Astrocytic YAP Protects the Optic Nerve and Retina in an Experimental Autoimmune Encephalomyelitis Model Through TGF-β Signaling

Astrocytic YAP Protects the Optic Nerve and Retina in an Experimental Autoimmune Encephalomyelitis Model Through TGF-β Signaling | 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 Astrocytic YAP Protects the Optic Nerve and Retina in an Experimental Autoimmune Encephalomyelitis Model Through TGF-β Signaling qian Wu, Xuemeng Miao, Jingjing Zhang, Ludan Xiang, Xiuchun Li, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-239628/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 Optic neuritis, inflammation of the optic nerve (ON), is one of the main symptoms in multiple sclerosis (MS) and leads to visual disability. Astrocytes are pivotal regulators of neuroinflammation in MS. However, it remains unclear that the detailed roles and mechanisms of astrocytes in the neuroinflammation and demyelination in optic neuritis of MS. Methods To assess the role of YAP in ON and retina in response to experimental autoimmune encephalomyelitis (EAE), mice that conditionally knockout (CKO) YAP in astrocytes, namely YAP GFAP -CKO mice, were successfully generated. Immunostaining, Nissl staining, Hematoxylin-Eosin (HE) staining, TUNEL staining, luxol fast blue (LFB) staining, electron microscopy (EM), qRT-PCR and gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) by RNA sequencing were used to examine the roles of YAP pathway in EAE based on these conditional knockout mice. Inhibitors including SRI-011381 [an agonist of transforming growth factor-β (TGF-β) pathway] and XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) were used to further explore the molecular mechanism of YAP in ON and retina of EAE mice. Additionally, microglia and retinal ganglion cells (RGCs) counts, and demyelination and astrocytes were assessed by immunohistological staining. Results We found that yes-associated protein (YAP) was significantly upregulated and activated in the astrocytes of ON in EAE. Conditional knockout YAP in astrocytes caused more severe inflammatory infiltration and demyelination in ON, and damage of the RGCs in EAE mice. Moreover, YAP deletion in astrocytes promoted the activation of astrocytes and microglia, but inhibited the proliferation of astrocytes of ON in EAE mice. Mechanically, TGF-β signaling pathway was significantly down-regulated after YAP deletion in astrocytes . Additionally, both qPCR and immunofluorescence assays confirmed the reduction of TGF-β 1 in YAP knockout ON astrocytes of EAE mice. Interestingly, SRI-011381 partially rescued the deficits in ON and retina of YAP knockout EAE mice. Finally, activation of YAP pathway relieved the neuroinflammation and demyelination in optic neuritis of EAE mice. Conclusions These results suggest astrocytic YAP may prevent the neuroinflammatory infiltration and demyelination through upregulation of TGF-β signaling and provide targets for the development of therapeutic strategies tailored for optic neuritis in MS. Neurobiology of Disease optic neuritis EAE astrocytes YAP TGF-β1 neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Supplementary Files Additionalfile1.pdf Additionalfile2.pdf Additionalfile3.pdf Additionalfile4.pdf Additionalfile5.pdf Additionalfile6.pdf Additionalfile7.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-239628","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":12739213,"identity":"0c958d56-6a55-49f3-895a-c4625e42c8a7","order_by":0,"name":"qian Wu","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"qian","middleName":"","lastName":"Wu","suffix":""},{"id":12739214,"identity":"c47c2c5a-6fb6-49b0-9a79-d04002c6db3c","order_by":1,"name":"Xuemeng Miao","email":"","orcid":"","institution":"Wenzhou Medical College - Chashan Campus: Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuemeng","middleName":"","lastName":"Miao","suffix":""},{"id":12739215,"identity":"2cac30ae-7ed1-42e4-beac-b3e345fd2b24","order_by":2,"name":"Jingjing Zhang","email":"","orcid":"","institution":"Wenzhou Medical College - 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(A) Immunohistochemistry detected the YAP expression in the ON of control and EAE mice. (B) Quantitative analysis of the number of YAP+ cells per mm2 as shown in (A) (n=8, per group). (C) Double immunostaining of YAP (green) and GFAP (red) in the ON of control and EAE mice. (D) Quantitative analysis of the number of nuclear YAP+ astrocytes per mm2 as shown in (C) (n=6, per group). (E) Double immunostaining of YAP (green) and Iba1 (red) in the ON of control and EAE mice. Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, Student’s t-test, compared with control mice, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/818ee673617492af1abe372e.jpg"},{"id":6353947,"identity":"f3410ee0-e0ad-42c8-bb61-169f57b630b5","added_by":"auto","created_at":"2021-02-25 15:04:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1161604,"visible":true,"origin":"","legend":"Ablation of YAP in astrocytes exacerbates the demyelination in ON of EAE mice. (A) LFB staining in the ON obtained from control, YAPf/f and YAPGFAP-CKO EAE mice. (B) Quantitative analysis of the demyelination score as shown in (A) (n=8, per group). (C) Immunostaining of MBP (green) in the ON obtained from control, YAPf/f and YAPGFAP-CKO EAE mice. (D) Quantitative analysis of MBP intensity as shown in (C) (n=12, per group). (E) The longitudinal section of the ON obtained from YAPf/f and YAPGFAP-CKO EAE mice under transmission electron microscopy consists of the myelinated axons and myelin sheath. (F) Quantitative analysis of the thickness of myelin sheath as shown in (E) (n=25, per group). Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, two-way ANOVA with Bonferroni’s post-tests, compared with control mice, *P\u003c0.05, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/e865f2c3d414d9d895b9c5dd.jpg"},{"id":6354390,"identity":"2e820c05-68fd-471a-b01e-84c8fc807069","added_by":"auto","created_at":"2021-02-25 15:07:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":908781,"visible":true,"origin":"","legend":"Ablation of YAP in astrocytes exacerbates severe inflammatory infiltration in ON of EAE mice. (A) HE staining of the ON obtained from control, YAPf/f and YAPGFAP-CKO EAE mice. (B) Quantitative analysis of the density of inflammatory caspases as shown in (A) (n=10, per group). (C) Immunostaining of CD45 (red) in the ON obtained from control mice, YAPf/f and YAPGFAP-CKO EAE mice. (D) Quantitative analysis of the number of CD45+ cells per mm2 as shown in (C) (n=9, per group). (E) Double immunostaining of Iba1 (green) and GFAP (red) in the ON obtained from control, YAPf/f and YAPGFAP-CKO EAE mice. (F) Quantitative analysis of GFAP intensity as shown in (E) (n=9, per group). (G) Quantitative analysis of the number of Iba1+ cells per mm2 as shown in (E) (n=12, per group). (H) Immunostaining of CD206 (green) in the ON obtained from control, YAPf/f and YAPGFAP-CKO EAE mice. (I) Quantitative analysis of the number of CD206+ cells per mm2 as shown in (H) (n=10, per group). Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, two-way ANOVA with Bonferroni’s post-tests, compared with control mice, *P\u003c0.05, **P\u003c0.01, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/72a9091fef82090af2cc38e0.jpg"},{"id":6353498,"identity":"eb55bed8-f000-4912-9870-cfe90ee980b2","added_by":"auto","created_at":"2021-02-25 15:01:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":747455,"visible":true,"origin":"","legend":"Ablation of YAP affect the proliferation of astrocytes and microglia in ON of EAE mice. (A-B) Double immunostaining of Ki67 (green) and GFAP (red) (A) or PH3 (green) and GFAP (red) (B) in the ON of control, YAPf/f and YAPGFAP-CKO EAE mice. (C-D) analysis of the percentage of Ki67+ (C) or PH3+ (D) cells over total astrocytes as shown in (A-B) (n=7, per group). (E-F) Double immunostaining of Ki67 (green) and Iba1(red) (E), or PH3 (green) and Iba1 (red) (F) in the ON of control, YAPf/f and YAPGFAP-CKO EAE mice. (G-H) Quantitative analysis of the percentage of Ki67+ (G) or PH3+ (H) cells over total microglia as shown in (E-F) (n=10, per group). Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, two-way ANOVA with Bonferroni’s post-tests, compared with control mice, *P\u003c0.05, **P\u003c0.01, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/5978f51257556626c3c85e4a.jpg"},{"id":6353499,"identity":"e3a65ba6-45b0-420e-8890-609ab649e2b1","added_by":"auto","created_at":"2021-02-25 15:01:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":650799,"visible":true,"origin":"","legend":"Ablation of YAP in astrocytes exacerbates inflammation and apoptosis in retina of EAE mice. (A, C, E) Immunostaining of Iba1 (green) (A), GFAP (red) (C) or Vimentin (cyan) (E) in the retina of control, YAPf/f and YAPGFAP-CKO EAE mice. (B) Quantitative analysis of the number of Iba1+ cells per mm2 as shown in (A) (n=7, per group). (D, F) Quantitative analysis of GFAP intensity (D) or Vimentin intensity (F) as shown in (C, E) (n=8, per group). (G) Representative images of Nissl staining in the retina of control, YAPf/f and YAPGFAP-CKO EAE mice. (H) Quantitative analysis of the number of Nissl+ cells per mm2 as shown in (G) (n=11, per group). (I, K) Immunostaining of RBPMS (green) (I) or NeuN (green) (K) in the retina of control, YAPf/f and YAPGFAP-CKO EAE mice. (J, L) Quantitative analysis of the number of RBPMS+ cells (J) or NeuN+ cells (L) per mm2 as shown in (I, K) (n=7, per group). (M) Immunostaining analysis of cell apoptosis by TUNEL staining in the retina of control, YAPf/f and YAPGFAP-CKO EAE mice. (N) Quantitative analysis of the number of TUNEL+ cells per mm2 as shown in (M) (n=9, per group). Data were mean ± SEM, two-way ANOVA with Bonferroni’s post-tests, compared with control mice, *P\u003c0.05, **P\u003c0.01, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/f73c8d757d2286b2de2a8c8f.jpg"},{"id":6353950,"identity":"860a1747-e795-45fb-aaf8-402970d263d4","added_by":"auto","created_at":"2021-02-25 15:04:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":544152,"visible":true,"origin":"","legend":"GSEA and GSVA in YAP+/+ vs YAP-/- astrocytes. (A) The 11 down-regulated gene set with statistically significant (FDR \u003c0.25) in YAPGFAP-CKO group. (B) The enrichment plot of TGF-β signaling gene sets. (C) The clustering heatmap of 49 genes in TGF-β signaling gene sets. Red, blue and white respectively represent high expression level, low expression level and no expression difference among the genes. (D) GSVA-derived clustering heatmap of differentially expressed gene sets. Red, blue and white respectively represent high expression level, low expression level and no expression difference among the genes.","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/482497f285ef6b5b7ee00e40.jpg"},{"id":6353507,"identity":"4c13c4a5-3b3f-4cf3-b0d4-37dbee9d5566","added_by":"auto","created_at":"2021-02-25 15:01:01","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":999860,"visible":true,"origin":"","legend":"Activation of TGF-β signaling reduces inflammatory infiltration and demyelination in the ON of EAE mice. (A) Immunostaining of TGF-β1 (green) and GFAP (red) in the ON of control, SRI-011381-treated YAPf/f and YAPGFAP-CKO EAE mice. Images of selected regions (white squares) were shown at higher magnification. (B) Quantitative analysis of the number of TGF-β1+ astrocytes per mm2 as shown in (A) (n=5, per group). (C) LFB staining in the ON of control, SRI-011381-treated YAPf/f and YAPGFAP-CKO EAE mice. (D) Quantitative analysis of the demyelination score of control, SRI-011381-treated YAPf/f and YAPGFAP-CKO EAE mice as shown in (C) (n=7, per group). (E, G, I, K) Immunostaining of MBP (green) (E), Iba1 (green) (G), GFAP (green) (I) or CD45 (green) (K) in the ON of control, SRI-011381-treated YAPf/f and YAPGFAP-CKO EAE mice. (F, J) Quantitative analysis of MBP (F) or GFAP (J) intensity as shown in (E, I) (n=6, per group). (H, L) Quantitative analysis of the number of Iba1+ cells (H) or CD45+ cells (L) per mm2 as shown in (G, K) (n=7, per group). Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, two-way ANOVA with Bonferroni’s post-tests, compared with control mice, **P\u003c0.01, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/1e23100b7968fe989c3b43ac.jpg"},{"id":6354391,"identity":"7baceb8a-3ab7-47fe-a7c0-aa9d0ad83f26","added_by":"auto","created_at":"2021-02-25 15:07:01","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":902513,"visible":true,"origin":"","legend":"XMU-MP-1 reduces inflammation and demyelination of ON, and the loss of RGCs in EAE mice. (A) LFB staining of the ON obtained from control and XMU-UP-1-treated EAE mice. (B) Quantitative analysis of the demyelination score of control and XMU-UP-1-treated EAE mice as shown in (A) (n=7, per group). (C, E, G, I) Immunostaining of MBP (green) (C), Iba1 (green) (E), GFAP (green) (G) or CD45 (green) (I) in the ON of control and XMU-UP-1-treated EAE mice. (D, H) Quantitative analysis of MBP intensity (D) or GFAP intensity (H) as shown in (C, G) (n=7, per group). (F, J) Quantitative analysis of the number of Iba1+ cells (F) or CD45+ cells (J) per mm2 as shown in (E, I) (n=6, per group). (K, M, O) Immunostaining of Iba1 (green) (K), GFAP (green) (M) or NeuN (green) (O) in the retina of control and XMU-UP-1-treated EAE mice. (L, P) Quantitative analysis of the number of Iba1+ cells (L) or NeuN+ cells (P) per mm2 as shown in (K, O) (n=5, per group). (N) Quantitative analysis of GFAP intensity as shown in (M) (n=7, per group). Images of selected regions (white squares) were shown at higher magnification. Data were mean ± SEM, Student’s t-test, compared with control mice, *P\u003c0.05, **P\u003c0.01, ***P\u003c0.001. Scale bars, 20 μm.","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/11934a5d289b88f82b789794.jpg"},{"id":13671998,"identity":"a472b5ed-7bcd-477a-821a-d75383df2723","added_by":"auto","created_at":"2021-09-17 11:11:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2068697,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/669c601d-1df7-4d38-9711-70f98d6142eb.pdf"},{"id":6353952,"identity":"3192978a-a371-45e8-9ce8-f52d6d09cbf1","added_by":"auto","created_at":"2021-02-25 15:04:01","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":4207443,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/29ad5ae59b6efb1c463e89fc.pdf"},{"id":6353955,"identity":"c6b89b3b-42d4-4a03-bc4e-3417aace53fe","added_by":"auto","created_at":"2021-02-25 15:04:01","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":3771351,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/2661ba7f46b4bf9c4e2fad40.pdf"},{"id":6353953,"identity":"26582512-398e-452f-8549-d544e5b0173f","added_by":"auto","created_at":"2021-02-25 15:04:01","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":9293546,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/8b0afc95847bc1f99f40c3c5.pdf"},{"id":6353509,"identity":"92d0e4cf-621d-4d4c-b688-a33000c201a1","added_by":"auto","created_at":"2021-02-25 15:01:01","extension":"pdf","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":6538975,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/9c87ac3ede5449228061e5fa.pdf"},{"id":6353957,"identity":"80f097e8-d454-423b-ba19-f83af1fb6c29","added_by":"auto","created_at":"2021-02-25 15:04:01","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":5460282,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/861235364b64fe828f70efb3.pdf"},{"id":6354393,"identity":"53ac1a99-0981-447b-be92-2dd6d6b6b54a","added_by":"auto","created_at":"2021-02-25 15:07:01","extension":"pdf","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":5701165,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/15b3956394650cd30dcfe9a8.pdf"},{"id":6353508,"identity":"79b656ea-4381-4850-b2f4-9ddee78d5cbe","added_by":"auto","created_at":"2021-02-25 15:01:01","extension":"pdf","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":143634,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-239628/v1/b36a637bf36bc380e1740742.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAstrocytic YAP Protects the Optic Nerve and Retina in an Experimental Autoimmune Encephalomyelitis Model Through TGF-β Signaling\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and \u003ca href='/article/rs-239628/latest.pdf' target='_blank'\u003e accessed as a PDF.\u003c/a\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"optic neuritis, EAE, astrocytes, YAP, TGF-β1, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-239628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-239628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003eOptic neuritis, inflammation of the optic nerve (ON), is one of the main symptoms in multiple sclerosis (MS) and leads to visual disability. Astrocytes are pivotal regulators of neuroinflammation in MS. However, it remains unclear that the detailed roles and mechanisms of astrocytes in the neuroinflammation and demyelination in optic neuritis of MS. \u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eTo assess the role of YAP in ON and retina in response to experimental autoimmune encephalomyelitis (EAE), mice that conditionally knockout (CKO) YAP in astrocytes, namely YAP GFAP -CKO mice, were successfully generated. Immunostaining, Nissl staining, Hematoxylin-Eosin (HE) staining, TUNEL staining, luxol fast blue (LFB) staining, electron microscopy (EM), qRT-PCR and gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) by RNA sequencing were used to examine the roles of YAP pathway in EAE based on these conditional knockout mice. Inhibitors including SRI-011381 [an agonist of transforming growth factor-β (TGF-β) pathway] and XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) were used to further explore the molecular mechanism of YAP in ON and retina of EAE mice. Additionally, microglia and retinal ganglion cells (RGCs) counts, and demyelination and astrocytes were assessed by immunohistological staining. \u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eWe found that yes-associated protein (YAP) was significantly upregulated and activated in the astrocytes of ON in EAE. Conditional knockout YAP in astrocytes caused more severe inflammatory infiltration and demyelination in ON, and damage of the RGCs in EAE mice. Moreover, YAP deletion in astrocytes promoted the activation of astrocytes and microglia, but inhibited the proliferation of astrocytes of ON in EAE mice. Mechanically, TGF-β signaling pathway was significantly down-regulated after YAP deletion in astrocytes . Additionally, both qPCR and immunofluorescence assays confirmed the reduction of TGF-β 1 in YAP knockout ON astrocytes of EAE mice. Interestingly, SRI-011381 partially rescued the deficits in ON and retina of YAP knockout EAE mice. Finally, activation of YAP pathway relieved the neuroinflammation and demyelination in optic neuritis of EAE mice. \u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eThese results suggest astrocytic YAP may prevent the neuroinflammatory infiltration and demyelination through upregulation of TGF-β signaling and provide targets for the development of therapeutic strategies tailored for optic neuritis in MS.\u003c/p\u003e","manuscriptTitle":"Astrocytic YAP Protects the Optic Nerve and Retina in an Experimental Autoimmune Encephalomyelitis Model Through TGF-β Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-25 15:00:58","doi":"10.21203/rs.3.rs-239628/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":"5c13c085-4c7d-4c57-bd2c-f67a18087324","owner":[],"postedDate":"February 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2623496,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2021-03-03T00:25:00+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-25 15:00:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-239628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-239628","identity":"rs-239628","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}