Spatiotemporal release and proteolytic activation of proBDNF and latent TGF-β1 in coordinating neuromuscular junction development

Brain-derived neurotrophic factor (BDNF) is the well-studied member of neurotrophin family that plays fundamental roles in many distinct processes in the nervous system, including neuronal outgrowth, survival, and differentiation, as well as synaptic formation, plasticity, and elimination. An important question of how BDNF could serve diverse functions, even in the same cell, remains unclear. BDNF appears to exert synapse-selective actions, implicating potential activity-dependent mechanisms of localized BDNF release in this process. Podosome-like structures (PLSs) are spatially enriched at the perforations of aneural and synaptic acetylcholine receptor (AChR) clusters in muscles, where they control the sitedirected vesicular trafficking of several integral membrane proteins in regulating synapse formation, remodeling, and maturation. We hypothesize that PLSs control the spatiotemporal release of BDNF at developing neuromuscular junctions (NMJs). Next, we will further investigate and address several crucial issues concerning proteolytic activation of BDNF precursor (proBDNF) to mature BDNF (mBDNF) extracellularly by matrix metalloproteinase (MMP) activity. We hypothesize that membrane-type 1 MMP (MT1-MMP)-mediated proteolytic activation of proBDNF differentially activates TrkB- and p75NTR/sortilinmediated signaling pathways in regulating different processes in outgrowth versus synaptogenic phase of neuronal development. Lastly, it is also important to know how the activity of transforming growth factor (TGF)-β1, a known Schwann cell-secreted factor in promoting neuromuscular synaptogenesis, is regulated to reverse the inhibitory effects of BDNF on agrin deposition and synaptic AChR cluster formation. In this project, advanced live-cell imaging will be performed to address three specific questions: (1) how the spatiotemporal release of BDNF proteins is regulated at the sites of AChR clusters at developing NMJs; (2) whether axonal growth, agrin synthesis/deposition, and nerve-induced AChR clustering are differentially regulated by mBDNF-TrkB and proBDNF-p75NTR/sortilin signaling pathways; (3) whether mechanical and/or proteolytic activation of latent TGF-β1 promote NMJ formation by reversing BDNF-induced inhibition. Taken together, answers to these questions will provide the support to a novel mechanism concerning BDNF vesicular trafficking, release, and proteolytic activation underlying the formation and maintenance of NMJs. Results of this study could also enhance our understanding of the molecular and cellular mechanisms underlying the diverse physiological functions of proBDNF and mBDNF in the nervous system.