Growing evidence indicates that brain-derived neurotrophic factor (BDNF) is produced in contracting skeletal muscles and is secreted as a myokine that plays an important role in muscle metabolism. However, the involvement of muscle-generated BDNF and the regulation of its vesicular trafficking, localization, proteolytic processing, and spatially restricted release during the development of vertebrate neuromuscular junctions (NMJs) remain largely unknown. In this study, we first reported that BDNF is spatially associated with the actin-rich core domain of podosome-like structures (PLSs) at topologically complex acetylcholine receptor (AChR) clusters in cultured Xenopus muscle cells. The release of spatially localized BDNF is tightly controlled by activity-regulated mechanisms in a calcium-dependent manner. Live-cell time-lapse imaging further showed that BDNF-containing vesicles are transported to and captured at PLSs in both aneural and synaptic AChR clusters for spatially restricted release. Functionally, BDNF knockdown or furin-mediated endoproteolytic activity inhibition significantly suppresses aneural AChR cluster formation, which in turn affects synaptic AChR clustering induced by nerve innervation or agrin-coated beads. Lastly, skeletal muscle-specific BDNF knockout (MBKO) mice exhibit structural defects in the formation of aneural AChR clusters and their subsequent recruitment to nerve-induced synaptic AChR clusters during the initial stages of NMJ development in vivo. Together, this study demonstrated the regulatory roles of PLSs in the intracellular trafficking, spatial localization, and activity-dependent release of BDNF in muscle cells and revealed the involvement of muscle-generated BDNF and its proteolytic conversion in regulating the initial formation of aneural and synaptic AChR clusters during early NMJ development in vitro and in vivo.

越来越多的证据表明，脑源性神经营养因子 （BDNF） 是在收缩的骨骼肌中产生的，并以肌因子的形式分泌，在肌肉代谢中起重要作用。然而，在脊椎动物神经肌肉接头 （NMJ） 发育过程中，肌肉产生的 BDNF 的参与及其囊泡运输、定位、蛋白水解加工和空间限制性释放的调节在很大程度上仍然未知。在这项研究中，我们首次报道了 BDNF 在空间上与培养的非洲爪蟾肌肉细胞中拓扑复杂的乙酰胆碱受体 （AChR） 簇的足小体样结构 （PLS） 富含肌动蛋白的核心结构域相关。空间定位的 BDNF 的释放以钙依赖性方式受到活性调节机制的严格控制。活细胞延时成像进一步显示，含有 BDNF 的囊泡被转运到无神经和突触 AChR 簇中的 PLS 并被捕获，以进行空间限制释放。在功能上，BDNF 敲低或弗林蛋白酶介导的内切蛋白水解活性抑制显着抑制神经 AChR 簇的形成，这反过来又影响神经支配或聚集蛋白包被的珠子诱导的突触 AChR 聚集。最后，骨骼肌特异性 BDNF 敲除 （MBKO） 小鼠在体内 NMJ 发育的初始阶段表现出无神经 AChR 簇的形成及其随后募集到神经诱导的突触 AChR 簇的结构缺陷。 总之，本研究证明了 PLSs 在肌肉细胞中 BDNF 的细胞内运输、空间定位和活性依赖性释放中的调节作用，并揭示了肌肉产生的 BDNF 及其蛋白水解转化在体外和体内早期 NMJ 发育过程中参与调节神经和突触 AChR 簇的初始形成。