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      Modulation of Agrin and RhoA Pathways Ameliorates Movement Defects and Synapse Morphology in MYO9A-Depleted Zebrafish

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          Abstract

          Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by impaired function of the neuromuscular junction (NMJ). This is due to defects in one of the many proteins associated with the NMJ. In three patients with CMS, missense mutations in a gene encoding an unconventional myosin protein, MYO9A, were identified as likely causing their disorder. Preliminary studies revealed a potential involvement of the RhoA/ROCK pathway and of a key NMJ protein, agrin, in the pathophysiology of MYO9A-depletion. In this study, a CRISPR/Cas9 approach was used to generate genetic mutants of MYO9A zebrafish orthologues, myo9aa/ab, to expand and refine the morphological analysis of the NMJ. Injection of NT1654, a synthetic agrin fragment compound, improved NMJ structure and zebrafish movement in the absence of Myo9aa/ab. In addition, treatment of zebrafish with fasudil, a ROCK inhibitor, also provided improvements to the morphology of NMJs in early development, as well as rescuing movement defects, but not to the same extent as NT1654 and not at later time points. Therefore, this study highlights a role for MYO9A at the NMJ, the first unconventional myosin motor protein associated with a neuromuscular disease, and provides a potential mechanism of action of MYO9A-pathophysiology.

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          Most cited references33

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          Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

          A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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            The role of the Rho GTPases in neuronal development.

            Our brain serves as a center for cognitive function and neurons within the brain relay and store information about our surroundings and experiences. Modulation of this complex neuronal circuitry allows us to process that information and respond appropriately. Proper development of neurons is therefore vital to the mental health of an individual, and perturbations in their signaling or morphology are likely to result in cognitive impairment. The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance. Given the importance of these molecules in these processes, it is therefore not surprising that mutations in genes encoding a number of regulators and effectors of the Rho GTPases have been associated with human neurological diseases. This review will focus on the role of the Rho GTPases and their associated signaling molecules throughout neuronal development and discuss how perturbations in Rho GTPase signaling may lead to cognitive disorders.
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              Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.

              During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.
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                Author and article information

                Journal
                Cells
                Cells
                cells
                Cells
                MDPI
                2073-4409
                07 August 2019
                August 2019
                : 8
                : 8
                : 848
                Affiliations
                [1 ]John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK
                [2 ]Interdisciplinary School of Health Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON K1H 8L1, Canada
                [3 ]Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON K1H 8L1, Canada
                [4 ]Institute of Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
                [5 ]Neurotune AG. Wagistrasse 27a, 8952 Schlieren, Switzerland
                [6 ]Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, UK
                [7 ]Institute of Neuroscience, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
                [8 ]Department of Neuropediatrics, Centre for Neuromuscular Disorders in Children, University Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany
                [9 ]Department of Neuropediatrics and Muscle Disorders, Faculty of Medicine, Medical Center—University of Freiburg, 79106 Freiburg, Germany
                [10 ]Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
                [11 ]Division of Neurology, Department of Medicine, The Ottawa Hospital, Ottawa, ON K1H 7X5, Canada
                Author notes
                [* ]Correspondence: hlochmuller@ 123456cheo.on.ca ; Tel.: +1-613-737-7600 (ext. 4014)
                Author information
                https://orcid.org/0000-0001-9579-3514
                https://orcid.org/0000-0003-2324-8001
                Article
                cells-08-00848
                10.3390/cells8080848
                6721702
                31394789
                ca2d94e0-4fca-4ccc-b721-4cb6f87feab4
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 01 July 2019
                : 02 August 2019
                Categories
                Article

                myosin ixa,unconventional myosin,nt1654,fasudil,neuromuscular junction,myo9aa,myo9ab

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