Abstract
A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 MuSK antibodies will also contribute to the reduced AChR density and neuromuscular dysfunction in myasthenia patients with MuSK antibodies.
Highlights
Myasthenia gravis (MG) is a rare but often severe disorder of neuromuscular transmission that causes fatigable muscle weakness
Over a decade has passed since the discovery of musclespecific tyrosine kinase (MuSK) antibodies in myasthenia [3] but, in vivo transfer and active immunization studies have confirmed the pathogenicity of MuSK antibodies, whether MuSK antibodies reduce MuSK function, reduce MuSK levels, or damage the structure and function of the neuromuscular junction by other mechanisms is still unclear
We could not find evidence for MuSK endocytosis, we show that the MuSK IgG antibodies block the essential interaction between lipoprotein receptor-related protein-4 (LRP4) and MuSK
Summary
Myasthenia gravis (MG) is a rare but often severe disorder of neuromuscular transmission that causes fatigable muscle weakness. MuSK antibodies were found to interfere with this signalling pathway in vitro, reducing agrin-induced AChR clustering in C2C12 myotubes [3]. In MG patients with AChR antibodies, IgG1 and IgG3 antibodies bind to the AChR and activate complement, cause internalisation of the AChRs, and/or block AChR function; the overall effect is to cause loss of AChRs or reduction in their function. MuSK IgG1-3 did impair agrin-induced AChR clustering in C2C12 myotubes, and both IgG1-3 and IgG4 MuSK antibodies dispersed preformed clusters in agrinindependent Dok7-overexpressing C2C12 cells. These results add new information to the complex mechanisms by which. IgG1-3 and IgG4 binding to MuSK cause MuSK antibodyinduced myasthenia
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