Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis

Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis | 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 Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis View ORCID Profile Takashi Matsumura , View ORCID Profile Masasuke Ryuda , Hitoshi Matsumoto , View ORCID Profile Takumi Kamiyama , View ORCID Profile Shu Kondo , View ORCID Profile Akira Nakamura , View ORCID Profile Yoichi Hayakawa , View ORCID Profile Ryusuke Niwa doi: https://doi.org/10.1101/2024.12.25.630336 Takashi Matsumura a Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba , Tsukuba 305-8577, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takashi Matsumura For correspondence: matsumura.takashi.ka{at}u.tsukuba.ac.jp ryusuke-niwa{at}tara.tsukuba.ac.jp Masasuke Ryuda b Analytical Research Center for Experimental Sciences, Saga University , Saga 840–8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Masasuke Ryuda Hitoshi Matsumoto c Department of Applied Biological Science , Saga 840–8502, Saga University , Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takumi Kamiyama a Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba , Tsukuba 305-8577, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takumi Kamiyama Shu Kondo d Department of Biological Science and Technology, Tokyo University of Science , Tokyo 162-8601, Japan e Invertebrate Genetics Laboratory, National Institute of Genetics , Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shu Kondo Akira Nakamura f Institute of Molecular Embryology and Genetics, Kumamoto University , Kumamoto 860-0811, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akira Nakamura Yoichi Hayakawa c Department of Applied Biological Science , Saga 840–8502, Saga University , Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoichi Hayakawa Ryusuke Niwa a Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba , Tsukuba 305-8577, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ryusuke Niwa For correspondence: matsumura.takashi.ka{at}u.tsukuba.ac.jp ryusuke-niwa{at}tara.tsukuba.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract All organisms are exposed to various stressors, which can sometimes lead to organismal death, depending on their intensity. While stress-induced organismal death has been observed in many species, the underlying mechanisms remain unclear. In this study, we investigated the molecular mechanisms of stress-induced organismal death in the fruit fly Drosophila melanogaster . We identified a chymotrypsin-like serine protease Phaedra1 ( Phae1 ) as a death mediator in D. melanogaster larvae. Phae1 expression was upregulated by lethal heat stress (40 °C) but not non-lethal heat stress (38 °C or lower). The most prominent induction of Phae1 occurred in the central nervous system (CNS). We found neuro-specific knockdown of Phae1 increased survival and reduced neuronal caspase activity following exposure to lethal heat stress, suggesting that the transcriptional upregulation of Phae1 in the CNS is essential for stress-induced organismal death. We next found via bioinformatic and biochemical analyses that the transcription factor Zeste (Z) bound the Phae1 enhancer region and that z loss-of-function impaired Phae1 induction in the CNS, increasing survival following lethal heat stress. In addition, we found via chemical screening that rapamycin, a chemical inhibitor of mechanistic target of rapamycin (mTOR), suppressed Phae1 expression. Neuro-specific knockdown of mTor reduced the protein levels of both Phae1 and Z, leading to an increase in survival following lethal heat stress. Together, these results indicate that heat stress-induced organismal death in D. melanogaster larvae is regulated by a genetically encoded transcriptional signaling pathway involving the mTOR-Z-Phae1 axis. Introduction In nature, living organisms must survive various stressors, such as low and high temperatures, dehydration, ultraviolet radiation, infection, fighting, crowding, and lack of food ( 1 – 8 ). Stress affects organismal physiology in part by inducing the expression of stress-responsive genes related to specific metabolic pathways, autophagy, and cell death ( 9 – 12 ). Organisms can survive stress levels that remain below their species-specific thresholds ( 13 , 14 ), but when stress exceeds these thresholds, it can lead to organismal death ( 15 ). Stress-induced organismal death occurs in various animals, including humans ( 16 , 17 ), often resulting from lethal stress exposure without any visible external damage ( 6 ). It remains unclear whether stress-induced organismal death is merely a secondary consequence of some sort of physiological breakdown or an active process driven by genetic mechanisms triggered by the excessive stress. A few pioneering studies using the nematode Caenorhabditis elegans suggested the involvement of genetic mechanisms in stress-induced organismal death. For example, nematodes exposed to lethal heat stress exhibit activation of the protease cascade along with a specific transcriptional pathway that promotes abnormal cell death ( 18 , 19 ). Despite these studies, however, the key mediators underlying stress-induced organismal death remain largely unknown. The fruit fly Drosophila melanogaster has long been a valuable model for studying the molecular mechanisms of animal physiology ( 20 ). In recent years, D. melanogaster has also emerged as an important model for investigating stress-induced injury pathways ( 21 ). To uncover the mechanisms underlying stress-induced organismal death, we first wanted to identify a reliable hallmark of organismal death. Such a hallmark would hopefully allow us to trace the origin of organismal death to a specific organ or tissue and then investigate the molecular regulatory events occurring there. In this study, we used an RNA-seq approach to identify the Drosophila gene Phaedral1 ( Phae1 ), which exhibits stress-specific transcriptional activation. We also examined the tissues where Phae1 is expressed and the mechanism underlying Phae1 expression. In stressed larvae, Phae1 is prominently expressed in the central nervous system (CNS). Lethal heat stress activates Phae1 expression via the transcription factor Zeste (Z). Furthermore, we found this Z-dependent induction of Phae1 expression in response to lethal heat stress is regulated by the mechanistic target of rapamycin (mTOR). We were able to confirm that this mTOR-Z-Phae1 pathway is essential for heat stress-induced organismal death, indicating that, at least in D. melanogaster , some forms of stress-induced organismal death are regulated by genetically-encoded transcriptional or signal transduction relays. Results Phaedra1 (Phae1) , encoding a serine protease, is a lethal stress-responsive gene Before investigating the molecular mechanism underlying stress-induced organismal death, we examined the survival of D. melanogaster larvae exposed to 30 minutes of heat stress at various temperatures. No larvae died from exposure to temperatures of 38 °C or lower, and only a few succumbed to a 30-min exposure to 39 °C. In contrast, almost all larvae died from a 30-min exposure to 40 °C, indicating that there is only a narrow 2 °C window between survivable and lethal stress ( Fig.1A ). We therefore used these two temperatures as non-lethal (38 °C) and lethal (40 °C) stressors. To identify genes involved in mediating heat stress-induced death, we looked for genes induced specifically by lethal stress in the larval fat body by performing a messenger RNA sequencing (mRNA-seq) transcriptome analysis comparing the response to non-lethal and lethal heat stress. We identified 78 genes upregulated in larvae exposed to lethal heat stress versus those exposed to non-lethal heat stress (fold change > 2, FDR < 0.000001). These 78 candidate genes included proteolysis-related genes (10 genes) and chitin metabolism-related genes (14 genes) (SI Appendix, Fig. S1A, Dataset S01). We next confirmed the specific lethal stress-induced upregulation of 23 of these candidate genes by quantitative reverse transcription PCR (qPCR) (SI Appendix, Fig. S1A, Dataset S01). Download figure Open in new tab Figure 1. Phae1 is a lethal heat stress-responsive death mediator gene. (A) Drosophila larval survival following 30 min of heat stress at the indicated temperatures. ** P < 0.01, **** P < 0.001 (Fisher’s exact test). N = 5 independent technical replicates, n = 25 independent biological replicates. (B) Control ( w 1118 ) and Phae1 mutant larval survival following a 30-min exposure to lethal heat stress at 40 °C. All values are means ± SE. **** P < 0.001 (Fisher’s exact test). N = 8 independent technical replicates, n = 25 independent biological replicates. (C) Phae1 mRNA levels after a 30-min heat stress exposure at the indicated temperatures. ** P < 0.01, **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 4 independent technical replicates. (D) Phae1 protein 3D structure. (E) Phae1 protease catalytic resides (Red area, His-Asp-Ser). (F) Survival of larvae with or without Phae1 protease function after exposure to lethal heat stress. **** P < 0.001 (Fisher’s exact test). N = 6 independent technical replicates, n = 25 independent biological replicates. (G) Survival of larvae with or without Phae1 overexpression following a 30-min exposure to the indicated temperature. All values are means ± SE. **** P < 0.001 (Fisher’s exact test). N = 4 independent technical replicates, n = 25 independent biological replicates. To screen these candidate mediator genes of stress-induced organismal death, we next exposed larvae with in vivo RNAi ( cg-GAL4>UAS-RNAi ) to lethal heat stress at 40 °C and measured their survival. In this screen, we found that knockdown of Phaedra1 ( Phae1 ), which encodes a chymotrypsin-like serine protease ( 22 ), increased larval survival (SI Appendix, Fig. S1B). This makes Phae1 a candidate death mediator gene. We confirmed that a Phae1 genetic null mutant ( Phae1 [SK1]/[Df] ) phenocopied the Phae1 RNAi result, with more mutant larvae surviving exposure to lethal heat stress than w 1118 control larvae ( Fig. 1B ). We also found that lethal (40 °C) but not nonlethal (38 °C) heat stress increased the expression of Phae1 ( Fig.1C ). After establishing a Phae1-GAL4>UAS-GFP ( Phae1>GFP ) transgenic reporter strain to visualize Phae1 expression in vivo , we found increased Phae1 expression upon exposure to various stressors, including low and high temperatures, the insecticides imidacloprid, methamidophos, and rotenone, as well as desiccation, but not starvation (SI Appendix Fig. S2). We next wondered whether Phae1 protease activity is responsible for stress-induced organismal death. Phae1 protein is predicted to have a typical serine protease active site, consisting of Histidine 76 , Aspartic acid 125 , and Serine 223 (His 76 , Asp 125 , Ser 223 ) ( 23 ) ( Fig. 1D and E ). Thus, we established a knock-in Phae1 strain with site-directed substitutions of these three amino acids to alanine (H76A:D125A:S223A) using the CRISPR-Cas9 technique ( 24 ). We chose alanine substitutions to minimize unfavorable steric effects ( 25 ). We used a single guide RNA (sgRNA) to replace the Phae1 CDS, resulting in HA-tagged Phae1 proteins with or without the enzymatically inactive (EI) mutations in the Phae1 active site ( Phae1 WT ::HA or Phae1 EI mutant ::HA ). As expected, more EI mutant ( Phae1 EI mutant ::HA ) larvae than wild-type larvae ( Phae1 WT ::HA ) survived following exposure to lethal stress, suggesting Phae1 protease activity is critical for stress-induced organismal death ( Fig. 1F ). We next asked whether forced overexpression of a Phae1 transgene could enhance stress-induced organismal death. When we overexpressed Phae1 in 3rd instar larvae ( hs-GAL4>UAS-Phae1 ), we observed a reduction in their survival compared with control larvae lacking Phae1 over-expression ( +>UAS-Phae1 and hs-GAL4>+ ) at each temperature; 25% of the larvae died at 35 °C, 50% of the larvae died at 36 °C, and all of the larvae died at 37 °C ( Fig. 1G ). These results are consistent with the hypothesis that Phae1 promotes heat stress-induced organismal death. Neuronal Phae1 affects survival following lethal stress Using public single-cell RNA-seq data in Flybase ( http://flybase.org/reports/FBgn0263234.htm ) ( 26 ), we found Phae1 is strongly expressed in several other larval tissues in addition to the fat body, including epidermal cells and the gut. We therefore used qPCR to determine whether Phae1 expression is also induced in these other tissues upon exposure to lethal heat stress. Compared to control conditions at 25 °C and non-lethal stress at 38 °C, the lethal stress condition at 40 °C led to an upregulation of Phae1 expression in various tissues ( Fig. 2A ). After calculating the fold change in Phae1 expression for each tissue following lethal heat stress, we found the largest increase in the central nervous system (CNS) ( Fig. 2B ). Download figure Open in new tab Figure 2. Neuronal Phae1 expression is specifically upregulated by heat stress. (A) Phae1 mRNA level in various tissues CNS; central nervous system, WD; wing disc, Hc; hemocyte, FB; fat boy, Gut, Car; carcass. * P < 0.05, ** P < 0.01, **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 6 independent technical replicates. (B) Phae1 mRNA fold change in various tissues. (C) Survival of larvae with or without tissue-specific Phae1 knockdown following a 30-min exposure to 40 °C (EEC; enteroendocrine cells, EC; enterocyte,). All values are means ± SE. * P < 0.05, ** P < 0.01, **** P < 0.001 (Fisher’s exact test). N = 6 independent technical replicates, n = 25 independent biological replicates. To clarify the importance of this increase in neuronal Phae1 expression, we used the GAL4-UAS system ( 20 ) to investigate the effects of tissue-specific Phae1 knockdown on larval survival following lethal heat stress. We found a dramatic increase in survival upon neuron-specific Phae1 knockdown, slight increases with tissue-specific knockdown in the epidermis, enteroendocrine cells (EEC), enterocyte (EC), fat body, and glial cells, and no change with other tissue-specific drivers compared to control larvae ( Fig. 2C , SI Appendix, Fig. S3A). We were also able to confirm with a second neuronal driver, elav-GAL4 , that neuro-specific Phae1 knockdown increased larval survival following lethal heat stress (SI Appendix, Fig. S3B). These results suggest neuronal Phae1 is the most important for stress-induced organismal death. Although Phae1 is similar in sequence with it paralogue Phaedra2 ( Phae2 ), which also encodes a chymotrypsin-type serine protease, neuro-specific Phae2 knockdown did not increase larval survival toward lethal heat stress (SI Appendix Fig. S3A). Thus, we focused only on Phae1 for the rest of this study. Phae1 promotes neuronal cell death along with caspase activation We next wondered how neuronal Phae1 promotes stress-induced organismal death. Stress exposure sometimes induces apoptosis-like cell death in dying animals ( 27 , 28 ), which inspired us to ask whether stress-induced cell death could be part of the mechanism underlying stress-induced organismal death. We first asked whether stress-induced cell death occurs in the D. melanogaster larval CNS after exposure to lethal heat stress and whether Phae1 is involved in the induction of stress-dependent cell death. In the CNS of control larvae ( w 1118 ), we observed a massive induction of cell death by lethal stress at 40 °C, but not by non-lethal stress at 38 °C ( Fig. 3A-C ). Phae1 knockout, however, reduced the number of TUNEL-positive dead cells (TUNEL + cells) following lethal stress ( Fig. 3C ’, 3D-F , 3M ), suggesting Phae1 regulates stress-induced cell death in the larval CNS. Download figure Open in new tab Figure 3. Phae1 is associated with cell death induction and caspase activation. (A-F) TUNEL-stained CNS from Drosophila larvae exposed or unexposed to heat stress. Dotted lines encircle the larval CNS. Scale bar: 200 μm. (C’) Magnified view of the CNS of control ( w 1118 ) larvae following exposure to lethal heat stress. (G-I) CNS of larvae either exposed or unexposed to heat stress stained with a cleaved Dcp-1 (cDcp-1) antibody. Dotted lines encircle the larval CNS. Scale bar: 200 μm. (I’) Magnified view of the CNS of control ( w 1118 ) larvae following exposure to lethal heat stress. (M) Quantification of TUNEL-positive cells. (N) Quantification of cDcp-1-positive cells. All values are means ± SE. * P < 0.05, **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 8 independent technical replicates. To determine whether Phae1 promotes cell death by regulating active caspase levels, we next quantified the levels of the active form of the D. melanogaster caspase 3 homolog Dcp-1 (cDcp-1) between control and Phae1 knockout larvae. Caspases are cysteine proteases associated with programmed cell death ( 29 ). Consistent with our earlier results, although we observed an increase in cDcp-1-positive cells (cDcp-1 + cells) in control larval brains following lethal heat stress ( Fig.3G -I, 3I’), Phae1 knockout prevented this stress-induced Dcp-1 activation ( Fig. 3J-L , 3N). Neuron-specific Phae1 knockdown inhibited both Dcp1-activation and cell death induction (SI Appendix, Fig. S4). We also found neuron-specific overexpression of the caspase inhibitor p35 increased larval survival following lethal heat stress (SI Appendix, Fig. S5). These results suggest Phae1-induced cell death, along with neuronal caspase activation, are responsible for heat stress-induced organismal death. The transcription factor Zeste (Z) regulates neuronal Phae1 expression following lethal heat stress Thus far, we have demonstrated a specific upregulation of Phae1 expression following lethal but not non-lethal heat stress, implying the existence of lethal stress-specific transcriptional regulation. To identify such a transcription factor regulating Phae1 expression, we used a luciferase-based reporter assay to analyze the cis -elements of the Phae1 promoter. First, we created luciferase reporter vectors containing various genomic regions upstream of the Phae1 gene. After transfecting these vectors into cultured D. melanogaster Schneider 2 (S2) cells, we measured Phae1 promoter-driven luciferase activity following lethal heat stress at 42 °C for 30-min according to the previously described procedure as follows ( 30 ). Heat stress triggered luciferase expression from the reporter vector containing the 550 base pairs (bp) upstream of Phae1 ( Phae1>Luc ). With enhancers of 540 bp or less, however, we did not observe any response to stress ( Fig. 4A ), suggesting the presence of a stress response element within the 10-bp span from 540 to 550 bp upstream of the Phae1 coding sequence. When we used this region to search the JASPAR transcription factor binding profile database ( 31 ), we found a predicted binding site for Zeste (Z) ( Fig. 4A , SI Appendix, Fig. S6). Z is a trihelix transcription factor ( 32 ) with a helix-turn-helix type DNA binding domain (SI Appendix, Fig. S7A) ( 33 ). Download figure Open in new tab Figure 4. The Z transcription factor regulates Phae1 expression following heat stress exposure. (A) Dual luciferase assay for identifying transcription factors that regulate Phae1 gene expression following heat stress exposure at 42 °C for 30-min. **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 8 independent technical replicates. (B) Gel shift assay confirming that Z protein binds the Phae1 enhancer. N = 3 independent technical replicates. (C) Phae1 mRNA levels in control ( w 1118 ) and z mutant larvae. **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). (D) Anti-Phae1 staining of the CNS of control ( w 111 ) and z mutant larvae exposed or unexposed to heat stress. Dotted lines encircle the larval CNS. Scale bar: 200 μm. (E) Quantification of Phae1 protein level. **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 3 independent technical replicates. To determine whether Z protein binds the Phae1 enhancer region, we performed an electrophoretic mobility shift assay (EMSA). The DNA fragment containing the wild-type Phae1 enhancer region formed a complex with recombinant Z protein (ZDBD, His-tagged protein), resulting in a band shift ( Fig. 4B ). The addition of anti-His tag antibody induced a super shift due to increased molecular weight, suggesting the interaction of the antibody with a DNA-Z complex. This apparent DNA-Z protein interaction was completely abolished by mutation of the Z binding motif within the Phae1 enhancer region, confirming the direct binding of Z to the Phae1 enhancer region. We also found that z mutation reduced Phae1 gene expression following lethal heat stress ( Fig. 4C , SI Appendix, Fig. S8). We next examined neuronal Phae1 protein levels in control ( w 1118 ) and z mutant ( z v77h ) larvae. Although we observed the typical increase in Phae1 protein in the CNS of control larvae exposed to lethal heat stress, this increase was abolished in the CNS of z mutant larvae ( Fig. 4D and 4E ). To further analyze Z protein in vivo , we used the CRISPR-Cas9 technique to produce a knock-in strain that added an N-terminal Ty1 tag (Z-Ty1) to the endogenous Z protein. Consistent with what we observed with z mRNA expression, we used this strain to confirm that lethal heat stress also increased neuronal Z protein levels ( Fig. 5B , SI Appendix, Fig. S9). We then performed an in vivo chromatin immunoprecipitation (ChIP) analysis with an anti-Ty1 antibody and nuclear extracts from the Z-Ty1 larval CNS. In this analysis, we found evidence that anti-Ty1 increased the enrichment of the Phae1 promoter region following lethal heat stress compared to control mouse IgG ( Fig. 5C ). We also found that both whole body knockout of z ( Fig. 5D ) and neuron-specific knockdown of z ( Fig. 5E ) increased the survival of larvae exposed to lethal heat stress. Both neuronal overexpression of either z or Phae1 , however, completely abolished this z mutation-induced increase in larval survival of heat stress (SI Appendix, Fig. S10). We therefore concluded that Z is a crucial transcription factor for inducing Phae1 expression after lethal heat stress. Download figure Open in new tab Figure 5. Z regulates larval survival and Phae1 induction following heat stress exposure. (A) CNS stained with anti-Ty1 tag antibody to detect endogenous Z protein. Scale bar: 200 μm. (B) Quantification of Z protein level. **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 5 independent technical replicates. (C) ChIP assay shows the relative precipitation of the Phae1 enhancer region in larval CNS following exposure to lethal heat stress. **** P < 0.001 (one-way ANOVA followed by Tukey’s HSD). N = 4 independent technical replicates. (D) Survival of control ( w 1118 ), Phae1 mutant, and z mutant larvae following a 30-min exposure to lethal heat stress at 40 °C. *** P < 0.001 (Fisher’s exact test). All values are means ± SE. N = 6 independent technical replicates, n = 25 independent biological replicates. (E) Survival of larvae with or without neuro-specific z knockdown following exposure to a 30-min lethal heat stress at 40 °C. **** P < 0.001 (Fisher’s exact test). All values are means ± SE. N = 3 independent technical replicates, n = 25 independent biological replicates. The neuronal mTOR-Z-Phae1 axis regulates heat stress-induced organismal death To further investigate the upstream signaling pathways that regulate Phae1 expression, we performed a small molecular inhibitor screen ( 34 ) in S2 cells using a luciferase reporter ( Phae1>Luc ) assay ( Fig. 6A ). We compared the effect of 96 different small molecule inhibitors (10 nM) on Phae1 transcriptional activity and found that rapamycin, the well-known inhibitor of mechanistic target of rapamycin (mTOR), produced the most significant reduction in Phae1>Luc activity ( Fig. 6B , Appendix Dataset 03). Rapamycin produced a dose-dependent suppression of Phae1>Luc activity ( Fig. 6C ), while also reducing TUNEL + cell death and caspase-3 activation in S2 cells following a 30-minute exposure to heat stress at 42 °C ( Fig. 6D and E ). These results suggest rapamycin suppresses heat stress-induced cell death by reducing Phae1 expression. Download figure Open in new tab Figure 6. The mTOR-Z-Phae1 axis genetically regulates heat stress-induced organismal death. (A-B) Chemical screen to identify signaling pathways upstream of Phae1 expression following heat stress exposure at 42 °C for 30-min. * P < 0.05 (Mann-Whitney-Wilcoxon test with Bonferroni correction). N = 3 independent technical replicates. (C) Dose-response curve for the effect of rapamycin on Phae1 expression. (D) The effect of rapamycin on the induction of cell death following a 30-min exposure to 42 °C. **** P < 0.001 (two-tailed Student’s t test). (E) The effect of rapamycin on caspase activation induced by a 30-minute exposure to 42 °C. **** P < 0.001 (two-tailed Student’s t test). (F) CNS stained with anti-Ty1 antibody, anti-Phae1 antibody, and Hoechst stain for detecting Z protein in the presence or absence of neuron-specific mTor knockdown. Scale bar: 200 μm. (G) A quantification of Z protein level in the presence or absence of neuron-specific mTOR knockdown. **** P < 0.001 (two-tailed Student’s t test). N = 5 independent technical replicates. (H) A quantification of Phae1 protein level in the presence or absence of neuron-specific mTor knockdown. **** P < 0.001 (two-tailed Student’s t test). N = 5 independent technical replicates. (I) z gene expression in the presence or absence of neuron-specific mTor knockdown. **** P < 0.001 (two-tailed Student’s t test). N = 3 independent technical replicates. (J) Survival of larvae in the presence or absence of neuron-specific mTor knockdown following exposure to lethal heat stress. **** P < 0.001 (Fisher’s exact test). All values are means ± SE. N = 4 independent technical replicates, n = 25 independent biological replicates. We next asked whether mTOR affects heat stress-induced organismal death by altering Phae1 expression. We found neuron-specific knockdown of mTor reduced Z and Phae1 protein levels in the larval CNS following exposure to lethal heat stress ( Fig. 6F ), indicating that mTOR signaling induces Phae1 expression by regulating Z levels ( Fig. 6G and H ). CNS-specific mTor knockdown also suppressed z expression ( Fig. 6I ) and increased larval survival following lethal heat stress ( Fig. 6J ), but this increased survival was completely abolished by neuron-specific overexpression of Phae1 . We therefore conclude that the mTOR-Z-Phae1 axis regulates heat stress-induced organismal death. Discussion This study identifies Phae1 as a key mediator promoting heat stress-induced organismal death. Phae1 was initially identified as a gene located near Drab6 , which encodes a Ras-like small GTPase involved in vesicle trafficking ( 23 ). Coincidentally, Phaedra was named after a woman in Greek mythology who committed suicide ( 35 ). Phae1 encodes a protein homologous to members of the kallikrein family ( 23 ) of secreted serine proteases, but its biological function in insects has not yet been otherwise reported. The Phae1 gene is evolutionally conserved in Diptera and Hymenoptera according to the MEROPS database ( https://www.ebi.ac.uk/merops/cgi-bin/sequence_features?mid=S01.B64 ) ( 36 ). Our experiment using Phae1 transgenes with three alanine substitutions in the putative Phae1 active site amino acid residues (H76A:D125A:S223A) indicated that Phae1 serine protease activity is critical for heat stress-induced organismal death. In mammals, some proteases of the kallikrein family are involved in the induction of autophagy and apoptosis ( 37 ). Since Phae1 is required for heat stress-induced neuronal cell death and caspase activation, it is likely that Phae1 shares similar functional properties with mammalian kallikrein proteases. In addition, Kallikrein-related peptidase-7 cleaves Caspase-14 and regulates its maturation ( 38 ). We therefore hope to examine in a future study whether any of the D. melanogaster caspases are substrates of Phae1. Our data suggested that the trihelix transcription factor Z binds the Phae1 enhancer, inducing Phae1 expression to regulate neuronal cell death following exposure to lethal heat stress. Z was originally identified as an eye color-related gene, because z mutant flies have brown eyes ( 39 ). Another previous study showed that Z regulates cell death induction in D. melanogaster salivary glands during pupariation, along with Ecdysone signaling ( 40 ). It is therefore possible that Z is a key regulator of cell death induction in various tissues. We also found that z expression is regulated by mTOR, which acts as a nutrient sensor ( 41 , 42 ). Starvation stress reduced Phae1 expression probably by suppressing mTOR (SI Appendix Fig. S2B). Neuronal mTOR regulates cell death induction in D. melanogaster ( 43 ), and mTOR inhibition suppresses a form of stress-dependent, apoptosis-like cell death along with caspase activation in mammalian cells ( 44 , 45 ). Consistent with these previous studies, our findings show that rapamycin and neuron-specific mTor knockdown reduce caspase activation, inhibit cell death, and increase larval survival following lethal heat stress. Together, our findings suggest that the genetically regulated mTOR-Z-Phae1 axis can determine whether an animal survives or succumbs to heat stress-induced death. Recent studies have proposed that stress-induced organismal death is genetically regulated ( 46 , 47 ). In this study, we found that lethal heat stress activates the expression of proteases and chitin metabolism-related genes, which are associated with tissue degeneration in dying insects ( 48 , 49 ). Thus, our results support the hypothesis that a specific genetic mechanism promotes heat stress-induced organismal death. To individuals in a group, stress-induced organismal death can offer the benefit of eliminating potentially dangerous individuals, such as those with infections, and reducing competition for space and food ( 50 ). In some organisms, such as the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans, such an “altruistic” sacrifice can increase survival under harsh conditions ( 51 , 52 ). The hypothesis that individual organismal death can contribute to a thriving population has not been completely proven in most animals, but mathematical simulations have suggested that stress-induced organismal death can be adaptative when the cost of cohort competition/danger is sufficiently large ( 53 ). In this respect, it will be interesting to examine whether and how the mTOR-Z-Phae1 axis contributes to D. melanogaster population fitness. It is noteworthy that genes like D. melanogaster Phae1 , z , and mTor are present in mammalian genomes. As mentioned above, Phae1 shows homology with mammalian kallikrein. The helix-turn-helix DNA binding domain of Z is also found in the mammalian nuclear apoptosis-inducing factor (NAIF) ( 54 ). Moreover, mTOR signaling is evolutionarily conserved in various animals, including mammals ( 55 ), and its inhibition can increase lifespan and stress resistance across several species ( 56 , 57 ). Therefore, it will be interesting to determine just how widely the mTOR-Z-Phae1 axis is conserved. Materials and Methods This study included RNA-seq, qRT-PCR, gel shift assays, multiple staining methods, and luciferase reporter assays. Detailed descriptions of the materials and methods used in this study are provided in the Supporting Information (SI). All primers are listed in Table S1, and all small molecule inhibitors appear in Table S2. Author Contributions T.M & R.N wrote manuscript, T.M performed experiments, M.R, supported experiments, S.K & A.N constructed transgenic lines, T.M, H.M, T.K, Y.H, R.N designed experiments. Competing Interest Statement The authors declare no competing interests. Acknowledgments We thank the following for their advice and generosity with their resources: N. Okamoto, K. Iwasaki, K. Takahashi, T. Yoshiga, M. Tokuda, and T. Tujita. We thank the Bloomington Stock Center (NIH P40OD018537), the Kyoto Stock Center, the Vienna Drosophila RNAi Center, and the Developmental Studies Hybridoma Bank (created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242) for fly stocks and antibodies. 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Share Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis Takashi Matsumura , Masasuke Ryuda , Hitoshi Matsumoto , Takumi Kamiyama , Shu Kondo , Akira Nakamura , Yoichi Hayakawa , Ryusuke Niwa bioRxiv 2024.12.25.630336; doi: https://doi.org/10.1101/2024.12.25.630336 Share This Article: Copy Citation Tools Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis Takashi Matsumura , Masasuke Ryuda , Hitoshi Matsumoto , Takumi Kamiyama , Shu Kondo , Akira Nakamura , Yoichi Hayakawa , Ryusuke Niwa bioRxiv 2024.12.25.630336; doi: https://doi.org/10.1101/2024.12.25.630336 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 Physiology Subject Areas All Articles Animal Behavior and Cognition (7646) Biochemistry (17728) Bioengineering (13917) Bioinformatics (42038) Biophysics (21489) Cancer Biology (18637) Cell Biology (25554) Clinical Trials (138) Developmental Biology (13403) Ecology (19941) Epidemiology (2067) Evolutionary Biology (24368) Genetics (15624) Genomics (22547) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88756) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9835) Zoology (2272)