Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging

Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging | 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 Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging Anupama Singh , Bhagyashree Kaduskar , Kirthi C Reddy , Caroline Kumsta , Ethan Bier , Malene Hansen , Sreekanth H Chalasani doi: https://doi.org/10.1101/2022.02.24.481867 Anupama Singh 1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies ; La Jolla, California 92037, USA 2 Sanford Burnham Prebys Medical Discovery Institute, Program of Development, Aging, and Regeneration ; La Jolla, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bhagyashree Kaduskar 3 Tata Institute for Genetics and Society, Section of Cell and Developmental Biology, University of California San Diego ; La Jolla, CA 92093, USA 4 Tata Institute for Genetics and Society, Center at inStem ; Bangalore, Karnataka 560065, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kirthi C Reddy 1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies ; La Jolla, California 92037, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Caroline Kumsta 2 Sanford Burnham Prebys Medical Discovery Institute, Program of Development, Aging, and Regeneration ; La Jolla, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ethan Bier 3 Tata Institute for Genetics and Society, Section of Cell and Developmental Biology, University of California San Diego ; La Jolla, CA 92093, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Malene Hansen 2 Sanford Burnham Prebys Medical Discovery Institute, Program of Development, Aging, and Regeneration ; La Jolla, CA, USA 5 Buck Institute for Research on Aging ; Novato, CA 94945, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: mhansen{at}buckinstitute.org schalasani{at}salk.edu Sreekanth H Chalasani 1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies ; La Jolla, California 92037, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: mhansen{at}buckinstitute.org schalasani{at}salk.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Age-related decline in intestinal barrier function impacts survival across species, but the underlying cell intrinsic and extrinsic factors remain unclear. Here, we demonstrate a role for neuropeptides in regulating aging-associated increases in intestinal leakiness. Adult-specific knockdown of insulin-like peptides daf-28 or ins-7 in C. elegans neurons or dilp3 in D. melanogaster neurons improves intestinal integrity and lifespan of these animals, respectively. Neuropeptide knockdowns activate intestinal DAF-16/FOXO transcription factor that likely increases expression of epithelial barrier components. Furthermore, age-associated changes in neuronal DAF-28 peptide secretion mirrors C. elegans reproductive span and overexpression of this peptide suppresses the improved intestinal health in long-lived germline-less animals. Collectively, we show that intestinal integrity is subject to neuronal regulation, and this neuropeptidergic axis may be modulated by the animal’s germline. Main Animal aging involves a coordinated decline in function of various tissues. While individual tissues deteriorate at different rates 1 , communication between them can profoundly affect aging across species (reviewed in 2 ). Consistently, an injured or diseased brain can trigger intestinal leakiness and reduce gastric motility, two characteristic features of intestinal aging, in both vertebrate and invertebrates 3 - 5 . Evidence of the role of inter-tissue signaling in animal aging is also seen in studies where transferring circulating polypeptides GDF11, myostatin and osteocalcin from young to older rodents has a rejuvenating effect on the latter 6 - 9 . However, little has been done to study whether age-associated changes in one tissue actively modulates aging in other distal tissues – an essential link in aging across species. In C. elegans , multiple conserved lifespan-modifying signaling pathways have been shown to function primarily in neurons, or their intestines, among other tissues 10 - 14 . Also, altering synaptic transmission from motor neurons or specific chemosensory neurons can improve animal lifespan 15 - 19 , confirming a role for neuronal signaling in animal aging. Similarly, their intestine has also been shown to play a crucial role in animal health and survival 11,20-22 . Despite this less is known about how changes in neuronal signaling affects aging in distal tissues like the intestine. Of the many age-associated changes in the intestine, barrier dysfunction is conserved between humans and laboratory animals 23 , 24 . This barrier dysfunction phenotype is readily quantified as the extent of intestinal leakiness in so-called “Smurf” assays. Here, the localization of a blue-dye co-ingested with food is monitored; the animal turns blue when its intestinal barrier is compromised, resulting in the dye spreading from its intestine into the body cavity 21 , 23 , 25 . Along with C. elegans , we also used the fruit fly D. melanogaster to probe the role of inter-tissue signals in affecting intestinal barriers in aged animals. Here, we leveraged neuron-selective RNA interference (RNAi) method to alter neuronal signaling and measured its impact on the aging outcomes in the intestine. Using C. elegans and D. melanogaster in this neuron-focused approach, we show a conserved role for neuronal peptides in modifying intestinal integrity during aging. We identify the specific neuronal peptides and their intestinal receptors that regulate age-related changes in intestinal leakiness in both nematodes and flies. Next, we show that blocking neuropeptide signaling, non-cell autonomously activated DAF-16/FOXO in the intestine. Additionally, DAF-16/FOXO has been shown to directly bind promoters of several epithelial barrier components, linking neuropeptide signaling with intestinal barrier function. Finally, we gained insights into the timing of this communication, and link reproductive activity with neuron-to-intestine insulin-like peptide signals and intestinal aging. Moreover, both C. elegans and Drosophila intestine bears many similarities to the human gastrointestinal tract 26 , 27 , implying that our findings could be conserved across species. Results Neuropeptide signaling selectively affects age-associated decline in intestinal integrity To test whether C. elegans neurons can influence age-associated changes in other tissues, we knocked down unc-13/Munc-13 (synaptic vesicle-priming for neurotransmitter release 28 ), snb-1/synaptobrevin (synaptic-vesicle associated vSNARE 29 ) or unc-31/CAPS (activator protein for peptide secretion 30 ) selectively in neurons of young adults (i.e., day 1 as reproductive adults) and monitored age-associated changes in other tissues ( Fig. 1A ). We first assessed intestinal health by measuring age-related increase in intestinal leakiness using the Smurf assay. Briefly, aged animals (day 15 of adulthood) were fed a blue dye mixed with bacteria for 3 hours, after which the dye-spread inside the body was monitored. We further evaluated intestinal leakiness by combining fluorescein with the blue dye, allowing us to obtain a more quantitative estimate of intestinal leakiness, besides the binary classification into Smurfs and non-Smurfs (see Methods). Neuron-specific feeding-RNAi was implemented using transgenic animals expressing sid-1 cDNA (SID-1 is membrane-associated, double-stranded RNA transporter), in a sid-1(qt9) null mutant 31 , using a pan-neuronal rgef-1 promoter (Y. Yang, C. Kumsta and M. Hansen, manuscript in preparation). We observed that neuronal unc-31/CAPS ( Fig. 1B , Supplementary Table 1 ), but not unc-13/Munc-13 or snb-1/synaptobrevin ( Extended DataFig. 1A, Supplementary Table 1 ) knockdowns reduced the age-related increase in intestinal leakiness, a phenotype we refer to as “HypoSmurfs”. Next, we tested whether neuropeptides also affected other intestinal functions like defecation and gastric motility. We found that neuronal unc-31/CAPS knockdowns had no effect on age-associated declines in defecation ( Extended Data Fig. 1B ) or gastric motility ( Extended Data Fig. 1C ). In addition, knocking down unc-13/Munc-13, snb-1/synaptobrevin , or unc-31/CAPS in neurons had no significant effect on the age-dependent declines in the rates of both body bends and pharyngeal pumping ( Extended Data Fig. 1D-E ). These results highlight the specificity of neuropeptides in selectively regulating intestinal barrier integrity (Smurf phenotype), without impacting age-associated changes in defecation, gastric motility, pharyngeal pumping, or body movements. Download figure Open in new tab Fig. 1. Blocking neuropeptides delays age-related intestinal leakiness in adult C. elegans and D. melanogaster models. (A) Schematic of experimental approach of reducing neurotransmitter and neuropeptide release in neurons to assess their effects on aging phenotypes in multiple C. elegans tissues. (B) FluoroSmurf phenotype of day 15 old animals during neuron-specific knockdown unc-31/CAPS feeding-RNAi, quantified n=2, N=54 (control), and N=48 ( unc-31/CAPS) , out of 8 trails, * P<0 . 05 , two-tailed t-test. Nematodes are aligned head (left) to tail (right), if not otherwise mentioned. Each dot represents relative fluorescent intensity in arbitrary units (a.u.) quantified from entire body of each animal, images acquired at constant exposure time. (C) FluoroSmurf phenotype of the four C. elegans pro-protein convertases, during their neuron-specific knockdown. Represented data quantified from n=1, N=45 (control), N=29 ( kpc-1/PC1) , N=36 ( egl-3/PC2) , N=26 ( aex-5/PC3) and N=28 ( bli-4/PC4) , out of 7 trails. * P<0 . 05, one-way ANOVA with Tukey’s multiple comparisons test. (D) The FluoroSmurf phenotype of transgenic animals pan-neuronally overexpressing egl-3/PC2 (OE). Represented data quantified from n=1, N=56 (wild type), N=58 ( egl-3 OE), out of 5 trails. **** P<0 . 0001 , two-tailed t-test. For (B-D), also see Supplementary Table 1 for data of independent trials. (E) Lifespan of non-Smurfs (non-leaky animals) compared to Smurfs (leaky intestine), classified after day10 Smurf assay in Wild-type (N2-CGC) animals (* P<0 . 0001 , log-rank test). Data is representative of two independent trials. See Supplementary Table 2 for lifespan statistics. (F) Lifespans of neuron-specific knockdown of unc-31/CAPS or egl-3/PC2 from day 1 of adulthood (* P<0 . 0001 , log-rank test). Data is representative of more than three independent trials. See Supplementary Table 1 for lifespan statistics. (G) Schematic of experimental approach for neuron-specific RNAi of cadps/CAPS or amon/PC2 , to assess lifespan and intestinal leakiness in the Drosophila . (H) Smurf phenotype and lifespans of neuron-specific RNAi of cadps/CAPS or amon/PC2 in flies (* P<0 . 01 , log-rank test for lifespan). Grey bar represents duration (∼10 days) of neuron-specific GAL4 activation at 29ºC. (I) Reduced % Smurf in old flies (∼55 days), during neuronal knockdown of cadps/CAPS compared to Gal4>+ control. Error bars are std. dev. of n=2. * P+ control. Error bars are std. dev. of n=3. * P<0 . 05 , two-tailed t-test. See Supplementary Table 3 for additional data of independent trials. Also see Extended Data Fig. 1-2 . To further investigate the role of neuropeptides in affecting intestinal integrity, we tested neuronal knockdowns of various components of the upstream peptide processing pathway, which is conserved from C. elegans to humans. Briefly, a pro-peptide is first synthesized and cleaved by one of four pro-protein convertases (i.e., KPC-1/PC1, EGL-3/PC2, AEX-5/PC3, and BLI-4/PC4) to generate peptide fragments. Basic amino acids from these peptide fragments are further removed by EGL-21/Carboxypeptidase E. The resulting peptides are then amidated by monooxygenase PAMN-1 and amidating lyase PGAL-1, before being packaged into dense core vesicles (DCVs) and released in an UNC-31/CAPS-dependent manner ( Extended Data Fig. 2A ) 32 . We found that knocking down the pro-protein convertase egl-3/PC2 , but not kpc-1/PC1, aex-5/PC3 , or bli-4/PC4 in neurons attenuated aging-induced increases in intestinal leakiness ( Fig. 1C ). In addition, neuronal knockdown of sbt-1/7B2 , the egl-3/PC2 chaperone 33 ( Extended Data Fig. 2B ) also reduced aging-induced intestinal leakiness, confirming a role for EGL-3/PC2 processing in regulating intestinal integrity. In addition, we also found that neuronal knockdown of the downstream egl-21 / Carboxypeptidase E ( Extended Data Fig. 2C ) similarly affected intestinal aging, suggesting an intestinal integrity-modifying role for EGL-21 peptidase. Consistent with these findings, over-expression of egl-3/PC2 in neurons increased intestinal leakiness in aged adults compared to controls, a phenotype we refer to as “HyperSmurfs” ( Fig. 1D ). However, knocking down the monooxygenase pamn-1 or amidating lyase pgal-1 in neurons had no effect on intestinal integrity in aged adults ( Extended Data Fig. 2D ), likely because of redundancy in the amidation step 34 . Collectively, these data suggest that C. elegans neurons use UNC-31/CAPS protein to release neuropeptides sequentially processed by EGL-3/PC2 and EGL-21/Carboxypeptidase E, whose function impairs intestinal integrity in aged animals. We next tested whether differences in intestinal integrity were correlated with C. elegans lifespan, similar to what has been observed in flies 23 . On day 10 of adulthood, we separated animals based on their Smurf phenotype into leaky or non-leaky groups and monitored their lifespans independently. Animals with intact intestines (non-Smurfs) were longer lived (* P<0 . 0001 , log-rank test) compared to animals with a leaky intestine (Smurfs), showing that intestinal integrity inversely correlates with lifespan ( Fig. 1E ) 25 . Consistently, we found that knocking down unc-31/CAPS and egl-3/PC2 in neurons extended lifespan by ∼15% (* P<0 . 0001 , log-rank test) and ∼20% (* P<0 . 0001 , log-rank test), respectively ( Fig. 1F , Supplementary Table 2 ), confirming that blocking neuropeptide processing or release improves intestinal integrity and lifespan. Next, we examined whether effects of neuropeptide signaling on intestinal integrity and lifespan were conserved between nematodes and flies ( Fig. 1G ). We monitored intestinal leakiness and animal lifespan in neuron-specific RNAi knockdowns of the Drosophila CAPS homolog, cadps 35 and PC2 homolog, amon 36 . We observed an ∼10% extension in lifespan (* P<0 . 01 , log-rank test) ( Fig. 1H , Supplementary Table 3 ) and reduced intestinal leakiness ( Fig. 1H-J , Supplementary Table 3 ) in aged flies in both neuronal knockdowns, suggesting that Amon/PC2-processed neuropeptides, secreted in Cadps-dependent manner, negatively affect intestinal barrier function and lifespan. Collectively, these data show a conserved role for C. elegans and D. melanogaster homologs of CAPS and PC2 proteins in intestinal and animal aging. Insulin-like neuropeptides exacerbate age-related decline in intestinal integrity While the C. elegans genome includes ∼150 neuropeptide genes that encode over 250 neuropeptides 32 , 37 , we focused our analysis on the 40 insulin-like peptides (ILPs) because of their known effects on growth, reproduction, and lifespan 38 - 41 . To identify candidate intestine-modifying ILPs, we knocked down individual insulin-like peptide genes in day 1 young adult neurons and monitored intestinal aging using Smurf assays ( Fig. 2A , S3A-D, Supplementary Table 4 ). We found that neuronal knockdowns of two beta-type insulin-like peptides, daf-28 and ins-7 , consistently exhibited the strongest HypoSmurf phenotypes ( Fig. 2B-C , S3B, Supplementary Table 1 ), identifying two intestine integrity-modifying neuropeptides. Consistent with this, neuronal over-expression of daf-28 or ins-7 reproducibly increased intestinal leakiness in aged adults ( Fig. 2D-E ), mimicking the HyperSmurf phenotype of neuronal egl-3/PC2 over-expression ( Fig. 1D ). Further, neuron-specific knockdown of daf-28 or ins-7 extended lifespan by ∼ 15% (* P<0 . 005 , log-rank test) and ∼30% (* P<0 . 0001 , log-rank test) respectively ( Fig. 2F-G , Supplementary Table 2 ). Together, our neuron-specific RNAi screen identified DAF-28 and INS-7 as the first insulin-like neuropeptides that function to antagonize intestinal integrity. Download figure Open in new tab Fig. 2. Conserved intestine-antagonizing role of insulin-like neuropeptides. (A) Schematic of experimental approach for neuron-specific knockdown of 35 (out of 40) insulin-like peptides to test their effect on intestinal leakiness in 15-day old C. elegans . (B) HypoSmurf phenotype of day 15 old animals during neuron-specific knockdown daf-28 . Represented data quantified from n=1, N=76 (control), N=61 (daf-28) , out of 3 trails. **** P<0 . 0001 , two-tailed t-test. (C) HypoSmurf phenotype of day 15 old animals during neuron-specific knockdown ins-7 . Represented data quantified from n=1, N=53 (control), N=60 (ins-7) , out of 9 trials. * P<0 . 05 , two-tailed t-test. (D) HyperSmurf phenotype of C. elegans pan-neuronally overexpressing daf-28 . Represented data quantified from n=2, N=62 (control), N=75 ( daf-28 OE), out of 6 trails. ** P<0 . 01 , two-tailed t-test. (E) HyperSmurf phenotype of C. elegans pan-neuronally overexpressing ins-7 . Represented data quantified from n=2, N=89 (control), N=95 ( ins-7 OE), out of 4 trails. ** P<0 . 01 , two-tailed t-test. For (B-E), also see Supplementary Table 1 for data of independent trials. (F) Lifespan extension during neuron-specific knockdown of daf-28 ( * P<0 . 005 , log-rank test), initiated from day 1 of adulthood. Data is representative of two independent trials. See Supplementary Table 2 for lifespan statistics. (G) Lifespan extension during neuron-specific knockdown of ins-7 (* P<0 . 0001 , log-rank test), initiated from day 1 of adulthood. Data is representative of three independent trials. See Supplementary Table 2 for lifespan statistics. (H) Neuron-specific RNAi of dilp3 in flies reduced age-related intestinal leakiness and extended lifespan (* P<0 . 001 , log-rank test for lifespan). Grey bar represents duration (∼10 days) of neuron specific GAL4 activation at 29ºC. See Supplementary Table 3 for additional data of independent trials. Also see Extended Data Fig. 3 . We next tested whether Drosophila insulin-like peptides also play a similar intestine-modifying function. The fly genome encodes eight insulin-like peptides (Dilp1-8), and three of these (Dilp2, 3, and 5) are secreted from insulin producing cells (IPCs) in the brain {reviewed in 42 , 43 }. We found that knocking down dilp3 (or dilp2, dilp5 , see Supplementary Table 3) specifically in adult neurons reduced the age-related Smurf phenotype and improved lifespan by ∼12% (* P<0 . 001 , log-rank test) ( Fig. 2H , Supplementary Table 3 ). Taken together, these data suggest a conserved intestine-modifying role of insulin-like neuropeptides, affecting intestinal integrity and lifespan in both nematodes and flies. Neuropeptide signals affect cellular integrity via intestinal insulin signaling In C. elegans , a single tyrosine kinase insulin/IGF-1-like receptor, DAF-2/InR, is known to integrate signals from all 40 insulin-like peptides ( Fig. 3A ) 44 . Moreover, both intestine-modifying neuropeptides, DAF-28 and INS-7, are known agonists of the DAF-2/InR 32 , 40 . We found that intestine-specific knockdown of daf-2/InR in day 1 young adults reduced intestinal leakiness on day 15 of adulthood ( Fig. 3B , Supplementary Table 1 ), similar to what was observed when genes encoding insulin-like neuropeptides, enzymes for their processing, or components of the release machinery were reduced in neurons. Additionally, we found that intestine-specific knockdown of the downstream phosphoinositide 3-kinase, age-1/PI3K 45 also reduced intestinal leakiness ( Extended Data Fig. 4A, Supplementary Table 1) , indicating a cell-autonomous role for insulin signaling in impairing intestinal leakiness. Moreover, intestine-specific knockdown of daf-2/InR or age-1/PI3K extended lifespan by ∼60% (* P<0 . 0001 , log-rank test) ( Fig. 3C , Supplementary Table 2) and ∼40% (* P<0 . 0001 , log-rank test) ( Extended Data Fig. 4B, Supplementary Table 2 ), respectively, confirming inverse correlation between intestinal leakiness and animal lifespan. We next tested whether the single insulin receptor in Drosophila , dInR, had a similar effect on age-related changes in intestinal leakiness and lifespan. We observed that midgut-specific knockdown of dInR resulted in reduced intestinal leakiness and increased lifespan by ∼10% (* P<0 . 001 , log-rank test), compared to controls ( Fig. 3D , Supplementary Table 3) . Cumulatively, our data underscores a conserved cell-autonomous role of DAF-2/dInR signaling in impairing intestinal integrity and animal lifespan. Download figure Open in new tab Fig. 3. Reduced neuropeptide signals are integrated by intestinal insulin signaling and downstream epithelial barrier components. (A) Schematic depicting the working hypothesis to test role of intestinal insulin signaling and the downstream DAF-16/FOXO transcription factor, in converging neuropeptide signals to impact intestinal barrier. (B) HypoSmurf phenotype of intestine-specific KD of daf-2/InR . Represented data quantified from n=1, N=31 (control), N=29 ( daf-2/InR) , out of 9 trails. ** P<0 . 01 , two-tailed t-test. Lower panel image was acquired at 10X exposure time. See Supplementary Table 1 for data of independent trials. (C) Lifespan extension during intestine-specific knockdown of daf-2/InR ( * P < 0 . 0001) . RNAi was initiated on day 1 of adulthood. Data is representative of six independent trials. See Supplementary Table 2 for lifespan statistics. (D) Midgut-specific RNAi of dInR reduced age-related intestinal leakiness and extended lifespan (* P<0 . 001 ) in flies. Grey bar represents duration (∼10 days) of midgut GAL4 activation at 29ºC. See Supplementary Table 3 for additional data of independent trials. (E) DAF-16/FOXO intestinal expression monitored using daf-16p::daf-16::gfp , during neuron-specific RNAi of indicated genes initiated on day 1 and imaged on day 5. Error bars are std. dev. of N>12, **** P<0 . 0001, one-way ANOVA with Dunnett’s multiple comparisons test. Data is representative of three independent trials. (F) MTL-1 expression during neuron-specific RNAi of egl-3/PC2 or daf-16/FOXO . Error bars are std. dev. of N>12, ** P<0 . 01 , * P<0 . 05, one-way ANOVA with Dunnett’s multiple comparisons test. Data is representative of two independent trials. (G) Schematic of experimental approach for testing the requirement of epithelial barrier components in regulating intestinal barrier integrity downstream of neuronal peptides. (H) Intestine-specific knockdown of daf-16/FOXO suppressed the HypoSmurf phenotype of neuronal unc-31/CAPS hairpin-RNAi transgenics. Represented data quantified from n=2, N=67 (control), N=75 (daf-16) , out of 5 trails. **** P<0 . 0001 , two-tailed t-test. (I) Intestine-specific knockdown of act-5 (actin) suppressed the HypoSmurf phenotype of neuronal unc-31/CAPS hairpin-RNAi transgenics. Represented data quantified from n=1, N=43 (control), N=36 (act-5) , out of 2 trails. * P<0 . 05 , two-tailed t-test. (J) Intestine-specific knockdown of ajm-1 (septate junction component) suppressed the HypoSmurf phenotype of neuronal unc-31/CAPS hairpin-RNAi transgenics. Represented data quantified from for n=1, N=24 (control), N=26 ( ajm-1) . * P<0 . 05 , two-tailed t-test. For (G-I), also see Supplementary Table 1 for data of independent trials. Also see Extended Data Fig. 4-5 . Download figure Open in new tab Fig. 4. The age-related secretion of the intestine-modifying DAF-28 neuropeptide patterns reproductive span in C. elegans . (A) daf-28p::daf-28::gfp reporter used as a tool to monitor the age-related synthesis and secretion of the DAF-28 peptide. (B-C) The age-related secretion of DAF-28 neuropeptide measured as number of GFP-positive coelomocytes (scavenging cells). Error bars are std. dev. for N>10 * ** P0 . 05, one-way ANOVA with Tukey’s multiple comparisons test. Data is representative of three independent trials. (D) Reproductive span of daf-28p::daf-28::gfp animals represented as average number of progenies produced each day of self-fertile period. Error bars are std. dev. for N=10, ** ** P<0 . 0001 , * ** P0 . 05, one-way ANOVA with Tukey’s multiple comparisons test. Data is representative of two independent trials. (E) Schematic depicting experimental approach for testing role of germline in modulating the neuron-to-intestine crosstalks. (F) HypoSmurf phenotype of germline-less glp-1(e2144ts) mutants. Represented data quantified from n=1, N=43 (control), N=67 (glp-1(e2144ts)) , out of 5 trails. *** P<0 . 001 , two-tailed t-test. (G) Pan-neuronal overexpression of daf-28 in glp-1(e2144ts) mutants suppressed their HypoSmurf phenotype. Represented data quantified from n=1, N=8 ( glp-1; control), N=17 ( glp-1; daf-28 OE), out of 3 trails. ** P<0 . 01 , two-tailed t-test. For (F-G), also see Supplementary Table 1 for data of independent trials. Also see Extended Data Fig. 6 . Reduction in insulin signaling activates DAF-16/FOXO, localizing it to the nucleus 46 , enhancing DAF-16/FOXO promoter occupancy 47 , 48 and transcriptional activation of a large number of genes 49 , 50 . We hypothesized that our neuron-specific knockdowns of insulin neuropeptides, or their processing and release machinery would result in reduced insulin signals and increased DAF-16/FOXO activation ( Fig. 3A ). To test this directly, we first monitored levels of DAF-16/FOXO using a daf-16p:daf-16::gfp translational reporter 51 . We found that neuron-specific knockdown of egl-3/PC2, daf-28 or ins-7 caused a significant increase in intestinal DAF-16/FOXO levels ( Fig. 3E ). Next, to validate the activation of DAF-16/FOXO, we monitored MTL-1 expression, a DAF-16/FOXO-regulated metallothionein protein, using an mtl-1p::mtl-1::rfp translational reporter 50 . We found that intestine-specific daf-2/InR knockdown upregulated MTL-1, while daf-16/FOXO knockdown suppressed its expression, confirming a cell autonomous role for insulin signaling in the intestine ( Extended Data Fig. 4C ). Consistent with the non-cell autonomous increase in intestinal DAF-16/FOXO, we observed that neuron-specific knockdown of egl-3/PC2 also upregulated intestinal MTL-1 ( Fig. 3F ), confirming the neuron-to-intestine signaling. Together, these results show that attenuating neuron-to-intestine peptide signaling, non-cell autonomously activates intestinal DAF-16/FOXO, that may reprogram the intestine to delay its age-related pathologies and improve survival outcomes. Neuropeptide signaling acts on intestinal epithelial barrier components in a DAF-16/FOXO-dependent manner We hypothesized that specific DAF-16/FOXO targets act in the intestine, downstream of neuropeptide signaling, to maintain epithelial barrier homeostasis. We mined a DAF-16/FOXO ChIP-sequencing dataset, in which DAF-16/FOXO was activated in daf-2(e1370) reduction-of-function mutants 47 , to identify genes with enriched DAF-16/FOXO recruitment (binding peaks distributed within ±1kb with respect to the transcriptional start site (TSS) 52 ). We identified several epithelial barrier components, including the intestine-specific actin; act-5 , tight-junction components; claudins, par-3 , and par-5 , the basolateral component; let-413 , and septate-junction components; ajm-1 and dlg-1 , among the differentially enriched DAF-16/FOXO targets in response to reduced insulin signaling ( Extended Data Fig. 5A ). To test whether these genes act downstream of neuropeptide signaling, we generated transgenic animals expressing the unc-31/CAPS hairpin RNAs pan-neuronally in the intestine-specific feeding RNAi transgenics ( Fig. 3G ). This transgenic strain allowed us to test whether an intestine-specific knockdown of a gene of interest can reverse the reduced intestinal leakiness evoked by the neuronal unc-31/CAPS knockdown ( Extended Data Fig. 5B ). We first confirmed the effectiveness of this strain, by feeding these animals bacteria expressing dsRNA for daf-16/FOXO . We found that knocking down daf-16/FOXO in the intestine increased its leakiness in the unc-31/CAPS hairpin-RNAi transgenics, confirming its sensitivity to feeding RNAi and a role for intestinal DAF-16/FOXO in promoting intestinal integrity ( Fig. 3H ). Next, we tested whether intestine-specific knockdown of epithelial components, identified as direct DAF-16/FOXO targets, had a role in regulating epithelial barrier function. We found that intestine-specific knockdown of act-5 (actin) ( Fig. 3I ), ajm-1 (septate junction component) ( Fig. 3J ), clc-1 (tight junction component) ( Extended Data Fig. 5C ) or let-413 (a basolateral component) ( Extended Data Fig. 5D ), significantly suppressed the HypoSmurf phenotype of unc-31/CAPS hairpin-RNAi transgenics. Together, these results show that blocking neuropeptide signaling engage key epithelial barrier components to maintain barrier integrity, possibly via a DAF-16/FOXO-dependent transcriptional mechanism. Download figure Open in new tab Fig. 5. Conserved neuropeptidergic modulation of intestinal integrity in C. elegans and D. melanogaster Model showing conserved components in the intestine-modifying neuropeptide signaling pathway. In C. elegans , we also show that intestine-modifying neuropeptide signaling impinges upon DAF-16/FOXO and components of the intestinal epithelial barrier. Also see text for details. Secretion of DAF-28 peptides correlates with reproductive aging and onset of intestinal aging As the age-related increase in intestinal leakiness is predominantly post-reproductive ( Extended Data Fig. 6A ), we speculated that the upstream neuropeptide secretion might also be affected by germline activity. Therefore, we turned our focus to quantify the age-related accumulation of GFP-tagged DAF-28 in coelomocytes 53 , 54 . DAF-28 neuropeptides are expressed in head (ASI, ASJ) and tail (PQR) sensory neurons and the distal intestine 39 and accumulates in the coelomocytes upon its secretion ( Fig. 4A-B ). Neuropeptide secretion has been previous quantified by monitoring the accumulation of fluorescently tagged peptides in coelomocytes 55 . Although the levels of DAF-28 peptides (measured as GFP fluorescence in head neurons) was comparable during the reproductive phase ( Extended Data Fig. 6B ), secretion of DAF-28 (quantified as the number of GFP-positive coelomocytes) exhibited an age-related surge and decline ( Fig. 4C ) . Interestingly, this DAF-28 secretion profile mirrored the reproductive span ( Fig. 4D ) 56 , 57 , consistent with its function in promoting reproduction 58 . When considering the relationships among reproductive aging, age-related DAF-28 secretion, and intestinal aging, we predicted that altering germline function could possibly impact intestinal aging either directly or by altering the secretion of DAF-28 peptides ( Fig. 4E ). To this end, we analyzed germline-less glp-1(e2144ts) mutants, which exhibited delayed intestinal leakiness ( Fig. 4F ). Importantly, glp-1(e2144ts) animals are longer lived, at least in part due to intestinal DAF-16/FOXO activation 11 , 59 ; however, the inter-tissue signals are not understood. We therefore tested if a neuronal insulin-peptide circuit could be part of the effects on the intestinal barrier function. In support of this notion, we found that neuronal over-expression of daf-28 was sufficient to hasten the observed delay in intestinal leakiness in germline-less glp-1(e2144ts) mutants ( Fig. 4G ). Collectively, our results suggest that the C. elegans germline activity may engage neurons to regulate the secretion of intestine-modifying neuropeptides. Discussion We find that neuron-specific knockdown of CAPS (the activator protein for peptide secretion) and the peptide-processing enzyme, prohormone convertase 2 (PC2), in young adults reduced age-related intestinal leakiness and extended survival in both C. elegans and D. melanogaster . Furthermore, using a neuron-specific genetic screen for insulin-like peptides, we identified antagonizing roles in the maintenance of intestinal barrier integrity for two C. elegans insulins, daf-28 and ins-7 , and three D. melanogaster dilp2, dilp3 , and dilp5 insulins. On the receiving end, knocking down daf-2/InR insulin receptor or the downstream age-1/PI3K cell-autonomously improved intestinal health, and lifespan. In C. elegans , we showed that reducing neuropeptide signaling non-cell-autonomously activated intestinal DAF-16/FOXO to potentially upregulate the transcription of genes associated with the intestinal epithelial barrier ( act-5, clc-1, let-413, dlg-1, ajm-1 , and others). Broadly, our results establish a conserved role for neuropeptides in affecting barrier function in both C. elegans and D. melanogaster intestines ( Fig. 5 ) Intestinal barriers are disrupted with age or in humans with Type 1 diabetes, or neurodegenerative conditions, but its genetic regulation is not well studied 60 , 61 . We show that neuronal peptides affect intestinal integrity in both nematodes and flies, and identify the proteolytic processing of pro-peptides by pro-protein convertases (PC2) 32 as a key step in this process. Consistently, both C. elegans egl-3/PC2 and D. melanogaster amon/PC2 mutants are deficient in bioactive neuropeptides 36 , 62 . Moreover, deficits in neuropeptide signaling result in developmental and growth defects in nematodes, flies, and mice 63 - 66 and, disease states in humans 67 , 68 . However, using a neuron-specific knockdown approach we show that knocking down peptide processing in young adults leads to improved intestinal health and increased longevity, in a DAF-16/FOXO-dependent manner. Furthermore, over-expressing egl-3/PC2 in C. elegans neurons worsened intestinal leakiness in aged adults, likely by increasing the level of bioactive peptides, which are then secreted to exacerbate intestinal integrity, likely by inducing insulin signaling. We suggest that EGL-3/PC2-mediated neuropeptide processing is a rate-determining step in intestinal aging. While genes involved in cytoskeleton organization are conserved DAF-16/FOXO direct targets in nematodes and flies 47 , 69 , its role in regulating epithelial barriers components is understudied. DAF-16/FOXO has increased promoter occupancy on genes associated with intestinal epithelial junctions, including act-5, let-413 and dlg-1 52 and we further validated a functional role for these proteins in modifying intestinal integrity, downstream of the neuron-to-intestine peptide signaling. Consistent with our findings in C. elegans , dFOXO binding to the Dlg1 promoter is greatly reduced in aged flies 70 , likely reducing Dlg1 expression and increasing barrier dysfunction in these animals. Together, we suggest that DAF-16/FOXO maintains the intestinal epithelial barrier function, by upregulating stress response genes like mtl-1 and, building blocks of epithelial junctions, like act-5, claudins, let-413, ajm-1, dlg-1 etc. Supporting this notion, previous studies in flies have shown that depleting septate junction components is sufficient to exacerbate barrier function in young flies, similar to the impact of physiological age 71 . Decline in germline function and reproductive capacity is one of the early aging phenotypes 72 . Nematode DAF-28 and fly Dilp3 are both required for proper germline maturation during development 58 , 73 , however these peptides become undetectable in post-reproductive adult nematodes and, flies 74 . Collectively, these data suggest an intriguing hypothesis – that germline activity modifies neuronal signaling, which in turn impacts intestinal aging. Consistent with this hypothesis, C. elegans adults lacking germline exhibit primitive chemotaxis behavior like those of developing larvae, as germline proliferation dictates animal behavior by altering responsiveness of olfactory neurons 75 . Also, a role for reproductive senescence in modifying neuronal signaling has been observed in multiple species including humans 76 - 78 . Here, we posit that besides modulating adult behaviors, germline signals govern the secretion of intestine-modifying neuropeptides to alter aging, primarily in the intestine. Consistent with this view, we observed that C. elegans glp-1 mutants, which lack a germline and are long-lived, displayed improved intestinal integrity, a phenotype that was significantly suppressed by neuronal overexpression of daf-28 . We suggest that the C. elegans germline engages neurons, possibly in an age-dependent manner, to produce intestine-modifying neuropeptides that becomes a trade-off for subsequent intestinal aging. In the long-term, these studies can provide scientific frameworks to establish the etiology of intestinal aging in mammals and altered brain-gut communications during age-related neuropathies. Materials and Methods Bacterial strains Escherichia coli OP50 was obtained from Dr. Cynthia Kenyon (formerly at UCSF), grown at 37ºC in Luria Bertani (LB) broth. For all RNAi experiments, HT115 E. coli were cultured under carbenicillin selection. All HT115 bacteria were grown for 6-8 hours at 37ºC in Luria Bertani (LB) broth, cultures were shaken at 220 rpm. C. elegans strains and husbandry C. elegans were maintained on nematode growth media plates seeded with E. coli OP50 at 20°C 79 . For RNAi experiments, animals were grown on HT115 bacteria from day 1 of gravid adulthood (unless otherwise indicated). All C. elegans strains used in this study are listed in Supplementary Table 5 . Bristol N2, both from Caenorhabditis Genetics Center (N2-CGC) and the Kenyon lab (N2-CK) were used as the wild-type animal (as indicated). For experiments with temperature-sensitive glp-1(e2144ts) mutants, both wild-type (N2-CK) and glp-1(e2144ts) stage 4 larva (L4) were shifted to 25ºC for egg-laying and until these progenies developed to day 1 of adulthood. Drosophila lines and husbandry All fly stocks were maintained at 25ºC or room temperature on standard fly food containing cornmeal-molasses agar media. Lines obtained from Bloomington Drosophila Stock Center (BDSC, Bloomington, IN): w1118 (#5905), elav C155 ; TubGal80 (#67058), UAS-RNAi- cadps (# 31984), UAS-RNAi- Amon (#28583), UAS-RNAi- dilp2 (#32475), UAS-RNAi- dilp3 (#33681), UAS-RNAi- dilp5 (#33683), and UAS-RNAi- InR (#51518), NP1-Gal4, TubGal80/ cyo 80 and UAS-GFP/MKRS were available in the Bier lab. Method details RNA interference in C. elegans Tissue-specific RNAi in the C. elegans model was implemented by either using feeding-RNAi method or hairpin-mediated RNAi 81 . Tissue-specific expression of the sid-1 cDNA (codes for membrane-associated, double-stranded RNA transporter), in a sid-1(qt9) null mutant, limits the RNAi activity to a particular tissue 82 . Neuron-specific or intestine-specific RNA interference (RNAi) transgenics are generated by expressing the sid-1 cDNA using a pan-neuronal rgef-1 promoter (Y. Yang, C. Kumsta and M. Hansen, manuscript in preparation) 31 or intestinal vha-6p promoter 83 , in a sid-1(qt9) null mutant. For hairpin-mediated RNAi, unc-31/CAPS was knocked down by pan-neuronally expressing unc-31 hairpin RNAs under h20 promoter 81 . For feeding-RNAi method, gene knockdown was implemented by feeding with E. coli HT115 bacterial clones expressing double-stranded RNA (dsRNA) targeting the gene of interest. unc-31/CAPS RNAi clone was generated for this study, other RNAi bacterial clones were obtained from the Ahringer RNAi library 84 or the Vidal RNAi library 85 , and daf-2, age-1 and daf-16 RNAi clones were provided by Dr. Andrew Dillin (UC Berkeley), the empty vector (L4440) for RNAi controls and daf-28 RNAi clone (from Vidal library) were provided by Drs. Andrew Fire (Stanford University) and Arnab Mukhopadhyay (National Institute of Immunology), respectively. All bacterial clones used in this study were verified by sequencing ( Supplementary Table 6 ). For feeding-RNAi experiments, HT115 bacterial clones were grown in Luria-Bertani (LB) liquid culture medium containing 0.1 mg ml −1 Carbenicillin (Carb; BioPioneer) for 6-8 hours, 140 μl aliquots of bacterial suspension were seeded onto 6 cm Petri plates with NGM containing 0.1 mg ml −1 Carbenicillin (NGM-Carb plates). Bacteria were allowed to grow for 1–2 days and induced for dsRNA expression by adding 140 μl of an isopropyl-[3-D-thiogalactoside solution containing 0.1M IPTG (Promega/Gold Biotechnology) and 500 mg ml −1 Carbenicillin added directly onto the bacterial lawn. For adult-only RNAi experiments, eggs laid by day 1 gravid adults were collected onto NGM plates seeded with OP50 bacteria for hatching and development till day 1 of adulthood. Day 1 gravid adults were transferred to NGM-Carb plates seeded with bacteria expressing control or dsRNA for indicated genes. Construction of unc-31 RNAi clone 1160-base-pair of unc-31 gDNA was amplified from wild-type genomic DNA using the forward primer 5’-CCACCGGTCTTCACCGCATTCGACAGTT-3’ and the reverse primer 5’-CTAGCTAGCCAA GCTGTAATCGTCGACTGA-3’ using NEB Taq Polymerase, cloned into AgeI and NheI restriction sites of pL4440 vector and transformed into E. coli HT115(DE3) cells. RNA interference in Drosophila Tissue-specific RNAi in the Drosophila model was implemented using the binary GAL4-UAS (Upstream Activating Sequences) system 86 . Tissue-specific Gal4 drivers limited the tissue-specific expression of RNAi 87 and timing of GAL4 activity was limited by a temperature sensitive form of GAL80 ( tubulin -GAL80ts), which inhibits GAL4 function below 25°C. In this study, for adult-only neuron-specific knockdown of cadps, amon, dilp2, dilp3 or dilp5 , neuron-specific Gal4: elav C155 ; TubGal80 flies 88 , were crossed to UAS-RNAi- cadps , UAS-RNAi- amon , UAS-RNAi- dilp2 , UAS-RNAi- dilp3 or UAS-RNAi- dilp5 flies, respectively and, 1-2 days old female F1 flies of desired genotype were shifted to 29ºC for 10 days (or indicated otherwise) for neuron-specific GAL4 activation. Similarly, for adult-only gut-specific knockdown of InR , NP1-Gal4, TubGal80/ cyo flies were crossed to UAS-RNAi- InR flies, and 3-5 days old female F1 flies of desired genotype were shifted to 29ºC for 10 days (or indicated otherwise) for gut-specific GAL4 activation. Lifespan assay in C. elegans Lifespan experiments were performed at 20ºC with ∼100 animals per condition, with 12–15 animals on each 6 cm NGM-Carb petri plates seeded with bacteria expressing empty control vector or dsRNA against the indicated gene. Feeding-RNAi was initiated on day 1 of adulthood and maintained throughout the lifespan analysis unless indicated otherwise. The L4 larval stage was recorded as day 0 of the lifespan and animals were transferred at least every other day to new NGM plates throughout the reproductive period. Animals were censored if they ruptured, escaped, desiccated on the edge of the plate, or suffered from internal hatching or contamination. OASIS2 software was used for statistical analyses and P-values were calculated using the log-rank (Mantel– Cox) test, a nonparametric test used to compare two survival functions through overall lifespan data 89 . See Supplementary Tables S2 for a summary of all lifespan experiments. Lifespan assay in D. melanogaster Lifespan experiments were performed at 25ºC with more than 50 animals per condition (protocol adapted from 90 . For neuron-specific RNAi experiments, 30-40 elav C155 ; TubGal80 virgin females were crossed to 15-25 males of desired UAS-RNAi line, or w1118 (wild type) and maintained at 25°C. Similarly, for gut-specific RNAi experiments, 30-40 NP1-Gal4, TubGal80/ cyo virgins were crossed to 15-25 males of UAS-RNAi- InR or w1118 and maintained at 25°C. F1 flies were collected and maintained at 25°C for a day, anaesthetized by CO 2 for sorting by sex and transferred to vials (10-15 flies/vial). Female flies were used for all lifespan experiments if not indicated otherwise. 1– 2-day old female flies were shifted to 29°C for 10 days (or indicated otherwise) for tissue-specific RNAi induction and then maintained at 25°C throughout lifespan. The dead flies were scored every alternate day and flies were transferred to fresh food vials twice a week (no CO 2 exposure). OASIS2 software 89 was used for statistical analyses and P values were calculated using the log-rank (Mantel–Cox) test. See Supplementary Table 3 for a summary of all lifespan experiments. Smurf and FluoroSmurf assay in C. elegans For Smurf assays, animals were aged on NGM-Carb plates supplemented with 120 μl of 5-Fluoro-2′-deoxyuridine solution containing 10 μl of 0.4M FUdR (Sigma-Aldrich, F0503) to prevent hatching of progenies and maintain synchronous nematode populations (Mitchell et al ., 1979). For Smurf assay, day 15 old nematodes (unless otherwise mentioned) were fed 250 μl of 5.0% wt/vol stock of blue food dye (Spectrum FD&C Blue #1 PD110) dissolved in overnight grown culture of OP50 bacteria 21 , in 96-well flat bottom plate. Following 3 hours of dye feeding at 20°C, animals were visualized under a microscope and classified into either Smurfs or non-Smurfs based on blue dye retention, and % Smurf was calculated. Animals with blue dye only in their germline were excluded from total population 21 . To quantify intestinal leakiness, we implemented modified FluoroSmurf assay by combining fluorescein with the blue dye. For these FluoroSmurf assays, worms were fed 125μl each of blue food dye and 2.5% wt/vol stock of Fluorescein sodium salt (Sigma-Aldrich, F6377), each dissolved in an overnight-grown culture of OP50 bacteria. Following 3 hours of dye feeding at 20°C, animals were first visualized under a microscope and classified into either Smurfs or non-Smurfs based on blue dye retention to calculate % Smurf. Next, animals were anaesthetized on NGM plates without food with M9 buffer containing 150mM NaN 3 , classified into Smurfs on non-Smurfs based on blue dye localization and imaged with a Leica DFC310 FX camera to visualize and measure fluorescein dye accumulation. Exposure time was kept constant within an experiment (3ms, 5ms, or 20ms), but varied between experiments and therefore absolute fluorescence of day 15 control animals also varied (higher absolute fluorescence at higher exposure). Fluorescein intensity was measured by outlining the entire animal using polygon tool of the FIJI/Image J software and, reported in an arbitrary unit relative to wild type/control. Age-related intestinal leakiness was monitored in cross-sectional Smurf assays, such that day 5, day 10, and day15 old nematodes populations were assayed on the same day. Smurf assay in D. melanogaster Longitudinal studies were conducted to monitor both lifespan and intestinal leakiness in aging fly population. Smurf assay was adapted from 91 . Briefly, 4 g of blue dye (Spectrum FD&C Blue #1 PD110) was dissolved in 10 ml distilled water. 7.8 ml of this stock was added to 100 ml of Drosophila food and distributed into 3 ml per vial. Care was taken to avoid any condensation and vials were set aside at room temperature and used the next day. On the day of Smurf assay, flies were transferred to blue-food and maintained at 25°C. After 24 hr of dye-feeding, flies were anaesthetized by CO 2 to monitor the blue dye localization under a microscope. As in nematodes, flies were classified as ‘Smurfs’ when the blue dye leaked into their body cavity. Following Smurf assays at multiple age-points, live flies were transferred back to fresh food vials for further lifespan analysis. C. elegans motility assay Thrashing rate was monitored at different age-points as indicated in the corresponding figures by counting the number of body bends per 30 seconds of randomly picked C. elegans swimming on NGM plate in a drop of M9 buffer. Movement of head was monitored under a stereo microscope, counted as one body bend when animal moved its anterior body from one side to another. Statistical comparisons were performed using two-way ANOVA, with Tukey’s multiple comparisons test, on data pooled from two independent experiments (n=2), taking 10 animals per condition in each set. C. elegans pharyngeal pumping assay At different age-points as indicated in the corresponding figures, the number of contractions of the pharyngeal terminal bulb were counted over 30 seconds. Animals were randomly picked and observed under a compound microscope, and a contraction was defined as posterior movement of the grinder in the pharyngeal terminal bulb. Statistical comparisons were performed using two-way ANOVA, with Tukey’s multiple comparisons test, on data pooled from two independent experiments (n=2), taking 10 animals per condition in each set. C. elegans defecation and gastric motility assay Posterior body was monitored for contraction of anal muscles (expulsion step, following posterior and anterior body-wall muscle contractions) under a compound microscope and length of defecation cycle was recorded as time in seconds, between two contractions of anal muscles 92 . In a new approach to measure gastric motility, animals were first fed OP50 bacteria mixed with blue dye for an hour and the presence of blue dye in the intestinal lumen was confirmed. Animals were then fed fresh OP50, and dye expulsion was monitored, visualized as loss of blue dye from the intestinal lumen. Using this approach, gastric motility of an animal was reported as a ratio of time taken for dye expulsion (in seconds) and length of defecation cycle (in seconds). Statistical comparisons were performed by two-tailed t test for unpaired samples, data pooled from three independent experiments (n=3), taking 10 animals per condition in each set. C. elegans reproductive span Animals were first synchronized by egg laying and raised on nematode growth media (NGM) plates seeded with E. coli OP50 at 20°C, until L4 stage. Then, 10 randomly picked L4 larvae were singly plated onto NGM plates with E. coli OP50. Adult worms were transferred every 24 hours onto fresh OP50 plates till the end of self-fertile period. To determine the number of viable progenies laid each day, number of L1 larvae were counted 24 hours after adult worms were moved, and 48 hours later for counting L1s that developed into adult worms. The reproductive span was reported as number of viable adult progenies generated each day of the self-fertile period. Coelomocyte assay to monitor peptide secretion in C. elegans Coelomocyte assay was performed to monitor age-related DAF-28 peptide secretion 53 . To determine the age-dependent secretion of DAF-28 insulin-like peptide, daf-28p::daf-28::gfp transgenics were aged simultaneously, such that day 1 to day 8 old animals were anaesthetized (150mM NaN 3 ) and imaged on the same day using a Leica DFC310 FX camera. Age-dependent secretion of DAF-28 insulin-like peptide was reported as the average number of GFP-positive coelomocytes per animal (at least 10 animals were monitored per age-point). To estimate the pre-secreted DAF-28 peptides synthesis, fluorescence was quantified from the ASI/ASJ head neurons. Generation of transgenic animals rgef-1p::egl-3c cDNA::pSM::mCherry The 1584 bp of egl-3 cDNA was amplified from wild-type cDNA using a forward primer (5’ ctaGCTAGCATGTCTCCATCGGATCCAC 3’) and reverse primer (5’ cggGGTACCTTAGTGGCTGCGTTTGTGG 3’) using Q5 ® High-Fidelity DNA Polymerase system (New England Biolabs, USA) and cloned into rgef-1p::pSM::mCherry vector using NheI and KpnI restriction sites. The recombinant plasmid was microinjected at a concentration of 100ng/μl into the syncytial gonad of wild-type N2 (CGC) worms. rgef-1p::daf-28(SM)::pSM::mCherry The 700 bp of daf-28 gDNA was amplified from daf-28p::daf-28::mCherry plasmid provided by Dr. Shohei Mitani (Tokyo Women’s Medical University) using a forward primer (5’ ctaGCTAGCATGAACTGCAAGCTCATCGC 3’) and reverse primer (5’ cggGGTACCTTAAAGAAGCAAACGTGGGCA 3’) using Q5 ® High-Fidelity DNA Polymerase system (New England Biolabs, USA) and cloned into rgef-1p::pSM::mCherry vector using NheI and KpnI restriction sites. The recombinant plasmid was microinjected at a concentration of 80ng/μl into the syncytial gonad of wild-type N2 (CGC) worms. rgef-1p::ins-7(2b)::pSM::mCherry The 764 bp of ins-7(2b) gDNA was amplified from wild-type genomic DNA using a forward primer (5’ ctaGCTAGCATGTATAAAGTACATTATTTTC 3’) and reverse primer (5’ cggGGTACCTTAAGGACAGCACTGTTTT 3’) using Q5 ® High-Fidelity DNA Polymerase system (New England Biolabs, USA) and cloned into rgef-1p::pSM::mCherry vector using NheI and KpnI restriction sites. The recombinant plasmid was microinjected at a concentration of 80ng/μl into the syncytial gonad of wild-type N2 (CGC) worms. Data availability The authors declare that all data supporting the findings of this study are available within this article. ChIP-Atlas database 52 was used to determine DAF-16 targets using data from DAF-16 ChIP sequencing study 47 available from the Gene Expression Omnibus (GSE63865 and GSM1558630). Code availability None were used in this study Funding National Institutes of Health grant MH096881 (SHC) National Institutes of Health grant AG038664 (MH) Breakthrough in Gerontology Award from the American Federation for Aging Research (MH) Postdoctoral fellowship from the Paul F. Glenn Center for Biology of Aging Research at the Salk Institute (AS) Author contributions Conceptualization: AS, MH, SHC Methodology: AS, BK, KCR, CK Investigation: AS, BK Visualization: AS, BK Funding acquisition: MH, SHC Project administration: AS, MH, SHC Supervision: EB, MH, SHC Writing – original draft: AS, SHC Writing – review & editing: AS, SHC, MH, CK, BK, EB, KCR Competing interests Authors declare that they have no competing interests. Data and materials availability All data are available in the main text or the supplementary materials. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sreekanth H Chalasani ( schalasani{at}salk.edu ). C. elegans strains and RNAi constructs generated in this study are available upon request from the Lead Contact with a completed Materials Transfer Agreement. Acknowledgements We thank Dr. Yongzhi Yang, Chung-Chih Liu, Andrew Davis, and Linnea Adams for technical assistance. We thank Drs. Andrew Dillin, Andrew Fire, Arnab Mukhopadhyay, Cynthia Kenyon, Mei Zhen, Shohei Mitani and Supriya Srinivasan, for kindly providing strains and reagents. We are also grateful to Drs. Rolf Bodmer, Karen Ocorr, and Annabel Guichard and, members of the Chalasani and Hansen labs for critical comments and insights. Some of the nematode strains used in this work were provided by the Caenorhabditis Genetics Center (University of Minnesota), which is supported by the NIH–Office of Research Infra-structure Program (P40 OD010440). 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Share Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging Anupama Singh , Bhagyashree Kaduskar , Kirthi C Reddy , Caroline Kumsta , Ethan Bier , Malene Hansen , Sreekanth H Chalasani bioRxiv 2022.02.24.481867; doi: https://doi.org/10.1101/2022.02.24.481867 Share This Article: Copy Citation Tools Conserved neuropeptidergic regulation of intestinal integrity in invertebrate models of aging Anupama Singh , Bhagyashree Kaduskar , Kirthi C Reddy , Caroline Kumsta , Ethan Bier , Malene Hansen , Sreekanth H Chalasani bioRxiv 2022.02.24.481867; doi: https://doi.org/10.1101/2022.02.24.481867 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7978) Biochemistry (18681) Bioengineering (14801) Bioinformatics (44255) Biophysics (22523) Cancer Biology (19646) Cell Biology (26805) Clinical Trials (138) Developmental Biology (13914) Ecology (20922) Epidemiology (2067) Evolutionary Biology (25377) Genetics (16119) Genomics (23428) Immunology (18638) Microbiology (42321) Molecular Biology (17983) Neuroscience (93101) Paleontology (694) Pathology (2974) Pharmacology and Toxicology (5072) Physiology (8083) Plant Biology (15941) Scientific Communication and Education (2094) Synthetic Biology (4545) Systems Biology (10199) Zoology (2378) window.__CF$cv$params={r:'a398a561ffa73983',t:'MTc4OTE1MDQwMg==',u:'01a091aca1777852ab8cc3f638cfc9a8',ut:'LJoaGcnJ4u8.pJhbEBoNtrXAqmgH1lYjZrjwxLm8rE0-1789150404-1.2.1.1-TOM39YTf0GioyYjNJMjqDyXd4ZB3qufZOEn5uc8c0gzjXHdEvqWjDsmK8nii3vQcVK_CgOzKdng4CsKjkIk_Xwincqey10jTgv5HouI1vOM',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();