Nazanin Ghazanfari

Patient autoantibodies deplete postsynaptic muscle‐specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice

The postsynaptic muscle‐specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody‐positive myasthenia gravis patients (MuSK IgG; 45 mg day−1 i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51–73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR β‐subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK‐rapsyn system of AChR clustering.

Muscle specific kinase : organiser of synaptic membrane domains

Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.

Pyridostigmine but not 3,4‐diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle‐specific kinase autoantibody

•  A mouse model of anti‐muscle‐specific kinase (MuSK) myasthenia gravis was used to study the effect of pyridostigmine (a cholinesterase inhibitor drug commonly used in myasthenia) on the disease process at the neuromuscular junction. •  In mice receiving injections of anti‐MuSK‐positive patient IgG, pyridostigmine treatment for 7–9 days did not prevent myasthenia, and even precipitated weakness. •  Pyridostigmine treatment potentiated the anti‐MuSK‐induced reductions in postsynaptic acetylcholine receptor density and endplate potential (EPP) amplitude. •  3,4‐Diaminopyridine, a drug that increases the number of quanta released (rather than the duration of each quantal response), elevated EPP amplitude without exacerbating the anti‐MuSK‐induced loss of acetylcholine receptors. •  The results suggest that cholinergic‐ and MuSK‐mediated signalling may converge postsynaptically to regulate the mature acetylcholine receptor scaffold.

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

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 µm2 and nerve terminal synaptophysin 168±14 µm2 (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 µm2 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.

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

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 15 days. 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.

Effects of the ß2-adrenoceptor agonist, albuterol, in a mouse model of anti-MuSK myasthenia gravis.

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.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

Myasthenic anti‐muscle‐specific‐kinase (MuSK) IgG was injected into mice to study its effect upon the MuSK signalling pathway and the homeostasis of postsynaptic acetylcholine receptor packing at the neuromuscular junction. Densities of MuSK, activated Src kinase, phosphorylated ACh receptors and rapsyn were all reduced at motor endplates while β‐dystroglycan was unaffected. Pulse‐labelling showed that the slow decline in junctional ACh receptor density could be explained largely by diminished retention of ACh receptors within the postsynaptic membrane scaffold. The results suggest that anti‐MuSK IgG reduces the density of MuSK, associated tyrosine phosphorylation and retention of junctional ACh receptors within the postsynaptic membrane.

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

Forced expression of muscle specific kinase slows postsynaptic acetylcholine receptor loss in a mouse model of MuSK myasthenia gravis

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.

The mouse passive‐transfer model of MuSK myasthenia gravis: disrupted MuSK signaling causes synapse failure

While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle‐specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre‐ and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti‐MuSK+ myasthenia gravis patients developed muscle weakness, associated with neuromuscular transmission failure. MuSK autoantibodies are predominantly of the IgG4 type. They suppress the kinase activity of MuSK and the phosphorylation of target proteins in the postsynaptic membrane. Loss of postsynaptic acetylcholine receptors is the primary cause of neuromuscular transmission failure. MuSK autoantibodies also disrupt the capacity of the motor nerve terminal to adaptively increase acetylcholine release in response to the reduced postsynaptic responsiveness to acetylcholine. The passive IgG transfer model of MuSK myasthenia gravis has been used to test candidate treatments. Pyridostigmine, a first‐line cholinesterase inhibitor drug, exacerbated the disease process, while 3,4‐diaminopyridine and albuterol were found to be beneficial in this mouse model.