Article

Muscle specific kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors

School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Experimental Neurology (Impact Factor: 4.7). 07/2012; 237(2):286-95. DOI: 10.1016/j.expneurol.2012.06.034
Source: PubMed

ABSTRACT

In myasthenia gravis muscle weakness is caused by autoantibodies against components of the neuromuscular junction. Patient autoantibodies against muscle specific kinase (MuSK) deplete MuSK from the postsynaptic membrane and reproduce signs of myasthenia gravis when injected into mice. Here we have examined the time-course of structural and functional changes that lead up to synaptic failure. C57Bl6J mice received daily injections of anti-MuSK patient IgG for 15days. Mice began to lose weight from day 12 and demonstrated whole-body weakness by day 14. Electromyography indicated synaptic impairment from day 6 in the gastrocnemius muscle and from day 10 in the diaphragm muscle. Confocal microscopy revealed linear declines in the area and density of postsynaptic acetylcholine receptors (3-5% per day) from day 1 through day 15 of the injection series in all five muscles examined. Intracellular recordings from the diaphragm muscle revealed comparable progressive declines in the amplitude of the endplate potential and miniature endplate potential of 3-4% per day. Neither quantal content nor the postsynaptic action potential threshold changed significantly over the injection series. The inverse relationship between the quantal amplitude of a synapse and its quantal content disappeared only late in the injection series (day 10). Our results suggest that the primary myasthenogenic action of anti-MuSK IgG is to cause wastage of postsynaptic acetylcholine receptor density. Consequent reductions in endplate potential amplitudes culminated in failure of neuromuscular transmission.

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Available from: William D Phillips, Apr 16, 2014
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    • "This results in progressive declines in endplate AChR labeling and endplate potential amplitude. Weight loss and whole body weakness develops by days 12–15 (Cole et al. 2008; Morsch et al. 2012). In this study, the acute (immediate) impact of MuSK MG IgG upon the motor endplate was assessed 24 h after the first IgG injection. "
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    ABSTRACT: We investigated the influence of postsynaptic tyrosine kinase signaling in a mouse model of muscle-specific kinase (MuSK) myasthenia gravis (MG). Mice administered repeated daily injections of IgG from MuSK MG patients developed impaired neuromuscular transmission due to progressive loss of acetylcholine receptor (AChR) from the postsynaptic membrane of the neuromuscular junction. In this model, anti-MuSK-positive IgG caused a reduction in motor endplate immunolabeling for phosphorylated Src-Y418 and AChR β-subunit-Y390 before any detectable loss of MuSK or AChR from the endplate. Adeno-associated viral vector (rAAV) encoding MuSK fused to enhanced green fluorescent protein (MuSK-EGFP) was injected into the tibialis anterior muscle to increase MuSK synthesis. When mice were subsequently challenged with 11 daily injections of IgG from MuSK MG patients, endplates expressing MuSK-EGFP retained more MuSK and AChR than endplates of contralateral muscles administered empty vector. Recordings of compound muscle action potentials from myasthenic mice revealed less impairment of neuromuscular transmission in muscles that had been injected with rAAV-MuSK-EGFP than contralateral muscles (empty rAAV controls). In contrast to the effects of MuSK-EGFP, forced expression of rapsyn-EGFP provided no such protection to endplate AChR when mice were subsequently challenged with MuSK MG IgG. In summary, the immediate in vivo effect of MuSK autoantibodies was to suppress MuSK-dependent tyrosine phosphorylation of proteins in the postsynaptic membrane, while increased MuSK synthesis protected endplates against AChR loss. These results support the hypothesis that reduced MuSK kinase signaling initiates the progressive disassembly of the postsynaptic membrane scaffold in this mouse model of MuSK MG.
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    • "Intriguingly, compensatory ACh release upregulation was found absent in this model as well. Similar synaptic electrophysiological defects were subsequently observed in mice passively transferred with total IgG from MuSK MG patients (Morsch et al., 2012; Viegas et al., 2012). Jointly, the passive and active MuSK MG mouse models show that MuSK autoantibodies (of the IgG4 subclass) are severely pathogenic and cause reduced MEPP amplitude without compensatory increased ACh release, paralleled by low MEPP frequency and extra EPP rundown. "
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    ABSTRACT: Study of the electrophysiological function of the neuromuscular junction (NMJ) is instrumental in the understanding of the symptoms and pathophysiology of myasthenia gravis (MG), an autoimmune disorder characterized by fluctuating and fatigable muscle weakness. Most patients have autoantibodies to the acetylcholine receptor at the NMJ. However, in recent years autoantibodies to other crucial postsynaptic membrane proteins have been found in previously 'seronegative' MG patients. Electromyographical recording of compound and single-fibre muscle action potentials provides a crucial in vivo method to determine neuromuscular transmission failure while ex vivo (miniature) endplate potential recordings can reveal the precise synaptic impairment. Here we will review these electrophysiological methods used to assess NMJ function and discuss their application and typical results found in the diagnostic and experimental study of patients and animal models of the several forms of MG. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jan 2015 · Experimental Neurology
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    • "The phrenic nerve-hemidiaphragm muscle preparation from the same mice was then used to record endplate potentials. As expected, the amplitudes of nerve-evoked EPPs and spontaneous mEPPs were rather low in anti-MuSK injected mice (∼0.3 mV), compared to values previously recorded in healthy mice (∼0.6 mV; [31], [38]). For mice injected with anti-MuSK IgG, treatment with albuterol (8 mg/kg/day) did not significantly change the amplitudes of the EPPs or mEPPs (Fig. 5A & B), the frequency of spontaneous mEPPs (Fig. 5C), nor the number of quanta released per nerve impulse (quantal content; Fig. 5D). "
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    ABSTRACT: The β2-adrenergic receptor agonist, albuterol, has been reported beneficial in treating several forms of congenital myasthenia. Here, for the first time, we examined the potential benefit of albuterol in a mouse model of anti-Muscle Specific Kinase (MuSK) myasthenia gravis. Mice received 15 daily injections of IgG from anti-MuSK positive patients, which resulted in whole-body weakness. At neuromuscular junctions in the tibialis anterior and diaphragm muscles the autoantibodies caused loss of postsynaptic acetylcholine receptors, and reduced the amplitudes of the endplate potential and spontaneous miniature endplate potential in the diaphragm muscle. Treatment with albuterol (8 mg/kg/day) during the two-week anti-MuSK injection series reduced the degree of weakness and weight loss, compared to vehicle-treated mice. However, the compound muscle action potential recorded from the gastrocnemius muscle displayed a decremental response in anti-MuSK-injected mice whether treated with albuterol or vehicle. Ongoing albuterol treatment did not increase endplate potential amplitudes compared to vehicle-treated mice nor did it prevent the loss of acetylcholine receptors from motor endplates. On the other hand, albuterol treatment significantly reduced the degree of fragmentation of endplate acetylcholine receptor clusters and increased the extent to which the remaining receptor clusters were covered by synaptophysin-stained nerve terminals. The results provide the first evidence that short-term albuterol treatment can ameliorate weakness in a robust mouse model of anti-MuSK myasthenia gravis. The results also demonstrate that it is possible for albuterol treatment to reduce whole-body weakness without necessarily reversing myasthenic impairment to the structure and function of the neuromuscular junction.
    Full-text · Article · Feb 2014 · PLoS ONE
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