The Neuromuscular Junction in Health and Disease: Molecular Mechanisms Governing Synaptic Formation and Homeostasis

Abstract

The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.

Keywords: Agrin; DOK7; MuSK; congenital myasthenic syndromes; myasthenia gravis; neuromuscular junction; sarcopenia; spinal muscular atrophy.

FIGURE 1

Fluorescence and electron microscopy images of the NMJ. (A) Fluorescence microscopy image of the NMJ showing the nerve terminal (green) innervating the muscle endplate (red) stained with fluorescently conjugated α-bungarotoxin that binds to the AChRs. (B) Super-resolution confocal microscopy image of the NMJ showing the postsynaptic muscle membrane and the junctional folds at the top of which AChRs are concentrated (image provided by Dr. J. Cheung). (C) Electron microscopy image of the NMJ. The presynaptic nerve terminal is filled with synaptic vesicles containing acetylcholine (*). The postsynaptic muscle membrane exhibits a high degree of folding which extends into the muscle sarcoplasm (arrows) in order to increase the total endplate surface. The NMJ is covered by terminal Schwann cells. Used with permission from Slater (2017).

FIGURE 2

Schematic representation of the AChR clustering and dispersal pathways. Upon the release of agrin by the nerve terminal, agrin binds to LRP4 resulting in MuSK activation (Kim et al., 2008) and recruitment of DOK7 and Crk/CrkL (Okada et al., 2006) that further stimulates MuSK activation. The signal is propagated downstream, which results in the clustering of the AChRs by the cytoplasmic anchoring protein rapsyn. By contrast, ACh disperses AChR clusters not stabilized by agrin signaling. A cyclin-dependent kinase (CdK5) mechanism is thought to drive this pathway through the interaction of rapsyn and the calcium-dependent protease calpain (Chen et al., 2007). Calpain activity promotes the cleavage of p35 to p25 (Patrick et al., 1999), an activator of CdK5. On the other hand, rapsyn behaves as a calpain suppressor, thus stabilizing AChR clusters. It is thought that on synaptogenic induction, new synthesized AChRs may be transported and delivered to the nascent postsynaptic sites for insertion, together with AChRs endocytosed from the spontaneous AChR clusters. Synapse specific transcription in subsynaptic nuclei by different transcription factors and specific promoter elements in synaptic genes such as GABP (Schaeffer et al., 1998) and Erm (Hippenmeyer et al., 2007) is also key to achieve a high concentration of AChRs in synaptic sites. VGNa+C, volgated-gated Na+ channel.

FIGURE 3

Schematic representation of key NMJ proteins. (A) Agrin (RefSeq NP_001292204.1) is a large proteoglycan (>200 KDa) with multiple domains that binds to laminins through the N-terminal domain, and to LRP4 and α-dystroglycan via C-terminus. Two of the three laminin G-like domains are required for binding to α-dystroglycan. Agrin mRNA undergoes cell-specific alternative splicing at different sites (Ferns et al., 1993). When agrin is produced by motor neurons, the Z site contain amino acid inserts specifically required for MuSK activation. (B) The binding of agrin to the N-terminal region of LRP4 induces a conformational change (active state) promoting the binding between LRP4 and the first IgG-like domain of MuSK and the formation of a tetrameric complex (Zhang et al., 2011). This results in MuSK activation via dimerization and trans-autophosphorylation of tyrosine residues within the cytoplasmic region (Schlessinger, 2000). The increase in the catalytic activity creates active binding sites for other proteins such as DOK7, leading to in full kinase activation and amplification of the signal downstream. The composition of LRP4 includes 8 Low density lipoprotein Class A (LDLa) domains (blue) at the N terminus, followed by 4 YWTD ß-propeller domains (gray) bounded by epidermal growth factor-like modules (yellow) and a short C-terminal domain (Springer, 1998). LRP4 self-associates and interacts with MuSK in the absence of agrin (inactive state), but is not capable of activating MuSK (Kim et al., 2008). (C) DOK7 (RefSeq NM_173660.4) is composed of an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain and a C-terminus. Dashes are used to mark the different exons. (D) Rapsyn (RefSeq NP_005046.2) is composed of a N-terminal myristoylation moiety (N-Myr) necessary for submembrane localization; seven tetratricopeptide (TRP) domains responsible for rapsyn self-association and MuSK binding (Ramarao et al., 2001; Lee et al., 2008); a coiled-coil domain that binds to the cytoplasmic loops of AChRs (Lee et al., 2008), and a RING-H2 domain that interacts with the dystroglycan complex and links with the cytoskeleton (Apel et al., 1995).

FIGURE 4

Simplified representation of the AChR pentamer and the N-glycosylation pathway of proteins and CMS-related genes (red color). (A) Adult (red) and fetal (green) acetylcholine receptors and glycosylation sites. The AChR is made up of five subunits arranged around a central pore. Each subunit is composed of an extracellular domain, four transmembrane domains (M1–M4) and a large cytoplasmic loop linking M3 and M4. Each subunit also contains a conserved N-glycosylation consensus sequence (Asn-X-Ser/Thr) located at the N-terminal region (Gehle et al., 1997), which is necessary for the correct assembly of AChR pentamers and efficient export to the cell surface. (B) The N-linked glycosylation pathway takes place in the endoplasmic reticulum (ER). The first step is the assembly of the core glycan (N-acetylglucosamine, glucose and mannose) on the lipid dolichol. Next, a number of cytosolic glycosyltransferases proceed to dolichol glycosylation on the cytoplasmic face of the ER: GFPT1 synthesizes UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine); DPAGT1 and the ALG13/14 complex add the first and second N-acetylglucosamine to dolichol. Additional sugar residues are incorporated by ALG2 and other enzymes. Then RFT1 flips the resulting product is flipped into the ER lumen where further sugar moieties are included until the glycan is transferred to asparagine residues of nascent proteins by the multimeric oligosaccharyl transferase complex (OST). DOLK, dolichol kinase; DPM, dolichol-phosphate mannose synthase; Fru-6-P, fructose-6-phosphate; GlcN-6-P, glucosamine-6-phosphate; Glu-6-P, glucose-6-phosphate; GMPPB, GDP-mannose pyrophosphrylase B.

FIGURE 5

Schematic representation of the mouse NMJ development. (A) Aneural AChR clusters are pre-patterned in the midbelly of the muscle fibers prior to the arrival of the nerve terminal. (B) Upon innervation of the pre-patterned clusters in the synaptic region, they become enlarged while aneural clusters located in the extrasynaptic region disappear. (C) As a result, AChR clusters are concentrated at a high density in the area underneath the nerve terminal maximizing the efficiency of neuromuscular transmission. (D) Postnatal maturation of AChR clusters and plaque to pretzel transformation. (E) Fragmentation of AChR clusters with aging.