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Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span

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Science  11 Dec 2015:
Vol. 350, Issue 6266, pp. 1375-1378
DOI: 10.1126/science.aac9257

Aging: All in the head—and the gut

The effects of hypoxia and caloric restriction, both of which extend life span in Caenorhabditis elegans, converge on the activation of an enzyme in cells of the intestine. Leiser et al. show that the life-extending effects of hypoxia begin in neurons with transcriptional activation by hypoxia-inducible factor–1 and increased serotonergic signaling. These effects lead to increased production of flavin-containing monooxygenase-2 (FMO-2) in the intestine, which increased longevity. Finding the relevant targets of FMO-2, which also accumulates in mammals under conditions that promote longevity, may elucidate further mechanisms that promote healthy aging.

Science, this issue p. 1375

Abstract

Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic 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 of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span–extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.

In nematodes, as in mammals, hypoxia-inducible factor (HIF) proteins have a central role in responding to changes in environmental oxygen (1). HIF proteins are transcription factors regulated by oxygen-dependent proteasomal degradation and are stabilized under low-oxygen conditions to modulate expression of hundreds of target genes to produce the hypoxic response (2). In mammals, constitutive stabilization of HIF through loss of the E3 ubiquitin ligase von Hippel-Lindau (VHL) protein leads to a disease characterized by angiomas and renal carcinomas (3), whereas in Caenorhabditis elegans, loss of the VHL homolog gene, vhl-1, improves proteostasis and increases life span (4, 5). This difference likely reflects the fact that somatic cells of adult C. elegans are postmitotic, with little or no potential for tumor development, and raises the possibility that specific targets of HIF-1 that promote healthy aging in C. elegans may function similarly in mammals.

To understand how hypoxic signaling slows aging in worms, we identified genes downstream of HIF-1 that promote longevity and health span. We took advantage of the large reduction in age-associated autofluorescence observed in animals in which vhl-1 is not expressed (vhl-1 knockout) (4) to screen for known HIF-1 target genes required for this phenotype (fig. S1). Our screen identified 24 RNA interference (RNAi) clones that substantially increased autofluorescence in vhl-1 animals, eight of which also reduced the long life span of vhl-1 mutant animals (table S1 and fig. S2). Six of these RNAi clones had no effect on the life span of the wild-type reference strain (N2 Bristol), which indicated that they may function specifically to enhance longevity when HIF-1 is stabilized.

Having established a set of HIF-1 target genes necessary for the full longevity effect of activation of HIF-1, we tested whether any of these genes were sufficient to enhance longevity and health span. We used the Mos1 transposase–mediated single-copy insertion system (6) to overexpress a single copy of each of the six genes from the ubiquitous eft-3 promoter (fig. S3). Depletion of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (fmo-2) by RNAi showed it to be required for full life-span extension in vhl-1 knockout animals (Fig. 1A). FMO-2 was also sufficient to extend life span on its own (Fig. 1B and fig. S3). Ubiquitous FMO-2 overexpression (FMO-2 OE) also improved multiple measures of health span, including enhanced maintenance of motility (measured by the ability to swim, or thrash, in liquid), pharyngeal pumping, and decreased age-associated autofluorescence (Fig. 1, C and D and fig. S4). FMO-2 OE animals did not show the decreased brood size or delay in development observed in animals lacking vhl-1; thus, these negative consequences of HIF-1 activation likely result from other HIF-1 targets and are separable from life-span and health-span extension (fig. S4).

Fig. 1 A screen for age-associated autofluorescence identifies FMO-2 as a modulator of longevity and health span in the hypoxic-response pathway.

(A) Life spans of vhl-1(ok161) animals on empty vector (EV), hif-1 RNAi, or fmo-2 RNAi. (B) Life spans of wild-type worms and worms overexpressing FMO-2 ubiquitously (eft-3 promoter, FMO-2 OE). (C and D) Thrashing, pumping, and autofluorescence measurements of wild-type, FMO-2 OE worms, and vhl-1(ok161) mutant worms during adulthood (days 10, 13, and 5, respectively). Statistically different (*P < 0.05) from wild type by individual t test for each strain. Error bars represent SEM; N ≥ 3 for all experiments.

Maintaining proteostasis is critical for healthy aging (7), and both dietary restriction (DR) and stabilization of HIF-1 enhance proteostasis in C. elegans (4, 8). To determine whether FMO-2 enhances proteostasis, we examined the effect of FMO-2 OE on resistance to proteotoxic stress. The most notable effect of FMO-2 OE was resistance to proteotoxic stress within the endoplasmic reticulum (ER), as evidenced by reduced growth inhibition in response to treatment of animals with tunicamycin (up to 10 μg/ml) and reduced mortality of animals treated with dithiothreitol (DTT, 7 mM) (Fig. 2, A and B). FMO-2 OE animals were also resistant to general proteotoxic stress induced by high temperature (Fig. 2C), reductive proteotoxic stress from 2-carboxyethyl phosphine hydrochloride treatment, and transgenic expression of an aggregation-prone polyglutamine peptide fused to yellow fluorescent protein (Q35::YFP) (9) (fig. S5).

Fig. 2 FMO-2 modulates proteostasis and longevity downstream of HIF-1 and DR.

(A to C) Control, fmo-2(ok2147), vhl-1(ok161), and FMO-2 OE resistance to tunicamycin (growth from egg), dithiothreitol [survival at the fourth larval stage (L4)], and heat (survival at L4). (D) Wild-type and fmo-2(ok2147) life spans on periodic DR (sDR). (E) fmo-2p::GFP reporter worms in fed and fasted conditions. (F) Quantitative measurements of fmo-2 fluorescence shown in (E). Statistically different (*P < 0.05) from wild type (**P < 0.05) from hif-1 by individual t test for each strain. Error bars represent SEM; N ≥ 3 for all experiments.

We examined the interaction between fmo-2 and other important longevity pathways. Life-span extension from stabilization of HIF-1 is genetically distinct from that regulated by both the insulin-like signaling pathway and DR (4, 5, 10). Life extension in FMO-2 OE animals appears not to require the rest of the hypoxic-response pathway, insulin-like signaling, or the phase II detoxification 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 transcription factors to promote longevity. Similarly, fmo-2 appears not to be necessary for life-span extension produced by known aging-related pathways, because loss of fmo-2 alone had only a modest effect on life span and did not prevent life-span extension in response to reduced insulin-like signaling caused by daf-2 RNAi or inhibition of mitochondrial respiration caused by isp-1 RNAi (fig. S7). However, fmo-2 was required for life-span extension induced by DR, when the technique of periodic feeding and fasting, or sDR, is used (11) (Fig. 2D). To further explore the possibility that FMO-2 acts in both the hypoxic response and DR, we confirmed that fmo-2 is transcriptionally induced by food deprivation by monitoring a reporter for fmo-2 transcription fused with green fluorescent protein (fmo-2p::GFP) (Fig. 2, E and F). Unlike that caused by hypoxia (12), induction of fmo-2 in response to fasting was not dependent upon HIF-1 (Fig. 2, E and F). This is consistent with our observation that life-span extension from DR does not require hif-1 (4) and raises the possibility that DR and the hypoxic response converge on FMO-2 to promote longevity through distinct signal transduction pathways.

The simplest way HIF-1 might increase fmo-2 expression is to bind the fmo-2 promoter directly and promote transcription. Previous reports, and our results with transcriptional reporters, both indicate that FMO-2 is expressed predominantly in the intestine (13). In agreement with this, overexpression of FMO-2 under an intestinal promoter was sufficient to promote longevity (Fig. 3A). To test whether HIF-1 also acts in the intestine to promote longevity, we used transgenic nematodes with a nondegradable HIF-1 variant (14), referred to hereafter as HIF-1S. Intestinal HIF-1S had no effect on longevity (fig. S8), whereas neuronal HIF-1S was sufficient to increase life span (Fig. 3B). Expressing HIF-1S in neurons was also sufficient to rescue additional defects in hif-1 knockout animals, including failure to develop in hypoxia (0.5% oxygen) (Fig. 3C and fig. S9), loss of vulval integrity during aging (fig. S10), and life-span extension from hypoxia during adulthood (fig. S11) (15, 16). Neuronal HIF-1S in animals lacking HIF-1 in other tissues was also sufficient to extend life span (Fig. 3D), which indicated that stabilization of HIF-1 in neurons alone is sufficient to extend life span in C. elegans, even without HIF-1 in other cell types.

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) Growth in 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 measurement 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-2 RNAi. Statistically different (*P < 0.05) from wild type or (**P < 0.05) from hif-1 by individual t test for each strain. Error bars represent SEM, N ≥ 3 for all experiments.

Neuronal overexpression of fmo-2 had no detectable effect on longevity (fig. S8). Thus, HIF-1 and FMO-2 appear to promote longevity and health span by acting in distinct tissues: HIF-1 in neurons and FMO-2 in intestine. Consistent with this model, transcription of fmo-2 was significantly induced in the intestine by stabilization of HIF-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 both quantitative reverse transcription polymerase chain reaction (QRT-PCR) and by fluorescence in a reporter strain (Fig. 3E and fig. S12). In agreement with these results, depletion of fmo-2 with RNAi prevented life-span extension in this strain (Fig. 3F), despite the inefficiency of RNAi in neurons (17), which indicated that neuronal HIF-1 signaling to intestinal FMO-2 is probably necessary for the longevity benefit.

Having established a connection between neuronal HIF-1 signaling and intestinal FMO-2 activation, we explored potential signal transduction pathways by depleting signaling components and transcription factors chosen on the basis of previous reports and in silico promoter analysis (18, 19). Although most of the factors examined had no effect (fig. S13), the serotonergic signaling pathway was both necessary and sufficient for the cell nonautonomous effect of HIF-1 signaling in neurons on expression of FMO-2 in intestine and subsequent longevity benefit of FMO-2. The 5-hydroxytryptamine7 receptor ser-7 and the rate-limiting enzyme in serotonin production, tph-1, were both required for the activation of FMO-2 in hypoxia and the longevity benefit from neuronal HIF-1S or vhl-1 mutation (Fig. 4, A to C, and fig. S14). In agreement with this, HIF-1S expressed under the serotonergic tph-1 promoter was sufficient to improve longevity to an extent comparable to that of pan-neuronal expression (Fig. 4D). A transcription factor with predicted binding to the fmo-2 promoter, HLH-30, was necessary for either hypoxia or starvation to fully induce transcription of an FMO-2 reporter in the intestine (Fig. 4E). HLH-30 is necessary for life-span extension by DR (20), and our results indicate that it is also required to achieve maximal life-span extension from HIF-1 stabilization (Fig. 4F), although expression of HIF-1S or deletion of vhl-1 still partially increase life span in animals depleted of hlh-30.

Fig. 4 The signaling pathway from neuronal HIF-1 to intestinal FMO-2 involves serotonin and HLH-30.

(A) Fluorescence images and quantification of fmo-2p::GFP reporter worms in normoxia (~21% O2) and hypoxia (0.1% O2) on control, ser-7, and tph-1RNAi. (B and C) Life spans of wild-type, vhl-1(ok161) mutant, and hif-1(ia04) with stabilized neuro-HIF-1S worms on control (EV, solid lines), ser-7 RNAi (B, dashed lines) and tph-1 RNAi (C, dashed lines). (D) Life spans of worms expressing HIF-1S under the pan-neuronal (unc-54p) and serotonergic (tph-1p) promoters. (E) Expression of fmo-2p::GFP reporter in fed, fasted, and hypoxic conditions under control and hlh-30 RNAi. (F) Life spans of 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 intestinal FMO-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.

Our results support a model in which the flavin-containing monooxygenase FMO-2 functions in the intestine to increase life span, improve health span, and enhance proteostasis in animals undergoing the hypoxic response or DR. Further, intestinal fmo-2 is regulated cell nonautonomously through serotonergic signaling originating in neurons, and subsequent activation of the transcription factor HLH-30 in the intestine (Fig. 4G). FMO-2 is thus an enzyme both necessary and sufficient for a majority of the beneficial effects of either of these longevity pathways. The FMO-2 substrates important for healthy aging in C. elegans remain unknown. It will also be of interest to directly assess whether FMOs may function in mammalian aging. There are five mammalian FMO proteins (FMO-1 to 5) (21), similar to the five C. elegans FMOs (13), and all of these proteins came from a single ancestral FMO (22). In mammals, there is relatively limited information on the specific functions of each FMO beyond tissue-specific expression patterns and the role of FMO-3 in a single human disease, fish-odor syndrome (23). Mammalian FMOs also have a major role in regulating cholesterol and fat metabolism (24, 25). Abundance of FMO proteins is increased in the tissues, particularly liver, of several long-lived mouse models including Snell 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 consistently induced mRNA under DR in mouse liver (27). Taken with the data presented here, these observations raise the possibility that activation of FMOs may be a conserved mechanism for enhancing protein homeostasis, improving health span, and extending life span and that appropriate activation of FMOs might promote healthy aging in mammals and people.

Supplementary Materials

www.sciencemag.org/content/350/6266/1375/suppl/DC1

Materials and Methods

Figs. S1 to S14

Tables S1 to S3

References (2833)

References and Notes

  1. Acknowledgments: We thank C. Dolphin for the vector to make the fmo-2p::GFP reporter strains and A. Mendenhall for advice and help with injecting worms. Strains were provided by the Caenorhabditis Genetics Center. This work was supported by NIH grant R01AG038518 and an award from the Samsung 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. were supported by NIH Training Grant T32AG000057. S.F.L. was also supported by an American Federation for Aging Research Postdoctoral Fellowship. Additional support was provided by the University of Washington Healthy Aging and Longevity Research Institute, the University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging (NIH grant P30AG013280), and an award to M.K. from the M. J. Murdock Charitable Trust. D.L.M. is an Ellison Medical Foundation New Scholar in Aging and receives support from National Institute on Aging, NIH, R00AGA0033050. Additional data are available in the supplementary materials.
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