{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T11:22:23Z","timestamp":1779362543268,"version":"3.51.4"},"posted":{"date-parts":[[2024,1,12]]},"group-title":"Neuroscience","reference-count":81,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,1,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Neuromuscular junctions (NMJs) are specialized synapses that mediate communication between motor neurons and skeletal muscles and are essential for movement. The degeneration of this system can lead to symptoms observed in neuromuscular and motor neuron diseases. Studying these synapses and their degeneration has proven challenging. Prior NMJ studies heavily relied upon the use of mouse, chick, or isolated primary human cells, which have demonstrated limited fidelity for disease modeling. To enable the study of NMJ dysfunction and model genetic diseases, we, and others, have developed methods to generate human NMJs from pluripotent stem cells (PSCs), embryonic stem cells, and induced pluripotent stem cells. However, published studies have highlighted technical limitations associated with these complex\n                  <jats:italic>in vitro<\/jats:italic>\n                  NMJ models. In this study, we developed a robust PSC-derived motor neuron and skeletal muscle co-culture method, and demonstrated its sensitivity in modeling motor neuron disease. Our method spontaneously and reproducibly forms human NMJs. We developed multiwell-multielectrode array (MEA) parameters to quantify the activity of PSC-derived skeletal muscles, as well as measured the electrophysiological activity of functional human PSC-derived NMJs. We further leveraged our method to morphologically and functionally assess NMJs from the familial amyotrophic lateral sclerosis (fALS) PSCs,\n                  <jats:italic>C9orf72<\/jats:italic>\n                  hexanucleotide (G4C2)n repeat expansion (HRE),\n                  <jats:italic>SOD1<\/jats:italic>\n                  <jats:sup>A5V<\/jats:sup>\n                  , and\n                  <jats:italic>TDP43<\/jats:italic>\n                  <jats:sup>G298S<\/jats:sup>\n                  to define the reproducibility and sensitivity of our system. We observed a significant decrease in the numbers and activity of PSC-derived NMJs developed from the different ALS lines compared to their respective controls. Furthermore, we evaluated a therapeutic candidate undergoing clinical trials and observed a variant-dependent rescue of functionality of NMJs. Our newly developed method provides a platform for the systematic investigation of genetic causes of NMJ neurodegeneration and highlights the need for therapeutic avenues to consider patient genotype.\n                <\/jats:p>","DOI":"10.1101\/2024.01.11.575304","type":"posted-content","created":{"date-parts":[[2024,1,12]],"date-time":"2024-01-12T19:25:10Z","timestamp":1705087510000},"source":"Crossref","is-referenced-by-count":2,"title":["Scalable, optically-responsive human neuromuscular junction model reveals convergent mechanisms of synaptic dysfunction in familial ALS"],"prefix":"10.64898","author":[{"given":"Daniel","family":"Chen","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0733-3591","authenticated-orcid":false,"given":"Polyxeni","family":"Philippidou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bianca de Freitas","family":"Brenha","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashleigh E.","family":"Schaffer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4064-9790","authenticated-orcid":false,"given":"Helen C.","family":"Miranda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024011600100937000_2024.01.11.575304v1.1","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/0736-5748(90)90058-A","article-title":"Changes in acetylcholine receptor distribution and binding properties at the neuromuscular junction during aging","volume":"8","year":"1990","journal-title":"Int. J. Dev. Neurosci"},{"key":"2024011600100937000_2024.01.11.575304v1.2","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.cophys.2018.05.007","article-title":"NMJ maintenance and repair in aging","volume":"4","year":"2018","journal-title":"Curr Opin Physiol"},{"key":"2024011600100937000_2024.01.11.575304v1.3","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1111\/joa.13463","article-title":"Small junction, big problems: Neuromuscular junction pathology in mouse models of amyotrophic lateral sclerosis (ALS)","volume":"241","year":"2022","journal-title":"J. Anat"},{"key":"2024011600100937000_2024.01.11.575304v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2014.03.019"},{"key":"2024011600100937000_2024.01.11.575304v1.5","doi-asserted-by":"publisher","DOI":"10.5966\/sctm.2013-0095"},{"key":"2024011600100937000_2024.01.11.575304v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.pneurobio.2012.05.007"},{"key":"2024011600100937000_2024.01.11.575304v1.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-physiol-022516-034255"},{"key":"2024011600100937000_2024.01.11.575304v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.11.008"},{"key":"2024011600100937000_2024.01.11.575304v1.9","doi-asserted-by":"publisher","DOI":"10.1089\/ten.TEB.2021.0204"},{"key":"2024011600100937000_2024.01.11.575304v1.10","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1111\/aor.14503","article-title":"FDA Modernization Act 2.0 allows for alternatives to animal testing","volume":"47","year":"2023","journal-title":"Artif. Organs"},{"key":"2024011600100937000_2024.01.11.575304v1.11","doi-asserted-by":"publisher","DOI":"10.1126\/science.adg6276"},{"key":"2024011600100937000_2024.01.11.575304v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.07.024"},{"key":"2024011600100937000_2024.01.11.575304v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/nature09229"},{"key":"2024011600100937000_2024.01.11.575304v1.14","doi-asserted-by":"publisher","DOI":"10.1126\/science.1158799"},{"key":"2024011600100937000_2024.01.11.575304v1.15","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1016\/j.bbrc.2013.07.060","article-title":"Differential effects of mutant SOD1 on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: role of chaperone network","volume":"438","year":"2013","journal-title":"Biochem. Biophys. Res. Commun"},{"key":"2024011600100937000_2024.01.11.575304v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.neures.2017.09.011"},{"key":"2024011600100937000_2024.01.11.575304v1.17","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/awaa309"},{"key":"2024011600100937000_2024.01.11.575304v1.18","doi-asserted-by":"crossref","unstructured":"Scaricamazza, S. , Salvatori, I. , Ferri, A. & Valle, C . Skeletal Muscle in ALS: An Unappreciated Therapeutic Opportunity? Cells 10, (2021).","DOI":"10.3390\/cells10030525"},{"key":"2024011600100937000_2024.01.11.575304v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2017.109"},{"key":"2024011600100937000_2024.01.11.575304v1.20","doi-asserted-by":"publisher","DOI":"10.1126\/science.1250127"},{"key":"2024011600100937000_2024.01.11.575304v1.21","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.43.2.382"},{"key":"2024011600100937000_2024.01.11.575304v1.22","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.62.3.715"},{"key":"2024011600100937000_2024.01.11.575304v1.23","doi-asserted-by":"publisher","DOI":"10.1126\/science.182.4112.588"},{"key":"2024011600100937000_2024.01.11.575304v1.24","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1039\/C7IB00144D","article-title":"Bioreactor model of neuromuscular junction with electrical stimulation for pharmacological potency testing","volume":"9","year":"2017","journal-title":"Integr. Biol"},{"key":"2024011600100937000_2024.01.11.575304v1.25","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1016\/j.stemcr.2015.02.010","article-title":"Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs","volume":"4","year":"2015","journal-title":"Stem Cell Reports"},{"key":"2024011600100937000_2024.01.11.575304v1.26","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1016\/j.jbiosc.2019.10.004","article-title":"Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons","volume":"129","year":"2020","journal-title":"J. Biosci. Bioeng"},{"key":"2024011600100937000_2024.01.11.575304v1.27","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E17-01-0046"},{"key":"2024011600100937000_2024.01.11.575304v1.28","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.molmet.2017.11.001","article-title":"Neurturin is a PGC-1\u03b11-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation","volume":"7","year":"2018","journal-title":"Mol Metab"},{"key":"2024011600100937000_2024.01.11.575304v1.29","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.167544"},{"key":"2024011600100937000_2024.01.11.575304v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejcb.2015.11.004"},{"key":"2024011600100937000_2024.01.11.575304v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2013.08.062"},{"key":"2024011600100937000_2024.01.11.575304v1.32","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.1501429"},{"key":"2024011600100937000_2024.01.11.575304v1.33","doi-asserted-by":"crossref","unstructured":"Santoso, J. W. et al. Engineering skeletal muscle tissues with advanced maturity improves synapse formation with human induced pluripotent stem cell-derived motor neurons. APL Bioeng 5, 036101 (2021).","DOI":"10.1063\/5.0054984"},{"key":"2024011600100937000_2024.01.11.575304v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2015.10.002"},{"key":"2024011600100937000_2024.01.11.575304v1.35","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/j.biomaterials.2018.02.047","article-title":"Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics","volume":"166","year":"2018","journal-title":"Biomaterials"},{"key":"2024011600100937000_2024.01.11.575304v1.36","doi-asserted-by":"crossref","unstructured":"Guo, X. et al. A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing. Adv. Ther. 3, (2020).","DOI":"10.1002\/adtp.202000133"},{"key":"2024011600100937000_2024.01.11.575304v1.37","doi-asserted-by":"publisher","DOI":"10.1039\/D1LC00497B"},{"key":"2024011600100937000_2024.01.11.575304v1.38","doi-asserted-by":"publisher","DOI":"10.7554\/elife.44530"},{"key":"2024011600100937000_2024.01.11.575304v1.39","doi-asserted-by":"crossref","unstructured":"Faustino Martins, J.-M. , et al. Self-Organizing 3D Human Trunk Neuromuscular Organoids. Cell Stem Cell 27, 498 (2020).","DOI":"10.1016\/j.stem.2020.08.011"},{"key":"2024011600100937000_2024.01.11.575304v1.40","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-24776-4"},{"key":"2024011600100937000_2024.01.11.575304v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.11.017"},{"key":"2024011600100937000_2024.01.11.575304v1.42","doi-asserted-by":"crossref","unstructured":"Lin, C.-Y. , et al. iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases. JCI Insight 4, (2019).","DOI":"10.1172\/jci.insight.124299"},{"key":"2024011600100937000_2024.01.11.575304v1.43","doi-asserted-by":"crossref","unstructured":"Vila, O. F. et al. Bioengineered optogenetic model of human neuromuscular junction. Biomaterials 276, 121033 (2021).","DOI":"10.1016\/j.biomaterials.2021.121033"},{"key":"2024011600100937000_2024.01.11.575304v1.44","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2013.02.012"},{"key":"2024011600100937000_2024.01.11.575304v1.45","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2011.391"},{"key":"2024011600100937000_2024.01.11.575304v1.46","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.12.032"},{"key":"2024011600100937000_2024.01.11.575304v1.47","doi-asserted-by":"publisher","DOI":"10.1016\/j.nbd.2011.09.014"},{"key":"2024011600100937000_2024.01.11.575304v1.48","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez Cruz , P. M., Cossins , J., Beeson , D. & Vincent , A. The Neuromuscular Junction in Health and Disease: Molecular Mechanisms Governing Synaptic Formation and Homeostasis. Front. Mol. Neurosci. 13, 610964 (2020).","DOI":"10.3389\/fnmol.2020.610964"},{"key":"2024011600100937000_2024.01.11.575304v1.49","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0046663"},{"key":"2024011600100937000_2024.01.11.575304v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/35074025"},{"key":"2024011600100937000_2024.01.11.575304v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/b978-1-4377-0310-8.00021-x"},{"key":"2024011600100937000_2024.01.11.575304v1.52","doi-asserted-by":"crossref","unstructured":"Moloney, E. B. , de Winter, F. & Verhaagen, J . ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease. Front. Neurosci. 8, 252 (2014).","DOI":"10.3389\/fnins.2014.00252"},{"key":"2024011600100937000_2024.01.11.575304v1.53","doi-asserted-by":"crossref","unstructured":"Butti, Z. , Pan, Y. E. , Giacomotto, J. & Patten, S. A . Reduced C9orf72 function leads to defective synaptic vesicle release and neuromuscular dysfunction in zebrafish. Commun Biol 4, 792 (2021).","DOI":"10.1038\/s42003-021-02302-y"},{"key":"2024011600100937000_2024.01.11.575304v1.54","doi-asserted-by":"crossref","unstructured":"L\u00e9pine, S. , Castellanos-Montiel, M. J. & Durcan, T. M . TDP-43 dysregulation and neuromuscular junction disruption in amyotrophic lateral sclerosis. Transl. Neurodegener. 11, 56 (2022).","DOI":"10.1186\/s40035-022-00331-z"},{"key":"2024011600100937000_2024.01.11.575304v1.55","doi-asserted-by":"crossref","unstructured":"Tovar-Y-Romo, L. B. , Ram\u00edrez-Jarqu\u00edn, U. N. , Lazo-G\u00f3mez, R. & Tapia, R . Trophic factors as modulators of motor neuron physiology and survival: implications for ALS therapy. Front. Cell. Neurosci. 8, 61 (2014).","DOI":"10.3389\/fncel.2014.00061"},{"key":"2024011600100937000_2024.01.11.575304v1.56","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.1038\/s41591-022-01956-3","article-title":"Transplantation of human neural progenitor cells secreting GDNF into the spinal cord of patients with ALS: a phase 1\/2a trial","volume":"28","year":"2022","journal-title":"Nat. Med"},{"key":"2024011600100937000_2024.01.11.575304v1.57","doi-asserted-by":"publisher","DOI":"10.1016\/j.coph.2012.02.002"},{"key":"2024011600100937000_2024.01.11.575304v1.58","first-page":"768","article-title":"Current Progress in the Creation","volume":"18","year":"2022","journal-title":"Characterization, and Application of Human Stem Cell-derived in Vitro Neuromuscular Junction Models. Stem Cell Rev Rep"},{"key":"2024011600100937000_2024.01.11.575304v1.59","doi-asserted-by":"crossref","unstructured":"Fralish, Z. , Lotz, E. M. , Chavez, T. , Khodabukus, A. & Bursac, N. Neuromuscular Development and Disease: Learning From and Models. Front Cell Dev Biol 9, 764732 (2021).","DOI":"10.3389\/fcell.2021.764732"},{"key":"2024011600100937000_2024.01.11.575304v1.60","doi-asserted-by":"crossref","unstructured":"Krakora, D. , Macrander, C. & Suzuki, M . Neuromuscular junction protection for the potential treatment of amyotrophic lateral sclerosis. Neurol. Res. Int. 2012, 379657 (2012).","DOI":"10.1155\/2012\/379657"},{"key":"2024011600100937000_2024.01.11.575304v1.61","doi-asserted-by":"crossref","unstructured":"Henriques, A. , Pitzer, C. & Schneider, A . Neurotrophic growth factors for the treatment of amyotrophic lateral sclerosis: where do we stand? Front. Neurosci. 4, 32 (2010).","DOI":"10.3389\/fnins.2010.00032"},{"key":"2024011600100937000_2024.01.11.575304v1.62","doi-asserted-by":"crossref","first-page":"12969","DOI":"10.1021\/acsomega.9b00973","article-title":"Toward Building the Neuromuscular Junction: In Vitro Models To Study Synaptogenesis and Neurodegeneration","volume":"4","year":"2019","journal-title":"ACS Omega"},{"key":"2024011600100937000_2024.01.11.575304v1.63","doi-asserted-by":"publisher","DOI":"10.1038\/nm.4214"},{"key":"2024011600100937000_2024.01.11.575304v1.64","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1016\/j.stemcr.2018.04.002","article-title":"Large-Scale Expansion of Human iPSC-Derived Skeletal Muscle Cells for Disease Modeling and Cell-Based Therapeutic Strategies","volume":"10","year":"2018","journal-title":"Stem Cell Reports"},{"key":"2024011600100937000_2024.01.11.575304v1.65","doi-asserted-by":"crossref","unstructured":"Joshi, J. , Rubart, M. & Zhu, W. Optogenetics: Background, Methodological Advances and Potential Applications for Cardiovascular Research and Medicine. Front Bioeng Biotechnol 7, 466 (2019).","DOI":"10.3389\/fbioe.2019.00466"},{"key":"2024011600100937000_2024.01.11.575304v1.66","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3863-06.2006"},{"key":"2024011600100937000_2024.01.11.575304v1.67","doi-asserted-by":"crossref","unstructured":"Hawrot, J. , Imhof, S. & Wainger, B. J . Modeling cell-autonomous motor neuron phenotypes in ALS using iPSCs. Neurobiol. Dis. 134, 104680 (2020).","DOI":"10.1016\/j.nbd.2019.104680"},{"key":"2024011600100937000_2024.01.11.575304v1.68","doi-asserted-by":"crossref","unstructured":"Dobrowolny, G. & Scicchitano, B. M . The Role of Skeletal Muscle in Neuromuscular Diseases: From Cellular and Molecular Players to Therapeutic Interventions. Cells 11, (2022).","DOI":"10.3390\/cells11071207"},{"key":"2024011600100937000_2024.01.11.575304v1.69","doi-asserted-by":"publisher","DOI":"10.2174\/1389450111007011250"},{"key":"2024011600100937000_2024.01.11.575304v1.70","doi-asserted-by":"publisher","DOI":"10.3389\/fncel.2017.00196"},{"key":"2024011600100937000_2024.01.11.575304v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2288-7"},{"key":"2024011600100937000_2024.01.11.575304v1.72","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaa9344"},{"key":"2024011600100937000_2024.01.11.575304v1.73","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.11.018"},{"key":"2024011600100937000_2024.01.11.575304v1.74","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.10.027"},{"key":"2024011600100937000_2024.01.11.575304v1.75","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2016.04.006"},{"key":"2024011600100937000_2024.01.11.575304v1.76","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/j.neuron.2016.04.005","article-title":"C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS\/FTD","volume":"90","year":"2016","journal-title":"Neuron"},{"key":"2024011600100937000_2024.01.11.575304v1.77","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.2315-06.2006"},{"key":"2024011600100937000_2024.01.11.575304v1.78","doi-asserted-by":"publisher","DOI":"10.1002\/brb3.143"},{"key":"2024011600100937000_2024.01.11.575304v1.79","doi-asserted-by":"crossref","first-page":"2196","DOI":"10.1007\/s10439-015-1259-x","article-title":"Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis","volume":"43","year":"2015","journal-title":"Ann. Biomed. Eng"},{"key":"2024011600100937000_2024.01.11.575304v1.80","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0000689"},{"key":"2024011600100937000_2024.01.11.575304v1.81","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3787"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.01.11.575304","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:47:07Z","timestamp":1768456027000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.01.11.575304"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,12]]},"references-count":81,"URL":"https:\/\/doi.org\/10.1101\/2024.01.11.575304","relation":{"has-version":[{"id-type":"doi","id":"10.7554\/eLife.95388.1","asserted-by":"object"}]},"subject":[],"published":{"date-parts":[[2024,1,12]]},"subtype":"preprint"}}