Regulation of acetylcholine receptor recruitment from aneural to synaptic clusters at developing NMJs by MT1-MMP and HSP90

Abstract

In the early phase of neuromuscular junction (NMJ) synaptogenesis, aggregation of acetylcholine receptors (AChR) and postsynaptic proteins occurs in the absence of nerve innervation, suggesting the muscle-intrinsic mechanism of AChR pre-patterning. Several lines of evidence have suggested the role of AChR pre-patterning in guiding axonal growth of motoneurons for NMJ development. However, whether AChRs from pre-patterns are contributed to the assembly of synaptic AChR clusters remains elusive. In this thesis work, I delineated the role of membrane-type 1 matrix metalloproteinase (MT1-MMP) and heat shock protein 90 (HSP90) in the process of AChR redistribution through diffusion-trapping and transcytosis mechanisms during NMJ formation. Firstly, the formation of podosome-like structures (PLSs) associated with aneural AChR clusters were induced in Xenopus muscle cells cultured on substratum coated with ubiquitous extracellular matrix (ECM) proteins. The PLS at perforated regions of AChR clusters is important in regulating the formation and remodeling of AChR clusters via focal ECM degradation. Immunostaining data further indicated that MT1-MMP was highly localized at the perforated regions of aneural AChR clusters. MT1-MMP plays an important role in the recruitment of aneural AChR clusters for the assembly of postsynaptic specializations upon synaptic induction. Structural defects of NMJs in embryonic MT1-MMP-/- mice further demonstrated the physiological role of MT1-MMP in normal NMJ development. To further understand the molecular mechanisms underlying MT1-MMP-regulated NMJ development, I next performed the combination of fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence (TIRF) microscopy, ECM degradation at AChR clusters was found to be mediated by site-directed vesicular trafficking and surface insertion of MT1-MMP through microtubule-capturing mechanisms. Lastly, I examined the mechanism of HSP90 in regulating AChR recruitment to the assembly of synaptic specializations at developing NMJs. Using 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) to pharmacologically inhibit HSP90 activity, aneural clusters were stabilized, together with the suppression of synaptic AChR clustering, upon agrin stimulation. This suggested that HSP90 is involved in regulating AChR redistribution for the assembly of postsynaptic apparatus. I further showed that actin depolymerizing factor (ADF)/cofilin-mediated vesicular trafficking was regulated by HSP90 to mediate AChR transcytosis during the formation of NMJs. Taken together, these findings unveil the molecular mechanism of AChR recruitment from aneural to synaptic AChR clusters during neuromuscular synaptogenesis.