Data_Sheet_1_Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor.docx

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.

近年来的研究证实，交感神经元可共同支配神经肌肉接头（neuromuscular junction）。这种共同支配对于神经肌肉接头的形态与功能维持至关重要。为深化我们对交感神经支配如何影响神经-肌肉突触稳态的认知，本研究采用活体成像、蛋白质组学、生化及显微学技术手段，对比了正常小鼠与交感神经切除小鼠的后肢肌肉。活体共聚焦显微成像结果显示，交感神经切除后小鼠肌纤维直径减小、乙酰胆碱受体（acetylcholine receptor）周转速率提升，且携带乙酰胆碱受体的内体/溶酶体囊泡数量增多。对交感神经切除骨骼肌的蛋白质组学分析表明，除线粒体、肌节及核糖体蛋白发生大规模表达变化外，囊泡运输标志物的相对丰度亦受交感神经切除的影响。免疫荧光染色与蛋白质印迹（Western blot）实验验证了上述发现，此外还证实肌纤维肌节区域及神经肌肉接头处，内体/溶酶体成熟与自噬标志物Rab7及p62出现局部上调与富集。综上，本研究数据为交感神经支配在肌肉与神经肌肉接头稳态中的调控作用提供了全新见解，其结果与整体肌肉及神经肌肉接头处内吞与自噬运输上调的结论相一致。