Agrin as a presynaptic differentiation inducer and its proteolytic regulation by MMPs

Abstract

Agrin, a heparan sulfate proteoglycan molecule, has been well studied for its critical role in promoting acetylcholine receptor (AChR) clustering during postsynaptic differentiation at the neuromuscular junction (NMJ). A previous study also suggested that agrin regulates growth cone guidance in spinal neuron cultures; however, the functional roles of agrin and its proteolytic regulation in presynaptic differentiation remain unclear. Matrix metalloproteinases (MMPs) play critical functions in the remodeling of extracellular matrix (ECM) proteins to control cell migration and motility. Interestingly, membrane-type 1 MMP (MT1-MMP) and secreted MMP-3 have previously been shown to extracellularly cleave agrin that affect synaptic structures. Using Xenopus primary cultures, this study aims to investigatehowagrin and its proteolytic regulation by MMP activity spatio-temporally modulate presynaptic differentiation at developing NMJs. Firstly, local application of agrin spatially induces the clustering of mitochondria and synaptic vesicles, two well-known presynaptic markers, along the neurites. The findings indicated that agrin is an effective presynaptic differentiation inducer. Secondly, pharmacological inhibition of MMP activity or reduced expression of MT1-MMP significantly inhibited agrin-induced presynaptic differentiation. We next observed that agrin is spatially enriched at nerve–muscle contact sites, which is coupled with ECM degradation and nerve-induced AChR clustering in nerve–muscle co-cultures. Since agrin is usually secreted globally along the neurites, localization of agrin at synaptic sites inferred that MMP proteolytic activity cleaves agrin that is present in extra-synaptic regions. Lastly, MMP inhibitors or morpholinomediated MT1-MMP knockdown also inhibited agrin deposition and the formation of nerve-induced AChR clusters at synaptic sites. Taken together, our results demonstrate a previously unappreciated role of agrin in presynaptic differentiation and the regulatory role of MT1-MMP in this process.