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


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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    • "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.
    Experimental Neurology 01/2015; 270. DOI:10.1016/j.expneurol.2015.01.007 · 4.70 Impact Factor
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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.
    PLoS ONE 02/2014; 9(2):e87840. DOI:10.1371/journal.pone.0087840 · 3.23 Impact Factor
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    • "At the time of sacrifice muscles were immediately dissected, embedded in OCT compound (ProSciTech Australia), snap frozen in liquid nitrogen and stored at −80°C. Cryosections were stained for immunofluorescence as described previously [23], [24]. Briefly, the TA muscle was sectioned longitudinally to the long axis of the fibers (20 µm). "
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    ABSTRACT: Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm(2) and nerve terminal synaptophysin 168±14 µm(2) (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm(2) area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.
    PLoS ONE 07/2013; 8(7):e67970. DOI:10.1371/journal.pone.0067970 · 3.23 Impact Factor
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