Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers

Abstract

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)-the connection between motor neurons and skeletal muscle-represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

Keywords: LRP4; MuSK; acetylcholine receptor; agrin; neuromuscular junctions; neurotrypsin; tyrosine kinase signaling.

Figure 1

Neuromuscular Junction (NMJ) superstructure and maturation. (A) Schematic representation of the overall nerve-muscle interface; enlargement of the NMJ superstructure showing clustered acetylcholine receptors (AChRs) juxtaposed to the motor neuron terminal “embraced” by a supporting terminal Schwann cell. (B) General scheme of “plaque-to-pretzel” maturation; diffuse AChR clusters coalesce into plaques which then “perforate” and re-structure to give rise to the typical NMJ pretzel-like architecture. (C) During prepatterning “immature” AchR plaques align perpendicular to muscle fiber length. These guide outreaching neuronal branches and restrict/restrain contact formation. Motor neurons stabilize pre-existing contacted AChR hot-spots, non-innervated clusters are dispersed.

Figure 2

Domain organization of key extracellular NMJ organizers. (A) Agrin: FS, Follistatin-like; LE, Laminin EGF-like; SEA, sperm, enterokinase and agrin; EGF, Epidermal growth factor like; LG, Laminin globular. Curly brackets show splicing sites and variants. Black arrowheads indicate Neurotrypsin cleavage sites α and β. Square brackets map missense pathogenic CMS mutations to the affected domains. (B) MuSK: Ig, Immunoglobulin-like; Fz-CRD, Frizzled-like cysteine-rich; TK, Tyrosine Kinase; Kr, Kringle; TM, transmembrane region. Square brackets map missense pathogenic CMS mutations to the affected domains, and red asterisks (*) indicate the epitopes recognized by autoantibodies in autoimmune MG. (C) LRP4: LDLa, Low density lipoprotein class A; EGF, Epidermal growth factor like; β: β-propeller; TM: transmembrane, IC: Intracellular. Square brackets map missense pathogenic CMS mutations. (D) Neurotrypsin: Kr, Kringle; SRCR, Scavenger receptor cysteine rich; SP, Serine-protease. The zymogen-activating region is indicated by a dashed arrow.

Figure 3

Structural information about agrin. The structure of the NtA, LG2, and LG3 domains of agrin are shown in correspondence to the overall domain organization. Reported human pathogenic mutations are indicated and mapped on available reference structures as red spheres. Splicing insertion sites y and z are boxed and shown in blue. Salient features of the LG3 domain are enlarged to show the residues mediating z8 binding to LRP4, and the Ca²⁺ binding site. Residue numbering refers to the human protein (uniprot: O00468), corresponding residues on the reference structures are as follows: LG2 residues Val1727 and Gly1709, correspond to Val1548 and Gly1530, respectively. LG3 residues Gly1875, Asn1892, Ile1894, Asp1940, Leu1957, Gln2007, and Asp2009, correspond to Gly1766, Asn1783, Ile1785, Asp1820, Leu1837, Gln1887, and Asp1889, respectively.

Figure 4

Structural information about MuSK. MuSK interacts with the Dok7 (top-left) phosphotyrosine-binding domain (PTB, white) via a phosphorylated tyrosine (Tyr553) on its intracellular juxtamembrane (JX) region (red). The tyrosine kinase (TK) domain (top right) mediates intracellular signaling after phosphorylation of Tyr750, Tyr754, and Tyr755 (shown in red), located on a regulatory loop. Extracellular interactions with LRP4 are mediated by MuSK Ig domains. A crystal structure of an Ig1-Ig2 MuSK dimer (bottom left) shows a hydrophobic dimerization interface (cyan) reliant upon aminoacids Met48 and Leu83. Residues Asp38 (blue) and Ile96 (white), on the exposed surfaces of the Ig1 domains, are respectively involved in congenital myasthenia and LRP4 binding. Disulphide bonds stabilize both the Ig1 and Ig2 domain structures, the former has an additional disulphide bridge. Unlike the Ig domains, the function of the Fz-CRD domain (bottom-right) is extensively debated. The crystal structure presents two alternative conformations packed in an asymmetric dimer. Residue numbering refers to the reference structures.

Figure 5

Structural information about LRP4. LRP4 interacts with agrin via its first β-propeller domain. The available crystal structure shows an overall tetrameric complex formed by each of the two molecules of agrin-LG3 (deep sky blue) simultaneously interacting with two LRP4 β-E1 monomers (light-gray) using two different binding sites. The primary agrin:LRP4 interface is mediated by the agrin z8-insert loop binding directly to the LRP4-β-E1 surface, shown in red. Additional secondary agrin:agrin and agrin:LRP4 contact points are highlighted in blue and yellow, respectively.

Figure 6

Structural information about Neurotrypsin. The NMR structure of the N-terminal Kr domain and the crystal structure of the Ca²⁺-binding rat SRCR3 domain (corresponding to human SRCR4 domain) are shown. At present, no other structural insights are available for this extracellular protease.

Figure 7

Possibility of Wnt-agrin:LRP4:MuSK cross-talk. (A) The interaction (left-panel) between Wnt's (red) and Frizzled domains (orange) relies on two opposed interfaces that accommodate contemporaneously a palmitate tail and a Zn-finger domain. Substituting (right-panel) the Frizzled domain with the MuSK Fz-CRD (light-green) shows how this domain of MuSK could accommodate the same type of opposed binding interfaces. (B) The crystal structure of the MuSK Fz-CRD (also shown in Figure 4) presents two alternative conformations; an “open” conformation (left), possibly capable of accommodating the Wnt palmitate tail, and a “closed” one, likely not conductive to Wnt binding (right). “open” and “closed” states are determined the by the stabilization of an α-helix from an otherwise disordered loop (shown in blue). (C) A superposition (left) of the agrin:LRP4 z8 loop complex (cyan) with an LRP6:Dkk1 N-terminal peptide complex (beige) shows the structural similarity of the interaction, hinting the possibility of cross-talk. Both the agrin z8-loop and the Dkk1 peptide interact with their target via a conserved NxI/V motif (top-right). A cross section view into the binding pocket of LRP4 (top) for the agrin-z8 loop (cyan) shows how it could also accommodate the Dkk1 peptide (beige) and allow for cross-talk between the two signaling pathways.

Figure 8

Summary scheme of LRP4:MuSK signaling. (A) Signaling events differentially induce AChR clustering toward either NMJ formation and motor neuron innervation or toward formation of aneural clusters. (B) Details of neural and aneural NMJ signaling via LRP4:MuSK activation. In canonical (agrin-dependent) NMJ signaling, binding of agrin to co-receptor LRP4 stimulates activation of MuSK and downstream effector functions (left). In this pathway, Neurotrypsin likely operates as a regulator through specific agrin cleavage at α and β sites; non-canonical (i.e., agrin-independent) events such as cross-talk with Wnt and BMP signaling pathways also trigger MuSK activation, possibly through direct binding of Wnt ligands and inhibitors to MuSK Fz-CRD or to LRP4 β-E domain clusters. The downstream effector functions differ into a spectrum that highlights numerous aneural regulatory events that could be particularly critical prior to motor neuron innervation and NMJ formation.