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

Abstract

Key points• 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.Abstract In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft.

Current Treatment, Emerging Translational Therapies, and New Therapeutic Targets for Autoimmune Myasthenia Gravis

“…Short-term treatment with the β2 adrenergic agonist albuterol was shown to improve weakness in a mouse model of anti-MuSK MG [151], suggesting that this class of agents might provide benefit in human MuSK MG. 3,4-Diaminopyridine is another symptomatic therapy with potential application in MuSK MG. Exploring use of this drug, which enhances AChR release at the motor nerve terminal, in MuSK MG is supported by preclinical models, experience with congenital MG with MuSK mutations, and case reports [152,153]. Other endplate targets with potential therapeutic application in MG include agrin potentiators and positive allosteric modulators of the skeletal muscle AChR [154,155].…”

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

“…AChR area was also markedly reduced in mice injected with IgG from anti-MuSK myasthenia gravis patients 17,21 . Myasthenic mice treated with the cholinesterase inhibitor drug, pyridostigmine, displayed a further significant reduction in endplate AChR area 20 .…”

“…13. Prepare a spreadsheet of data sample means, calculate and plot standard deviations and standard errors as histograms or scatterplots 20,22 . Note that the value of n generally represents the number of mice per sample group for statistical purposes.…”

“…Frequency histograms of pooled data revealed roughly normal distributions for the area of endplate AChR and synaptophysin ( Figure 6A' and B'). However a normal distribution of synaptic areas cannot be assumed in disease states such as myasthenia gravis 16,20 . This may affect the choice of the statistical test.…”

“…Qualitatively similar findings were reported by other research groups 10,19 . The same NMJ measurement methods have since been used to assess the impact of three drugs for treating anti-MuSK myasthenia gravis in this mouse model 20,21 .…”