Phosphatidylinositol Transfer Protein-1 Integrates Insulin/IGF-1 and TOR Signaling to Negatively Regulate Lifespan and Healthspan in Caenorhabditis elegans

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24 Phosphatidylinositol transfer protein -1 (pitp-1) is involved in phosphoinositide 25 turnover. The role of pitp-1 in promoting healthy longevity remains unknown. Our 26 previous work showed that the phosphoinositide turnover genes dagl-1 and dgk-5 27 regulates lifespan, as overexpression of dagl-1 or knockdown of dgk-5 prolongs 28 lifespan and enhances oxidative stress resistance through TOR signaling. As pitp-1 is a 29 key component of this pathway, we investigated its role in lifespan regulation and the 30 underlying mechanisms, aiming to clarify whether it represents a critical regulator of 31 healthy longevity and how it coordinates conserved si gnaling pathways to regulate 32 aging. 33

34 C. elegans mutants, RNAi-mediated knockdown, and transgenic overexpression 35 were applied to assess lifespan, motility, stress resistance. Temporal and tissue-specific 36 RNAi were applied to identify the critical time window and tissue for pitp-1-mediated 37 lifespan regulation. TOR signaling was measured by phosphorylated S6 kinase and 38 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 3 puromycin incorporation, and transcriptomic analysis identified affected pathways. 39

40 pitp-1 negatively regulates lifespan and healthspan in Caenorhabditis elegans . 41 Genetic deletion or RNAi -mediated knockdown of pitp-1 extends lifespan, attenuates 42 age-related motility decline, and increases oxidative stress resistance. Temporal and 43 spatial analyses reveal that suppression of pitp-1 in neurons during early adulthood is 44 sufficient to promote healthy longevity. Mechanistically, these beneficial effects upon 45 pitp-1 reduction are mediated by suppressing TOR signaling. Conversely, pitp-1 46 overexpression shortens lifespan and impairs healthspan via TOR activation. Moreover, 47 pitp-1 is transcriptionally repressed by DAF-16 downstream of insulin/IGF-1 signaling 48 (IIS), and contributes to IIS-mediated lifespan extension. 49

50 These findings identify pitp-1 as a novel regulator of healthy aging that integrates 51 IIS and TOR pathways, providing new insights into conserved mechanisms for 52 promoting healthy longevity. 53 54

55 Aging is an inevitable biological process with a progressive decline in 56 physiological integrity, ultimately increasing vulnerability to stress and leading to death. 57 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 4 It is a major risk factor for diverse aging-related diseases, including cancer, 58 neurodegenerative disorders, metabolic syndromes , and substantially causes global 59 healthcare burdens [1]. Thus, identifying genes and molecular mechanisms to promote 60 healthy longevity is urgent for improving the quality of life in expanding aging 61 populations. 62 Target of rapamycin (TOR) signaling and insulin/IGF -1 signaling (IIS) are 63 evolutionarily conserved nutrient-sensing pathways in the regulation of aging [2, 3, 4]. 64 TOR signaling integrates metabolic cues to control protein synthesis, growth , and 65 metabolism [2, 5]. Suppression of TOR activity reduces phosphorylated S6 kinase (p-66 S6K) levels, lowers protein translation, dampens anabolic signaling , extends lifespan 67 and enhance stress resistance across species [6, 7] . Similarly, reduced IIS signaling 68 suppresses the PI3K–PDK–AKT kinase cascade and prevents the phosphorylation of 69 DAF-16/FOXO by phosphorylated AKT (p -AKT), which allows DAF-16/FOXO to 70 translocate into nucleus and orchestrate gene expression program that protect s against 71 cellular damage , improves stress resistance , maintains homeostasis and promotes 72 healthy longevity. Notably, these two pathways are functionally interconnected. IIS-73 activated p -AKT inhibits the TSC1/2, which prevents Rheb from the repression by 74 TSC1/2 and activates TOR complex 1 (TORC1) [3, 8]. Conversely, TOR complex 2 75 (TORC2) can phosphorylate and activate AKT [9]. This crosstalk fine -tunes cellular 76 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 5 and physiological responses to metabolic conditions, highlighting a coordinated 77 regulatory network that governs the aging process. Understanding the integrat ion of 78 TOR and IIS signaling offers crucial insights into the molecular logic of aging and 79 provides promising targets for interventions aimed at extending lifespan and delaying 80 age-associated physiological decline. 81 Phosphatidylinositol transfer proteins (PITPs) are conserved lipid transfer proteins 82 that mediate the transport of phosphatidylinositol (PI) and phosphatidic acid (PA) 83 between the endoplasmic reticulum (ER) and plasma membranes (PM), playing 84 essential roles in phosphoinositide (PPI) turnover and maintaining PIP2 homeostasis 85 [10, 11] . PITPs are conserved across species with homologs among C. elegans, 86 Drosophila, and mammals [11, 12], and classified into two evolutionarily conserved 87 classes of PITPs, class I and class II [12]. In C. elegans, pitp-1, the sole class II PITP, 88 is mainly expressed in sensory neurons and regulates chemotaxis in response to 89 environmental cues [13]. It also facilitates rapid recovery of feeding behavior after 90 hypoxia by limiting diacylglycerol (DAG) availability and suppressing PKC activity in 91 mod-1-expressing neurons [14]. In Drosophila, the class II PITP ortholog rdgB is 92 essential for PPI cycling during phototransduction in photoreceptor cells [15]. In 93 mammals, Nir2, homologous to pitp-1, binds PA and enhances the MAPK a nd 94 PI3K/AKT signaling pathways in response to growth factor stimulation [16]. 95 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 6 Suppression of Nir2 reduces breast cancer cell mi gration and metastasis [17]. Despite 96 the roles of PITPs in neuronal signaling and cancer biology, whether pitp-1 contributes 97 to lifespan regulation remains unknown. 98 Our previous study demonstrated that overexpression of dagl-1/inaE (DAG lipase) 99 or knockdown of dgk-5/rdgA (DAG kinase) extends lifespan and enhances oxidative 100 stress resistance in C. elegans and Drosophila, likely through reduced levels of PA and 101 subsequent inhibition of TOR signaling [6]. In addition, the phospholipase C β (PLCβ) 102 homolog egl-8, another PPI cycle component, has been shown to regulate lifespan in C. 103 elegans, as the null mutant egl-8(n488) exhibits extended longevity [18]. These findings 104 suggest that components of the PPI cycle may play a role in lifespan regulation. 105 Intriguingly, our previous multiple stress genetic screening by oxidative stress and 106 starvation to find the mutants with increased stress tolerance as longevity candidates 107 not only identified the mutant with inaE up-regulation in Drosophila [6] but also 108 uncovered the mutant of rdgB (unpublished data), the ortholog of pitp-1. This prompts 109 us to examine whether pitp-1 participates in lifespan regulation and stress response in 110 C. elegans. Here, we demonstrated the reduction of pitp-1 promotes healthy longevity 111 via modulating TOR signaling. Furthermore, pitp-1 acts downstream of DAF -16 and 112 contributes to IIS -mediated lifespan regulation , suggesting that pitp-1 functions as a 113 potential integrator of IIS and TOR signaling crosstalk. The transcriptomic analysis 114 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 7 supports this notion, as pitp-1 reduction is associated with gene expression changes that 115 resemble those predicted for reduced TOR and IIS signaling activity. To gether, our 116 findings uncover pitp-1 as a novel regulator of healthy longevity and provide new 117 insights into conserved mechanisms of lifespan regulation. 118 119

120 C. elegans strains 121 C. elegans were maintained at 20°C on NGM agar plates seeded with E. coli OP50 122 using standard protocols. The following alleles and strains were used in this study: 123 Wild-type Bristol N2, JN1297: pitp-1(pe1297), pitp-1(tm1500), TU3311: uIs60 [unc-124 119p::YFP + unc -119p::sid-1], TU3401: sid-1(pk3321) ; uIs69 [pCFJ90 (myo -125 2p::mCherry) + unc-119p::sid-1], WM118: rde-1(ne300); neIs9 [myo-3::HA::RDE-1 126 + rol -6(su1006)], VP303: rde-1(ne219); kbIs7 [nhx -2p::rde-1 + rol -6(su1006)], 127 XE1375: wpIs36 [unc -47p::mCherry] I. wpSi1 [unc -47p::rde-1::SL2::sid-1 + Cbr -128 unc-119(+)]; eri -1(mg366); rde -1(ne219); lin -15B(n744), XE1474: wpSi6 [dat -129 1p::rde-1::SL2::sid-1 + Cbr-unc-119(+)]; eri-1(mg366); rde-1(ne219); lin-15B(n744), 130 XE1581: wpSi10 [unc-17p::rde-1::SL2::sid-1 + Cbr-unc-119(+)] ; eri-1(mg366); rde-131 1(ne219); lin -15B(n744), XE1582: wpSi11[eat-4p::rde-1::SL2::sid-1 + Cbr -unc-132 119(+)]; eri -1(mg366); rde -1(ne219); lin -15B(n744), N2 [Ppfkb-1.1::GFP], N2 133 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 8 [Ppfkb-1.1::pfkb-1.1::GFP], CB1370: daf-2(e1370), CF1038: daf-16(mu86), RB1828: 134 dgk-5(ok2366), VC1383: dgk-5(gk691), RB1206: rsks-1(ok1255), TG38: aak-2(gt33), 135 RB2325: sens-1(ok3157), sesn-1(tm2872), TJ356: zIs356[daf-16p::daf-16a/b::GFP + 136 rol-6(su1006)], CF1553: muIs84 [sod-3p::GFP + rol-6(su1006)], N2 [pitp-1p::GFP; 137 myo-2p::mRFP], N2 [pitp-1p::pitp-1::GFP; myo -2p::mRFP], N2 [pitp-1p::pitp-1; 138 myo-2p::mRFP]. Strains were obtained from Caenorhabditis Genetics Center (CGC) 139 and National BioResource Project (NBRP), unless noted otherwise. TU3401, TU3311, 140 VP303, WM118, TJ356, RB2325 and sesn-1(tm2872) were provided by Dr. Chang-Shi 141 Chen (National Cheng Kung University, Taiwan). XE1375, XE1474, XE1581, XE1582 142 were provided by Dr. Tsui-Ting Ching (National Yang Ming Chiao Tung University, 143 Taiwan). For generation of N2 [pitp-1p::GFP; myo -2p::mRFP], N2 [pitp-1p::pitp-144 1::GFP; myo-2p::mRFP] and N2 [pitp-1p::pitp-1; myo-2p::mRFP], a plasmid DNA 145 mix consisting of 80 ng/µL of pitp-1 constructs and 20 ng/µL of co -injection marker, 146 [myo-2p::mRFP], were microinjected into the gonad of young adult N2 hermaphrodite 147 animals. For generation of N2 [ pitp-1p::ptr-23; myo -2p::tdtomato], a plasmid DNA 148 mix consisting of 80 ng/µL of [ pitp-1p::ptr-23] and 20 ng/µL of co -injection marker, 149 [myo-2p::tdtomato], were microinjected into the gonad of young adult N2 150 hermaphrodite animals. Individual F2 progenies were isolated to establish independent 151 lines. Microinjection of N2 worms with co-injection marker, [myo-2p::mRFP] or [myo-152 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 9 2p::tdtomato], alone did not affect the mean lifespan of wild-type animals when grown 153 on OP50 or HT115(DE3) bacteria (data not shown). 154 155 RNA interference assay 156 E. coli HT115(DE3) (Caenorhabditis Genetics Center, Cat#HT115) transformed 157 with either empty vector (L4440) or plasmid expressing double -stranded RNA for 158 desired gene were cultured at 37°C overnight in LB supplemented with 100 µg/ml 159 ampicillin and 100 µg/ml tetracycline. Bacteria were seeded on nematode growth 160 medium (NGM) plates containing 100 µg/ml ampicillin and 1 mM IPTG. The RNAi 161 clones picked from Julie Ahringer’s library (Source BioS cience) were confirmed by 162 sequencing using M13 forward primer (5' - TGTAAAACGACGGCCAGT-3'). RNAi 163 clones made in this paper were constructed by inserting the cDNA of genes into the 164 L4440 vector. For whole life RNAi, synchronized L1 were transferred to RNAi p lates 165 at 20°C. For adult only RNAi, worms were transferred from L4440 plates to RNAi 166 plates at late L4 to young adult stage. Similarly, for RNAi from different adult age, 167 worms were transferred from L4440 plates to RNAi plates at desired adult age. 168 169 Lifespan assay 170 The worms used for lifespan assays were well fed and maintained at 20°C for at 171 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 10 least three generations. All the lifespan assays were performed without using 5-fluoro-172 2’-deoxyuridine (FUdR). Synchronized worms were placed on NGM plates seeded 173 with OP50 at 20°C. For RNAi conditions, synchronized worms were placed on NGM 174 plates seeded with HT115(DE3) bacteria containing empty vector and were transferred 175 to RNAi plates at desired age. For rapamycin treatment condition, rapamycin was 176 dissolved in dimethyl sulfoxide (DMSO). The rapamycin solution was added into NGM 177 plates with the final concentration of 100 µM rapamycin. The final concentration of 178 DMSO for each plate was adjusted to 0.2%, including the control. All the plates used 179 for treating rapamycin were used within 3 days. Synchronized worms were transferred 180 from control plates to rapamycin plates at late L4 to young adult stage. When worms 181 reached adulthood, worms were transferred to fresh plates with desired bacteria at a 182 density of 25-35 worms per plate and were continually transferred to fresh plates every 183 day until egg- laying ceased. After day 8 of adulthood, living worms were scored every 184 1-2 days and transferred to fresh plate every 3 -7 days until all the worms were d ead. 185 Worms which did not move and did not respond to gently touch by platinum picker 186 were scored as dead. Worms which exploded, crawled off plates, bagged or were 187 accidentally killed were censored. Statistical analysis of lifespan data was performed 188 by OASIS 2 [19]. 189 190 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 11 RNA extraction and quantitative real-time PCR 191 Synchronized worms were harvested at the desired stage and total RNA was 192 extracted with REzol (Protech, PT -KP200CT). Worms were lysed by three times 193 freezing and thawing by liquid nitrogen. RNA was purified by adding chloroform 194 (Sigma, C2432) and precipitated by adding isopropanol (VWR, 0918). Extracted RNA 195 was further purified by RQ1 RNase -Free DNase (Promega, # M6101). 1 µg of total 196 RNA added with random primer (Promega, C118A) and M-MLV reverse transcriptase 197 (Promega, M1701) w as used for synthesis of cDNA accordin g to the manufacturer's 198 instructions. Quantitative real -time PCR was set up by using Power SYBR™ Green 199 PCR master mix (ABI, 4367659) and performed the reactions by ABI StepOnePlus 200 Real-Time PCR System. The relative expression levels were calculated by Ct which 201 was normalized by the internal control, act-1. The p-values were calculated by unpaired 202 student t -test. qPCR primers used in this study are listed below. act-1, F: 5’ - 203 CGCCAACACTGTTCTTTCCG-3’; R: 5’ -CTTGATCTTCATGGTTGATGGGG-3’. 204 pitp-1, F: 5’ -GGACAAGGTTCAAGATCGCC-3’; R: 5’ -205 CTCACGGGAAAGAGCAACCA-3’. Y54F10AR.1, F: 5’ -206 CATCCAGACTCCACTCC-3’; R: 5’ -CGTATGCGCGTAGTTTTCGAC-3’. 207 Y71G12B.17, F: 5’ -GGTCTCCTATACGCAGTGTCG-3’; R: 5’ -208 CGGACGCAGTGTGTTACTTG-3’. sod-3, F: 5’-GGGAGCACGCCTACTACTTG-3’; 209 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 12 R: 5’ -AGCATTGGCAAATCTCTCGC-3’. dod-24, F: 5’ -210 TGTCCAACACAACCTGCATT-3’; R: 5’-TGTGTCCCGAGTAACAACCA-3’. 211 212 Body bending assay 213 Synchronized worms at the desired stage were harvested from the NGM -OP50 214 agar plate to the M9 buffer. Each worm was allowed to adapt to the environment for 1 215 min, and the number of body bends was counted in 30 seconds under stereomicroscope. 216 A body bend was considered as a change of direction of the cephalic region of a worm, 217 denoted by the presence of the pharyngeal bulb towards the right side. Bending rates 218 was calculated as body bend per second. The p-values were calculated by Two -way 219 ANOV A. 220 221 Paralysis assay 222 Paralysis assay were performed on fresh NGM plates. Worms at day 14 adulthood 223 were placed on NGM plates and gently touched by platinum-made picker several times. 224 If worms cannot escape from original location but still can move their head or pump 225 their pharynx, these worms were cons idered as paralyzed worms. Worms that can 226 escape form original location after touch or dead worms were not counted as paralyzed 227 worms. The p-values were calculated by One-way ANOV A or unpaired student t-test. 228 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 13 229 Body size measurement 230 Synchronized worms at the desired stage were harvested from the NGM -OP50 231 agar plate to 2% agarose pad. Photos were taken with a CCD camera ( Nikon DS-Ri2) 232 attached to a stereoscopic microscope (Nikon SMZ1500). Open Lab ver.2.2.5 software 233 (Improvision) were used to measure the body size of each worm. The p-values were 234 calculated by One-way ANOV A. 235 236 Oxidative stress assay 237 The oxidative stress assay was conducted at 20°C. Young adult hermaphrodites 238 were transferred to 160 µM or 240 µM juglone (5-hydroxyl-1,4-naphthoquinone, sigma, 239 481-39-0) containing NGM plates which were seeded with bacteria but without adding 240 FUdR to induce oxidative stress. The number of dead worms w as recorded every 2-6 241 hours until all worms were dead or censored from analysis because of worms exploded, 242 crawled off plates, bagged or accidentally killed. The survival curves were performed 243 by the percentage of death and the p-values were calculated by log-rank test. Statistical 244 analysis of lifespan data was performed by OASIS 2 [19]. 245 246 Gene Expression Omnibus (GEO) analysis 247 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 14 Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) is an open 248 functional genomics database of high -throughput resource. In th is study, we 249 downloaded the microarray data of GSE21784, GSE53890, GSE106672 and 250 GSE77109 from GEO database. The microarray data of GSE21784 contained three 251 biological repeats of synchronized populations of C. elegans at three points during 252 aging. The microarray data of GSE53890 contained several samples of adult human 253 brain samples from frontal cortical regions at different age s. The microarray data of 254 GSE106672 contained four biological repeats of synchronized N2 (Bristol), daf-255 2(e1370). The microarray data of GSE77109 contained 2 replicates, each was collected 256 on day 4 of adulthood, fed by HT1115 bacteria. Gene expression was analyzed by 257 GEO2R to compare two or more groups of samples. The p-values were calculated by 258 unpaired student t-test. 259 260 Western blot 261 Synchronized worms were harvested at the desired stage . 300-500 worms per 262 sample were washed three times by M9 buffer and collected into 1.5mL tubes. After 263 removing supernatants, WCE buffer (20 mM HEPES, pH 7.4, 0.2 M NaCl, 0.5 % Triton 264 X-100, 5% glycerol, 1 mM EDTA, 10 mM −glycerophosphate, 2 mM NA3VO4, 1mM 265 NaF, 1 mM DTT) with 1x cocktail protease inhibitor (Roche) and 1x phosphatase 266 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 15 inhibitor (Roche) was added to each sample. Samples were added with 0.5 mm ZrO 267 beads and homogenized by the B ullet Blender. The concentration of extracted 268 supernatants was detected by Bradford protein assay. The quantified proteins were 269 mixed with 6X sample buffer dye (100 mM tris -HCl, pH 6.8, 4% SDS, 0.2% 270 bromophenol blue, 200 mM 2-mercaptoethanol, 20% glycerol, 8M urea) and denatured 271 at 95°C. 30 µg proteins were loaded in 10% SDS-PAGE for protein electrophoresis and 272 transferred to NC-membrane by Bio-Rad system. The NC membrane was incubated in 273 5% BSA in 1x TBST as the blocking buffer. Immunoblotting was performed by 274 incubating with anti-pS6K (Cell Signaling, Billerica, MA, USA, #9209, 1:500 dilution 275 in 5% BSA /1xTBST), anti -p-AKT (Cell Signaling, #9271, 1:1000 dilution in 5% 276 BSA/1xTBST), anti -puromycin (Merck Millipore, #MABE343, 1:5000 in 5% 277 milk/1XTBST), anti --actin ( GeneTex, GTX109639, 1:10 000 dilution in 5% 278 milk/1xTBST), or anti -GAPDH (Epitomics, #S0011, 1:2000 in 5% milk/1XTBST). 279 The membrane was washed three times with 1xTBST and incubated with the secondary 280 antibody (Peroxidase -conjugated AffiniPure Goat Anti -Rabbit IgG (H+L), Jackson, 281 111-035-003, 1:10000 in 5 % BSA/1xTBST for phosphorylated proteins or 5 % 282 milk/1xTBST for other proteins). After three times washing by 1xTBS T, membrane 283 was incubated with chemiluminescent HRP substrate (Millipore, WBKLS0500) and 284 detected the chemiluminescent signals by ImageQuant LAS 4000 mini. The protein 285 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 16 image was quantified by Image J to calculate the fold changes by normalizing each 286 measurement to its control. The p-values were calculated by unpaired student t-test. 287 288 Puromycin incorporation assay 289 To evaluate global protein synthesis in C. elegans , we employed a puromycin 290 incorporation assay adapted with modifications from previously pub lished protocols 291 [20]. Synchronized worms were aged to day 5 of adulthood and collected using M9 292 buffer. Approximately 500 animals were washed twice with M9 and then resuspended 293 in S-basal medium. For puromycin treatment, OP50 bacteria were grown overnight and 294 subsequently concentrated 10-fold in S-basal. Worms were incubated in a 1 mL mixture 295 composed of 750 µL S-basal, 200 µL of the concentrated OP50 suspension, and 50 µL 296 of 10 mg/mL puromycin (Sigma, SI-P8833), yielding a final puromycin concentration 297 of 0.5 mg/mL. Worms were then incubated in a mixture at 200 rpm for 4 hours at room 298 temperature. Following treatment, worms were washed three times with ice -cold S-299 basal, chilled on ice, and snap-frozen in liquid nitrogen. Protein lysates were prepared 300 using RIPA buffer, and pur omycin-labeled proteins were detected by anti -puromycin 301 via Western blotting as described previously. After blot stripping, β-actin was probed 302 and used as a loading control. 303 304 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 17 SOD-3 and DAF-16 reporter assay 305 The transgenic strains TJ356 and CF1553 were used in this study. Synchronized 306 worms fed with EV or RNAi clones were harvested at the desired stage. Photos were 307 taken with a CCD camera ( Nikon DS -Ri2) attached to a stereoscopic microscope 308 (Nikon SMZ1500) with the X-Cite® 120Q excitation light source (excitation at 470 nm 309 and emission at 535 nm). The mean fluorescence intensity was measured by ImageJ 310 software (NIH). The p-values were calculated by unpaired student t-test. 311 312 RNAseq 313 Synchronized worms were harvested to day 3 of adulthood. The extracted RNA samples 314 were DNase treated and assigned RNA Integrity Number (RIN) quality control. The 315 RNA sample with RIN>7.0 can be used to perform next generation RNA sequencing 316 (150 bp, paired -end, ~20 million reads/sample, ~6G total) . Gene expression level is 317 measured by transcript abundance. HISAT2 software was used to read alignment and 318 StringTie software was used to assemble RNA-Seq alignments into potential transcripts 319 in this experiment. Differential expression analysis was performed using DEGseq2 320 software. |FoldChange| > 1.5 and q-value < 0.05 are taken as the differentially expressed 321 gene screening standard. The gene ontology (GO) enrichment analysis and the KEGG 322 pathway analysis were performed by DA VID (https://david.ncifcrf.gov/). The RNAseq 323 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 18 raw data can be accessed by the GEO accession number GSE309580. 324 325

326 Reduction of pitp-1 Promotes Healthy Longevity in C. elegans 327 To examine whether pitp-1 regulates lifespan in C. elegans , we obtained two 328 different pitp-1 mutants, pitp-1(pe1297) and pitp-1(tm1500), for lifespan measurement. 329 Both pitp-1 mutants exhibit significant lifespan extension and lowered pitp-1 mRNA 330 levels compared to wild -type N2 worms (Fig. 1A , 1B and Supplementary Table S1). 331 Similarly, RNAi knockdown of pitp-1 from day-1 adult (D1A) stage significantly 332 extends lifespan compared to the control worms fed with empty vector ( EV) (Fig. 1C, 333 1D and Supplementary Table S1 ). These results indicate that reduction of pitp-1 334 expression promotes longevity in C. elegans. 335 pitp-1 is the single class II PITP gene with a conserved PITP domain in C. elegans. 336 Given that the reduction of pitp-1 extends lifespan, we wondered whether inhibition of 337 the class I PITP genes , Y54F10AR.1 or Y71G12B.17, would have a similar effect on 338 lifespan. However, knockdown of Y54F10AR.1 or Y71G12B.17, alone or in 339 combination, did not prolong lifespan (Supplementary Fig. 1A and Supplementary 340 Table S1). qPCR confirmed that Y54F10AR.1 knockdown specifically reduced its own 341 mRNA levels without affecting Y71G12B.17 or pitp-1 expression, and vice versa for 342 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 19 Y71G12B.17 knockdown (Supplementary Fig. 1B–D). These results suggest that only 343 the reduced expression of class II PITP gene pitp-1 plays a key role in promoting 344 longevity in C. elegans. 345 We next examined whether reduction of pitp-1 confers benefits on healthspan. 346 Aging associates with motility decline and often culminates in paralysis. Thus, we 347 measured locomotor capacity by quantifying body bending rates at day 1 and day 10 of 348 adulthood (D1A and D10A). While pitp-1 mutants exhibited slightly reduced bending 349 rates compared to N2 at D1A, these mu tants retained markedly higher bending rates 350 than age-matched N2 at D10A (Fig. 1E, 1F), indicating the mutants do not display early-351 life hyperactivity but shows improved preservation of motility upon aging. RNAi 352 knockdown of pitp-1 produced similar benefits (Fig. 1H, 1I). Additionally, we assessed 353 age-associated paralysis at D14A as another indicator of motor function. Paralysis at 354 D14A was significantly reduced in both mutants and RNAi-treated worms (Fig. 1G, 1J), 355 demonstrating a marked delay in par alysis onset. We next examined oxidative stress 356 tolerance, another longevity-associated phenotype. We challenged both pitp-1 mutants 357 and RNAi-treated worms with juglone (5-hydroxy-1,4-naphthoquinone), a pro-oxidant 358 compound that induces intracellular oxidative damage, and found both mutants and the 359 RNAi-treated worms exhibited significantly increased survival (Fig. 1K , 1L and 360 Supplementary Table S2). Long-lived organisms frequently display smaller body size. 361 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 20 Consistent with this notion, both pitp-1 mutants were significantly smaller than wild -362 type animals (Supplementary Fig. 1E). Together, these results demonstrate that 363 reduction of pitp-1 accompanies with multiple longevity -associated phenotypes , 364 including improved motility, less paralysis, enhanced oxidative stress resistance, and 365 reduced body size, supporting its role in promoting healthspan. 366 367 Knockdown of pitp-1 before post -reproductive age is essential for enhanced 368 longevity 369 The timing of longevity intervention is critical, as different lifespan -regulating 370 pathways show distinct temporal requirements. For example, in C. elegans, knockdown 371 of daf-2 during reproductive adulthood is important to extend lifespan [21]. Similarly, 372 overexpression of dFOXO during reproductive adulthood promotes longevity in 373 Drosophila [22]. In addition, the geroprotective benefits of TOR inhibition via long -374 term rapamycin treatment can be achieved with a short -term exposure to rapamycin 375 during early adulthood in Drosophila and mice [23]. These data suggest the importance 376 of investigating the precise time window for longevity assurance. 377 To delineate the temporal requirement of pitp-1 reduction for extended lifespan , 378 we initiated pitp-1 RNAi knockdown from different stages: L1 stage, late L4 stage 379 (adult-only, AO), and D5A (Fig. 1M). Knockdown of pitp-1 from L1 or AO both 380 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 21 significantly extended lifespan similar ly, suggesting that pitp-1 reduction from L1 381 larval development is dispensable and from AO is sufficient for promoting longevity 382 (Fig. 1N and Supplementary Table S1). In contrast, pitp-1 RNAi initiated at D5A with 383 effective pitp-1 mRNA reduction produced only a marginal increase for lifespan 384 without significance (Fig. 1N, 1O), indicating a temporal restriction for the longevity 385 effect. To further narrow the time window, we treated worms with pitp-1 RNAi starting 386 from D3A, D4A, D5A, or D7A (Supplementary Fig. 1F, 1G and Supplementary Table 387 S1). pitp-1 knockdown from D3A or D4A significantly extended lifespan, whereas pitp-388 1 knockdown initiated at D5A or later failed to prolong lifespan (Fig. 1N; 389 Supplementary Fig. 1G and Supplementary Table S1). Interestingly, the longevity effect 390 of pitp-1 knockdown was diminished when RNAi treated from D4A, suggesting that an 391 optimal time window from L4 to D3A during early reproductive age . A previous 392 microarray revealed that pitp-1 expression declines with age in C. elegans [24], with 393 significantly lower levels at D6A and D15A compared to L4 (Supplementary Fig. 2A). 394 This age-associated pitp-1 downregulation may explain why pitp-1 knockdown from 395 post-reproductive age no longer influences lifespan. Interestingly, analysis of human 396 microarray data revealed similar age-dependent expression changes in pitp-1 human 397 orthologs [25]. Specifically, PITPNM2 and PITPNM3 expression was significantly 398 reduced in the prefrontal cortex of extremely old individuals (>90 years) compared to 399 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 22 younger adults (<40 years) (Supplementary Fig. 2B–2E), whereas PITPNM1 showed a 400 slight, non-significant decline (Supplementary Fig. 2F –2G). Together, these findings 401 suggest that the longevity effect of pitp-1 suppression is temporally restricted to a 402 critical window prior to the post -reproductive stage, and that age -related reduction of 403 pitp-1/PITPNMs expression may represent a conserved, protective feature of aging. 404 405 The reduction of neuronal pitp-1 is critical for lifespan extension 406 In addition to the temporal aspect, the spatial effect also plays an important role 407 on longevity. For instance, increased neuronal or intestinal, but not muscular, DAF-16 408 activity is sufficient for lifespan extension in C. elegans [26]. In addition, neuronal 409 TORC1 is essential for TOR-mediated aging regulation [27]. To evaluate the effect of 410 tissue-specific pitp-1 reduction on longevity, we performed RNAi knockdown in 411 various tissue -specific RNAi strains. Neuronal knockdown of pitp-1 significantly 412 extended lifespan (Fig. 2A , 2B and Supplementary Table S3 ), whereas pitp-1 413 knockdown in the intestine or muscle had no effect (Fig. 2C , 2D and Supplementary 414 Table S3), indicating neuron is a crucial tissue for pitp-1-mediated lifespan regulation. 415 To further investigate which neuron circuits may participate in the longevity upon pitp-416 1 reduction, we performed pitp-1 knockdown in either GABAergic, glutamatergic, 417 cholinergic or dopaminergic neuron al circuit -specific strains individually for the 418 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 23 lifespan measurement. Interestingly, except for dopaminergic neurons, knockdown of 419 pitp-1 in either GABAergic, glutamatergic, or cholinergic neurons is sufficient to 420 extend lifespan (Fig. 2E-2H and Supplementary Table S3). These findings indicate that 421 neuronal pitp-1 suppression, particularly in certain specific neuron types, plays a central 422 role in mediating its longevity effect. 423 424 Overexpression of pitp-1 decreases lifespan and impairs healthspan 425 To examine whether increased pitp-1 has the opposite effects, we generated pitp-426 1 overexpressing transgenic worms, one line with pitp-1::GFP fusion construct under 427 pitp-1 promoter, N2[pitp-1p::pitp-1::GFP; myo-2p::mRFP] (named N2 PITP-1 OE 1) 428 with the control line, N2[pitp-1p::GFP] (named N2 control). To exclude possible GFP 429 effects, we also generated pitp-1 overexpressing without GFP fusion transgenic worms, 430 N2[pitp-1p::pitp-1; myo -2p::mRFP] (named N2 PITP -1 OE 2 ). Confocal imaging 431 confirmed the neuronal expression of the pitp-1::GFP fusion protein (Fig. 3A) , 432 consistent with previous findings [13]. Both strains showed about 3 -4-fold increase in 433 pitp-1 mRNA (Fig. 3B). Opposite to pitp-1 reduction, both N2 PITP-1 OE lines 434 exhibited significantly shortened lifespan (Fig. 3C and Supplementary Table S1 ), 435 reduced motility by at D10A (Fig. 3D), and increased paralysis at D14A (Fig. 3E), 436 indicating deteriorated aging and health. Importantly, lifespan shortening in N2 PITP-437 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 24 1 OE worms was fully rescued by pitp-1(RNAi) (Fig. 3F), and overexpression of pitp-438 1 in pitp-1 mutants abolished their extended lifespan (Fig. 3G and Supplementary Table 439 S4). These findings demonstrate that pitp-1 acts as a negative regulator of healthy 440 longevity in C. elegans. 441 442 pitp-1 negatively regulates lifespan through modulating TOR signaling 443 Our previous study demonstrated that reduced dgk-5 extends lifespan through 444 downregulation of TOR signaling [6]. Since pitp-1 and dgk-5 function in the same 445 pathway and that reduced expression of either gene leads to longevity, we hypothesized 446 that pitp-1 may also regulate lifespan via TOR sig naling. Supporting this notion, 447 knockdown of pitp-1 did not further enhance the extended lifespan of dgk-5 mutants 448 (Supplementary Fig. 3A, 3B and Supplementary Table S4), suggesting pitp-1 and dgk-449 5 act through a common mechanism. Moreover, both pitp-1 mutants and RNAi-treated 450 worms exhibited significantly reduced p -S6K levels (Fig. 4A -4D), indicating 451 diminished TOR signaling. In contrast, overexpression of pitp-1 markedly increased p-452 S6K abundance (Fig. 4E and 4F), suggesting enhanced TOR activation. Furthermore, 453 the elevated TOR signaling in pitp-1-overexpressing worms was suppressed by RNAi 454 targeting let-363/TOR or its upstream activator raga-1 (Supplementary Fig. 3C and 3D). 455 Re-expression of pitp-1 in the pitp-1 mutant background restored p -S6K levels 456 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 25 (Supplementary Fig. 3E, 3F), supporting a role for pitp-1 as an upstream positive 457 regulator of TOR signaling. Since TOR activation promotes protein translation, we 458 further examined the effect of pitp-1 on global translation. Both pitp-1 mutants and 459 RNAi-treated animals showed significantly reduced puromycin labeling (Fig. 4G –4J), 460 indicating decreased protein translation. Conversely, overexpression of pitp-1 markedly 461 enhanced puromycin incorporation (Fi g. 4K, 4L), representing elevated translational 462 output. These results support that pitp-1 positively regulates TOR activity and 463 downstream protein synthesis. Furthermore, the extended lifespan of pitp-1 mutants 464 was not further prolonged by either genetic or pharmacological inhibition of TOR (Fig. 465 4M, 4N, supplementary Fig. 3G, 3H and Supplementary Table S4). Similarly, pitp-1 466 RNAi in the rsks-1/S6K mutant background failed to further extend the prolonged 467 lifespan (supplementary Fig. 3I and Supplementary Table S4). Moreover, suppression 468 of TOR signaling by either let-363(RNAi) or rapamycin treatment rescued the lifespan 469 shortening and reduced motility caused by pitp-1 overexpression (Fig. 4O , 4P, 470 supplementary Fig. 3J and Supplementary Table S4 ). Collectively, these results 471 demonstrate that pitp-1 negatively regulates lifespan through modulation of TOR 472 signaling. 473 474 Several TOR regulators are involved in pitp-1-mediated lifespan regulation 475 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 26 TOR activity is modulated by amino acids, growth factors, and energy stress via 476 distinct regulators (Supplementary Fig. 3 K). To identify upstream regulators linking 477 pitp-1 to TOR activity, we performed lifespan epistasis tests. RNAi knockdown of raga-478 1 or rheb-1 rescued the shortened lifespan in pitp-1-overexpressing animals (Fig. 4Q , 479 4R and Supplementary Table S4), suggesting that both Rag and Rheb GTPases are 480 involved in pitp-1-mediated lifespan regulation. Conversely, knockdown of pitp-1 still 481 extended lifespan in the AMPK -deficient strain aak-2(gt33) (Supplementary Fig. 3L 482 and Supplementary Table S4), suggesting that pitp-1 regulates lifespan independent of 483 AMPK. Sestrin, a negative regulator of Rag GTPases, is known to inhibit the amino 484 acid sensing arm of TORC1 and promotes longevity in C. elegans [28]. Given the role 485 of Rag GTPases in pitp-1-mediated lifespan regulation, we next investigated whether 486 sestrin is also required for this effect. Notably, the lifespan extension induced by pitp-487 1 knockdown was abolished in sesn-1 mutant animals (Supplementary Fig. 3M, 3N and 488 Supplementary Table S4 ), indicating that sestrin is required for pitp-1-mediated 489 lifespan extension. Together, these findings reveal that the sestrin–Rag GTPase axis and 490 Rheb GTPase, upstream regulators of TOR , are involved in pitp-1-mediated lifespan 491 regulation. 492 493 pitp-1 is involved in insulin/IGF-1 signaling-mediated lifespan regulation 494 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 27 Because AKT lies upstream of Rheb -TOR, we first detected the p -AKT levels in 495 long-lived pitp-1 mutants to check IIS involvement in pitp-1-mediated lifespan 496 regulation. Both pitp-1 mutants exhibit significantly reduced p -AKT levels compared 497 to N2 worms (Fig. 5A and 5B), suggesting that IIS may be involved in pitp-1-mediated 498 lifespan extension. However, knockdown of pitp-1 did not promote DAF -16::GFP 499 nuclear translocation nor increase expression of DAF-16 target gene sod-3 500 (Supplementary Fig. 4A -4C). These results indicate that pitp-1 knockdown does not 501 promote DAF-16 transcription activity. To further clarify if DAF-16 is required for pitp-502 1 knockdown-mediated lifespan extension , we performed lifespan assays in the daf-503 16(mu86) null mutant by pitp-1 knockdown. Knockdown of pitp-1 still extended 504 lifespan in daf-16(mu86) mutant (Fig. 5C and Supplementary Table S4), indicating that 505 DAF-16 is not required for the longevity effect by pitp-1 suppression. 506 Interestingly, two DAF-16 binding sites were identified in the pitp-1 promoter [29], 507 raising the possibility that pitp-1 may act downstream to DAF-16 and be regulated by 508 DAF-16. To test this, we analyzed pitp-1 promoter-driven GFP expression following 509 RNAi of daf-2 or daf-16. Knockdown of daf-2 significantly reduced pitp-1::GFP 510 expression, while daf-16 RNAi elevated its expression (Fig. 5D and 5E). Moreover, co-511 treatment with daf-2 and daf-16 RNAi restored the decreased pitp-1 GFP intensity 512 caused by daf-2 knockdown (Fig. 5D and 5E) . Similarly, qPCR confirmed that pitp-1 513 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 28 transcript levels were downregulated in daf-2(e1370) but upregulated in daf-16(mu86) 514 mutants (Fig. 5F). Consistently, a previous microarray study also reported reduced pitp-515 1 expression in daf-2(e1370) (Supplementary Fig. 4D) [30]. These data indicate that 516 pitp-1 is transcriptionally repressed by DAF-16. Accordingly, pitp-1 knockdown failed 517 to further extend lifespan in daf-2(e1370) mutant (Fig. 5G and Supplementary Table 518 S4), likely due to their already reduced pitp-1 expression levels. Conversely, 519 overexpression of pitp-1 partially suppressed the lifespan extension induced by daf-520 2(RNAi) or age-1(RNAi) (Fig. 5H, Supplementary Fig. 4E and Supplementary Table 521 S5), further supporting the notion that pitp-1 acts as a downstream effector negatively 522 regulated by IIS. Together, these results suggest that pitp-1 functions downstream of 523 DAF-16 and contributes to IIS-mediated lifespan regulation. 524 525 Transcriptome-wide analyses uncover signaling shifts and longevity mechanisms 526 upon pitp-1 suppression 527 Since the longevity effect of pitp-1 suppression is temporally restricted, and likely 528 involves complex transcriptional reprogramming and pathway cross-talk, we 529 performed RNA sequencing on D3A worms with reduced pitp-1 expression to gain a 530 comprehensive understanding of transcriptomic changes. Transcriptomic profiles were 531 analyzed using Qiagen Ingenuity Pathway Analysis (IPA) and Over -Representation 532 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 29 Analysis (ORA) (Fig. 6A). IPA canonical pathway analysis revealed downregulation of 533 IIS, PI3K/AKT and TOR in pitp-1 mutant and pitp-1(RNAi)-treated worms (Fig. 6B), 534 consistent with our mechanism study findings. In addition, PTEN signaling, the 535 negative regulator of IIS, was activated upon pitp-1 suppression, supporting pitp-1 536 reduction leads to attenuation of IIS. In contrast, AMPK signaling was not activated, 537 which is consistent with our previous results showing that pitp-1 reduction does not 538 promote longevity through AMPK activation (Supplementary Fig. 3L). Similarly, IPA 539 upstream regulator analysis suggests that the transcriptomic changes we observed may 540

from decreased upstream activity of mTOR or insulin signaling (Fig. 6C) . 541 Together, these results reinforce the critical roles of IIS and TOR signaling in mediating 542 pitp-1-dependent longevity. 543 In addition to IIS and TOR, eIF2 signaling was also significantly downregulated 544 in pitp-1-reduced worms (Fig. 6B) . As a key regulator of translation initiation, 545 inhibition of eIF2B enhance s proteostasis and extends lifespan [20, 31]. Consistently, 546 pitp-1 suppression reduced protein synthesis (Fig. 4G -4J), accompanied by extended 547 lifespan. Together, these observations suggest that pitp-1 reduction promotes longevity, 548 perhaps by improving protein homeostasis. 549 We also found that pitp-1 suppression downregulated CDP-DAG biosynthesis 550 pathway and 3-phosphoinositide biosynthesis (Fig. 6B), suggesting PPI cycle activity 551 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 30 may be downregulated. Consistently, IPA Diseases and Bio-functions analysis (Fig. 6D) 552 revealed decreased lipid metabolism , amino acid metabolism, and protein synthesis, 553 likely reflecting TOR suppression. This metabolic reduction may also explain the 554 downregulation of biofunctions such as "size of body" and "growth of organism" (Fig. 555 6D). These results align with our earlier observation that pitp-1 mutants exhibit smaller 556 body size (Supplementary Fig. 1E). 557 In addition to IPA, we employed Over-Representation Analysis (ORA) to identify 558 downstream gene targets upon pitp-1 reduction. Using a cutoff of >1.5-fold change and 559 adjusted p-value < 0.05, we identified 10 genes significantly upregulated upon pitp-1 560 suppression (Fig. 6E). GO and KEGG pathway analyses revealed that the “SCF -561 dependent, ubiquitin -mediated proteasomal protein cata bolic process” , “ubiquitin-562 mediated proteolysis” and “Protein processing in endoplasmic reticulum” was 563 significantly overrepresented (Fig. 6 F, 6G). This result aligns with our IPA analysis 564 showing reduced global protein synthesis (Fig. 6D) and our puromycin incorporation 565 assay (Fig. 4G-4J). Given that enhancing proteolytic systems, including the ubiquitin–566 proteasome pathway, promotes longevity [32], our findings suggest that pitp-1 567 reduction may promote healthy longevity by maintaining proteostasis. 568 In summary, these transcriptomic analyses reinforce that pitp-1 reduction 569 promotes longevity through coordinated suppression IIS , TOR signaling , reducing 570 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 31 anabolic activity and enhancing proteostasis. Our data support a model in which DAF-571 16 represses pitp-1 transcription u nder reduced IIS, partially contributing to IIS -572 mediated longevity. Reduced pitp-1 attenuates TOR activity via the AKT–RHEB axis, 573 thereby promoting longevity. This is the first study to identify pitp-1 as a novel lifespan 574 regulator and highlights its involvement in IIS –TOR crosstalk, offering new insights 575 into aging regulation and potential anti-aging interventions. 576 577

578 PITP is a critical regulator in PPI turnover, but its role in aging remains unclear. 579 In this study, we identify a novel function for pitp-1, a Class II PITP, in regulating 580 lifespan and healthspan in C. elegans. We show that pitp-1 is transcriptionally repressed 581 by DAF-16 and acts as a pro -aging factor by promoting TOR signaling. Notably, our 582 spatial and temporal analyses reveal that both neuronal specificity and early adulthood 583 timing are essential for pitp-1-mediated lifespan regulation. These findings position 584 pitp-1 as a critical regulator that connects the IIS and TOR pathways in aging control, 585 with its pro-aging function constrained by specific neuronal and temporal contexts. 586 Our temporal analysis highlighted a critical window during early adulthood , 587 particularly the early reproductive stages , as essential for pitp-1-mediated lifespan 588 regulation, with adult-onset knockdown sufficient to promote healthy longevity without 589 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 32 interfering with development. This time window is consistent with the concept that 590 interventions in nutrient-sensing pathways are most effective during this stage [21, 22, 591 23]. These parallels highlight that pitp-1 reduction aligns with these conserved 592 longevity-regulating pathways during a critical early-adult temporal window. Moreover, 593 our analysis of public gene expression datasets revealed a natural, age -associated 594 decline in pitp-1/PITPNMs expression in worms and humans (Supplementary Fig. 2) 595 [24, 25] , suggesting that this downregulation may represent a conserved protective 596 mechanism against aging. 597 Spatially, neuronal knockdown of pitp-1 is sufficient to promote longevity, 598 emphasizing the central role of the nervous system in systemic aging regulation where 599 IIS and TOR signaling exert their lifespan-modulating effects [26, 27, 33]. Consistently, 600 neuronal inhibition of RAGA-1 from hatching or D1A extends lifespan, supporting the 601 temporal flexibility of neuronal TOR suppression in promoting longevity [34]. In 602 addition, other PPI cycle genes , inaE/dagl/dagl-1 and egl-8/PLC, expressed in 603 neurons also regulate lifespan through the TOR pathway [6, 35] . These findings 604 reinforce neuronal modulation of PI signaling impacts systemic aging via TOR, in line 605 with the role we propose for pitp-1. 606 Moreover, suppression of pitp-1 in either glutamatergic, cholinergic, or 607 GABAergic neurons each extended lifespan, suggesting that pitp-1 exerts its pro-aging 608 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 33 function through multiple excitatory and inhibitory neuronal circuits. Inhibition of age-609 related increases in n eural excitation, particularly in glutamatergic and cholinergic 610 neurons, extend s lifespan [36]. Chronic hyperexcitability of glutamatergic neurons 611 accelerates aging by PLCβ–IP3R pathway overactivation, and suppressing this pathway 612 restores normal lifespan [36, 37] . Additionally, mTOR hyperactivation enhance s 613 synaptic responses in glutamatergic and GABAergic neurons, while rapamycin 614 treatment normalizes glutamatergic overexcitation and restores neurotransmitter 615 balance [38]. These data suggest that PLCβ–IP3R pathway or mTOR suppression in 616 excitatory and inhibitory neurons promotes neural homeostasis and healthy aging, 617 aligning with our findings. Taken together, pitp-1 emerges as a neuronal regulator of 618 longevity acting through TOR, potentially by modulating neuronal exci tability and 619 neurotransmission. Future studies should clarify the role of distinct circuits and how 620 pitp-1 coordinates PPI turnover to systemic metabolic responses upon aging. 621 eIF2 is a central regulator of translation initiation, whose activity is inhibited by 622 phosphorylation of eIF2 α, leading to global translational repression and proteostasis 623 maintenance [20, 31]. Our IPA analysis revealed that pitp-1 downregulation reduces 624 eIF2 signaling, consistent with reduced global translation. Moreover, prior studies have 625 reported bidirectional crosstalk between eIF2 and mTORC1 : mTORC1 inhibition can 626 activate GCN2 to phosphorylate eIF2α, whereas eIF2α phosphorylation and ATF4 627 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 34 translation can inhibit mTORC1 by REDD1 and Sestrin2 induction [39, 40]. In line 628 with this, we found that pitp-1 downregulation not only represses TOR signaling but 629 also requires sestrin for lifespan extension (Supplementary Fig. 3M, 3N), suggesting 630 the involvement of the eIF2α–ATF4–Sestrin axis. This pathway acts independently of 631 AMPK [40], consistent with our data (Supplementary Fig. 3L), and may inhibit TOR 632 by restraining Rag GT Pase–mediated activation [28]. Together, our findings suggest 633 that the observed dow nregulation of eIF2 signaling upon pitp-1 reduction may 634 potentially contribute to TOR inhibition and healthy longevity, while further studies are 635 needed to establish a direct causal role in the future. 636 In this study, we also found that pitp-1 suppression in C. elegans not only promotes 637 longevity but also results in reduced body size, a phenotype often linked to altered 638 nutrient signaling. In addition to reduced TOR or IIS activity, the two nutrient-sensing 639 pathways known to influence body size, our transcriptomic analysis further revealed a 640 downregulation of YAP and TAZ in pitp-1-suppressed worms, as predicted by upstream 641 regulator analysis using IPA (Fig. 6C). This finding is consistent with recent studies in 642 mammalian systems showing that inhibition of PITP α/β activates the Hippo pathway, 643 leading to suppression of YAP -mediated transcription, reduced cell proliferation, and 644 enhanced cancer cell death [41]. Our observation of reduced YAP/TAZ activity and 645 smaller body size upon pitp-1 suppression may reflect a conserved mechanism, where 646 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 35 diminished PI4P-mediated suppression of the Hippo pathway contributes to reduced 647 growth. Importantly, these findings raise the possibility that modulation of the Hippo 648 pathway or direct regulation of its downstream transcription factors YAP/TAZ may 649 represent a novel and promising strategy to promote healthy aging . In particular, 650 investigating how pitp-1 interfaces with the Hippo pathway may uncover a previously 651 unrecognized lipid-signaling mechanism with relevance to both growth regulation and 652 age-associated functional decline. 653 In addition to downregulation of canonical nutrient -sensing pathways , eIF2 654 signaling and Hippo pathway, our IPA analysis revealed a suppression of Huntington’s 655 disease (HD) signaling upon pitp-1 reduction (Fig. 6B). Given that mutated Huntingtin 656 (Htt) enhances mTORC1 activity through Rheb interaction and aberrant 657 PI3K/AKT/mTOR signaling contributes to HD pathogenesis [42], these findin gs are 658 consistent with our model that pitp-1 modulates lifespan through Rheb-TOR signaling 659 and shows downregulation of HD -associated signaling. Notably, HD is characterized 660 by dysfunction of both GABAergic and glutamatergic neurons, which are central to 661 motor impairment and excitotoxicity [43]. Strikingly, suppression of pitp-1 in either 662 GABAergic or glutamatergic neurons was sufficient to extend lifespan in C. elegans 663 (Fig. 2), suggesting that pitp-1 may act through conserved neuronal circuits also 664 implicated in HD pathogenesis. This raises the intriguing possibility that pitp-1 665 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 36 suppression could not only promote healthy longevity but also provide therapeutic 666 potential for neurodegenerative diseases such as HD. Future investigations exploring 667 whether pitp-1 modulation can mitigate HD-related phenotypes in mammalian models 668 may uncover promising therapeutic avenues for neurodegenerative disease intervention. 669 Consistently, our parallel study in Drosophila revealed that downregulation of 670 rdgB, the orthologue of pitp-1, also promotes healthy longevity and reduces TOR 671 activity (data not shown). These findings strongly suggest that the pro -aging role of 672 PITPs and their regulation of TOR signaling are evolutionarily conserved. Thus, the 673 mechanisms uncovered here may extend beyond nematodes and flies, raisi ng the 674 exciting possibility that PITP modulation could exert similar effects on aging and 675 healthspan in mammals, including humans. 676 677

678 Our findings uncover pitp-1 as a previously unappreciated regulator of aging, 679 acting at the intersection of IIS and TOR signaling in a neuron- and age-specific manner. 680 This study establishes pitp-1 as a critical node coordinating nutrient-sensing pathways 681 to regulate healthy longevity and highlights its potential as a target for aging-associated 682 interventions. While our results reveal its role in IIS-TOR cross talk, it also suggest that 683 pitp-1 may influence additional pathways implicated in growth control, proteostasis, 684 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 37 and neurodege neration, including Hippo, eIF2, and Huntington’s disease –related 685 signaling. These findings warrant further investigation, particular in other species such 686 as Drosophila or mammals, future studies to assess the conservation of this regulatory 687 axis and its relevance for both healthy aging and anti-neurodegenerative diseases. 688 689 Data availability 690 All the RNAseq raw data can be accessed by the GEO accession number GSE309580. 691 692 Abbreviations 693 pitp-1: phosphatidylinositol transfer protein-1 694 PITPs: phosphatidylinositol transfer proteins 695 PI: phosphatidylinositol 696 PA: phosphatidic acid 697 ER: endoplasmic reticulum 698 PM: plasma membrane 699 PPI: phosphoinositide 700 DAG: diacylglycerol 701 IIS: insulin/IGF-1 signaling 702 TOR: target of rapamycin 703 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 38 p-S6K: phosphorylated S6K 704 p-AKT: phosphorylated AKT 705 TORC1: TOR complex 1 706 TORC2: TOR complex 2 707 PLCβ: phospholipase C β 708 EV: empty vector 709 GEO: Gene Expression Omnibus 710 RNAseq: RNA sequencing 711 RIN: RNA Integrity Number 712 IPA: ingenuity pathway analysis 713 ORA: over-representation analysis 714 GO: gene ontology 715 CGC: Caenorhabditis Genetics Center 716 NBRP: National BioResource Pproject 717 NGM: nematode growth medium 718 FUdR: 5-fluoro-2’-deoxyuridine 719 DMSO: dimethyl sulfoxide 720 Juglone: 5-hydroxyl-1,4-naphthoquinone: 721 DTT: Dithiothreitol 722 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 39 NSTC: National Science and Technology Council 723 724

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V ogel KR, Ainslie GR, Gibson KM. mTOR inhibitors rescue premature lethality 835 and attenuate dysregulat ion of GABAergic/glutamatergic transcription in murine 836 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 45 succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA 837 metabolism. J Inherit Metab Dis. 2016;39(6):877-86. 838 39. Quentin T, Steinmetz M, Poppe A, Thoms S. Metformin differentially a ctivates 839 ER stress signaling pathways without inducing apoptosis. Dis Model Mech. 840 2012;5(2):259-69, Wengrod J, Wang D, Weiss S, Zhong H, Osman I, Gardner LB. 841 Phosphorylation of eIF2alpha triggered by mTORC1 inhibition and PP6C activation is 842 required for au tophagy and is aberrant in PP6C -mutated melanoma. 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The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 46 43. Garret M, Du Z, Chazalon M, Cho YH, Baufreton J. Alteration of GABAergic 856 neurotransmission in Huntington's disease. CNS Neurosci Ther. 2018;24(4):292 -300, 857 Hsu YT, Chang YG, Chern Y . Insights into GA BA(A)ergic system alteration in 858 Huntington's disease. Open Biol. 2018;8(12), Estrada Sanchez AM, Mejia -Toiber J, 859 Massieu L. Excitotoxic neuronal death and the pathogenesis of Huntington's disease. 860 Arch Med Res. 2008;39(3):265-76. 861 862 863 864 865 866 867 868 869 870 871 872 873 874 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 47 Figures 875 Figure 1 876 877 Figure 1. Reduction of pitp-1 extends lifespan and promotes healthspan. (A) Two 878 independent pitp-1 mutants displayed significantly extended lifespan. (B) qPCR 879 confirmed reduced pitp-1 mRNA levels in pitp-1 mutants. (C) Knockdown of pitp-1 by 880 RNAi from day-1 adult (D1A) extended lifespan. (D) qPCR confirmed reduced pitp-1 881 mRNA expression upon pitp-1(RNAi). (E, F) Both pitp-1 mutants exhibited increased 882 motility and ameliorated motility declines at D10A. (G) Both of the pitp-1 mutants 883 exhibited less paralyzed worms at D14A. (H, I) Knockdown of pitp-1 increased motility 884 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 48 and ameliorated motility declines at D10A. (J) Knockdown of pitp-1 by RNAi 885 ameliorated age-induced paralysis at D14A. (K, L) pitp-1 mutants RNAi-treated worm 886 showed enhanced resistance to juglone -induced oxidative stress. (M) Schematic 887 diagram of RNAi treatment timeline. (N) pitp-1 knockdown during adulthood extended 888 lifespan. (O) qPCR confirmed that pitp-1(RNAi) from D5A reduces pitp-1 mRNA 889 levels. P-values were calculated by log-rank t-test in (A, C, K, L, N), and by One -way 890 ANOV A in (B, F, G), and by Two-way ANOV A in (E, H), and by unpaired student’s t-891 test in (D, I, J, O). Survival curves are representative of three independent experiments, 892 except oxidative stress assays, which were repeated twice with consistent results . 893 Statistical significance was determined by log-rank test for lifespan assays, ANOV A for 894 multiple comparisons, and unpaired Student’s t-test where applicable. 895 896 897 898 899 900 901 902 903 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 49 Figure 2 904 905 906 Figure 2. Reduction of pitp-1 in pan-neuronal tissue and specific neuronal circuits 907 extends lifespan. (A, B) Neuron-specific knockdown of pitp-1 in TU3401 and TU3311 908 from adult hood increased lifespan. (C, D ) Intestine-specific or Muscle-specific 909 knockdown of pitp-1 from adulthood did not alter lifespan. (E–G) Knockdown of pitp-910 1 specifically in GABAergic neuron ( XE1375), in glutamatergic neuron ( XE1582), or 911 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 50 in cholinergic neuron (XE1581) from adulthood extended lifespan. (H) Knockdown of 912 pitp-1 specifically in dopaminergic neuron ( XE1474) showed no significant lifespan 913 increase. Survival curves are representative of three independent experiments. 914 Statistical significance was determined by log-rank test. 915 916 Figure 3 917 918 Figure 3. Overexpression of pitp-1 decreases lifespan and impairs healthspan. (A) 919 Confocal images showing pitp-1 expression (GFP) and co-injection marker (mRFP) in 920 PITP-1 OE 1 . (B) qPCR confirmed elevated pitp-1 mRNA levels in the pitp-1 921 overexpression strains. (C) Overexpression of pitp-1 reduced lifespan in both 922 transgenic lines. (D , E ) PITP-1-overexpressing worms displayed decreased b ody 923 bending rate at D10A and increased paralysis at D14A compared to control line. (F) 924 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 51 The reduced lifespan in N2 PITP-1 OE 1 can be rescued by pitp-1(RNAi) knockdown. 925 (G) Overexpression of PITP -1 OE 1 reverted the extended lifespan in both pitp-1 926 mutants. Survival curves are representative of three independent experiments. 927 Statistical significance was determined by log-rank test for lifespan assays, ANOV A for 928 multiple comparisons. 929 930 Figure 4 931 932 Figure 4. pitp-1 negatively regulates lifespan through modulating TOR signaling. 933 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 52 (A–F) pitp-1 mutants or RNAi knockdown reduced phosphorylated S6K levels, 934 whereas PITP -1 overexpression increased S6K phosphorylation. (G –L) puromycin 935 incorporation assays showed reduced prot ein synthesis in pitp-1 mutants or RNAi 936 animals and elevated levels in PITP -1-overexpressing strains. (M, N) Genetic or 937 pharmacological inhibition of TOR by let-363(RNAi) or rapamycin did not further 938 extend the longevity of pitp-1 mutants. (O, P) TOR inhibition rescued the shortened 939 lifespan caused by PITP -1 overexpression. (Q, R) Knockdown of upstream TOR 940 regulators raga-1 or rheb-1 rescued the reduced lifespan of PITP -1-overexpressing 941 worms. Survival curves are representative of three independent experiments. Statistical 942 significance was assessed by log -rank test for lifespan assays, ANOV A for multiple 943 comparisons, and unpaired Student’s t-test where applicable. 944 945 946 947 948 949 950 951 952 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 53 Figure 5 953 954 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 54 Figure 5. Integration of pitp-1 in insulin/IGF -1 signaling –mediated lifespan 955 regulation. (A, B) pitp-1 mutants exhibited reduced p-AKT levels compared to N2. (C) 956 Knockdown of pitp-1 extended lifespan in daf-16(mu86). (D–F) Transcriptional 957 reporter and qPCR analyses showed that pitp-1 expression is negatively regulated by 958 IIS: daf-2 knockdown reduced expression, while daf-16 knockdown increased it. 959 Known DAF-16 targets (sod-3, dod-24) were used as positive controls. (G) Knockdown 960 of pitp-1 did not further extend lifespan in daf-2(e1370). (H) PITP-1 overexpression 961 partially blocks the longevity effect by daf-2(RNAi) knockdown. Survival curves are 962 representative of three independent experiments. Statistical significance was 963 determined by log-rank test for lifespan assays, ANOV A for multiple comparisons. 964 965 966 967 968 969 970 971 972 973 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 55 Figure 6 974 975 Figure 6. Transcriptomic and pathway analysis upon pitp-1 reduction. RNA-seq 976 and pathway enrichment analyses revealed downregulation of insulin/TOR signaling 977 and upregulation of proteolysis -related genes in pitp-1 mutants and RNAi -treated 978 worms. (A) Schematic diagram of RNA-seq samples collection and data analysis. (B) 979 The results of QIAGEN IPA canonical pathway analysis upon pitp-1 reduction. (C) The 980 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 56

of QIAGEN IPA upstream regulator analysis upon pitp-1 reduction. (D) The 981

of QIAGEN IPA disease and biofunctions analysis upon pitp-1 reduction. (E) 982 ORA analysis identified potential up -regulated downstream target genes which might 983 involve in pitp-1 reduction-mediated longevity. (F) GO terms enrichment analysis of 984 the up-regulated genes upon pitp-1 reduction (FC> 1.5; adjusted p<0.05*). The vertical 985 coordinates were the enriched GO terms, and the horizontal coordinates were the 986 numbers of the up-regulated genes in these GO terms. The blue columns represent the 987 biological process GO terms. The green columns represent the molecular function GO 988 terms. GO terms enrichment analysis was conducted by DA VID. (G) KEGG enrichment 989 analysis of the changed genes upon pitp-1 reduction (FC> 1.5; adjusted p<0.05*). 990 KEGG enrichment analysis was conducted by DA VID. (H) The working model for pitp-991 1 reduction-mediated lifespan regulation in C. elegans. 992 993 994 995 996 997 998 999 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 57 Supplementary Figure 1 1000 1001 Supplementary Figure 1. The reduction of class II PITP, pitp-1, reveals longevity-1002 related phenotypes. (A) Knockdown of pitp-1, but not other class I PITP homologs, 1003 extended lifespan in N2. (B-D) qPCR confirmed RNAi against class I PITP homologs 1004 specifically reduced their own transcript levels without affecting pitp-1. (E) pitp-1 1005 mutants exhibited reduced body size. (F) Schematic diagram of RNAi treatment 1006 timelines. (G) pitp-1 knockdown during the reproductive stage promotes longevity. 1007 Survival curves are representative of three independent experiments . Statistical 1008 significance was determined by log-rank test for lifespan assays, ANOV A for multiple 1009 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 58 comparisons. 1010 1011 1012 Supplementary Figure 2 1013 1014 Supplementary Figure 2. GEO shows reduced class II PITP expression in old age. 1015 (A) Whole-genome microarray data from C. elegans [24] revealed significant 1016 reductions in both pitp-1 splice variants at day -6 and day -15 adults compared to L4 1017 larvae (One-way ANOV A). (B-G) Microarray analysis of human frontal cortex [25] 1018 showed that expression of PITPNM2 (232950_at) and PITPNM3 (230076_at) was 1019 significantly lower in individuals >90 years (extremely old) compared to those <40 1020 years (young) in both sexes, while PITPNM1 showed a slight, non-significant decrease 1021 (unpaired Student’s t-test). 1022 1023 1024 1025 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 59 Supplementary Figure 3 1026 1027 Supplementary Figure 3. pitp-1 negatively regulates lifespan by modulating TOR 1028 signaling. (A–B) RNAi knockdown of pitp-1 did not further prolong the extended 1029 lifespan in two dgk-5 mutants. (C, D) The elevated p -S6K levels in PITP -1 1030 overexpression strains were reverted by genetic inhibition of TOR signaling (let-363, 1031 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 60 raga-1). (E, F) The reduced p-S6K levels in two pitp-1 mutants were blocked by PITP-1032 1 overexpression. (G, H) Genetic or pharmacological inhibition of TOR did not further 1033 enhance the extended lifespan in pitp-1(tm1500) mutant. (I) Knockdown of pitp-1 did 1034 not further prolong the enhanced lifespan in rsks-1 mutants. (J) Genetic knockdown of 1035 TOR by let-363(RNAi) rescued the motility defect caused by PIT P-1 overexpression. 1036 (K) Schematic diagram of TOR upstream regulators RAG, RHEB, AMPK. (L) 1037 Knockdown of pitp-1 extended lifespan in aak-2(gt33). (M, N) sesn-1 mutation blocked 1038 the longevity effect of pitp-1(RNAi) knockdown. Survival curves are representative of 1039 three independent experiments. Statistical significance was determined by log-rank test 1040 for lifespan assays, ANOV A for multiple comparisons. 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 61 Supplementary Figure 4 1051 1052 Supplementary Figure 4. The role of pitp-1 in IIS-mediated lifespan regulation. 1053 (A) Knockdown of pitp-1 did not promote DAF -16 nuclear translocation. TJ356[ daf-1054 16p::daf-16a/b::GFP + rol -6(su1006)] was used as a DAF -16 reporter strain. Red 1055 arrows indicated DAF-16::GFP translocated into the nucleus and forms GFP puncta by 1056 daf-2(RNAi) as the positive control. (B, C) Knockdown of pitp-1 did not increase sod-1057 3 expression. CF1553[ sod-3p::GFP + rol -6(su1006)] was used as a sod-3 reporter 1058 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 62 strain. (D) Whole-genome microarray data from C. elegan s [30] revealed pitp-1 1059 expression was significantly reduced in daf-2(e1370). (E) PITP -1 overexpression 1060 partially blocked the longevity effect by age-1(RNAi) knockdown. Survival curves are 1061 representative of three independent experiments. Statistical significance was 1062 determined by log-rank test for lifespan assays, ANOV A for multiple comparisons, and 1063 unpaired Student’s t-test where applicable. 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 63 Supplementary Tables 1078 1079 1080 Trials strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure N2 OP50 16.3 ± 0.5 - - 99 pitp-1(pe1297) OP50 20.3 ± 0.5 24.5 p<0.001*** 82 pitp-1(tm1500) OP50 19.8 ± 0.5 21.5 p<0.001*** 100 N2 OP50 16.0 ± 0.4 - - 103 pitp-1(pe1297) OP50 20.3 ± 0.6 26.9 p<0.001*** 84 pitp-1(tm1500) OP50 18.5 ± 0.5 15.6 p<0.001*** 126 N2 OP50 17.0 ± 0.5 - - 107 pitp-1(pe1297) OP50 21.3 ± 0.6 25.3 p<0.001*** 85 pitp-1(tm1500) OP50 20.3 ± 0.5 19.4 p<0.001*** 117 Trials strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure N2 EV 16.7 ± 0.5 - - 105 N2 pitp-1(RNAi) 19.5 ± 0.5 16.8 p<0.001*** 98 N2 EV 16.4 ± 0.5 - - 106 N2 pitp-1(RNAi) 18.5 ± 0.5 12.8 p<0.001*** 95 N2 EV 17.0 ± 0.5 - - 95 N2 pitp-1(RNAi) 19.7 ± 0.5 15.9 p<0.001*** 102 Trials strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure N2 EV 16.7 ± 0.5 - - 112 N2 pitp-1(RNAi) WL 18.5 ± 0.5 10.8 p=0.013* 120 N2 pitp-1(RNAi) AO 18.9 ± 0.5 13.2 p=0.003** 105 N2 pitp-1(RNAi) from D3A 19.8 ± 0.5 18.6 p<0.001*** 88 N2 pitp-1(RNAi) from D4A 18.6 ± 0.5 11.4 p=0.022* 126 N2 pitp-1(RNAi) from D5A 16.9 ± 0.5 1.2 P=0.927 104 N2 pitp-1(RNAi) from D7A 17.0 ± 0.6 1.8 P=0.491 98 N2 EV 16.6 ± 0.5 - - 110 N2 pitp-1(RNAi) WL 18.2 ± 0.5 9.6 p=0.017* 124 N2 pitp-1(RNAi) AO 18.2 ± 0.5 9.6 p=0.018* 121 N2 pitp-1(RNAi) from D3A 18.3 ± 0.5 10.2 p=0.021* 105 N2 pitp-1(RNAi) from D4A 18.2 ± 0.5 9.6 p=0.042* 107 N2 pitp-1(RNAi) from D5A 16.5 ± 0.6 -0.6 p=0.591 100 N2 pitp-1(RNAi) from D7A 16.6 ± 0.6 0.0 p=0.795 89 N2 EV 17.6 ± 0.5 - - 109 N2 pitp-1(RNAi) WL 19.9 ± 0.5 13.1 p=0.009** 95 N2 pitp-1(RNAi) AO 20.6 ± 0.5 17.0 p<0.001*** 128 N2 pitp-1(RNAi) from D3A 20.2 ± 0.5 14.8 p=0.002** 93 N2 pitp-1(RNAi) from D4A 19.1 ± 0.6 8.5 p=0.047* 95 N2 pitp-1(RNAi) from D5A 18.1 ± 0.5 2.8 p=0.727 92 N2 pitp-1(RNAi) from D7A 18.2 ± 0.5 3.4 p=0.866 83 Trials strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figures EV 18.6 ± 0.4 - - 127 pitp-1 (RNAi) 20.8 ± 0.4 11.8 p<0.001*** 146 Y54F10AR.1 (RNAi) 18.8 ± 0.4 1.1 p= 0.743 130 Y71G12B.17 (RNAi) 19.5 ± 0.4 4.8 p= 0.104 122 Y54F10AR.1+ Y71G12B.17 (RNAi) 19.0 ± 0.4 2.2 p= 0.590 143 EV 17.6 ± 0.3 - - 170 Y54F10AR.1 (RNAi) 16.9 ± 0.3 -4.0 p=0.17 184 Y71G12B.17 (RNAi) 17.5 ± 0.3 -0.6 p=0.777 218 Y54F10AR.1+ Y71G12B.17 (RNAi) 17.5 ± 0.3 -0.6 p=0.385 195 EV 18.0 ± 0.5 - - 128 Y54F10AR.1 (RNAi) 17.6 ± 0.5 -2.2 p= 0.400 117 Y71G12B.17 (RNAi) 17.8 ± 0.6 -1.1 p= 0.704 94 Y54F10AR.1+ Y71G12B.17 (RNAi) 17.3 ± 0.5 -3.9 p= 0.167 94 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure N2 Control OP50 16.8 ± 0.6 - - 85 N2 PITP-1 OE 1 OP50 15.6 ± 0.5 -7.1 p=0.048* 90 N2 PITP-1 OE 2 OP50 14.6 ± 0.6 -13.1 p=0.015* 98 N2 Control OP50 16.2 ± 0.6 - - 78 N2 PITP-1 OE 1 OP50 14.0 ± 0.6 -13.6 p=0.006** 78 N2 PITP-1 OE 2 OP50 14.6 ± 0.5 -9.9 p=0.031* 85 N2 Control OP50 17.1 ± 0.4 - - 106 N2 PITP-1 OE 1 OP50 15.7 ± 0.5 -8.2 p=0.022* 105 N2 PITP-1 OE 2 OP50 15.5 ± 0.4 -9.4 p<0.001*** 100 1st 2nd 3rd V V 2nd 3rd Table S1. Lifespan analysis upon pitp-1 inhibition and overexpression. 1st 1st N2 V 2nd N2 1st 2nd 3rd V 1st 2nd V 3rd 3rd N2 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 64 1081 Change (%) p-value N2 OP50 7.0 ± 0.4 - - 87 pitp-1(pe1297) OP50 9.3 ± 0.4 32.9 p<0.001*** 135 pitp-1(tm1500) OP50 8.8 ± 0.4 25.7 p=0.001** 131 N2 OP50 17.6 ± 1.0 - - 90 pitp-1(pe1297) OP50 23.7 ± 0.9 34.7 p<0.001*** 135 pitp-1(tm1500) OP50 20.2 ± 1.0 14.8 p=0.042* 113 N2 OP50 7.6 ± 0.3 - - 140 pitp-1(pe1297) OP50 12.1 ± 0.6 59.2 p<0.001*** 128 pitp-1(tm1500) OP50 10.4 ± 0.4 36.8 p<0.001*** 125 N2 OP50 19.2 ± 0.9 - - 130 pitp-1(pe1297) OP50 29.9 ± 0.9 55.7 p<0.001*** 128 pitp-1(tm1500) OP50 23.5 ± 1.0 22.4 p<0.001*** 121 Change (%) p-value EV 7.4 ± 0.3 - - 108 pitp-1(RNAi) 9.8 ± 0.4 32.4 p<0.001*** 109 EV 17.5 ± 0.9 - - 111 pitp-1(RNAi) 23.0 ± 1.0 31.4 p<0.001*** 109 EV 8.8 ± 0.4 - - 106 pitp-1(RNAi) 11.5 ± 0.5 30.7 p<0.001*** 105 EV 18.1 ± 0.9 - - 106 pitp-1(RNAi) 22.4 ± 1.0 23.8 p<0.001*** 105 n Figure 1st 240uM Juglone180uM Juglone Trialconc. strain Treatment Mean lifespan ± SEM (Hours) Compared with N2 240uM Juglone V 180uM Juglone Trialconc. strain Treatment Mean lifespan ± SEM (Hours) Compared with EV Table S2. Oxidative stress survival upon pitp-1 inhibition. 2nd 240uM Juglone N2 180uM Juglone N2 n Figure 1st 240uM Juglone N2 V 180uM Juglone N2 2nd preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 65 1082 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 14.5 ± 0.4 - - 109 pitp-1 (RNAi) 17.4 ± 0.4 20 p<0.001*** 102 EV 13.9 ± 0.3 - - 101 pitp-1 (RNAi) 16.2 ± 0.4 16.5 p<0.001*** 100 EV 14.0 ± 0.3 - - 105 pitp-1 (RNAi) 16.5 ± 0.4 17.9 p<0.001*** 106 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 17.7 ± 0.5 - - 107 pitp-1 (RNAi) 20.8 ± 0.6 17.5 p<0.001*** 102 EV 16.9 ± 0.4 - - 104 pitp-1 (RNAi) 19.5 ± 0.5 15.4 p<0.001*** 103 EV 19.1 ± 0.5 - - 109 pitp-1 (RNAi) 21.2 ± 0.6 11 p<0.001*** 106 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 14.5 ± 0.3 - - 123 pitp-1 (RNAi) 14.5 ± 0.3 0 p=0.819 123 EV 14.0 ± 0.3 - - 95 pitp-1 (RNAi) 14.1 ± 0.3 0.7 p=0.997 107 EV 14.0 ± 0.3 - - 111 pitp-1 (RNAi) 14.1 ± 0.4 0.7 p=0.593 109 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 15.5 ± 0.5 - - 109 pitp-1 (RNAi) 16.0 ± 0.5 3.2 p=0.345 120 EV 15.4 ± 0.4 - - 111 pitp-1 (RNAi) 15.7 ± 0.5 2.6 p=0.195 106 EV 15.9 ± 0.6 - - 92 pitp-1 (RNAi) 16.3 ± 0.5 2.5 p=0.827 93 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 13.9 ± 0.2 - - 127 pitp-1 (RNAi) 15.1 ± 0.2 8.6 p= 0.004** 146 EV 14.8 ± 0.2 - - 119 pitp-1 (RNAi) 16.2 ± 0.3 9.5 p= 0.004** 118 EV 14.0 ± 0.3 - - 135 pitp-1 (RNAi) 15.2 ± 0.1 8.6 p= 0.029* 118 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 12.5 ± 0.3 - - 112 pitp-1 (RNAi) 13.8 ± 0.6 10.4 p=0.002** 110 EV 13.2 ± 0.1 - - 106 pitp-1 (RNAi) 14.4 ± 0.4 9.1 p=0.030* 102 EV 13.3 ± 0.4 - - 110 pitp-1 (RNAi) 14.7 ± 0.3 10.5 p=0.003** 104 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 11.8 ± 0.6 - - 106 pitp-1 (RNAi) 12.7 ± 0.3 7.6 p= 0.004** 114 EV 12.4 ± 0.3 - - 119 pitp-1 (RNAi) 13.2 ± 0.4 6.5 p= 0.011* 127 EV 14.0 ± 0.4 - - 186 pitp-1 (RNAi) 15.0 ± 0.3 7.1 p= 0.012* 155 Trials Strain Treatment Mean lifespan±SEM (Day) Change (%) p-value n Figure EV 12.9 ± 0.3 - - 108 pitp-1 (RNAi) 13.6 ± 0.3 5.4 p=0.239 111 EV 13.5 ± 0.3 - - 106 pitp-1 (RNAi) 14.0 ± 0.3 3.7 p=0.097 105 1st TU3401 V 2nd TU3401 3rd TU3401 1st TU3311 V 2nd TU3311 3rd TU3311 1st VP303 V 2nd VP303 3rd VP303 1st WM118 V 2nd WM118 3rd WM118 1st XE1375 2nd XE1375 V 3rd XE1375 1st XE1582 V 2nd XE1582 3rd XE1582 2nd XE1474 V Table S3. Tissue-specific lifespan analysis upon pitp-1 inhibition. 3rd XE1581 1st XE1474 1st XE1581 V 2nd XE1581 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 66 1083 Change (%) p-value Change (%) p-value N2 Control 17.5 ± 0.5 - - - - 98 N2 PITP-1 OE 1 16.0 ± 0.5 -8.6 p=0.012* - - 98 N2 Control 19.4 ± 0.6 10.9 p=0.001** - - 101 N2 PITP-1 OE 1 18.9 ± 0.5. 8 p=0.015* -2.6 p=0.224 102 N2 Control 16.9 ± 0.4 - - - - 101 N2 PITP-1 OE 1 15.4 ± 0.5 -8.9 p=0.024* - - 102 N2 Control 19.0 ± 0.6 12.4 p<0.001*** - - 100 N2 PITP-1 OE 1 18.6 ± 0.5 10.1 p=0.003** -2.1 p=0.204 99 N2 Control 16.8 ± 0.4 - - - - 101 N2 PITP-1 OE 1 15.3 ± 0.4 -8.9 p=0.017* - - 99 N2 Control 18.6 ± 0.4 10.7 p=0.003** - - 100 N2 PITP-1 OE 1 17.8 ± 0.4 6 p=0.065 -4.3 p=0.177 102 Change (%) p-value Change (%) p-value N2 control OP50 15.4 ± 0.5 - - - - 99 N2 PITP-1 OE OP50 13.4 ± 0.4 -13.0 p= 0.002** -13.0 p= 0.002** 101 pitp-1(pe1297) control OP50 17.8 ± 0.4 15.6 p=0.001** - - 98 pitp-1(pe1297) PITP-1 OE OP50 14.3 ± 0.5 -7.1 p=0.217 -19.7 p<0.001*** 101 pitp-1(tm1500) control OP50 17.1 ± 0.5 11.0 p=0.006** - - 100 pitp-1(tm1500) PITP-1 OE OP50 14.2 ± 0.5 -8.0 p=0.049* -17.0 p<0.001*** 98 N2 control OP50 15.8 ± 0.5 - - - - 100 N2 PITP-1 OE OP50 14.1 ± 0.4 -10.8 p= 0.002** -10.8 p= 0.002** 100 pitp-1(pe1297) control OP50 18.8 ± 0.4 19.0 p<0.001*** - - 100 pitp-1(pe1297) PITP-1 OE OP50 14.6 ± 0.5 -7.6 p=0.062 -22.3 p<0.001*** 99 pitp-1(tm1500) control OP50 18.3 ± 0.5 15.8 p<0.001*** - - 100 pitp-1(tm1500) PITP-1 OE OP50 14.6 ± 0.5 -7.6 p=0.034* -20.2 p<0.001*** 101 N2 control OP50 15.5 ± 0.5 - - - - 99 N2 PITP-1 OE OP50 14.2 ± 0.5 -8.4 p= 0.031* -8.4 p= 0.031* 102 pitp-1(pe1297) control OP50 17.9 ± 0.5 15.5 p<0.001*** - - 100 pitp-1(pe1297) PITP-1 OE OP50 14.9 ± 0.4 -3.9 p=0.131 -16.8 p<0.001*** 101 pitp-1(tm1500) control OP50 17.5 ± 0.4 12.9 p=0.006** - - 101 pitp-1(tm1500) PITP-1 OE OP50 14.6 ± 0.4 -5.8 p=0.024* -16.6 p<0.001*** 100 Change (%) p-value Change (%) p-value N2 control EV 15.3 ± 0.5 - - - - 99 N2 control rheb-1(RNAi) 18.7 ± 0.5 22.2 p<0.001*** - - 99 N2 control raga-1(RNAi) 19.6 ± 0.5 28.1 p<0.001*** - - 98 N2 PITP-1OE 1 EV 13.5 ± 0.4 -11.8 p=0.005** -11.8 p=0.005** 100 N2 PITP-1OE 1 rheb-1(RNAi) 18.9 ± 0.5 23.5 p<0.001*** 1.1 p=0.625 98 N2 PITP-1OE 1 raga-1(RNAi) 19.0 ± 0.5 24.2 p<0.001*** -3.1 p=0.320 97 N2 control EV 15.8 ± 0.5 - - - - 98 N2 control rheb-1(RNAi) 18.3 ± 0.5 15.8 p<0.001*** - - 98 N2 control raga-1(RNAi) 18.2 ± 0.4 15.2 p<0.001*** - - 100 N2 PITP-1OE 1 EV 13.6 ± 0.4 -13.9 p<0.001*** -13.9 p<0.001*** 98 N2 PITP-1OE 1 rheb-1(RNAi) 18.2 ± 0.4 15.2 p<0.001*** -0.5 p=0.793 98 N2 PITP-1OE 1 raga-1(RNAi) 18.2 ± 0.4 15.2 p<0.001*** 0.0 p=0.767 100 N2 control EV 15.9 ± 0.5 - - - - 98 N2 control rheb-1(RNAi) 17.9 ± 0.4 12.6 p=0.005** - - 98 N2 control raga-1(RNAi) 18.2 ± 0.4 13.2 p=0.002** - - 97 N2 PITP-1OE 1 EV 14.0 ± 0.4 -11.9 p=0.001** -11.9 p=0.001** 98 N2 PITP-1OE 1 rheb-1(RNAi) 18.0 ± 0.5 11.9 p=0.006** -0.6 p=0.954 97 N2 PITP-1OE 1 raga-1(RNAi) 17.9 ± 0.5 12.6 p=0.005** -0.6 p=0.698 95 Change (%) p-value Change (%) p-value EV 17.2 ± 0.4 - - - - 101 pitp-1(RNAi) 19.0 ± 0.5 10.5 p=0.002** 10.5 p=0.002** 99 EV 18.8 ± 0.5 9.3 p=0.007** - - 93 pitp-1(RNAi) 18.6 ± 0.5 8.1 p=0.008** -1.1 p=0.690 103 EV 19.5 ± 0.5 13.4 p<0.001*** - - 92 pitp-1(RNAi) 18.7 ± 0.5 8.7 p=0.015* -0.5 p=0.320 96 EV 17.7 ± 0.5 - - - - 88 pitp-1(RNAi) 20.3 ± 0.6 14.7 p<0.001*** 14.7 p<0.001*** 85 EV 19.6 ± 0.6 10.7 p=0.007** - - 81 pitp-1(RNAi) 19.0 ± 0.6 7.3 p=0.019* -3.1 p=0.557 81 EV 20.7 ± 0.5 16.9 p<0.001*** - - 92 pitp-1(RNAi) 19.3 ± 0.6 9.0 p=0.004** -1.5 p=0.171 96 EV 17.8 ± 0.4 - - - - 100 pitp-1(RNAi) 20.9 ± 0.5 17.4 p<0.001*** 17.4 p<0.001*** 104 EV 20.1 ± 0.6 12.9 p<0.001*** - - 86 pitp-1(RNAi) 19.4 ± 0.5 9.0 p=0.004** -3.5 p=0.171 87 EV 20.1 ± 0.5 12.9 p<0.001*** - - 101 pitp-1(RNAi) 19.5 ± 0.5 9.6 p=0.003** -3.0 p=0.481 105 Change (%) p-value Change (%) p-value EV 18.5 ± 0.5 - - - - 107 let-363(RNAi) 21.4 ± 0.6 15.7 p<0.001*** 15.7 p<0.001*** 103 EV 21.6 ± 0.6 16.8 p<0.001*** - - 104 let-363(RNAi) 20.8 ± 0.6 12.4 p<0.001*** -3.7 p=0.246 103 EV 20.8 ± 0.6 12.4 p<0.001*** - - 100 let-363(RNAi) 20.4 ± 0.6 10.3 p=0.001** -1.9 p=0.798 100 EV 17.4 ± 0.5 - - - - 107 let-363(RNAi) 20.6 ± 0.5 18.4 p<0.001*** 18.4 p<0.001*** 103 EV 20.9 ± 0.5 20.1 p<0.001*** - - 106 let-363(RNAi) 20.3 ± 0.6 16.7 p<0.001*** -2.9 p=0.712 104 EV 20.4 ± 0.5 17.2 p<0.001*** - - 104 let-363(RNAi) 20.0 ± 0.6 14.9 p<0.001*** -2.0 p=0.925 100 EV 17.3 ± 0.5 - - - - 98 let-363(RNAi) 20.1 ± 0.5 16.2 p<0.001*** 16.2 p<0.001*** 97 EV 20.4 ± 0.4 17.9 p<0.001*** - - 97 let-363(RNAi) 19.7 ± 0.5 13.9 p<0.001*** -3.4 p=0.880 95 EV 19.9 ± 0.5 15.0 p<0.001*** - - 93 let-363(RNAi) 19.5 ± 0.6 12.7 p<0.001*** -2.0 p=0.769 96 Change (%) p-value Change (%) p-value DMSO 16.7 ± 0.5 - - - - 114 100 uM rapamycin 18.8 ± 0.6 12.6 p= 0.007** 12.6 p= 0.007** 95 DMSO 19.4 ± 0.6 16.2 p<0.001*** - - 106 100 uM rapamycin 18.2 ± 0.7 9.0 p=0.013* -6.2 p=0.107 91 DMSO 19.4 ± 0.6 16.2 p<0.001*** - - 98 100 uM rapamycin 18.2 ± 0.6 9.0 p=0.036* -6.2 p=0.078 100 DMSO 17.2 ± 0.5 - - - - 99 100 uM rapamycin 19.6 ± 0.6 14.0 p<0.001*** 14.0 p<0.001*** 101 DMSO 20.6 ± 0.6 19.8 p<0.001*** - - 90 100 uM rapamycin 19.2 ± 0.6 11.6 p=0.001** -6.8 p=0.040* 98 DMSO 20.0 ± 0.5 16.3 p<0.001*** - - 112 100 uM rapamycin 19.2 ± 0.5 11.6 p=0.002** -4.0 p=0.258 113 DMSO 17.1 ± 0.5 - - - - 93 100 uM rapamycin 19.4 ± 0.5 13.5 p<0.001*** 13.5 p<0.001*** 103 DMSO 20.5 ± 0.6 19.9 p<0.001*** - - 108 100 uM rapamycin 19.2 ± 0.6 12.3 p<0.001*** -6.3 p=0.078 91 DMSO 20.3 ± 0.6 18.7 p<0.001*** - - 91 100 uM rapamycin 19.2 ± 0.5 12.3 p<0.001*** -5.4 p=0.091 117 Change (%) p-value Change (%) p-value EV 17.1 ± 0.5 - - - - 111 pitp-1(RNAi) 19.3 ± 0.5 12.9 p<0.001*** 12.9 p<0.001*** 120 EV 22.5 ± 0.6 31.6 p<0.001*** - - 114 pitp-1(RNAi) 20.6 ± 0.7 20.5 p<0.001*** -8.4 p=0.119 96 EV 17.1 ± 0.4 - - - - 110 pitp-1(RNAi) 20.5 ± 0.5 19.9 p<0.001*** 19.9 p<0.001*** 108 EV 22.1 ± 0.5 29.2 p<0.001*** - - 109 pitp-1(RNAi) 21.4 ± 0.6 25.1 p<0.001*** -3.2 p=0.874 109 EV 17.4 ± 0.5 - - - - 89 pitp-1(RNAi) 20.8 ± 0.6 19.5 p<0.001*** 19.5 p<0.001*** 94 EV 21.8 ± 0.6 25.3 p<0.001*** - - 97 pitp-1(RNAi) 20.5 ± 0.7 17.8 p<0.001*** -6.0 p=0.6808 94 Change (%) p-value Change (%) p-value EV 17.4 ± 0.4 - - - - 130 let-363(RNAi) 20.3 ± 0.4 16.7 p<0.001*** 16.7 p<0.001*** 128 EV 15.3 ± 0.4 -12.1 p<0.001*** - - 125 let-363(RNAi) 20.2 ± 0.5 16.1 p<0.001*** 32.0 p<0.001*** 128 EV 17.3 ± 0.4 - - - - 146 let-363(RNAi) 20.6 ± 0.5 19.1 p<0.001*** 19.1 p<0.001*** 146 EV 15.8 ± 0.4 -8.7 p<0.001*** - - 147 let-363(RNAi) 20.1 ± 0.5 16.2 p=0.003** 27.2 p<0.001*** 125 EV 17.5 ± 0.4 - - - - 132 let-363(RNAi) 20.1 ± 0.4 14.9 p<0.001*** 14.9 p<0.001*** 130 EV 15.5 ± 0.4 -11.4 p<0.001*** - - 128 let-363(RNAi) 19.9 ± 0.4 13.7 p=0.003** 28.4 p<0.001*** 130 Change (%) p-value Change (%) p-value Vehicle 15.9 ± 0.4 - - - - 105 rapamycin 18.4 ± 0.6 15.7 p<0.001*** 15.7 p<0.001*** 107 Vehicle 13.9 ± 0.4 -12.6 p<0.001*** - - 103 rapamycin 18.5 ± 0.6 16.4 p<0.001*** 33.1 p<0.001*** 106 Vehicle 15.9 ± 0.4 - - - - 108 rapamycin 18.1 ± 0.5 13.8 p<0.001*** 13.8 p<0.001*** 106 Vehicle 13.6 ± 0.4 -14.5 p<0.001*** - - 107 rapamycin 18.1 ± 0.5 13.8 p<0.001*** 33.1 p<0.001*** 108 Vehicle 15.9 ± 0.4 - - - - 100 rapamycin 18.0 ± 0.5 13.2 p<0.001*** 13.2 p<0.001*** 101 Vehicle 14.0 ± 0.4 -11.9 p<0.001*** - - 108 rapamycin 18.0 ± 0.5 13.2 p<0.001*** 28.6 p<0.001*** 107 Change (%) p-value Change (%) p-value EV 16.8 ± 0.6 - - - - 97 pitp-1(RNAi) 19.1 ± 0.6 13.7 p=0.002** 13.7 p=0.002** 95 EV 14.9 ± 0.4 -11.3 p<0.001*** - - 97 pitp-1(RNAi) 16.2 ± 0.5 -3.6 p=0.153 8.7 p=0.015* 97 EV 16.3 ± 0.5 - - - - 96 pitp-1(RNAi) 18.7 ± 0.6 14.7 p=0.002** 14.7 p=0.002** 98 EV 14.3 ± 0.4 -12.3 p<0.001*** - - 96 pitp-1(RNAi) 15.7 ± 0.3 -3.7 p=0.013* 9.8 p=0.022* 96 Change (%) p-value Change (%) p-value EV 18.6 ± 0.4 - - - - 152 pitp-1(RNAi) 20.0 ± 0.4 9.1 p= 0.003** 9.1 p= 0.003** 144 EV 17.3 ± 0.4 -7.0 p= 0.003** - - 134 pitp-1(RNAi) 17.7 ± 0.4 -4.8 p= 0.059 2.3 p= 0.258 134 EV 17.6 ± 0.4 -5.4 p= 0.022* - - 136 pitp-1(RNAi) 17.8 ± 0.4 -4.3 p= 0.089 1.1 p= 0.540 137 EV 17.8 ± 0.4 - - - - 112 pitp-1(RNAi) 20.8 ± 0.4 16.9 p<0.001*** 16.9 p<0.001*** 111 EV 16.5 ± 0.4 -7.3 p=0.005** - - 119 pitp-1(RNAi) 16.9 ± 0.4 -5.1 p=0.036* 2.4 p=0.396 121 EV 16.6 ± 0.4 -6.7 p=0.004** - - 122 pitp-1(RNAi) 16.8 ± 0.4 -5.6 p=0.132 1.2 p=0.160 122 Change (%) p-value Change (%) p-value EV 16.7 ± 0.4 - - - - 130 pitp-1(RNAi) 18.6 ± 0.5 11.4 p=0.003** 11.4 p=0.003** 130 EV 13.6 ± 0.4 -18.6 p<0.001*** - - 140 pitp-1(RNAi) 16.2 ± 0.4 -3.0 p=0.298 19.1 p<0.001*** 139 EV 16.9 ± 0.5 - - - - 128 pitp-1(RNAi) 19.0 ± 0.5 12.4 p=0.001** 12.4 p=0.001** 127 EV 14.6 ± 0.4 -13.6 p<0.001*** - - 128 pitp-1(RNAi) 16.7 ± 0.5 -1.2 p=0.637 14.4 p=0.002** 127 EV 17.2 ± 0.5 - - - - 126 pitp-1(RNAi) 19.4 ± 0.5 12.8 p=0.001** 12.8 p=0.001** 126 EV 15.3 ± 0.4 -11.0 p<0.001*** - - 126 pitp-1(RNAi) 16.9 ± 0.4 -1.7 p=0.055 10.5 p=0.013* 124 Change (%) p-value Change (%) p-value EV 16.6 ± 0.5 - - - - 96 pitp-1(RNAi) 18.6 ± 0.5 12.0 p=0.007** 12.0 p=0.007** 95 EV 39.4 ± 1.4 137.3 p<0.001*** - - 106 pitp-1(RNAi) 38.8 ± 1.4 133.7 p<0.001*** -1.5 p=0.864 107 EV 16.6 ± 0.5 - - - - 98 pitp-1(RNAi) 18.4 ± 0.5 10.8 p=0.021* 10.8 p=0.021* 99 EV 36.8 ± 0.9 121.7 p<0.001*** - - 109 pitp-1(RNAi) 36.6 ± 1.1 120.5 p<0.001*** -0.5 p=0.515 109 EV 16.4 ± 0.5 - - - - 96 pitp-1(RNAi) 18.6 ± 0.6 13.4 p=0.004** 13.4 p=0.004** 95 EV 36.9 ± 1.2 125.0 p<0.001*** - - 110 pitp-1(RNAi) 36.6 ± 1.3 123.2 p<0.001*** -0.8 p=0.845 110 2nd EV V pitp-1(RNAi) 3rd EV pitp-1(RNAi) n Figure 1st EV pitp-1(RNAi) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control EV compared to control pitp-1(RNAi) n Figure 1st 2nd V Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 control compared to control 1st 2nd V 3rd 3rd Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 control compared to control RNAi n Figure n Figure 1st N2 dgk-5(ok2366) dgk-5(gk691) Trials Strain Treatment Mean lifespan±SEM(Day) compared to N2 EV compared to EV 2nd N2 dgk-5(ok2366) dgk-5(gk691) 3rd N2 Vdgk-5(ok2366) dgk-5(gk691) n Figure 1st N2 pitp-1(pe1297) pitp-1(tm1500) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 EV compared to EV 2nd N2 Vpitp-1(pe1297) pitp-1(tm1500) 3rd N2 pitp-1(pe1297) pitp-1(tm1500) n Figure 1st N2 pitp-1(pe1297) pitp-1(tm1500) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 control compared to control 2nd N2 pitp-1(pe1297) pitp-1(tm1500) 3rd N2 Vpitp-1(pe1297) pitp-1(tm1500) 2nd N2 V rsks-1(ok1255) 3rd N2 rsks-1(ok1255) n Figure 1st N2 rsks-1(ok1255) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 EV compared to EV 2nd N2 control N2 PITP-1OE1 3rd N2 control N2 PITP-1OE1 n Figure 1st N2 control V N2 PITP-1OE1 Trials Strain Treatment Mean lifespan±SEM(Day) compared to control EV compared to EV 2nd N2 control N2 PITP-1OE1 3rd N2 control V N2 PITP-1OE1 n Figure 1st N2 control N2 PITP-1OE1 Trials Strain Treatment Mean lifespan±SEM(Day) compared to control Vehicle compared to Vehicle n Figure 1st N2 V aak-2(gt33) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 EV compared to EV n Figure 1st N2 sesn-1(ok3157) sesn-1(tm2872) 2nd N2 aak-2(gt33) Trials Strain Treatment Mean lifespan±SEM(Day) compared to control N2 EV compared to EV 2nd N2 Vsesn-1(ok3157) sesn-1(tm2872) Trials Strain Treatment Mean lifespan±SEM(Day) compared to N2 EV daf-16(mu86) 3rd N2 daf-16(mu86) compared to EV n Figure 1st N2 daf-16(mu86) Table S4. Genetic epistasis tests of pitp-1 regulation on lifespan 2nd N2 daf-2(e1370) 3rd N2 V daf-2(e1370) n Figure 1st N2 daf-2(e1370) Trials Strain Treatment Mean lifespan±SEM(Day) compared to N2 EV compared to EV 2nd N2 V preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 67 1084 1085 1086 Acknowledgments 1087 We thank the C. elegans Core Facility of the National Core Facility for 1088 biopharmaceuticals, National Science and Technology Council (NSTC), in Taiwan for 1089 technical support, and the assistance from Dr. Ao-Lin Hsu. We thank the technical 1090 support from Ya -Hsien Chou at the confocal imaging core at National Tsing Hua 1091 University sponsored by NSTC. 1092 1093 Funding 1094 We thank the grant funding support from NSTC ( 108-2311-B-007-007 ;109 -1095 2311-B-007-002 ;110-2320-B-007-003-; 111-2320-B-007-006-MY3, 114 -2320-B-1096 007-002-) to H-D Wang and (111-2320-B-400-018-MY3; 114-2320-B-400-022-MY3) 1097 Change (%) p-value Change (%) p-value Change (%) p-value EV 16.4 ± 0.5 - - - - - - 109 daf-2(RNAi) 32.4 ± 1.0 97.6 p<0.001*** 97.6 p<0.001*** - - 112 EV 14.7 ± 0.5 -10.4 p=0.010* - - - - 107 daf-2(RNAi) 23.4 ± 1.2 42.7 p<0.001*** 59.2 p<0.001*** -27.8 p<0.001*** 110 EV 15.9 ± 0.5 - - - - - - 108 daf-2(RNAi) 27.5 ± 0.8 73.0 p<0.001*** 73.0 p<0.001*** - - 111 EV 14.2 ± 0.4 -10.7 p<0.001*** - - - - 105 daf-2(RNAi) 21.1 ± 0.8 32.7 p<0.001*** 48.6 p<0.001*** -23.3 p<0.001*** 105 EV 15.9 ± 0.4 - - - - - - 105 daf-2(RNAi) 28.1 ± 0.9 76.7 p<0.001*** 76.7 p<0.001*** - - 109 EV 13.4 ± 0.4 -15.7 p<0.001*** - - - - 104 daf-2(RNAi) 21.1 ± 0.9 32.7 p<0.001*** 57.5 p<0.001*** -24.9 p<0.001*** 109 Change (%) p-value Change (%) p-value Change (%) p-value EV 16.9 ± 0.5 - - - - - - 108 age-1(RNAi) 29.3 ± 1.1 73.4 p<0.001*** 73.4 p<0.001*** - - 103 EV 15.0 ± 0.4 -11.2 p=0.001** - - - - 110 age-1(RNAi) 23.5 ± 0.9 39.1 p<0.001*** 56.7 p<0.001*** -19.8 p<0.001*** 108 EV 17.4 ± 0.4 - - - - - - 107 age-1(RNAi) 26.7 ± 0.6 53.4 p<0.001*** 53.4 p<0.001*** - - 107 EV 15.3 ± 0.4 -12.1 p<0.001*** - - - - 108 age-1(RNAi) 21.3 ± 0.5 22.4 p<0.001*** 39.2 p<0.001*** -20.2 p<0.001*** 108 EV 17.5 ± 0.4 - - - - - - 111 age-1(RNAi) 27.9 ± 0.9 59.4 p<0.001*** 59.4 p<0.001*** - - 113 EV 15.6 ± 0.4 -10.9 p<0.001*** - - - - 108 age-1(RNAi) 22.3 ± 0.7 27.4 p<0.001*** 42.9 p<0.001*** -20.1 p<0.001*** 109 Figure 1st N2 control N2 PITP-1OE1 Trials Strain Treatment Mean lifespan±SEM(Day) compared to control EV compared to EV 3rd N2 control V N2 PITP-1OE1 compared to age-1(RNAi) n Figure 1st N2 control N2 PITP-1OE1 Trials Strain Treatment Mean lifespan±SEM(Day) compared to control EV Table S5. Lifespan epistasis analysis of pitp-1 overexpression with IIS inhibition 2nd N2 control N2 PITP-1OE1 compared to EV 2nd N2 control N2 PITP-1OE1 3rd N2 control V N2 PITP-1OE1 compared to daf-2(RNAi) n preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 68 to C -H Yuh . The postdoc fellowship support (114Q101CE1, 113Q101CE1, 1098 112Q101CE1) from National Tsing Hua University to Y-H Lin is acknowledged. 1099 1100 Author contribution 1101 H. D. Wang, C. H. Yuh, and Y . H. Lin contributed to the conception, design of the 1102 study. Y . H. Lin and H. D. Wang wrote the manuscript. H. D. Wang and C. H. Yuh 1103 contributed to funding acquisition. Y . H. Lin, Y . H. Liao, S. B. Liao, T. Y . Lin and P. J. 1104 Hsu contributed to the acquisition of data and helped the data analysis. Y . H. Lin, H. D. 1105 Wang, C. S. Chen, T. T. Ching and C. H. Yuh contributed to the development of 1106 methodology. C. H. Yuh, Y . H. Lin and H. D. Wang contributed to analyze 1107 transcriptomic profiles. C. S. Chen, T. T. Ching and O. I. Wagner offered RNAi clones 1108 or C. elegans strains. Y . H. Lin, T. T. Ching, M . M. Shanmugam and O . I. Wagner 1109 contributed to establish overexpression construct and microinject transgenic strains. Y . 1110 H. Lin, H. D. Wang, C. S. Chen and C. H. Yuh contributed to the interpretation of data. 1111 1112 Corresponding authors 1113 Correspondence to Horng-Dar Wang ([email protected]) and Chiou-Hwa Yuh 1114 ([email protected]) 1115 1116 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint 69 Ethics declarations 1117 Ethics approval and consent to participate 1118 Not applicable. The analysis of PITPNM1, PITPNM2, and PITPNM3 expressions was 1119 from the RNAseq raw data in the published GEO, no human tissue was used. 1120 1121 Consent for publication 1122 Not applicable. 1123 1124 Competing interests 1125 The authors have declared that no competing interests exist. 1126 1127 1128 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 2, 2025. ; https://doi.org/10.1101/2025.11.28.691094doi: bioRxiv preprint