Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing mitophagy

Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing mitophagy | 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 Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing mitophagy Jin Wang, Yuyuan Ma, Kaichuang Yang, Weihua Zhang, Weiyu Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9225741/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 Background: The resultant microglial activation and sustained neuroinflammatory response are key contributors to secondary injury, promoting blood-brain barrier disruption, cerebral edema, and progressive neuronal loss. Although NEDD4L has been implicated in inflammation and immunity, the functions of NEDD4L in acute brain injury and microglia-specific responses remain largely unknown in intracerebral hemorrhage( ICH). Methods: Using proteomic sequencing, single-cell RNA sequencing, Immunofluorescence Staining. flow cytometry analysis, we investigated cellular distribution and expression of NEDD4L in ICH. Genetically engineered mouse models, neurological function testing, flow cytometry analysis, engineering extracellular vesicle (EV) isolation and TUNEL staining, we investigated the role of NEDD4L in ICH. Protein interactions was used for mechanism analysis. Results: We identified a substantial upregulation of the ubiquitin ligase NEDD4L in isolated microglia following ICH, and demonstrated that its increase is mediated by STAT2-dependent transcriptional mechanisms. Microglial subpopulations with high NEDD4L expression exhibit a pronounced pro-inflammatory phenotype. Moreover, knockout of NEDD4L specifically in microglia conferred neuroprotection and attenuated innate immune responses by the cytosolic DNA-sensing pathway. Mechanistically, NEDD4L promotes the ubiquitination of Parkin, resulting in the leakage of mitochondrial DNA into the cytoplasm and subsequent activation of the cGAS–Sting inflammatory cascade. Engineered extracellular vesicles of NEDD4L-specific siRNA successfully mitigated neuroinflammation, tissue damage, and functional deficits—in both ICH and TBI mouse models. Conclusions: we unveil a novel role for NEDD4L in modulating microglia-driven neuroinflammation after acute brain injury and highlight its function in activating the mtDNA–cGAS–Sting signaling axis. brain injury microglia neuroinflammation NEDD4L cGAS-Sting mitophagy Full Text Additional Declarations No competing interests reported. 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-9225741","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617436540,"identity":"cfa29fc1-9901-42b8-b898-7dffeeecb199","order_by":0,"name":"Jin Wang","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Wang","suffix":""},{"id":617436542,"identity":"a99e7500-714a-4717-8f6b-073fbb0790a5","order_by":1,"name":"Yuyuan Ma","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital, 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University","correspondingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Wu","suffix":""},{"id":617436563,"identity":"c1c2aaae-218f-46b5-ad74-4f2693da6b05","order_by":11,"name":"Bing Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYFCCAxCKsYGN8UFCRQ1pWpgNHpw5RpJ1bGySD1uYCauTbzz+7NPNtsP2zDPS0ioSG9gY+Nu7E/BqYWw4Yzw7t+1wYuOMtGM3EnfIMEicObsBrxZmhjPMzEAtCYyz09tuJJ5hYzCQyMWvhY3h+GOQFnuQloLENmbCWngYDhiDtDA2zk47xkCUFgmGM8bMOefSExvnP0uWSDhzjIegX+RnAB2WU2Ztb9hzzPDjj4oaOf72XvxaGCQOAIONrZnBsAHmUoKAH6T0Tx2DPGGlo2AUjIJRMFIBADBCTWthDXFNAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Provincial People's Hospital, Hangzhou Medical College","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Lei","suffix":""}],"badges":[],"createdAt":"2026-03-25 16:54:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9225741/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9225741/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108182306,"identity":"168d7b28-c624-43d0-bc60-872f0d405793","added_by":"auto","created_at":"2026-04-30 08:59:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2195035,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9225741/v1_covered_3984099f-d836-495f-b375-319d78352717.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing 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mitophagy","lastPublishedDoi":"10.21203/rs.3.rs-9225741/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9225741/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eThe resultant microglial activation and sustained neuroinflammatory response are key contributors to secondary injury, promoting blood-brain barrier disruption, cerebral edema, and progressive neuronal loss. Although NEDD4L has been implicated in inflammation and immunity, \u0026nbsp;the functions of NEDD4L in acute brain injury and microglia-specific responses remain largely unknown in intracerebral hemorrhage( ICH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Using proteomic sequencing, single-cell RNA sequencing, Immunofluorescence Staining. flow cytometry analysis, we investigated cellular distribution and expression of NEDD4L in ICH. Genetically engineered mouse models, neurological function testing, flow cytometry analysis, engineering extracellular vesicle (EV) isolation and TUNEL staining, we investigated the role of NEDD4L in ICH. Protein interactions was used for mechanism analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eWe identified a substantial upregulation of the ubiquitin ligase NEDD4L in isolated microglia following ICH, and demonstrated that its increase is mediated by STAT2-dependent transcriptional mechanisms. Microglial subpopulations with high NEDD4L expression exhibit a pronounced pro-inflammatory phenotype. Moreover, knockout of NEDD4L specifically in microglia conferred neuroprotection and attenuated innate immune responses by the cytosolic DNA-sensing pathway. Mechanistically, NEDD4L promotes the ubiquitination of Parkin, resulting in the leakage of mitochondrial DNA into the cytoplasm and subsequent activation of the cGAS–Sting inflammatory cascade. Engineered extracellular vesicles of NEDD4L-specific siRNA successfully mitigated neuroinflammation, tissue damage, and functional deficits—in both ICH and TBI mouse models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003ewe unveil a novel role for NEDD4L in modulating microglia-driven neuroinflammation after acute brain injury and highlight its function in activating the mtDNA–cGAS–Sting signaling axis.\u003c/p\u003e","manuscriptTitle":"Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing mitophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 01:30:05","doi":"10.21203/rs.3.rs-9225741/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":"c745ffb2-138e-4dcf-98da-1f5863b62cd6","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T18:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 01:30:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9225741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9225741","identity":"rs-9225741","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}