Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction

Abstract

The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission.

Figure 1. Developmental expression of synaptic extracellular matrix (ECM) proteins at the rodent neuromuscular junction

By embryonic (E) day 15, skeletal myofibers have formed and are innervated the nerve terminals of motor neurons at the neuromuscular junction. The nerve terminal is capped by a non-myelinating Schwann cell. Skeletal myofibers express a number of ECM proteins, including laminin (LN), collagen IV, fibronectin, perlecan, ColQ and nidogen, while motor nerves express ECM proteins including agrin (z8, 11, or 19) and neuregulin. Some ECM proteins are localized within the synaptic (SYN) basal lamina, which exists only at the neuromuscular junction, while other ECM proteins are present within the extrasynaptic (ESYN) basal lamina that surrounds the remainder of the skeletal myofiber membrane. ESYN and SYN composition shifts during neuromuscular development as synaptic ultrastructure develops, such that unique laminin and collagen chains become expressed exclusively in the synaptic basal lamina in the postnatal (P) animal.

Figure 2. Synaptic extracellular matrix (ECM) proteins and their receptors at the neuromuscular junction

ECM proteins within the synaptic basal lamina at the neuromuscular junction include agrin, laminins, perlecan, collagen IVs, and ColQ. Potential ECM-receptor and ECM-ECM interactions are indicated in blue. Agrin contains splice sites in the N-terminus to allow for secreted and transmembrane forms and A/y and B/z splice sites in the C-terminus that allow heparin binding and AChR clustering, respectively. Laminins are heterotrimeric proteins comprised of an α, β, and γ chain. At the NMJ, the uniquely synaptic laminin chains are laminin β2, laminin α4, and laminin α5. Collagen IVs are heterotrimeric proteins of different three alpha chains. The NMJ contains two synaptic collagen IV trimers ((α3, α4, α5) and (α5)₂(α6)). Legend: EGF, Epidermal growth factor-like repeat; FS, Follistatin-like domain; Ig, Immunoglobulin domain; LF, Laminin four domain (of the β chain); LB, Laminin B domain; L4, Laminin domain IV (of the γ or α chain); LE, Laminin EGF-like domain; LG, Laminin-like globular domain; LN, Laminin N-terminal domain; LA, LDL receptor domain class A; NtA, N-terminal agrin-laminin binding domain; PRAD, Proline-rich attachment domain; S/T, Serine/threonine-rich mucin-like domain; SP, Signal peptide; SEA, Sea urchin sperm protein, enterokinase, and agrin domain; TM, Transmembrane domain; AChE, Acetylcholinesterase; NCAM, Neural cell adhesion molecule; FGF, Fibroblast growth factor, HB-GAM, Heparin-binding growth-associated molecule; BMP, Bone morphogenetic protein; Lrp4, Low-density lipoprotein receptor-related protein 4; MuSK, Muscle-specific kinase; PDGF, Platelet-derived growth factor, LDL, Low density lipoprotein; HSPG, Heparan sulfate proteoglycan; VGCC, Voltage-gated calcium channel.

Figure 3. Synaptic ECM Signaling in Neuromuscular Development

Genetic evidence in mice and humans points to three central ECM-mediated groups of signals that are essential for proper neuromuscular development in muscle, those involving synaptic agrin, synaptic laminins and synaptic collagens. Signals from neuron-specific splice forms of agrin (for example z8) in the synaptic basal lamina (BL) work via the MuSK-LRP4 complex in the membrane to induce downstream tyrosine phosphorylation signals that drive synaptogenesis, which is agrin-independent, and to organize synaptic topography and induce synaptic stabilization, which are agrin-dependent. Down-stream signals are mediated or modulated by a variety of cytoplasmic effectors, including structural and adaptor/signaling proteins (Dok7, Dvl, Pak1, Tid1, APC, Rapsyn), proteases (Calpain), tyrosine kinases (Abl, Src, Fyn), tyrosine phosphatases (Shp2), serine/threonine kinases (Cdk5, CaMKII and CK2), small G proteins (Rho, Rac) and a geranylgeranyltransferase (GGT). Calcium entry stimulated by acetylcholine works, in part via Calpain, Cdk5 and CamKII to destabilize synaptic membranes. Laminins, particularly Laminin 421 and 521, in the synaptic BL signal via dystroglycan and possibly integrins in muscle to induce changes in synaptic ultrastructure and to maintain synaptic stability. Ultrastructural changes at the synapse are mediated, in part, by cytoplasmic dystroglycan-binding proteins including utrophin, syntrophins and dystrobrevins. These proteins help to link the ECM outside the muscle membrane to filamentous (F-) actin in the cytoskeleton. Laminin 421 and 521 also bind to the voltage-gated calcium channels in the presynaptic membrane to organize active zones. Collagens IV in the synaptic BL and collagen XIII in the postsynaptic membrane may signal via integrins to induce synaptic maturation and stability. Integrins may bind and signal through a variety of intracellular proteins (for example Focal adhesion kinase (FAK), vinculin, paxillin,talin).