Regulation of postsynaptic apparatus maturation at neuromuscular junctions by caveolin-3 and TRPC1

Abstract

During the maturation of neuromuscular junctions (NMJs), the postsynaptic muscle membrane undergoes extensive topological changes to form a complex structure optimized for synaptic transmission in motor function. While previous studies have shown that the transformation occurs independent of innervation, the detailed molecular mechanisms remain elusive. In this thesis, I investigated the roles of caveolin-3 and transient receptor potential canonical channel 1 (TRPC1) in the transformation of acetylcholine receptor (AChR) clusters during postsynaptic maturation. Firstly, I examined the presence of membrane infoldings, a hallmark of mature NMJs, at aneural AChR clusters spontaneously formed in cultured muscle cells. AChRs within the infoldings were redistributed over time, and the spatial arrangement of postsynaptic proteins eventually resembled that observed at the junctional folds of NMJs in vivo. Intact lipid rafts and precisely regulated caveolin-3 expression were required for membrane infolding development at aneural AChR clusters and agrin-induced synaptic AChR clustering in vitro, as well as junctional fold development at maturing NMJs in vivo. Next, I focused on the role of mechanobiological regulation in AChR cluster maturation, particularly how the substrate stiffness affects the formation podosome-like structures (PLSs) at perforated AChR clusters. TRPC1 expression and elevated calcium levels were detected at PLS-enriched perforations, which promoted calpain-mediated talin proteolysis for integrin β1 activation and PLS assembly. Mechanosensitive TRPC1 activity and expression was further shown to regulate the localization and activity-dependent release of muscle-specific brain-derived neurotrophic factor. Collectively, these findings reveal previously unappreciated roles of caveolin-3 and TRPC1 in regulating the structural maturation of postsynaptic apparatus at developing NMJs.