A bioassay for neuromuscular junction-restricted complement activation by myasthenia gravis acetylcholine receptor antibodies

Journal of Neuroscience Methods, vol. 373, pp. 109551

Abstract

BACKGROUND: Myasthenia gravis (MG) is an autoimmune neuromuscular disorder hallmarked by fluctuating fatigable muscle weakness. Most patients have autoantibodies against acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). These are thought to have three possible pathogenic mode-of-actions: 1) cross-linking and endocytosis of AChRs, 2) direct block of AChRs and 3) complement activation. The relative contributions of these mechanisms to synaptic block and muscle weakness of individual patients cannot be determined. It likely varies between patients and perhaps also with disease course, depending on the nature of the circulating AChR antibodies. NEW METHOD: We developed a new bioassay which specifically enables functional characterization and quantification of complement-mediated synaptic damage at NMJs, without interference of the other pathogenic mechanisms. To this end, we pre-incubated mouse hemi-diaphragm muscle-nerve preparations with mAb35-hG1, a humanized rat AChR monoclonal and subsequently exposed the preparation to normal human serum as a complement source. NMJ-restricted effects were studied. RESULTS: Clearly NMJ-restricted damage occurred. With immunohistology we showed complement deposition at NMJs, and synaptic electrophysiological measurements demonstrated transmission block. In whole-muscle contraction experiments we quantified the effect and characterized its onset and progression during the incubation with normal human serum. COMPARISON WITH EXISTING METHODS: With this new assay the complement-mediated component of myasthenic NMJ pathology can be studied separately. CONCLUSIONS: Our assay will be of importance in detailed mechanistic studies of local complement activation at NMJs, investigations of new complement inhibitors, and laboratory pre-screening of therapeutic efficacy for individual MG patients to optimize care with clinically approved complement inhibitors.

Authors 4

1. Jaap J. Plomp corresponding Aachen Departments of Neurology

   Leiden University Medical Center

   Affiliation as printed

   Departments of Neurology. Electronic address: j.j.plomp@lumc.nl

   Departments of Neurology

2. Maartje G. M. Huijbers Aachen Human Genetics

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Departments of Neurology; Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands

   Departments of Neurology

   Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands

3. Jan J.G.M. Verschuuren
   Affiliation as printed

   Departments of Neurology

4. Anna Borodovsky

   Alnylam Pharmaceuticals (United States)

   Affiliation as printed

   Alnylam Pharmaceuticals, Cambridge, MA, USA