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Molecular mechanisms underlying maturation and maintenance of the vertebrate neuromuscular junction

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The vertebrate neuromuscular junction (NMJ), a peripheral synapse formed between motoneuron and skeletal muscle, is characterized by a protracted postnatal period of maturation and life-long maintenance. In neuromuscular disorders such as congenital myasthenic syndromes (CMSs), disruptions of NMJ maturation and/or maintenance are frequently observed. In particular, defective neuromuscular transmission associated with structural and molecular abnormalities at the pre- and postsynaptic membranes, as well as at the synaptic cleft, has been reported in these patients. Here, we review recent advances in the understanding of molecular and cellular events that mediate NMJ maturation and maintenance. The underlying regulatory mechanisms, including key molecular regulators at the presynaptic nerve terminal, synaptic cleft, and postsynaptic muscle membrane, are discussed.

Introduction

Structural and functional maturation of established synapses during development controls the consolidation and refinement of neural circuits. For example, experience-dependent neuronal plasticity in the brain requires maturation and maintenance of a subset of newly formed synapses, while others are eliminated [1]. The peripheral synapse neuromuscular junction (NMJ) has long been used as a model system for studying the general principles of synapse development owing to its large size and experimental accessibility [2]. The vertebrate NMJ utilizes acetylcholine (ACh) as its neurotransmitter, which binds to ion-channel ACh receptors (AChRs) highly concentrated on the postsynaptic membrane. Notably, the NMJ goes through a unique series of maturation processes that differ from those of the central synapse 2, 3. First, whereas maturation of the central synapse normally occurs within hours, the NMJ takes weeks to refine its molecular and structural organization to achieve its mature form, which exhibits efficient neurotransmission. Furthermore, the stability of the NMJ increases on maturation, and the synaptic structure, once mature, persists throughout postnatal life, thus enabling life-long effective motor performance 2, 3. This is in contrast to the rapid turnover of central synapses; for example, mature dendritic spines (where excitatory synapses reside) can be rapidly eliminated and replaced by newly formed ones when neural circuits are reconstructed [1].

Abnormalities in NMJ maturation or maintenance are among the most common pathological causes of congenital myasthenic syndromes (CMSs), a heterogeneous group of hereditary neuromuscular diseases characterized by muscle weakness and a reduced safety margin for synaptic transmission [4]. Thus, delineation of the molecular mechanisms underlying NMJ maturation and maintenance is important for an understanding of the pathogenesis of CMSs and to provide significant insights for potential therapeutic approaches. Here we summarize the molecular and cellular events mediating NMJ maturation and maintenance, and review current knowledge on the regulation of these processes.

Section snippets

Structural and molecular changes during NMJ maturation and maintenance

Remarkable changes occur at the postsynaptic membrane during postnatal maturation of the NMJ 2, 3. First, alterations in AChR clusters take place at the levels of morphology, subunit composition and stability. The neonatal plaque-like AChR clusters with uniform receptor density are transformed into multi-perforated elaborate branches that display a pretzel-like shape (Figure 1a and Box 1) 5, 6, 7. The embryonic-specific γ-subunit of AChR is replaced by the ɛ-subunit during early postnatal

Agrin: a master organizer of the NMJ

Before the arrival of approaching motoneuron terminals, AChR clusters and postsynaptic-like structures are spontaneously formed at the center of muscle fibers, a process known as muscle prepatterning (Box 3) 16, 17. Although the initial formation of prepatterned structures is independent of nerve terminals, subsequent postsynaptic differentiation requires trans-synaptic activity. Nerve-derived agrin, a large heparin sulfate proteoglycan synthesized and released from the nerve terminal, is a

ECM proteins at the synaptic cleft control both pre- and postsynaptic maturation

The synaptic basal lamina, composed of a distinct set of ECM proteins, extends through the synaptic cleft into the junctional folds. The synaptic basal lamina comprise four main types of ECM proteins: heparan sulfate proteoglycans (e.g., the neural agrin), laminins, collagens and nidogens [14]. As described above, neural agrin is an indispensible organizer for postsynaptic assembly. The other three core species, synthesized and deposited by muscle fibers, play important roles in the maturation

MuSK-mediated postsynaptic signaling pathways

Agrin-induced activation of the MuSK receptor is the most extensively studied signaling pathway in the assembly and maintenance of the postsynaptic apparatus. Agrin activates MuSK through binding to the MuSK co-receptor low-density lipoprotein receptor-related protein 4 (LRP4) 44, 45. Structural analysis has revealed that a tetrameric complex formed by two agrin–LRP4 heterodimers mediates MuSK dimerization and activation 46, 47. Activation of MuSK also requires its interaction with the

The dystrophin–glycoprotein complex controls postsynaptic maturation and maintenance

The dystrophin–glycoprotein complex (DGC) is a multi-molecular complex linking AChR clusters to both the extracellular basal lamina and the intracellular cytoskeleton [98]. The main components of the DGC include a transmembrane module containing α- and β-dystroglycan, the associated cytoplasmic actin-binding proteins dystrophin and utrophin, and two groups of cytoplasmic proteins, α-dystrobrevins and syntrophins [98]. Some of the components are expressed along the entire muscle fiber, whereas

Protein kinases and other synaptic regulators implicated in NMJ maturation and maintenance

Two serine/threonine kinases, protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), are involved in NMJ maturation and maintenance. PKC activity is important for the plaque-to-pretzel transition of AChRs and for motor axon withdrawal during synapse elimination [122]. CaMKII promotes the insertion of recycled AChRs into synaptic sites, and thus contributes to the maintenance of synaptic receptor density [123]. PKC and CaMKII are also implicated in suppression of the

Synaptic transcription during NMJ maturation and maintenance

Synapse-specific genes are locally transcribed at specialized nuclei located beneath motoneuron terminals (known as subsynaptic nuclei). A number of synaptic genes, including those encoding AChR subunits, utrophin and AChE, contain an Ets-binding promoter sequence (the N-box element) to target their synaptic transcription through binding to Ets transcription factors. A mutation of the N-box of the gene encoding AChRɛ is associated with CMSs, suggesting that N-box-directed subsynaptic

Concluding remarks

Studies on the prototypic synapse NMJ have shed light on the general principles of synapse assembly. Nonetheless, the molecular mechanisms underlying NMJ maturation and maintenance are only beginning to be understood. We have reviewed molecular regulators at pre- and postsynaptic sites and the synaptic cleft, discussed the regulation of postsynaptic maturation and maintenance (Table 1 and Figure 2), and summarized some outstanding questions that remain to be addressed (Box 4). It is noteworthy

Acknowledgments

We apologize to authors whose work could not be discussed or cited because of space limitations. This study was supported in part by the Research Grants Council of Hong Kong (6421/05 M, 661007, 661109, 660810 and HKUST6/CRF/08), the Area of Excellence Scheme of the University Grants Committee (AoE/B-15/01-II), the Hong Kong Jockey Club, the National Natural Science Foundation of China (81101015), and the Bureau of Science and Information Technology of Guangzhou Municipality (2011J2200048).

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