The flight response impairs cytoprotective mechanisms by activating the insulin pathway

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Abstract

An animal’s stress response requires different adaptive strategies depending on the nature and duration of the stressor. Whereas acute stressors, such as predation, induce a rapid and energy-demanding fight-or-flight response, long-term environmental stressors induce the gradual and long-lasting activation of highly conserved cytoprotective processes1,2,3. In animals across the evolutionary spectrum, continued activation of the fight-or-flight response weakens the animal’s resistance to environmental challenges4,5. However, the molecular and cellular mechanisms that regulate the trade-off between the flight response and long-term stressors are poorly understood. Here we show that repeated induction of the flight response in Caenorhabditis elegans shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, an invertebrate analogue of adrenaline and noradrenaline. Tyramine stimulates the insulin–IGF-1 signalling (IIS) pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. By contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release and thereby allow the induction of cytoprotective genes. These findings demonstrate that a neural stress hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defence systems and accelerates ageing.

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Fig. 1: The flight response and tyramine impair the resistance to subsequent stressors.
Fig. 2: RIM neurons show opposing activity patterns during the flight response and during exposure to environmental stressors.
Fig. 3: The GPCR TYRA-3 is required in the intestine for tyraminergic modulation of the stress response.
Fig. 4: Tyramine signalling inhibits stress-dependent nuclear translocation of DAF-16.

Data availability

All the data are publicly available via the Open Science Framework: https://osf.io/mj3n9/?view_only=b6c7ff8697544e71b725767f17e19628.

Code availability

All custom software created and used in this work is available upon request.

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Acknowledgements

Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank C. Bargmann, M. Nonet, A. Dillin, H. Tissenbaum, D. Albrecht, J. Hawk, C. Weist, M. Madhav, A. Thackeray, C. Benard, M. Gorczyca, A. Bizet and W. Joyce for strains and technical support; and A. Garelli, G. Spitzmaul, A. Byrne, V. Budnik, M. Walhout, M. Belew, M. Ailion and T. Shpilka for discussions. This work was supported by grants from UNS (PGI 24/B216 to D.R., PGI 24/ZB62 to M.J.D.R.), ANPCYT (PICT 2014 3118 to D.R.) and CONICET (PIP11220150100182CO to D.R. and M.J.D.R.), and grant GM084491 from the National Institutes of Health to M.J.A.

Author information

M.J.D.R., T.V., J.F., J.G., M.G.B., N.A., D.R. and M.J.A. designed the experiments. Stress resistance and lifespan assays: M.J.D.R., T.V., J.F., M.G.B., N.A., D.R. Optogenetic and calcium imaging: J.F. and J.G. Microscopy and image analysis: M.J.D.R., T.V., M.G.B., N.A., D.R. Molecular biology and strain generation: M.J.D.R., T.V., J.F., J.D., D.R. and M.J.A. conceived the study and wrote the paper.

Correspondence to Diego Rayes or Mark J. Alkema.

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The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Mario de Bono, Veena Prahlad, Nektarios Tavernarakis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 C. elegans faces different types of environmental stress.

Like other animals, C. elegans is exposed to different forms of stress in its environment that occur either abruptly (for example, predation) or more progressively (food shortage, osmotic stress, oxidation, high or low environmental temperatures). Stress can induce behavioural and/or cellular responses. The stress response to environmental stressors such as heat, starvation and oxidative stress features a central role for the DAF-2–insulin–IGF-1 signalling (IIS) pathway and the activation of DAF-16/FOXO and HSF-1 transcription factors. In response to acute challenges, such as touch, C. elegans can engage in an escape response during which it moves rapidly away from a life-threatening stimulus.

Extended Data Fig. 2 The flight response and tyramine decrease lifespan and impair resistance to stressors.

a, Oxidation and heat survival indexes of animals pre-exposed to different tap stimulus protocols (from 1 to 40 plate taps every 5 min). n = 4 and n = 3 independent experiments for oxidation and heat, respectively. At least 40 animals were analysed in each independent experiment. Repeated triggering of the flight response (>20–25 times) before exposure to oxidative or heat stress reduced animal survival. b, Top, scheme for the sequential stress experiments. Pre-stressors: 1 h 1 mM Fe2+ (Ox), 0.5 h 35 °C (°C), 8 h food deprivation (Fst), or tap stimulus every 5 min for 2.5 h (Tap). After 1 h of recovery, survival to heat stress (4 h 35 °C) was evaluated. Bottom, representative Kaplan–Meier survival curves of pre-stressed nematodes exposed to subsequent thermal stress (heat); two sided log-rank test. Curves are representative of three independent replicates with similar results (n = 3), 40–80 animals per condition per experiment. c, Heat survival index in the absence and presence of exogenous TA during pre-exposure to the mild stressor followed by 1 h recovery and exposure to the second strong stressor in the absence of TA after pre-treatment. For 0 mM TA conditions, one-way ANOVA followed by Holm–Sidak post-hoc test versus pre-heat, n = 4, 40–80 animals per condition. For 10 mM TA conditions, two-tailed Student’s t-test (versus same condition without TA), n = 3, 40–80 animals per condition. Exogenous tyramine during mild stressor exposure inhibits hormesis. d, Oxidation and heat survival indexes of tdc-1(n3420) mutant animals pre-exposed to the same stressors as in b. n = 4, 40–80 animals per condition. Unlike wild-type animals, tdc-1 mutants displayed no hormetic effects after pre-treatment with mild environmental stressors. e, Pre-incubation with exogenous tyramine (TA) in the absence of environmental pre-stressors did not produce significant differences in subsequent oxidative stress and thermal resistance. Animals were exposed to 10 mM of exogenous TA for 3 h. After 1 h recovery, resistance to heat (4 h at 35 °C) and oxidative (2 h, 3 mM Fe2+) stress was evaluated. n = 4 independent experiments, 80–100 animals per condition per experiment. Two-tailed Student’s t-test (versus same condition without pre-exposure to TA). f, Left, representative Kaplan–Meier survival curves of naive and pre-stressed (tap) animals exposed to heat (35 °C). The experiment was independently repeated three times (n = 3) with similar results (35–50 animals per condition per experiment), two tailed log-rank test. Right, survival indexes of naive and pre-stressed, wild-type and tdc-1 mutant worms upon exposure to heat stress. n = 3, 35–50 animals per condition, two-tailed Student’s t-test. The flight response impairs survival to heat exposure in wild-type worms but not tyramine-deficient mutants. g, Resistance of wild-type animals exposed to oxidation or heat in the absence and presence of exogenous tyramine (10 mM). n = 4, 60–80 animals per condition. Two-tailed Student’s t-test (versus same condition without TA). Exogenous tyramine reduces oxidation and heat resistance. h, Scatter dot plots (line shows mean). Left, average locomotion rate of animals grown in the presence of exogenous tyramine (10 mM) for 4, 7 or 10 days. Right, average numbers of eggs in the adult uterus (36 h post-L4) in the presence of exogenous tyramine (10 mM, 36 h of exposure). n for each condition shown. Two-tailed Student’s t-test (versus same condition without TA). Exogenous tyramine does not significantly affect locomotion or egg-laying, even upon extended exposure. i, Representative Kaplan–Meier lifespan curves of wild-type and tdc-1 mutant animals. Two-tailed log-rank test. n = 3, 40–80 animals per condition per experiment. Tyramine-deficient animals have an increased lifespan compared to wild-type. j, Representative Kaplan–Meier lifespan curves in the absence or presence of 10 mM exogenous TA. Two tailed log-rank test. n = 3, 40–80 animals per condition per experiment. Exogenous tyramine reduces lifespan. All bars show mean ± s.e.m. Source data

Extended Data Fig. 3 Tyramine-deficient animals are resistant to environmental stress.

a, Tyramine and octopamine biosynthesis pathway. Tyramine is synthesized from tyrosine by tyrosine decarboxylase (TDC-1) in the RIM and RIC neurons; octopamine is synthesized from tyramine by tyramine β-hydroxylase (TBH-1) in the RIC neurons13. Whereas tdc-1 null mutants are deficient in both tyramine and octopamine, tbh-1 null mutants are deficient only in octopamine. b, c, Survival percentages of wild-type, tyramine- and octopamine-deficient (tdc-1), octopamine-deficient (tbh-1) and tyramine receptor (tyra-3) null mutants exposed to oxidation induced by FeSO4 (b, 1 h, 15 mM) and H2O2 (c, 3 h, 5 mM). Mean ± s.e.m., number of independent experiments shown, 60–80 animals per condition per experiment. One-way ANOVA followed by Holm–Sidak post-hoc test for multiple comparisons versus wild-type. d, Survival to heat (4 h at 35 °C) of wild-type, tdc-1, tbh-1, tdc-1;zfIs29[Ptbh-1::TDC-1] (tyramine- but not octopamine-deficient) and the quadruple tyramine-receptor mutant QW833 (lgc-55;ser-2 tyra-2 tyra-3). Mean ± s.e.m., number of independent experiments shown, 60–80 animals per condition per experiment. One-way ANOVA followed by Holm–Sidak post-hoc test for multiple comparisons. tdc-1 mutants have improved survival to thermal stress. Octopamine-deficient mutants (tbh-1) were slightly more heat resistant than wild-type animals, albeit not at the level of tyramine- and octopamine-deficient tdc-1 mutants. Moreover, rescue of tdc-1 expression in only octopaminergic neurons (tdc-1; Ptbh-1::TDC-1) failed to reduce thermoresistance of tdc-1 mutants. In addition, quadruple tyramine receptor mutants show heat resistance levels similar to that of tdc-1. These results indicate that the lack of tyramine underlies the oxidative and thermal resistant phenotype of tdc-1 mutants. e, Scatter dot plot (line at the mean) showing pharyngeal pumping rates (pumps per min) of wild-type, tdc-1, tbh-1 and tph-1 null mutants. tph-1 (tryptophan hydroxylase) mutant animals, which lack serotonin, have a reduced pharyngeal pumping and were used as a control. Because tdc-1 mutants have no obvious defects in pharyngeal pumping, dietary restriction is not likely to be the cause of the enhanced stress resistance and increased longevity. n = 30 animals per condition. One-way ANOVA followed by Holm–Sidak post-hoc test versus wild type. Source data

Extended Data Fig. 4 Optogenetic activation of the flight response reduces the resistance to environmental stressors.

a, Velocity traces and survival curves during strong oxidative stress (3 mM Fe2+) for tdc-1 mutants in the absence (grey) or presence (red) of a mechanical stimulus (tap). n = 7 independent experiments, 40 animals per condition. Velocity remains constant over the 2.5-h duration of recording in the absence of a stimulus, but increases rapidly in response to a mechanical plate tap (top). Tap delivery does not reduce resistance to oxidative stress in tdc-1 mutants (bottom). Black squares, tap delivery. Red line, tap; grey line, no tap; shaded regions, s.e.m. b, Optogenetic activation of mechanosensory neurons induces a flight response that results in velocity increases. Animals were exposed to 5-s pulses of 617-nm light every 5 min (top); n = 8 independent experiments. Optogenetic induction of the flight response reduced resistance to strong oxidation (3 mM Fe2+) (bottom). Dark blue line, survival curve of animals raised with ATR (n = 10 independent experiments); light blue line, animals raised without ATR (n = 9), 40 animals for each experiment. Shaded regions indicate s.e.m., blue squares indicate light delivery. Strain used: QW1649 zfIs144[Pmec-4::Chrimson::wCherry, pL15EK]. c, Survival indexes of animals exposed to oxidative (1 h, 3 mM Fe2+) or heat stress (4 h at 35 °C) with or without vibrational stimulus (plate tapping every 5 min). Tap impaired environmental stress resistance in the wild type, but not in tdc-1 mutants. Mean ± s.e.m. from n = 4 independent experiments, 60–90 animals per condition per experiment. Two-tailed Student’s test versus wild type. d, Percentage of animals suppressing head movements in response to anterior touch in unstressed animals and animals subjected to a tap stimulus every 5 min for 2.5 h. Tyramine release in response to mechanical stimulation induces fast reversal and suppression of head movements13,14. Animals that were subjected to 30 taps administered every 5 min still suppressed head movements in response to anterior touch, indicating that tyramine continues to be released during the tapping protocol and that RIM neuronal activity is not affected. Mean ± s.e.m. from n = 5 independent experiments, 20 animals per condition per experiment. Two-tailed Student’s t-test. e, Survival curves of animals exposed to heat stress (7 h at 35 °C) with simultaneous optogenetic activation of mechanosensory neurons (QW1649: zfis144[Pmec-4::Chrimson::wCherry +pL15EK]). Animals expressing Chrimson in mechanosensory neurons were cultivated in the presence or absence of ATR and subjected to 5-s, 617-nm light pulses every 5 min at 35 °C. Blue squares indicate light delivery. Mean ± s.e.m. Optogenetic activation of mechanosensory neurons reduced heat resistance in animals raised on ATR (n = 6 independent experiments) compared to animals raised without ATR (n = 5 independent experiments), 40 animals per experiment. f, Stress survival analysis of animals grown in the presence or absence of ATR without light stimulation. Ox: 1 h, 3 mM Fe2+; heat: 4 h, 35 °C. ATR does not modify resistance to these environmental stressors. Mean ± s.e.m. from n = 3 independent experiments, 40–50 animals per condition per experiment. No significant differences were observed, indicating that ATR does not affect stress resistance; two-tailed Student’s t-test. g, h, Stress survival of animals expressing HisCl in RIM neurons (RIM::HisCl). Animals were exposed to 10 mM histamine (Hist) before and during oxidation (1 h, 15 mM Fe2+, g) or heat (4 h, 35 °C, h). Specific silencing of RIM neurons leads to increased resistance to environmental stress. Mean ± s.e.m. Numbers of independent experiments for each condition are shown, 80–100 animals per condition per experiment. Two-tailed Student’s t-test versus same strain in the absence of histamine. i, j, Ca2+ responses upon oxidative stress (i; GCaMP: n = 36 animals; mCherry: n = 15) and food deprivation (j; GCaMP: n = 30; mCherry: n = 6). Grey trace, mCherry fluorescence insensitive to calcium. Mean ± s.e.m.; one-way ANOVA, compared to initial time point, Dunnett’s multiple comparison. k, Overall RIM Ca2+ levels (ΔF/F0) increase upon refeeding (with E. coli) of animals that have been starved overnight. Mean ± s.e.m., n = 36 for each data point, six independent experiments. Fluorescence increase is initiated within 10 min of food addition, indicating that RIM activity recovers quickly and is probably not due to changes in expression of GCaMP. One-way ANOVA, compared to initial time point, Dunnett’s multiple comparison. Source data

Extended Data Fig. 5 Tyramine modulates the stress response through activation of TYRA-3.

a, Resistance of wild-type and tyramine receptor mutant animals exposed to heat (4 h, 35 °C). Only tyra-3 mutants are as resistant as tdc-1 mutants to heat stress. Number of independent experiments performed for each condition shown, 80–100 animals per condition per experiment. One-way ANOVA followed by Holm–Sidak post-hoc test for multiple comparisons. b, Representative survival curves of wild-type, tdc-1 and tyra-3 mutants exposed to starvation. Animals were removed from food at the L4 stage. n = 3 independent replicates with similar results, 40–80 animals per condition per experiment. c, Resistance of wild-type, tdc-1 and tyra-3 mutant animals exposed to oxidation, heat or starvation in the absence or presence of exogenous TA (10 mM). Detrimental effects of exogenous TA on stress resistance are abolished in tyra-3 mutant animals. Mean ± s.e.m., n = 4 for oxidation and heat and n = 3 for starvation, 60–80 animals per condition. For conditions without TA, one-way ANOVA followed by Holm–Sidak post-hoc test versus wild-type; for conditions with TA, two-tailed Student’s t-test (versus same strain without TA). Source data

Extended Data Fig. 6 tyra-3 acts in the intestine to inhibit stress resistance.

a, Top, gene structure of tyra-3. Coding sequences, black boxes. Bottom, confocal image of a transgenic animal expressing mCherry driven by Ptyra-3short promoter. mCherry expression is limited to a subset of head and tail neurons (and vulval cells). Scale bar, 200 µm. An mCherry reporter driven by Ptyra-3long is expressed in neurons and intestine (Fig. 3b). b, Survival of tyra-3 mutants expressing tyra-3 cDNA driven by Ptyra-3long, Ptyra-3short, Prgef-1 or Pelt-2 upon exposure to heat stress with or without tyramine (10 mM). Mean ± s.e.m., n = 5, 80–100 animals per condition. For conditions without TA, one-way ANOVA followed by Holm–Sidak post-hoc test versus wild-type; for conditions with TA, two-tailed Student’s t-test versus same strain without TA. Expression of tyra-3 in the intestine, but not in neurons, was sufficient to restore stress sensitivity and the negative effect of exogenous tyramine on heat resistance. c, Representative Kaplan–Meier survival curves of wild-type worms, tyra-3 null mutants and animals expressing tyra-3 solely in the intestine (Pelt-2::tyra-3). Animals were food-deprived as L4s in the absence or presence of 10 mM TA. n = 3 independent replicates, 40–80 animals per condition per experiment. Two-tailed log-rank test. Expression of tyra-3 in the intestine restores the negative effect of exogenous TA on starvation resistance. d, Survival indexes to heat exposure (4 h, 35 °C) of animals pre-exposed to vibrational stimulus (tap). Intestinal expression of tyra-3 restores the detrimental effect of tapping on the stress response. Mean ± s.e.m., n = 3, 30–40 animals per condition. One-way ANOVA followed by Holm–Sidak post-hoc test for multiple comparisons. Source data

Extended Data Fig. 7 Tyraminergic inhibition of stress response depends on the DAF-2 insulin receptor.

a, Survival percentage of animals exposed to oxidative stress (3 h, 15 mM Fe2+) or heat (7 h, 35 °C). tdc-1;daf-2 and tyra-3;daf-2 double mutants are as resistant as daf-2 single mutants. Mean ± s.e.m., n = 3 (heat), n = 4 (oxidation), 80–100 animals per condition per experiment. One-way ANOVA followed by Holm–Sidak’s test for multiple comparisons compared to daf-2 mutants. b, Survival percentage of animals exposed to oxidative stress (3 h, 15 mM Fe2+, top) or heat (7 h, 35 °C, bottom) with or without TA (10 mM). Exogenous TA does not impair heat or oxidative stress resistance in daf-2 mutants. Mean ± s.e.m., n = 3 (heat), n = 4 (oxidation), 80–100 animals per condition per experiment. Two-tailed Student’s t-test versus each strain without TA. Source data

Extended Data Fig. 8 The flight response inhibits stress-dependent nuclear translocation of DAF-16.

a, Left, fluorescence images of young adults expressing the translational reporter Pdaf-16::DAF-16a/b::GFP upon exposure to mild stressors as in Fig. 1: 1 h, 1 mM Fe2+ (Ox), 0.5 h 35 °C (°C), 8 h food deprivation (Fst), or tap every 5 min for 2.5 h (Tap). Right, scatter dot plot (line shows mean) of the number of cells with nuclear DAF-16 per animal. n for each condition is shown. One-way ANOVA, Dunnett’s post-hoc test compared to naive. Scale bar, 150 µm. b, Top, DAF-16 localization upon heat exposure (37 °C, 1 h) in wild-type and tdc-1 mutant animals exposed to tap every 5 min. Bottom, scatter dot plot of the number of cells with nuclear DAF-16 per animal (normalized to naive animals (left)). n for each condition is shown. Two-tailed Student’s t-test versus same strain without tapping. Scale bar, 150 µm. Repetitive induction of the flight response impairs DAF-16 localization to the nucleus in wild-type but not in tdc-1 mutant animals. c, Top, DAF-16 localization upon heat exposure (37 °C, 1 h) in RIM::ChR2 transgenic animals raised with or without ATR subjected to 5-s, 617-nm light pulses every 5 min. Bottom, scatter dot plot of the number of cells with nuclear DAF-16 per animal (normalized to ATR-treated animals). n for each condition is shown. Two-tailed Student’s t-test. Scale bar, 150 µm. Source data

Extended Data Fig. 9 Tyramine signalling mutants show ectopic activation of stress response genes.

a, Left, representative fluorescence images (40× magnification) of the pharynx (top) and intestine (bottom) of animals expressing Psod-3::GFP on different genetic backgrounds after 20 min of exposure to 5 mM Fe2+ followed by 45 min recovery on NGM plates plus food. Scale bar, 100 μm. Right, corresponding quantification of the fluorescence level per animal in the pharynx and intestine. Scatter dot plot (line shows mean) with relative expression of sod-3 normalized to naive animals. n for each condition is shown. One-way ANOVA (Kruskal–Wallis test) and Dunn’s post-hoc test. b, Survival percentage of animals exposed to oxidative stress (1 h, 15 mM Fe2+) or heat (4 h, 35 °C). tdc-1;daf-16 and tyra-3;daf-16 double mutants were compared to the corresponding single mutants. Mean ± s.e.m. For oxidation resistance experiments, n = 6; for heat resistance experiments, n = 4; 80 animals per condition per experiment. One-way ANOVA, Holm–Sidak post-hoc test versus daf-16 mutants. Two-tailed Student’s t-test was used to compare tdc-1;daf-16 and tyra-3;daf-16 double mutants with tdc-1 and tyra-3 single null mutants, respectively. tdc-1;daf-16 and tyra-3;daf-16 double mutants show intermediate resistance phenotypes, so tyraminergic control of the stress response does not depend exclusively on daf-16. c, Left, representative fluorescence images (20× magnification) of young adult animals expressing Phsp16.2::GFP on different genetic backgrounds after 15 min of heat (35 °C) followed by 45 min recovery at 20 °C. Scale bar, 150 μm. Right, corresponding quantification of the fluorescence level per animal in pharynx and intestine. Scatter dot plot with relative expression of Phsp16.2::GFP normalized to naive animals. n for each condition shown. One-way ANOVA (Kruskal–Wallis test) and Dunn’s post-hoc test versus naive. d, Left, representative fluorescence images (20× magnification) of young adult animals expressing Pgst-4::GFP on different genetic backgrounds under basal conditions (20 °C on NGM plates seeded with OP50). Scale bar, 100 μm. Right, corresponding quantification of fluorescence per worm. Scatter dot plot with the relative expression of Pgst-4::GFP normalized to naive animals. n for each condition shown. One-way ANOVA and Dunnett’s post-hoc test versus naive. These experiments indicate that distinct DAF-2-dependent transcription factors are activated in tdc-1 and tyra-3 mutants: DAF-16/sod-3, HSF-1/hsp16.2 and SKN-1/gst-4. Source data

Extended Data Fig. 10 Inhibition of stress resistance by tyramine requires intestinal hid-1.

Survival percentages of young adult hid-1 mutant animals, expressing either endogenous (endog. rescue), neuronal (neu. rescue) or intestinal (int. rescue) rescue constructs, exposed to oxidation (3 h, 15 mM FeSO4) (left) or heat (4 h, 35 °C) (right) in the absence or presence of 10 mM TA. Mean ± s.e.m. from number of independent experiments shown, 80–100 animals per condition per experiment. Two-tailed Student’s t-test versus same strain without TA. hid-1 expression in the intestine in hid-1 mutants restores the negative effect of tyramine on oxidative and heat stress resistance. Source data

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De Rosa, M.J., Veuthey, T., Florman, J. et al. The flight response impairs cytoprotective mechanisms by activating the insulin pathway. Nature 573, 135–138 (2019) doi:10.1038/s41586-019-1524-5

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