Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

BackgroundDevelopment of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.
Principal FindingsWe report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.
ConclusionMeltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

背景：神经肌肉接头（neuromuscular junction, NMJ）的发育起始于肌肉中央区域突触后特化结构的形成，随后运动神经末梢抵达突触后区域的对侧位置。此后突触前、后组分得以稳定并重塑，最终形成成熟突触。此前尚无关于解整合素金属蛋白酶（A Disintegrin And Metalloprotease, ADAM）家族蛋白在神经肌肉接头发育中作用的相关报道。
主要研究结果：本研究报道了解整合素金属蛋白酶家族成员Meltrin β（ADAM19）参与神经肌肉接头的发育过程。与野生型胚胎膈肌相比，Meltrin β缺陷型胚胎膈肌中，乙酰胆碱受体α亚基mRNA的分布区域更为宽泛，运动神经末梢的过度出芽现象更为显著。基因芯片分析（microarray analysis）显示，Meltrin β缺陷型肌肉中，Ephrin-A5 mRNA在肌肉突触区域的偏好性分布出现异常。Ephrin-A5基因敲除小鼠（knockout mice）中同样观察到运动神经末梢过度出芽的表型，这促使我们探究Meltrin β与ephrin-A5-Eph信号通路在神经肌肉接头发育中的潜在关联。在发育中的运动神经元内，Meltrin β与EphA4存在相互作用，且二者均定位于神经肌肉接头。共表达Meltrin β与EphA4可强烈阻断ephrin-A5-EphA4复合物的囊泡内吞作用（vesicular internalization），且该过程不依赖Meltrin β的蛋白酶活性（protease activity），这提示Meltrin β对ephrin-A5-Eph信号通路具有调控作用。
结论：Meltrin β在神经肌肉接头的发育过程中发挥调控作用。Ephrin-Eph复合物的内吞作用（endocytosis）是ephrin与Eph之间高效接触依赖性排斥反应的必要条件。我们提出，Meltrin β通过负向调控ephrin-A5-EphA复合物的内吞作用，稳定ephrin-A5与EphA4之间的相互作用，这一机制有助于在发育过程中对神经肌肉接头进行精细调控。