Using Xenopus tissue cultures for the study of myasthenia gravis pathogenesis.
Myasthenia gravis (MG), the most common autoimmune disease of neuromuscular junction (NMJ), is heterogeneous in terms of pathophysiology, which is determined by the pathogenic antigen of autoantibodies targeting to synaptic proteins at the NMJs. Currently, patients suspected with MG are routinely screened for the presence of autoantibodies against acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) using a cell-based assay (CBA) that involves the expression of target synaptic membrane protein in heterologous cell lines. However, some autoantibodies may only show reactivity for binding to densely clustered AChR in the physiological conformation, while AChR clustering is known to involve signaling events orchestrated by over a dozen of postsynaptic proteins. To improve the existing serological diagnosis of MG, this study explored the possibility of using the well-established Xenopus primary culture system as a novel CBA for MG. Here, by examining the pathogenic effects of four MG human plasma samples, we found that the samples from both seropositive and seronegative MG patients effectively induced the disassembly of aneural AChR clusters in cultured Xenopus muscle cells, as well as the nerve-induced AChR clusters in the nerve-muscle co-cultures. Importantly, the disassembly of AChR clusters was spatio-temporally correlated to the disappearance of actin depolymerizing factor (ADF)/cofilin, an actin regulator involved in AChR trafficking and clustering. Taken together, this study develops a reliable CBA using Xenopus primary cultures for screening the pathogenicity of human MG plasma samples, and providing a platform for investigating the pathogenic mechanisms underlying the endocytic trafficking and degradation of AChRs at NMJs in MG patients.
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
Hwee Li
HL
Yeo
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
Jorain Yu Ni
JY
Lim
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599, Singapore.
Yuki
Y
Fukami
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599, Singapore.
Nobuhiro
N
Yuki
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore. Electronic address: leechiwai@nuhs.edu.sg.
Chi Wai
CW
Lee
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001323:Autoantibodies / Q000097:blood; D006801:Humans; D009157:Myasthenia Gravis / Q000209:etiology* / Q000276:immunology / Q000378:metabolism; D009469:Neuromuscular Junction / Q000276:immunology / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000276:immunology / Q000378:metabolism; D046509:Tissue Culture Techniques; D014981:Xenopus / Q000196:embryology / Q000235:genetics
D051339:Actin Depolymerizing Factors; D001323:Autoantibodies; D011950:Receptors, Cholinergic; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases
ADF/cofilin; Acetylcholine receptor; Cell-based assay; Myasthenia gravis; Neuromuscular junction; Xenopus