α−Calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles

Abstract Objective α-Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the central and peripheral nervous systems involved in the stabilization of muscle-type cholinergic receptors nicotinic subunits (AChR). However, the underlying mechanism by which this neuropeptide regulates skeletal muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear. Methods To shed light on the regulation of NMJ stablity by CGRP, we used immunofluorescence, confocal microscopy, and western blot analyses of denervated muscles induced by sciatic nerve transection. For the investigation of protein metabolism, rates of protein synthesis and proteolysis were estimated in rat isolated muscles and intracellular signaling pathways and gene expression were assessed in muscles and C2C12 cells. Results CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca 2+ -dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP. Conclusion Taken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions.