This study investigates the role of neuronal innervation in regulating the secretion of neurotrophic myokines and exosomes from skeletal muscle. Using an engineered neuromuscular tissue model, the researchers found that innervated muscles expressed higher levels of mRNAs encoding neurotrophic myokines such as interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF), and fibronectin type III domain-containing protein 5 (FNDC5). Upon glutamate stimulation, innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs (miRNAs) compared to neuron-free muscles. These muscle-derived factors enhanced branching, axonal transport, and spontaneous network activities of primary hippocampal neurons in vitro. The study highlights the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggests that the engineered neuromuscular tissue model is a valuable platform for producing neurotrophic molecules.This study investigates the role of neuronal innervation in regulating the secretion of neurotrophic myokines and exosomes from skeletal muscle. Using an engineered neuromuscular tissue model, the researchers found that innervated muscles expressed higher levels of mRNAs encoding neurotrophic myokines such as interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF), and fibronectin type III domain-containing protein 5 (FNDC5). Upon glutamate stimulation, innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs (miRNAs) compared to neuron-free muscles. These muscle-derived factors enhanced branching, axonal transport, and spontaneous network activities of primary hippocampal neurons in vitro. The study highlights the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggests that the engineered neuromuscular tissue model is a valuable platform for producing neurotrophic molecules.