Local protein synthesis of neuronal MT1-MMP for agrin-induced presynaptic development.

Upon the stimulation of extracellular cues, a significant number of proteins are synthesized distally along the axon. Although local protein synthesis is crucial for various stages throughout neuronal development, its involvement in presynaptic differentiation at developing neuromuscular junctions remains unknown. By using axon severing and microfluidic chamber assays, we first showed that treatment of a protein synthesis inhibitor, cycloheximide, inhibits agrin-induced presynaptic differentiation in cultured Xenopus spinal neurons. Newly synthesized proteins are prominently detected, as revealed by the staining of click-reactive cell-permeable puromycin analog O-propargyl-puromycin, at agrin bead-neurite contacts involving the mTOR/4E-BP1 pathway. Next, live-cell time-lapse imaging demonstrated the local capturing and immobilization of ribonucleoprotein granules upon agrin bead stimulation. Given that our recent study reported the roles of membrane-type 1 matrix metalloproteinase (MT1-MMP) in agrin-induced presynaptic differentiation, here we further showed that MT1-MMP mRNA is spatially enriched and locally translated at sites induced by agrin beads. Taken together, this study reveals an essential role for axonal MT1-MMP translation, on top of the well-recognized long-range transport of MT1-MMP proteins synthesized from neuronal cell bodies, in mediating agrin-induced presynaptic differentiation.

Neuronal MT1-MMP mediates ECM clearance and Lrp4 cleavage for agrin deposition and signaling in presynaptic development.

Agrin is a crucial factor that induces postsynaptic differentiation at neuromuscular junctions (NMJs), but how secreted agrin is locally deposited in the context of extracellular matrix (ECM) environment and its function in presynaptic differentiation remain largely unclear. Here, we report that the proteolytic activity of neuronal membrane-type 1 matrix metalloproteinase (MT1-MMP; also known as MMP14) facilitates agrin deposition and signaling during presynaptic development at NMJs. Firstly, agrin deposition along axons exhibits a time-dependent increase in cultured neurons that requires MMP-mediated focal ECM degradation. Next, local agrin stimulation induces the clustering of mitochondria and synaptic vesicles, two well-known presynaptic markers, and regulates vesicular trafficking and surface insertion of MT1-MMP. MMP inhibitor or MT1-MMP knockdown suppresses agrin-induced presynaptic differentiation, which can be rescued by treatment with the ectodomain of low-density lipoprotein receptor-related protein 4 (Lrp4). Finally, neuronal MT1-MMP knockdown inhibits agrin deposition and nerve-induced acetylcholine receptor clustering in nerve-muscle co-cultures and affects synaptic structures at Xenopus NMJs in vivo Collectively, our results demonstrate a previously unappreciated role of agrin, as well as dual functions of neuronal MT1-MMP proteolytic activity in orchestrating agrin deposition and signaling, in presynaptic development.

MMP-mediated modulation of ECM environment during axonal growth and NMJ development.

Motor neurons, skeletal muscles, and perisynaptic Schwann cells interact with extracellular matrix (ECM) to form the tetrapartite synapse in the peripheral nervous system. Dynamic remodeling of ECM composition is essential to diversify its functions for distinct cellular processes during neuromuscular junction (NMJ) development. In this review, we give an overview of the proteolytic regulation of ECM proteins, particularly by secreted and membrane-type matrix metalloproteinases (MMPs), in axonal growth and NMJ development. It is suggested that MMP-2, MMP-9, and membrane type 1-MMP (MT1-MMP) promote axonal outgrowth and regeneration upon injury by clearing the glial scars at the lesion site. In addition, these MMPs also play roles in neuromuscular synaptogenesis, ranging from spontaneous formation of synaptic specializations to activity-dependent synaptic elimination, via proteolytic cleavage or degradation of growth factors, neurotrophic factors, and ECM molecules. For instance, secreted MMP-3 has been known to cleave agrin, the main postsynaptic differentiation inducer, further highlighting the importance of MMPs in NMJ formation and maintenance. Furthermore, the increased level of several MMPs in myasthenia gravis (MG) patient suggest the pathophysiological mechanisms of MMP-mediated proteolytic degradation in MG pathogenesis. Finally, we speculate on potential major future directions for studying the regulatory functions of MMP-mediated ECM remodeling in axonal growth and NMJ development.