Molecular control of neuromuscular junction development

Abstract

Skeletal muscle innervation is a multi-step process leading to the neuromuscular junction (NMJ) apparatus formation. The transmission of the signal from nerve to muscle occurs at the NMJ level. The molecular mechanism that orchestrates the organization and functioning of synapses is highly complex, and it has not been completely elucidated so far. Neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates (EP). Acetylcholine receptor (AChR) clusterization at the end plates is required for an accurate synaptic transmission. This review will focus on some mechanisms responsible for accomplishing the correct distribution of AChRs at the synapses. Recent evidences support the concept that a dual transcriptional control of AChR genes in subsynaptic and extrasynaptic nuclei is crucial for AChR clusterization. Moreover, new players have been discovered in the agrin-MuSK pathway, the master organizer of postsynaptical differentiation. Mutations in this pathway cause neuromuscular congenital disorders. Alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting. Indeed, recent evidences demonstrate how NMJ misfunctioning has a crucial role at the onset of age-associated sarcopenia.

Fig. 1

The agrin–MuSK–Lrp4 and neuregulin–ErbB pathways induce NMJ assembly positive signals. a Agrin is released by the motor axon terminal and induces AChR clustering, phosphorylation, and stabilization at the postsynaptic membrane. Lrp4 associates with MuSK in the absence of agrin. Agrin binds to the preformed MuSK–Lrp4 complex by interacting with Lrp4 and promotes MuSK transphosphorylation and activation. Once phosphorylated, MuSK recruits the adapter protein Dok-7 which binds Crk and CrkL and stimulates further MuSK phosphorylation and kinase activity. This induces phosphorylation and stabilization of nascent AChR clusters. Rapsyn is a coeffector in AChR assembly which anchorates AChRs at the muscle membrane. b Neuregulin (NGR-1) is released by the nerve and induces AChR transcription in synaptic nuclei. NRG-1 acts by binding tyrosine kinases receptors ErbBs. ErbB phosphorylation induced by NRG stimulates ERK and JNK kinase activity which phosphorylates GABP-α and GABP-β transcription factors. GABP-α heterodimerizes with GABP-β and binds DNA at the N-box thereby enhancing transcription of AChR genes

Fig. 2

The ACh–AChR interaction triggers NMJ assembly negative signals. Global AChR transcription is inhibited by ACh released by the nerve. AChR gene transcription suppression occurs through myogenin inhibition. The myogenic transcription factor myogenin activates muscle-specific genes including AChRs by binding the E-box element. When the action potential reaches the axon terminals, it induces the opening of the voltage-gated Ca²⁺ channels on the presynaptic nerve membrane. The Ca²⁺ influx into the neuron leads synaptic vesicles to fuse with the presynaptic membrane and to release the neurotransmitter acetylcholine (ACh) in the synaptic cleft. From here, ACh diffuses and binds to acetylcoline receptors (AChRs) localized on the postsynaptic membrane on the muscle fiber. This binding makes AChRs permeable to ions so that Na⁺ flows into the fiber while K⁺ flows out of the muscle cytosol. In this way, the muscle membrane locally depolarizes, and this local depolarization opens voltage-gated Na⁺ channels on the membrane, allowing a propagation of the depolarization (action potential) that spreads to involve the entire plasma membrane. The action potential induced by ACh release leads to the release of Ca²⁺ from the sarcoplasmic reticulum into the cytosol which causes muscle contraction. The high concentration of Ca²⁺ inside the cytosol also activates CaMKII and PKC kinases which mediate signaling pathways leading to myogenin phosphorylation and inactivation. When myogenin is inactive, AChR expression is suppressed. In addition to myogenin phosphorylation, a second mechanism, which represses myogenin, is activated by Ca²⁺ signaling upon innervation and leads to the activation of three transcription repressors (MSY-3, DACH2, and HDAC9). These repressors bind myogenin promoter and inhibit its transcriptional activation. The binding motifs for MSY-3, DACH2, and HDAC9 (respectively HCE, SIX, and MEF2) are located on adjacent sequences on the myogenin promoter