title: A stem-cell based bioassay to critically assess the pathology of dysfunctional neuromuscular junctions.
abstract: Pluripotent stem cells can be directed to differentiate into motor neurons and assessed for functionality in vitro. An emerging application of this technique is to model genetically inherited diseases in differentiated motor neurons and to screen for new therapeutic targets. The neuromuscular junction (NMJ) is essential to the functionality of motor neurons and its dysfunction is a primary hallmark of motor neuron disease. However, mature NMJs that possess the functional and morphological characteristics of those formed in vivo have so far not been obtained in vitro. Here we describe the generation and analysis of mature NMJs formed between embryonic stem cell-derived motor neurons (ESCMNs) and primary myotubes. We compared the formation and maturation of NMJs generated by wild-type (NCAM+/+) ESCMNs to those generated by neural cell adhesion molecule null (NCAM-/-) ESCMNs in order to definitively test the sensitivity of this assay to identify synaptic pathology. We find that co-cultures using NCAM-/- ESCMNs replicate key in vivo NCAM-/- phenotypes and reveal that NCAM influences neuromuscular synaptogenesis by controlling the mode of synaptic vesicle endocytosis. Further, we could improve synapse formation and function in NCAM-/- co-cultures by chronic treatment with nifedipine, which blocks an immature synaptic vesicle recycling pathway. Together, our results demonstrate that this ESCMN/myofiber co-culture system is a highly sensitive bioassay for examining molecules postulated to regulate synaptic function and for screening therapeutics that will improve the function of compromised NMJs.
authors.affiliation: Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Chipman
authors.affiliation: Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada; Brain Repair Centre, Life Science Research Centre, Halifax, Nova Scotia, Canada.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada; Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; Brain Repair Centre, Life Science Research Centre, Halifax, Nova Scotia, Canada.
authors.forename: Victor F
authors.initials: VF
authors.lastname: Rafuse
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019002:CD56 Antigen / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D015815:Cell Adhesion Molecules; D002454:Cell Differentiation; D002642:Chick Embryo; D018920:Coculture Techniques; D055724:Electrophysiological Phenomena; D053595:Embryonic Stem Cells / Q000166:cytology; D004705:Endocytosis; D005260:Female; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000166:cytology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D009469:Neuromuscular Junction / Q000473:pathology*; D009543:Nifedipine / Q000737:chemistry; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology; D013234:Stem Cells / Q000166:cytology*; D013569:Synapses / Q000378:metabolism
chemical_list: D019002:CD56 Antigen; D015220:Calcium Channels; D015815:Cell Adhesion Molecules; C501063:Ncam1 protein, mouse; D009543:Nifedipine