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

我们已经提供了用麝香蛋白主动免疫在动物身上复制重症肌无力的证据。接下来，我们关注马斯克抗体是如何导致MG的。麝香抗体在MG发病中的作用一直受到质疑，因为MG患者的二头肌Musk阳性患者的AChRs数量没有减少，补体也没有沉积在NMJ。AChR抗体引起MG的机制已被很好地阐明，但已揭示的机制不能简单地应用于伴有Musk抗体的MG。麝香抗体已被鉴定为主要是IgG4亚类，不能激活补体。我们发现，在麝香抗体的存在下，集聚蛋白诱导的AChR聚集被强烈地阻断，而抗体与纯化的麝香产品的吸收阻止了这种阻断作用。这些结果表明，麝香抗体有效地抑制了集聚蛋白诱导的AChR聚簇的形成。有趣的是，来自患有EAMG的兔的麝香抗体的单价Fab片段也通过聚集蛋白抑制了AChR在C2C12细胞上的聚集，这表明这种抑制不一定需要补体介导的机制。然后，我们用荧光显微镜观察了瘫痪兔和正常兔的比目鱼肌中AChR在NMJ的表达减少。此外，我们的瘫痪兔的NMJ的结构以及运动终末的大小和分支都显著减少。在注射麝香蛋白诱导的兔EAMG模型中，NMJ的电子显微镜观察显示，突触皱折的复杂性显著降低，但没有破坏，这里所述的EAMG模型类似于人类MG和麝香抗体的表型。我们的结果表明，麝香抗体抑制了维持成熟NMJ突触结构所需的顺行和逆行信号。

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.