A Centenarian Single Nucleotide Polymorphism in collagen gene COL25A1 promotes longevity in C. elegans

A Centenarian Single Nucleotide Polymorphism in collagen gene COL25A1 promotes longevity in C. elegans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Centenarian Single Nucleotide Polymorphism in collagen gene COL25A1 promotes longevity in C. elegans Anita Goyala, Cyril Statzer, Ji Young Cecilia Park, Ines Neundorf, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6035585/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in npj Aging → Version 1 posted 8 You are reading this latest preprint version Abstract Before human genome sequencing, a genome-wide study of sibling centenarian pairs identified a longevity-associated locus on chromosome 4. Here, we mapped the genes in this locus and identified a collagen gene, COL25A1. Introducing an SNP linked to longevity that changes a serine predicted to be phosphorylated to leucine in COL25A1 , into col-99 , the C. elegans ortholog, extended lifespan. These col-99(gk694263 [S106L] ) SNP-mutants exhibited enhanced innate immune-related transcriptional responses, and their lifespan extension was abolished by inhibiting the p38 MAPK pathway. YAP-1, a transcriptional co-activator responsive to extracellular matrix changes, was essential for this longevity. Mechanistically, we propose that this SNP modifies furin-mediated cleavage of this transmembrane collagen in vitro, and expressing the cleaved extracellular domain of COL-99 alone was sufficient to prolong lifespan. These findings reveal a potential mechanism by which a human centenarian-associated SNP in COL25A1 influences furin cleavage and shedding of the collagen ectodomain to promote healthy longevity. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Molecular biology Centenarians SNP col-99 COL25A1 p38 MAPK yap-1 extracellular matrix exceptional longevity C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Centenarians not only achieve exceptional longevity but also often exhibit delayed onset of age-related diseases, providing valuable insights into the biological mechanisms of healthy aging 1 . While lifestyle factors, such as diet and physical activity, contribute to extended lifespans—as observed in the Okinawan population 2 —the genetic determinants that interact with these factors remain less understood. Genome-wide association studies (GWAS) have begun to uncover genetic variations associated with longevity 3 . Notably, studies like the New England Centenarian Study and the Long Life Family Study have identified genetic loci that may contribute to extended lifespan and reduced disease incidence among centenarians 4 . One such study by Puca and colleagues 5 identified a significant locus on chromosome 4 (marker D4S1564) associated with exceptional longevity in a cohort of 308 individuals from 137 long-lived families. However, this study was prior to the human genome being sequenced, and thus, the specific genes and variants within this locus were not characterized. Here, we focus on the chromosome 4 locus identified by Puca et al. and investigate the collagen gene COL25A1 as a candidate for influencing longevity 5 . We utilized Caenorhabditis elegans as a model organism to assess the role of a centenarian-associated single nucleotide polymorphism (SNP), S106L, introduced into col-99 , the ortholog of human COL25A1 6–8 . We demonstrate that this SNP promotes lifespan extension in C. elegans . Mechanistically, we propose that the S106L mutation alters the cleavage rate of COL-99 in vitro , and the cleaved extracellular domain of COL-99 is sufficient to extend lifespan. Additionally, our findings indicate that genes involved in the innate immune response are upregulated in col-99(gk694263 [S106L] ) mutants and that the p38 mitogen-activated protein kinase (MAPK) pathway and the transcriptional co-activator yes-associated protein 1 (YAP-1) are crucial for this lifespan extension. Our study provides novel insights into the genetic mechanisms regulating aging by identifying a specific genetic variant that influences lifespan through modulation of the innate immune response and MAPK signaling pathways. These findings may contribute to developing targeted interventions for age-related diseases and enhance our understanding of the genetic factors underlying human longevity. Results Introduction of centenarian COL25A1 SNP in C. elegans homolog col-99 In 2001, Puca and colleagues performed a linkage study in centenarian sibling pairs to identify genetic loci associated with exceptional longevity 5 . Interestingly, they found a 3 cM locus on chromosome 4 that was strongly associated with longevity 5 . However, the human genome was not sequenced at the time of publication, and this region was not characterized. Therefore, we investigated this locus and found 108 ensemble features, including 33 protein-coding genes, 14 linc RNA, 2 miRNA, 2 snoRNA, and 9 snRNA ( Figure 1a, S1a, Supplementary Table 1 ). To identify causally implicated protein-coding genes, we cross-referenced genes to identify those with longevity-associated SNPs in centenarian GWAS or with studies showing that altering gene function would increase lifespan in any organism ( Figure S1a, Supplementary Table 1 ). Strikingly, we found a collagen gene, COL25A1 , which was previously shown to be significantly associated with human healthy aging (“Wellderly phenotype"), but without mechanistic validation 9 . In a subsequent literature search, we found that COL25A1 exhibited several SNPs associated with exceptional lifespan and/or healthy aging ( Figure 1b, Supplementary Table 1) . Most of the molecular alterations of these longevity-associated SNPs were predicted to result in the loss or reduction of the function of COL25A1 . To determine whether the loss of COL25A1 is beneficial, we investigated the effect of a loss-of-function mutation in the COL25A1 orthologue col-99 in C. elegans . However, the col-99(ok1204) deletion mutant was short-lived with a mean lifespan of 18.4 days, approximately 5 days less than the wild type ( Figure 1b; blue line, Supplementary Table 2 ). Overexpression of full-length COL-99 protein tagged with GFP, on the other hand, did not affect the lifespan ( Figure 1b; green line, Supplementary Table 2 ). Assuming COL25A1 is responsible for the above-mentioned linkage, these findings indicated that the effect of SNPs in COL25A1 on longevity has a more complex mechanism. S106L SNP in col-99 extends lifespan in C. elegans To investigate this unknown mechanism, we tried to map all human SNPs in the C. elegans col-99 gene. However, due to the vast evolutionary distance and the repetitive nature of collagen, we could only reliably map three sites with some confidence (human S116L → C. elegans S106, G225C → G215, R402C → R393) ( Figure 1c ). We focused on the S116L SNP in COL25A1 (S106L in col-99 in C. elegans ) as it is proximal to a conserved furin cleavage site with the potential to impact domain processing ( Figure 1c) . First, to determine if the human S116L SNP introduction into col-99 in C. elegans had any effect on lifespan, we obtained C. elegans harboring this S106L SNP in col-99 (hereafter referred to as col-99(gk694263 [S106L] ) mutants) and outcrossed ten times (10X) to remove any potential background mutations. We then performed lifespan studies with 4X, 8X, and 10X outcrossed col-99(gk694263 [S106L] ) mutants and found all outcrossed col-99(gk694263 [S106L] ) mutants increased lifespans ( Figure 2a, S1b, Supplementary Table 2 ) by approximately 26% compared to wild-type control, indicating not only that S106L in humans might indeed be causative for prolonged life, but also re-assuring our mapping of this SNP between the species. To independently validate that the increased lifespan is due to the single mutation in col-99 , we CRISPR-generated the S106L SNP mutation into wild-type animals (hereafter referred to as col-99(syb4350 [S106L] ) ). Reassuringly, we found a similar extension in the lifespan of col-99(syb4350 [S106L] ) compared to the wild type ( Figure S1c, Supplementary Table 2) . The col-99(gk694263 [S106L] ) mutants superficially looked wild type but were slightly delayed developmentally by a few hours, with larval L4 stage ( Figure S1d, Supplementary Table 2 ). Furthermore, the longevity of col-99(gk694263 [S106L] ) mutants did not correlate with oxidative or heat stress resilience ( i.e., col-99(gk694263 [S106L] ) mutants were more susceptible to 14 mM arsenite or 32 ℃ treatment; Figures S1e-f, Supplementary Table 2 ). The extracellular domain of COL-99 is sufficient for increased longevity COL25A1 in humans and COL-99 in C. elegans are both single-pass type II transmembrane collagen proteins with predicted furin cleavage sites 7,10 . For COL25A1 , this site is reported to be important for shedding, creating the so-called CLAC (collagen-like Alzheimer amyloid plaque component) fragment 11 . Besides its association with Alzheimer’s Disease, the function of this shredded collagen fragment is not understood, although it seems to play a role in muscle development and innervation 12,13 . Similarly, in C. elegans, col-99 was important for axonal guidance, and the first and third predicted furin cleavage sites seem important for its function 6,8 . As S116L (and S106L) are close to the conserved (first) furin cleavage site, we hypothesized that the S106L mutation in C. elegans might affect furin cleavage, leading to a change in the extracellular domain shedding rate. To test this hypothesis, we expressed the extracellular domain of COL-99 under its endogenous promoter in C. elegans . The expression of a shed extracellular domain of COL-99 prolonged the lifespan of C. elegans by approximately 11% already ( Figure 2b, Supplementary Table 2 ), consistent with the longevity effect of the S106L SNP in col-99(gk694263 [S106L] ) mutant. Although the furin recognition site is often described as R-X-[K/R]-R⇓, the full recognition sequence is longer and amino acids up to 14 amino acids N-terminally (P14) and six amino acids C-terminally (P6′) from the cleavage site are described to influence furin activity (see Figure 3a for clarification of P1/P1’ nomenclature and positioning in COL-99 and COL25A1) 14 . To measure the effect of an S to L mutation at the P2’ site in C. elegans, we synthesized short fluorescently quenched peptides containing the furin cleavage site and a serine or leucine residue, respectively. The quenching was resolved upon cleavage, and an increased fluorescence was observed ( Figure 2c ). Contrary to our expectation, the mutant showed a roughly halved Vmax (29 vs 52) compared to the wild-type sequence, indicating cleavage of S106L by furin, but at a slightly decreased rate. Given the abundance of furin, this small difference can hardly explain the observed effect in vivo, especially as it seems to contradict our finding from the expression of the extracellular domain alone, which suggested benefits from increased cleavage. Besides its location near the furin cleavage site, both sites are also predicted to be phosphorylated ( Figure 3a ). Although extracellular phosphorylation is not completely understood yet, its importance has become more evident over the last few years 15–18 . Bioinformatically, several phosphorylation sites in COL25A1 have been predicted 19 . Furthermore, it was shown at least twice that phosphorylation might affect furin cleavage 20 . We therefore speculated that phosphorylation of S106 might inhibit COL-99 shedding, thereby reducing the extracellular concentration of the extracellular domain of COL-99. The described SNP might prevent this phospho‑dependent inhibition, thus increasing shedding. Consequently, a CRISPR mutant of col-99 introducing a phosphomimetic S106D exchange ( col-99(syb4352 [S106D] ) ) did not significantly affect lifespan (3 out of 5 independent trials; Figure 3b, Supplementary Table 2 ). Taken together, this suggests a model wherein phosphorylation of serine 106 influences furin cleavage and release of the extracellular domain of COL-99 to alter downstream processes to increase lifespan. Innate immune response is upregulated in col-99(gk694263 [S106L] ) mutants To identify the downstream mechanisms affected by the S106L mutation in col-99(gk694263 [S106L] ) , we performed transcriptomic profiling of the col-99(gk694263 [S106L] ) mutant and wild type at the young adult stage. The introduction of col-99 SNP drove limited transcriptomic effects ( Figure 4a, Supplementary Table 3 ). Interestingly, the col-99 SNP did drive a ~20% increase in the col-99 transcript level ( Figure 4b ), suggesting an autoregulated overexpression. Gene ontology term analysis revealed a downregulation of genes involved in oxidative phosphorylation, while innate immune response genes were upregulated ( Figure 4c, Supplementary Table 3 ). ATF-7 is the master regulator of immune response in C. elegans 21 , and we found it to be upregulated by col-99(gk694263 [S106L] ) expression ( Figure S2a, Supplementary Table 3 ). However, in our epistatic lifespan analysis, we observed that atf-7 was not required for lifespan extension in col-99(gk694263 [S106L] ) mutants ( Figure S2b, Supplementary Table 2 ). Arginine kinase (creatine kinase) argk-1 was also upregulated in col-99(gk694263 [S106L] ) mutants ( Supplementary Table 3 ). We verified this increase in col-99(gk694263 [S106L] ) mutants with a reporter strain argk-1 p::GFP using fluorescence microscopy ( Figure S2c ). Previously, ARGK-1 overexpression increased lifespan via AMPK, which triggers the activation of the innate immune response 22,23 . Therefore, we checked for the requirement of argk-1 in lifespan extension by col-99(gk694263 [S106L] ) mutants. We assayed the lifespan of argk-1(ok2993); col-99(gk694263 [S106L] ) double mutants but found that the extended lifespan of col-99(gk694263 [S106L] ) mutant was independent of argk-1 ( Figure S2d, Supplementary Table 2 ). Since DAF-16 is also an important modulator of innate immune response and lifespan 24 , we tested the nuclear localization of DAF-16 in col-99(gk694263 [S106L] ) mutants. However, the differences in DAF-16 nuclear localization were insignificant ( Figure S2e ). Taken together, our approach in testing these genes differentially expressed in RNA-sequencing did not identify underlying pathways driving longevity. Next, we used a candidate RNAi screen approach to identify the pathways mediating col-99(gk694263 [S106L] ) lifespan extension. Given the strong innate immune transcriptional signature, we tested two immune-response reporter genes, clec-85 and T24B8.5, that were also differentially regulated by col-99(gk694263 [S106L] ) ( Figures 4d, S2f, Supplementary Table 3 ). The transcriptional reporter driving GFP under the T24B8.5 promoter ( T24B8.5p ::GFP 25 ) showed a two-fold upregulation in the fluorescent intensity in col-99(gk694263 [S106L] ) mutants compared to wild-type, making it suitable for screening. Given that col-99(gk694263 [S106L] ) induces T24B8.5p ::GFP in vivo , knocking down genes that are required for mediating col-99(gk694263 [S106L] ) -dependent T24B8.5 upregulation may reveal underlying pathways. Therefore, we screened through a targeted candidate library including known immunogenic-, collagen-, and mechano-receptors using the T24B8.5p ::GFP reporter strain. We started with genes in the canonical innate immune response pathway (P38 Map Kinase pmk-1 26 , Toll and Interleukin 1 Receptor tir-1 27 , ATF cAMP-dependent transcription factor atf-7 28 , and Toll-like receptor tol-1 29 ) and found that RNAi targeting tir-1 suppressed the col-99(gk694263 [S106L] ) -mediated T24B8.5p ::GFP induction ( Figure 4d ), indicating that this immune receptor was required. However, it is unlikely that the extracellular domain of COL-99 would bind the TIR-1 receptor directly for signaling, suggesting additional upstream signaling. To identify the potential direct binding partners/receptors of shed COL-99 extracellular domain, we turned to the COL-99 collagen receptors, the Discoidin Domain Receptors ( ddr-1 and ddr-2 ), and basement membrane component nidogen homolog nid-1 , which are putative binding partners of the cleaved COL-99 ectodomain and are involved in col-99 -mediated neuronal axon guidance 6 . However, knockdown of ddr-1, ddr-2, or nid-1 did not suppress col-99(gk694263 [S106L] )- mediated T24B8.5p ::GFP induction ( Figure 4d ). COL-99 is expressed in the hypodermis, and the furin cleavage and shedding of the COL-99 ectodomain are required for neuronal axon guidance 6 . Thus, a neuronal receptor may be required. We tested neuronal and mechanosensitive transient receptor potential (TRP) channel trpa-1/ TRPA and osm-9/ TRPV, which are shown to be involved in longevity 30,31 . We found that osm-9 was required for col-99(gk694263 [S106L] ) -mediated T24B8.5p ::GFP induction ( Figure 4d ). OSM-9 is a mechanosensitive receptor upstream of YAP-1 (yes-associated protein), a mechanoresponsive transcriptional co-activator activated upon stiffness changes in the extracellular collagen network and implicated in the control of lifespan 32–34 . In addition, yap-1 is functionally implicated in Touch Receptor Neuron asymmetric neurite extension 35 and pathogen resistance in C. elegans 36 , consistent with a potential role for neuronal regulation and innate immunity. We found that the knockdown of yap-1 also suppressed col-99(gk694263 [S106L] ) -mediated T24B8.5p ::GFP induction ( Figure 4d ). Taken together, we identified two receptors, tir-1 and osm-9 , and the co-transcriptional activator yap-1 required for col-99(gk694263 [S106L] ) induction of the transcriptional innate immune response program. By contrast, yap-1 and tir-1 were not required for col-99(gk694263 [S106L] ) induction of the unfolded protein response (UPR) gene hsp-4 ( Figure S2g-h ), suggesting that our reporter screen findings need to be validated with functional lifespan assays. The P38 MAPK pathway is required for longevity extension by col-99(gk694263 [S106L] ) Given the distinct downstream pathways we recovered for col-99(gk694263 [S106L] ) , we aimed to determine the pathway that mediates col-99(gk694263 [S106L] ) longevity. For instance, our reporter assays revealed that the COL-99 receptors ddr-1 and ddr-2 were not required for immune and UPR responses; this does not rule out their role in lifespan regulation. However, we found that loss of ddr-1, or ddr-2, or the double mutant ddr-1; ddr-2 did not suppress col-99(gk694263 [S106L] ) -induced longevity ( Figure S3a, Supplementary Table 2 ), suggesting that DDR-1 and DDR-2 are the COL-99 receptor for axonal guidance 37 but not for longevity. Another likely candidate for COL-99 ectodomain receptor is TIR-1, and we found that tir-1 was required for col-99(gk694263 [S106L] ) -induced lifespan extension ( Figure 5a, Supplementary Table 2 ). Since tir-1 is upstream of the p38 MAPK signaling cascade, we tested whether the downstream protein p38 MAPK ( pmk-1 ) was required for lifespan extension. Although RNAi of pmk-1 was not sufficient to block T24B8.5p ::GFP induction in col-99(gk694263 [S106L] ) mutants, knockdown of pmk-1 abolished the longevity of col-99(gk694263 [S106L] ) mutants ( Figure 5b, Supplementary Table 2 ). We confirmed these lifespan results of col-99(gk694263 [S106L] ) with the CRISPR-generated mutant CED04 col-99(syb4350 [S106L] ) , and also showed no adverse effects with the phosphomimetic CED05 col-99(syb4352 [S106D] ) mutants in combination with tir-1 or pmk-1 knockdown ( Figure S3b-d, Supplementary Table 2 ). Consistently, tir-1 and pmk-1 were required for lifespan extension in the CED06 col-99(syb4332 [COL-99EXT] ) mutant expressing the ectodomain of COL-99 ( Figure 5c-d, Supplementary Table 2 ). Furthermore, in line with the requirement of pmk-1 for lifespan extension, col-99(gk694263 [S106L] ) mutants showed higher p38 MAPK/PMK-1 phosphorylation ( Figure 5e, Supplementary Table 2 ), an event generally known to drive increased ATF-7 transcription factor levels. Despite this association, ATF-7 was not required for the extended lifespan in col-99(gk694263 [S106L] ) mutants ( Figure S2b, Supplementary Table 2 ). This led us to investigate other potential mediators, and we discovered that YAP-1 played a critical role in facilitating the longevity effects of col-99(gk694263 [S106L] ) mutants ( Figure 5f, Supplementary Table 2 ). Collectively, our data support a model in which a non-canonical immune response pathway involving osm-9, tir-1, pmk-1, and yap-1 is activated upon blunting the phosphorylation of COL-99 at the S106 site, and thus altered furin-mediated cleavage resulting in the subsequent release of the COL-99 ectodomain ( Figures 5g, S4 ). Discussion Previously, in at least three centenarian cohorts and one healthy aging cohort, several SNPs in collagen XXV associated with healthy human aging have been identified 9 , 38 – 40 . Despite these associations, causal mechanisms linking specific genetic variants to healthy longevity remain largely unvalidated. In this study, we addressed this gap by demonstrating that a rare coding variant in the collagen gene COL25A1 , specifically the S116L SNP in the COL25A1 ortholog, is sufficient to extend the lifespan in C. elegans . By introducing the S106L orthologous mutation into col-99 , we observed an approximately 26% increase in lifespan, suggesting a conserved role of this variant in longevity. Mechanistically, the S106L mutation influences furin-mediated cleavage of transmembrane COL-99, altering the release of its extracellular domain. This extracellular domain is sufficient to promote lifespan extension, implicating the remodeling of extracellular matrix (ECM) in aging processes. Our results also reveal that the lifespan extension in col-99(gk694263 [S106L] ) mutants depends on the p38 MAPK pathway and the transcriptional co-activator YAP-1. Interestingly, this effect is independent of ATF-7, a typical downstream target of p38 MAPK involved in immune responses 28 , indicating alternative signaling pathways are at play. The involvement of YAP-1, known for its roles in mechanotransduction and cellular proliferation 41 and mechanotransduction-mediated longevity 42 , suggests that ECM changes can influence longevity through mechanoresponsive signaling pathways. The activation of innate immune response genes further supports a model where ECM alterations modulate immune signaling to impact aging. Our findings suggest that similar mechanisms may operate in humans, given the conservation of these pathways. Proteomic analyses of centenarians have revealed elevated levels of proteins involved in angiogenesis and cell junctions, suggesting enhanced tissue homeostasis 43 . Additionally, single-cell transcriptomics of peripheral blood mononuclear cells (PBMCs) from centenarians has indicated improved immune function, characterized by increased T cell populations 44 . Notably, serum proteomic signatures have shown enrichment of collagen proteins, such as COL28A1 and COL6A3, in centenarians 45 , highlighting the potential role of collagen in longevity. Identifying COL25A1 variants affecting furin cleavage and ECM dynamics opens new avenues for research into the genetic regulation of human aging. Future studies should focus on elucidating where COL-99 serine 106 is phosphorylated, i.e. , in the Golgi or the extracellular space. Furthermore, our hypothetical model ( Figure S4 ) shows genetic interactions, but the direct interactions between the COL-99 extracellular domain and its signaling partners are needed to fully understand the mechanisms underlying this lifespan extension. One limitation of our study is the use of C. elegans as a model organism, which, while highly informative, may not fully recapitulate the complexity of human aging. Additionally, the direct applicability of the S116L variant in humans requires further validation in mammalian systems. Interestingly, several human genetic studies associate COL25A1 variants with healthy aging, longevity, and age-related diseases (Fig. 1 c , Supplementary Table 1). For instance, SNPs in COL25A1 are associated with Alzheimer’s disease 46 , 47 , which maps to the Aβ-binding side in COL25A1, suggesting a failure of the extracellular domain of COL25A1 to scoop up Aβ oligomers 46 . Similarly, SNPs in COL25A1 are associated with congenital cranial dysinnervation disorder 48 , 49 , which can lead to a loss of the most outer collagen domain (COL3) in COL25A1 expressed from the muscle that is required to bind to receptors protein tyrosine phosphatases σ and δ (PTP σ/δ), a process essential for intramuscular motor innervation 50 . Previous comparisons of centenarians with disease GWAS studies found an overlap of SNPs in genes associated with diseases, such as Alzheimer’s and coronary artery diseases, and other SNPs in the same gene associated with longevity 51 , 52 . However, these risk alleles in these genes for diseases are depleted in centenarians and/or compensated by a constellation of multiple beneficial genetic variants in centenarians 51 , 53 , 54 . These observations suggest that centenarians in given disease pathways have resilience variants tipping the balance to health and not down towards disease progression. In line with this idea, mapping proteins associated with human healthspan, COL25A1 is in the first central node together with LRP1, TOMM40, and CREBBP, which are also implicated in Alzheimer’s disease and potentially under dietary restrictions might signal through NOTCH to promote human healthspan 55 . Further GWAS studies revealed SNPs in COL25A1 are associated with resistance to COVID-19 infections 56 and higher spermidine levels 57 , a polyamine compound inducing autophagy and increasing lifespan of model organisms 58 , suggesting some implications in resilience. In summary, our research identifies a causal role for a coding variant in COL25A1 in extending lifespan via modulation of ECM remodeling and immune signaling pathways. These insights advance our understanding of the genetic contributors to longevity and may inform the development of therapeutic strategies targeting age-related diseases. Material and methods Strain maintenance All strains were maintained at 20 ℃, on NGM (nematode growth media) plates seeded with Escherichia coli OP50. The strains used in this study are: N2 (wild-type Bristol), RB1165 col-99(ok1204) IV, COL-99 overexpression VH2847 hdIs73 [GFP::COL-99, pha-1(+)], AU78 agIs219 [T24B8.5p::GFP:: unc-54 3’UTR + ttx-3 p::GFP:: unc-54 3’UTR] III, yap-1(tm1416) , VC1518 atf-7(gk715) III, MAH547 sqEx82 [ argk-1p ::GFP + rol-6 (su1006)], MAH205 argk-1(ok2993 ) V, TJ356 zIs356 [ daf-16 p::daf-16a/b::GFP + rol-6 (su1006)], SAL146 pha-1 (e2123) III; denEx24 [ clec-85 p::GFP + pha-1 (+)], SJ4005 zcIs4 [ hsp-4 ::GFP] V, RB970 ddr-1(ok874) , RB788 ddr-2(ok574) , VH1681 ddr-1(ok874); ddr-2(ok574) X; evIs111 [ rgef ::GFP] V, VC40561 col-99(gk694263 [S106L] ) SNP mutant [4 times, 8 times, and 10 times outcrossed] 59 . col-99 (SNP) is interchangeably used in figures for col-99(gk694263 [S106L] ) mutant, which is originally the VC40561 strain obtained from the Million mutation project. The strain has a missense mutation G→A at position 144813 on chromosome IV, generating col-99 ( gk694263 ). Strains generated in this study : Strains generated by CRISPR gene editing (performed by SUNY Biotech) - F29C4.8j.1 is the isoform of col-99 which was modified to obtain the following transgenic strains: CED04 PHX4350 [ col-99(syb4350 [S106L] ) ] ( col-99 S106L mutant): Precise one base pair missense mutation (TCG→TTG) was introduced in col-99 gene using CRISPR. It resulted in the expression of nonpolar leucine amino acid (L) at the 106th position, instead of polar serine (S) in wild-type COL-99 (termed as S106L). CED05 PHX4352 [ col-99(syb4352 [S106D] ) ] ( col-99 S106D mutant): The mutation TCG→GAC was induced in col-99 gene using CRISPR gene editing, which resulted in the expression of phosphomimetic aspartic acid residue (D) at 106th position, instead of serine (S) in wild-type COL-99 (termed as S106D). CED06 PHX4332 [ col-99(syb4332 [COL-99EXT] ) ] ( col-99 extracellular domain): Precisely 3075 bp (95 aa) were deleted after the first methionine coding codon ATG in col-99 gene so that first conserved Furin cleavage site “RRVR” at 104th position 8 in the non-collagenous extracellular domain of COL-99 was expressed, leading to expression of only the COL-99 extracellular domain (COL-99 EXT). Strains generated by genetic cross : col-99(gk694263 [S106L] ) ; T24B8.5p::GFP, col-99(gk694263 [S106L] ) ; yap-1(tm1416) , col-99(gk694263 [S106L] ) ; atf-7(gk715) , col-99(gk694263 [S106L] ) ; argk-1 p::GFP, col-99(gk694263 [S106L] ) ; argk-1(ok2993) , col-99(gk694263 [S106L] ) ; DAF-16::GFP, col-99(gk694263 [S106L] ) ; clec-85 p::GFP, col-99(gk694263 [S106L] ) ; hsp-4 p::GFP, col-99(gk694263 [S106L] ) ; ddr-1(ok874); ddr-2(ok574) , col-99(gk694263 [S106L] ) ; ddr-2(ok574) , col-99(gk694263 [S106L] ); ddr-1(ok874) . Genotyping col-99(gk694263 [S106L] ) SNP mutation : A quick and reliable method of SNP detection by endpoint PCR was followed as described previously 60 . Briefly, the principle of allele variant detection is based on SuperSelective (SS) primers design. A SS primer generally consists of three regions- anchor, bridge, and foot. An “anchor” is a 5’ long sequence that hybridizes to the template DNA, followed by an intervening region, called “bridge” which is non-complementary to the template. “Foot” region is a 3’ short sequence complementary to the template, with a terminal mismatch residue. SuperSelective (SS) primers were designed for genotyping a single point mutation in col-99(gk694263 [S106L] ) , consisting of a 16 bp anchor, a 6 bp optimal bridge, and a 7 bp foot region with terminal mismatch nucleotide G→A for wild-type and mutant allele. When primers were annealed to the template DNA, the anchor region hybridized while the bridge region, being non-complementary, formed a bubble-like structure separating the foot region from the anchor. Since the foot region is too short, any single nucleotide mismatch would inhibit the binding of primers and, hence, prevent primer extension. So, when PCR was run with wild-type gDNA as a template, only primers designed for wild-type allele would bind to wild-type gDNA and yield a specific band (expected size of the amplicon is 348 bp) at an annealing temperature of 62 ℃, while a combination of mutant primers with wild-type gDNA should not yield any band. Reaction conditions : Components Volume (𝛍L) Template (gDNA) (approx. 5 ng) 2 5X GoTaq Flexi Buffer 2.5 10 mM dNTPs 0.5 500 nM Primer 1 1.25 500 nM Primer 2 1.25 Go Taq Polymerase 0.25 MQ 17.25 PCR cycle : Steps Temperature (℃) Time Initial Denaturation 98 30 sec Denaturation 98 10 sec 30 cycles Annealing 62 15 sec Elongation 72 40 sec Extension 72 2 min In-silico prediction of phosphorylation sites in collagen Full-length sequences of COL-99 and COL25A1 were subjected to the NetPhos 3.1 algorithm ( https://services.healthtech.dtu.dk/services/NetPhos-3.1/ ) and analyzed for serine phosphorylation. Only predictions with a score higher than 0.5 and in the vicinity of the furin cleavage site near the S106 / S116 SNP in question were considered 61 , 62 . Automated Lifespan Assay The automated lifespan assay was performed as described by Stroustrup et al 63 . Briefly, C. elegans populations were subjected to population lysis, and the eggs were transferred to OP50 plates. At the L4 stage, the animals were transferred to either OP50 or RNAi plates that contained 50 µM FUdR. At day two of adulthood, eggs were removed through repeated washing, and the animals were transferred to lifespan plates of the same composition, featuring a tight-fitting lid (BD Falcon Petri Dishes, 50x9 mm). The plates were placed in the Epson V800 flatbed scanners and continuously imaged for 60 days. Peptide synthesis The internally quenched fluorogenic peptides S106S (Abz-VAKSRRVRNS-Lys[DNP]) and S106L (Abz-VAKSRRVRNL-Lys[DNP]) were synthesized by manual and automated solid-phase peptide synthesis (SPPS) (peptide synthesizer from MultiSynTech, Syro I) using the Fmoc (fluorenylmethoxycarbonyl)/t-Bu (tert-butyl) strategy on Rinkamid resin (loading 0.48 mmol, 0.015 mmol scale). First, Fmoc-Lys(DNP)-OH was manually loaded on the resin, and subsequently, the resin was elongated by the peptide synthesizer. All amino acids were coupled using 8 eq each of N,N′-diisopropylcarbodiimide (DIC), and ethyl cyanohydroxyiminoacetate (Oxyma) dissolved in DMF. Fmoc was cleaved off using 20% piperidine in DMF. The final peptides were cleaved by treating the resin for 3 h at room temperature with a mixture of concentrated (conc.) trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5, v/v/v). Following, the peptides were precipitated in ice-cold diethyl ether, washed five times, and freeze-dried. Purification was achieved by preparative reversed-phase (RP)-HPLC (Hitachi Elite LaChrom) on a VP250/16 NUCLEODUR 100-5 C18ec column (Macherey Nagel) using linear gradients from 20–70% B in A (A = 0.1% TFA in water; B = 0.1% TFA in ACN) over 60 min. All peptides were identified by RP-HPLC-ESI-MS (column: CC 125/4.6 Nucleodur 100-5 C18e (Macherey-Nagel) using a gradient from 10‑60% of acetonitrile (ACN) in H 2 O with 0.1% formic acid (ESI-MS, Thermo Scientific LTQ-XL). Final purity of all compounds was > 95%. Furin Cleavage Assay Using an Internally Quenched Fluorogenic Peptides The activity of furin towards the fluorogenic substrate containing an N-terminal Abz group and a C-terminal Lys-DNP group was measured by the increase in fluorescence at 420 nm that occurs upon cleavage of the peptide bond (|) in the peptide sequence Abz-VAKSRRVR|N(S/L)[-Lys[DNP]. Furin (New England Biolabs, #P8077) concentration was kept constant at a total of 1 unit in the total assay volume of 100 µl. Peptide concentrations were determined based on the absorption of the DNP moieties at 365 nM, calculated based on the extinction coefficient 17’300 M − 1 cm − 1 64 , and used in concentrations between 1 and 2000 nM. The reaction was performed in 50 mM Hepes (pH 7.5), 10 mM CaCl, 1 mM β-Mercaptoethanol at 25 ℃ and measured in a Fluoromax 4P (HORIBA) with the following settings: Excitation 320 nm (5 nm Bandpass); Emission 420 nm (5 nm Bandpass); Integration time 0.5 s; Interval time 1.0 s; total time 300 s. Initial velocity was determined by linear fit in Origin (OriginLabs) and plotted against substrate concentrations. The resulting titration curve was fitted by the Hill equation for evaluation of maximal velocity and approximate Km of the peptide. Representative data from three different assays are shown. Manual Lifespan Assay Gravid adults were treated with hypochlorite solution to obtain eggs. These were grown on OP50-seeded NGM plate till the L4 stage. At the L4 stage, 50 animals were transferred to OP50 NGM plates containing FuDR, in replicas of four plates. For RNAi lifespan, animals were grown on the respective RNAi NGM plates seeded with corresponding bacteria (L4440/ tir-1 / pmk-1 ) until the L4 stage and then transferred to FuDR-containing RNAi plates at the L4 stage. Survival of the animals was scored by prodding their tail once with platinum wire every alternate day. Unhealthy animals showing vulval bursting or those crawling to the sides/lid of the plates were censored from the population. Lifespan Scoring was started on Day 7 of adulthood, L4 being taken as t = 0 timepoint. The survival curve was plotted using GraphPad Prism. The lifespans were performed in at least two biological replicates at 20 ℃. Statistical analysis was performed using the online software OASIS 65 . A lifespan summary for individual experiments is provided in Supplementary Table 2. C. elegans Total RNA sample preparation for Transcriptomics Synchronized populations of the strains Wild-type (N2), and col-99(gk694263 [S106L] ) SNP mutant were grown on OP50 until the young adult stage (approximately 10 x 90 mm NGM petri plates with 500–800 animals feeding OP50 per plate). Young adult animals were collected in M9 buffer, washed twice with the buffer, and then flash frozen the pellet for storage at -80 ℃ until RNA isolation. RNA isolation was done using the Qiagen RNeasy mini kit (#74004) method according to the manufacturer’s protocol. Three biological replicates were collected for the experiment. RNA Sequencing and Analysis RNA concentration and purity were assessed via a Nanodrop spectrophotometer and an Agilent 2100 Bio-Analyzer. cDNA libraries for sequencing were prepared using the Illumina TruSeq RNA Library Prep Kit. Libraries were pooled and sequenced with 150 bp paired-end reads to a target depth of 20 million reads per sample. Reads were aligned to WBcel235 reference genome using the align function and annotated to the corresponding GTF using the featureCounts function from the Rsubread R package 66 (v. 2.3.5). Differentially expressed genes were determined using the edgeR 67 (v. 3.30.2) and limma 68 (v. 3.44.2) R packages. Briefly, genes were filtered based on minimum expression level (> 5 counts per million in at least 3 samples). Normalization was carried out using the trimmed mean of M-values method as implemented in calcNormFactors from edgeR. Data were modeled and differentially determined using the limma voom workflow to generate linear models with empirical Bayes moderation. Gene set overrepresentation analysis was performed using the enrichGO function from the clusterProfiler 69 (v. 3.15.0) R package, using lists of genes with either significantly increased or decreased expression and compared to a background list of all genes detected in the dataset. Raw reads are available at the NIH Sequence Read Archive (SRA) under accession number PRJNA1198094. Oxidative Stress Assay Young adult wild-type or col-99(gk694263 [S106L] ) SNP mutant animals grown on OP50 NGM plates were collected in M9 buffer and washed three times with M9 buffer by centrifugation. Approx. 30–40 animals were pipetted into the wells of a 96-well round-bottom plate. Animals were either swimming in 30 µL of M9 buffer (filled in the first two rows of the 96-well round bottom plate) or 14 mM sodium arsenite solution (in the rest of the 6 rows). Every column of the plate was denoted with one condition or strain. Stress resistance was measured as a function of the motility of the animals scored for 3 days in an automated wormtracker (wMicroTracker by NemaMetrix) platform. The resulting quantified motility output was analyzed and plotted using an R Script generated in our lab (Ewaldlab-LSD @ github). The experiment was done in three biological replicates. Automated Heat stress survival assay Synchronized wild-type or col-99(gk694263 [S106L] ) SNP mutant populations were grown until the young adult stage on the OP50 NGM plate. At the young adult stage, animals were shifted to a 32 ℃ (or 35 ℃) incubator for initiating heat stress. The incubator was equipped with scanners that were set to scan the plates every half an hour and capture the images of the animals for 3 days. Survival data of the animals was recorded and generated automatically after we manually decoded images of the animals from misleading object data points. The survival curve was plotted using an R script developed in our lab (Ewaldlab-LSD @ github). The experiment was done in two biological replicates at 32 ℃ and once at 35 ℃. Developmental Time Assay Ten gravid adult Day 1 animals were allowed to lay eggs on OP50-seeded NGM plates for 4 h and then sacrificed. After 48 h, the number of animals was estimated in different life cycle stages. Three plates per condition (technical replicates) were kept to assess the developmental stages. The total number of animals in a particular stage was plotted in a bar graph. Two-way ANOVA statistical analysis was performed in GraphPad Prism. Fluorescent Microscopy Gravid adults were treated with sodium hypochlorite solution to obtain eggs. The eggs were grown on OP50 or respective RNAi bacteria seeded NGM plate until young adults. Approx. 30 young adult animals were mounted onto a 2% agarose pad and anesthetized with 20 mM levamisole for imaging. For hsp-4 p::GFP, young adults were heat-shocked at 33 ℃ for 2 h and imaged after recovery from shock for 6 h at 20 ℃. Animals were stacked together and captured at 10X with one or two fields of view and then stitched together later using ImageJ. Quantifying the total fluorescence intensity per number of pixels was done by running a Python script, GreenIntensityCalculator (publicly available in Github-Ewaldlab), in ImageJ. Data is plotted as mean, and error bars represent SEM. Two-tailed Welch’s t -test statistical analysis of the quantified data was done using GraphPad Prism software (GraphPad Prism 9.0). The experiment was performed in three biological batches, with n > 80 total animals per condition. DAF-16 nuclear localization assay The synchronized population was grown until the L4 stage on OP50 NGM plates. At the L4 stage, approximately 30 animals were assessed manually for DAF-16 nuclear localization and categorized as none, low, intermediate, or high based on the intensity of the nuclear localization throughout the body. The percentage of animals showing DAF-16 nuclear localization was plotted in GraphPad Prism 9.0. Western Blot Young adult animals were harvested in the M9 buffer and washed three times with the M9 buffer to remove excess bacteria. The pellet was flash-frozen and stored at -80 ℃ until further use. To isolate protein from the animals, the pellet was resuspended in Buffer ‘C’ solution (50 mM HEPES, 100 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 10% Glycerol) with protease inhibitor cocktail (#11697498001, Merck) and then subjected to the homogenizer. The tube was then centrifuged to pellet the insoluble fraction and separate the soluble supernatant in another tube. Protein in the sample was estimated using the Bradford assay (#5000006, Bio-Rad). Approx. 30 ug of protein was run in duplicate lanes on SDS-PAGE gel. After blotting the protein on the PVDF membrane (#IPVH00010, Merck-Millipore), it was blocked in a 5% BSA blocking buffer for 1 hour at room temperature while shaking. Primary P-PMK-1 antibody (#9215S, Cell Signalling Technology) was used in dilution 1:5000, and alpha-tubulin (#T9026, Sigma) was used in dilution 1:10,000 in the blocking buffer overnight at 4 ℃. The Next day, the blots were washed three times with 1X TBST buffer and put in secondary anti-rabbit (#7074S, Cell Signalling Technology) (for P-PMK-1) and anti-mouse (#7076S, Cell Signalling Technology) (for alpha-tubulin) antibodies in a dilution 1:10,000 for 1 h at room temperature. Then, the blots were washed three times with TBST buffer and developed in a chemiluminescent chamber. Clarity Western ECL Blotting Substrate (# 1705060S , Bio-Rad) was used to develop the blot. Declarations Data Availability All source and raw data are available in Supplementary Tables 1-3 and the RNA-sequencing raw reads are available at the NIH Sequence Read Archive (SRA) under accession number PRJNA1198094. Acknowledgment We thank Garif Yalak for help with the collagen phosphorylation predictions, Valerio Izzi for sharing his analysis of the COL25A1 variants in LUSC patients, Harald Hutter for the col-99, ddr-1, and ddr-2 mutants, and WormBase for curated gene and phenotype information. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Funding from the Swiss National Science Foundation Funding from the Swiss National Science Foundation SNF P3 Project 190072 to CYE, CS, and AG. Author contributions All authors participated in analyzing and interpreting the data. CYE and AG designed the C. elegans experiments. JG performed an in vitro furin cleavage assay. MM performed the analysis of RNA sequencing. CS performed the automated lifespan analysis. JYCP performed a few col-99(gk694263 [S106L] ) SNP crosses and conducted some reporter assays. AG performed all other lifespan assays and other experiments. AG, JYCP, JG, and CYE wrote the manuscript in consultation with the other authors. Conflict of interest The authors have no competing interests to declare. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare the following personal relationship which may be considered as potential competing interests: co-author Michael R. MacArthur and Sarah Mitchell, Editor-in-Chief of npj Aging, are married. Dr. Sarah Mitchell was not involved in the journal’s review of, or decisions related to, this manuscript. With no relation to the present manuscript, CYE declares to be a co-founder and shareholder of Avea Life AG and Lichi3 GmbH and is employed by Novartis. Correspondence should be addressed to C. Y. E. References Borras, C. et al. Centenarians: An excellent example of resilience for successful ageing. Mech. Ageing Dev. 186 , 111199 (2020). Willcox, B. J., Willcox, D. C. & Suzuki, M. Demographic, phenotypic, and genetic characteristics of centenarians in Okinawa and Japan: Part 1—centenarians in Okinawa. Mech. 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Supplementary Files SupplementaryTable1centenarianchroIVmapped.xlsx SupplementaryTable2rawdatasummary.xlsx SupplementaryTable3RNASeq.csv SupplementaryFigures.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in npj Aging → Version 1 posted Editorial decision: Revision requested 27 May, 2025 Reviews received at journal 26 May, 2025 Reviews received at journal 15 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers agreed at journal 29 Apr, 2025 Reviewers invited by journal 26 Apr, 2025 Submission checks completed at journal 26 Apr, 2025 First submitted to journal 15 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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MacArthur","email":"","orcid":"","institution":"Princeton University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"R.","lastName":"MacArthur","suffix":""},{"id":448503539,"identity":"174cc4dd-a1fb-4dce-8ecc-09ca972febb2","order_by":5,"name":"Jan M. Gebauer","email":"","orcid":"","institution":"University of Cologne","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"M.","lastName":"Gebauer","suffix":""},{"id":448503540,"identity":"d4cc335e-6f78-413d-b662-d9ad23468a22","order_by":6,"name":"Collin Y. Ewald","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYFAC5sYDCRBGg8QHBgYQm42AFsYGqBbGBskZDAZEaoExpHmI0cLf3thw4OEOBjmD4wcbb9tU/MmTb2B+9gCfFokzBxsOJJ5hMDY4k9hsnXPGoJixgc3cAJ8WA4lEoJa2/4nbgKR0bptBYjMDD5sEEVoY6redf9gmbfnPAMgmUkuC2Q2gLYwNBok9hLRA/NLGYLj/xsNmy55jxokzmNnM8Grhb28++PBnG4O8ZH/ywRs/auQS57c3P8OrBQtgJlH9KBgFo2AUjAJMAABtKUyekHu9oQAAAABJRU5ErkJggg==","orcid":"","institution":"ETH Zürich","correspondingAuthor":true,"prefix":"","firstName":"Collin","middleName":"Y.","lastName":"Ewald","suffix":""}],"badges":[],"createdAt":"2025-02-15 09:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6035585/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6035585/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41514-025-00264-7","type":"published","date":"2025-09-30T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81606494,"identity":"67d9b229-e082-41a5-8e4b-c8776820f30c","added_by":"auto","created_at":"2025-04-29 06:01:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1335626,"visible":true,"origin":"","legend":"\u003cp\u003eMapping longevity SNP from humans in \u003cem\u003eC. elegans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea) Depiction of the human chromosome IV with the putative longevity region highlighted in red. The overlapping open reading frames located in this region are displayed in separate rows, and \u003cem\u003eCOL25A1\u003c/em\u003e is highlighted in red (for details, see Supplementary Table 1). b) Lifespan curve showing short-lived \u003cem\u003ecol-99(ok1204)\u003c/em\u003e mutant compared to wild-type. Automated Lifespan was performed using n\u0026gt;87. The log-rank test was performed for statistical analysis. c) Schematic showing the parallels drawn between human \u003cem\u003eCOL25A1\u003c/em\u003e and \u003cem\u003eC. elegans col-99\u003c/em\u003e gene and the associated SNPs. Grey lines indicate observed SNPs in \u003cem\u003eCOL25A1\u003c/em\u003e; Red boxes indicate SNPs/mutations associated with diseases, while SNPs marked in green are associated with longevity. Dark blue bars indicated collagen domains. Predicted furin sites are marked by dashed lines; orange clamps indicate cysteine-knots. SNPs in dark grey are potential longevity-associated SNPs matched on \u003cem\u003ecol-99\u003c/em\u003e. Light blue boxes are known for deletion/stop mutation in \u003cem\u003eC. elegans\u003c/em\u003e. The Brown box indicates the transmembrane domain.\u003c/p\u003e","description":"","filename":"Figure1revised.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/310535fcfe0c23c1d199d155.jpg"},{"id":81606496,"identity":"538ebf79-f86e-4014-814e-17ef4d0feb67","added_by":"auto","created_at":"2025-04-29 06:01:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1274597,"visible":true,"origin":"","legend":"\u003cp\u003eCOL-99 S106L influences longevity in \u003cem\u003eC. elegans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea) Lifespan curve showing an extension of life span in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant. b) Lifespan curve showing the extracellular domain (CED06 \u003cem\u003ecol-99(syb4332\u003c/em\u003e[COL-99EXT]\u003cem\u003e)\u003c/em\u003e) is sufficient to increase lifespan. The lifespans were performed in at least two biological replicates at 20 ℃. Kaplan-Meier Log-Rank test was performed for statistical analysis using the online software OASIS. A lifespan summary for individual experiments is provided in Supplementary Table 2. c) \u003cem\u003ein-vitro\u003c/em\u003efurin cleavage assay shows differential cleavage of COL-99 peptide. Representative data from three different assays are shown.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/e25ec826529d8ca87d594c00.jpg"},{"id":81606486,"identity":"1a46c540-612e-4cb0-9e84-7b147aa7f386","added_by":"auto","created_at":"2025-04-29 06:01:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285237,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphorylation may alter shedding of COL-99\u003c/p\u003e\n\u003cp\u003ea) Phosphorylation potential of serine residue in the SNP next to the furin cleavage site (highlighted in green) in COL25A1 and COL-99. Bold “Serines” are SNP S106 and S116, respectively. Prediction by NetPhos 3.1. b) Lifespan curve showing incapability of phosphomimetic variant of \u003cem\u003ecol-99\u003c/em\u003eS106D (CED05 \u003cem\u003ecol-99(syb4352\u003c/em\u003e[S106D]\u003cem\u003e)\u003c/em\u003e) to extend lifespan.The lifespan was performed in at least two biological replicates at 20 ℃. Kaplan-Meier Log-Rank test was performed for statistical analysis using the online software OASIS. A lifespan summary for individual experiments is provided in Supplementary Table 2.\u003c/p\u003e","description":"","filename":"Figure3col99.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/979885d61793db5e8b74a864.jpg"},{"id":81607395,"identity":"961f0762-6705-4e03-a324-9a964e083dc7","added_by":"auto","created_at":"2025-04-29 06:09:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2540044,"visible":true,"origin":"","legend":"\u003cp\u003eDefense response genes are upregulated in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutants.\u003c/p\u003e\n\u003cp\u003ea) Logarithmic scale representing the distribution of differentially expressed genes in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003eSNP mutant versus wild-type. b) The graph shows an increase in the expression of \u003cem\u003ecol-99\u003c/em\u003e transcripts in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant compared to wild-type (in terms of FKPM values). c) GO term analysis showing downregulated and upregulated genes in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003eSNP mutant to wild-type. RNA sequencing was performed with three biological batches. d) Targeted screening to find regulators of T24B8.5 GFP expression. Data is plotted as mean, and error bars represent SEM. Two-tailed Welch’s\u003cem\u003e t\u003c/em\u003e-test statistical analysis. The experiment was performed in three biological batches. \u003cem\u003ecol-99\u003c/em\u003e(SNP) is \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/442cebb11ed6712528b364f8.jpg"},{"id":81606476,"identity":"fec846d5-75d6-43ab-b2d5-725a1160b0cc","added_by":"auto","created_at":"2025-04-29 06:01:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1969917,"visible":true,"origin":"","legend":"\u003cp\u003ep38 MAPK is responsible for extending lifespan in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutants.\u003c/p\u003e\n\u003cp\u003ea, b) Survival curve showing the dependence of the increased lifespan of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant on\u003cem\u003e tir-1\u003c/em\u003e(A), \u003cem\u003epmk-1\u003c/em\u003e(B). c, d) Survival curve showing the dependence of the increased lifespan of CED06 \u003cem\u003ecol-99(syb4350\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant on \u003cem\u003etir-1\u003c/em\u003e( c) and \u003cem\u003epmk-1\u003c/em\u003e (d). e) Western Blot showing activation of PMK-1 in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003eSNP mutant. f) Survival curve showing the dependence of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003eSNP on \u003cem\u003eyap-1\u003c/em\u003e for extension of lifespan. g) Hypothetical model of signal relay from the extracellular domain of COL-99 to activate the p38 MAPK pathway, which signals to \u003cem\u003eyap-1\u003c/em\u003e to extend longevity in the \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant. Genes displayed are unlikely to be in the same cell. Please, also see Figure S4. The lifespans were performed in at least two biological replicates at 20 ℃. Kaplan-Meier Log-Rank test was performed for statistical analysis using the online software OASIS. The western blot was done twice. A lifespan summary and raw western blots for individual experiments are provided in Supplementary Table 2. \u003cem\u003ecol-99\u003c/em\u003e(SNP) is \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5revised.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/01142907cb55ec87617e1c50.jpg"},{"id":92883695,"identity":"eaa6e5c1-01e8-4515-aa93-6e078e327462","added_by":"auto","created_at":"2025-10-06 16:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9093261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/db695b51-1396-4f2d-a810-547e71f75766.pdf"},{"id":81607394,"identity":"d0e58dce-34e5-4840-b1c1-5d5875d578a0","added_by":"auto","created_at":"2025-04-29 06:09:35","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8864289,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1centenarianchroIVmapped.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/68c9b9737ac48a43619c5c07.xlsx"},{"id":81607390,"identity":"58aecfcd-d7a4-46ec-ad6a-b9a31fb5724f","added_by":"auto","created_at":"2025-04-29 06:09:35","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2943210,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2rawdatasummary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/1c3cb60e4454c2b5f9686d35.xlsx"},{"id":81607598,"identity":"c9091ae5-09ea-410a-821d-cf3657d3facf","added_by":"auto","created_at":"2025-04-29 06:17:35","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1799535,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3RNASeq.csv","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/0b27b80418336ad5a6555e34.csv"},{"id":81606481,"identity":"84b0aa15-21ed-4f09-9bb0-e29ceb0005b8","added_by":"auto","created_at":"2025-04-29 06:01:35","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2196784,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6035585/v1/1073380f83348b3b4307727f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Centenarian Single Nucleotide Polymorphism in collagen gene COL25A1 promotes longevity in C. elegans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCentenarians not only achieve exceptional longevity but also often exhibit delayed onset of age-related diseases, providing valuable insights into the biological mechanisms of healthy aging \u003csup\u003e1\u003c/sup\u003e. While lifestyle factors, such as diet and physical activity, contribute to extended lifespans\u0026mdash;as observed in the Okinawan population \u003csup\u003e2\u003c/sup\u003e\u0026mdash;the genetic determinants that interact with these factors remain less understood.\u003c/p\u003e\n\u003cp\u003eGenome-wide association studies (GWAS) have begun to uncover genetic variations associated with longevity \u003csup\u003e3\u003c/sup\u003e. Notably, studies like the New England Centenarian Study and the Long Life Family Study have identified genetic loci that may contribute to extended lifespan and reduced disease incidence among centenarians \u003csup\u003e4\u003c/sup\u003e. One such study by Puca and colleagues \u003csup\u003e5\u003c/sup\u003e identified a significant locus on chromosome 4 (marker D4S1564) associated with exceptional longevity in a cohort of 308 individuals from 137 long-lived families. However, this study was prior to the human genome being sequenced, and thus, the specific genes and variants within this locus were not characterized.\u003c/p\u003e\n\u003cp\u003eHere, we focus on the chromosome 4 locus identified by Puca et al. and investigate the collagen gene \u003cem\u003eCOL25A1\u003c/em\u003e as a candidate for influencing longevity \u003csup\u003e5\u003c/sup\u003e. We utilized \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e as a model organism to assess the role of a centenarian-associated single nucleotide polymorphism (SNP), S106L, introduced into\u003cem\u003e col-99\u003c/em\u003e, the ortholog of human \u003cem\u003eCOL25A1 \u003c/em\u003e\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e. We demonstrate that this SNP promotes lifespan extension in \u003cem\u003eC. elegans\u003c/em\u003e. Mechanistically, we propose that the S106L mutation alters the cleavage rate of COL-99 \u003cem\u003ein vitro\u003c/em\u003e, and the cleaved extracellular domain of COL-99 is sufficient to extend lifespan. Additionally, our findings indicate that genes involved in the innate immune response are upregulated in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants and that the p38 mitogen-activated protein kinase (MAPK) pathway and the transcriptional co-activator yes-associated protein 1 (YAP-1) are crucial for this lifespan extension.\u003c/p\u003e\n\u003cp\u003eOur study provides novel insights into the genetic mechanisms regulating aging by identifying a specific genetic variant that influences lifespan through modulation of the innate immune response and MAPK signaling pathways. These findings may contribute to developing targeted interventions for age-related diseases and enhance our understanding of the genetic factors underlying human longevity.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eIntroduction of centenarian \u003cem\u003eCOL25A1\u003c/em\u003e SNP in \u003cem\u003eC. elegans\u003c/em\u003e homolog \u003cem\u003ecol-99\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn 2001, Puca and colleagues performed a linkage study in centenarian sibling pairs to identify genetic loci associated with exceptional longevity \u003csup\u003e5\u003c/sup\u003e. Interestingly, they found a 3 cM locus on chromosome 4 that was strongly associated with longevity \u003csup\u003e5\u003c/sup\u003e. However, the human genome was not sequenced at the time of publication, and this region was not characterized. Therefore, we investigated this locus and found 108 ensemble features, including 33 protein-coding genes, 14 linc RNA, 2 miRNA, 2 snoRNA, and 9 snRNA (\u003cstrong\u003eFigure 1a, S1a, Supplementary Table 1\u003c/strong\u003e). To identify causally implicated protein-coding genes, we cross-referenced genes to identify those with longevity-associated SNPs in centenarian GWAS or with studies showing that altering gene function would increase lifespan in any organism (\u003cstrong\u003eFigure S1a, Supplementary Table 1\u003c/strong\u003e). Strikingly, we found a collagen gene, \u003cem\u003eCOL25A1\u003c/em\u003e, which was previously shown to be significantly associated with human healthy aging (\u0026ldquo;Wellderly phenotype\u0026quot;), but without mechanistic validation \u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn a subsequent literature search, we found that \u003cem\u003eCOL25A1\u003c/em\u003e exhibited several SNPs associated with exceptional lifespan and/or healthy aging (\u003cstrong\u003eFigure 1b, Supplementary Table 1)\u003c/strong\u003e. Most of the molecular alterations of these longevity-associated SNPs were predicted to result in the loss or reduction of the function of \u003cem\u003eCOL25A1\u003c/em\u003e. To determine whether the loss of \u003cem\u003eCOL25A1\u003c/em\u003e is beneficial, we investigated the effect of a loss-of-function mutation in the \u003cem\u003eCOL25A1\u003c/em\u003e orthologue \u003cem\u003ecol-99\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e. However, the\u003cem\u003e col-99(ok1204)\u003c/em\u003e deletion mutant was short-lived with a mean lifespan of 18.4 days, approximately 5 days less than the wild type (\u003cstrong\u003eFigure 1b; blue line, Supplementary Table 2\u003c/strong\u003e). Overexpression of full-length COL-99 protein tagged with GFP, on the other hand, did not affect the lifespan (\u003cstrong\u003eFigure 1b; green line, Supplementary Table 2\u003c/strong\u003e). Assuming \u003cem\u003eCOL25A1\u003c/em\u003e is responsible for the above-mentioned linkage, these findings indicated that the effect of SNPs in \u003cem\u003eCOL25A1\u003c/em\u003e on longevity has a more complex mechanism. \u003c/p\u003e\n\u003ch2\u003eS106L SNP in \u003cem\u003ecol-99\u003c/em\u003e extends lifespan in \u003cem\u003eC. elegans\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eTo investigate this unknown mechanism, we tried to map all human SNPs in the \u003cem\u003eC. elegans\u003c/em\u003e \u003cem\u003ecol-99\u003c/em\u003e gene. However, due to the vast evolutionary distance and the repetitive nature of collagen, we could only reliably map three sites with some confidence (human S116L \u0026rarr; \u003cem\u003eC. elegans \u003c/em\u003eS106, G225C \u0026rarr; G215, R402C \u0026rarr; R393) (\u003cstrong\u003eFigure 1c\u003c/strong\u003e). We focused on the S116L SNP in \u003cem\u003eCOL25A1\u003c/em\u003e (S106L in \u003cem\u003ecol-99\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e) as it is proximal to a conserved furin cleavage site with the potential to impact domain processing (\u003cstrong\u003eFigure 1c)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFirst, to determine if the human S116L SNP introduction into \u003cem\u003ecol-99\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e had any effect on lifespan, we obtained \u003cem\u003eC. elegans\u003c/em\u003e harboring this S106L SNP in \u003cem\u003ecol-99 \u003c/em\u003e(hereafter referred to as \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants) and outcrossed ten times (10X) to remove any potential background mutations. We then performed lifespan studies with 4X, 8X, and 10X outcrossed \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants and found all outcrossed \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants increased lifespans (\u003cstrong\u003eFigure 2a, S1b, Supplementary Table 2\u003c/strong\u003e) by approximately 26% compared to wild-type control, indicating not only that S106L in humans might indeed be causative for prolonged life, but also re-assuring our mapping of this SNP between the species.\u003c/p\u003e\n\u003cp\u003eTo independently validate that the increased lifespan is due to the single mutation in \u003cem\u003ecol-99\u003c/em\u003e, we CRISPR-generated the S106L SNP mutation into wild-type animals (hereafter referred to as \u003cem\u003ecol-99(syb4350\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e ). Reassuringly, we found a similar extension in the lifespan of \u003cem\u003ecol-99(syb4350\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e compared to the wild type (\u003cstrong\u003eFigure S1c, Supplementary Table 2)\u003c/strong\u003e. The \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants superficially looked wild type but were slightly delayed developmentally by a few hours, with larval L4 stage (\u003cstrong\u003eFigure S1d, Supplementary Table 2\u003c/strong\u003e). Furthermore, the longevity of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants did not correlate with oxidative or heat stress resilience (\u003cem\u003ei.e., col-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants were more susceptible to 14 mM arsenite or 32 ℃ treatment; \u003cstrong\u003eFigures S1e-f, Supplementary Table 2\u003c/strong\u003e).\u003c/p\u003e\n\u003ch2\u003eThe extracellular domain of COL-99 is sufficient for increased longevity\u003c/h2\u003e\n\u003cp\u003eCOL25A1 in humans and COL-99 in \u003cem\u003eC. elegans\u003c/em\u003e are both single-pass type II transmembrane collagen proteins with predicted furin cleavage sites \u003csup\u003e7,10\u003c/sup\u003e. For \u003cem\u003eCOL25A1\u003c/em\u003e, this site is reported to be important for shedding, creating the so-called CLAC (collagen-like Alzheimer amyloid plaque component) fragment \u003csup\u003e11\u003c/sup\u003e. Besides its association with Alzheimer\u0026rsquo;s Disease, the function of this shredded collagen fragment is not understood, although it seems to play a role in muscle development and innervation \u003csup\u003e12,13\u003c/sup\u003e. Similarly, in \u003cem\u003eC. elegans,\u003c/em\u003e \u003cem\u003ecol-99 \u003c/em\u003ewas important for axonal guidance, and the first and third predicted furin cleavage sites seem important for its function \u003csup\u003e6,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs S116L (and S106L) are close to the conserved (first) furin cleavage site, we hypothesized that the S106L mutation in \u003cem\u003eC. elegans\u003c/em\u003e might affect furin cleavage, leading to a change in the extracellular domain shedding rate. To test this hypothesis, we expressed the extracellular domain of COL-99 under its endogenous promoter in \u003cem\u003eC. elegans\u003c/em\u003e. The expression of a shed extracellular domain of COL-99 prolonged the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e by approximately 11% already (\u003cstrong\u003eFigure 2b,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e), consistent with the longevity effect of the S106L SNP in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e) \u003c/em\u003emutant. \u003c/p\u003e\n\u003cp\u003eAlthough the furin recognition site is often described as R-X-[K/R]-R\u0026dArr;, the full recognition sequence is longer and amino acids up to 14 amino acids N-terminally (P14) and six amino acids C-terminally (P6\u0026prime;) from the cleavage site are described to influence furin activity (see Figure 3a for clarification of P1/P1\u0026rsquo; nomenclature and positioning in COL-99 and COL25A1) \u003csup\u003e14\u003c/sup\u003e. To measure the effect of an S to L mutation at the P2\u0026rsquo; site in \u003cem\u003eC. elegans,\u003c/em\u003e we synthesized short fluorescently quenched peptides containing the furin cleavage site and a serine or leucine residue, respectively. The quenching was resolved upon cleavage, and an increased fluorescence was observed (\u003cstrong\u003eFigure 2c\u003c/strong\u003e). Contrary to our expectation, the mutant showed a roughly halved Vmax (29 vs 52) compared to the wild-type sequence, indicating cleavage of S106L by furin, but at a slightly decreased rate. Given the abundance of furin, this small difference can hardly explain the observed effect \u003cem\u003ein vivo,\u003c/em\u003e especially as it seems to contradict our finding from the expression of the extracellular domain alone, which suggested benefits from increased cleavage.\u003c/p\u003e\n\u003cp\u003eBesides its location near the furin cleavage site, both sites are also predicted to be phosphorylated (\u003cstrong\u003eFigure 3a\u003c/strong\u003e). Although extracellular phosphorylation is not completely understood yet, its importance has become more evident over the last few years \u003csup\u003e15\u0026ndash;18\u003c/sup\u003e. Bioinformatically, several phosphorylation sites in COL25A1 have been predicted \u003csup\u003e19\u003c/sup\u003e. Furthermore, it was shown at least twice that phosphorylation might affect furin cleavage \u003csup\u003e20\u003c/sup\u003e. We therefore speculated that phosphorylation of S106 might inhibit COL-99 shedding, thereby reducing the extracellular concentration of the extracellular domain of COL-99. The described SNP might prevent this phospho‑dependent inhibition, thus increasing shedding. Consequently, a CRISPR mutant of \u003cem\u003ecol-99\u003c/em\u003e introducing a phosphomimetic S106D exchange (\u003cem\u003ecol-99(syb4352\u003c/em\u003e[S106D]\u003cem\u003e)\u003c/em\u003e) did not significantly affect lifespan (3 out of 5 independent trials; \u003cstrong\u003eFigure 3b, Supplementary Table 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTaken together, this suggests a model wherein phosphorylation of serine 106 influences furin cleavage and release of the extracellular domain of COL-99 to alter downstream processes to increase lifespan.\u003c/p\u003e\n\u003ch2\u003eInnate immune response is upregulated in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e\u003cem\u003e \u003c/em\u003emutants\u003c/h2\u003e\n\u003cp\u003eTo identify the downstream mechanisms affected by the S106L mutation in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e, we performed transcriptomic profiling of the \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutant and wild type at the young adult stage. The introduction of \u003cem\u003ecol-99 \u003c/em\u003eSNP drove limited transcriptomic effects (\u003cstrong\u003eFigure 4a, Supplementary Table 3\u003c/strong\u003e). Interestingly, the \u003cem\u003ecol-99 \u003c/em\u003eSNP did drive a ~20% increase in the \u003cem\u003ecol-99\u003c/em\u003e transcript level (\u003cstrong\u003eFigure 4b\u003c/strong\u003e), suggesting an autoregulated overexpression. Gene ontology term analysis revealed a downregulation of genes involved in oxidative phosphorylation, while innate immune response genes were upregulated (\u003cstrong\u003eFigure 4c, Supplementary Table 3\u003c/strong\u003e). ATF-7 is the master regulator of immune response in \u003cem\u003eC. elegans \u003c/em\u003e\u003csup\u003e21\u003c/sup\u003e, and we found it to be upregulated by \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e expression (\u003cstrong\u003eFigure S2a, Supplementary Table 3\u003c/strong\u003e). However, in our epistatic lifespan analysis, we observed that \u003cem\u003eatf-7 \u003c/em\u003ewas not required for lifespan extension in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e) \u003c/em\u003emutants (\u003cstrong\u003eFigure S2b, Supplementary Table 2\u003c/strong\u003e). Arginine kinase (creatine kinase) \u003cem\u003eargk-1\u003c/em\u003e was also upregulated in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants (\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e). We verified this increase in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants with a reporter strain \u003cem\u003eargk-1\u003c/em\u003ep::GFP using fluorescence microscopy (\u003cstrong\u003eFigure S2c\u003c/strong\u003e). Previously, ARGK-1 overexpression increased lifespan via AMPK, which triggers the activation of the innate immune response \u003csup\u003e22,23\u003c/sup\u003e. Therefore, we checked for the requirement of \u003cem\u003eargk-1 \u003c/em\u003ein lifespan extension by \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants. We assayed the lifespan of \u003cem\u003eargk-1(ok2993); col-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e double mutants but found that the extended lifespan of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutant was independent of \u003cem\u003eargk-1 \u003c/em\u003e(\u003cstrong\u003eFigure S2d, Supplementary Table 2\u003c/strong\u003e). Since DAF-16 is also an important modulator of innate immune response and lifespan \u003csup\u003e24\u003c/sup\u003e, we tested the nuclear localization of DAF-16 in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants. However, the differences in DAF-16 nuclear localization were insignificant (\u003cstrong\u003eFigure S2e\u003c/strong\u003e). Taken together, our approach in testing these genes differentially expressed in RNA-sequencing did not identify underlying pathways driving longevity. \u003c/p\u003e\n\u003cp\u003eNext, we used a candidate RNAi screen approach to identify the pathways mediating \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e lifespan extension. Given the strong innate immune transcriptional signature, we tested two immune-response reporter genes, \u003cem\u003eclec-85\u003c/em\u003e and \u003cem\u003eT24B8.5,\u003c/em\u003e that were also differentially regulated by \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e (\u003cstrong\u003eFigures 4d, S2f, Supplementary Table 3\u003c/strong\u003e). The transcriptional reporter driving GFP under the \u003cem\u003eT24B8.5 \u003c/em\u003epromoter (\u003cem\u003eT24B8.5p\u003c/em\u003e::GFP \u003csup\u003e25\u003c/sup\u003e) showed a two-fold upregulation in the fluorescent intensity in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants compared to wild-type, making it suitable for screening. Given that \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e induces \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP \u003cem\u003ein vivo\u003c/em\u003e, knocking down genes that are required for mediating \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-dependent T24B8.5 upregulation may reveal underlying pathways. Therefore, we screened through a targeted candidate library including known immunogenic-, collagen-, and mechano-receptors using the \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP reporter strain. \u003c/p\u003e\n\u003cp\u003eWe started with genes in the canonical innate immune response pathway (P38 Map Kinase \u003cem\u003epmk-1 \u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e, Toll and Interleukin 1 Receptor \u003cem\u003etir-1 \u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e, ATF cAMP-dependent transcription factor \u003cem\u003eatf-7 \u003c/em\u003e\u003csup\u003e28\u003c/sup\u003e, and Toll-like receptor \u003cem\u003etol-1\u003c/em\u003e \u003csup\u003e29\u003c/sup\u003e) and found that RNAi targeting \u003cem\u003etir-1\u003c/em\u003e suppressed the \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-mediated \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP induction (\u003cstrong\u003eFigure 4d\u003c/strong\u003e), indicating that this immune receptor was required. However, it is unlikely that the extracellular domain of COL-99 would bind the TIR-1 receptor directly for signaling, suggesting additional upstream signaling. To identify the potential direct binding partners/receptors of shed COL-99 extracellular domain, we turned to the COL-99 collagen receptors, the Discoidin Domain Receptors (\u003cem\u003eddr-1\u003c/em\u003e and \u003cem\u003eddr-2\u003c/em\u003e), and basement membrane component nidogen homolog \u003cem\u003enid-1\u003c/em\u003e, which are putative binding partners of the cleaved COL-99 ectodomain and are involved in \u003cem\u003ecol-99\u003c/em\u003e-mediated neuronal axon guidance \u003csup\u003e6\u003c/sup\u003e. However, knockdown of \u003cem\u003eddr-1, ddr-2,\u003c/em\u003e or \u003cem\u003enid-1\u003c/em\u003e did not suppress \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)-\u003c/em\u003emediated \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP induction (\u003cstrong\u003eFigure 4d\u003c/strong\u003e). COL-99 is expressed in the hypodermis, and the furin cleavage and shedding of the COL-99 ectodomain are required for neuronal axon guidance \u003csup\u003e6\u003c/sup\u003e. Thus, a neuronal receptor may be required. We tested neuronal and mechanosensitive transient receptor potential (TRP) channel \u003cem\u003etrpa-1/\u003c/em\u003eTRPA and \u003cem\u003eosm-9/\u003c/em\u003eTRPV, which are shown to be involved in longevity \u003csup\u003e30,31\u003c/sup\u003e. We found that \u003cem\u003eosm-9\u003c/em\u003e was required for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-mediated \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP induction (\u003cstrong\u003eFigure 4d\u003c/strong\u003e). OSM-9 is a mechanosensitive receptor upstream of YAP-1 (yes-associated protein), a mechanoresponsive transcriptional co-activator activated upon stiffness changes in the extracellular collagen network and implicated in the control of lifespan \u003csup\u003e32\u0026ndash;34\u003c/sup\u003e. In addition, \u003cem\u003eyap-1\u003c/em\u003e is functionally implicated in Touch Receptor Neuron asymmetric neurite extension \u003csup\u003e35\u003c/sup\u003e and pathogen resistance in \u003cem\u003eC. elegans\u003c/em\u003e \u003csup\u003e36\u003c/sup\u003e, consistent with a potential role for neuronal regulation and innate immunity. We found that the knockdown of \u003cem\u003eyap-1\u003c/em\u003e also suppressed \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-mediated \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP induction (\u003cstrong\u003eFigure 4d\u003c/strong\u003e). Taken together, we identified two receptors, \u003cem\u003etir-1\u003c/em\u003e and \u003cem\u003eosm-9\u003c/em\u003e, and the co-transcriptional activator \u003cem\u003eyap-1\u003c/em\u003e required for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e) \u003c/em\u003einduction of the transcriptional innate immune response program. By contrast, \u003cem\u003eyap-1\u003c/em\u003e and \u003cem\u003etir-1\u003c/em\u003e were not required for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e induction of the unfolded protein response (UPR) gene \u003cem\u003ehsp-4 \u003c/em\u003e(\u003cstrong\u003eFigure S2g-h\u003c/strong\u003e), suggesting that our reporter screen findings need to be validated with functional lifespan assays.\u003c/p\u003e\n\u003ch2\u003eThe P38 MAPK pathway is required for longevity extension by \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eGiven the distinct downstream pathways we recovered for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e, we aimed to determine the pathway that mediates \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e longevity. For instance, our reporter assays revealed that the COL-99 receptors \u003cem\u003eddr-1\u003c/em\u003e and \u003cem\u003eddr-2\u003c/em\u003e were not required for immune and UPR responses; this does not rule out their role in lifespan regulation. However, we found that loss of \u003cem\u003eddr-1, \u003c/em\u003eor\u003cem\u003e ddr-2, \u003c/em\u003eor the double mutant \u003cem\u003eddr-1; ddr-2\u003c/em\u003e did not suppress \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-induced longevity (\u003cstrong\u003eFigure S3a, Supplementary Table 2\u003c/strong\u003e), suggesting that DDR-1 and DDR-2 are the COL-99 receptor for axonal guidance \u003csup\u003e37\u003c/sup\u003e but not for longevity. Another likely candidate for COL-99 ectodomain receptor is TIR-1, and we found that \u003cem\u003etir-1\u003c/em\u003e was required for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e-induced lifespan extension (\u003cstrong\u003eFigure 5a, Supplementary Table 2\u003c/strong\u003e). Since \u003cem\u003etir-1\u003c/em\u003e is upstream of the p38 MAPK signaling cascade, we tested whether the downstream protein p38 MAPK (\u003cem\u003epmk-1\u003c/em\u003e) was required for lifespan extension. Although RNAi of \u003cem\u003epmk-1\u003c/em\u003e was not sufficient to block \u003cem\u003eT24B8.5p\u003c/em\u003e::GFP induction in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants, knockdown of \u003cem\u003epmk-1\u003c/em\u003e abolished the longevity of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants (\u003cstrong\u003eFigure 5b, Supplementary Table 2\u003c/strong\u003e). We confirmed these lifespan results of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e with the CRISPR-generated mutant CED04 \u003cem\u003ecol-99(syb4350\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e, and also showed no adverse effects with the phosphomimetic CED05 \u003cem\u003ecol-99(syb4352\u003c/em\u003e[S106D]\u003cem\u003e)\u003c/em\u003e mutants in combination with \u003cem\u003etir-1\u003c/em\u003e or \u003cem\u003epmk-1\u003c/em\u003e knockdown (\u003cstrong\u003eFigure S3b-d, Supplementary Table 2\u003c/strong\u003e). Consistently, \u003cem\u003etir-1\u003c/em\u003e and \u003cem\u003epmk-1 \u003c/em\u003ewere required for lifespan extension in the CED06 \u003cem\u003ecol-99(syb4332\u003c/em\u003e[COL-99EXT]\u003cem\u003e)\u003c/em\u003e mutant expressing the ectodomain of COL-99 (\u003cstrong\u003eFigure 5c-d, Supplementary Table 2\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eFurthermore, in line with the requirement of \u003cem\u003epmk-1\u003c/em\u003e for lifespan extension, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants showed higher p38 MAPK/PMK-1 phosphorylation (\u003cstrong\u003eFigure 5e, Supplementary Table 2\u003c/strong\u003e), an event generally known to drive increased ATF-7 transcription factor levels. Despite this association, ATF-7 was not required for the extended lifespan in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants (\u003cstrong\u003eFigure S2b, Supplementary Table 2\u003c/strong\u003e). This led us to investigate other potential mediators, and we discovered that YAP-1 played a critical role in facilitating the longevity effects of \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants (\u003cstrong\u003eFigure 5f, Supplementary Table 2\u003c/strong\u003e). Collectively, our data support a model in which a non-canonical immune response pathway involving \u003cem\u003eosm-9, tir-1, pmk-1,\u003c/em\u003e and \u003cem\u003eyap-1\u003c/em\u003e is activated upon blunting the phosphorylation of COL-99 at the S106 site, and thus altered furin-mediated cleavage resulting in the subsequent release of the COL-99 ectodomain (\u003cstrong\u003eFigures 5g, S4\u003c/strong\u003e). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreviously, in at least three centenarian cohorts and one healthy aging cohort, several SNPs in collagen XXV associated with healthy human aging have been identified \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Despite these associations, causal mechanisms linking specific genetic variants to healthy longevity remain largely unvalidated. In this study, we addressed this gap by demonstrating that a rare coding variant in the collagen gene \u003cem\u003eCOL25A1\u003c/em\u003e, specifically the S116L SNP in the \u003cem\u003eCOL25A1\u003c/em\u003e ortholog, is sufficient to extend the lifespan in \u003cem\u003eC. elegans\u003c/em\u003e. By introducing the S106L orthologous mutation into \u003cem\u003ecol-99\u003c/em\u003e, we observed an approximately 26% increase in lifespan, suggesting a conserved role of this variant in longevity.\u003c/p\u003e \u003cp\u003eMechanistically, the S106L mutation influences furin-mediated cleavage of transmembrane COL-99, altering the release of its extracellular domain. This extracellular domain is sufficient to promote lifespan extension, implicating the remodeling of extracellular matrix (ECM) in aging processes. Our results also reveal that the lifespan extension in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutants depends on the p38 MAPK pathway and the transcriptional co-activator YAP-1. Interestingly, this effect is independent of ATF-7, a typical downstream target of p38 MAPK involved in immune responses \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, indicating alternative signaling pathways are at play.\u003c/p\u003e \u003cp\u003eThe involvement of YAP-1, known for its roles in mechanotransduction and cellular proliferation \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and mechanotransduction-mediated longevity \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, suggests that ECM changes can influence longevity through mechanoresponsive signaling pathways. The activation of innate immune response genes further supports a model where ECM alterations modulate immune signaling to impact aging.\u003c/p\u003e \u003cp\u003eOur findings suggest that similar mechanisms may operate in humans, given the conservation of these pathways. Proteomic analyses of centenarians have revealed elevated levels of proteins involved in angiogenesis and cell junctions, suggesting enhanced tissue homeostasis \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Additionally, single-cell transcriptomics of peripheral blood mononuclear cells (PBMCs) from centenarians has indicated improved immune function, characterized by increased T cell populations \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Notably, serum proteomic signatures have shown enrichment of collagen proteins, such as COL28A1 and COL6A3, in centenarians \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, highlighting the potential role of collagen in longevity. Identifying \u003cem\u003eCOL25A1\u003c/em\u003e variants affecting furin cleavage and ECM dynamics opens new avenues for research into the genetic regulation of human aging. Future studies should focus on elucidating where COL-99 serine 106 is phosphorylated, \u003cem\u003ei.e.\u003c/em\u003e, in the Golgi or the extracellular space. Furthermore, our hypothetical model (\u003cb\u003eFigure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e) shows genetic interactions, but the direct interactions between the COL-99 extracellular domain and its signaling partners are needed to fully understand the mechanisms underlying this lifespan extension.\u003c/p\u003e \u003cp\u003eOne limitation of our study is the use of \u003cem\u003eC. elegans\u003c/em\u003e as a model organism, which, while highly informative, may not fully recapitulate the complexity of human aging. Additionally, the direct applicability of the S116L variant in humans requires further validation in mammalian systems.\u003c/p\u003e \u003cp\u003eInterestingly, several human genetic studies associate \u003cem\u003eCOL25A1\u003c/em\u003e variants with healthy aging, longevity, and age-related diseases (Fig.\u0026nbsp;1\u003cb\u003ec\u003c/b\u003e, Supplementary Table\u0026nbsp;1). For instance, SNPs in \u003cem\u003eCOL25A1\u003c/em\u003e are associated with Alzheimer\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, which maps to the Aβ-binding side in COL25A1, suggesting a failure of the extracellular domain of COL25A1 to scoop up Aβ oligomers \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Similarly, SNPs in \u003cem\u003eCOL25A1\u003c/em\u003e are associated with congenital cranial dysinnervation disorder \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, which can lead to a loss of the most outer collagen domain (COL3) in COL25A1 expressed from the muscle that is required to bind to receptors protein tyrosine phosphatases σ and δ (PTP σ/δ), a process essential for intramuscular motor innervation\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Previous comparisons of centenarians with disease GWAS studies found an overlap of SNPs in genes associated with diseases, such as Alzheimer\u0026rsquo;s and coronary artery diseases, and other SNPs in the same gene associated with longevity \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, these risk alleles in these genes for diseases are depleted in centenarians and/or compensated by a constellation of multiple beneficial genetic variants in centenarians \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. These observations suggest that centenarians in given disease pathways have resilience variants tipping the balance to health and not down towards disease progression.\u003c/p\u003e \u003cp\u003eIn line with this idea, mapping proteins associated with human healthspan, COL25A1 is in the first central node together with LRP1, TOMM40, and CREBBP, which are also implicated in Alzheimer\u0026rsquo;s disease and potentially under dietary restrictions might signal through NOTCH to promote human healthspan \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Further GWAS studies revealed SNPs in \u003cem\u003eCOL25A1\u003c/em\u003e are associated with resistance to COVID-19 infections \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and higher spermidine levels \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, a polyamine compound inducing autophagy and increasing lifespan of model organisms \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, suggesting some implications in resilience.\u003c/p\u003e \u003cp\u003eIn summary, our research identifies a causal role for a coding variant in COL25A1 in extending lifespan via modulation of ECM remodeling and immune signaling pathways. These insights advance our understanding of the genetic contributors to longevity and may inform the development of therapeutic strategies targeting age-related diseases.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eStrain maintenance\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll strains were maintained at 20 ℃, on NGM (nematode growth media) plates seeded with \u003cem\u003eEscherichia coli\u003c/em\u003e OP50.\u003c/p\u003e \u003cp\u003eThe strains used in this study are: N2 (wild-type Bristol), RB1165 \u003cem\u003ecol-99(ok1204)\u003c/em\u003e IV, COL-99 overexpression VH2847 \u003cem\u003ehdIs73\u003c/em\u003e [GFP::COL-99, pha-1(+)], AU78 \u003cem\u003eagIs219\u003c/em\u003e [T24B8.5p::GFP::\u003cem\u003eunc-54\u003c/em\u003e 3\u0026rsquo;UTR\u0026thinsp;+\u0026thinsp;\u003cem\u003ettx-3\u003c/em\u003ep::GFP::\u003cem\u003eunc-54\u003c/em\u003e 3\u0026rsquo;UTR] III, \u003cem\u003eyap-1(tm1416)\u003c/em\u003e, VC1518 \u003cem\u003eatf-7(gk715)\u003c/em\u003e III, MAH547 \u003cem\u003esqEx82\u003c/em\u003e [\u003cem\u003eargk-1p\u003c/em\u003e::GFP\u0026thinsp;+\u0026thinsp;\u003cem\u003erol-6\u003c/em\u003e(su1006)], MAH205 \u003cem\u003eargk-1(ok2993\u003c/em\u003e) V, TJ356 \u003cem\u003ezIs356\u003c/em\u003e [\u003cem\u003edaf-16\u003c/em\u003ep::daf-16a/b::GFP\u0026thinsp;+\u0026thinsp;\u003cem\u003erol-6\u003c/em\u003e(su1006)], SAL146 \u003cem\u003epha-1\u003c/em\u003e(e2123) III; \u003cem\u003edenEx24\u003c/em\u003e [\u003cem\u003eclec-85\u003c/em\u003ep::GFP\u0026thinsp;+\u0026thinsp;\u003cem\u003epha-1\u003c/em\u003e(+)], SJ4005 \u003cem\u003ezcIs4\u003c/em\u003e [\u003cem\u003ehsp-4\u003c/em\u003e::GFP] V, RB970 \u003cem\u003eddr-1(ok874)\u003c/em\u003e, RB788 \u003cem\u003eddr-2(ok574)\u003c/em\u003e, VH1681 \u003cem\u003eddr-1(ok874); ddr-2(ok574)\u003c/em\u003e X; \u003cem\u003eevIs111\u003c/em\u003e[\u003cem\u003ergef\u003c/em\u003e::GFP] V, VC40561 \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant [4 times, 8 times, and 10 times outcrossed] \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ecol-99\u003c/em\u003e(SNP) is interchangeably used in figures for \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e mutant, which is originally the VC40561 strain obtained from the Million mutation project. The strain has a missense mutation G\u0026rarr;A at position 144813 on chromosome IV, generating \u003cem\u003ecol-99\u003c/em\u003e(\u003cem\u003egk694263\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStrains generated in this study\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStrains generated by CRISPR gene editing (performed by SUNY Biotech)\u003c/span\u003e- F29C4.8j.1 is the isoform of \u003cem\u003ecol-99\u003c/em\u003e which was modified to obtain the following transgenic strains: CED04 PHX4350 [\u003cem\u003ecol-99(syb4350\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e] (\u003cem\u003ecol-99\u003c/em\u003e S106L mutant): Precise one base pair missense mutation (TCG\u0026rarr;TTG) was introduced in \u003cem\u003ecol-99\u003c/em\u003e gene using CRISPR. It resulted in the expression of nonpolar leucine amino acid (L) at the 106th position, instead of polar serine (S) in wild-type COL-99 (termed as S106L).\u003c/p\u003e \u003cp\u003eCED05 PHX4352 [\u003cem\u003ecol-99(syb4352\u003c/em\u003e[S106D]\u003cem\u003e)\u003c/em\u003e] (\u003cem\u003ecol-99\u003c/em\u003e S106D mutant): The mutation TCG\u0026rarr;GAC was induced in \u003cem\u003ecol-99\u003c/em\u003e gene using CRISPR gene editing, which resulted in the expression of phosphomimetic aspartic acid residue (D) at 106th position, instead of serine (S) in wild-type COL-99 (termed as S106D).\u003c/p\u003e \u003cp\u003eCED06 PHX4332 [\u003cem\u003ecol-99(syb4332\u003c/em\u003e[COL-99EXT]\u003cem\u003e)\u003c/em\u003e] (\u003cem\u003ecol-99\u003c/em\u003e extracellular domain): Precisely 3075 bp (95 aa) were deleted after the first methionine coding codon ATG in \u003cem\u003ecol-99\u003c/em\u003e gene so that first conserved Furin cleavage site \u0026ldquo;RRVR\u0026rdquo; at 104th position \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e in the non-collagenous extracellular domain of COL-99 was expressed, leading to expression of only the COL-99 extracellular domain (COL-99 EXT).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStrains generated by genetic cross\u003c/span\u003e: \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; T24B8.5p::GFP, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eyap-1(tm1416)\u003c/em\u003e, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eatf-7(gk715)\u003c/em\u003e, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eargk-1\u003c/em\u003ep::GFP, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eargk-1(ok2993)\u003c/em\u003e, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; DAF-16::GFP, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eclec-85\u003c/em\u003ep::GFP, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003ehsp-4\u003c/em\u003ep::GFP, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eddr-1(ok874); ddr-2(ok574)\u003c/em\u003e, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e; \u003cem\u003eddr-2(ok574)\u003c/em\u003e, \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e); ddr-1(ok874)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenotyping\u003c/b\u003e \u003cb\u003ecol-99(gk694263\u003c/b\u003e\u003cb\u003e[S106L]\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e \u003cb\u003eSNP mutation\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eA quick and reliable method of SNP detection by endpoint PCR was followed as described previously \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Briefly, the principle of allele variant detection is based on SuperSelective (SS) primers design. A SS primer generally consists of three regions- anchor, bridge, and foot. An \u0026ldquo;anchor\u0026rdquo; is a 5\u0026rsquo; long sequence that hybridizes to the template DNA, followed by an intervening region, called \u0026ldquo;bridge\u0026rdquo; which is non-complementary to the template. \u0026ldquo;Foot\u0026rdquo; region is a 3\u0026rsquo; short sequence complementary to the template, with a terminal mismatch residue. SuperSelective (SS) primers were designed for genotyping a single point mutation in \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e, consisting of a 16 bp anchor, a 6 bp optimal bridge, and a 7 bp foot region with terminal mismatch nucleotide G\u0026rarr;A for wild-type and mutant allele. When primers were annealed to the template DNA, the anchor region hybridized while the bridge region, being non-complementary, formed a bubble-like structure separating the foot region from the anchor. Since the foot region is too short, any single nucleotide mismatch would inhibit the binding of primers and, hence, prevent primer extension. So, when PCR was run with wild-type gDNA as a template, only primers designed for wild-type allele would bind to wild-type gDNA and yield a specific band (expected size of the amplicon is 348 bp) at an annealing temperature of 62 ℃, while a combination of mutant primers with wild-type gDNA should not yield any band.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eReaction conditions\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVolume (\u0026#120525;L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemplate (gDNA) (approx. 5 ng)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5X GoTaq Flexi Buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 mM dNTPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 nM Primer 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 nM Primer 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGo Taq Polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePCR cycle\u003c/span\u003e:\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteps\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemperature (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial Denaturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDenaturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30 cycles\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnnealing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 sec\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElongation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 sec\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cb\u003eIn-silico\u003c/b\u003e \u003cb\u003eprediction of phosphorylation sites in collagen\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFull-length sequences of COL-99 and COL25A1 were subjected to the NetPhos 3.1 algorithm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/NetPhos-3.1/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/NetPhos-3.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and analyzed for serine phosphorylation. Only predictions with a score higher than 0.5 and in the vicinity of the furin cleavage site near the S106 / S116 SNP in question were considered \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAutomated Lifespan Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe automated lifespan assay was performed as described by Stroustrup \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Briefly, \u003cem\u003eC. elegans\u003c/em\u003e populations were subjected to population lysis, and the eggs were transferred to OP50 plates. At the L4 stage, the animals were transferred to either OP50 or RNAi plates that contained 50 \u0026micro;M FUdR. At day two of adulthood, eggs were removed through repeated washing, and the animals were transferred to lifespan plates of the same composition, featuring a tight-fitting lid (BD Falcon Petri Dishes, 50x9 mm). The plates were placed in the Epson V800 flatbed scanners and continuously imaged for 60 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePeptide synthesis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe internally quenched fluorogenic peptides S106S (Abz-VAKSRRVRNS-Lys[DNP]) and S106L (Abz-VAKSRRVRNL-Lys[DNP]) were synthesized by manual and automated solid-phase peptide synthesis (SPPS) (peptide synthesizer from MultiSynTech, Syro I) using the Fmoc (fluorenylmethoxycarbonyl)/t-Bu (tert-butyl) strategy on Rinkamid resin (loading 0.48 mmol, 0.015 mmol scale). First, Fmoc-Lys(DNP)-OH was manually loaded on the resin, and subsequently, the resin was elongated by the peptide synthesizer. All amino acids were coupled using 8 eq each of N,N\u0026prime;-diisopropylcarbodiimide (DIC), and ethyl cyanohydroxyiminoacetate (Oxyma) dissolved in DMF. Fmoc was cleaved off using 20% piperidine in DMF. The final peptides were cleaved by treating the resin for 3 h at room temperature with a mixture of concentrated (conc.) trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5, v/v/v). Following, the peptides were precipitated in ice-cold diethyl ether, washed five times, and freeze-dried. Purification was achieved by preparative reversed-phase (RP)-HPLC (Hitachi Elite LaChrom) on a VP250/16 NUCLEODUR 100-5 C18ec column (Macherey Nagel) using linear gradients from 20\u0026ndash;70% B in A (A\u0026thinsp;=\u0026thinsp;0.1% TFA in water; B\u0026thinsp;=\u0026thinsp;0.1% TFA in ACN) over 60 min. All peptides were identified by RP-HPLC-ESI-MS (column: CC 125/4.6 Nucleodur 100-5 C18e (Macherey-Nagel) using a gradient from 10‑60% of acetonitrile (ACN) in H\u003csub\u003e2\u003c/sub\u003eO with 0.1% formic acid (ESI-MS, Thermo Scientific LTQ-XL). Final purity of all compounds was \u0026gt;\u0026thinsp;95%.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFurin Cleavage Assay Using an Internally Quenched Fluorogenic Peptides\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe activity of furin towards the fluorogenic substrate containing an N-terminal Abz group and a C-terminal Lys-DNP group was measured by the increase in fluorescence at 420 nm that occurs upon cleavage of the peptide bond (|) in the peptide sequence Abz-VAKSRRVR|N(S/L)[-Lys[DNP]. Furin (New England Biolabs, #P8077) concentration was kept constant at a total of 1 unit in the total assay volume of 100 \u0026micro;l. Peptide concentrations were determined based on the absorption of the DNP moieties at 365 nM, calculated based on the extinction coefficient 17\u0026rsquo;300 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1 64\u003c/sup\u003e, and used in concentrations between 1 and 2000 nM. The reaction was performed in 50 mM Hepes (pH 7.5), 10 mM CaCl, 1 mM β-Mercaptoethanol at 25 ℃ and measured in a Fluoromax 4P (HORIBA) with the following settings: Excitation 320 nm (5 nm Bandpass); Emission 420 nm (5 nm Bandpass); Integration time 0.5 s; Interval time 1.0 s; total time 300 s. Initial velocity was determined by linear fit in Origin (OriginLabs) and plotted against substrate concentrations. The resulting titration curve was fitted by the Hill equation for evaluation of maximal velocity and approximate Km of the peptide. Representative data from three different assays are shown.\u003c/p\u003e \u003cp\u003e \u003cb\u003eManual Lifespan Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGravid adults were treated with hypochlorite solution to obtain eggs. These were grown on OP50-seeded NGM plate till the L4 stage. At the L4 stage, 50 animals were transferred to OP50 NGM plates containing FuDR, in replicas of four plates. For RNAi lifespan, animals were grown on the respective RNAi NGM plates seeded with corresponding bacteria (L4440/\u003cem\u003etir-1\u003c/em\u003e/\u003cem\u003epmk-1\u003c/em\u003e) until the L4 stage and then transferred to FuDR-containing RNAi plates at the L4 stage. Survival of the animals was scored by prodding their tail once with platinum wire every alternate day. Unhealthy animals showing vulval bursting or those crawling to the sides/lid of the plates were censored from the population. Lifespan Scoring was started on Day 7 of adulthood, L4 being taken as t\u0026thinsp;=\u0026thinsp;0 timepoint. The survival curve was plotted using GraphPad Prism. The lifespans were performed in at least two biological replicates at 20 ℃. Statistical analysis was performed using the online software OASIS \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. A lifespan summary for individual experiments is provided in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003eTotal RNA sample preparation for Transcriptomics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSynchronized populations of the strains Wild-type (N2), and \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant were grown on OP50 until the young adult stage (approximately 10 x 90 mm NGM petri plates with 500\u0026ndash;800 animals feeding OP50 per plate). Young adult animals were collected in M9 buffer, washed twice with the buffer, and then flash frozen the pellet for storage at -80 ℃ until RNA isolation. RNA isolation was done using the Qiagen RNeasy mini kit (#74004) method according to the manufacturer\u0026rsquo;s protocol. Three biological replicates were collected for the experiment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA Sequencing and Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA concentration and purity were assessed via a Nanodrop spectrophotometer and an Agilent 2100 Bio-Analyzer. cDNA libraries for sequencing were prepared using the Illumina TruSeq RNA Library Prep Kit. Libraries were pooled and sequenced with 150 bp paired-end reads to a target depth of 20\u0026nbsp;million reads per sample. Reads were aligned to WBcel235 reference genome using the align function and annotated to the corresponding GTF using the featureCounts function from the Rsubread R package \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e (v. 2.3.5). Differentially expressed genes were determined using the edgeR \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e (v. 3.30.2) and limma \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e (v. 3.44.2) R packages. Briefly, genes were filtered based on minimum expression level (\u0026gt;\u0026thinsp;5 counts per million in at least 3 samples). Normalization was carried out using the trimmed mean of M-values method as implemented in calcNormFactors from edgeR. Data were modeled and differentially determined using the limma voom workflow to generate linear models with empirical Bayes moderation. Gene set overrepresentation analysis was performed using the enrichGO function from the clusterProfiler \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e (v. 3.15.0) R package, using lists of genes with either significantly increased or decreased expression and compared to a background list of all genes detected in the dataset. Raw reads are available at the NIH Sequence Read Archive (SRA) under accession number PRJNA1198094.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxidative Stress Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eYoung adult wild-type or \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant animals grown on OP50 NGM plates were collected in M9 buffer and washed three times with M9 buffer by centrifugation. Approx. 30\u0026ndash;40 animals were pipetted into the wells of a 96-well round-bottom plate. Animals were either swimming in 30 \u0026micro;L of M9 buffer (filled in the first two rows of the 96-well round bottom plate) or 14 mM sodium arsenite solution (in the rest of the 6 rows). Every column of the plate was denoted with one condition or strain. Stress resistance was measured as a function of the motility of the animals scored for 3 days in an automated wormtracker (wMicroTracker by NemaMetrix) platform. The resulting quantified motility output was analyzed and plotted using an R Script generated in our lab (Ewaldlab-LSD @ github). The experiment was done in three biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAutomated Heat stress survival assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSynchronized wild-type or \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP mutant populations were grown until the young adult stage on the OP50 NGM plate. At the young adult stage, animals were shifted to a 32 ℃ (or 35 ℃) incubator for initiating heat stress. The incubator was equipped with scanners that were set to scan the plates every half an hour and capture the images of the animals for 3 days. Survival data of the animals was recorded and generated automatically after we manually decoded images of the animals from misleading object data points. The survival curve was plotted using an R script developed in our lab (Ewaldlab-LSD @ github). The experiment was done in two biological replicates at 32 ℃ and once at 35 ℃.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDevelopmental Time Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTen gravid adult Day 1 animals were allowed to lay eggs on OP50-seeded NGM plates for 4 h and then sacrificed. After 48 h, the number of animals was estimated in different life cycle stages. Three plates per condition (technical replicates) were kept to assess the developmental stages. The total number of animals in a particular stage was plotted in a bar graph. Two-way ANOVA statistical analysis was performed in GraphPad Prism.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescent Microscopy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGravid adults were treated with sodium hypochlorite solution to obtain eggs. The eggs were grown on OP50 or respective RNAi bacteria seeded NGM plate until young adults. Approx. 30 young adult animals were mounted onto a 2% agarose pad and anesthetized with 20 mM levamisole for imaging. For \u003cem\u003ehsp-4\u003c/em\u003ep::GFP, young adults were heat-shocked at 33 ℃ for 2 h and imaged after recovery from shock for 6 h at 20 ℃. Animals were stacked together and captured at 10X with one or two fields of view and then stitched together later using ImageJ. Quantifying the total fluorescence intensity per number of pixels was done by running a Python script, GreenIntensityCalculator (publicly available in Github-Ewaldlab), in ImageJ. Data is plotted as mean, and error bars represent SEM. Two-tailed Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test statistical analysis of the quantified data was done using GraphPad Prism software (GraphPad Prism 9.0). The experiment was performed in three biological batches, with n\u0026thinsp;\u0026gt;\u0026thinsp;80 total animals per condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDAF-16 nuclear localization assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe synchronized population was grown until the L4 stage on OP50 NGM plates. At the L4 stage, approximately 30 animals were assessed manually for DAF-16 nuclear localization and categorized as none, low, intermediate, or high based on the intensity of the nuclear localization throughout the body. The percentage of animals showing DAF-16 nuclear localization was plotted in GraphPad Prism 9.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blot\u003c/b\u003e \u003c/p\u003e \u003cp\u003eYoung adult animals were harvested in the M9 buffer and washed three times with the M9 buffer to remove excess bacteria. The pellet was flash-frozen and stored at -80 ℃ until further use. To isolate protein from the animals, the pellet was resuspended in Buffer \u0026lsquo;C\u0026rsquo; solution (50 mM HEPES, 100 mM KCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EGTA, 10% Glycerol) with protease inhibitor cocktail (#11697498001, Merck) and then subjected to the homogenizer. The tube was then centrifuged to pellet the insoluble fraction and separate the soluble supernatant in another tube. Protein in the sample was estimated using the Bradford assay (#5000006, Bio-Rad). Approx. 30 ug of protein was run in duplicate lanes on SDS-PAGE gel. After blotting the protein on the PVDF membrane (#IPVH00010, Merck-Millipore), it was blocked in a 5% BSA blocking buffer for 1 hour at room temperature while shaking. Primary P-PMK-1 antibody (#9215S, Cell Signalling Technology) was used in dilution 1:5000, and alpha-tubulin (#T9026, Sigma) was used in dilution 1:10,000 in the blocking buffer overnight at 4 ℃. The Next day, the blots were washed three times with 1X TBST buffer and put in secondary anti-rabbit (#7074S, Cell Signalling Technology) (for P-PMK-1) and anti-mouse (#7076S, Cell Signalling Technology) (for alpha-tubulin) antibodies in a dilution 1:10,000 for 1 h at room temperature. Then, the blots were washed three times with TBST buffer and developed in a chemiluminescent chamber. Clarity Western ECL Blotting Substrate (#\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1705060S\u003c/span\u003e, Bio-Rad) was used to develop the blot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eAll source and raw data are available in Supplementary Tables 1-3 and the RNA-sequencing raw reads are available at the NIH Sequence Read Archive (SRA) under accession number PRJNA1198094.\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eWe thank Garif Yalak for help with the collagen phosphorylation predictions, Valerio Izzi for sharing his analysis of the \u003cem\u003eCOL25A1\u003c/em\u003e variants in LUSC patients, \u0026nbsp;Harald Hutter for the \u003cem\u003ecol-99, ddr-1,\u003c/em\u003e and \u003cem\u003eddr-2\u003c/em\u003e mutants, and WormBase for curated gene and phenotype information. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Funding from the Swiss National Science Foundation Funding from the Swiss National Science Foundation SNF P3 Project 190072 to CYE, CS, and AG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eAll authors participated in analyzing and interpreting the data. CYE and AG designed the \u003cem\u003eC. elegans\u003c/em\u003e experiments. JG performed an \u003cem\u003ein vitro\u003c/em\u003e furin cleavage assay. MM performed the analysis of RNA sequencing. CS performed the automated lifespan analysis. JYCP performed a few \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003e SNP crosses and conducted some reporter assays. AG performed all other lifespan assays and other experiments. AG, JYCP, JG, and CYE wrote the manuscript in consultation with the other authors.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare the following personal relationship which may be considered as potential competing interests: co-author Michael R. MacArthur and Sarah Mitchell, Editor-in-Chief of npj Aging, are married. Dr. Sarah Mitchell was not involved in the journal’s review of, or decisions related to, this manuscript. With no relation to the present manuscript, CYE declares to be a co-founder and shareholder of Avea Life AG and Lichi3 GmbH and is employed by Novartis. Correspondence should be addressed to C. Y. E. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBorras, C. \u003cem\u003eet al.\u003c/em\u003e Centenarians: An excellent example of resilience for successful ageing. \u003cem\u003eMech. Ageing Dev. \u003c/em\u003e\u003cstrong\u003e186\u003c/strong\u003e, 111199 (2020).\u003c/li\u003e\n\u003cli\u003eWillcox, B. J., Willcox, D. C. \u0026amp; Suzuki, M. Demographic, phenotypic, and genetic characteristics of centenarians in Okinawa and Japan: Part 1\u0026mdash;centenarians in Okinawa. \u003cem\u003eMech. Ageing Dev. \u003c/em\u003e\u003cstrong\u003e165\u003c/strong\u003e, 75\u0026ndash;79 (2017).\u003c/li\u003e\n\u003cli\u003eDeelen, J. \u003cem\u003eet al.\u003c/em\u003e Genome-wide association meta-analysis of human longevity identifies a novel locus conferring survival beyond 90 years of age. \u003cem\u003eHum. Mol. 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Biol. \u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, 284\u0026ndash;287 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Centenarians, SNP, col-99, COL25A1, p38 MAPK, yap-1, extracellular matrix, exceptional longevity, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-6035585/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6035585/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBefore human genome sequencing, a genome-wide study of sibling centenarian pairs identified a longevity-associated locus on chromosome 4. Here, we mapped the genes in this locus and identified a collagen gene, \u003cem\u003eCOL25A1.\u003c/em\u003eIntroducing an SNP linked to longevity that changes a serine predicted to be phosphorylated to leucine in \u003cem\u003eCOL25A1\u003c/em\u003e, into \u003cem\u003ecol-99\u003c/em\u003e, the \u003cem\u003eC. elegans\u003c/em\u003e ortholog, extended lifespan. These \u003cem\u003ecol-99(gk694263\u003c/em\u003e[S106L]\u003cem\u003e)\u003c/em\u003eSNP-mutants exhibited enhanced innate immune-related transcriptional responses, and their lifespan extension was abolished by inhibiting the p38 MAPK pathway. YAP-1, a transcriptional co-activator responsive to extracellular matrix changes, was essential for this longevity. Mechanistically, we propose that this SNP modifies furin-mediated cleavage of this transmembrane collagen \u003cem\u003ein vitro,\u003c/em\u003e and expressing the cleaved extracellular domain of COL-99 alone was sufficient to prolong lifespan. These findings reveal a potential mechanism by which a human centenarian-associated SNP in \u003cem\u003eCOL25A1\u003c/em\u003e influences furin cleavage and shedding of the collagen ectodomain to promote healthy longevity.\u003c/p\u003e","manuscriptTitle":"A Centenarian Single Nucleotide Polymorphism in collagen gene COL25A1 promotes longevity in C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 06:01:24","doi":"10.21203/rs.3.rs-6035585/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-27T07:55:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T14:45:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T16:28:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115432150514041627687815497447726236831","date":"2025-05-15T13:14:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72536842970314363893850475380484483898","date":"2025-04-29T17:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-27T02:58:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-26T16:47:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Aging","date":"2025-04-15T04:38:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b377e5a9-b7aa-4609-90a6-a49e97382ea7","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47731795,"name":"Biological sciences/Biochemistry"},{"id":47731796,"name":"Biological sciences/Cell biology"},{"id":47731797,"name":"Biological sciences/Genetics"},{"id":47731798,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-10-06T16:00:26+00:00","versionOfRecord":{"articleIdentity":"rs-6035585","link":"https://doi.org/10.1038/s41514-025-00264-7","journal":{"identity":"npj-aging","isVorOnly":false,"title":"npj Aging"},"publishedOn":"2025-09-30 15:57:10","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-04-29 06:01:24","video":"","vorDoi":"10.1038/s41514-025-00264-7","vorDoiUrl":"https://doi.org/10.1038/s41514-025-00264-7","workflowStages":[]},"version":"v1","identity":"rs-6035585","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6035585","identity":"rs-6035585","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}