Roles of neuronal MT1-MMP and C9orf72 in regulating agrin-induced presynaptic differentiation

Abstract

Agrin has long been studied for its role in inducing postsynaptic differentiation at the neuromuscular junction (NMJ). Although agrin is known to regulate growth cone guidance in cultured spinal neurons, its functional roles in presynaptic differentiation have remained elusive. In this thesis research project, I aimed to explore the roles of agrin in inducing presynaptic differentiation and how matrix metalloproteinases (MMPs) and the chromosome 9 open reading frame 72 (C9orf72) regulate presynaptic differentiation at developing NMJs.

By using Xenopus primary cultures, I first investigated how MMP proteolytic activity spatiotemporally modulates presynaptic differentiation during NMJ development. Firstly, agrin deposition along the neurites was spatiotemporally controlled via MMP-mediated focal degradation of extracellular matrix proteins. Secondly, local agrin stimulation by polystyrene beads induced presynaptic differentiation, as shown by the spatial clustering of mitochondria and synaptic vesicles, and regulated membrane-type 1 (MT1)-MMP vesicular trafficking and surface insertion. Moreover, agrin bead-induced presynaptic differentiation was inhibited by either the broad-spectrum MMP inhibitor treatment or morpholino (MO)-mediated MT1-MMP knockdown. Lastly, MT1-MMP MO inhibited agrin deposition and acetylcholine receptor clustering at the nerve-muscle contact sites in vitro and altered the synaptic structures of Xenopus NMJs in vivo. Most importantly, these inhibitory effects were rescuable by ecto-domain low-density lipoprotein receptor-related protein 4 (Lrp4) treatment, suggesting that agrin-induced presynaptic differentiation is regulated by MT-MMP-mediated Lrp4 cleavage.

Mutations in the chromosome 9 open reading frame 72 (C9orf72) protein are recognized as the most common cause of amyotrophic lateral sclerosis (ALS). However, the exact roles of C9orf72 in the pathogenic mechanisms remain unclear. Therefore, by using MO-mediated knockdown approach in Xenopus spinal neurons, the second part of this project investigated whether C9orf72 loss-of-function contributes to the deficits in presynaptic differentiation and motor neuron development. Firstly, C9orf72 MO promoted axonal retraction and inhibited agrin-induced presynaptic differentiation in old cultured neurons. C9orf72 MO-mediated knockdown also inhibited NMJ formation in vitro. As C9orf72 is previously found to interact with cofilin, the observed phenotypic defects may be contributed by the altered actin dynamics by C9orf72 knockdown.

Taken together, the results have demonstrated the previously unappreciated role of agrin in presynaptic differentiation, and the regulatory roles of neuronal MT1-MMP and C9orf72 in the formation of NMJs in health and disease.