Analysis of pathogenic roles of MuSK antibodies in myasthenia gravis using the experimental animal models.

我们已经提供了用MuSK蛋白主动免疫在动物中复制重症肌无力的证据。接下来我们关注MuSK抗体如何引起MG。MuSK抗体在MG中的致病作用受到质疑，因为AChR的数量没有减少，并且补体没有沉积在MuSK阳性MG患者的肱二头肌NMJ处。AChR抗体引起MG的机制已被很好地描述，但所揭示的机制不能简单地应用于MuSK抗体引起的MG。MuSK抗体已被鉴定为主要的IgG4亚类，其不激活补体。我们发现，在MuSK抗体的存在下，聚集蛋白诱导的AChR聚集被强烈阻断，而用纯化的MuSK产品吸收抗体阻止了这种阻断作用。这些结果表明，MuSK抗体有效地抑制了聚集蛋白诱导的AChR聚集的形成。有趣的是，来自患有EAMG的兔的MuSK抗体的单价Fab片段也抑制C2C12细胞上聚集蛋白引起的AChR聚集，这表明补体介导的机制不一定是这种抑制所必需的。然后，我们研究了减少表达的乙酰胆碱受体在NMJ在比目鱼肌麻痹和正常兔通过荧光显微镜。此外，在我们的麻痹兔的NMJ的结构，以及运动终末的大小和分支，显着减少。注射MuSK蛋白诱导的EAMG家兔NMJ的电镜观察表明，回旋突触折叠的复杂性显著丧失，但没有破坏，这里引用的EAMG模型类似于MG和MuSK抗体的人的表型。我们的研究结果表明，MuSK抗体抑制顺行和逆行信号所需的维持成熟NMJ的突触结构。

We have provided the evidence that active immunization with MuSK protein reproduces myasthenia gravis in animals. Next we focus on how MuSK antibodies cause MG. The pathogenic roles of MuSK antibodies in MG have been questioned as the number of AChRs is not reduced and complement is not deposited at the NMJ of biceps brachii muscles MuSK-positive patients with MG. The mechanisms of MG caused by AChR antibodies are well delineated, but the revealed mechanisms are not able to simply apply to MG with MuSK antibodies. MuSK antibodies have been identified as predominantly IgG4 subclass, which does not activate complement. We found that agrin-induced clustering of AChR was strongly blocked in the presence of MuSK antibodies, whereas absorption of the antibodies with purified MuSK products prevented this blocking effect. These results showed that the MuSK antibodies effectively inhibited the formation of agrin-induced AChR clustering. Intriguingly, the monovalent Fab fragments of MuSK antibodies from rabbits with EAMG also inhibited AChR clustering by agrin on C2C12 cells, indicating that complement-mediated mechanisms are not necessarily required for such inhibition. We then examined the reduced expression of AChR at NMJ in soleus muscles of paretic and normal rabbits by fluorescence microscopy. In addition, the structure of NMJ in our paretic rabbits, as well as the size and branching of the motor terminals, were significantly reduced. Electron microscopic observations of NMJ in rabbits with EAMG induced by injection of MuSK protein demonstrated a significant loss of complexity of convoluted synaptic folds but no destruction, and EAMG model cited here resembles the phenotype of humans with MG and MuSK antibodies. Our results showed that MuSK antibodies inhibit both anterograde and retrograde signals required for maintaining the synaptic structures in mature NMJ.