Focal Loss of the Paranodal Domain Protein Neurofascin155 in the Internal Capsule Impairs Cortically Induced Muscle Activity in Vivo.

Focal Loss of the Paranodal Domain Protein Neurofascin155 in the Internal Capsule Impairs Cortically Induced Muscle Activity in Vivo. | 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 Short report Focal Loss of the Paranodal Domain Protein Neurofascin155 in the Internal Capsule Impairs Cortically Induced Muscle Activity in Vivo. Kazuo Kunisawa, Nobuhiko Hatanaka, Takeshi Shimizu, Kenta Kobayashi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-84417/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Nov, 2020 Read the published version in Molecular Brain → Version 1 posted 10 You are reading this latest preprint version Abstract Paranodal axoglial junctions are essential for rapid nerve conduction and the organization of axonal domains in myelinated axons. Neurofascin155 (Nfasc155) is a glial cell adhesion molecule that is also required for the assembly of these domains. Previous studies have demonstrated that general ablation of Nfasc155 disorganizes these domains, reduces conduction velocity, and disrupts motor behaviors. Multiple sclerosis (MS), a typical disorder of demyelination in the central nervous system, is reported to have autoantibody to Nfasc155. However, the impact of focal loss of Nfasc155, which may occur in MS patients, remains unclear. Here, we examined whether restricted focal loss of Nfasc155 affects the electrophysiological properties of the motor system in vivo. Adeno-associated virus type5 (AAV5) harboring EGFP-2A-Cre was injected into the glial-enriched internal capsule of floxed-Neurofascin (NfascFlox/Flox) mice to focally disrupt paranodal junctions in the cortico-fugal fibers from the motor cortex to the spinal cord. Electromyograms (EMGs) of the triceps brachii muscles in response to electrical stimulation of the motor cortex were successively examined in these awake mice. EMG analysis showed significant delay in the onset and peak latencies after AAV injection compared to control (Nfasc+/+) mice. Moreover, EMG half-widths were increased, and EMG amplitudes were gradually decreased by 13 weeks. Similar EMG changes have been reported in MS patients. These findings provide physiological evidence that motor outputs are obstructed by focal ablation of paranodal junctions in myelinated axons. Our findings may open a new path toward development of a novel biomarker for an early phase of human MS, as Nfasc155 detects microstructural changes in the paranodal junction. Molecular Biology paranodal junction multiple sclerosis electromyogram motor system Neurofascin155 Figures Figure 1 Figure 2 Full Text Cite Share Download PDF Status: Published Journal Publication published 23 Nov, 2020 Read the published version in Molecular Brain → Version 1 posted Review # 1 received at journal 14 Oct, 2020 Editorial decision: Major revision 14 Oct, 2020 Review # 2 received at journal 09 Oct, 2020 Reviewer # 2 agreed at journal 30 Sep, 2020 Reviewer # 1 agreed at journal 24 Sep, 2020 Editor assigned by journal 23 Sep, 2020 Reviewers invited by journal 23 Sep, 2020 First submitted to journal 22 Sep, 2020 Submission checks completed at journal 22 Sep, 2020 Editor invited by journal 22 Sep, 2020 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. 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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-84417","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short report","associatedPublications":[],"authors":[{"id":2848965,"identity":"f537f436-55e4-404e-888b-dc2da77a48d2","order_by":0,"name":"Kazuo Kunisawa","email":"","orcid":"","institution":"National Institutes of Physiological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazuo","middleName":"","lastName":"Kunisawa","suffix":""},{"id":2848966,"identity":"8aef7244-8d62-4835-a0ba-e20abffe1df0","order_by":1,"name":"Nobuhiko Hatanaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACAwiVwMDPwNgAFTtASAszRItkA8laDAgoRABz9vMHPxfUpMkb325ue/iDwU6egfEsft2WPcnM0jOO5Rhuu3Ow3ZiHIdmwgeFcAn6HHUhmkOZhq2DcdiOxTZqBgRmo/IwBfi3nHzP/5vlXYb95RmKb5A+GeiK03Ehmk+Zty0ncIJHYJsHDcJgYLY/NrHn70pJn3EgE+sXguGEbQb+cT3x8m+dbsm3/jPRnD39UVMvzSxAIMWTABk4MbBJniNYB0gIC/D3EaxkFo2AUjIIRAQCCEUXG3d1oQgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8793-1438","institution":"Division of System Neurophysiology, National Institute for Physiological Sciences, 38 2 Nishigonaka, M 27 yodaiji, Okazaki, 444-8585, Japan.","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nobuhiko","middleName":"","lastName":"Hatanaka","suffix":""},{"id":2848967,"identity":"b37514c6-38dd-4a19-bb15-502907361b10","order_by":2,"name":"Takeshi Shimizu","email":"","orcid":"","institution":"National Institute for Physiological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Shimizu","suffix":""},{"id":2848968,"identity":"1b6cfb2b-297b-44c6-9429-a35619c1db1e","order_by":3,"name":"Kenta Kobayashi","email":"","orcid":"","institution":"National Institute for Physiological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Kobayashi","suffix":""},{"id":2848969,"identity":"8639f3fc-b990-4dd3-b20f-cf8f76d43219","order_by":4,"name":"Yasuyuki Osanai","email":"","orcid":"","institution":"National Institute for Physisological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuyuki","middleName":"","lastName":"Osanai","suffix":""},{"id":2848970,"identity":"3ac829ce-72b3-45d3-8926-d3ba85232c4a","order_by":5,"name":"Akihiro Mouri","email":"","orcid":"","institution":"Fujita Health University: Fujita Ika Daigaku","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Mouri","suffix":""},{"id":2848971,"identity":"9eedaf18-9b66-4ed4-9dbf-09443d700f58","order_by":6,"name":"Qian Shi","email":"","orcid":"","institution":"University of Texas Health Science Center at Tyler: The University of Texas Health Science Center at Tyler","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Shi","suffix":""},{"id":2848972,"identity":"9af0a8f0-4568-4ea8-bb89-7f6765a2cb88","order_by":7,"name":"Manzoor A. Bhat","email":"","orcid":"","institution":"University of Texas Health Science Center at Tyler: The University of Texas Health Science Center at Tyler","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manzoor","middleName":"A.","lastName":"Bhat","suffix":""},{"id":2848973,"identity":"e9aafdbb-511b-48ca-9c11-a4b52dbf37b3","order_by":8,"name":"Atsushi Nambu","email":"","orcid":"","institution":"National Institute of Physiological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Nambu","suffix":""},{"id":2848974,"identity":"7dd80aaa-76a4-4d67-ab82-9141d920f9b9","order_by":9,"name":"Kazuhiro Ikenaka","email":"","orcid":"","institution":"National Institute for Physiological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Ikenaka","suffix":""}],"badges":[],"createdAt":"2020-09-27 20:40:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-84417/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-84417/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13041-020-00698-y","type":"published","date":"2020-11-23T15:01:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2746931,"identity":"71f781a0-ad20-4c22-b7c6-8a0702fd3782","added_by":"auto","created_at":"2020-10-02 15:36:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124301,"visible":true,"origin":"","legend":"AAV-Cre-induced loss of Nfasc155 in the internal capsule. (a) Schematic diagrams showing the design of the AAV-EGFP-2A-Cre constructs. The 2A peptide is cleaved just after translation, and EGFP and Cre recombinase are expressed independently. (b, c) Time course and schematic drawing of our experiments. To examine whether site-directed loss of paranodal junctions causes a delay in cortically evoked electromyograms (EMGs), a pair of bipolar stimulating electrodes was chronically implanted into the motor cortex (MCx), and EMG recording electrodes were placed in the triceps brachii muscles contralateral to the cortical stimulation electrodes in 6-week-old NfasccKO and Nfasc+/+ mice. AAV5-EGFP-2A-Cre was injected into the internal capsule (IC) of the mice at week 0. Evoked EMGs by cortical stimulation (Stim.) were recorded every week before (week 0) and after (from week 1 to week 13) AAV injection. (d) Representative micrograph of AAV-mediated EGFP expression (green) in the internal capsule 12 weeks after AAV injection. White dotted lines indicate the boundaries of the internal capsule (IC). Scale bar: 250 μm. (e) Doubly staining by in situ hybridization for PLP mRNA (blue) and immunostaining with an anti-GFP antibody (brown) in the IC 12 weeks after AAV injection. Arrowheads indicate double positive cells for both PLP mRNA and GFP. The inset is a magnified view of the cell indicated by the arrow. Scale bar: 50 μm (25 μm in inset). (f) Immunofluorescence staining with an anti-Caspr (green) and anti-Na+ channel (red) antibodies in sections containing the IC of Nfasc+/+ (left) or NfasccKO (right) mice 12 weeks after AAV injection. Each inset shows a magnified view of the immunolabeling indicated by the arrow. Scale bar: 20 μm (5 μm in inset). (g, h) Quantification of the number (g) and length (h) of Caspr-positive paranodes in Nfasc+/+ and NfasccKO mice injected with AAV5-EGFP-2A-Cre vector (Student’s t-test, number [t = 4.623, df = 4, **p \u003c 0.01], length [t = 5.319, df = 4, **p \u003c 0.01]; n = 3 mice each). (i) Quantification of the number of Na+ channel-positive nodes in Nfasc+/+ and NfasccKO mice injected with AAV5-EGFP-2A-Cre vector (Student’s t-test, t = 1.504, df = 4, p = 0.2069; n = 3 mice each). All data are shown as mean ± SEM.","description":"","filename":"Fig1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-84417/v1/Fig1.JPG"},{"id":2746933,"identity":"9420c47c-8a95-40f1-9b32-ea1d4d178a44","added_by":"auto","created_at":"2020-10-02 15:36:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90305,"visible":true,"origin":"","legend":"Cortically evoked EMGs in NfasccKO and Nfasc+/+ mice injected with AAV5-EGFP-2A-Cre. (a) Representative EMGs in response to electrical stimulation (0.6 mA, 100 μs duration, single pulse, 1400 ms interval, averaged 100 times) of the motor cortex in Nfasc+/+ mice 12 weeks after AAV injection. Onset latency, peak latency and half-width of the EMGs are indicated. Amplitude of the EMGs is defined as the area over the mean during the response exceeding the level of the mean + 2SD (blue area, see also Methods). (b-e) Cortically evoked population EMGs of Nfasc+/+ (n = 5, blue line) and NfasccKO (n = 7, red line) at week 0 (b), 7 (c), 10 (d) and 13. The light-shaded colors represent ± SEM. (f-i) Ratio (%) of onset latency (f), peak latency (g), half-width (h) and amplitude (i) of cortically evoked EMGs in NfasccKO mice compared to those of Nfasc+/+mice before (week 0) and after (from week 1 to week 13) AAV injection (two-way ANOVA followed by Sidak multiple comparison test; onset latency (genotype [F (1,137) = 21.41, **p \u003c 0.01], week [F (13,137) = 2.480, **p \u003c 0.01], genotype × week [F (13,137) = 2.480, **p \u003c 0.01]), peak latency (genotype [F (1,125 = 20.19, **p \u003c 0.01), week [F (13,125) = 1.179, p = 0.3024], genotype × week [F (13,125) = 1.179, p = 0.3024]], half-width (genotype [F (1,127) = 9.162, **p \u003c 0.01], week [F (13,127) = 0.8992, p = 0.5558], genotype × week [F (13,127) = 0.9034, p = 0.5515]), amplitude (genotype [F (1,126) = 3.182, p = 0.0769], week [F (13,126) = 0.4220, p = 0.9595], genotype × week [F (13,126) = 0.4221, p = 0.9594])). All data are expressed as mean ± SEM.","description":"","filename":"Fig2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-84417/v1/Fig2.JPG"},{"id":13597865,"identity":"40ffaa2f-3eb5-4e8f-8e96-fab19346b6f3","added_by":"auto","created_at":"2021-09-17 05:33:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":351110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-84417/v1/5c346d21-a46b-4b88-8021-6868c06b0e69.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFocal Loss of the Paranodal Domain Protein Neurofascin155 in the Internal Capsule Impairs Cortically Induced Muscle Activity in Vivo.\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-84417/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"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":"molecular-brain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbrj","sideBox":"Learn more about [Molecular Brain](http://molecularbrain.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mbrj/default.aspx","title":"Molecular Brain","twitterHandle":"@molecularbrain","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"paranodal junction, multiple sclerosis, electromyogram, motor system, Neurofascin155","lastPublishedDoi":"10.21203/rs.3.rs-84417/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-84417/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParanodal axoglial junctions are essential for rapid nerve conduction and the organization of axonal domains in myelinated axons. Neurofascin155 (Nfasc155) is a glial cell adhesion molecule that is also required for the assembly of these domains. Previous studies have demonstrated that general ablation of Nfasc155 disorganizes these domains, reduces conduction velocity, and disrupts motor behaviors. Multiple sclerosis (MS), a typical disorder of demyelination in the central nervous system, is reported to have autoantibody to Nfasc155. However, the impact of focal loss of Nfasc155, which may occur in MS patients, remains unclear. Here, we examined whether restricted focal loss of Nfasc155 affects the electrophysiological properties of the motor system in vivo. Adeno-associated virus type5 (AAV5) harboring EGFP-2A-Cre was injected into the glial-enriched internal capsule of floxed-Neurofascin (NfascFlox/Flox) mice to focally disrupt paranodal junctions in the cortico-fugal fibers from the motor cortex to the spinal cord. Electromyograms (EMGs) of the triceps brachii muscles in response to electrical stimulation of the motor cortex were successively examined in these awake mice. EMG analysis showed significant delay in the onset and peak latencies after AAV injection compared to control (Nfasc+/+) mice. Moreover, EMG half-widths were increased, and EMG amplitudes were gradually decreased by 13 weeks. Similar EMG changes have been reported in MS patients. These findings provide physiological evidence that motor outputs are obstructed by focal ablation of paranodal junctions in myelinated axons. Our findings may open a new path toward development of a novel biomarker for an early phase of human MS, as Nfasc155 detects microstructural changes in the paranodal junction.\u003c/p\u003e","manuscriptTitle":"Focal Loss of the Paranodal Domain Protein Neurofascin155 in the Internal Capsule Impairs Cortically Induced Muscle Activity in Vivo.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-02 15:36:31","doi":"10.21203/rs.3.rs-84417/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-10-14T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2020-10-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-10-09T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-09-30T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-09-24T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-09-23T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-23T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-09-22T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-22T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-22T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-brain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbrj","sideBox":"Learn more about [Molecular Brain](http://molecularbrain.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mbrj/default.aspx","title":"Molecular Brain","twitterHandle":"@molecularbrain","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"737157e8-4484-48f3-873b-92f1d6d88743","owner":[],"postedDate":"October 2nd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":667332,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2020-11-29T15:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-84417","link":"https://doi.org/10.1186/s13041-020-00698-y","journal":{"identity":"molecular-brain","isVorOnly":false,"title":"Molecular Brain"},"publishedOn":"2020-11-23 15:01:41","publishedOnDateReadable":"November 23rd, 2020"},"versionCreatedAt":"2020-10-02 15:36:31","video":"","vorDoi":"10.1186/s13041-020-00698-y","vorDoiUrl":"https://doi.org/10.1186/s13041-020-00698-y","workflowStages":[]},"version":"v1","identity":"rs-84417","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-84417","identity":"rs-84417","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}