Protein Quality Control Failure Enables Toxic Gain-of-Function by TDP43 C-Terminal Fragments

Protein Quality Control Failure Enables Toxic Gain-of-Function by TDP43 C-Terminal Fragments | 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 Research Article Protein Quality Control Failure Enables Toxic Gain-of-Function by TDP43 C-Terminal Fragments Winnie M. Mirembe, Kanchan Budhathoki, Yasar Arfat T Kasu, Kimberly Castro, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8921356/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract C-terminal fragments (CTFs) of TDP43 accumulate in neurodegenerative disease, but whether pathology arises from loss of normal TDP43 function or from toxic gain-of-function effects of the fragments remain unresolved. To address this, we examined two disease-associated fragments, TDP43 219 and TDP43 247 , in cultured neurons and in vivo in the presence of intact endogenous TDP43, thereby isolating fragment-specific effects. TDP43 247 formed prominent cytoplasmic inclusions in neurons, whereas TDP43 219 remained largely soluble, reflecting differences in their susceptibility to protein quality control pathways. Proteomic profiling revealed that proteins detected in soluble CTF fractions were enriched for RNA splicing and post-transcriptional regulatory processes, whereas proteins detected in insoluble CTF fractions were enriched for translation-associated and metabolic pathways. Notably, accumulation of aggregation-prone TDP43 247 disrupted pre-mRNA splicing of STMN2 and UNC13A , inducing cryptic exon inclusion and resulting in loss of Stathmin-2 protein. In vivo expression of TDP43 247 in the mouse motor cortex induced motor impairments despite the continued presence of endogenous TDP43. Together, these findings demonstrate that an elevated aggregate load of TDP43 CTFs can drive neuronal dysfunction through a toxic gain-of-function mechanism, linking impaired clearance to RNA-processing defects in the presence of intact full-length TDP43. aggregation amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease) frontal temporal lobar degeneration (FTLD) proteasome TAR DNA‐binding protein 43 (TDP‐43) (TARDBP) pre-mRNA splicing Stathmin-2 Full Text Additional Declarations No competing interests reported. Supplementary Files FigS2mouseweight.tif TableS1plasmidsandoligos.docx TableS2ProteinsinteractingwithsolubleCTFs.xlsx TableS3ProteinsinteracingwithinsoloubleCTFs.xlsx FigS5hindlimbclasping.tif FigS3survival.tif FigS4Rotarod.tif FigS1stereotaxicsurgery.tif Uncroppedgelsandblots.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8921356","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596835398,"identity":"db214ff5-65b5-470d-a3cf-bc9737785ae1","order_by":0,"name":"Winnie M. 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Fragments","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"aggregation, amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease), frontal temporal lobar degeneration (FTLD), proteasome, TAR DNA‐binding protein 43 (TDP‐43) (TARDBP), pre-mRNA splicing, Stathmin-2","lastPublishedDoi":"10.21203/rs.3.rs-8921356/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8921356/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eC-terminal fragments (CTFs) of TDP43 accumulate in neurodegenerative disease, but whether pathology arises from loss of normal TDP43 function or from toxic gain-of-function effects of the fragments remain unresolved. To address this, we examined two disease-associated fragments, TDP43\u003csup\u003e219\u003c/sup\u003e and TDP43\u003csup\u003e247\u003c/sup\u003e, in cultured neurons and \u003cem\u003ein vivo\u003c/em\u003e in the presence of intact endogenous TDP43, thereby isolating fragment-specific effects. TDP43\u003csup\u003e247\u003c/sup\u003e formed prominent cytoplasmic inclusions in neurons, whereas TDP43\u003csup\u003e219\u003c/sup\u003e remained largely soluble, reflecting differences in their susceptibility to protein quality control pathways. Proteomic profiling revealed that proteins detected in soluble CTF fractions were enriched for RNA splicing and post-transcriptional regulatory processes, whereas proteins detected in insoluble CTF fractions were enriched for translation-associated and metabolic pathways. Notably, accumulation of aggregation-prone TDP43\u003csup\u003e247\u003c/sup\u003e disrupted pre-mRNA splicing of \u003cem\u003eSTMN2\u003c/em\u003e and \u003cem\u003eUNC13A\u003c/em\u003e, inducing cryptic exon inclusion and resulting in loss of Stathmin-2 protein. \u003cem\u003eIn vivo\u003c/em\u003e expression of TDP43\u003csup\u003e247\u003c/sup\u003e in the mouse motor cortex induced motor impairments despite the continued presence of endogenous TDP43. Together, these findings demonstrate that an elevated aggregate load of TDP43 CTFs can drive neuronal dysfunction through a toxic gain-of-function mechanism, linking impaired clearance to RNA-processing defects in the presence of intact full-length TDP43.\u003c/p\u003e","manuscriptTitle":"Protein Quality Control Failure Enables Toxic Gain-of-Function by TDP43 C-Terminal Fragments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 10:46:04","doi":"10.21203/rs.3.rs-8921356/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d715e0a3-37d1-45ca-a591-9c90a66952de","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T05:56:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 10:46:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8921356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8921356","identity":"rs-8921356","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}