The hypoxic response extends lifespan through a bioaminergic and peptidergic neural circuit

The hypoxic response extends lifespan through a bioaminergic and peptidergic neural circuit | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The hypoxic response extends lifespan through a bioaminergic and peptidergic neural circuit View ORCID Profile Elizabeth S. Kitto , View ORCID Profile Shijiao Huang , View ORCID Profile Mira Bhandari , View ORCID Profile Cassie Tian , View ORCID Profile Rebecca L. Cox , View ORCID Profile Safa Beydoun , Emily Wang , Danielle Shave , Hillary A. Miller , View ORCID Profile Sarah A. Easow , Ella Henry , Megan L. Schaller , View ORCID Profile Scott F. Leiser doi: https://doi.org/10.1101/2025.05.04.652087 Elizabeth S. Kitto 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth S. Kitto For correspondence: leiser{at}umich.edu Shijiao Huang 2 Department of Biochemistry and Molecular Biophysics, Kansas State University , Manhattan, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shijiao Huang For correspondence: leiser{at}umich.edu Mira Bhandari 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mira Bhandari Cassie Tian 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cassie Tian Rebecca L. Cox 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rebecca L. Cox Safa Beydoun 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Safa Beydoun Emily Wang 3 Department of Cellular and Developmental Biology; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danielle Shave 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hillary A. Miller 4 Cellular and Molecular Biology Program, University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sarah A. Easow 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah A. Easow Ella Henry 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Megan L. Schaller 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Scott F. Leiser 1 Molecular and Integrative Physiology Department; University of Michigan , Ann Arbor, United States 5 Department of Internal Medicine; University of Michigan , Ann Arbor, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Scott F. Leiser For correspondence: leiser{at}umich.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract A coordinated response to stress is crucial for promoting the short- and long-term health of an organism. The perception of stress, frequently through the nervous system, can lead to physiological changes that are fundamental to maintaining homeostasis. Activating the response to low oxygen, or hypoxia, extends healthspan and lifespan in C. elegans . However, despite some positive impacts, negative effects of the hypoxic response in specific tissues prevent translation of their benefits in mammals. Thus, it is imperative to identify which components of this response promote longevity. Here, we interrogate the cell-nonautonomous hypoxic response signaling pathway. We find that HIF-1-mediated signaling in ADF serotonergic neurons is both necessary and sufficient for lifespan extension. Signaling through the serotonin receptor SER-7 in the GABAergic RIS interneurons is necessary in this process. Our findings also highlight the involvement of additional neural signaling molecules, including the neurotransmitters tyramine and GABA, and the neuropeptide NLP-17, in mediating longevity effects. Finally, we demonstrate that oxygen- and carbon-dioxide-sensing neurons act downstream of HIF-1 in this circuit. Together, these insights develop a circuit for how the hypoxic response cell-nonautonomously modulates aging and suggests valuable targets for modulating aging in mammals. Introduction The global population is aging at an unprecedented rate, posing significant challenges for healthcare systems worldwide 1 . The increasing burden of age-related diseases such as cardiovascular disease, diabetes, and neurodegeneration 2 highlights the pressing need to improve healthspan, defined as the disease-free period of life 3 . Work over the past several decades has led to a better understanding of the conserved molecular and cellular mechanisms that regulate lifespan. One promising avenue has been the identification of environmental stressors that, when applied in a controlled manner, activate stress-response pathways to promote longevity. These interventions, including calorie restriction, exercise, hypoxia, heat shock, and cold exposure, extend lifespan and healthspan 4 – 7 . These stress-response pathways induce hormesis, a phenomenon in which a low dose of a stressor results in adaptive beneficial effects on cellular function 8 , 9 . Hormesis and the underlying stress-responses that promote longevity converge on cellular pathways, including oxidative stress, insulin signaling, autophagy, and protein homeostasis 10 , to promote longevity. Multicellular organisms secrete a highly conserved set of neuromodulators including neurotransmitter and neuropeptide signals from the central nervous system. These signals integrate information about the organism’s external environment and internal state, and coordinate a physiological response in peripheral tissues. Within the context of aging, bioamine neurotransmitters contribute to the activation of many well-studied stress response mechanisms that promote longevity. For example, serotonin signaling modulates the mitochondrial unfolded protein response in C. elegans 11 , and regulates how food perception contributes to dietary restriction-mediated longevity in worms 12 and flies 13 . Dopamine also modifies the longevity benefits of dietary restriction in invertebrates 12 , 14 , and polymorphisms in the dopamine D4 receptor are associated with longevity in humans 15 . Finally, adrenaline and its invertebrate analog, tyramine, have been linked to changes in the aging rate in both nematodes 14 , 16 – 18 and mammals 19 . Hormone signals such as insulin 20 – 22 , growth factors 23 , 24 , and GnRH 14 , 25 also contribute to longevity pathways across taxa. This growing body of work has led to great interest in manipulating longevity-promoting neural circuits to improve health 26 . While significant research effort focuses on pathways like autophagy, insulin signaling, and proteostasis in the context of aging, the role of hypoxia remains relatively underexplored. Currently, no longevity-promoting agents have been reported to target the hypoxic response. The hypoxic response is highly conserved across species 27 – 29 , highlighting the relevance of this pathway in other organisms, including humans. In C. elegans , activating the hypoxic response by knocking down or mutating the E3 ubiquitin ligase von hippel-lindau-1 (VHL-1) 30 protein or stabilizing the hypoxia-inducible factor-1 (HIF-1) transcription factor 6 , 31 is sufficient to extend lifespan. All of these interventions—environmental hypoxia, VHL-1 knockdown, and HIF-1 stabilization—require the intestinal enzyme flavin-containing monooxygenase-2 ( fmo-2) to extend lifespan in C. elegans 31 . Hypoxic conditions also extend lifespan in a short-lived progeria mouse model 32 , and epidemiological studies indicate a correlation between hypoxia exposure and longevity 33 , 34 . However, the physiological changes induced by the hypoxic response are broad and involve adaptations such as increased vascularization, metabolic rewiring, and changes in cell survival pathways. In mammals, some of these same adaptations can be detrimental, as mutations in components of the hypoxic response have been linked to conditions like cancer and cardiovascular disease 35 – 38 . This presents a key challenge: while hypoxia-induced longevity in a post-mitotic invertebrate model like C. elegans is promising, some of the mechanisms involved may not be directly translatable to mammals without causing deleterious side effects. Therefore, a mechanistic understanding of the individual cells, neural circuits, and pathways that mediate the beneficial but not detrimental effects of hypoxia on aging is essential to determine whether this circuit could be leveraged to improve human health. Our previous work in C. elegans identified that stabilization of HIF-1 in neurons is sufficient to extend lifespan through the serotonin receptor, SER-7. This pathway eventually leads to the induction of fmo-2 , a longevity gene expressed in the intestine 31 , 39 . In this study, we uncover key neural components of the hypoxic response longevity circuit. Within this circuit, we identify individual cells, signals, and receptors necessary and/or sufficient to extend lifespan downstream of the hypoxic response. More specifically, we find serotonin signaling in the ADF serotonergic neurons is both necessary and sufficient to extend lifespan through the hypoxic response. This pathway signals through the serotonin receptor SER-7 in the RIS interneuron, which is also essential for hypoxia-mediated longevity. We further demonstrate additional neurotransmitters (GABA and tyramine), and a neuropeptide (NLP-17) are critical for mediating these longevity effects. Finally, we identify that oxygen sensing neurons (URX, AQR, PQR and BAG) act downstream of neuronal HIF-1 in this circuit. Our insights into this longevity pathway provide a mechanistic understanding of how the hypoxic response delays aging and improves health. Results Serotonin signaling through the ADF neuron and the SER-7 receptor are necessary and sufficient for the hypoxic response to extend lifespan Induction of the hypoxic response by targeted genetic manipulations can increase lifespan in C. elegans. These manipulations include decreasing activity of the VHL-1 E3 ubiquitin ligase that targets the transcription factor HIF-1 for degradation under hypoxia 30 or by a mutation that stabilizes HIF-1 (HIF-1 P621A ) 6 , 31 ( Fig. 1A ). We previously found that stabilizing HIF-1 in serotonergic neurons is sufficient to extend lifespan 31 . To determine which serotonergic neurons initiate HIF-1-mediated longevity, we generated strains with a nondegradable HIF-1 variant (HIF-1 P621A ) 31 , 40 expressed under promoters specific to each of C. elegans’ three primary serotonergic neuron types—the ADF, NSM, and HSN neurons 41 . These transgenic worms were then crossed into the hif-1 null background to ensure that HIF stabilization in one neuron type could not feedback to modify HIF-1 activity in other cells. When we measured the lifespan of each strain relative to WT and hif-1 knockout controls, we observed that stabilizing HIF-1 in the ADF or NSM neurons significantly extended lifespan by 26% and 23%, respectively ( Fig. 1B-C ). HIF-1 stabilization in the HSN neurons had a smaller effect but significantly extended lifespan by 9% ( Fig. 1D ). This result indicates that modifying signaling in any serotonergic neuron is sufficient to induce some level of hypoxic response-mediated longevity. These data suggest that serotonergic neurons can partially substitute for each other to promote longevity in this pathway. In summary, the ADF and/or NSM serotonergic neurons play an essential role in hypoxic response-mediated longevity. Download figure Open in new tab Figure 1. ADF serotonergic neurons are necessary and sufficient to extend lifespan downstream of the hypoxic response. ( A ) Diagram of the conserved hypoxic response and genetic approaches to activate it, vhl-1 knockdown and HIF-1 stabilization. ( B-D ) Survival curves of WT, hif-1(ia4) knockout, hif-1(ia4); ADF::HIF-1S ( B ), hif-1(ia4); NSM::HIF-1S ( C ), and hif-1(ia4); HSN::HIF-1S ( D ) worms. N ≥ 256 ( B ), N ≥ 188 ( C ), and N ≥ 188 ( D ) worms per condition. ( E ) Survival curves of WT and ADF: tph-1 knockout worms on empty vector (EV) or vhl-1 RNAi. N ≥ 143 worms per condition. ( F ) Survival curves of WT and NSM: tph-1 knockout worms on empty vector (EV) or vhl-1 RNAi. N ≥ 193 worms per condition. ( G ) Survival curves of WT and tph-1(mg280); ADF: tph-1 rescue worms on empty vector (EV) or vhl-1 RNAi. N ≥ 178 worms per condition. ( H ) Survival curves of WT and tph-1(mg280); NSM: tph-1 rescue worms on empty vector (EV) or vhl-1 RNAi. N ≥ 160 worms per condition. ( I ) Gene expression of fmo-2 in WT and hif-1(ia4); ADF:HIF-1S worms. Significance in panels B-H is from a log-rank test comparing median survival. Significance in panel I is from a Student’s t-test (unpaired, two-tailed). In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. To further test the necessity of the most impactful (for lifespan) serotonergic neurons in hypoxia-mediated longevity, we used transgenic animals in which tph-1 , the rate-limiting enzyme in serotonin synthesis, was knocked out exclusively in the ADF or the NSM serotonergic neurons 12 , 42 . We then measured the lifespans of these strains on empty vector (EV) and vhl-1 RNAi to determine the necessity of ADF and NSM serotonergic signaling in vhl-1 -mediated longevity. Successful RNAi knockdown was confirmed with qPCR validation ( Fig. S1A ). We found that serotonin synthesis in ADF but not NSM neurons was required for vhl-1 knockdown to extend lifespan ( Fig. 1E-F ). This indicates that while HIF stabilization in multiple serotonergic neurons can extend lifespan, ADF serotonin signaling is required for lifespan extension in response to vhl-1 knockdown. Download figure Open in new tab Supplemental Figure 1. t ph-1 is required for vhl-1 mediated longevity. ( A ) Gene expression of vhl-1 in worms on vhl-1 RNAi compared to control worms on empty vector (EV) RNAi for two generations. N ≥ 200 worms per replicate, or 1200 worms per condition. The top of the bar represents the mean of the population and error bars indicate standard error of the mean (SEM). ( B ) Survival curve of WT, vhl-1(ok161) , tph-1(mg280), and vhl-1(ok161); tph-1(mg280) worms. N ≥ 338 worms per condition. Significance is from a log-rank test comparing median survival. In all panels, NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. Three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. To test if serotonin production in a single neuron type is sufficient for the hypoxic response to extend lifespan, we rescued tph-1 expression in a tph-1 null background using promoters specific to the ADF or the NSM neurons 12 . The lifespans of these strains on EV or vhl-1 RNAi were then measured. As expected and consistent with our previous publication 31 , the lifespan of the tph-1 knockout strain was not extended by vhl-1 knockdown ( Fig. S1B ). Consistent with the ADF neuron’s requirement for hypoxic response-mediated longevity ( Fig. 1E-F ), serotonin synthesis in the ADF neurons was sufficient for vhl-1 knockdown to extend lifespan while NSM serotonin synthesis was not ( Fig. 1G-H ). The enzyme fmo-2 is induced in the intestine under hypoxia and is required for lifespan extension by environmental hypoxia, HIF-1 stabilization, or vhl-1 depletion 31 . To test whether ADF-specific HIF-1 stabilization also induces fmo-2 , we measured fmo-2 transcription in this strain using qPCR. We found that ADF HIF-1 stabilization significantly induced fmo-2 expression relative to WT controls ( Fig. 1I ), suggesting a model in which ADF signaling extends lifespan through the same mechanism as vhl-1 knockdown and environmental hypoxia. Together, these data indicate that while HIF-1 stabilization in any serotonergic neuron is sufficient to extend lifespan, only ADF serotonin production is necessary and sufficient for vhl-1- mediated longevity. This suggests that under normal physiological conditions the serotonergic ADF neurons propagate a signal in response to whole body stabilized HIF-1. After identifying ADF neurons as the most central serotonergic neurons in hypoxic response-mediated longevity, we next sought to determine the downstream receptor responding to serotonin signaling from ADF neurons. C. elegans have six known serotonin receptors—SER-1, SER-4, SER-5, SER-7, MOD-1, and LGC-50. Of these six receptors, our previous findings showed that ser-7 expression is required for hypoxia to induce fmo-2 and extend lifespan 31 . SER-7 is a G protein-coupled receptor (GPCR) with high sequence identity to the 5-HT 7 receptor in mammals 43 , 44 . SER-7 is thought to be expressed in 27 of C. elegans’ 302 neurons 45 and contributes to many aspects of physiology such as reproduction and pharyngeal pumping 43 . Consequently, broad manipulation of SER-7 signaling is not an ideal method for exclusively modifying aging. To identify more specific targets for lifespan extension within this pathway, we asked whether ser-7 expression in any subset of neurons is sufficient to rescue the hypoxic response in a ser-7 null background. We created transgenic strains with ser-7 expression under the control of 11 different cell-specific promoters in a ser-7 null background. These promoters were selected to rescue ser-7 expression in the following neuronal populations: whole-body rescue ( ser-7p::ser-7) , interneuron rescue ( glr-1p::ser-7) , bioaminergic neuron rescue ( cat-1p::ser-7) , glutamatergic neuron rescue ( eat-4p::ser-7 ), GABAergic neuron rescue ( unc-47p::ser-7 ), cholinergic neuron rescue ( unc-17p::ser-7 ), sensory neuron rescue ( osm-6p::ser-7 ), GABAergic motor neuron rescue ( unc-25p::ser-7 ), cholinergic motor neuron rescue ( acr-2p::ser-7 ), M3 and M4 neuron rescue ( ceh-28p::ser-7 ), and intestinal rescue ( vha-6p::ser-7 ) in a ser-7 null background. To measure whether ser-7 expression restores a WT-like hypoxic response, we injected each construct into ser-7 null worms crossed with a single-copy transcriptional reporter for fmo-2 (fmo-2p::mCherry) . Because fmo-2 is induced by and required for hypoxia-mediated longevity 31 , we utilized its induction as an efficient screening tool to identify components of this longevity pathway. We first confirmed that ser-7 knockout attenuates vhl-1- mediated fmo-2 induction ( Fig. 2A ). The whole-body rescue of SER-7 expression under the endogenous ser-7 promoter restored fmo-2 induction to 91% of WT controls ( Fig. 2A ). Of the 10 cell-specific ser-7 rescues, expression in interneurons, bioaminergic neurons, glutamatergic neurons, or GABAergic neurons fully rescued vhl-1 -mediated fmo-2 induction to ≥ 100% of the WT control response ( Fig. 2A , pink bars). ser-7 rescue in GABAergic motor neurons or in sensory neurons showed a partial rescue (defined as a 50-99% increase from fmo-2 induction in the ser-7 knockout) ( Fig. 2A , orange bars). In contrast, rescuing ser-7 in cholinergic motor neurons, cholinergic neurons, M3 and M4 neurons, or in the intestine did not rescue ( Fig. 2A , gray bars). Taken together, 6 of the 10 cell-specific ser-7 rescues at least partially restored vhl-1- mediated fmo-2 induction, including some that have no known overlap in ser-7 expression patterns. Collectively, these data suggest that serotonin receptor ser-7 expression in multiple neurons may be sufficient to convey the hypoxic signal to the intestine and induce fmo-2 . Download figure Open in new tab Figure 2. ser-7 expression in the GABAergic RIS neuron is required for hypoxia to extend lifespan. ( A ) Quantification of fmo-2p::mCherry (WT positive control), fmo-2p::mCherry; ser-7(tm1325) knockout (negative control), and fmo-2p::mCherry; ser-7(tm1325) knockouts with ser-7 rescued in the whole body ( ser-7p::ser-7) , interneurons ( glr-1p::ser-7) , bioaminergic neurons ( cat-1p::ser-7) , glutamatergic neurons ( eat-4p::ser-7 ), GABAergic neurons ( unc-47p::ser-7 ), GABAergic motor neurons ( unc-25p::ser-7 ), sensory neurons ( osm-6p::ser-7 ), cholinergic motor neurons ( acr-2p::ser-7 ), cholinergic neurons ( unc-17p::ser-7 ), M3 & M4 neurons ( ceh-28p::ser-7 ), and the intestine ( vha-6p::ser-7 ) on empty vector (EV, WT control shown) or vhl-1 RNAi (all strains shown). Bar height indicates the mean fluorescence on vhl-1 RNAi of each genotype normalized to the empty vector control value from that genotype. The dashed line indicates the vhl-1- mediated fmo-2 induction of WT positive control. N ≥ 45 worms per condition. Error bars indicate SEM. Significance is from a two-way ANOVA ( fmo-2 induction ∼ genotype*RNAi) and post-hoc Tukey HSD test (unpaired, two-tailed). Stars signify results from this post-hoc test comparing the fmo-2 induction of each strain on EV RNAi the fmo-2 induction of that strain on vhl-1 RNAi. ( B ) A table showing the 10 ser-7 expressing candidate neurons and their expression patterns among the ser-7 cell-specific strains that successfully rescued fmo-2 induction in Fig. 2A . Pink rows indicate rescues that full restored vhl-1 mediated fmo-2 induction, orange rows indicate rescues that partially restored vhl-1- mediated fmo-2 induction, and gray rows signify unsuccessful rescue constructs from the data in Fig. 2A . ( C ) Survival curve of WT and RIS ablation ( Ex[srsx-18p::caspase-3(p12)-nz]; Ex[srsx-18p::cz-caspase-3 (p17)]; srsx-18p::GFP ) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 192 worms per condition. ( D ) Survival curve of WT and ser-7 (tm1325); ser-7 rescue in the RIS neuron ( flp-11p::ser-7 ) worms on EV and vhl-1 RNAi. N ≥ 144 worms per condition. Significance in panels C-D is from a log-rank test comparing median survival. In all panels, NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. We compiled a table containing the top candidates for the ser-7 neuron based on which rescue constructs restored ser-7 expression (simplified table in Fig. 2B , full table in Fig. S2A ). We hypothesized that neurons present in many of the successful rescue constructs would be most important in the hypoxia longevity circuit. Conversely, we hypothesized that candidate neurons within rescues that did not restore a WT-like hypoxia response are less likely to be important ( Fig. 2B ; Fig. S2A ). We acknowledge that this analysis strategy is biased toward neurons rescued in a greater proportion of the transgenic strains, which could lead to false negatives. From this analysis, we identified 10 neurons that were rescued in 3-4 of the 6 successful rescue constructs ( Fig. 2B ). Five of these neurons (DVB, FLP, M1, RME_LR, and RME_DV) were also present in unsuccessful rescue constructs, so they were not considered for follow-up experiments. Of the remaining five neurons (RIS, AUA, AVC, LUA, and VD_DD), the top candidate was the RIS neuron, because RIS was present in the highest number (4 as shown in Fig. 2B ) of successful rescue constructs. Download figure Open in new tab Supplemental Figure 2. s e r -7 expression in each ser-7 expressing neuron is rescued by at least one construct, and ser-7 is required for vhl-1- mediated longevity. ( A ) A table showing all ser-7 expressing candidate neurons, and which constructs in Fig. 2A restored ser-7 expression in these neurons. Pink rows indicate rescues that fully restored vhl-1 mediated fmo-2 induction, orange rows indicate rescues that partially restored vhl-1- mediated fmo-2 induction, and gray rows signify unsuccessful rescue constructs from the data in panel A. ( B ) Survival curve of WT, and ser-7(tm1325) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 259 worms per condition. Significance is from a log-rank test comparing median survival. Cox Regression for an interaction between the effect of ser-7 (tm1325) knockout and vhl-1 RNAi knockdown on lifespan. p = 0.003, **. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. Three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. To validate whether the RIS neuron is a key signaling cell in hypoxia-mediated longevity, we first genetically ablated the RIS neuron to test its necessity. Our results showed that vhl-1 RNAi does not extend lifespan of RIS ablated worms, indicating that the RIS neuron is completely required for vhl-1 knockdown to extend lifespan ( Fig. 2C ). To test the sufficiency of RIS signaling, we next rescued ser-7 expression in the RIS neuron in the ser-7 null background under the flp-11 promoter. flp-11 is expressed in the RIS neuron 46 and uv1 neuroendocrine cells 47 that do not endogenously express ser-7 45 . We first confirmed that ser-7 knockout worms are partially required for vhl-1 RNAi to extend lifespan ( Fig. S2B , Cox Regression, p = 0.003, **). Significantly, ser-7 rescue in the RIS neuron ( ser-7; flp-11p::ser- 7) restores the lifespan extension by vhl-1 RNAi to the same degree as WT worms ( Fig. 2D ). These data suggest that ser-7 expression in the RIS neuron only is sufficient for vhl-1- mediated longevity. However, one limitation of this approach is that non-physiological expression of ser-7 in the uv1 cells could play a role in restoration of a WT-like hypoxic response. Together, these data are consistent with a model where hypoxic conditions stabilize HIF-1 in the ADF neurons, which modify serotonin signaling to the SER-7 receptor on the RIS interneuron. Tyraminergic signaling from the RIM neuron and the tyramine receptor TYRA-3 are required for vhl-1 knockdown to extend lifespan After identifying neuron subtypes involved in serotonin signaling downstream of the hypoxic response, we next set out to identify whether other neurotransmitters act in the pathway. To answer this question, we obtained mutants deficient in the production of each type of neurotransmitter in C. elegans ( Fig. 3A ) and measured their lifespan on control and vhl-1 RNAi. Out of the six neurotransmitters we tested, blocking the production of GABA ( unc-25 , Fig. 3B ) or tyramine + octopamine ( tdc-1, Fig. 3C ) attenuated longevity on vhl-1 RNAi. The requirement of GABA is consistent with our finding that ser-7 expression on the GABAergic RIS neuron is also necessary for hypoxic response-mediated longevity ( Fig. 2 ), connecting the serotonergic and GABAergic components of this circuit. Download figure Open in new tab Figure 3. GABA and tyramine synthesis are required for the hypoxic response to extend lifespan. ( A ) Summary table of the necessity of each C. elegans neurotransmitter for vhl-1 RNAi - mediated longevity. ( B-E ) Survival curves of WT and unc-25(e156) (GABA deficiency) ( B ), tdc-1(n3419) (tyramine + octopamine deficiency) ( C ), tbh-1(n3247) (octopamine deficiency) ( D ), and RIC ablation ( Ex[tbh-1p::caspase-3(p12)-nz]; Ex[tbh-1p::cz-caspase-3 (p17)]; tbh-1p::GFP ) ( E ) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 310 ( B ), ≥ 201 ( C ), ≥ 258 ( D ), and ≥ 258 ( E ) worms per condition. Significance in panels B-E is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. Tyramine (analogous to mammalian adrenaline 48 ) is synthesized from tyrosine by tyrosine decarboxylase ( tdc-1 ) and can be further converted to octopamine (analogous to mammalian noradrenaline) by tyramine β-hydroxylase ( tbh-1 ) in C. elegans 49 . Therefore, the tdc-1 knockout is deficient in both tyramine and octopamine synthesis ( Fig. 3C ) and the tbh-1 knockout ( Fig. 3D ) is only deficient in octopamine synthesis. Since worms lacking only octopamine ( tbh-1 , Fig. 3D ) were long-lived on vhl-1 RNAi but worms lacking both tyramine and octopamine ( tdc-1 , Fig. 3C ) were not, we can conclude that tyramine is required for the hypoxic response to extend lifespan. To further test whether tyramine but not octopamine is involved in this pathway, we generated a strain in which the primary octopamine-producing neuron, RIC 49 , is genetically ablated. We found that the RIC neuron is not required for vhl-1- mediated longevity ( Fig. 3E ), further supporting that tyramine but not octopamine is a key neurotransmitter in the hypoxic response longevity circuit. In addition to octopamine ( tbh-1, Fig. S3C ), we also observed that acetylcholine ( unc-17 , Fig. S3A ) and glutamate ( eat-4 , Fig. S3B ) were not required for lifespan extension by vhl-1 RNAi. Our lifespans of the dopamine synthesis mutant cat-2 indicated that dopamine is inconsistently required for the hypoxic response to extend lifespan. In three biological replicates, we observed a full requirement, partial requirement, and no requirement for dopamine synthesis in vhl-1 RNAi-mediated longevity. When analyzed together, the net result suggests that dopamine is partially required for vhl-1- mediated longevity ( Fig. S3C , Cox Regression for interaction between genotype and vhl-1 RNAi, p < 0.0001, ****). We did not follow up on dopamine due to the inconsistency. Together, the results from this neurotransmitter screen support the conclusion that in addition to serotonin, both GABA and tyramine play a role in hypoxic response-mediated longevity. Download figure Open in new tab Supplemental Figure 3. Acetylcholine, glutamate, and dopamine synthesis are not fully required for vhl-1 RNAi to extend lifespan. ( A-C ) Survival curves of WT and unc-17(e113) (acetylcholine deficiency) ( A ), eat-4(ky5) (glutamate deficiency) ( B ), and cat-2(n4547) (dopamine deficiency) ( C ) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 213 ( A ), ≥ 274 ( B ), and ≥ 270 ( C ) worms per condition. ( C ) Cox Regression for an interaction between genotype and vhl-1 RNAi, p 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. Nematodes synthesize GABA in 26 of 302 neurons while tyramine is only synthesized in two cells 50 . As a result, identifying highly specific components of tyraminergic signaling is more feasible than mapping GABAergic signaling components. Therefore, we sought to determine the tyramine-producing neuron(s) and tyramine receptor(s) that contribute to hypoxic response-mediated longevity. In C. elegans , the two canonically tyraminergic cell types (the RIM neuron and the uv1 neuroendocrine cells) express tdc-1 but not tbh-1 50 . To determine which tyraminergic cell(s) act in the hypoxic response pathway, we expressed tdc-1 under promoters specific to either RIM or uv1 in a tdc-1 null background 14 . We then measured whether the lifespans of these tyramine rescue strains could be extended by vhl-1 RNAi. Our results showed that vhl-1 RNAi extended lifespan in the RIM rescue strain ( Fig. 4A ), but not in the uv1 rescue ( Fig. 4B ). This finding demonstrates that tyramine synthesis in the RIM neuron is sufficient for vhl-1 RNAi to extend lifespan. Notably, both tyramine rescue constructs lived longer than WT controls ( Fig. 4A-B ). This could indicate that altering tyramine signaling modifies lifespan both within and independently of the vhl-1- mediated longevity pathway. Download figure Open in new tab Figure 4. The RIM neuron and the tyramine receptor tyra-3 act in the hypoxic response-mediated longevity pathway. ( A-B ) Survival curves of WT, RIM rescue ( tdc-1 (n3419); Ex[ ocr-4p::tdc-1]) ( A ), and uv1 tdc-1 rescue ( tdc-1 (n3419); Ex[ gcy-13p::tdc-1]) ( B ) strains on empty vector (EV) and vhl-1 RNAi. N ≥ 126 ( A ) and N ≥ 140 ( B ) worms per condition. ( C ) Survival curves of TU3311( unc-119p::sid-1) and vhl-1(ok161); TU3311( unc-119p::sid-1 ) worms on empty vector (EV) and tyra-3 RNAi. N ≥ 215 worms per condition. Significance in all panels is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. To identify the tyramine receptor(s) necessary in the hypoxic response-mediated longevity pathway, we used RNAi to knockdown each of C. elegans’ four known tyramine receptors 51 – 54 ( Fig. 4C ). Since tyramine receptors are mostly expressed in neurons, and RNAi uptake into some neurons is less efficient than other cell types, we used a strain with enhanced neuronal RNAi uptake (TU3311, unc-119p::sid-1 ) crossed into vhl-1 knockout worms. This neural enhanced RNAi uptake strain was not used previously in this work for vhl-1 RNAi experiments because vhl-1 RNAi knockdown extends lifespan in WT worms 31 . The generated vhl-1; unc-119p::sid-1 strain was long-lived compared to the control strain unc-119p::sid-1 ( Fig. 4C ). Successful RNAi knockdown was validated via qPCR ( Fig. S4A ). Our lifespan results show that the tyramine receptor tyra-3 was fully required for vhl-1- mediated longevity ( Fig. 4C ), but the tyramine receptors tyra-2 and ser-2 were not ( Fig. S4B -C ). Another tyramine receptor, Igc-55, showed a partial requirement for vhl-1 -mediated longevity ( Fig. S4D ). Although there was a lifespan extension by vhl-1 on Igc-55 RNAi ( Fig. S4D ), we did observe a significant interaction between lgc-55 knockdown and genotype on lifespan (Cox Regression, p < 0.0001, ****), indicating a partial requirement. In summary, these data indicate that tyramine signaling from the RIM neuron to the TYRA-3 receptor is required for hypoxic response-mediated longevity, while the LGC-55 receptor may play a partial role. Interestingly, TYRA-3 is highly expressed on the low oxygen/high carbon dioxide-sensing BAG neuron as well as in the intestine 45 , 55 , the site of fmo-2 induction during hypoxia 31 . Download figure Open in new tab Supplemental Figure 4. t y ra -2 and ser-2 are not necessary for vhl-1- mediated longevity, while lgc-55 is partially required. ( A ) Gene expression of tyra-3, tyra-2, ser-2, and lgc-55 in TU3311( unc-119p::sid-1) worms raised on RNAi targeting each gene compared to control worms raised on empty vector (EV) RNAi for two generations. N ≥ 300 worms per replicate, or 900 worms per condition. The top of the bar represents the mean of the population and error bars indicate standard error of the mean (SEM). ( B-D ) Survival curves of TU3311( unc-119p::sid-1) and vhl-1(ok161); TU3311( unc-119p::sid-1 ) worms on empty vector (EV) and tyra-2 ( B ), ser-2 ( C ), or lgc-55 ( D ) RNAi. Cox Regression for an interaction between lgc-55 knockdown and vhl-1 knockout on lifespan in the TU3311background strain. p 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. Oxygen and carbon dioxide sensing neurons act downstream of serotonin signaling Given that tyra-3 is highly expressed in the canonical low-oxygen sensing neuron BAG 45 , we next explored whether neurons responsible for sensing high- and low-oxygen conditions are essential for hypoxic response-mediated longevity. C. elegans have four oxygen sensory neurons: the URX, PQR, and AQR neurons detect high levels of oxygen 55 , the BAG neuron responds to low levels of oxygen 56 and high levels of carbon dioxide 57 ( Fig. 5A ). We hypothesized that these oxygen sensing cells may perceive hypoxic conditions and help initiate the hypoxic response. Alternatively, these oxygen sensing neurons could modify behavior in response to hypoxia but not play a role in longevity. We found that vhl-1 knockdown was unable to extend lifespan when we genetically ablated the three high-oxygen sensing neurons (URX, AQR, PQR, Fig. 5B ). Interestingly, the low O 2 /high CO 2 responsive BAG neurons were also required for vhl-1- mediated longevity ( Fig. 5C ). We next crossed the URX/AQR/PQR ablation worms or BAG ablation worms into C. elegans with HIF-1 stabilized in the ADF neurons. Ablation of either the URX/PQR/AQR or the BAG neurons abrogated lifespan extension in the ADF HIF-1 stabilized worms. This suggests that oxygen sensing neurons act downstream of ADF HIF-1 stabilization in this pathway ( Fig. 5D-E ). Download figure Open in new tab Figure 5: High- and low-oxygen sensing neurons are important for hypoxic response-mediated longevity. ( A ) Diagram of oxygen sensing neurons in C. elegans . ( B ) Survival curves of WT and URX/PQR/AQR ablated worms ( qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+) ]) on empty vector (EV) and vhl-1 RNAi. N ≥ 279 worms per condition. ( C ) Survival curves of WT and BAG ablated worms ( Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP ) on empty vector (EV) and vhl-1 RNAi. N ≥ 188 worms per condition. ( D ) Survival curves of WT, hif-1(ia4); ADF-HIF-1S, URX/PQR/AQR ( qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+) ]) ablation, and URX/PQR/AQR ( qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+) ]) ablation; hif-1(ia4); ADF-HIF-1S worms. N ≥ 150 worms per condition. ( E ) Survival curves of WT, hif-1(ia4); ADF-HIF-1S, BAG ( Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP ) ablation, and BAG ( Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP ) ablation; hif-1(ia4); ADF-HIF-1S worms. N ≥ 186 worms per condition. Significance in all panels is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. The neuropeptide NLP-17 and its receptors contribute to vhl-1- mediated longevity After identifying neurons, neurotransmitters, and neuroreceptors acting in the hypoxic response longevity circuit, we wondered how this neural circuit ultimately propagates information about hypoxic conditions to the intestine and induces the pro-longevity gene fmo-2 . The C. elegans nervous system does not directly innervate the intestine. Instead, neurosignaling molecules bind to receptors on peripheral tissues through packaging in dense-core vesicles and subsequent release into and diffusion through pseudocoelomic fluid 58 , 59 . These dense-core vesicles are packaged with a wide variety of neuropeptide signals, although there is also evidence that bioaminergic neurotransmitters like serotonin, dopamine, and adrenaline/noradrenaline can also signal through this dense-core vesicle mechanism 59 – 61 . We first tested if inducing the hypoxic response through vhl-1 RNAi extends lifespan in a mutant strain lacking the dense-core vesicle packaging gene unc-31 . We observed that unc-31 was required for vhl-1 knockdown to extend lifespan ( Fig. 6A ). To examine whether neuropeptide signals carried by dense-core vesicles are involved, we knocked down two neuropeptide processing enzymes— egl-3 and egl-21 62 . Successful knockdown was validated with qPCR ( Fig. S5A ). Interestingly, egl-21 but not egl-3 was required for vhl-1- mediated longevity ( Fig. 6B-C ). This could be because egl-21 is the only enzyme known to perform the carboxypeptidase E cleavage step of neuropeptide processing 63 , while there are four known proprotein convertases with similar function to egl-3 62 , 64 . Download figure Open in new tab Figure 6. The neuropeptide nlp-17 and its receptors npr-37 and npr-43 are required for hypoxic response-mediated longevity. ( A ) Survival curves of unc-31(e169) and WT worms on empty vector (EV) and vhl-1 RNAi. N ≥ 242 worms per condition. ( B-C ) Survival curves of TU3311 ( unc-119p::sid-1) and vhl-1(ok161); TU3311 ( unc-119p::sid-1) worms on empty vector (EV) and egl-3 ( B ) or egl-21 ( C ) RNAi. N ≥ 209 worms ( B ) and N ≥ 185 worms ( C ) per condition. ( D ) Summary table of two sets of neuropeptide ligands/receptors that blunted vhl-1- mediated lifespan. ( E-G ) Survival curves of TU3311 ( unc-119p::sid-1) and vhl-1(ok161); TU3311 ( unc-119p::sid-1) worms on empty vector (EV) and npr-37 ( E ), npr-43 ( F ), or nlp-17 ( G ) RNAi. N ≥ 288 worms ( E ), N ≥ 309 worms ( F ), and N ≥ 309 worms ( G ) per condition. Significance in panels A-C and E-G are from log-rank test comparing median survival. Cox Regression for a significant interaction between npr-43 knockdown and vhl-1 knockout on lifespan, p <0.01, **. Cox regression for a significant interaction between nlp-17 knockdown and vhl-1 knockout on lifespan, p 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. We next tested a group of 6 genes corresponding to neuropeptide/receptor binding pairs that blunted hypoxic response-mediated fmo-2 induction ( Fig. 6D ). These genes, along with their corresponding receptors, were targeted for RNAi and tested for lifespan in TU3311 ( unc-119p::sid-1 ) and TU3311 ( unc-119p::sid-1 ); vhl-1 (ok161) strains. We found that the npr-3 receptor and it’s ligands, flp-15 and flp-21 were not required for vhl-1 -mediated longevity. ( Fig. S5B -D ). However, knocking down the neuropeptide nlp-17 partially blocked vhl-1 -mediated longevity ( Fig. 6E , Cox regression p < 0.05 ). Consistently, knocking down nlp-17’ s receptors also completely ( npr-37 , Fig. 6F ) or partially ( npr-43 , Fig. 6G , Cox regression p < 0.001 ) blocked vhl-1- mediated longevity. Successful knockdown of npr-37 and npr-43 was validated with qPCR ( Fig. S5A ). The nlp-17 ligand result was validated using a genetic knockout ( nlp-17 (ok3461) ), which completely prevented vhl-1 knockdown from extending lifespan ( Fig. S5E ). Together, these data suggest a role for NLP-17 signaling in hypoxic response-mediated longevity. Download figure Open in new tab Supplemental Figure 5. qPCR validation of RNAi hits from Figure 5, and neuropeptide screen hits that did not validate. ( A-B ) Gene expression of ( A ) egl-3, egl-21, npr-37, and npr-43 in TU3311 ( unc-119p::sid-1) worms raised on RNAi targeting each gene compared to control worms raised on empty vector (EV) RNAi for two generations. N ≥ 300 worms per replicate, or 900 worms per condition. The top of the bar represents the mean and error bars represent SEM. ( B-D ) Survival curves of TU3311 ( unc-119p::sid-1) and vhl-1(ok161); TU3311 ( unc-119p::sid-1) worms on empty vector (EV) and npr-3 ( B ), flp-15 ( C ), or flp-21 ( D ) RNAi. N ≥ 191 worms ( B ), N ≥ 181 worms ( C ), and N ≥ 183 worms ( D ) per condition. ( E ) Survival curves of nlp-17(ok3461) on empty vector (EV) or vhl-1 RNAi. N ≥ 275 worms per condition. Significance in panels B-E are from log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, 2-3 replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. Taken together, these results support a model in which HIF-1 stabilization in the ADF serotonergic neurons leads to modified serotonin signaling from the ADF to the SER-7 expressing and GABA-producing RIS neuron ( Fig. 7 ). Oxygen sensing neurons act downstream of HIF-1 stabilization in the ADF neuron in the hypoxic response longevity circuit. Tyramine produced by the RIM neuron and neuropeptide (NLP-17) signaling are also required for the hypoxic response to extend lifespan, although it remains unclear whether these signals act upstream, downstream, or in parallel to the serotonergic and GABAergic components in this pathway. Download figure Open in new tab Figure 7. Working model of the hypoxia-mediated longevity pathway. Discussion In this study, we interrogated a complex signaling pathway through which genetic induction of the hypoxic response extends lifespan in C. elegans . Within this pathway, we demonstrate that: 1) stabilization of HIF-1 in the ADF serotonergic neurons modifies signaling to the SER-7 receptor on the GABAergic and neuropeptidergic RIS neuron ( Fig. 1 - 2 ); 2) tyraminergic signaling through the RIM neuron and the TYRA-3 receptor ( Fig. 3 - 4 ) are required for hypoxic response-mediated longevity; 3) oxygen and carbon-dioxide sensory neurons act downstream of HIF-1 stabilization and serotonergic ADF neurons to extend lifespan ( Fig. 5 ); and 4) in addition to neurotransmitter signals, the neuropeptide NLP-17 and its receptors NPR-37 and NPR-43 are required for the hypoxic response to extend lifespan ( Fig. 6 ). Ultimately, these neural signals converge on the intestine, to activate FMO-2 and extend lifespan (working model summarized in Fig. 7 ). The hypoxic response is highly conserved across species, highlighting the potential relevance of this pathway in other organisms, including humans. In nematodes, activating the hypoxic response promotes health and longevity. However, the physiological changes induced by the hypoxic response are broad and involve adaptations that can drive tumorigenesis in mammals. Therefore, a mechanistic understanding of the individual cells, signals, and circuits that mediate the beneficial effects of hypoxia is essential. In this work, the discovery that HIF-1 stabilization in the ADF neurons extends lifespan by 26% demonstrates the potential for targeted manipulations within this pathway to have large effects on lifespan. Additionally, our data identify a previously unknown role for tyramine/adrenaline in the hypoxic response as well as establish NLP-17, a neuropeptide with no previous known function, as a signal in vhl-1- mediated longevity. This work also demonstrates that the oxygen sensing AQR, PQR, URX, and BAG neurons act downstream of the serotonergic ADF neuron in the hypoxic response, indicating an interaction between internal and external oxygen sensing mechanisms in longevity. Together, the mapping of these cells, signaling molecules, and receptors in the hypoxic response lays the foundation to discover druggable targets that selectively modulate aging in humans without negative side effects. Other studies in C. elegans have also identified neural networks involving the hypoxic response. For example, previous work found a role for HIF-1 in hypoxia-mediated behaviors, such as food-dependent hyperoxia (excessive oxygen) avoidance 65 . Hyperoxia avoidance requires hif-1 expression both in the neurons and the tyraminergic uv1 neuroendocrine cells. Interestingly, serotonin signaling from the ADF neuron is also required for WT-like hyperoxia avoidance 65 . While this HIF-1-mediated behavioral circuit has parallels with the HIF-1-mediated longevity circuit interrogated in this work, these pathways do diverge: the key tyraminergic cell type implicated in hyperoxia avoidance is the uv1 gonadal neuroendocrine cells, while the RIM tyraminergic neurons played a role in longevity ( Fig. 4 ). Together, the partial overlap between this behavioral and longevity circuit implies that modifying neural signaling to induce the hypoxic response and extend lifespan may also alter behavioral phenotypes. Future research in this area should interrogate exactly where these and any other HIF-1-mediated circuits overlap and diverge. While this study identifies many neural signals required for vhl-1 knockdown or knockout to extend lifespan, one key limitation of this work is the potential differences between genetic and environmental methods of inducing the hypoxic response. While vhl-1 knockdown or knockout leads to HIF-1 stabilization by blocking its proteasomal degradation, it also results in hydroxylated but stable HIF-1. This contrasts with environmental hypoxia, in which HIF-1 remains stable because it cannot be hydroxylated. While HIF-1 is stabilized and localized to the nucleus in both cases, there are differences in transcriptional outcomes between stable hydroxylated and unhydroxylated states 66 , 67 . Additionally, we did not explore the effects of alternative genetic activators of the hypoxic response such as PHD/EGL mutants, which may provide further insight into how different manipulations of the hypoxic response impact longevity. The circuit-mapping approaches employed in this work are also impacted by limitations in cell-specific genetic modifications and in the use of RNAi knockdown. For example, cell-specific rescue constructs can sometimes lead to unintended rescues in other cell types due to cell-nonautonomous signaling. Because all serotonin-producing neurons also express the serotonin reuptake transporter mod-5 , serotonin produced by one cell in our rescue strains could be taken up by other serotonin-producing neurons, leading to unintended signaling effects. This may also be true of the uv1 and RIM tyraminergic rescue strains, although little is known about tyramine reuptake in C. elegans . Additionally, although RNAi experiments were validated using sequence confirmation and qPCR, not all RNAi hits were corroborated with genetic knockouts. This leaves open the possibility that RNAi-induced knockdown effects differ from complete genetic ablation, or that production of a given RNAi may modify bacterial metabolism in a way that indirectly modifies the hypoxic response in C. elegans . Finally, while the use of RNAi knockdown and genetic knockouts establishes the necessity of many signals within the hypoxia-mediated longevity circuit, the exact directionality of these signals remains unclear. It is possible that increased, decreased, or pulsatile changes in signaling through these bioamines and neuropeptides are required for the hypoxic response to extend lifespan. Future work in this area could use tools to measure or modify neuronal activity, such as calcium imaging or optogenetics, to begin answering these questions. While many individual neurosignaling components are essential for the hypoxic response to extend lifespan, their epistasis is unclear. Future work in this area should focus on manipulating various components of this network in a manner that we would expect to mimic hypoxia and extend lifespan. This will allow us to narrow down which signals are necessary only to adapt to hypoxia, and which are sufficient to extend lifespan in a normoxic environment. One notable target for further exploration is the SER-7 expressing RIS neuron, which plays a role in sleep 68 and stress resistance 69 . Moreover, optogenetically increasing RIS activity can extend lifespan under normoxic conditions 70 . While our understanding of this circuit is still incomplete, our findings point to several promising signaling components that could be targeted for longevity interventions. Manipulating serotonin and tyramine/adrenaline signaling pathways may offer strategies for extending lifespan, with minimal pleiotropic effects if precisely controlled. These neural targets have great potential for longevity therapeutics due to 1) the small number of neurons that can control aging-related pathways in the entire organism; 2) the availability of FDA-approved pharmaceuticals that target individual bioaminergic transporters and receptors 71 – 73 ; and 3) the high conservation of neurotransmitter biology between invertebrates and mammals 50 , 62 , 74 . Further research into how these pathways can be modulated in a targeted manner could spur development of interventions that promote healthy aging and delay the onset of age-related diseases. Methods Strains and Growth Conditions C. elegans were cultured according to established protocols 31 . In brief, worms were grown at 20 °C on standard solid nematode growth media (NGM). Throughout their lifespan, the worms were fed E. coli OP50, except during RNA interference (RNAi) experiments, where E. coli HT115 was used to deliver double-stranded RNA. Transfers of worms were carried out using a platinum wire, unless stated otherwise. The RNAi strains employed are listed in Supplementary Table 1, while the strains used in the experiments are provided in Supplementary Table 2. Genotypes were verified through PCR, and RNAi imaging results were confirmed by sequencing and quantitative PCR (qPCR) before proceeding with the experiments. Generating transgenic strains Neuron-specific stabilized HIF-1 strains We used the following promoters to drive cDNA of HIF-1 (P621A)::SL2::GFP in three serotonergic neuronal populations: ADF neurons ( srh-142p:: HIF-1 (P621A)), NSM neurons ( ceh-2p:: HIF-1 (P621A)), HSN neurons ( ham-2p:: HIF-1 (P621A)). All plasmids were verified via restriction digest and sanger sequencing, and ApE files are available upon request. Plasmids were microinjected to hif-1(ia4) KO strain by Suny Biotech using the co-injection marker myo-2p ::GFP and 2-3 transgenic lines were tested in each experiment. Neuron-specific tph-1 rescues The pKA805[ srh-142p ::TPH-1] and pKA807[ ceh-2p:: TPH-1] constructs were generously provided by Dr. Kaveh Ashrafi. These constructs were injected at ∼50 ng/µL) with fluorescent co-injection marker myo-2p ::mNeonGreen (15 ng/µL) or sur-5p :: sur-5:: NLSGFP (20 ng/µL) and junk DNA (up to 100 ng/µL) into gonads of day 1 gravid adult hermaphrodites. Standard protocols were followed to isolate and obtain stable over-expression mutants 75 . ser-7 rescue strains Plasmid construction and microinjection was conducted by Suny Biotech to generate all ser-7 rescue constructs. In designing the plasmids, we used the following promoters to drive cDNA of SER-7::SL2::GFP in different neuronal populations: full rescue ( ser-7p::ser-7) , interneuron rescue ( glr-1p::ser-7) , bioaminergic neuron rescue ( cat-1p::ser-7) , glutamatergic neuron rescue ( eat-4p::ser-7 ), GABAergic neuron rescue ( unc-47p::ser-7 ), GABAergic motor neuron rescue( unc-25p::ser-7 ), sensory neuron rescue ( osm-6p::ser-7 ), cholinergic motor neuron rescue( acr-2p::ser-7 ), cholinergic neuron rescue ( unc-17p::ser-7 ), M3 & M4 neuron rescue ( ceh-28p::ser-7 ), and intestinal rescue ( vha-6p::ser-7 ). All plasmids were verified via restriction digest and sanger sequencing, and ApE files are available upon request. Plasmids were microinjected by Suny Biotech using the co-injection marker myo-2p:: GFP and 2-3 transgenic lines were tested in each experiment. RIS, RIC, and BAG neuronal ablation strains For RIS neuronal ablation strains, we purchased donor plasmid mec-18p ::caspase-3 (p12)::nz [TU#813] (Plasmid #16082) and mec-18p cz::caspase-3 (p17) [TU#814] from Addgene (Plasmid #16083), and used Gibson cloning (NEB) to replace mec-18p with srsx-18p . Three constructs srsx-18p ::caspase-3(p12)::nz, srsx-18p ::cz::caspase-3(p17), and srsx-18p ::GFP were co-injected with fluorescent co-injection marker myo-3p ::GFP (20 ng/µL) into the wild-type N2 strain to generate RIS genetic ablation strains. Similarly, for RIC ablation strain, tbh-1p was constructed into TU#813 and TU#814 to replace mec-18p . Three constructs tbh-1p ::caspase-3(p12)::nz, tbh-1p ::cz::caspase-3(p17), and tbh-1p ::GFP were co-injected with fluorescent co-injection marker myo-3p ::GFP (20 ng/µL) into the wild-type N2 strain to generate RIC genetic ablation strains. For BAG ablation strain, gcy-31p was constructed into TU#813 and TU#814 to replace mec-18p . Three constructs gcy-31p ::caspase-3(p12)::nz, gcy-31p ::cz::caspase-3(p17), and gcy-31p ::GFP were co-injected with fluorescent co-injection marker myo-3p ::GFP (20 ng/µL) into the wild-type N2 strain to generate BAG genetic ablation strains. All plasmids were verified via restriction digest and sanger sequencing. ApE files available upon request. Plasmid construction and microinjection was conducted by Suny Biotech. RIM and uv1 tdc-1 rescue strains Plasmid construction and microinjection was conducted by Suny Biotech to generate both tdc-1 rescue constructs. In designing the plasmids, we used the ocr-4 promoter to drive cDNA of TDC-1::SL2::GFP in the uv1 neuroendocrine cells. To express cDNA of TDC-1::SL2::GFP in the RIML neuron, we used the gcy-13 promoter. All plasmids were verified via restriction digest and sanger sequencing, and ApE files are available upon request. Plasmids were microinjected by Suny Biotech using the co-injection marker myo-2 p::GFP. RNAi Knockdown For all RNAi knockdowns, worms were exposed to the RNAi treatment for two generations to achieve optimal knockdown. All RNAi clones were sourced from the Vidal RNAi library. Each RNAi clone was sequence-verified. Quantitative PCR For RNAi validation experiments, RNA was isolated from day 1 adult worms that had been grown on RNAi for two generations. For measuring fmo-2 induction in the hif-1(ia4); ADF::HIF-1S strain, worms were grown on OP50, synchronized, and collected at day 1 of adulthood. RNA extraction was performed using the Direct-zol RNA Miniprep Kit (Zymo), and cDNA synthesis was carried out with the Biorad iScript cDNA Synthesis Kit. Gene expression was assessed using the SYBR Green quantitative RT-PCR (qRT-PCR) system (BioRad), with mRNA levels normalized to the housekeeping genes cdc-42 and Y45FD10.4. Primers for RNAi validation were designed to target the 3’ UTR of each gene to avoid amplification of the bacterially produced RNAi. Gene expression was quantified using standard curves. Lifespan Measurements Lifespan assays were conducted following previously described protocols 31 . Briefly, 10-15 gravid adults were transferred to NGM plates for a three-hour timed egg lay, after which they were removed. Once the progeny reached day 1 of adulthood, 60-80 worms were placed on NGM plates containing 33 µL of 150 mM fluorodeoxyuridine (FUdR) and 100 µL of 50 mg/mL ampicillin per 100 mL of NGM. FUdR inhibits progeny development, while ampicillin prevents bacterial contamination. NGM + FUdR + Ampicillin plates were seeded with concentrated bacteria (5x for OP50-fed lifespan assays). At least two plates per strain and condition were used for each lifespan replicate. Worms were considered dead when they failed to respond to a gentle touch with a platinum wire under a dissection microscope. Lifespan data were recorded at least three times a week until all worms were dead. To prevent escape, a barrier of 75 µL of 100 mM palmitic acid (Sigma-Aldrich) dissolved in 100% ethanol was applied along the edges of each lifespan plate. Data were analyzed using R version 4.3.1 and visualized in Adobe Illustrator 2022. RNAi Lifespans RNAi lifespan assays were performed similarly to standard lifespan assays, with modifications to the initial timed egg lay (TEL) procedure and food concentrations. To ensure maximal RNAi knockdown, worms were first TEL’ed for 3 hours on RNAi plates. The progeny from this TEL were left on RNAi plates to develop into gravid adults and then used for a second TEL under the same RNAi conditions. Progeny from the second-generation TEL were then used for the lifespan assay. RNAi bacteria (HT115) are ampicillin-resistant and can grow slowly on AMP-containing lifespan plates. To maintain consistent bacterial availability throughout the lifespan assay, RNAi plates were seeded with bacteria at 2x concentration, starting from an optical density (OD 600 ) of 3.0. Fluorescent Slide Microscopy Fluorescent images for this study were captured using a Leica M165F fluorescent microscope, controlled by Leica Application Suite X (LASX) software. A minimum of 15 worms per condition were imaged at a magnification of at least 70x. For image quantification, individual worms were carefully separated to ensure they did not touch. Custom R code 76 was then used to create a pixel mask of each worm from the brightfield image and measure the fluorescent intensity of that region in the corresponding fluorescent image. The background fluorescence was subtracted from the mean fluorescence values. Data were analyzed using R version 4.3.1 and visualized in Adobe Illustrator 2022. Statistical Analysis The height of all bar plots represents the mean value for each condition, with error bars indicating the standard error of the mean (SEM). For comparisons involving more than two conditions, one-way ANOVA followed by post-hoc Tukey HSD tests (2-tailed, unpaired) were used to assess interactions between variables and determine statistical differences between conditions. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. For lifespan assays, statistical comparisons of survivorship curves were performed using the survfit survival analysis function in R. The log-rank test was used to compare two survival curves. To assess interactions across multiple experimental variables and for comparisons involving more than two survival curves, a Cox regression analysis was conducted using the survivalMPL package in R. For experiments with multiple biological replicates, p-values were adjusted using the Bonferroni correction. Exact sample sizes (Ns) for each experiment are provided in the source data files, and minimum sample sizes for each plot are specified in the figure legends. Acknowledgements and Funding Funding Sources: National Institutes of Health grant F31AG084146-01 (ESK) National Science Foundation Graduate Research Fellowship Program DGE1841052 (ESK) National Institutes of Health grant R01AG058717 (SFL) Funder Information Declared National Institutes of Health , F31AG084146-01 , R01AG058717 National Science Foundation Graduate Research Fellowship Program , DGE1841052 Footnotes * Indicates co-first authors References 1. ↵ Burch , J.B. , Augustine , A.D. , Frieden , L.A. , Hadley , E. , Howcroft , T.K. , Johnson , R. , Khalsa , P.S. , Kohanski , R.A. , Li , X.L. , Macchiarini , F. , et al. ( 2014 ). Advances in geroscience: impact on healthspan and chronic disease . J Gerontol A Biol Sci Med Sci 69 Suppl 1 , S1 – 3 . doi: 10.1093/gerona/glu041 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Kaeberlein , M . ( 2013 ). Longevity and aging . F1000Prime Reports 5 . doi: 10.12703/P5-5 . OpenUrl CrossRef PubMed 3. ↵ Kirkland , J.L. , and Peterson , C . 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