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Cell nonautonomous activation offlavin-containing monooxygenasepromotes longevity and health spanScott F. Leiser,1 Hillary Miller,1* Ryan Rossner,1* Marissa Fletcher,1 Alison Leonard,1

Melissa Primitivo,1 Nicholas Rintala,1 Fresnida J. Ramos,1

Dana L. Miller,2 Matt Kaeberlein1†

Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health spanin nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal tothe intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotoninbiosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine.Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point ofconvergence for two distinct longevity pathways. FMOs are conserved in eukaryotes andinduced by multiple life span–extending interventions in mice, which suggests that theseenzymes may play a critical role in promoting health and longevity across phyla.

In nematodes, as in mammals, hypoxia-inducible factor (HIF) proteins have a centralrole in responding to changes in environ-mental oxygen (1). HIF proteins are transcrip-tion factors regulated by oxygen-dependent

proteasomal degradation and are stabilized underlow-oxygen conditions to modulate expression ofhundreds of target genes to produce the hypoxicresponse (2). In mammals, constitutive stabiliza-tion of HIF through loss of the E3 ubiquitin ligasevon Hippel-Lindau (VHL) protein leads to a dis-ease characterized by angiomas and renal carci-nomas (3), whereas in Caenorhabditis elegans,loss of the VHL homolog gene, vhl-1, improvesproteostasis and increases life span (4, 5). Thisdifference likely reflects the fact that somaticcells of adult C. elegans are postmitotic, withlittle or no potential for tumor development, andraises the possibility that specific targets ofHIF-1 that promote healthy aging in C. elegansmay function similarly in mammals.To understand how hypoxic signaling slows

aging inworms, we identified genes downstreamof HIF-1 that promote longevity and health span.We took advantage of the large reduction in age-associated autofluorescence observed in animalsin which vhl-1 is not expressed (vhl-1 knockout)(4) to screen for known HIF-1 target genes re-quired for this phenotype (fig. S1). Our screenidentified 24 RNA interference (RNAi) clonesthat substantially increased autofluorescencein vhl-1 animals, eight of which also reducedthe long life span of vhl-1 mutant animals(table S1 and fig. S2). Six of these RNAi cloneshad no effect on the life span of the wild-type

reference strain (N2 Bristol), which indicatedthat they may function specifically to enhancelongevity when HIF-1 is stabilized.Having established a set of HIF-1 target genes

necessary for the full longevity effect of activa-tion of HIF-1, we tested whether any of these

genes were sufficient to enhance longevity andhealth span. We used the Mos1 transposase–mediated single-copy insertion system (6) tooverexpress a single copy of each of the six genesfrom the ubiquitous eft-3 promoter (fig. S3). De-pletion of the xenobiotic detoxification enzymeflavin-containing monooxygenase-2 (fmo-2) byRNAi showed it to be required for full life-spanextension in vhl-1 knockout animals (Fig. 1A).FMO-2 was also sufficient to extend life span onits own (Fig. 1B and fig. S3). Ubiquitous FMO-2overexpression (FMO-2 OE) also improved multi-ple measures of health span, including enhancedmaintenance of motility (measured by the abilityto swim, or thrash, in liquid), pharyngeal pump-ing, and decreased age-associated autofluores-cence (Fig. 1, C and D and fig. S4). FMO-2 OEanimals did not show the decreased brood sizeor delay in development observed in animalslacking vhl-1; thus, these negative consequencesof HIF-1 activation likely result from other HIF-1targets and are separable from life-span andhealth-span extension (fig. S4).Maintaining proteostasis is critical for healthy

aging (7), and both dietary restriction (DR) andstabilization of HIF-1 enhance proteostasis inC. elegans (4, 8). To determine whether FMO-2enhances proteostasis, we examined the effectof FMO-2 OE on resistance to proteotoxic stress.The most notable effect of FMO-2 OE was resist-ance to proteotoxic stress within the endoplasmicreticulum (ER), as evidenced by reduced growthinhibition in response to treatment of animals

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1Department of Pathology, University of Washington, Seattle,WA 98195, USA. 2Department of Biochemistry, University ofWashington, Seattle, WA 98195, USA.*These authors contributed equally to this work. †Correspondingauthor. E-mail: kaeber@uw.edu

Fig. 1. A screen for age-associated autofluorescence identifies FMO-2 as a modulator of longevityand health span in the hypoxic-response pathway. (A) Life spans of vhl-1(ok161) animals on emptyvector (EV), hif-1RNAi, or fmo-2RNAi. (B) Life spans of wild-typeworms andworms overexpressing FMO-2ubiquitously (eft-3 promoter, FMO-2 OE). (C and D) Thrashing, pumping, and autofluorescence measure-ments of wild-type, FMO-2OEworms, and vhl-1(ok161)mutantworms during adulthood (days 10, 13, and 5,respectively). Statistically different (*P < 0.05) from wild type by individual t test for each strain. Error barsrepresent SEM; N ≥ 3 for all experiments.

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with tunicamycin (up to 10 mg/ml) and reducedmortality of animals treated with dithiothreitol(DTT, 7mM) (Fig. 2, A and B). FMO-2 OE animalswere also resistant to general proteotoxic stressinduced by high temperature (Fig. 2C), reductiveproteotoxic stress from 2-carboxyethyl phosphinehydrochloride treatment, and transgenic expressionof an aggregation-prone polyglutamine peptidefused to yellow fluorescent protein (Q35::YFP) (9)(fig. S5).We examined the interaction between fmo-2

and other important longevity pathways. Life-span extension from stabilization of HIF-1 is ge-netically distinct from that regulated by both theinsulin-like signaling pathway and DR (4, 5, 10).Life extension in FMO-2 OE animals appears notto require the rest of the hypoxic-response path-way, insulin-like signaling, or the phase II detox-ification pathway, because it was not lost in hif-1,daf-16, or skn-1 mutants, respectively (fig. S6).Thus, FMO-2 does not act through these tran-scription factors to promote longevity. Similarly,fmo-2 appears not to be necessary for life-spanextension produced by known aging-relatedpathways, because loss of fmo-2 alone had onlya modest effect on life span and did not pre-vent life-span extension in response to reducedinsulin-like signaling caused by daf-2 RNAi orinhibition of mitochondrial respiration causedby isp-1 RNAi (fig. S7). However, fmo-2 was re-quired for life-span extension induced by DR,when the technique of periodic feeding andfasting, or sDR, is used (11) (Fig. 2D). To furtherexplore the possibility that FMO-2 acts in boththe hypoxic response and DR, we confirmedthat fmo-2 is transcriptionally induced by fooddeprivation by monitoring a reporter for fmo-2transcription fused with green fluorescent pro-tein (fmo-2p::GFP) (Fig. 2, E and F). Unlike thatcaused by hypoxia (12), induction of fmo-2 inresponse to fasting was not dependent uponHIF-1 (Fig. 2, E and F). This is consistent withour observation that life-span extension fromDR does not require hif-1 (4) and raises the pos-sibility that DR and the hypoxic response con-verge on FMO-2 to promote longevity throughdistinct signal transduction pathways.The simplest way HIF-1 might increase fmo-2

expression is to bind the fmo-2 promoter direct-ly and promote transcription. Previous reports,and our results with transcriptional reporters,both indicate that FMO-2 is expressed predomi-nantly in the intestine (13). In agreement withthis, overexpression of FMO-2 under an intes-tinal promoter was sufficient to promote lon-gevity (Fig. 3A). To test whether HIF-1 also actsin the intestine to promote longevity, we usedtransgenic nematodes with a nondegradableHIF-1 variant (14), referred to hereafter asHIF-1S.Intestinal HIF-1S had no effect on longevity(fig. S8), whereas neuronal HIF-1S was sufficientto increase life span (Fig. 3B). Expressing HIF-1S

in neurons was also sufficient to rescue addi-tional defects in hif-1 knockout animals, includ-ing failure to develop in hypoxia (0.5% oxygen)(Fig. 3C and fig. S9), loss of vulval integrity dur-ing aging (fig. S10), and life-span extension from

hypoxia during adulthood (fig. S11) (15, 16). Neu-ronal HIF-1S in animals lacking HIF-1 in othertissues was also sufficient to extend life span(Fig. 3D), which indicated that stabilization ofHIF-1 in neurons alone is sufficient to extend lifespan in C. elegans, even without HIF-1 in othercell types.Neuronal overexpression of fmo-2 had no

detectable effect on longevity (fig. S8). Thus,HIF-1 and FMO-2 appear to promote longevityand health span by acting in distinct tissues:HIF-1 in neurons and FMO-2 in intestine. Con-sistent with this model, transcription of fmo-2was significantly induced in the intestine bystabilization ofHIF-1 in neurons in a background

where hif-1 is knocked out in all other tissues[hif-1(ia04); neuro-HIF-1S] as measured by bothquantitative reverse transcription polymerasechain reaction (QRT-PCR) and by fluorescence ina reporter strain (Fig. 3E and fig. S12). In agree-ment with these results, depletion of fmo-2 withRNAi prevented life-span extension in this strain(Fig. 3F), despite the inefficiency of RNAi in neu-rons (17), which indicated that neuronal HIF-1signaling to intestinal FMO-2 is probably neces-sary for the longevity benefit.Having established a connection between neu-

ronal HIF-1 signaling and intestinal FMO-2 acti-vation, we explored potential signal transductionpathways by depleting signaling components

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Fig. 2. FMO-2 modulates proteo-stasis and longevity downstreamof HIF-1 and DR. (A to C) Con-trol, fmo-2(ok2147), vhl-1(ok161),and FMO-2 OE resistance to tuni-camycin (growth from egg), dithio-threitol [survival at the fourth larvalstage (L4)], andheat (survival at L4).(D)Wild-type and fmo-2(ok2147) lifespansonperiodicDR (sDR). (E) fmo-2p::GFP reporter worms in fed andfasted conditions. (F) Quantitativemeasurements of fmo-2 fluores-cence shown in (E). Statisticallydifferent (*P < 0.05) fromwild type(**P<0.05) from hif-1 by individualt test for each strain. Error bars rep-resent SEM;N≥3 for all experiments.

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and transcription factors chosen on the basis ofprevious reports and in silico promoter analysis(18, 19). Although most of the factors examinedhad no effect (fig. S13), the serotonergic signal-ing pathway was both necessary and sufficientfor the cell nonautonomous effect of HIF-1 sig-naling in neurons on expression of FMO-2 inintestine and subsequent longevity benefit ofFMO-2. The 5-hydroxytryptamine7 receptor ser-7and the rate-limiting enzyme in serotonin pro-duction, tph-1, were both required for the acti-vation of FMO-2 in hypoxia and the longevitybenefit from neuronal HIF-1S or vhl-1 mutation(Fig. 4, A to C, and fig. S14). In agreement withthis, HIF-1S expressed under the serotonergictph-1 promoter was sufficient to improve longevityto an extent comparable to that of pan-neuronalexpression (Fig. 4D). A transcription factor withpredictedbinding to the fmo-2promoter, HLH-30,was necessary for either hypoxia or starvationto fully induce transcription of an FMO-2 reporterin the intestine (Fig. 4E). HLH-30 is necessary for

life-span extension by DR (20), and our resultsindicate that it is also required to achieve maxi-mal life-span extension from HIF-1 stabilization(Fig. 4F), although expression ofHIF-1S or deletionof vhl-1 still partially increase life span in animalsdepleted of hlh-30.Our results support a model in which the

flavin-containing monooxygenase FMO-2 func-tions in the intestine to increase life span, im-prove health span, and enhance proteostasis inanimals undergoing the hypoxic response or DR.Further, intestinal fmo-2 is regulated cell non-autonomously through serotonergic signalingoriginating in neurons, and subsequent activa-tion of the transcription factor HLH-30 in theintestine (Fig. 4G). FMO-2 is thus an enzyme bothnecessary and sufficient for a majority of thebeneficial effects of either of these longevitypathways. The FMO-2 substrates important forhealthy aging in C. elegans remain unknown. Itwill also be of interest to directly assess whetherFMOs may function in mammalian aging. There

are five mammalian FMO proteins (FMO-1 to 5)(21), similar to the five C. elegans FMOs (13), andall of these proteins came from a single ancestralFMO (22). In mammals, there is relatively lim-ited information on the specific functions of eachFMO beyond tissue-specific expression patternsand the role of FMO-3 in a single human disease,fish-odor syndrome (23). Mammalian FMOs alsohave a major role in regulating cholesterol andfat metabolism (24, 25). Abundance of FMO pro-teins is increased in the tissues, particularly liver,of several long-lived mouse models includingSnell dwarf mice, Ames dwarf mice, growth-hormone receptor knockout mice, Little mice,dietary-restricted mice, and rapamycin-fed mice(26). Indeed, FMO-3 mRNA is the most consist-ently induced mRNA under DR in mouse liver(27). Taken with the data presented here, theseobservations raise the possibility that activationof FMOs may be a conserved mechanism forenhancing protein homeostasis, improving healthspan, and extending life span and that appropriate

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Fig. 3. Neuronal HIF-1 activates intestinal fmo-2 to increase longevity. (A)Life spans of worms overexpressing FMO-2 under an intestinal (vha-6p)promoter. (B) Life spans of control worms and worms expressing HIF-1S

under neuronal (unc-14p) and ubiquitous (hif-1p) promoters. (C) Growthin hypoxia (0.5% oxygen) of wild-type, hif-1(ia04), and hif-1(ia04)::neuro-HIF-1S worms after 6 days from egg. (D) Life spans of control, hif-1(ia04),

and hif-1(ia04) worms with stabilized neuronal HIF-1. (E) QPCR measure-ment of fmo-2 transcript in multiple strains. (F) Life spans of hif-1(ia04)and hif-1(ia04) worms with stabilized neuro-HIF-1S in control (EV) and fmo-2RNAi. Statistically different (*P < 0.05) from wild type or (**P < 0.05) fromhif-1 by individual t test for each strain. Error bars represent SEM, N ≥ 3 forall experiments.

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activation of FMOsmight promote healthy agingin mammals and people.

REFERENCES AND NOTES

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17. R. S. Kamath, M. Martinez-Campos, P. Zipperlen, A. G. Fraser,J. Ahringer, Genome Biol. 2, RESEARCH0002 (2001).

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E. A. Shephard, Pharmacogenetics 14, 117–130 (2004).23. C. T. Dolphin et al., Nat. Genet. 17, 491–494 (1997).24. S. Veeravalli et al., Biochem. Pharmacol. 90, 88–95 (2014).25. D. M. Shih et al., J. Lipid Res. 56, 22–37 (2015).26. M. J. Steinbaugh, L. Y. Sun, A. Bartke, R. A. Miller, Am. J.

Physiol. Endocrinol. Metab. 303, E488–E495 (2012).27. W. R. Swindell, BMC Genomics 10, 585 (2009).

ACKNOWLEDGMENTS

We thank C. Dolphin for the vector to make the fmo-2p::GFP reporterstrains and A. Mendenhall for advice and help with injecting worms.Strains were provided by the CaenorhabditisGenetics Center. This workwas supported by NIH grant R01AG038518 and an award from theSamsung Advanced Institute of Technology, Samsung Electronics Co.,

to M.K. and NIH grant K99AG045200 to S.F.L. S.F.L. and F.J.R. weresupported by NIH Training Grant T32AG000057. S.F.L. was alsosupported by an American Federation for Aging Research PostdoctoralFellowship. Additional support was provided by the University ofWashington Healthy Aging and Longevity Research Institute, theUniversity of Washington Nathan Shock Center of Excellence in theBasic Biology of Aging (NIH grant P30AG013280), and an award toM.K. from the M. J. Murdock Charitable Trust. D.L.M. is an EllisonMedical Foundation New Scholar in Aging and receives support fromNational Institute on Aging, NIH, R00AGA0033050. Additional dataare available in the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/350/6266/1375/suppl/DC1Materials and MethodsFigs. S1 to S14Tables S1 to S3References (28–33)

30 June 2015; accepted 3 November 2015Published online 19 November 201510.1126/science.aac9257

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Fig. 4.The signaling pathway from neuronalHIF-1 to intestinal FMO-2 involves serotoninand HLH-30. (A) Fluorescence images andquantification of fmo-2p::GFP reporter wormsin normoxia (~21% O2) and hypoxia (0.1% O2)on control, ser-7, and tph-1RNAi. (B and C) Lifespans of wild-type, vhl-1(ok161) mutant, andhif-1(ia04) with stabilized neuro-HIF-1S wormson control (EV, solid lines), ser-7 RNAi (B, dashedlines) and tph-1 RNAi (C, dashed lines). (D) Lifespans of worms expressing HIF-1S under thepan-neuronal (unc-54p) and serotonergic (tph-1p) promoters. (E) Expression of fmo-2p::GFPreporter in fed, fasted, and hypoxic conditionsunder control and hlh-30 RNAi. (F) Life spansof wild-type, vhl-1(ok161) mutant, and hif-1(ia04)with stabilized neuro-HIF-1S worms on control (EV, solid lines) and hlh-30 RNAi (dashed lines) (G) Model of hypoxic response and DR converging on intestinalFMO-2. In graphs, statistical difference (*P < 0.05) from wild type by individual t test for each strain. Error bars represent SEM, N ≥ 3 for all experiments.

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health spanCell nonautonomous activation of flavin-containing monooxygenase promotes longevity and

Ramos, Dana L. Miller and Matt KaeberleinScott F. Leiser, Hillary Miller, Ryan Rossner, Marissa Fletcher, Alison Leonard, Melissa Primitivo, Nicholas Rintala, Fresnida J.

originally published online November 19, 2015DOI: 10.1126/science.aac9257 (6266), 1375-1378.350Science 

, this issue p. 1375Sciencelongevity, may elucidate further mechanisms that promote healthy aging.longevity. Finding the relevant targets of FMO-2, which also accumulates in mammals under conditions that promoteeffects lead to increased production of flavin-containing monooxygenase-2 (FMO-2) in the intestine, which increased

1 and increased serotonergic signaling. These−in neurons with transcriptional activation by hypoxia-inducible factor show that the life-extending effects of hypoxia begin et al.on the activation of an enzyme in cells of the intestine. Leiser

, convergeCaenorhabditis elegansThe effects of hypoxia and caloric restriction, both of which extend life span in and the gut−−Aging: All in the head

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REFERENCES

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