Human in vitro neuromuscular junction model to functionally dissect the pathogenic mechanism of anti-AChR autoantibody-positive myasthenia gravis

Abstract Background Myasthenia gravis is a rare autoimmune disease mediated by autoantibodies directed against acetylcholine receptors (AChRs) at the neuromuscular junction. These autoantibodies cause dysfunction through AChR blockade, AChR degradation due to crosslinking and internalisation, and complement activation. Methods A novel in vitro model of the human neuromuscular junction was established on a microfluidic platform to investigate the effect of anti-AChR autoantibodies on complement activation and neuromuscular transmission and the mechanism of action of complement inhibition in myasthenia gravis. The NeuroMuscle TM platform enabled the connection of human induced pluripotent stem-cell-derived motor neuron spheroids with three-dimensional cultures of skeletal muscle fibres, forming functional neuromuscular junctions. Functional connectivity was assessed by glutamate stimulation of motor neuron spheroids and monitoring of calcium transients in genetically encoded calcium indicator protein 6 (GCaMP6)-transduced muscle fibres. Results Incubation of in vitro neuromuscular junction tissues with sera from patients with anti-AChR autoantibody-positive myasthenia gravis, in contrast to healthy controls, induced a significant increase in membrane attack complex (MAC) deposition and complement split products, accompanied by a notable reduction in calcium transients. Treatment with zilucoplan, a complement component 5 (C5) inhibitor, prevented complement activation and preserved neuromuscular junction functional integrity. The model demonstrated that complement-mediated damage is a major driver of neuromuscular junction functional impairment in the myasthenia gravis patient sera tested in this study. Furthermore, the study explored the reversibility of neuromuscular junction damage, revealing that shortening the delay before initiating complement inhibitor treatment in the in vitro neuromuscular junction tissues enhances the reversibility of neuromuscular transmission. Conclusions These findings offer a mechanistic rationale for the observed clinical response in patients with anti-AChR autoantibody-positive myasthenia gravis treated with C5 inhibitors. The in vitro neuromuscular junction model provides a robust platform for studying the mechanistic pathways of complement-mediated damage and evaluating therapeutic interventions for myasthenia gravis.