Fibroadipogenic Progenitor Regulates Neuromuscular Junction by Secreting Novel Protein Granzyme E

Muscle fibroadipogenic progenitor (FAP) cells, which are muscular mesenchymal cells that originate from lateral plate mesoderm have been proposed to act as a critical regulator for adult muscle homeostasis1–7, including the maturation and proper functioning of the neuromuscular junction (NMJ)3, Prx-Bap1 paper. However, the mechanism and intercellular crosstalk by which FAPs regulate the stability and functionality of neuromuscular system remains unknown. Here we show that FAPs not only locally but also systemically regulate the neuromuscular system through the secretion of a serine-type endopeptidase Granzyme E which may imply the previously unidentified endocrine function of FAPs. Local transplantation of wild-type FAPs into the neuromuscular disease model (Prrx1Cre;Bap1f/f, hereafter, cKO) can readily prevent neuromuscular defects, including degeneration of the neuromuscular junction and loss of motor neurons. These effects are found not only in transplanted hindlimb muscles but also in the contralateral hindlimb and even forelimb muscles. Notably, subcutaneous administration of microparticles encapsulating FAP-conditioned media into cKO mice was sufficient to restore normal neuromuscular functions. By analyzing the transcriptomic and secretomic profiles of FAPs, we identified a novel protein, Granzyme E, which is specifically expressed in and secreted by FAPs, and which indispensably regulates the structure and function of NMJ and motor neuron survival. Our study has defined a unique mechanism of Granzyme E-dependent, systemic control of the neuromuscular system by FAPs, which would provide a comprehensive understanding on the neuromuscular systems and their crosstalk with non-neuronal cells. These findings may provide a therapeutic benefit to treat NMJ-related diseases. Overall design: Identification of downstream targets of Bap1 and Smn in FAP

肌肉纤维脂肪生成祖细胞（Muscle fibroadipogenic progenitor, FAP）是起源于侧板中胚层的肌肉间充质细胞，已有研究提出其为成年肌肉稳态的关键调控因子1–7，可参与神经肌肉接头（neuromuscular junction, NMJ）的成熟与正常功能维持3，相关研究见于Prx-Bap1论文。但FAP调控神经肌肉系统稳定性与功能的具体机制及细胞间互作通路仍未明确。本研究发现，FAP可通过分泌丝氨酸型内肽酶颗粒酶E（Granzyme E），同时在局部与全身层面调控神经肌肉系统，这一结果提示FAP存在此前未被发现的内分泌功能。将野生型FAP局部移植至神经肌肉疾病模型（Prrx1Cre;Bap1f/f，下文简称cKO小鼠）中，可有效预防神经肌肉缺陷的发生，包括神经肌肉接头退变与运动神经元丢失。该保护效应不仅见于移植的后肢肌肉，还可波及对侧后肢乃至前肢肌肉。值得注意的是，向cKO小鼠皮下注射包裹FAP条件培养基的微球，即可恢复其正常神经肌肉功能。通过分析FAP的转录组与分泌组谱，本研究鉴定出一种新型蛋白质——颗粒酶E（Granzyme E），该蛋白特异性表达于FAP并由其分泌，且对神经肌肉接头的结构与功能、运动神经元存活均不可或缺。本研究阐明了FAP依赖颗粒酶E的神经肌肉系统全身性调控机制，可为理解神经肌肉系统及其与非神经细胞的互作提供全新视角。上述发现或可为神经肌肉接头相关疾病的治疗提供潜在治疗策略。实验整体设计：鉴定FAP中Bap1与Smn的下游靶标。