Early mitochondrial gene dysregulation precedes motor neuron degeneration in genomically humanised FUS mutant mice

Early mitochondrial gene dysregulation precedes motor neuron degeneration in genomically humanised FUS mutant mice | 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 Early mitochondrial gene dysregulation precedes motor neuron degeneration in genomically humanised FUS mutant mice Georgia Price, Graciana de Azambuja, Chloe Williams, David Thompson, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9043045/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, with mutations in the Fused in Sarcoma ( FUS ) gene accounting for some of the most aggressive forms of the disease. Although FUS mutations are rare, accounting for only around 5% of all familial ALS cases, they are an important paradigm for study because FUS is an RNA binding protein and disruption to RNA metabolism is prominent across ALS subtypes. Here we present two novel, genomically humanised gene-targeted FUS mouse models expressing ALS patient mutations P525L or Q519Ifs. These humanised models express only human FUS protein under physiological control of the endogenous mouse promoter. Humanised knock-in hFUS P525L mice developed a mid-life onset reduction in motor performance with neuromuscular denervation, motor neuron loss, and unilateral limb muscle weakness. Pre-symptomatic transcriptomic changes suggested metabolic impairments as an early phenotype in skeletal muscle. We also directly compared phenotypes between hFUS P525L and hFUS Q519Ifs expressing mice. hFUS Q519Ifs expressing mice were more severely affected, including a pronounced developmental phenotype which varied in severity on different genetic backgrounds. The phenotypes of these new FUS-ALS models highlight the potential of fully humanised knock-in mice to aid in unravelling early disease mechanisms, and ultimately to assist in the development of therapies targeted towards the human FUS gene and protein in ALS. Amyotrophic lateral sclerosis (ALS) FUS humanised transcriptomics metabolism muscle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryFigs.pptx SupplementaryDocument1RNAseqcode.docx SupplementaryDataFile1.xlsx SupplementaryDataFile2.xlsx SupplementaryVideo1AxonalTransportFUSP525Lmice.mp4 Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-9043045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603959750,"identity":"6f16d9f3-f0c3-4e48-b77f-71dd0cfa1799","order_by":0,"name":"Georgia Price","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK; Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK","correspondingAuthor":false,"prefix":"","firstName":"Georgia","middleName":"","lastName":"Price","suffix":""},{"id":603959751,"identity":"2d5332f8-0222-4a9b-8062-51ee31777201","order_by":1,"name":"Graciana de Azambuja","email":"","orcid":"","institution":"VIB-KU Leuven Center for Neuroscience (CNL), Department of Neurosciences, KU Leuven, Leuven Brain Institute, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Graciana","middleName":"","lastName":"de Azambuja","suffix":""},{"id":603959752,"identity":"1d94d0f8-cee9-456d-9583-5806bd901b46","order_by":2,"name":"Chloe Williams","email":"","orcid":"","institution":"Department of Medical and Translational Biology, Faculty of Medicine, Umeå University, Sweden","correspondingAuthor":false,"prefix":"","firstName":"Chloe","middleName":"","lastName":"Williams","suffix":""},{"id":603959753,"identity":"91948b64-d031-4f92-8442-00fd7dc243e1","order_by":3,"name":"David Thompson","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK; Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Thompson","suffix":""},{"id":603959754,"identity":"5afe1a3f-96fb-43ae-b7dd-a6a3f9b8b08b","order_by":4,"name":"Charlotte Tibbit","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Tibbit","suffix":""},{"id":603959755,"identity":"918103fd-5326-44b8-89ad-a85ff6518ece","order_by":5,"name":"Zeinab Ali","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK","correspondingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Ali","suffix":""},{"id":603959756,"identity":"bdf8d05a-d423-4f9d-9a5b-262fc516541f","order_by":6,"name":"Adele Austin","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK","correspondingAuthor":false,"prefix":"","firstName":"Adele","middleName":"","lastName":"Austin","suffix":""},{"id":603959757,"identity":"90f2c915-a117-4cc8-a594-b1ae9c71435b","order_by":7,"name":"Mhoriam Ahmed","email":"","orcid":"","institution":"UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, UCL, London, UK","correspondingAuthor":false,"prefix":"","firstName":"Mhoriam","middleName":"","lastName":"Ahmed","suffix":""},{"id":603959758,"identity":"bb4c2f89-e38b-472d-80da-c724c2633f08","order_by":8,"name":"Andrew Tosolini","email":"","orcid":"","institution":"UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, UCL, London, UK; Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia; School of Biomedical Sciences, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, St Lucia, Australia","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Tosolini","suffix":""},{"id":603959759,"identity":"41ef0e24-c03c-44bf-b221-885c98966f90","order_by":9,"name":"Anny Devoy","email":"","orcid":"","institution":"Dementia Research Institute, King's College London, London, UK; UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, UCL, London, UK","correspondingAuthor":false,"prefix":"","firstName":"Anny","middleName":"","lastName":"Devoy","suffix":""},{"id":603959760,"identity":"2a51be6f-6ab8-4a33-8a84-a7b27cef2d16","order_by":10,"name":"Gemma F Codner","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK","correspondingAuthor":false,"prefix":"","firstName":"Gemma","middleName":"F","lastName":"Codner","suffix":""},{"id":603959761,"identity":"eefe985b-33df-470b-8b20-c23815dd1f83","order_by":11,"name":"Lydia Teboul","email":"","orcid":"","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK","correspondingAuthor":false,"prefix":"","firstName":"Lydia","middleName":"","lastName":"Teboul","suffix":""},{"id":603959762,"identity":"ab364850-fab5-4fdf-bd6d-cf69bd31c4b9","order_by":12,"name":"Linda Greensmith","email":"","orcid":"","institution":"UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, UCL, London, UK","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Greensmith","suffix":""},{"id":603959763,"identity":"622157f8-fb36-444d-ad81-43e2a5162f3e","order_by":13,"name":"Giampietro Schiavo","email":"","orcid":"","institution":"Department of Neuromuscular Diseases and UCL Queen Square Motor Neuron Disease Centre, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom; UK Dementia Research Institute, University College London, London, United Kingdom.","correspondingAuthor":false,"prefix":"","firstName":"Giampietro","middleName":"","lastName":"Schiavo","suffix":""},{"id":603959764,"identity":"ce904bb1-e223-4d7b-95dd-d4d3cbfd8499","order_by":14,"name":"Sandrine da Cruz","email":"","orcid":"","institution":"VIB-KU Leuven Center for Neuroscience (CNL), Department of Neurosciences, KU Leuven, Leuven Brain Institute, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Sandrine","middleName":"da","lastName":"Cruz","suffix":""},{"id":603959765,"identity":"c85ab97a-e061-4f14-b761-35387e813cad","order_by":15,"name":"Elizabeth MC Fisher","email":"","orcid":"https://orcid.org/0000-0003-2850-9936","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK; UCL Queen Square Motor Neuron Disease Centre, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, UCL, London, UK","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"MC","lastName":"Fisher","suffix":""},{"id":603959766,"identity":"352ffed2-84a8-48b7-99e7-428967529200","order_by":16,"name":"Thomas J Cunningham","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDADNjBisGFgYIcIGOBSyYOmJY2BgZlYLRBdDIcJa7Fn705g+Nl2J49Puv3ag5855xP7mxkYP/xgOGyM0xaesxsYe9ueFbPJnCk37N12O3HGYQZmyR6Gw2Y4tUjkbmBmbDuc2CaRkybBC9SyAegwaaALbYjSIvl32zmQFubfRGpJPybNu+0ASAsbyBbcDjtzdsPBnnNgW9ikZbclG884zNhm2WOQjtP77O29Gx/8KDucOH9G+jPJt9vsZPvbmw/f+FFhbdiASw8QHIBaCIsJxgY8sYJq4QOilI2CUTAKRsHIAwCYzFKbblePrwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4751-3140","institution":"Mary Lyon Centre at MRC Harwell, Harwell, UK; UCL Institute of Prion Diseases, MRC Prion Unit at UCL, UCL, London, UK","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"J","lastName":"Cunningham","suffix":""}],"badges":[],"createdAt":"2026-03-05 17:38:19","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9043045/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-9043045/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105326416,"identity":"8dadb6fd-71ba-4550-810a-d648fdea266b","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5557309,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of fully humanised \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003ea \u003c/strong\u003eSanger sequencing showing the position of the missense mutations in exon 15 and the amino acid substitution at position 525L. \u003cstrong\u003eb \u003c/strong\u003e\u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice have significantly worse survival than \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e control mice (mixed sex group, p=0.0015, Log-rank (Mantel-Cox) test).\u003cstrong\u003e c \u003c/strong\u003ePercentage (%) of motor neurons with nuclear FUS staining at 12 months in \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L \u003c/em\u003e\u003c/sup\u003emice (n=3 biological replicates per genotype, mixed sex group, Welch’s unpaired \u003cem\u003et \u003c/em\u003etest).\u003cstrong\u003e d \u003c/strong\u003eLumbar spinal cord sections from 12-month-old \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice (mixed sex group) immunostained with ChAT (motor neurons in red) and FUS (green). Scale bars = 50 and 20 μm. White arrows indicate motor neurons with nuclear FUS depletion. \u003cstrong\u003ee \u003c/strong\u003e\u003cem\u003eFUS\u003c/em\u003e RNA expression in the spinal cord of aged (21-month-old) mice (n=5-8 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test).\u003cstrong\u003e f\u003c/strong\u003e FUS protein expression in spinal cord from aged (15-month-old) mice (n=3 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003eg\u003c/strong\u003e Male and female \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were smaller than \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003econtrols (male mice p=0.0002, female mice p=0.0004, Mixed-effects analysis (Harwell), p\u0026lt;0.0001 male and female mice, Two-way ANOVA (VIB-KU-Leuven)). \u003cstrong\u003eh\u003c/strong\u003e EchoMRI data indicating fat and lean mass in male and female \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice compared to\u003cem\u003e hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice (n=6-14 biological replicates per genotype, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). Data are shown as mean average ± SEM, ns=not significant, *=p\u0026lt;0.05, **=p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/3976ce7bf69442042da6e29c.png"},{"id":105326415,"identity":"ca49ddfc-391f-4458-9111-65f116d4ab74","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2798577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice display ALS relevant phenotypes including a mid-life onset degenerative motor phenotype, tibialis anterior (TA) muscle denervation and motor neuron loss \u003cstrong\u003ea\u003c/strong\u003e Longitudinal combined fore- and hindlimb grip strength (in grams) of \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice bred at MRC Harwell (n=6-14 biological replicates per genotype, male and female mice, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). \u003cstrong\u003eb\u003c/strong\u003e Lumbar spinal cord motor neuron numbers in 21-month-old \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eversus\u003cem\u003e hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e mice bred at MRC Harwell (n=5 biological replicates per genotype, male mice, Mann-Whitney test). \u003cstrong\u003ec \u003c/strong\u003eBar graphs represent mean hindlimb grip strength (in grams) of mice at 6, 12, 16, and 18 months of age housed at VIB-KU Leuven (n=8–12 biological replicates per genotype, male and female mice, Two-way ANOVA with Tukey’s multiple comparisons test). \u003cstrong\u003ed\u003c/strong\u003e Lumbar spinal cord motor neuron counts in \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice bred at VIB-KU Leuven compared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003econtrols, at 12 and 20 months of age (n=3 biological replicates per genotype, mixed sex group, Two-way ANOVA with Tukey’s multiple comparisons test).\u003cstrong\u003e e \u003c/strong\u003eTA muscles from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eL\u003c/sup\u003e mice immunostained with alpha-bungarotoxin (endplate in green) and synaptophysin/neurofilament heavy chain (pre-synapse in red). Scale bars = 50 and 20 µm.\u003cstrong\u003e f \u003c/strong\u003eNMJ innervation in 12-month-old TA muscles from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice\u003csup\u003e \u003c/sup\u003ecompared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003econtrols (mixed sex group, n=3 biological replicates per genotype, Mann-Whitney test). \u003cstrong\u003eg \u003c/strong\u003eDistance travelled by male mice\u003cstrong\u003e \u003c/strong\u003ein the open field (n=6-14 biological replicates per genotype, male mice, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). \u003cstrong\u003eh \u003c/strong\u003eDistance travelled by female mice in the open field experiment (n=7-10 biological replicates per genotype, female mice, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). Data are shown as mean average ± SEM, ns=not significant, *=p\u0026lt;0.05, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, ****=p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/bebf5bdac64160c9c219f369.png"},{"id":105326419,"identity":"2db13082-e58b-44d9-a968-563a99052695","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1953733,"visible":true,"origin":"","legend":"\u003cp\u003eRNA sequencing data highlights widespread gene dysregulation, in both the tibialis anterior (TA) muscle and spinal cord of pre-symptomatic \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003ea\u003c/strong\u003e Volcano plots of differential gene expression in 4-month-old \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eversus \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e mice in spinal cord and \u003cstrong\u003eb \u003c/strong\u003eTA muscle (n=4 biological replicates per genotype, male mice); purple circles = padj\u0026lt;0.1. \u003cstrong\u003ec \u003c/strong\u003eSignificant\u0026nbsp;and strong positive correlation in fold change of 156 overlapping differentially expressed genes in\u0026nbsp;\u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u0026nbsp;\u003c/em\u003e\u003c/sup\u003emice between the spinal cord and TA muscle\u0026nbsp;(r=0.882, p=3.81e-52,\u0026nbsp;Pearson correlation\u0026nbsp;test);\u0026nbsp;purple circles = padj\u0026lt;0.1 in both tissues,\u0026nbsp;orange diamonds\u0026nbsp;=\u0026nbsp;padj\u0026lt;0.1 in one tissue and nominal p\u0026lt;0.05 in the other tissue. \u003cstrong\u003ed\u0026nbsp;\u003c/strong\u003eVolcano plot\u0026nbsp;of differential gene\u0026nbsp;expression in \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e versus \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e spinal cord tissue (at 3 months, n=4 biological replicates per genotype, male mice);\u0026nbsp;purple circles = padj\u0026lt;0.1. \u003cstrong\u003ee \u003c/strong\u003eSignificant and strong\u0026nbsp;negative\u0026nbsp;correlation\u0026nbsp;in fold change\u0026nbsp;of\u0026nbsp;195\u0026nbsp;genes identified as differentially expressed from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eversus \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003espinal cord (padj\u0026lt;0.1) plotted against\u0026nbsp;values\u0026nbsp;observed\u0026nbsp;from\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;versus\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;spinal cord\u0026nbsp;(r=-0.737, p=1.11e-34, Pearson correlation test);\u0026nbsp;purple circles =\u0026nbsp;padj\u0026lt;0.1 in \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/- \u003c/em\u003e\u003c/sup\u003emice, orange filled\u0026nbsp;diamonds\u0026nbsp;= nominal p\u0026lt;0.05 in\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice\u0026nbsp;and\u0026nbsp;orange\u0026nbsp;open\u0026nbsp;diamonds\u0026nbsp;= p\u0026gt;0.05 in\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice.\u0026nbsp;\u003cstrong\u003ef\u0026nbsp;\u003c/strong\u003eSignificant\u0026nbsp;and strong\u0026nbsp;negative correlation\u0026nbsp;in fold change of 340 genes identified as\u0026nbsp;differentially expressed from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eversus \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT \u003c/em\u003e\u003c/sup\u003eTA (padj\u0026lt;0.1)\u0026nbsp;plotted against values identified from \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e versus \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e spinal cord \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;(r=-0.525,\u0026nbsp;p=8.01e-24,\u0026nbsp;Pearson correlation test);\u0026nbsp;purple circles =\u0026nbsp;padj\u0026lt;0.1 in \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/- \u003c/em\u003e\u003c/sup\u003emice, orange filled diamonds\u0026nbsp;= nominal p\u0026lt;0.05 in\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice and\u0026nbsp;orange open diamonds = p\u0026gt;0.05 in\u0026nbsp;\u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/be6e1e21d0b333877c326587.png"},{"id":105326417,"identity":"4b2ee756-4213-41dd-adc3-86dc8a7caab2","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2928344,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis highlights mitochondrial dysfunction, particularly affecting the\u0026nbsp;tibialis anterior (TA)\u0026nbsp;muscle in\u0026nbsp;\u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice\u0026nbsp;\u003cstrong\u003ea\u0026nbsp;\u003c/strong\u003eSchematic showing the localisation of differentially expressed mitochondria-associated genes\u0026nbsp;identified\u0026nbsp;in RNA sequencing from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e versus \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT \u003c/em\u003e\u003c/sup\u003emice, blue box represents downregulated genes (made using\u0026nbsp;BioRender). \u003cstrong\u003eb,c,d\u0026nbsp; \u003c/strong\u003eVolcano\u0026nbsp;plots\u0026nbsp;highlighting downregulated mitochondrial genes (blue triangles) identified from overrepresentation analyses (52\u0026nbsp;padj\u0026lt;0.1 genes from TA data; 2 padj\u0026lt;0.1 genes from cord data). All are downregulated from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e TA\u0026nbsp;muscle \u003cstrong\u003e(b).\u003c/strong\u003e The majority are downregulated from \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e spinal cord including a significant bias towards downregulation (47/51) of mitochondrial genes identified from TA that did not meet the padj\u0026lt;0.1 significant threshold in spinal cord (51 padj\u0026gt;0.1 genes; one-sided exact binomial test; n=51, k=46, p= 9.16E-09) \u003cstrong\u003e(c).\u003c/strong\u003e A significant bias towards upregulation was observed in the mitochondrial gene set from \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e versus \u003cem\u003eFus\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e spinal cord data (53 padj\u0026gt;0.1 genes; one-sided exact binomial test; n=53, k=41, p=4.09E-05) \u003cstrong\u003e(d)\u003c/strong\u003e. Note that 53/54 genes from this curated list were expressed in cord tissue.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/a4da0129adcd49c26d7b756d.png"},{"id":105728094,"identity":"01090e2d-5b35-4967-9dbb-fb0f82770db9","added_by":"auto","created_at":"2026-03-30 11:09:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1251671,"visible":true,"origin":"","legend":"\u003cp\u003eSkeletal muscle phenotypes in \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice\u003cstrong\u003e a\u003c/strong\u003e RT-qPCR data shows muscle-specific \u003cem\u003eFUS\u003c/em\u003e expression changes in the tibialis anterior (TA), extensor digitorum longus (EDL) and soleus muscle of 4-month-old \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e mice (n=3 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003eb\u003c/strong\u003e Number of type II fibres in the TA muscle of \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice as a percentage of total fibres in 15-month-old mice compared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e controls (n=3 biological replicates per genotype, male mice, Mann-Whitney test).\u003cstrong\u003e c \u003c/strong\u003eTA muscles dissected from 21-month-old\u003cem\u003e hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice were significantly smaller than \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e controls (n=5-9 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003ed\u003c/strong\u003e Relative mitochondrial DNA content (\u003cem\u003emt-Nd1\u003c/em\u003e) in TA muscles of \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice aged 12 months old compared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT \u003c/em\u003e\u003c/sup\u003emice (n=3-6 biological replicates per genotype, male mice, unpaired \u003cem\u003et \u003c/em\u003etest). \u003cstrong\u003ee \u003c/strong\u003eMaximum tetanic force (in grams) of TA muscles in 21-month-old mice, averaged from both legs (n=5-7 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test) \u003cstrong\u003ef\u003c/strong\u003e Maximum tetanic force readings (in grams) from the TA of the weakest leg of \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e21-month-old\u003cem\u003e \u003c/em\u003emice (n=5-7 biological replicates per genotype, male mice, unpaired\u003cem\u003e t\u003c/em\u003e test). \u003cstrong\u003eg\u003c/strong\u003e The difference in readings between legs (within each animal) was larger in \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice (n=5-7 biological replicates per genotype, male mice, Mann-Whitney test). \u003cstrong\u003eh \u003c/strong\u003eNumber of motor units in EDL averaged from both legs in 21-month-old mice (n=5 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003ei \u003c/strong\u003eNumber of motor units in the EDL from the weakest leg of \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e21-month-old mice (n=5 biological replicates per genotype, male mice, unpaired \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003ej \u003c/strong\u003eThe difference in number of motor units in the EDL between legs (within each animal) in 21-month-old mice (n=5 biological replicates per genotype, male mice, Mann-Whitney test). Data are shown as mean average ± SEM, ns=not significant, *=p\u0026lt;0.05, **=p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/6f8a23a91e60bd2f4ffccedb.png"},{"id":105326421,"identity":"22906e0e-158e-4f7b-87d6-e60cf8ba782e","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3446752,"visible":true,"origin":"","legend":"\u003cp\u003eMice expressing the \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emutation exhibit a more severe phenotype than mice expressing the \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mutation \u003cstrong\u003ea\u003c/strong\u003e Schematic depicting the FUS protein expressed in wildtype, \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003eb\u003c/strong\u003e Survival of \u003cem\u003emFus/mFus, mFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L \u003c/em\u003e\u003c/sup\u003eand\u003cem\u003e mFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs\u003c/em\u003e\u003c/sup\u003e mice up to 14 months of age (n=36-45 biological replicates per genotype, mixed sex group, Log-rank (Mantel-Cox) test). \u003cstrong\u003ec\u003c/strong\u003e Weight of mice expressing either \u003cem\u003eFUS\u003c/em\u003e mutation compared to wildtype (n=12-17 biological replicates per genotype, male and female mice, Mixed-effects analysis with Tukey's multiple comparisons post-hoc test. Red asterisks (*) denote significant differences between \u003cem\u003emFus/mFus \u003c/em\u003eand \u003cem\u003emFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e mice. Blue asterisks (*) denote significant differences between \u003cem\u003emFus/mFus \u003c/em\u003eand \u003cem\u003emFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs \u003c/em\u003e\u003c/sup\u003emice. Black asterisks (*) denote significant differences between \u003cem\u003emFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003emFus/hFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice). \u003cstrong\u003ed\u003c/strong\u003e Combined fore- and hindlimb grip strength data (in grams) (n=6-12 biological replicates per genotype, male and female mice, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). \u003cstrong\u003ee \u003c/strong\u003eOpen\u003cstrong\u003e \u003c/strong\u003efield data showing time the mouse spent in the centre of the arena (n=6-12 biological replicates per genotype, male and female mice, Mixed-effects analysis with Šídák's multiple comparisons post-hoc test). \u003cstrong\u003ef\u003c/strong\u003e Weight of TA muscles dissected from 14-month-old male and female mice expressing either \u003cem\u003eFUS\u003c/em\u003e mutation and wildtype (n=5 biological replicates per genotype, Ordinary One-way ANOVA with Tukey's multiple comparisons post-hoc test). \u003cstrong\u003eg\u003c/strong\u003e Number of lumbar spinal cord motor neurons in mice aged 14 months old (n=5 biological replicates per genotype, male mice, Ordinary One-way ANOVA with Tukey's multiple comparisons post-hoc test). \u003cstrong\u003eh\u003c/strong\u003e Immunofluorescence staining of FUS (green) in lumbar spinal cord from 14-month-old male mice. Scale bar = 50 µm. Data are shown as mean average ± SEM, ns=not significant, *=p\u0026lt;0.05, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, ****=p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/3490f71eaca57462ec3ed0e1.png"},{"id":105752215,"identity":"89911edc-d5e4-433e-8153-6a472432cdba","added_by":"auto","created_at":"2026-03-30 15:56:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26252933,"visible":true,"origin":"","legend":"","description":"","filename":"EarlymitochondrialgenedysregulationprecedesmotorneurondegenerationingenomicallyhumanisedFUSmutantmice18.3.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2_covered_f8fa3c58-9197-4310-9ade-044098377d1b.pdf"},{"id":105326434,"identity":"a69b5e26-12c5-43d9-9d55-96c78dad5f81","added_by":"auto","created_at":"2026-03-24 19:05:05","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163903138,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigs.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/5e9605e95581adb17b3c6710.pptx"},{"id":105565140,"identity":"1b774344-125e-4dee-9750-aeef647cd5b7","added_by":"auto","created_at":"2026-03-27 12:52:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":89400,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDocument1RNAseqcode.docx","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/7939298d9b7bc51921b62f55.docx"},{"id":105326423,"identity":"6053ff8c-f7b3-4fc7-a17d-975d8a79d7df","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5536422,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataFile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/7918527616042d194620ad7e.xlsx"},{"id":105326420,"identity":"d7cd609b-1c25-4d50-bf66-9d3c8420cf74","added_by":"auto","created_at":"2026-03-24 19:05:00","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":75029,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataFile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/a452f51f0034848276f5a4e1.xlsx"},{"id":105326424,"identity":"936ae4a7-d924-4bad-a131-2d445e92555f","added_by":"auto","created_at":"2026-03-24 19:05:01","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11583992,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo1AxonalTransportFUSP525Lmice.mp4","url":"https://assets-eu.researchsquare.com/files/rs-9043045/v2/5395cdb85dc0e9f5e055cd82.mp4"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Early mitochondrial gene dysregulation precedes motor neuron degeneration in genomically humanised FUS mutant mice","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":true,"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":"Amyotrophic lateral sclerosis (ALS), FUS, humanised, transcriptomics, metabolism, muscle","lastPublishedDoi":"10.21203/rs.3.rs-9043045/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9043045/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, with mutations in the Fused in Sarcoma (\u003cem\u003eFUS\u003c/em\u003e) gene accounting for some of the most aggressive forms of the disease. Although \u003cem\u003eFUS\u003c/em\u003e mutations are rare, accounting for only around 5% of all familial ALS cases, they are an important paradigm for study because FUS is an RNA binding protein and disruption to RNA metabolism is prominent across ALS subtypes. Here we present two novel, genomically humanised gene-targeted \u003cem\u003eFUS\u003c/em\u003e mouse models expressing ALS patient mutations P525L or Q519Ifs. These humanised models express only human FUS protein under physiological control of the endogenous mouse promoter. Humanised knock-in \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice developed a mid-life onset reduction in motor performance with neuromuscular denervation, motor neuron loss, and unilateral limb muscle weakness. Pre-symptomatic transcriptomic changes suggested metabolic impairments as an early phenotype in skeletal muscle. We also directly compared phenotypes between \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eP525L\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs\u003c/em\u003e\u003c/sup\u003e expressing mice. \u003cem\u003ehFUS\u003c/em\u003e\u003csup\u003e\u003cem\u003eQ519Ifs \u003c/em\u003e\u003c/sup\u003eexpressing mice were more severely affected, including a pronounced developmental phenotype which varied in severity on different genetic backgrounds. The phenotypes of these new FUS-ALS models highlight the potential of fully humanised knock-in mice to aid in unravelling early disease mechanisms, and ultimately to assist in the development of therapies targeted towards the human \u003cem\u003eFUS\u003c/em\u003e gene and protein in ALS.\u003c/p\u003e","manuscriptTitle":"Early mitochondrial gene dysregulation precedes motor neuron degeneration in genomically humanised FUS mutant mice","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-03-24 19:04:55","doi":"10.21203/rs.3.rs-9043045/v2","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}},{"code":1,"date":"2026-03-10 17:00:14","doi":"10.21203/rs.3.rs-9043045/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":"424d1aa5-2807-458c-8874-1fcbc517dee5","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T17:00:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 19:04:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-9043045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9043045","identity":"rs-9043045","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}