To build a synapse: signaling pathways in neuromuscular junction assembly

Abstract

Synapses, as fundamental units of the neural circuitry, enable complex behaviors. The neuromuscular junction (NMJ) is a synapse type that forms between motoneurons and skeletal muscle fibers and that exhibits a high degree of subcellular specialization. Aided by genetic techniques and suitable animal models, studies in the past decade have brought significant progress in identifying NMJ components and assembly mechanisms. This review highlights recent advances in the study of NMJ development, focusing on signaling pathways that are activated by diffusible cues, which shed light on synaptogenesis in the brain and contribute to a better understanding of muscular dystrophy.

Fig. 1.

Neuromuscular synapse formation in vertebrates. (A) In mice, muscle fibers of the diaphragm form primitive, aneural AChR clusters prior to the arrival of phrenic nerve terminals. The clusters are distributed in a broad, poorly defined region in the middle of muscle fibers, a phenomenon called prepatterning (a). Innervation leads to the appearance of large AChR clusters in the synaptic region and to the disappearance of primitive clusters in non-synaptic areas (b). (B) In zebrafish, adaxial, slow muscle cells form aneural AChR clusters on the medial surface prior to innervation (a). Subsequently, adaxial muscle cells migrate outwards, and their original position is filled with fast muscle cells that form nerve-induced AChR clusters where the aneural clusters used to be (b).

Fig. 2.

Coordinated action of positive and negative signals in NMJ assembly. At least three cellular mechanisms contribute to the high density of AChRs at the NMJ. First, AChR might redistribute from primitive clusters to the synaptic area, either by lateral movement, by diffusion in the plasma membrane or by endo- and exocytosis. Second, muscle fibers are multi-nucleated cells, and only the nuclei beneath the postsynaptic membrane (synaptic nuclei) are actively transcribing the AChR subunit genes, contributing to synapse-specific AChR expression. Third, AChR turnover rate is reduced at mature NMJ or when clustered (as shown by the half-life of AChRs at the NMJ at 8-14 days compared with 17-24 hours for non-clustered or embryonic AChR). Motor nerves activate muscle fibers by releasing ACh, a negative signal, which activates AChR. Muscle activation stimulates the serine/threonine kinases cyclin-dependent kinase 5 (Cdk5) and Ca²⁺/calmodulin-dependent kinase II (CaMKII) to inhibit AChR clustering, to suppress AChR expression and to destabilize AChR clusters in entire muscle fibers. At the same time, nerves also release positive signals, such as agrin, which counteract the effects of negative signals, resulting in a high AChR concentration at the NMJ.

Fig. 3.

Intracellular pathways activated by agrin for AChR clustering. Agrin interacts with Lrp4 to increase its interaction with Musk and the dimerization of Musk and thus Musk activation. Subsequently, interactions between the kinase and distinct proteins, such as Dok7, which are crucial for its catalytic activity and for downstream signaling, are increased. Concomitantly, the tyrosine kinase Abl and and the metalloenzyme geranylgeranyl transferase I (GGT) are activated. GGT facilitates Rho GTPase activation, which, via multiple pathways, regulates the actin dynamics involved in AChR trafficking, membrane insertion and immobilization. Agrin also stimulates the association of AChR with rapsyn and Apc, which link the receptor directly or indirectly to the cytoskeleton. Rapsyn stability is increased at the synapse by the chaperone Hsp90β, and rapsyn interacts with and inhibits calpain, and thus antagonizes the AChR cluster-dispersing effect of ACh. Agrin signaling is regulated by Musk endocytosis, the E3 ubiquitin ligase Pdzrn3, lipid microdomains (Kishi et al., 2005; Stetzkowski-Marden et al., 2006; Willmann et al., 2006; Zhu et al., 2006) and intracellular calcium (Megeath and Fallon 1998). Many pathways illustrated here have been identified in cultured muscle and non-muscle cells, and their role in vivo remains to be studied. For example, mice lacking Shp2, a cytoplasmic tyrosine phosphatase, are viable and form normal NMJs (Dong et al., 2006), although many in vitro studies suggest a crucial role in AChR clustering. See text for details. Stars indicate protein-protein interactions that are increased by agrin. Red lipid bilayers indicate lipid rafts.

Fig. 4.

NMJ assembly in Drosophila and in C. elegans. (A) The Drosophila NMJ is a glutamatergic synapse. The Wnt ligands Wingless (Wg) and Wnt5 are released from presynaptic boutons. Binding to the Wnt receptor Frizzled on the postsynaptic membrane, Wg is thought to stimulate a Frizzled nuclear import (FNI) pathway for gene expression. This pathway might be crucial for both pre- and postsynaptic differentiation. Wg also activates Frizzled on the presynaptic membrane to promote presynaptic assembly via a pathway dependent on Gsk3β and MAP1B. Conversely, Wnt5 directly binds to and activates the receptor tyrosine kinase Derailed (Drl) on the postsynaptic membrane for postsynaptic assembly. (B) The C. elegans DA9 neuron, which is positioned in the ventral region of the worm, sends a dendrite anteriorly (green) and an axon (blue) that first migrates posteriorly; this soon turns toward the dorsal region and travels anteriorly in the dorsal region. It does not form synapses until it reaches a more anterior region, leaving an asynaptic domain in the axon. LIN-44/Wnt is produced by hypodermal cells and activates LIN-17/Frizzled in the axon to suppress presynaptic assembly.

Fig. 5.

A working model of signaling pathways in postsynaptic assembly. (A) Prior to innervation, Wnt interacts with Musk to activate the Wnt-Musk pathways necessary for forming aneural clusters and for guiding motor growth cones to the middle region of muscle fibers. At the same time, Wnt might also activate Wnt canonical and non-canonical pathways to regulate Musk-dependent pathways. (B) Upon innervation, neural agrin binds to Lrp4 and activates Musk to initiate the agrin-Lrp4-Musk pathway (see Fig. 3) for AChR clustering at the synapse. Wnt might regulate agrin signaling by directly binding to Musk or Lrp5/Lrp6-Frizzled. Dashed lines indicate pathways that remain to be determined.

Fig. 6.

Retrograde mechanisms in NMJ formation. (A) In Drosophila, muscle cells release TGFβ/Gbb for both post- and presynaptic assembly. Wnt released from presynaptic neurons activates the Frizzled-dependent pathway for presynaptic differentiation. (B) In rodents, muscle fibers release Gdnf, which activates the receptor tyrosine kinase Ret for presynaptic differentiation. FGF, synaptic laminin and synaptic collagens orchestrate the complex temporal control of presynaptic differentiation. The interaction of laminin β2 with P/Q type calcium channels might be required for nerve terminal maturation. Muscle fibers also regulate presynaptic differentiation via a pathway that requires muscle- but not motoneuron-generated β-catenin or β1 integrin.