed by quantitative RT-PCR (SI Appendix, Fig. S9C). (E) UBL-5 protein levels were elevated in the posterior intestine by germ-cell removal. Animals expressing a UBL-5::GFP translational fusion were germ-cell (Z2 and Z3) ablated at the L1 stage and were imaged at day 1 of adulthood. (F) ubl-5 was specifically required for long lifespan of germline-deficient (glp-1) animals. ubl-5 RNAi was initiated at the time of hatching. Changes in mean lifespan are shown in parentheses. n >60 for each line. P values were determined using the log-rank test: ns, not significant; *P < 0.05; **P < 0.001. (G) dve-1 was specifically required for long lifespan of germline-less (glp-1) animals. dve-1 was knocked down by feeding RNAi bacteria from L1 to L3. Animals then were transferred to plates containing control RNAi bacteria. Changes in mean lifespan are shown in parentheses. n >60 for each line. P values were determined using the log-rank test: ns, not significant; *P < 0.05; **P < 0.001. dve-1 RNAi also prevented germline loss from extending life when the L3 animals were transferred to OP-50 bacteria or when dve-1 RNAi bacteria were diluted with control bacteria (see SI Appendix, SI Discussion and Fig. S9 A and B).

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The Transsulfuration Pathway Promotes Life Extension by Activating SKN-1/Nrf2. Next, we asked how the transsulfuration pathway and

H2S might extend life. One candidate target was SKN-1, the C. elegans ortholog of mammalian Nrf2. Nrf2 is a key oxidative stress-response transcription factor that activates antioxidant and phase II detoxification genes, which in turn detoxify free radicals and conjugate electrophiles (32, 33). SKN-1 overexpression is known to extend lifespan in C. elegans (34). In addition, SKN-1 is required for the lifespan extension caused by caloric restriction (35), and it contributes to the life extension caused by impaired insulin/IGF-1 signaling (34). Both ROS and H2S are known to activate Nrf2 (36); in particular, H2S has been shown to extend worm lifespan via increased SKN-1/Nrf2 activity (37). We tested skn-1 and found, as have others recently (38), that expression of the glutathione-S-

Fig. 3. H2S activates SKN-1 to extend life. (A) The SKN-1 target gene gst-4 was induced in the intestine by germ-cell loss. Animals expressing GFP driven by the gst-4 promoter (Pgst-4::GFP) were germ-cell ablated at L1 and were imaged at day 1 of adulthood. (B) NAC treatment attenuated Pgst-4::GFP induction in germline-less glp-1(ts) mutants. n, number of animals from three independent experiments. P values were determined using the χ2 test and the two-way ANOVA test (between groups): **P < 0.001. (C) Germline loss enhanced SKN-1 expression in intestinal nuclei. Wild-type and germline-less glp-1(ts) animals expressing SKN-1::GFP were imaged at day 1 of adulthood. Arrows indicate the nuclei of intestinal cells. (D) NAC decreased SKN-1 nuclear enrichment. Animals were treated with 8 mM NAC from L1 and were examined at day 1 of adulthood. n, number of animals from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups): ns, not significant: **P < 0.001. (E) cbs-1 RNAi decreased Pgst-4::GFP induction in germline-less glp-1(ts) mutants. n, number of animals from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups): **P < 0.001. [C. elegans has a second cystathionine beta-synthase gene, cbs-2. However, cbs-2 RNAi did not prevent germline loss from inducing Pgst-4::GFP (SI Appendix, Fig. S9 D and E). Thus, cbs-2 does not appear to act with cbs-1 in this pathway.] (F) SKN-1 was specifically required for the lifespan extension of germline-less animals. skn-1 was inactivated by the zu135 mutation. Changes in mean lifespan are shown in parentheses. n >60 for each line. P values were determined using the log-rank test: **P < 0.001. (G) elt-2 and mdt-15 were required for the extended lifespan of germline-less glp-1(ts) animals. elt-2 and mdt-15 RNAi were initiated at the L1 stage. Changes in mean lifespan are shown in parentheses. n >60 for each line. P values were determined using the log-rank test: ns, not significant; *P < 0.05; **P < 0.001.

Wei and Kenyon

PNAS | Published online May 2, 2016 | E2835


transferase reporter Pgst-4::GFP, a direct target of SKN-1, was induced upon loss of germ cells in C. elegans in a skn-1– dependent fashion (Fig. 3 A and B and SI Appendix, Fig. S2 A and B). A second SKN-1 reporter, Pgst-7::GFP, behaved similarly to Pgst-4::GFP in these and other experiments (see SI Appendix, SI Discussion, and Fig. S9 F–H). In addition, loss of germ cells triggered SKN-1 nuclear localization specifically in intestinal cells (Fig. 3 C and D). We asked whether Pgst-4::GFP induction and SKN-1 nuclear accumulation were sensitive to NAC and found that they were (Fig. 3 B and D). In contrast, Pgst-4::GFP induction was not sensitive to vitamin C (SI Appendix, Fig. S2C). Thus, SKN-1 was likely activated by the transsulfuration pathway and H2S rather than by ROS. Consistent with this interpretation, RNAi-inhibition of the transsulfuration-pathway gene cbs-1 reduced Pgst-4::GFP expression (Fig. 3E). We then tested whether skn-1 was required for germline loss to extend life. As shown previously (34), skn-1 RNAi shortened the lifespan of intact wild-type animals (SI Appendix, Fig. S2D), and, in agreement with recent, independent reports (25, 38), we found that germline loss did not extend life in skn-1(zu135) loss-of-function mutants (Fig. 3F). Because germline loss triggered a strong SKN-1 response in the intestine, we hypothesized that skn-1 acts in the intestine to extend life. In an extensive genetic analysis, we either inactivated or overexpressed tissue-specific isoforms of skn-1 in different


indeed required for germline loss to extend life (Fig. 2F). Testing dve-1 was problematic, because dve-1 RNAi caused ∼90% of the glp-1(−) mutants to remain small and often to rupture during midlife (SI Appendix, Fig. S1 J and K). Interestingly, the frequency of rupture (SI Appendix, Fig. S1J) and the size of the animals (SI Appendix, Fig. S1K) were affected to a greater extent in germline-deficient glp-1 mutants than in wild-type animals, suggesting a specific role of DVE-1 in the germline pathway. Consistent with this idea, when applied transiently or diluted to avoid rupture, dve-1 RNAi specifically shortened the lifespan of glp-1 mutants (Fig. 2G and SI Appendix, Fig. S9 A and B).

tissues and demonstrated this effect of skn-1 (SI Appendix, SI Discussion and Figs. S2 E–I and S3A). SKN-1 Is Activated by a Noncanonical Pathway. How does H2S activate SKN-1? Oxidative damage (39), impaired-insulin/IGF-1 signaling (34), and pathogen infection (40) all activate SKN-1 via the p38 MAP kinase PMK-1 and its upstream regulator SEK-1 kinase. However, pmk-1 makes only a small contribution to the longevity of germline-deficient animals (41). Similarly, the pmk-1(km25) deletion mutation only partially reduced Pgst-4::GFP expression in germline-deficient animals, and the sek-1(km4) mutation did not reduce it at all (SI Appendix, Fig. S3B). Likewise, the long lifespan of germline-deficient animals was reduced only partially by pmk-1(km25) and was not affected by sek-1(km4) (SI Appendix, Fig. S3 C and D). We inhibited other known and potential SKN-1 regulators as well, including nekl-2 and bli-3, but found no effect on Pgst-4::GFP (SI Appendix, Fig. S3E). Thus, germline loss and the resulting rise in H2S appear to activate gst-4 expression via a noncanonical pathway, only partially dependent on the p38 MAP kinase pathway. The C. elegans WD40-repeat protein WDR-23 appears to act downstream of PMK-1 as a negative regulator of SKN-1 (42, 43). As expected, wdr-23 RNAi treatment increased Pgst-4::GFP expression in intact animals; however, it did not further increase expression in glp-1(−) mutants (SI Appendix, Fig. S3F). This lack of additivity suggests that WDR-23 may be part of the germline longevity pathway. We identified two additional proteins that may function with SKN-1 to activate Pgst-4::GFP in germline-deficient animals: ELT-2, a GATA transcription factor, and MDT-15, a transcriptional-mediator subunit required for the expression of many SKN-1–up-regulated genes (44). elt-2 and mdt-15 RNAi both blocked Pgst-4::GFP induction (SI Appendix, Fig. S3E) as well as the extended lifespans of germline-defective animals (Fig. 3G). Neither gene knockdown prevented nuclear SKN-1 accumulation (SI Appendix, Fig. S3G), suggesting that ELT-2 and MDT-15 may function in parallel to SKN-1, e.g., as nuclear SKN-1 coactivators. The finding that skn-1 and the mito-UPR are induced by different redox systems suggested that their induction by germcell loss would be independent of one another. Consistent with this idea, induction of the mito-UPR reporter Phsp-6::GFP was not affected by knocking down skn-1, elt-2, or mdt-15 (SI Appendix, Fig. S4 A and E). Conversely, neither ubl-5 nor dve-1 RNAi prevented germline loss from activating the SKN-1 target gene Pgst-4::GFP (SI Appendix, Fig. S4B). KRI-1 Is Required for Germ-Cell Loss to Increase ROS and H2S Levels in Somatic Tissues. Several genes and processes are known to pro-

mote life extension in germline-deficient animals, and we asked whether any might be required to activate the SKN-1–dependent oxidative-stress response or the mito-UPR. Loss of the germline extends life only if the somatic gonad is present (12, 20). We tested the requirement for the somatic gonad and for components of the DAF-12/NHR sterol-signaling pathway, which mediates somatic-gonad signaling (20, 21). We also tested genes required for autophagy and genes affecting lipid metabolism that are required for germline loss to extend life. However, none of these genes, tissues, or processes was required for the DHE-sensitive ROS signal we observed or for gst-4 expression. Likewise, genes required for daf-2 inhibition to generate life-extending ROS (6) were not required (SI Appendix, SI Discussion and Figs. S4 C, D, and F–I, S5 A and B, and S9 C and D). We also examined the gene kri-1, which encodes an intestinal cytoskeletal protein required for many molecular and cellular events that occur in the intestine to extend lifespan when the germ line is removed (27, 45). We found that the kri-1(ok1251) loss-of-function mutation abolished the DHE ROS signal (Fig. 4A) as well as the mito-UPR signal (Phsp-6::GFP) in day-1 adults E2836 |

(Fig. 4B and SI Appendix, Fig. S5C). kri-1 also was partially required for the elevated production of H2S and for activation of Pgst-4::GFP expression in germline-defective worms (Fig. 4 C and D). KRI-1 controls the nuclear localization of DAF-16/FOXO in germline-deficient animals (27), so we asked whether kri-1 might act through DAF-16 to influence the DHE/ROS signal. However, the daf-16–null mutation mu86 did not prevent germline removal from increasing the DHE/ROS signal or the expression of Phsp-6::GFP or Pgst-4::GFP (Fig. 4 A and D and SI Appendix, Fig. S4E) in day-1 adults. Thus, KRI-1 acts independently of DAF-16 to influence SKN-1 and the mito-UPR. KRI-1 Regulates a Dynamic Pattern of ROS in Somatic Tissues. To learn more about KRI-1’s role in ROS generation, we asked whether the DHE/ROS signal we observed, which was cytoplasmic, might have a mitochondrial origin. This notion seemed plausible, because mitochondria are major sites of ROS production, and H2S also can be produced in mitochondria (36, 46). In addition, DHE can be oxidized by mitochondrial superoxide. To assay mitochondrial ROS levels more directly, we treated germline-deficient glp-1 mutants with a reduced (oxidizable) form of MitoTracker-Red that accumulates in functional mitochondria (MitoTracker Red H2-CMXRos; Invitrogen) (47). This sensor has been shown to detect mitochondrial ROS in worms (6). We found that H2-CMXRos, as well as DHE, generated an ROS signal in worms treated with the mitochondrial superoxide generator paraquat (SI Appendix, Fig. S5 D and E). H2-CMXRos also produced an elevated mitochondrial ROS signal in germlinedeficient worms, a signal that was attenuated by NAC antioxidant treatment (Fig. 4 E and F). We carried out a genetic experiment to ask whether mitochondrial superoxide was likely to play a role in this pathway. Inactivating the two C. elegans mitochondrial superoxide dismutase genes (sod-2 and sod-3) is known to raise mitochondrial ROS levels and extend life (48, 49). We found that the extended lifespans of germ cell-ablated animals were increased only slightly further by sod-2; sod-3 double mutation (SI Appendix, Fig. S5F). This lack of additivity is consistent with the interpretation that mitochondrial superoxide promotes the life extension of germlinedeficient animals, although we note that alternative, more complex interpretations are also possible. Although both DHE and H2-CMXRos detected elevated ROS levels in germline-deficient animals, we observed, quite unexpectedly, that the two ROS signals were different in their timing and their genetic dependency. The H2-CMXRos signal appeared earlier than the DHE signal, during the L4 stage of development, and then subsided by day 1 of adulthood, at which time the DHE signal reached its peak (Fig. 4G and SI Appendix, Fig. S5G). Moreover, kri-1 was required for germline loss to trigger the adult-specific DHE pattern but was not required for the mitochondrial H2-CMXRos ROS signal in L4 animals (Fig. 4G). Thus, KRI-1 is not required for ROS generation per se in germline-less animals but is required for an adult-specific form of ROS that triggers a DHE signal. If kri-1’s role in life extension involves ROS production, then the adult-specific DHE signal should be important. We investigated this idea by treating adults with the antioxidant NAC after the rise and fall of the H2-CMXRos signal. We found that adult-specific NAC treatment prevented activation of the SKN-1 and mito-UPR reporters Pgst-4::GFP and Phsp-6::GFP (SI Appendix, Fig. S5 H and I), which took place only in adults. In addition, adult-only NAC treatment shortened the lifespan of germline-defective animals (SI Appendix, Fig. S5J). These findings argue that the redox state of the adult, as reflected in the kri-1–dependent DHE signal, plays a role in the induction of life-extending pathways. Mitochondrial Biogenesis Is Enhanced by Loss of Germ Cells and by Low Levels of Paraquat. To begin to investigate the mechanism

by which germ-cell loss elevates ROS levels in the soma, we Wei and Kenyon


examined the somatic mitochondria, the initial sites of ROS accumulation, more closely. To visualize mitochondria, we created transgenic glp-1(ts) worms expressing GFP fused to a mitochondrial localization signal (mito-GFP). We found that the mito-GFP signal was elevated in the intestines of L4 germline-deficient animals (Fig. 5A and SI Appendix, Fig. S6A). To assess mitochondrial abundance in a different way, we dissected the intestines of glp-1 and wild-type worms away from the rest of the animal and measured relative mitochondrial and nuclear DNA levels using RT-PCR. In each of two experiments, we observed a modest but significant increase in mitochondrial DNA in L4 and day-1 adult germline-deficient animals relative to intact controls (Fig. 5B). We also examined a marker indicative of mitochondrial biogenesis, expression of the nuclear gene cts-1, which encodes mitochondrial citrate synthase. Expression of this gene also was increased in day-1 adults when germ cells were removed (Fig. 5C and SI Appendix, Fig. S6B). Interestingly, neither mitochondrial biogenesis marker was affected by antioxidant (NAC) treatment or by the kri-1(ok1251) mutation (Fig. 5 A and C), suggesting that mitochondrial biogenesis occurs independently of, and potentially upstream of, ROS generation. Together these findings are consistent with the idea that an increase in mitochondrial abundance in response to germline loss might cause or contribute to enhanced ROS levels.

SKN-1 is known to activate antioxidant and cell-protective gene expression. We were curious to know whether induction of SKN-1 and the mito-UPR has these effects in germline-defective animals. Indeed, in contrast to their elevated levels of DHEreactive ROS at day 1 of adulthood, germline-deficient worms exhibited much lower levels of DHE-reactive ROS accumulation later in life than did age-matched controls (Fig. 5D). The ability of germline loss to reduce the midlife accumulation of DHEreactive ROS was prevented by blocking the early-adult induction of ROS through kri-1 mutation (Fig. 5E) or by inactivating either SKN-1 or the mito-UPR (Fig. 5 E and F). The idea that mitochondria-associated redox signaling serves to restore cellular homeostasis also was demonstrated by a timecourse analysis of the mito-UPR reporter Phsp-6::GFP, the oxidative stress reporter Pgst-4::GFP, and the mitochondrial biogenesis marker Pcts-1::GFP, because expression of these reporters was higher in germline-deficient animals than in intact wild-type animals during early adulthood (day 1) but fell below wild-type expression levels later during adulthood (SI Appendix, Fig. S6 C–E). Together these experiments suggested that early induction of ROS and H2S serves to reduce the level of ROS, and potentially the level of oxidative damage, later in life.

KRI-1–Dependent Redox Signaling Improves ROS Homeostasis. The

KRI-1 Is Required for DHE-Sensitive ROS Production in Animals Treated with Paraquat. Like germline loss, paraquat elevates mi-

mito-UPR is known to restore mitochondrial homeostasis, and

tochondrial ROS levels. As predicted, like germline loss, paraquat

Wei and Kenyon

PNAS | Published online May 2, 2016 | E2837


Fig. 4. KRI-1 regulates redox signaling in response to germ-cell loss. (A) kri-1 was required for DHE-reactive ROS generation in germline-less animals, but daf16 was not. n, number of animals from three independent experiments. P values were determined using the χ2 test and two-way ANOVA (between groups): ns, not significant; *P < 0.05; **P < 0.001. (B) kri-1 loss prevented mito-UPR induction in glp-1 mutant animals. Wild-type and mutant animals expressing Phsp6::GFP were examined at day 1 of adulthood. n, number of animals from three independent experiments. P values were determined using the χ2 test and twoway ANOVA test (between groups): ns, not significant: **P < 0.001. (C) kri-1 mutation reduced H2S production in germline-mutant (glp-1) worms. Young adults were lysed, and various amounts of lysate were spotted on lead acetate paper. (D) kri-1 was required for expression of the SKN-1 target gene gst-4 in germ cell-ablated animals, but daf-16 was not. Wild-type and mutant animals expressing Pgst-4::GFP were Z2- and Z3-ablated at L1 and were examined at day 1 of adulthood. Data are from three independent experiments. n, number of animals examined. P values were determined using the χ2 test and two-way ANOVA (between groups): ns, not significant: **P < 0.001. (E) Mitochondria-specific ROS (mito-ROS) was elevated in germline-less glp-1(ts) animals at the L4 developmental stage. ROS was visualized using MitoTracker Red H2-CMXRos. Quantification is shown in F. (F) Mito-ROS was attenuated by NAC treatment. Animals were treated with 8 mM NAC from the time of hatching, and L4 animals were treated with H2-CMXRos. Data from five independent experiments are shown. n, number of animals examined. P values were determined using the χ2 test and two-way ANOVA (between groups): **P < 0.001. (G) The mito-ROS signal detected with H2-CMXRos peaked earlier than the DHE-ROS signal. kri-1 was required for the DHE signal but not for the mito-ROS signal. kri-1 and kri-1; glp-1 mutants were treated with H2-CMXRos or DHE at the indicated time points, and the fraction of animals with strong ROS induction was quantified.

Appendix, Fig. S7B). This finding also supports the hypothesis that DHE and H2-CMXRos detect different forms of ROS in worms. Paraquat also stimulated H2S production, and this production was kri-1 dependent as well (SI Appendix, Fig. S7C). Thus, we hypothesized that kri-1 and the downstream pathways it activates would be required for low levels of paraquat to extend life. We tested a range of low doses of paraquat and found that, even at optimal concentrations, paraquat produced only a small lifespan extension in kri-1 and also in skn-1 mutants (SI Appendix, Fig. S7D). Low (0.2 mM) paraquat also activated the mito-UPR reporter Phsp-6::GFP (30), and this activation was blocked by dve-1 RNAi and by ubl-5 RNAi (SI Appendix, Fig. S7E). Likewise, dve-1 RNAi treatment prevented low (0.2 mM) levels of paraquat from extending lifespan (SI Appendix, Fig. S7F). Thus, KRI-1, SKN-1, and the mito-UPR all promote longevity in response to a different source of ROS, low levels of paraquat. Interestingly, as with germline loss, the mitochondrial unfolded-protein sensor CLPP-1 is not required for mitoUPR induction by low levels of paraquat (30), a finding that is consistent with the interpretation that low levels of paraquat and germline ablation activate similar life-extending processes. We found that paraquat treatment enhanced the mito-GFP signal (SI Appendix, Fig. S6F). This finding seemed paradoxical, because germline loss enhanced mitochondrial biogenesis even in NAC-treated animals, which had reduced levels of ROS. One possibility is that germline loss activates a feed-forward loop, first triggering an ROS-independent increase in mitochondrial biogenesis with an attendant increase in ROS, which in turn triggers further increases in mitochondrial biogenesis, potentially via mito-UPR activation. KRI-1–Regulated Redox Signaling in Response to Mild Respiration Inhibition. ROS plays a key role in the longevity of mild respi-

Fig. 5. KRI-1–dependent redox signaling improves ROS homeostasis. (A) Germ-cell loss increased mitochondrial content in a kri-1– and ROSindependent manner. Animals expressing mito-GFP in the intestine were Z2- and Z3-ablated at L1 and were examined at day 1 of adulthood (See SI Appendix, Fig. S6A). NAC treatment was initiated at the time of hatching. Data are from three independent experiments. n, number of animals examined. P values were determined using the χ2 test and two-way ANOVA (between groups): ns, not significant; **P < 0.001. (B) Intestinal mitochondrial DNA content was increased upon loss of germ cells. Intestines of 20 wild-type animals and 20 glp-1 mutants were dissected from L4 larvae or day-1 adult animals, and mitochondrial DNA levels were evaluated using quantitative RT-PCR. Student’s t test: **P < 0.001. (C ) The mitochondrial biogenesis marker Pcts-1::GFP was induced by germ-cell ablation (also see SI Appendix, Fig. S6B). Control (N2), kri-1(−) mutants, and animals treated with NAC were Z2- and Z3-ablated at L1 and were quantified at day 1 of adulthood. Data are from three independent experiments. n, number of animals examined. P values were determined using the χ2 test and two-way ANOVA (between groups): ns, not significant; **P < 0.001. (D) Germline-less animals exhibited lower ROS levels later in life. Animals were treated with DHE at indicated time points, and the fraction of worms showing a strong DHE signal was plotted. Error bars show the SEM of data from three independent experiments (n > 100). Student’s t test: ns, not significant; *P < 0.05; **P < 0.001. (E) KRI-1 and SKN-1/Nrf2 were required for germline loss to reduce ROS levels later in life. Wild-type and glp-1(ts) germline-less animals in kri-1– or skn-1–mutant backgrounds were treated with DHE at day 10 of adulthood. Data are from two independent experiments. n, number of animals examined. χ2 test: ns, not significant; **P < 0.001. (F) The mito-UPR is required for germline loss to reduce ROS levels later in life. Wild-type and germline-less glp-1(ts) animals were subjected to dve-1 RNAi from the time of hatching to L3 and then were shifted to (non-RNAi) OP-50 bacteria (SI Appendix, SI Experimental Procedures). Animals were treated with DHE at day 10 of adulthood. Data are from three independent experiments. n, number of animals examined. χ2 test: ns, not significant; **P < 0.001.

treatment induced a mitochondrial H2-CMXRos signal independently of kri-1 (SI Appendix, Fig. S7A). However, surprisingly, paraquat’s ability to induce a DHE signal was kri-1 dependent (SI E2838 |

ration mutants (2, 3), as does the mito-UPR (31, 50). Therefore, we asked whether kri-1 and skn-1 might also be involved in this pathway. Indeed, we found that kri-1 RNAi prevented the isp-1(qm150) respiration mutation from extending life and that skn-1 RNAi greatly attenuated life extension (SI Appendix, Fig. S8 A–C). The elt-2 transcription factor is required for isp-1 mutants to live long (51), and we found mdt-15, the other putative SKN-1 coactivator identified in this study, was required as well (SI Appendix, Fig. S8D). Thus, multiple components of the ROS-dependent lifeextension pathway induced by germline loss are also activated by mutations that inhibit respiration. The detailed mechanism by which respiration inhibition and germline loss activate this redox-response may differ, however, because gcn-2, which is required to activate the mito-UPR when respiration is inhibited by isp-1 mutation (52), had no effect on mito-UPR induction in response to germline loss (SI Appendix, Figs. S1L and S9C). Aβ Toxicity and Environmental Stress Resistance. Expression of human Aβ-42 in C. elegans muscle paralyzes worms as they reach adulthood, and these Aβ animals have been used to model aspects of proteotoxicity (53). We found that laser ablation of the germ-cell precursors significantly delayed the onset of Aβ paralysis (SI Appendix, Fig. S8 E and F). The skn-1(−) mutation and dve-1 RNAi both prevented the delayed paralysis (SI Appendix, Fig. S8 E and F). Unexpectedly, kri-1 mutation had only a partial effect. KRI-1 is thought to be an intestinal protein, so it would be interesting to learn whether the germline’s effect on paralysis is caused by SKN-1 and mito-UPR activity in the intestine or in another tissue. Like many long-lived mutants, germline-deficient worms are stress resistant. We found that loss of kri-1, skn-1, or dve-1 reduced the oxidative stress resistance of glp-1 mutants (SI Appendix, Fig. S8 G and H). The finding that both SKN-1 and the mito-UPR contribute to the stress resistance of germline-defective animals could explain the puzzling finding that the stress-resistance regulator DAF-16 is required for the longevity of germline-defective animals but not for their heat resistance (54). Wei and Kenyon

Germline Loss Triggers a Dynamic Pattern of ROS in Somatic Tissues.

A major finding of this study is that loss of the germ line triggers a cell-nonautonomous increase in ROS within the soma, particularly in the intestine. By monitoring ROS using two different sensors, H2-CMXRos and DHE, we were able to observe two temporally distinct ROS waves. The first, a mitochondrial signal detected with H2-CMXRos but not DHE, peaked during the L4 stage of development. This ROS signal coincided with an increase in mitochondrial biogenesis. Thus, this first ROS wave may result simply from an overall increase in intestinal respiration. How and why germline loss stimulates mitochondrial biogenesis is unclear. Perhaps the organism interprets germline removal as a signal that germline maturation is incomplete and in response attempts to generate more energy for germline biosynthesis by increasing electron transport levels. Currently it is not possible to assay respiration levels directly in a tissuespecific fashion in germline-deficient vs. intact animals, and dissociating the intestine from the rest of the animal would likely alter any intercellular signaling responsible for changes in intestinal respiration. However, this energy-rebalancing hypothesis remains the simplest interpretation of the data and is consistent with a metabolic shift toward fat accumulation, likely a reservoir for excess energy, in these animals. When germline-deficient animals reach adulthood, the mitochondrial H2-CMXRos signal subsides and is replaced by a new ROS signal detected in the cytoplasm by DHE (SI Appendix, Fig. S10). This finding was unexpected. Both these sensors are able to detect superoxide, and both produce ROS signals in L4 and day1 adult animals treated with the superoxide generator paraquat (SI Appendix, Fig. S5 D and E). Nevertheless, our findings suggest that in C. elegans these two sensors detect different forms of ROS. Consistent with this interpretation, KRI-1 is required for the DHE signal but not for the H2-CMXRos signal in germline-deficient animals and in intact, wild-type animals treated with paraquat. At this point, we do not know the identity of the relevant ROS (or potentially reactive nitrogen species), and this information is notoriously difficult to acquire in living animals. Our findings do, however, underscore the value of using multiple ROS sensors to study redox regulation, even sensors assumed to have the same biological activity. Our findings with timed application of antioxidants indicate that adult-specific ROS activates life-extending pathways. Therefore, it is important to learn how this DHE-reactive species arises. We propose that this DHE signal coincides with the disappearance of the L4 mitochondrial H2-CMXRos signal because KRI-1 converts superoxide generated in the mitochondria to a second form of ROS, one that oxidizes DHE. Such a conversion is clearly possible because, directly or indirectly, KRI-1 is able to convert the superoxide generated by paraquat to a DHE-reactive ROS signal (SI Appendix, Fig. S10). This interpretation, in which the L4 mitochondrial ROS produced in response to germline loss leads ultimately to life extension, is consistent with the finding Wei and Kenyon


that germ-cell ablation is not additive with the life extension produced by genetic loss of mitochondrial superoxide dismutase activity. However, it remains possible that germ-cell loss directly triggers one form of ROS during development and then triggers another form, one that extends life, during adulthood. The kri-1(ok1251) mutation we analyzed is a null mutation; however, it does not completely prevent ROS and H2S levels from rising in paraquat-treated animals (SI Appendix, Fig. S7 B and C) and in glp-1 mutants (Fig. 4 A and C). Perhaps the loss of kri-1 decreases ROS and H2S levels below a threshold required to activate pathways that extend life. ROS Activates the Mito-UPR. The finding that vitamin C, an antioxidant that does not affect the transsulfuration pathway, reduced the longevity of germline-defective animals suggested that ROS itself promotes life extension. Moreover, our timed antioxidant experiments indicate that adult-specific ROS plays this role. We took a candidate approach to ask what life-extension pathway might be activated by this ROS and so identified the mito-UPR. MitoUPR activation can be sufficient to extend life, and the pathway promotes longevity in response to the inhibition of respiration in worms and flies (7, 31). We found that in animals lacking germ cells, the mito-UPR is activated specifically in the intestines of day-1 adults and that it is required for life extension. This activation was blocked by vitamin C, suggesting that it was induced by ROS. The mito-UPR is so-named because it can be activated by unfolded proteins. However, germline loss may activate the mito-UPR in another way, because neither the mitochondrial protease CLPP-1 nor the mitochondrial peptide transporter HAF-1 (29) is required for its induction (Fig. 2D and SI Appendix, Fig. S1M). Instead, as described above, in germline-deficient animals, the mito-UPR may be activated by ROS produced as the mitochondrial biogenesis response proceeds, thereby ensuring an adequate balance of nuclear-to-mitochondrial protein synthesis. The mito-UPR can be activated in the intestine when respiration is inhibited in neurons (31). Our findings show that neurons are not the only cells capable of activating the mito-UPR cell nonautonomously; reproductive tissues can do so as well. How this intertissue signaling occurs is not known in either case, but it is possible that the animal responds to a perceived energy shortage by producing the same signal or mitokine. Perhaps the intestine, which is an active metabolic organ that also serves as the animal’s adipose tissue, is poised to respond to changes in energy levels in a variety of other tissues. H2S and SKN-1 Activation. Recently, the longevity response to dietary restriction was shown to require the transsulfuration pathway (11). This pathway produces a number of sulfur-containing compounds, including H2S, which is sufficient to extend lifespan quite dramatically in worms (10, 37). We found that, as does dietary restriction, germline loss triggers an increase in H2S and that blocking the transsulfuration pathway blocked H2S production and shortened the lifespans of germline-defective worms. Because the superoxide generator paraquat also generated H2S, we propose that H2S is produced in germline-defective animals in response to elevated mitochondrial superoxide. Because H2S is sufficient to extend C. elegans’ lifespan (10), it seems likely that H2S, and not another product of the transsulfuration pathway, mediates life extension. However, it is important to note that we have not demonstrated a specific role for H2S directly. Activation of the mito-UPR was independent of the transsulfuration pathway. Again, we used a candidate approach to look for life-extension pathways that might be H2S dependent. Activation of SKN-1/Nrf2, a conserved oxidative- and xenobiotic-response regulator, can extend C. elegans’ lifespan, and SKN-1/Nrf2 is required for caloric restriction and insulin/IGF-1 signaling impairment to extend life (34, 35). Moreover, H2S has been shown to extend lifespan through SKN-1 activity in worms PNAS | Published online May 2, 2016 | E2839


Discussion ROS and H2S both play key roles in the induction of cell-protective processes that extend life in response to signals arising from environmental stress. Our findings suggest that C. elegans deploys both these substances to slow the aging of somatic tissues when its germ line is compromised (SI Appendix, Fig. S10). The precise origin of these species is not known; they may originate as a consequence of increased mitochondrial biogenesis during development. During adulthood, their effects bifurcate, with ROS activating the mito-UPR and H2S, and/or potentially other products of the transsulfuration pathway, activating SKN-1/Nrf2. Remarkably, this subnetwork also appears to influence the longevity response to redox species generated by low levels of paraquat or by a mild inhibition of respiration (SI Appendix, Fig. S10).

(37). We found that SKN-1 was activated in germline-deficient animals and that it was required for germline loss to extend life. The skn-1 dependence of the germline longevity pathway also was reported recently by others (38) while our manuscript was in preparation. Interestingly, SKN-1/Nrf2 was activated in a transsulfuration-dependent, and thus presumably H2S-dependent, fashion. H2S is known to activate Nrf2, but it seemed interesting that SKN-1/Nrf2 was not activated by the elevated levels of (DHE-reactive) ROS present in the germline-deficient animals, because Nrf2 is a classic oxidative stress-responsive transcription factor. SKN-1/Nrf2 activation occurs specifically at day 1 of adulthood, because it is blocked by adult-only NAC treatment. Perhaps the type of ROS that oxidizes DHE in our system is not capable of activating SKN-1. It is also possible that Nrf2 activation in other systems is sometimes attributed to ROS’ oxidative action when, in fact, ROS is acting indirectly, by stimulating the production of H2S. How might H2S activate SKN-1/Nrf2? Directly or indirectly, the ultimate target of this SKN-1–activation pathway is likely the WD-40 protein WDR-23. This activation must take place in the intestine, as indicated by our functional analysis of tissue-specific skn-1 alleles and the intestine-specific pattern of SKN-1’s nuclear localization and downstream gene expression. In general, WDR23 is thought to promote SKN-1 proteasomal degradation under unstressed conditions by forming a complex with SKN-1 and the ubiquitin ligase CUL-4/DBB1 in the nucleus (42, 43). Oxidative stress is hypothesized to disrupt the SKN-1–WRD-23 interaction, thereby stabilizing SKN-1. However, known upstream regulators of WDR-23, such as the p38 MAP kinase PMK-1 and the MAPKK SEK-1 (34, 39, 40), have only a modest role in this system. This finding is interesting and may potentially be linked to the fact that in this situation the more direct effector of SKN-1 activity is likely H2S rather than ROS. The Mysterious Role of KRI-1. Another major finding of this study is that the KRI-1 protein, which is required for many life-extending processes observed following germline loss, acts at an early step in this pathway to promote the formation of a life-extending form of ROS and for production of H2S (SI Appendix, Fig. S10). We see this effect not only in germline-deficient animals but also in intact animals treated with paraquat, which also have elevated levels of mitochondrial ROS. KRI-1 is localized to the C. elegans intestine (27), providing an attractive explanation for the intestinal localization of the lifeextending adult-specific DHE-sensitive ROS signal and downstream mito-UPR and SKN-1 activation, all of which are kri-1 dependent. Likewise, the tissue specificity of SKN-1 activation suggests that the transsulfuration pathway is activated only, or predominantly, in the intestine. How KRI-1 might influence cellular redox chemistry is not known. KRI-1 is a putative adaptor protein associated with the cytoskeleton, although it has been observed in the nucleus as well (27). Mammalian KRIT-1 suppresses integrin signaling (55, 56), but we found that integrin RNAi did not suppress the kri-1 mutant phenotype. Interestingly, the punctate pattern of cytoplasmic ROS within the intestine suggests that the oxidized DHE signal may emanate from lysosome-related gut granules, which become more prominent in glp-1 mutants (57) and possibly are less abundant in kri-1 mutants, which appear pale under the dissecting microscope. A possible role for lysosomes would be an interesting subject for future investigations. In mammalian cells grown in culture the loss of the KRI-1 ortholog KRIT1 raises the level of ROS, as assayed by DHE (58). This result seems paradoxical, because in germline-deficient worms loss of KRI-1 decreases the DHE signal. Although it is possible that worm KRI-1 and mammalian KRIT1 proteins influence the response to ROS in different ways, it is also possible that this apparent difference reflects the kinetics of the response: E2840 |

At older ages, kri-1 mutant worms also have elevated levels of DHE-reactive ROS relative to wild-type animals. A Stress-Responsive Subnetwork for Life Extension. It seems remarkable that ROS and H2S should both activate essential lifeextension pathways in response to loss of the germ line. This complexity could be rationalized by postulating that germline loss unleashes a variety of intercellular signals, each responding to the loss of a different aspect of germline cell biology. However, what appears to be the same redox response is elicited by paraquat, which, as far as we know, specifically generates mitochondrial superoxide. Likewise, many components of this system, the mito-UPR (31) and kri-1 and skn-1 (this study), are part of the mechanism by which the inhibition of respiration (which also generates ROS) extends life. Thus, this entire subnetwork could potentially be activated under different conditions to extend life. In summary, these findings add to a growing body of evidence that ROS and H2S (and potentially other products of the transsulfuration pathway) are general components of biological longevity pathways, and they broaden the scope of this ROS dependence to an endogenous physiological perturbation, germline loss. Interestingly, although a more detailed, side-by-side comparison is warranted, the data so far suggest that ROS can act in different ways to extend life in response to different stimuli. For example, an acute decrease in insulin/IGF-1 signaling produces a transient ROS signal that promotes life extension (6). This pathway has been reported to involve AMP kinase and altered proline metabolism, neither of which seems to be involved in the germline pathway (SI Appendix, SI Discussion). Likewise, we find that this KRI-1/SKN-1/mito-UPR–dependent pathway plays a key role in the process by which ROS promotes life extension in respiratory-chain mutants (2, 3), even though only one of these pathways (the germline pathway) is daf-16 dependent, and only one pathway (the one activated by respiration mutations) requires the HIF-1 transcription factor (2). Whether these differences are profound or instead are simply the consequence of perturbing an interconnected regulatory network in slightly different ways is an important unanswered question.

Materials and Methods Strains. C. elegans strains were maintained at 15 °C on standard nematode growth medium plates seeded with OP-50 bacteria. Experiments were conducted at 20 °C if not stated otherwise. The strain background was N2 (Bristol), to which all other strains were outcrossed at least four times. glp-1(e2144) III is a ts mutation that arrests germline proliferation when animals are grown from L1 to L4 at 25 °C. When using glp-1(e2144), the glp-1(+) control animals were temperature-shifted in parallel. [Please note that this allele, glp-1(e2144), was previously mislabeled as glp-1(e2141) in many C. elegans labs, including ours.] A list of C. elegans strains used in this study is shown in SI Appendix. Laser Ablation of Germ Cells. Gravid worms were dissolved using a bleaching solution (20% alkaline hypochlorite, 0.25M NaOH in H2O) for about 10 min. Embryos were washed at least three times with M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl) and were autoclaved; then 1 mM MgSO4 was added. Embryos were allowed to hatch in M9 buffer overnight to obtain a synchronized L1 population. L1 worms were paralyzed with 1 mM levamisole, placed on agarose pads on slides, and covered by a glass coverslip. Germ-cell precursors Z2 and Z3 (or, in some cases the somatic gonad precursors Z1 and Z4) were ablated using a MicroPoint laser-ablation system (Andor Technology). Drug Treatment. NAC, vitamin C, and paraquat (methyl viologen dichloride) were dissolved in water and added to agar plates already containing bacteria (food) at the indicated concentrations 1 d before worms were added. For lifespan assays, the animals were transferred to new drug plates every 3 or 4 d. If not stated otherwise, NAC and vitamin C treatments were initiated from hatching, and paraquat was initiated at the L4 stage. ROS and H2S Detection. Mitochondrial ROS was detected using the mitochondrial-localized ROS sensor MitoTracker Red H2CMXRos as described (6). Briefly, animals were incubated in M9 buffer containing 5 μM H2CMXRos for

Wei and Kenyon

1. Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40(4):869–907. 2. Lee SJ, Hwang AB, Kenyon C (2010) Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity. Curr Biol 20(23): 2131–2136. 3. Yang W, Hekimi S (2010) A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol 8(12):e1000556. 4. Schulz TJ, et al. (2007) Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab 6(4): 280–293. 5. Pan Y, Schroeder EA, Ocampo A, Barrientos A, Shadel GS (2011) Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling. Cell Metab 13(6):668–678. 6. Zarse K, et al. (2012) Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal. Cell Metab 15(4): 451–465. 7. Owusu-Ansah E, Song W, Perrimon N (2013) Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. Cell 155(3):699–712. 8. Wang D, Malo D, Hekimi S (2010) Elevated mitochondrial reactive oxygen species generation affects the immune response via hypoxia-inducible factor-1alpha in longlived Mclk1+/- mouse mutants. J Immunol 184(2):582–590. 9. Ristow M, et al. (2009) Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci USA 106(21):8665–8670. 10. Miller DL, Roth MB (2007) Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans. Proc Natl Acad Sci USA 104(51):20618–20622. 11. Hine C, et al. (2015) Endogenous hydrogen sulfide production is essential for dietary restriction benefits. Cell 160(1-2):132–144. 12. Hsin H, Kenyon C (1999) Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399(6734):362–366. 13. Arantes-Oliveira N, Apfeld J, Dillin A, Kenyon C (2002) Regulation of life-span by germ-line stem cells in Caenorhabditis elegans. Science 295(5554):502–505. 14. Flatt T, et al. (2008) Drosophila germ-line modulation of insulin signaling and lifespan. Proc Natl Acad Sci USA 105(17):6368–6373. 15. Leopold AC, Niedergang-Kamien E, Janick J (1959) Experimental Modification of Plant Senescence. Plant Physiol 34(5):570–573. 16. Mason JB, Cargill SL, Anderson GB, Carey JR (2009) Transplantation of young ovaries to old mice increased life span in transplant recipients. J Gerontol A Biol Sci Med Sci 64(12):1207–1211. 17. Zhang G, et al. (2013) Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature 497(7448):211–216. 18. Min KJ, Lee CK, Park HN (2012) The lifespan of Korean eunuchs. Curr Biol 22(18): R792–R793. 19. Boulias K, Horvitz HR (2012) The C. elegans microRNA mir-71 acts in neurons to promote germline-mediated longevity through regulation of DAF-16/FOXO. Cell Metab 15(4):439–450. 20. Yamawaki TM, et al. (2010) The somatic reproductive tissues of C. elegans promote longevity through steroid hormone signaling. PLoS Biol 8(8):e1000468. 21. Shen Y, Wollam J, Magner D, Karalay O, Antebi A (2012) A steroid receptor-microRNA switch regulates life span in response to signals from the gonad. Science 338(6113): 1472–1476. 22. Goudeau J, et al. (2011) Fatty acid desaturation links germ cell loss to longevity through NHR-80/HNF4 in C. elegans. PLoS Biol 9(3):e1000599. 23. Lapierre LR, Gelino S, Meléndez A, Hansen M (2011) Autophagy and lipid metabolism coordinately modulate life span in germline-less C. elegans. Curr Biol 21(18): 1507–1514. 24. Hansen M, Hsu AL, Dillin A, Kenyon C (2005) New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLoS Genet 1(1):119–128. 25. Vilchez D, et al. (2012) RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489(7415):263–268. 26. McCormick M, Chen K, Ramaswamy P, Kenyon C (2012) New genes that extend Caenorhabditis elegans’ lifespan in response to reproductive signals. Aging Cell 11(2):192–202. 27. Berman JR, Kenyon C (2006) Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 124(5):1055–1068. 28. Yoneda T, et al. (2004) Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J Cell Sci 117(Pt 18):4055–4066. 29. Haynes CM, Ron D (2010) The mitochondrial UPR - protecting organelle protein homeostasis. J Cell Sci 123(Pt 22):3849–3855.

30. Runkel ED, Liu S, Baumeister R, Schulze E (2013) Surveillance-activated defenses block the ROS-induced mitochondrial unfolded protein response. PLoS Genet 9(3): e1003346. 31. Durieux J, Wolff S, Dillin A (2011) The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144(1):79–91. 32. An JH, Blackwell TK (2003) SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev 17(15):1882–1893. 33. Itoh K, Mimura J, Yamamoto M (2010) Discovery of the negative regulator of Nrf2, Keap1: A historical overview. Antioxid Redox Signal 13(11):1665–1678. 34. Tullet JM, et al. (2008) Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 132(6):1025–1038. 35. Bishop NA, Guarente L (2007) Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature 447(7144):545–549. 36. Wallace JL, Wang R (2015) Hydrogen sulfide-based therapeutics: Exploiting a unique but ubiquitous gasotransmitter. Nat Rev Drug Discov 14(5):329–345. 37. Miller DL, Budde MW, Roth MB (2011) HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans. PLoS One 6(9):e25476. 38. Steinbaugh MJ, et al. (2015) Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence. eLife 4:e07836. 39. Inoue H, et al. (2005) The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev 19(19):2278–2283. 40. Hoeven Rv, McCallum KC, Cruz MR, Garsin DA (2011) Ce-Duox1/BLI-3 generated reactive oxygen species trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans. PLoS Pathog 7(12):e1002453. 41. Alper S, et al. (2010) The Caenorhabditis elegans germ line regulates distinct signaling pathways to control lifespan and innate immunity. J Biol Chem 285(3):1822–1828. 42. Choe KP, Przybysz AJ, Strange K (2009) The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol Cell Biol 29(10):2704–2715. 43. Hasegawa K, Miwa J (2010) Genetic and cellular characterization of Caenorhabditis elegans mutants abnormal in the regulation of many phase II enzymes. PLoS One 5(6): e11194. 44. Oliveira RP, et al. (2009) Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8(5):524–541. 45. Ghazi A, Henis-Korenblit S, Kenyon C (2009) A transcription elongation factor that links signals from the reproductive system to lifespan extension in Caenorhabditis elegans. PLoS Genet 5(9):e1000639. 46. Fu M, et al. (2012) Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production. Proc Natl Acad Sci USA 109(8):2943–2948. 47. Poot M, Pierce RH (1999) Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry. Cytometry 35(4):311–317. 48. Van Raamsdonk JM, Hekimi S (2009) Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans. PLoS Genet 5(2):e1000361. 49. Van Raamsdonk JM, Hekimi S (2010) Reactive oxygen species and aging in Caenorhabditis elegans: Causal or casual relationship? Antioxid Redox Signal 13(12): 1911–1953. 50. Mouchiroud L, et al. (2013) The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Cell 154(2):430–441. 51. Shore DE, Carr CE, Ruvkun G (2012) Induction of cytoprotective pathways is central to the extension of lifespan conferred by multiple longevity pathways. PLoS Genet 8(7): e1002792. 52. Baker BM, Nargund AM, Sun T, Haynes CM (2012) Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2. PLoS Genet 8(6): e1002760. 53. Link CD (2005) Invertebrate models of Alzheimer’s disease. Genes Brain Behav 4(3): 147–156. 54. Libina N, Berman JR, Kenyon C (2003) Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell 115(4):489–502. 55. Glading AJ, Ginsberg MH (2010) Rap1 and its effector KRIT1/CCM1 regulate betacatenin signaling. Dis Model Mech 3(1-2):73–83. 56. Zawistowski JS, Serebriiskii IG, Lee MF, Golemis EA, Marchuk DA (2002) KRIT1 association with the integrin-binding protein ICAP-1: A new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. Hum Mol Genet 11(4):389–396. 57. O’Rourke EJ, Soukas AA, Carr CE, Ruvkun G (2009) C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab 10(5):430–435. 58. Goitre L, et al. (2010) KRIT1 regulates the homeostasis of intracellular reactive oxygen species. PLoS One 5(7):e11786.

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PNAS | Published online May 2, 2016 | E2841


ACKNOWLEDGMENTS. We thank Dr. Keith Blackwell at Harvard Medical School and Dr. Christopher Link at the University of Colorado for strains and reagents; Dr. Sivan Henis-Korenblit, then a postdoctoral researcher in the C.K. laboratory, for creating the glp-1(e2144); skn-1(zu135) double-mutant strain and for her unpublished discovery, in ∼2006, that skn-1 is required for the longevity of glp-1 mutants; Richard Parenteau and Hildegard Mack for help with experiments; members of the C.K. laboratory for helpful discussions; Rex Kerr for advice regarding statistics; and Dr. Hao Li for University of California, San Francisco laboratory space after August 2015. Some strains were provided by the Caenorhabditis Gene Center, which is funded by NIH Office of Research Infrastructure Programs Grant P40 OD010440. This study was funded by a postdoctoral fellowship from the American Heart Association (to Y.W.) and by NIH Grant R01 AG032435 (to C.K.).


30 min, washed extensively with M9 buffer, and imaged and quantified using a Zeiss Axio Scope microscopy platform. For DHE assays, animals raised on agar plates were washed in M9 buffer and then were incubated in M9 buffer containing 3 μM DHE for 30 min. Worms were washed extensively in M9 buffer before the ROS signal was imaged and quantified using a Zeiss Axio Scope microscopy platform. H2S was measured using lead acetate paper (11). Briefly, worms were lysed in PBS supplemented with 10 mM Cys and 10 μM–6 mM pyridoxal 5′-phosphate hydrate. Worm lysate then was spotted on lead acetate paper overnight or until a signal appeared. Lead acetate paper was prepared by soaking filter paper in a 20-mM lead acetate solution. For additional experimental procedures and statistical analyses, please see SI Appendix, SI Experimental Procedures.

Roles for ROS and hydrogen sulfide in the longevity response to germline loss in Caenorhabditis elegans.

In Caenorhabditis elegans, removing germ cells slows aging and extends life. Here we show that transcription factors that extend life and confer prote...
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