MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion

Human Molecular Genetics, Apr 2018

O’Connor, Emily, Phan, Vietxuan, Cordts, Isabell, Cairns, George, Hettwer, Stefan, Cox, Daniel, Lochmüller, Hanns, Roos, Andreas

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MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion

Human Molecular Genetics MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion Emily O'Connor 2 Vietxuan Phan 1 Isabell Cordts 2 George Cairns 0 Stefan Hettwer 3 Daniel Cox 2 Hanns Lochmu¨ ller 2 Andreas Roos 1 2 0 Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne NE1 3BZ , UK 1 Leibniz-Institut fu ̈ r Analytische Wissenschaften-ISAS e.V , Dortmund 44227 , Germany 2 John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne NE1 3BZ , UK 3 Neurotune AG , 8952 Schlieren , Switzerland Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterized by compromised function of the neuromuscular junction, manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterized. On the basis of the role of MYO9A as an actinbased molecular motor and as a negative regulator of RhoA, we hypothesized that loss of MYO9A may affect the neuronal cytoskeleton, leading to impaired intracellular transport. To investigate this, we used MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells), revealing altered expression of a number of cytoskeletal proteins important for neuron structure and intracellular transport. On the basis of these findings, the effect on protein transport was determined using a vesicular recycling assay which revealed impaired recycling of a neuronal growth factor receptor. In addition, an unbiased approach utilizing proteomic profiling of the secretome revealed a key role for defective intracellular transport affecting proper protein secretion in the pathophysiology of MYO9A-related CMS. This also led to the identification of agrin as being affected by the defective transport. Zebrafish with reduced MYO9A orthologue expression were treated with an artificial agrin compound, ameliorating defects in neurite extension and improving motility. In summary, loss of MYO9A affects the neuronal cytoskeleton and leads to impaired transport of proteins, including agrin, which may provide a new and unexpected treatment option. Introduction The neuromuscular junction (NMJ) is a tightly controlled functional unit, with highly specialized pre- and post-synaptic regions that must function in a coordinated manner for effective NMJ transmission to be achieved. The complex organization of the neuronal cytoskeleton is crucial for both NMJ formation and functionality. Actin in particular is a highly important component of the cytoskeleton as it enables complex and dynamic movement of cargo for junctional signalling and formation by using members of the myosin superfamily. Rho-GTPases and Rho-GEFs (GDP/GTP nucleotide exchange factors) are critical in the control of actin dynamics and disturbed Rho has already been implicated in the vulnerability of the peripheral nervous system ( 1,2 ). Other cytoskeletal components, also important for NMJ functionality, include microtubules which facilitate the long-distance transport necessary for motor neurons and neurofilaments that provide pre-dominantly structural support to neurons but are implicated in a range of peripheral neuropathies ( 3–5 ). Various mutations in critical NMJ proteins are known to cause primary defects in neuromuscular transmission and lead to the clinical picture of congenital myasthenic syndromes (CMS). The main symptom of patients with CMS is fatigable muscle weakness that usually starts in childhood and can disrupt the skeletal, respiratory, bulbar and ocular muscles depending on the protein involved. CMS constitute a group of genetically heterogenic disorders and causative genes can be broadly categorized as pre-synaptic, synaptic or post-synaptic. Recently, we expanded the catalogue of known pre-synaptic CMS causative genes by describing recessive missense mutations in the unconventional myosin encoding gene, MYO9A, present in three patients showing a severe neonatal phenotype with both respiratory and bulbar involvement ( 6 ). Unconventional myosins are actin based molecular motors that are present in almost all eukaryotic cells. There are two class 9 myosin proteins expressed in humans; MYO9A and MYO9B. MYO9A follows the same basic structural composition as other unconventional myosins, containing a force generating myosin motor domain in the head region which binds actin, as well as an extended tail region that contains a Rho-GAP (Rho GTPase-activating protein) domain. The presence of this Rho-GAP domain, unique to class 9 myosins, infers the ability to negatively regulate the activity of Rho by converting it from its active GTP-bound state to an inactive GDP-bound state, with the pathway summarized in Supplementary Material, Figure S1A ( 7 ). Because of direct interaction with actin and influence of actin via the Rho-GAP domain, MYO9A ha (...truncated)


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O’Connor, Emily, Phan, Vietxuan, Cordts, Isabell, Cairns, George, Hettwer, Stefan, Cox, Daniel, Lochmüller, Hanns, Roos, Andreas. MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion, Human Molecular Genetics, 2018, pp. 1434-1446, Volume 27, Issue 8, DOI: 10.1093/hmg/ddy054