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The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans

The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans ARTICLE Received 4 Mar 2013 | Accepted 8 Jul 2013 | Published 8 Aug 2013 DOI: 10.1038/ncomms3267 The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans 1 2 1,3 1 4 Louis R. Lapierre , C. Daniel De Magalhaes Filho , Philip R. McQuary , Chu-Chiao Chu , Orane Visvikis , 1 1,3 1 1 4 2 Jessica T. Chang , Sara Gelino , Binnan Ong , Andrew E. Davis , Javier E. Irazoqui , Andrew Dillin & Malene Hansen Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity- extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans. Program of Development and Aging, Del E Webb Neuroscience, Aging and Stem Cell Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California, USA. The Howard Hughes Medical Institute, The Glenn Center for Aging Research, The Salk Institute for Biological Studies, La Jolla, California, USA. Graduate School of Biomedical Sciences, Sanford-Burnham Medical Research Institute, La Jolla, California, USA. Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Correspondence and requests for materials should be addressed to M.H. (email: [email protected]). NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 9–11 ging is modulated by multiple conserved signal trans- C. elegans via TOR, as observed in mammalian cells . duction pathways, such as the insulin/insulin-like Consistent with this possibility, we observed green fluorescent Agrowth factor-1 (IGF-1) and target of rapamycin (TOR) protein (GFP)-tagged HLH-30 to be enriched prominently in the signalling pathways. Studies in model organisms, including the nuclei of intestinal cells in adult glp-1(e2141) mutants (Fig. 1). nematode Caenorhabditis elegans, have identified key regulatory Similarly, we observed nuclear localization of HLH-30 in wild- roles for several transcription factors in these pathways . type (WT; N2 Bristol) animals subjected to tor RNA interference However, the degree to which these distinct longevity pathways (RNAi) (Fig. 2). These observations suggest that HLH-30 localizes converge on common downstream mechanisms remains an to the nucleus in C. elegans intestinal cells under conditions important topic of investigation. Macroautophagy (hereafter associated with induction of autophagy, including TOR 9–11 referred to as autophagy) is a cellular recycling process that has inhibition, similar to TFEB in mammalian cells . emerged as a crucial mechanism for lifespan extension in many different species . During autophagy, cytosolic components are sequestered in vesicles called autophagosomes, which then HLH-30 regulates the expression of orthologous TFEB targets. fuse with lysosomes, allowing the contents to be enzymatically We next investigated the expression of hlh-30 and orthologues of degraded and recycled. Notably, long-lived C. elegans mutants TFEB target genes in glp-1(e2141) mutants and in WT animals with reduced insulin/IGF-1 signalling, TOR signalling, germline with reduced TOR levels. We found that hlh-30 mRNA levels signalling, food intake or mitochondrial respiration, all exhibit were markedly increased in both glp-1(e2141) and tor(RNAi) increased levels of autophagy and require autophagy genes for mutants when compared with WT animals (Figs 1b and 2b). lifespan extension . However, the mechanism(s) by which these Moreover, glp-1(e2141) mutants showed increased expression of long-lived animals regulate the autophagy process is not fully several nematode orthologues of human TFEB target genes, understood. including genes involved in autophagosome formation and We recently reported that the forkhead transcription factor autophagic flux, such as lgg-1/LC3 and sqst-1/SQSTM1/p62, and PHA-4/FOXA regulates the expression of several genes important genes with lysosomal functions, such as lmp-1/LAMP-1, subunits for autophagy in response to germline removal or TOR inhibition of vacuolar ATPases (vha-15, vha-16 and vha-17), sulphatases in C. elegans . Moreover, PHA-4/FOXA and autophagy genes (sul-2/ASRA and sul-3/ASRA) and cathepsins (ctsa/cathepsin A, 4,5 are essential for lifespan extension in these animals , as has been cpr-1/cathepsin B and asp-1/cathepsin D) (Fig. 1b). Several of 6,7 observed in dietary-restricted C. elegans . However, PHA-4/ these autophagy-related and lysosomal genes were similarly FOXA may not be a general transcriptional regulator of auto- changed in adult WT animals subjected to tor RNAi (Fig. 2b). phagy, because inhibition of PHA-4/FOXA has only a minor Changes in the expression of these genes may be transcriptionally effect on the long lifespan of insulin/IGF-1 signalling mutants or regulated by hlh-30, because glp-1(e2141); hlh-30(tm1978) double animals with reduced mitochondrial respiration . Thus, it is not mutants or hlh-30(tm1978) single mutants fed bacteria expressing clear whether induction of autophagy in these longevity pathways tor dsRNA, all showed a profound reduction in transcription of is mediated by a common transcriptional regulator. most of the putative target genes (Figs 1c and 2c). The The basic helix-loop-helix (HLH) transcription factor EB glp-1(e2141) mutants fed bacteria expressing hlh-30 dsRNA only (TFEB) was recently identified as a key transcriptional regulator during adulthood showed decreased expression of most target of the autophagy process in mammals . In mammalian cells, genes (Supplementary Fig. S1a), whereas inhibition of hlh-30 by TFEB translocates to the nucleus in response to nutrient RNAi in WT animals had a modest or no effect on the expression 9–11 deprivation, at least in part, via a TOR-dependent mechanism of these genes (Supplementary Fig. S1b). We also found that to ensure the transcriptional induction of many autophagy-related hlh-30(tm1978) single mutants displayed decreased expression of and lysosomal genes .Inthisstudy,weasked whether C. elegans some (for example, atg-18), but not all, of these genes (Supplemen- contains a functional orthologue of TFEB, and if so, whether the tary Fig. S1c), consistent with a previously published microarray orthologue has a role in lifespan determination. To this end, we analysis . Notably, most of the genes examined contain one or examined HLH-30 (W02C12.3, www.wormbase.org), which is a more E-box motifs in their promoters (Supplementary Table S1); member of a group of 42 HLH transcription factors in C. elegans this sequence overlaps with that of the CLEAR (Coordinated and has the highest homology to TFEB in the DNA-binding Lysosomal Expression And Regulation) binding motif, which is domain and in an acidic activation domain . We report that present in the promoters of many lysosomal gene targets of HLH-30 regulates the expression of multiple autophagy-related TFEB . Collectively, these data suggest that HLH-30 functionally and lysosomal genes, and modulates the autophagy process in mimics TFEB by inducing the transcription of autophagy-related C. elegans. HLH-30 is required for the long lifespan observed in all and lysosomal genes. C. elegans longevity mutants tested, is nuclear localized in these long-lived models, and the overexpression of HLH-30 extends the C. elegans lifespan. Taken together, these observations suggest that HLH-30 modulates autophagy and lysosomal function.To increased HLH-30 activity may be a universal mechanism for directly determine whether HLH-30 regulates autophagy, we used longevity. Finally, we find that TFEB levels are increased in the liver transgenic WT and glp-1(e2141) animals expressing GFP repor- nuclear extracts of dietary-restricted mice, raising the possibility ters that allow distinct steps of the autophagy process to be that HLH-30/TFEB modulates lifespan in a conserved manner. examined. First, we used a strain expressing GFP-tagged LGG-1/ LC3 (ref. 15), a protein that localizes to preautophagosomal and autophagosomal membranes, to investigate autophagosome Results formation. Compared with WT animals, glp-1(e2141) mutants HLH-30 locates to the nucleus in animals with reduced TOR contained more GFP::LGG-1-positive punctae, reflecting an activity. Our recent work showed that autophagy is tran- increased number of autophagosomes . However, significantly scriptionally activated in long-lived, germline-less C. elegans, such fewer GFP::LGG-1-positive punctae were detected in both as temperature-sensitive glp-1/Notch receptor mutants, possibly hypodermal seam cells and intestinal cells of hlh-30 RNAi- via a reduction in TOR activity . Therefore, we used glp-1(e2141) treated glp-1(e2141) animals compared with that in control mutants raised at the restrictive temperature to investigate animals (Fig. 1d, e), suggesting that hlh-30 is required for whether HLH-30 has a role in autophagy regulation in autophagy induction in glp-1(e2141) mutants. 2 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE 75% WT WT glp-1 ** ** ** 1.5 50% ** ** glp-1 25% 0.5 0% WT glp-1 Control hlh-30 RNAi Control hlh-30 RNAi ** WT glp-1 12 ** Control ** ** ** ** ** 6 ** ** ** hlh-30 RNAi Autophagy Fusion H Sulphatases Cathepsins Control hlh-30 Pumps RNAi ** glp-1 glp-1; hlh-30 Control ** ** 1.5 1 25 ** * 20 0.5 ** ** ** ** ** ** ** * ** * hlh-30 RNAi Autophagy Fusion H Sulphatases Cathepsins Control hlh-30 Pumps RNAi Figure 1 | HLH-30 regulates autophagy in germline-less C. elegans. (a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25 C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, B50 animals each, mean s.d., **Po0.01, Student’s t-test). Magnification  100; scale bar, 100mm. (b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WTand glp-1(e2141) animals raised at 25 C (mean s.d. of three biological replicates, *Po0.05, **Po0.01, Student’s t-test). (c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25 C (mean s.d. of three biological replicates, *Po0.05, **Po0.01, Student’s t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA. (d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 C and analysed at the L3 larval stage (mean s.e.m. ofB300 seam cells andB25 intestines, **Po0.01, Student’s t-test). (f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25 C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean s.d. of B10 animals, **Po0.01, Student’s t-test) and SQST-1::GFP foci (mean s.d. of B30 animals, **Po0.01, Student’s t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification,  200; scale bar, 100mm. To monitor the lysosomal compartment, we used a strain impairment of autophagy turnover. Finally, to monitor auto- expressing GFP-tagged LMP-1/LAMP1 (ref. 16), a glycoprotein phagic flux we used a strain expressing GFP-tagged SQST-1/p62 localized to the lysosomal membrane. Germline-less glp-1(e2141) (ref. 17), an adaptor protein that targets autophagic cargo for animals displayed slightly elevated expression of LMP-1/LAMP1 degradation. SQST-1 was expressed at low levels in adult animals, (Supplementary Fig. S2a), indicative of enhanced lysosomal where it localized to distinct subcellular sites, referred to as foci. biogenesis. This is consistent with our previous observation Interestingly, we found the number of SQST-1::GFP foci was that germline-less animals display increased autophagosome modestly increased in certain tissues of glp-1(e2141) mutants, formation . Notably, the expression of the LMP-1::GFP reporter including in some intestinal cells (Fig. 1g and Supplementary was robustly increased in the absence of hlh-30 (Fig. 1f and Fig. S2c). This may reflect a higher steady-state level of cargo Supplementary Fig. S2b), as would be expected following the delivery to autophagosomes and increased autophagic flux in NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 3 & 2013 Macmillan Publishers Limited. All rights reserved. Relative expression Relative expression HLH-30::GFP hlh-30 hlh-30 atg-18 atg-18 lgg-1 lgg-1 atg-9 atg-9 sqst-1 p62 sqst-1 vps-11 vps-11 vps-18 vps-18 lmp-1 lmp-1 vha-15 vha-15 vha-16 vha-16 vha-17 vha-17 % Of animals with nuclear localization of HLH-30 sul-1 sul-1 sul-2 sul-2 sul-3 sul-3 ctsa* ctsa* cpr-1 cpr-1 asp-1 asp-1 No. of GFP::LGG-1 foci/ glp-1; SQST-1::GFP glp-1; LMP-1::GFP seam cell No. of SQST-1::GFP aggregates Relative LMP-1::GFP No. of GFP::LGG-1 foci/ in distal intestine/animal fluorescence per worm proximal intestinal cells ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 HLH-30-regulated genes are required for the long lifespan of WT + control glp-1 animals (for example, atg-18 and lgg-1 (ref. 4)), we next 75% 100 WT + tor RNAi determined whether HLH-30 has a role in C. elegans lifespan. We hlh-30 + control performed survival analyses on adult WT and glp-1 animals fed ** hlh-30 + tor RNAi 50% bacteria, expressing hlh-30 dsRNA. Although hlh-30 RNAi administered during adulthood did not affect the lifespan of 25% WT animals, this treatment significantly shortened the long lifespan of glp-1(e2141) mutants (Fig. 3 and Supplementary Table 0% S2). Likewise, we found glp-1(e2141); hlh-30(tm1978) double Control tor 010 20 30 40 mutants to have mean lifespans similar to hlh-30(tm1978) single RNAi Days of adulthood mutants (Supplementary Fig. S3a and Supplementary Table S2). In addition, RNAi-mediated inhibition of the lysosomal gene Control 4 vha-16, but not of lmp-1, substantially reduced the long lifespan WT of glp-1(e2141) mutants (Supplementary Fig. S3b-d and 3 tor RNAi * * Supplementary Table S3), supporting the notion that a subset of ** 2 * HLH-30-regulated genes, with functions relevant to autophagy, may contribute to lifespan extension. Similar to germline removal, reduced TOR activity extends lifespan at least partly via an autophagy-dependent mechanism . To test whether hlh-30 has a role in TOR-mediated longevity in C. elegans, we fed bacteria expressing tor dsRNA to adult Autophagy Fusion H Sulfatases Cathepsins WT animals or mutants lacking hlh-30 bacteria. As expected, Pumps tor RNAi significantly extended the lifespan of WT animals; however, the same treatment failed to prolong the lifespan of Control hlh-30(tm1978) mutants (Fig. 2d and Supplementary Table S4), hlh-30 tor RNAi suggesting that hlh-30 acts downstream of TOR. Consistent with this notion, we found that hlh-30 was required for the develop- 1 * mental arrest of C. elegans induced by treatment with tor RNAi for multiple generations (Supplementary Fig. S4a, b). Inhibition of hlh-30 also prevented dauer formation (Supplementary Fig. S4c), a developmental stage in which autophagy plays a critical role , indicating that developmental arrest may be governed by Autophagy Fusion H Sulfatases Cathepsins Pumps hlh-30, possibly via autophagy regulation. Taken together, these observations demonstrate that HLH-30 is a novel longevity- Figure 2 | HLH-30 is required for TOR inhibition to extend C. elegans modulating transcription factor in C. elegans, which likely acts lifespan. (a) Nuclear localization of HLH-30 was quantified in day 1 adult downstream of TOR. As HLH-30 regulates several autophagy- animals expressing HLH-30::GFP. Animals were fed bacteria expressing related genes with effects on longevity, it is possible that HLH-30 control or tor dsRNA from hatching and raised at 20 C (three biological affects lifespan via regulation of autophagy in both germline-less replicates, B50 animals each, mean s.d., **Po0.01, Student’s t-test). animals and animals with reduced TOR function. (b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA HLH-30 is required for lifespan extension in other mutants.In from hatching (20 C). Data are mean s.d. of biological triplicates. addition to germline removal and TOR inhibition, several other *Po0.05, **Po0.01; Student’s t-test. csta* is a cathepsin A orthologue mechanistically distinct and conserved longevity pathways (cosmid C08H9.1 (ref. 30)).(d) Lifespan analysis of WT animals and modulate aging in C. elegans: dietary restriction (DR; the feeding- hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from deficient mutant eat-2 (ref. 19)), inhibition of insulin/IGF-1 day 1 of adulthood was carried out at 20 C. See Supplementary Table S3 signalling (the receptor mutant daf-2 (ref. 20)), impaired mito- for details of lifespan analyses and replicate experiments. chondrial respiration (the ubiquinone synthesis mutant clk-1 (ref. 21)) and reduced translation (the ribosomal S6 kinase mutant rsks-1 (refs 22,23)). We therefore investigated whether specific tissues of these animals. The number of SQST-1::GFP foci hlh-30 has a role in lifespan determination in these longevity was significantly increased following hlh-30 RNAi treatment models. To do these experiments, we initiated hlh-30 RNAi during (Fig. 1g and Supplementary Fig. S2d), consistent with a block in adulthood (for eat-2(ad1116), daf-2(e1370) and rsks-1(sv31) autophagic turnover of SQST-1/p62. In glp-1 animals with mutants) or during the fourth larval stage (for clk-1(e2519) reduced HLH-30 levels, the modest increase in the expression mutants). We found that hlh-30 RNAi significantly shortened the of both LMP-1/LAMP1 and SQST-1/p62 occurred, despite a lifespan of these mutants (Fig. 3c–f and Supplementary Table S2), marked reduction in their mRNA levels (Supplementary Fig. suggesting a common role for HLH-30 in their longevity. S1b), implying that HLH-30 is necessary for the turnover of these To further investigate this possibility, we introduced the proteins. Taken together, these results indicate that inhibition of HLH-30::GFP transgene into eat-2, daf-2, clk-1 and rsks-1 HLH-30 impairs the coordination of the autophagy process at mutants. Consistent with a regulatory role for hlh-30,we several steps, and suggest that HLH-30 regulates autophagy in a observed that HLH-30 was nuclear localized in the intestine of manner similar to TFEB . eat-2(ad1116), daf-2(e1370), clk-1(e2519) and rsks-1(sv31) animals (Fig. 4a). Interestingly, although hlh-30 mRNA levels were significantly increased in these four long-lived mutants compared glp-1 and TOR mutants require HLH-30 for lifespan with WT animals (Fig. 4b), fewer of the TFEB target gene extension. As we previously reported that several of the putative orthologues with functions in autophagy (for example, atg-9) 4 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. Relative expression Relative expression % Of animals with nuclear localization of HLH-30 tor tor hlh-30 hlh-30 atg-18 atg-18 lgg-1 lgg-1 atg-9 atg-9 sqst-1 sqst-1 vps-11 vps-11 % Alive vps-18 vps-18 lmp-1 lmp-1 vha-15 vha-15 vha-16 vha-16 vha-17 vha-17 sul-1 sul-1 sul-2 sul-2 sul-3 sul-3 ctsa* ctsa* cpr-1 cpr-1 asp-1 asp-1 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE 100 100 Control Control hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 WT glp-1 010 20 30 010 20 30 40 Days of adulthood Days of adulthood Control Control 100 100 hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 eat-2 daf-2 0 0 010 20 30 0 20 40 60 80 100 Days of adulthood Days of adulthood Control 100 Control hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 clk-1 rsks-1 010 20 30 40 010 20 30 40 Days of adulthood Days of adulthood Figure 3 | HLH-30 is required for the long lifespan of multiple longevity mutants. Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments. were upregulated in eat-2(ad1116), daf-2(e1370), clk-1(e2519) and (Supplementary Fig. S6b), suggesting that the long life of rsks-1(sv31) mutants (Supplementary Fig. S6a) than that in the HLH-30-overexpressing animals may be mediated through par- glp-1(e2141) (Fig. 1b) and tor(RNAi) (Fig. 2b) mutants. These tially overlapping mechanisms. Notably, HLH-30-overexpressing results suggest that HLH-30 may function differently in the animals had increased numbers of GFP-positive, LGG-1-positive distinct longevity mutants. As autophagy has been shown to have punctae (Fig. 4d), and inhibition of the autophagy gene atg-18 a role in the longevity of eat-2(ad1116) (refs 7,24,25), daf- reduced the lifespan of these animals (Supplementary Table S8). 2(e1370) (refs 7,15,26) and clk-1(e2519) (Supplementary Table S5) Thus, HLH-30 may extend lifespan, at least in part, by inducing mutants, and possibly also in rsks-1(sv31) mutants autophagy. Collectively, our data provide evidence that HLH-30 (Supplementary Fig. S5 and Supplementary Table S6), these is a conserved transcription factor with a novel universal role in findings suggest that autophagy could be differentially regulated modulating C. elegans lifespan. in the C. elegans longevity models. These results also indicate that HLH-30 may regulate additional genes with roles in longevity that are not necessarily related to autophagy. Dietary restriction regulates TFEB in mice. To determine whether TFEB, the mammalian orthologue of HLH-30, has a conserved role in lifespan determination, we asked whether Overexpression of HLH-30 extends C. elegans lifespan. Our TFEB is regulated under conditions that modulate longevity in results indicated a requirement for hlh-30 in multiple C. elegans mammals. For this, we examined mice placed on a dietary- longevity models. To determine whether HLH-30 expression was restricted diet, an effective non-genetic intervention that extends sufficient to extend C. elegans lifespan, we generated strains stably lifespan in mammals . In two key nutrient-responsive tissues, overexpressing HLH-30::GFP from transgenic, integrated arrays the liver and pancreas, we found TFEB mRNA levels to be and measured the lifespan of two independent lines. We observed significantly increased in male and female mice subjected to DR that the mean lifespan of the transgenic animals was B15–20% compared with control mice fed ad libitum (AL) (Supplementary longer than that of WT animals (Fig. 4c and Supplementary Fig. S8). Consistent with these observations, TFEB protein levels Table S7). These long-lived animals showed a partial induction were also significantly higher in nuclear extracts of livers from of the genes regulated in glp-1(e2141) and tor(RNAi) mutants dietary-restricted mice compared with control animals (Fig. 4f), NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 5 & 2013 Macmillan Publishers Limited. All rights reserved. % Alive % Alive % Alive % Alive % Alive % Alive ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 of mammalian transcription factor TFEB . Our studies reveal hlh-30 as a key transcription factor required for lifespan extension 75% ** ** in all six longevity models analysed: germline removal, TOR ** ** ** 50% ** inhibition, DR, reduced insulin/IGF-1 signalling, reduced mito- ** 3 * chondrial respiration and reduced translation, suggesting a 25% central role for HLH-30 in lifespan determination in C. elegans. Our results are consistent with HLH-30 being a functional 0% orthologue of TFEB, because the expression of orthologues of WT eat-2 daf-2 clk-1 rsks-1 WT eat-2 daf-2 clk-1 rsks-1 many TFEB target genes with functions in multiple steps of the autophagy process were regulated by HLH-30 in both germline- WT HLH-30 OE#1 less glp-1 mutants and animals with reduced TOR levels. Many of 1.4 HLH-30 OE#2 ** 1.2 the conserved hlh-30-regulated genes have predicted functions in autophagosome formation, cargo degradation, fusion and lyso- 0.8 0.6 somal degradation, including proton-translocating ATPases/ 0.4 pumps, and degradative enzymes, such as cathepsins and 0.2 arylsulphatases. Thus, HLH-30 appears to display a broad role 010 20 30 WT HLH-30 OE in the transcription of multiple genes with functions in several Days of adulthood steps of the autophagy process. Consistent with this notion, the AL DR Ruvkun laboratory independently reported, while this study was Liver 2.0 under revision, a conserved role for HLH-30/TFEB in autophagy - TFEB 38 kDa - 1.5 gene expression . It remains to be investigated whether such - Actin 38 kDa - HLH-30-regulated genes are direct or indirect targets of HLH-30. 1.0 Our experiments with different reporter proteins indicated 0.5 ** that induction of autophagosome formation and lysosomal 0.0 1 degradation were impaired by the loss of hlh-30, implying that AL DR AL DR HLH-30 regulates different stages of the autophagy process. FM AL DR We found that hlh-30 had a critical role in six mechanistically distinct C. elegans longevity models, many of which had previously been reported to rely, at least in part, on increased autophagy for Figure 4 | HLH-30 and TFEB are similarly regulated in nematode and their lifespan extension: germline ablation, TOR inhibition, DR, reduced insulin/IGF-1 signalling and reduced mitochondrial mouse longevity models. (a) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary respiration. This may also be the case for animals with reduced levels of the S6 kinase RSKS-1, as we found that the long lifespan of restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals these animals was dependent on at least one autophagy gene, atg- ± 18, and we observed an increase in autophagic events in adult (mean s.d. of three biological replicates, *Po0.05, Student’s t-test). (b) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, animals (Supplementary Fig. S5). The observation that hlh-30 is daf-2, clk-1 and rsks-1 mutants (mean s.d. of four biological replicates, upregulated and is required for the long lifespan in these C. elegans B50 animals each, **Po0.01, analysis of variance). (c) Lifespan analysis of longevity paradigms is notable, because no downstream transcrip- WT and transgenic animals overexpressing HLH-30::GFP, and raised and tion factor common to these models has so far been described. In addition, the Ruvkun and Ballabio laboratories recently reported maintained on OP50 was carried out at 20 C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments. (d) GFP::LGG-1 that hlh-30 is required for lifespan extension induced by a loss-of- function mutation of the transcription factor mxl-3 (Max-like 3) punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n4300, s.e.m., **Po0.01, Student’s and for starvation-induced longevity in C. elegans.Although it remains to be investigated how these mechanistically different t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment longevity models activate HLH-30, TOR regulation is a candidate 9–11,28 mechanism . (data not shown). (e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to The identification of HLH-30 as a key transcription factor that ± regulates multiple genes with functions throughout the autophagy DR for 5.5 weeks starting at 3 months of age (mean s.e.m. of B20 mice per group, *Po0.05, Student’s t-test). (f) TFEB protein was detected by process supports the concept that increased autophagic flux is western blotting of nuclear fractions from the livers of five female and five likely critical to ensure long lifespan. Indeed, we found that knockdown of vha-16, a lysosomal ATPase, significantly male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein shortened the long lifespan of germline-less glp-1 animals, as previously observed for genes important for early steps in the levels in Fig. 4f were quantified by densitometry and normalized to actin (mean s.d., *Po0.05, Student’s t-test). autophagy process . Another lysosomal gene, C08H9.1/cathepsin A was previously reported to be required for the long lifespan of daf-2/insulin/IGF-1 mutants , supporting the notion that as observed in all the long-lived C. elegans models we examined lysosomal function has an important role in distinct longevity (Figs 1a, 2b and 4b). Taken together, these results raise the mechanisms. Nonetheless, knockdown of the lysosomal possibility that mammalian TFEB may have a broad and glycoprotein lmp-1 did not shorten glp-1-mediated lifespan conserved role in lifespan extension. extension, implying that not all lysosomal hlh-30-regulated genes contribute to lifespan. Although our data are consistent with HLH-30 mediating lifespan extension in multiple longevity Discussion pathways, at least in part, by inducing autophagy, it is possible In this study, we show that the HLH transcription factor HLH-30 that other HLH-30-regulated mechanisms, such as lipid 28,29 has a role in the determination of lifespan, and demonstrate that metabolism , are also concomitantly engaged. As C. elegans HLH-30 regulates autophagy in C. elegans, similar to the function longevity pathways engage distinct sets of transcription factors ,it 6 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. % Alive % Of animals with nuclear localization of HLH-30 TFEB relative expression hlh-30 relative expression No. of GFP::LGG-1 foci/ seam cell Nuclear hepatic TFEB level NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE will also be interesting to investigate how the activities of HLH-30 experiments with 10–15 L3 worms or day 1 adults were imaged for each experi- ment. The number of GFP::LGG-1-positive punctae were counted in the seam cells, and other lifespan-modulating transcription factors, for example, intestinal cells and muscle in one 0.6mm slice. The Z-position of the seam cells and 4,6 PHA-4/FOXA , are coordinated in specific longevity models. intestinal cells was chosen at a position where the nucleus could be clearly seen. We found that at least two aspects of C. elegans HLH-30 Statistical analysis (analysis of variance) was performed with GraphPad Prism 5.0 function were conserved in a mammalian model of longevity, software (GraphPad, La Jolla, CA). namely, DR . Expression of TFEB was increased in the pancreas and liver of dietary-restricted mice, tissues known to respond to Lifespan analysis. Synchronized animals, raised at 20 C and fed OP50 E. coli starvation by autophagy induction , and nuclear TFEB protein until adulthood, were transferred onto plates seeded with RNAi clones from the 35 36 Ahringer library and viability was measured every 2–3 days, as described . levels were enhanced in the livers from dietary-restricted mice. It B100 animals were tested in each experiment. The plasmid expressing dsRNA will be interesting to investigate the transcriptional activity of against let-363 (tor) was previously described . Statistical analysis (log-rank) was TFEB in response to DR in mammals. Taken together, these performed with STATA (StataCorp, College Station, TX). results support the intriguing possibility that TFEB might have a conserved role in lifespan modulation in higher organisms. Quantitative PCR in C. elegans. Synchronized animals were raised at 20 C, In conclusion, our study demonstrates novel, essential and, unless otherwise noted, and fed OP50 E. coli bacteria until they reached adulthood. possibly, conserved roles for the transcription factor HLH-30/ For each strain, animals were collected with M9 solution at day 1 of adulthood for TFEB in modulation of longevity by mechanisms that rely, at analysis. For RNAi experiments, wild-type (N2, WT) and glp-1(e2141) animals were raised at 25 C on OP50 E. coli bacteria and were transferred onto plates least partly, on the autophagy process. HLH-30 is the first trans- freshly seeded with control bacteria or bacteria expressing dsRNA against the gene cription factor reported to function in all known autophagy- of interest. Animals were incubated at 20 C for 48 h, then collected and washed dependent C. elegans longevity paradigms, strengthening the twice with M9 solution. emerging concept that increased autophagy can contribute to Nematodes (B1,000) were flash frozen in liquid N and RNA was extracted with Trizol (Invitrogen/Life Technologies, Carlsbad, CA) as described . long lifespan. Accumulating evidence suggests that failure of the Concentration and purity of RNA samples were determined with a NanoDrop autophagy–lysosomal pathway contributes to the pathogenesis spectrophotometer and samples were stored at  80 C. Reverse transcription was 32,33 of a variety of age-related disorders . Thus, HLH-30 and performed on 1mg RNA per sample using iScript Supermix (Bio-Rad, Hercules, TFEB may represent attractive targets for the development of CA). Samples were diluted 1/100 and complementary DNA standards (1/25–1/400) were prepared as serial dilutions from a mixture of the relevant cDNAs. Diluted therapeutic agents for such diseases. samples and custom-designed primers (IDT, San Diego, CA) were mixed with SYBR Green (Roche, Indianapolis, IN) and samples were analysed using a Roche Methods LightCycler 480 (Roche). Relative mRNA levels of target genes were normalized Animals. C. elegans strains (a list is available in Supplementary Table S9) were against the geometric mean of the housekeeping genes act-1, cyn-1, cdc-42 and maintained on OP50 Escherichia coli at 20 C and were handled using standard pmp-3. Primer sequences can be found in Supplementary Table S10. Nematode methods . All mice procedures were carried out ethically according to the guidelines orthologues of the TFEB target genes analysed in this study were selected based on 8,14 of the Institutional Animal Care and Use Committee Conduct and have been their significance in previous studies . Each biological sample was analysed in approved by the Salk Institute Institutional Animal Care and Use Committee duplicate or triplicate for each gene assayed. The mRNA levels of nematode genes Conduct Department. ± are presented as mean s.d. and statistical analysis of biological triplicates was performed by two-tailed Student’s t-test or analysis of variance using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA). Construction of HLH-30 transgenic strains. An expression plasmid for hlh-30p::hlh-30::gfp was created by Gateway cloning technology lambda (Gateway System, Life Technologies) using pDONR P4-P1R-hlh-30p (2 kb promoter, Open Quantitative PCR in mice. Three-month-old C57BL/6 J male and female mice Biosystems, Vidal promoterome library), pDONR201-hlh-30 open reading frame 40,41 were subjected to short-term DR according to previously described methods . (Vidal ORFeome library, isoform W02C12.3a) and pKA674/pDEST-MB14-GFP The animal room was maintained at 22 C on a 12:12 h light:dark cycle. Mice were destination vector (a generous gift from Kaveh Ashrafi, UCSF). Upon sequencing housed individually and were allowed water AL throughout the experiment. All of the hlh-30 open reading frame, the codons for amino acids 123 and 255 were mice were initially fed with AIN-93 M AL pellets (Bio-Serv, Frenchtown, NJ). After found to diverge from the corresponding RefSeq file (NP_500462.1) and were 1 week of habituation, daily food intake was measured for each mouse over a restored to the WT sequence using a QuikChange Site-Directed Mutagenesis kit period of 10 days. Hypophagic or hyperphagic mice were excluded from the study, (Agilent Technologies, Santa Clara, CA). Transgenic animals expressing an extra- and the remaining mice were randomly assigned to an AL group that had free chromosomal array of hlh-30p::hlh-30::gfp were created by gonadal microinjection of access to standard AIN-93 M AL pellets, or to a DR group that received AIN-93 the plasmid pKA674(hlh-30p::hlh-30::gfp)(10ngml ) into young gravid adult WT 40% pellets (Bio-Serv) in an amount corresponding to 60% of their regular daily animals using pRF4/rol-6(su1006) (100 ngml ) as a selection marker. Integration food intake. DR animals were fed once daily at 1800 h. Body weights were was subsequently performed by g-irradiation, followed by outcrossing four times to monitored to confirm the efficiency of DR and, as expected, body weight loss in N2 WT animals. The nuclear localization of HLH-30::GFP was visualized with a Leica both genders stabilized after 2.5 weeks of DR. The DR or AL feeding regimens were fluorescence dissecting scope equipped with a Leica DFC310 FX camera in animals continued for further 3 weeks. On the day of killing, food was removed from cages within 5 min of mounting on a 2% agarose pad, because HLH-30::GFP translocates to of mice fed AL at 1100 h to minimize intragroup variation related to food intake, the nucleus during starvation and under stress caused by mounting (data not shown). and mice from both groups were killed between 1500 and 1600 h by isoflurane anaesthesia followed by decapitation. Organs were collected immediately after death, snap-frozen in liquid nitrogen and were stored at  80 C until analysis. Autophagy measurements. Animals expressing GFP::LGG-1 were synchronized Total RNA was extracted from frozen mouse tissues using the Qiazol/ and raised at the appropriate temperature on media plates seeded with bacteria chloroform method and RNeasy columns (Qiagen, Valencia, CA). In brief, frozen expressing control or hlh-30 dsRNA. For the HLH-30-overexpressing animals, tissues were grounded and homogenized in Qiazol using RNA/DNAse-free heterozygous F1 progeny from cross between strains MAH236 and MAH240 (see disposable pestles (VWR, San Francisco, CA). After chloroform extraction, the Supplementary Information) was analysed. The number of GFP::LGG-1 punctae RNA-containing phase was transferred to RNeasy columns and RNA purification were counted in seam and intestinal cells at L3–L4 larval stage as described .Worms was achieved following the manufacturer’s instructions. RNA purity and were analysed at  1,000 magnification on a Zeiss Imager Z1 after mounting on concentration were determined on a NanoDrop spectrophotometer and samples a 2% agarose pad in M9 medium containing 0.1% NaN . SQST-1::GFP foci and were stored at  80 C. Reverse transcription was performed with 1mg of RNA per LMP-1::GFP signal were imaged in animals on media plates with a Leica DFC310 FX sample using the Qiagen Quantitect Reverse Transcription kit (Qiagen), according camera. SQST-1::GFP foci were counted visually in the posterior intestine (from to the manufacturer’s recommendations. For quantitative PCR (qPCR), cDNAs vulva to the tail), and LMP-1::GFP fluorescence was quantified using ImageJ 1.45 were diluted 1/20 and mixed with the relevant primers (see Supplementary software (National Institute of Health, Bethesda, MD). Statistical analysis was done Table S10) and SYBR Green. qPCR was performed using the standard curve using GraphPad Prism 5.0 (GraphPad, La Jolla, CA). method on cDNAs from 10 females and 11 males in the AL group and 9 females and 10 males in the DR group. All biological samples were tested in triplicate Quantification of GFP::LGG-1 punctae by confocal microscopy. Day 1 adult WT (technical repeats) for each gene. Expression of TFEB was normalized to the or rsks-1(sv31) animals expressing GFP::LGG-1 fed OP50 E. coli bacteria and geometric mean of RPL23 and ARBP housekeeping genes. We validated the use of grown at 20 C were mounted on a 2% agarose pad containing 0.1% 1.5 M NaN . the housekeeping genes in our experimental conditions using Normfinder and Worms were imaged using a LSM Zeiss 710 scanning confocal microscope. Genorm algorithms . Normal distribution of data was verified by the Z-stacks were taken at 0.6-mm slices. GFP excitation/emission was limited to Kolmogorov–Smirnov test, and groups were compared by two-tailed Student’s 493/512 nm to eliminate background autofluorescence from the worms. Four t-test using GraphPad Prism 5.0. (GraphPad, La Jolla, CA) NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 7 & 2013 Macmillan Publishers Limited. All rights reserved. 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How to cite this article: Lapierre, L. R. et al. The TFEB orthologue HLH-30 regulates 26. Hars, E. S. et al. Autophagy regulates ageing in C. elegans. Autophagy 3, 93–95 autophagy and modulates longevity in Caenorhabditis elegans. Nat. Commun. 4:2267 (2007). doi: 10.1038/ncomms3267 (2013). 8 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans

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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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Abstract

ARTICLE Received 4 Mar 2013 | Accepted 8 Jul 2013 | Published 8 Aug 2013 DOI: 10.1038/ncomms3267 The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans 1 2 1,3 1 4 Louis R. Lapierre , C. Daniel De Magalhaes Filho , Philip R. McQuary , Chu-Chiao Chu , Orane Visvikis , 1 1,3 1 1 4 2 Jessica T. Chang , Sara Gelino , Binnan Ong , Andrew E. Davis , Javier E. Irazoqui , Andrew Dillin & Malene Hansen Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity- extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans. Program of Development and Aging, Del E Webb Neuroscience, Aging and Stem Cell Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California, USA. The Howard Hughes Medical Institute, The Glenn Center for Aging Research, The Salk Institute for Biological Studies, La Jolla, California, USA. Graduate School of Biomedical Sciences, Sanford-Burnham Medical Research Institute, La Jolla, California, USA. Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Correspondence and requests for materials should be addressed to M.H. (email: [email protected]). NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 9–11 ging is modulated by multiple conserved signal trans- C. elegans via TOR, as observed in mammalian cells . duction pathways, such as the insulin/insulin-like Consistent with this possibility, we observed green fluorescent Agrowth factor-1 (IGF-1) and target of rapamycin (TOR) protein (GFP)-tagged HLH-30 to be enriched prominently in the signalling pathways. Studies in model organisms, including the nuclei of intestinal cells in adult glp-1(e2141) mutants (Fig. 1). nematode Caenorhabditis elegans, have identified key regulatory Similarly, we observed nuclear localization of HLH-30 in wild- roles for several transcription factors in these pathways . type (WT; N2 Bristol) animals subjected to tor RNA interference However, the degree to which these distinct longevity pathways (RNAi) (Fig. 2). These observations suggest that HLH-30 localizes converge on common downstream mechanisms remains an to the nucleus in C. elegans intestinal cells under conditions important topic of investigation. Macroautophagy (hereafter associated with induction of autophagy, including TOR 9–11 referred to as autophagy) is a cellular recycling process that has inhibition, similar to TFEB in mammalian cells . emerged as a crucial mechanism for lifespan extension in many different species . During autophagy, cytosolic components are sequestered in vesicles called autophagosomes, which then HLH-30 regulates the expression of orthologous TFEB targets. fuse with lysosomes, allowing the contents to be enzymatically We next investigated the expression of hlh-30 and orthologues of degraded and recycled. Notably, long-lived C. elegans mutants TFEB target genes in glp-1(e2141) mutants and in WT animals with reduced insulin/IGF-1 signalling, TOR signalling, germline with reduced TOR levels. We found that hlh-30 mRNA levels signalling, food intake or mitochondrial respiration, all exhibit were markedly increased in both glp-1(e2141) and tor(RNAi) increased levels of autophagy and require autophagy genes for mutants when compared with WT animals (Figs 1b and 2b). lifespan extension . However, the mechanism(s) by which these Moreover, glp-1(e2141) mutants showed increased expression of long-lived animals regulate the autophagy process is not fully several nematode orthologues of human TFEB target genes, understood. including genes involved in autophagosome formation and We recently reported that the forkhead transcription factor autophagic flux, such as lgg-1/LC3 and sqst-1/SQSTM1/p62, and PHA-4/FOXA regulates the expression of several genes important genes with lysosomal functions, such as lmp-1/LAMP-1, subunits for autophagy in response to germline removal or TOR inhibition of vacuolar ATPases (vha-15, vha-16 and vha-17), sulphatases in C. elegans . Moreover, PHA-4/FOXA and autophagy genes (sul-2/ASRA and sul-3/ASRA) and cathepsins (ctsa/cathepsin A, 4,5 are essential for lifespan extension in these animals , as has been cpr-1/cathepsin B and asp-1/cathepsin D) (Fig. 1b). Several of 6,7 observed in dietary-restricted C. elegans . However, PHA-4/ these autophagy-related and lysosomal genes were similarly FOXA may not be a general transcriptional regulator of auto- changed in adult WT animals subjected to tor RNAi (Fig. 2b). phagy, because inhibition of PHA-4/FOXA has only a minor Changes in the expression of these genes may be transcriptionally effect on the long lifespan of insulin/IGF-1 signalling mutants or regulated by hlh-30, because glp-1(e2141); hlh-30(tm1978) double animals with reduced mitochondrial respiration . Thus, it is not mutants or hlh-30(tm1978) single mutants fed bacteria expressing clear whether induction of autophagy in these longevity pathways tor dsRNA, all showed a profound reduction in transcription of is mediated by a common transcriptional regulator. most of the putative target genes (Figs 1c and 2c). The The basic helix-loop-helix (HLH) transcription factor EB glp-1(e2141) mutants fed bacteria expressing hlh-30 dsRNA only (TFEB) was recently identified as a key transcriptional regulator during adulthood showed decreased expression of most target of the autophagy process in mammals . In mammalian cells, genes (Supplementary Fig. S1a), whereas inhibition of hlh-30 by TFEB translocates to the nucleus in response to nutrient RNAi in WT animals had a modest or no effect on the expression 9–11 deprivation, at least in part, via a TOR-dependent mechanism of these genes (Supplementary Fig. S1b). We also found that to ensure the transcriptional induction of many autophagy-related hlh-30(tm1978) single mutants displayed decreased expression of and lysosomal genes .Inthisstudy,weasked whether C. elegans some (for example, atg-18), but not all, of these genes (Supplemen- contains a functional orthologue of TFEB, and if so, whether the tary Fig. S1c), consistent with a previously published microarray orthologue has a role in lifespan determination. To this end, we analysis . Notably, most of the genes examined contain one or examined HLH-30 (W02C12.3, www.wormbase.org), which is a more E-box motifs in their promoters (Supplementary Table S1); member of a group of 42 HLH transcription factors in C. elegans this sequence overlaps with that of the CLEAR (Coordinated and has the highest homology to TFEB in the DNA-binding Lysosomal Expression And Regulation) binding motif, which is domain and in an acidic activation domain . We report that present in the promoters of many lysosomal gene targets of HLH-30 regulates the expression of multiple autophagy-related TFEB . Collectively, these data suggest that HLH-30 functionally and lysosomal genes, and modulates the autophagy process in mimics TFEB by inducing the transcription of autophagy-related C. elegans. HLH-30 is required for the long lifespan observed in all and lysosomal genes. C. elegans longevity mutants tested, is nuclear localized in these long-lived models, and the overexpression of HLH-30 extends the C. elegans lifespan. Taken together, these observations suggest that HLH-30 modulates autophagy and lysosomal function.To increased HLH-30 activity may be a universal mechanism for directly determine whether HLH-30 regulates autophagy, we used longevity. Finally, we find that TFEB levels are increased in the liver transgenic WT and glp-1(e2141) animals expressing GFP repor- nuclear extracts of dietary-restricted mice, raising the possibility ters that allow distinct steps of the autophagy process to be that HLH-30/TFEB modulates lifespan in a conserved manner. examined. First, we used a strain expressing GFP-tagged LGG-1/ LC3 (ref. 15), a protein that localizes to preautophagosomal and autophagosomal membranes, to investigate autophagosome Results formation. Compared with WT animals, glp-1(e2141) mutants HLH-30 locates to the nucleus in animals with reduced TOR contained more GFP::LGG-1-positive punctae, reflecting an activity. Our recent work showed that autophagy is tran- increased number of autophagosomes . However, significantly scriptionally activated in long-lived, germline-less C. elegans, such fewer GFP::LGG-1-positive punctae were detected in both as temperature-sensitive glp-1/Notch receptor mutants, possibly hypodermal seam cells and intestinal cells of hlh-30 RNAi- via a reduction in TOR activity . Therefore, we used glp-1(e2141) treated glp-1(e2141) animals compared with that in control mutants raised at the restrictive temperature to investigate animals (Fig. 1d, e), suggesting that hlh-30 is required for whether HLH-30 has a role in autophagy regulation in autophagy induction in glp-1(e2141) mutants. 2 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE 75% WT WT glp-1 ** ** ** 1.5 50% ** ** glp-1 25% 0.5 0% WT glp-1 Control hlh-30 RNAi Control hlh-30 RNAi ** WT glp-1 12 ** Control ** ** ** ** ** 6 ** ** ** hlh-30 RNAi Autophagy Fusion H Sulphatases Cathepsins Control hlh-30 Pumps RNAi ** glp-1 glp-1; hlh-30 Control ** ** 1.5 1 25 ** * 20 0.5 ** ** ** ** ** ** ** * ** * hlh-30 RNAi Autophagy Fusion H Sulphatases Cathepsins Control hlh-30 Pumps RNAi Figure 1 | HLH-30 regulates autophagy in germline-less C. elegans. (a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25 C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, B50 animals each, mean s.d., **Po0.01, Student’s t-test). Magnification  100; scale bar, 100mm. (b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WTand glp-1(e2141) animals raised at 25 C (mean s.d. of three biological replicates, *Po0.05, **Po0.01, Student’s t-test). (c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25 C (mean s.d. of three biological replicates, *Po0.05, **Po0.01, Student’s t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA. (d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 C and analysed at the L3 larval stage (mean s.e.m. ofB300 seam cells andB25 intestines, **Po0.01, Student’s t-test). (f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25 C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean s.d. of B10 animals, **Po0.01, Student’s t-test) and SQST-1::GFP foci (mean s.d. of B30 animals, **Po0.01, Student’s t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification,  200; scale bar, 100mm. To monitor the lysosomal compartment, we used a strain impairment of autophagy turnover. Finally, to monitor auto- expressing GFP-tagged LMP-1/LAMP1 (ref. 16), a glycoprotein phagic flux we used a strain expressing GFP-tagged SQST-1/p62 localized to the lysosomal membrane. Germline-less glp-1(e2141) (ref. 17), an adaptor protein that targets autophagic cargo for animals displayed slightly elevated expression of LMP-1/LAMP1 degradation. SQST-1 was expressed at low levels in adult animals, (Supplementary Fig. S2a), indicative of enhanced lysosomal where it localized to distinct subcellular sites, referred to as foci. biogenesis. This is consistent with our previous observation Interestingly, we found the number of SQST-1::GFP foci was that germline-less animals display increased autophagosome modestly increased in certain tissues of glp-1(e2141) mutants, formation . Notably, the expression of the LMP-1::GFP reporter including in some intestinal cells (Fig. 1g and Supplementary was robustly increased in the absence of hlh-30 (Fig. 1f and Fig. S2c). This may reflect a higher steady-state level of cargo Supplementary Fig. S2b), as would be expected following the delivery to autophagosomes and increased autophagic flux in NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 3 & 2013 Macmillan Publishers Limited. All rights reserved. Relative expression Relative expression HLH-30::GFP hlh-30 hlh-30 atg-18 atg-18 lgg-1 lgg-1 atg-9 atg-9 sqst-1 p62 sqst-1 vps-11 vps-11 vps-18 vps-18 lmp-1 lmp-1 vha-15 vha-15 vha-16 vha-16 vha-17 vha-17 % Of animals with nuclear localization of HLH-30 sul-1 sul-1 sul-2 sul-2 sul-3 sul-3 ctsa* ctsa* cpr-1 cpr-1 asp-1 asp-1 No. of GFP::LGG-1 foci/ glp-1; SQST-1::GFP glp-1; LMP-1::GFP seam cell No. of SQST-1::GFP aggregates Relative LMP-1::GFP No. of GFP::LGG-1 foci/ in distal intestine/animal fluorescence per worm proximal intestinal cells ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 HLH-30-regulated genes are required for the long lifespan of WT + control glp-1 animals (for example, atg-18 and lgg-1 (ref. 4)), we next 75% 100 WT + tor RNAi determined whether HLH-30 has a role in C. elegans lifespan. We hlh-30 + control performed survival analyses on adult WT and glp-1 animals fed ** hlh-30 + tor RNAi 50% bacteria, expressing hlh-30 dsRNA. Although hlh-30 RNAi administered during adulthood did not affect the lifespan of 25% WT animals, this treatment significantly shortened the long lifespan of glp-1(e2141) mutants (Fig. 3 and Supplementary Table 0% S2). Likewise, we found glp-1(e2141); hlh-30(tm1978) double Control tor 010 20 30 40 mutants to have mean lifespans similar to hlh-30(tm1978) single RNAi Days of adulthood mutants (Supplementary Fig. S3a and Supplementary Table S2). In addition, RNAi-mediated inhibition of the lysosomal gene Control 4 vha-16, but not of lmp-1, substantially reduced the long lifespan WT of glp-1(e2141) mutants (Supplementary Fig. S3b-d and 3 tor RNAi * * Supplementary Table S3), supporting the notion that a subset of ** 2 * HLH-30-regulated genes, with functions relevant to autophagy, may contribute to lifespan extension. Similar to germline removal, reduced TOR activity extends lifespan at least partly via an autophagy-dependent mechanism . To test whether hlh-30 has a role in TOR-mediated longevity in C. elegans, we fed bacteria expressing tor dsRNA to adult Autophagy Fusion H Sulfatases Cathepsins WT animals or mutants lacking hlh-30 bacteria. As expected, Pumps tor RNAi significantly extended the lifespan of WT animals; however, the same treatment failed to prolong the lifespan of Control hlh-30(tm1978) mutants (Fig. 2d and Supplementary Table S4), hlh-30 tor RNAi suggesting that hlh-30 acts downstream of TOR. Consistent with this notion, we found that hlh-30 was required for the develop- 1 * mental arrest of C. elegans induced by treatment with tor RNAi for multiple generations (Supplementary Fig. S4a, b). Inhibition of hlh-30 also prevented dauer formation (Supplementary Fig. S4c), a developmental stage in which autophagy plays a critical role , indicating that developmental arrest may be governed by Autophagy Fusion H Sulfatases Cathepsins Pumps hlh-30, possibly via autophagy regulation. Taken together, these observations demonstrate that HLH-30 is a novel longevity- Figure 2 | HLH-30 is required for TOR inhibition to extend C. elegans modulating transcription factor in C. elegans, which likely acts lifespan. (a) Nuclear localization of HLH-30 was quantified in day 1 adult downstream of TOR. As HLH-30 regulates several autophagy- animals expressing HLH-30::GFP. Animals were fed bacteria expressing related genes with effects on longevity, it is possible that HLH-30 control or tor dsRNA from hatching and raised at 20 C (three biological affects lifespan via regulation of autophagy in both germline-less replicates, B50 animals each, mean s.d., **Po0.01, Student’s t-test). animals and animals with reduced TOR function. (b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA HLH-30 is required for lifespan extension in other mutants.In from hatching (20 C). Data are mean s.d. of biological triplicates. addition to germline removal and TOR inhibition, several other *Po0.05, **Po0.01; Student’s t-test. csta* is a cathepsin A orthologue mechanistically distinct and conserved longevity pathways (cosmid C08H9.1 (ref. 30)).(d) Lifespan analysis of WT animals and modulate aging in C. elegans: dietary restriction (DR; the feeding- hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from deficient mutant eat-2 (ref. 19)), inhibition of insulin/IGF-1 day 1 of adulthood was carried out at 20 C. See Supplementary Table S3 signalling (the receptor mutant daf-2 (ref. 20)), impaired mito- for details of lifespan analyses and replicate experiments. chondrial respiration (the ubiquinone synthesis mutant clk-1 (ref. 21)) and reduced translation (the ribosomal S6 kinase mutant rsks-1 (refs 22,23)). We therefore investigated whether specific tissues of these animals. The number of SQST-1::GFP foci hlh-30 has a role in lifespan determination in these longevity was significantly increased following hlh-30 RNAi treatment models. To do these experiments, we initiated hlh-30 RNAi during (Fig. 1g and Supplementary Fig. S2d), consistent with a block in adulthood (for eat-2(ad1116), daf-2(e1370) and rsks-1(sv31) autophagic turnover of SQST-1/p62. In glp-1 animals with mutants) or during the fourth larval stage (for clk-1(e2519) reduced HLH-30 levels, the modest increase in the expression mutants). We found that hlh-30 RNAi significantly shortened the of both LMP-1/LAMP1 and SQST-1/p62 occurred, despite a lifespan of these mutants (Fig. 3c–f and Supplementary Table S2), marked reduction in their mRNA levels (Supplementary Fig. suggesting a common role for HLH-30 in their longevity. S1b), implying that HLH-30 is necessary for the turnover of these To further investigate this possibility, we introduced the proteins. Taken together, these results indicate that inhibition of HLH-30::GFP transgene into eat-2, daf-2, clk-1 and rsks-1 HLH-30 impairs the coordination of the autophagy process at mutants. Consistent with a regulatory role for hlh-30,we several steps, and suggest that HLH-30 regulates autophagy in a observed that HLH-30 was nuclear localized in the intestine of manner similar to TFEB . eat-2(ad1116), daf-2(e1370), clk-1(e2519) and rsks-1(sv31) animals (Fig. 4a). Interestingly, although hlh-30 mRNA levels were significantly increased in these four long-lived mutants compared glp-1 and TOR mutants require HLH-30 for lifespan with WT animals (Fig. 4b), fewer of the TFEB target gene extension. As we previously reported that several of the putative orthologues with functions in autophagy (for example, atg-9) 4 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. Relative expression Relative expression % Of animals with nuclear localization of HLH-30 tor tor hlh-30 hlh-30 atg-18 atg-18 lgg-1 lgg-1 atg-9 atg-9 sqst-1 sqst-1 vps-11 vps-11 % Alive vps-18 vps-18 lmp-1 lmp-1 vha-15 vha-15 vha-16 vha-16 vha-17 vha-17 sul-1 sul-1 sul-2 sul-2 sul-3 sul-3 ctsa* ctsa* cpr-1 cpr-1 asp-1 asp-1 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE 100 100 Control Control hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 WT glp-1 010 20 30 010 20 30 40 Days of adulthood Days of adulthood Control Control 100 100 hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 eat-2 daf-2 0 0 010 20 30 0 20 40 60 80 100 Days of adulthood Days of adulthood Control 100 Control hlh-30 RNAi hlh-30 RNAi 75 75 50 50 25 25 clk-1 rsks-1 010 20 30 40 010 20 30 40 Days of adulthood Days of adulthood Figure 3 | HLH-30 is required for the long lifespan of multiple longevity mutants. Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments. were upregulated in eat-2(ad1116), daf-2(e1370), clk-1(e2519) and (Supplementary Fig. S6b), suggesting that the long life of rsks-1(sv31) mutants (Supplementary Fig. S6a) than that in the HLH-30-overexpressing animals may be mediated through par- glp-1(e2141) (Fig. 1b) and tor(RNAi) (Fig. 2b) mutants. These tially overlapping mechanisms. Notably, HLH-30-overexpressing results suggest that HLH-30 may function differently in the animals had increased numbers of GFP-positive, LGG-1-positive distinct longevity mutants. As autophagy has been shown to have punctae (Fig. 4d), and inhibition of the autophagy gene atg-18 a role in the longevity of eat-2(ad1116) (refs 7,24,25), daf- reduced the lifespan of these animals (Supplementary Table S8). 2(e1370) (refs 7,15,26) and clk-1(e2519) (Supplementary Table S5) Thus, HLH-30 may extend lifespan, at least in part, by inducing mutants, and possibly also in rsks-1(sv31) mutants autophagy. Collectively, our data provide evidence that HLH-30 (Supplementary Fig. S5 and Supplementary Table S6), these is a conserved transcription factor with a novel universal role in findings suggest that autophagy could be differentially regulated modulating C. elegans lifespan. in the C. elegans longevity models. These results also indicate that HLH-30 may regulate additional genes with roles in longevity that are not necessarily related to autophagy. Dietary restriction regulates TFEB in mice. To determine whether TFEB, the mammalian orthologue of HLH-30, has a conserved role in lifespan determination, we asked whether Overexpression of HLH-30 extends C. elegans lifespan. Our TFEB is regulated under conditions that modulate longevity in results indicated a requirement for hlh-30 in multiple C. elegans mammals. For this, we examined mice placed on a dietary- longevity models. To determine whether HLH-30 expression was restricted diet, an effective non-genetic intervention that extends sufficient to extend C. elegans lifespan, we generated strains stably lifespan in mammals . In two key nutrient-responsive tissues, overexpressing HLH-30::GFP from transgenic, integrated arrays the liver and pancreas, we found TFEB mRNA levels to be and measured the lifespan of two independent lines. We observed significantly increased in male and female mice subjected to DR that the mean lifespan of the transgenic animals was B15–20% compared with control mice fed ad libitum (AL) (Supplementary longer than that of WT animals (Fig. 4c and Supplementary Fig. S8). Consistent with these observations, TFEB protein levels Table S7). These long-lived animals showed a partial induction were also significantly higher in nuclear extracts of livers from of the genes regulated in glp-1(e2141) and tor(RNAi) mutants dietary-restricted mice compared with control animals (Fig. 4f), NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 5 & 2013 Macmillan Publishers Limited. All rights reserved. % Alive % Alive % Alive % Alive % Alive % Alive ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 of mammalian transcription factor TFEB . Our studies reveal hlh-30 as a key transcription factor required for lifespan extension 75% ** ** in all six longevity models analysed: germline removal, TOR ** ** ** 50% ** inhibition, DR, reduced insulin/IGF-1 signalling, reduced mito- ** 3 * chondrial respiration and reduced translation, suggesting a 25% central role for HLH-30 in lifespan determination in C. elegans. Our results are consistent with HLH-30 being a functional 0% orthologue of TFEB, because the expression of orthologues of WT eat-2 daf-2 clk-1 rsks-1 WT eat-2 daf-2 clk-1 rsks-1 many TFEB target genes with functions in multiple steps of the autophagy process were regulated by HLH-30 in both germline- WT HLH-30 OE#1 less glp-1 mutants and animals with reduced TOR levels. Many of 1.4 HLH-30 OE#2 ** 1.2 the conserved hlh-30-regulated genes have predicted functions in autophagosome formation, cargo degradation, fusion and lyso- 0.8 0.6 somal degradation, including proton-translocating ATPases/ 0.4 pumps, and degradative enzymes, such as cathepsins and 0.2 arylsulphatases. Thus, HLH-30 appears to display a broad role 010 20 30 WT HLH-30 OE in the transcription of multiple genes with functions in several Days of adulthood steps of the autophagy process. Consistent with this notion, the AL DR Ruvkun laboratory independently reported, while this study was Liver 2.0 under revision, a conserved role for HLH-30/TFEB in autophagy - TFEB 38 kDa - 1.5 gene expression . It remains to be investigated whether such - Actin 38 kDa - HLH-30-regulated genes are direct or indirect targets of HLH-30. 1.0 Our experiments with different reporter proteins indicated 0.5 ** that induction of autophagosome formation and lysosomal 0.0 1 degradation were impaired by the loss of hlh-30, implying that AL DR AL DR HLH-30 regulates different stages of the autophagy process. FM AL DR We found that hlh-30 had a critical role in six mechanistically distinct C. elegans longevity models, many of which had previously been reported to rely, at least in part, on increased autophagy for Figure 4 | HLH-30 and TFEB are similarly regulated in nematode and their lifespan extension: germline ablation, TOR inhibition, DR, reduced insulin/IGF-1 signalling and reduced mitochondrial mouse longevity models. (a) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary respiration. This may also be the case for animals with reduced levels of the S6 kinase RSKS-1, as we found that the long lifespan of restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals these animals was dependent on at least one autophagy gene, atg- ± 18, and we observed an increase in autophagic events in adult (mean s.d. of three biological replicates, *Po0.05, Student’s t-test). (b) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, animals (Supplementary Fig. S5). The observation that hlh-30 is daf-2, clk-1 and rsks-1 mutants (mean s.d. of four biological replicates, upregulated and is required for the long lifespan in these C. elegans B50 animals each, **Po0.01, analysis of variance). (c) Lifespan analysis of longevity paradigms is notable, because no downstream transcrip- WT and transgenic animals overexpressing HLH-30::GFP, and raised and tion factor common to these models has so far been described. In addition, the Ruvkun and Ballabio laboratories recently reported maintained on OP50 was carried out at 20 C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments. (d) GFP::LGG-1 that hlh-30 is required for lifespan extension induced by a loss-of- function mutation of the transcription factor mxl-3 (Max-like 3) punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n4300, s.e.m., **Po0.01, Student’s and for starvation-induced longevity in C. elegans.Although it remains to be investigated how these mechanistically different t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment longevity models activate HLH-30, TOR regulation is a candidate 9–11,28 mechanism . (data not shown). (e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to The identification of HLH-30 as a key transcription factor that ± regulates multiple genes with functions throughout the autophagy DR for 5.5 weeks starting at 3 months of age (mean s.e.m. of B20 mice per group, *Po0.05, Student’s t-test). (f) TFEB protein was detected by process supports the concept that increased autophagic flux is western blotting of nuclear fractions from the livers of five female and five likely critical to ensure long lifespan. Indeed, we found that knockdown of vha-16, a lysosomal ATPase, significantly male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein shortened the long lifespan of germline-less glp-1 animals, as previously observed for genes important for early steps in the levels in Fig. 4f were quantified by densitometry and normalized to actin (mean s.d., *Po0.05, Student’s t-test). autophagy process . Another lysosomal gene, C08H9.1/cathepsin A was previously reported to be required for the long lifespan of daf-2/insulin/IGF-1 mutants , supporting the notion that as observed in all the long-lived C. elegans models we examined lysosomal function has an important role in distinct longevity (Figs 1a, 2b and 4b). Taken together, these results raise the mechanisms. Nonetheless, knockdown of the lysosomal possibility that mammalian TFEB may have a broad and glycoprotein lmp-1 did not shorten glp-1-mediated lifespan conserved role in lifespan extension. extension, implying that not all lysosomal hlh-30-regulated genes contribute to lifespan. Although our data are consistent with HLH-30 mediating lifespan extension in multiple longevity Discussion pathways, at least in part, by inducing autophagy, it is possible In this study, we show that the HLH transcription factor HLH-30 that other HLH-30-regulated mechanisms, such as lipid 28,29 has a role in the determination of lifespan, and demonstrate that metabolism , are also concomitantly engaged. As C. elegans HLH-30 regulates autophagy in C. elegans, similar to the function longevity pathways engage distinct sets of transcription factors ,it 6 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. % Alive % Of animals with nuclear localization of HLH-30 TFEB relative expression hlh-30 relative expression No. of GFP::LGG-1 foci/ seam cell Nuclear hepatic TFEB level NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3267 ARTICLE will also be interesting to investigate how the activities of HLH-30 experiments with 10–15 L3 worms or day 1 adults were imaged for each experi- ment. The number of GFP::LGG-1-positive punctae were counted in the seam cells, and other lifespan-modulating transcription factors, for example, intestinal cells and muscle in one 0.6mm slice. The Z-position of the seam cells and 4,6 PHA-4/FOXA , are coordinated in specific longevity models. intestinal cells was chosen at a position where the nucleus could be clearly seen. We found that at least two aspects of C. elegans HLH-30 Statistical analysis (analysis of variance) was performed with GraphPad Prism 5.0 function were conserved in a mammalian model of longevity, software (GraphPad, La Jolla, CA). namely, DR . Expression of TFEB was increased in the pancreas and liver of dietary-restricted mice, tissues known to respond to Lifespan analysis. Synchronized animals, raised at 20 C and fed OP50 E. coli starvation by autophagy induction , and nuclear TFEB protein until adulthood, were transferred onto plates seeded with RNAi clones from the 35 36 Ahringer library and viability was measured every 2–3 days, as described . levels were enhanced in the livers from dietary-restricted mice. It B100 animals were tested in each experiment. The plasmid expressing dsRNA will be interesting to investigate the transcriptional activity of against let-363 (tor) was previously described . Statistical analysis (log-rank) was TFEB in response to DR in mammals. Taken together, these performed with STATA (StataCorp, College Station, TX). results support the intriguing possibility that TFEB might have a conserved role in lifespan modulation in higher organisms. Quantitative PCR in C. elegans. Synchronized animals were raised at 20 C, In conclusion, our study demonstrates novel, essential and, unless otherwise noted, and fed OP50 E. coli bacteria until they reached adulthood. possibly, conserved roles for the transcription factor HLH-30/ For each strain, animals were collected with M9 solution at day 1 of adulthood for TFEB in modulation of longevity by mechanisms that rely, at analysis. For RNAi experiments, wild-type (N2, WT) and glp-1(e2141) animals were raised at 25 C on OP50 E. coli bacteria and were transferred onto plates least partly, on the autophagy process. HLH-30 is the first trans- freshly seeded with control bacteria or bacteria expressing dsRNA against the gene cription factor reported to function in all known autophagy- of interest. Animals were incubated at 20 C for 48 h, then collected and washed dependent C. elegans longevity paradigms, strengthening the twice with M9 solution. emerging concept that increased autophagy can contribute to Nematodes (B1,000) were flash frozen in liquid N and RNA was extracted with Trizol (Invitrogen/Life Technologies, Carlsbad, CA) as described . long lifespan. Accumulating evidence suggests that failure of the Concentration and purity of RNA samples were determined with a NanoDrop autophagy–lysosomal pathway contributes to the pathogenesis spectrophotometer and samples were stored at  80 C. Reverse transcription was 32,33 of a variety of age-related disorders . Thus, HLH-30 and performed on 1mg RNA per sample using iScript Supermix (Bio-Rad, Hercules, TFEB may represent attractive targets for the development of CA). Samples were diluted 1/100 and complementary DNA standards (1/25–1/400) were prepared as serial dilutions from a mixture of the relevant cDNAs. Diluted therapeutic agents for such diseases. samples and custom-designed primers (IDT, San Diego, CA) were mixed with SYBR Green (Roche, Indianapolis, IN) and samples were analysed using a Roche Methods LightCycler 480 (Roche). Relative mRNA levels of target genes were normalized Animals. C. elegans strains (a list is available in Supplementary Table S9) were against the geometric mean of the housekeeping genes act-1, cyn-1, cdc-42 and maintained on OP50 Escherichia coli at 20 C and were handled using standard pmp-3. Primer sequences can be found in Supplementary Table S10. Nematode methods . All mice procedures were carried out ethically according to the guidelines orthologues of the TFEB target genes analysed in this study were selected based on 8,14 of the Institutional Animal Care and Use Committee Conduct and have been their significance in previous studies . Each biological sample was analysed in approved by the Salk Institute Institutional Animal Care and Use Committee duplicate or triplicate for each gene assayed. The mRNA levels of nematode genes Conduct Department. ± are presented as mean s.d. and statistical analysis of biological triplicates was performed by two-tailed Student’s t-test or analysis of variance using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA). Construction of HLH-30 transgenic strains. An expression plasmid for hlh-30p::hlh-30::gfp was created by Gateway cloning technology lambda (Gateway System, Life Technologies) using pDONR P4-P1R-hlh-30p (2 kb promoter, Open Quantitative PCR in mice. Three-month-old C57BL/6 J male and female mice Biosystems, Vidal promoterome library), pDONR201-hlh-30 open reading frame 40,41 were subjected to short-term DR according to previously described methods . (Vidal ORFeome library, isoform W02C12.3a) and pKA674/pDEST-MB14-GFP The animal room was maintained at 22 C on a 12:12 h light:dark cycle. Mice were destination vector (a generous gift from Kaveh Ashrafi, UCSF). Upon sequencing housed individually and were allowed water AL throughout the experiment. All of the hlh-30 open reading frame, the codons for amino acids 123 and 255 were mice were initially fed with AIN-93 M AL pellets (Bio-Serv, Frenchtown, NJ). After found to diverge from the corresponding RefSeq file (NP_500462.1) and were 1 week of habituation, daily food intake was measured for each mouse over a restored to the WT sequence using a QuikChange Site-Directed Mutagenesis kit period of 10 days. Hypophagic or hyperphagic mice were excluded from the study, (Agilent Technologies, Santa Clara, CA). Transgenic animals expressing an extra- and the remaining mice were randomly assigned to an AL group that had free chromosomal array of hlh-30p::hlh-30::gfp were created by gonadal microinjection of access to standard AIN-93 M AL pellets, or to a DR group that received AIN-93 the plasmid pKA674(hlh-30p::hlh-30::gfp)(10ngml ) into young gravid adult WT 40% pellets (Bio-Serv) in an amount corresponding to 60% of their regular daily animals using pRF4/rol-6(su1006) (100 ngml ) as a selection marker. Integration food intake. DR animals were fed once daily at 1800 h. Body weights were was subsequently performed by g-irradiation, followed by outcrossing four times to monitored to confirm the efficiency of DR and, as expected, body weight loss in N2 WT animals. The nuclear localization of HLH-30::GFP was visualized with a Leica both genders stabilized after 2.5 weeks of DR. The DR or AL feeding regimens were fluorescence dissecting scope equipped with a Leica DFC310 FX camera in animals continued for further 3 weeks. On the day of killing, food was removed from cages within 5 min of mounting on a 2% agarose pad, because HLH-30::GFP translocates to of mice fed AL at 1100 h to minimize intragroup variation related to food intake, the nucleus during starvation and under stress caused by mounting (data not shown). and mice from both groups were killed between 1500 and 1600 h by isoflurane anaesthesia followed by decapitation. Organs were collected immediately after death, snap-frozen in liquid nitrogen and were stored at  80 C until analysis. Autophagy measurements. Animals expressing GFP::LGG-1 were synchronized Total RNA was extracted from frozen mouse tissues using the Qiazol/ and raised at the appropriate temperature on media plates seeded with bacteria chloroform method and RNeasy columns (Qiagen, Valencia, CA). In brief, frozen expressing control or hlh-30 dsRNA. For the HLH-30-overexpressing animals, tissues were grounded and homogenized in Qiazol using RNA/DNAse-free heterozygous F1 progeny from cross between strains MAH236 and MAH240 (see disposable pestles (VWR, San Francisco, CA). After chloroform extraction, the Supplementary Information) was analysed. The number of GFP::LGG-1 punctae RNA-containing phase was transferred to RNeasy columns and RNA purification were counted in seam and intestinal cells at L3–L4 larval stage as described .Worms was achieved following the manufacturer’s instructions. RNA purity and were analysed at  1,000 magnification on a Zeiss Imager Z1 after mounting on concentration were determined on a NanoDrop spectrophotometer and samples a 2% agarose pad in M9 medium containing 0.1% NaN . SQST-1::GFP foci and were stored at  80 C. Reverse transcription was performed with 1mg of RNA per LMP-1::GFP signal were imaged in animals on media plates with a Leica DFC310 FX sample using the Qiagen Quantitect Reverse Transcription kit (Qiagen), according camera. SQST-1::GFP foci were counted visually in the posterior intestine (from to the manufacturer’s recommendations. For quantitative PCR (qPCR), cDNAs vulva to the tail), and LMP-1::GFP fluorescence was quantified using ImageJ 1.45 were diluted 1/20 and mixed with the relevant primers (see Supplementary software (National Institute of Health, Bethesda, MD). Statistical analysis was done Table S10) and SYBR Green. qPCR was performed using the standard curve using GraphPad Prism 5.0 (GraphPad, La Jolla, CA). method on cDNAs from 10 females and 11 males in the AL group and 9 females and 10 males in the DR group. All biological samples were tested in triplicate Quantification of GFP::LGG-1 punctae by confocal microscopy. Day 1 adult WT (technical repeats) for each gene. Expression of TFEB was normalized to the or rsks-1(sv31) animals expressing GFP::LGG-1 fed OP50 E. coli bacteria and geometric mean of RPL23 and ARBP housekeeping genes. We validated the use of grown at 20 C were mounted on a 2% agarose pad containing 0.1% 1.5 M NaN . the housekeeping genes in our experimental conditions using Normfinder and Worms were imaged using a LSM Zeiss 710 scanning confocal microscope. Genorm algorithms . Normal distribution of data was verified by the Z-stacks were taken at 0.6-mm slices. GFP excitation/emission was limited to Kolmogorov–Smirnov test, and groups were compared by two-tailed Student’s 493/512 nm to eliminate background autofluorescence from the worms. Four t-test using GraphPad Prism 5.0. (GraphPad, La Jolla, CA) NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications 7 & 2013 Macmillan Publishers Limited. All rights reserved. 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How to cite this article: Lapierre, L. R. et al. The TFEB orthologue HLH-30 regulates 26. Hars, E. S. et al. Autophagy regulates ageing in C. elegans. Autophagy 3, 93–95 autophagy and modulates longevity in Caenorhabditis elegans. Nat. Commun. 4:2267 (2007). doi: 10.1038/ncomms3267 (2013). 8 NATURE COMMUNICATIONS | 4:2267 | DOI: 10.1038/ncomms3267 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved.

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Nature Communications – Springer Journals

Published: Aug 8, 2013

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