Examining synapse formation at the neuromuscular junction

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Although synapse formation is a significant process of neuronal development its understanding is still incomplete. In the central nervous system, various proteins such as neuroligin, cadherins and Eph receptors have been linked to the process in-vitro. Whether and how much, these proteins affect synaptogenesis in vivo, however, has not yet been solved. At the skeletal neuromuscular junction (NMJ), the synapse between motoneurons and their target muscle, the picture is much clearer.

The intricate NMJ is structured to enable rapid, focal signalling of the nerve impulse to the muscle. Each muscle fibre is innervated at the motor end plate by a single motor axon, whose soma lies in the spinal cord or brain stem. This ensures that no synaptic integration occurs at the NMJ as each action potential in the nerve causes a single action potential in the muscle. At the muscle, each axon branches to innervate tens to hundreds of muscle fibres. At its target fibre, each axonal branch loses its myelin sheath and forms an array of fine terminal branches or boutons. The boutons cluster in the shallow gutters of the muscle cell surface and are capped by Schwann cell processes.

At the contact region both the nerve axon and the muscle fibre are highly specialized. The axon terminal has clusters of synaptic vesicles containing the neurotransmitter acetylcholine (ACh). Each vesicle cluster aligns with a patch of dense material on the presynaptic membrane, forming the active zone. At the active zone, the vesicles fuse with the plasma membrane and release the ACh by exocytosis. The high number of mitochondria present supplies the energy needed for the synthesis, storage, and release of neurotransmitter. The postsynaptic terminal holds the shallow gutters on the fibre surface into which the nerve terminals fit. The junctional folds that indent the gutters are precisely aligned with the presynaptic active zones. The membrane thickening that covers the crests of the folds and extends partly down their sides is the ACh receptor (AChR), a rich chemoreceptive surface.

A basal lamina crosses the synaptic cleft between the pre- and post-synaptic terminal, extending into the junctional folds. The synaptic basal lamina is part of the continuous sheath that surrounds the muscle fibre and is attached to the Schwann cell's basal lamina. Although the synaptic basal lamina is morphologically identical to the extra-synaptic basal lamina to which it is fused, it is biochemically specialized as it contains acetylcholinesterase (AChE). AChE inactivates the neurotransmitter, the components responsible for the strong adhesion of nerve to muscle and the factors that mediate developmental interactions. The Schwann cells found at the NMJ are also specialized: the pre-terminal Schwann cells form myelin while the synapse-associated Schwann cells cap the nerve terminals, protecting them from chemical and mechanical insults.

In order to understand the intricacy of the NMJ it is necessary to comprehend how the connectivity is formed during development. Experiments on the regeneration of damaged muscles and neurons have shown that synapse formation at the NMJ is a mutually inductive event. The neurons promote postsynaptic differentiation in the muscle fibres and myofibres promote presynaptic differentiation in the motor axon terminals. Even when muscle regeneration is prevented, the axons still return to the precise original location on the basal lamina and form an axon terminus with synaptic vesicles.

These observations led to the research for molecules in the basal lamina that direct where on the muscle a postsynaptic specialization will form. From the time of initial contact between an axon and a muscle fibre to the formation of a completely functional synapse, more than forty molecules are clustered at the synapse. One of the first to cluster is the AChR on the muscle-cell membrane. AChR subunits are first synthesized as myoblasts and thenfuse and differentiate into myotubes.

In a past experiment, cultured chick myotubes were used to analyse the AChRâ€"clustering process of protein from the basal lamina of the electric ray Torpedo californica, which is rich in cholinergic synapses. This enabled the isolation and partial sequencing of the protein agrin. Agrin, as shown by antibody staining, is stably associated with the basal lamina at the NMJ. As inclusion of anti-agrin antibodies in nerve-muscle co-cultures inhibited AChR aggregation it was concluded that agrin mediates aggregation of AChRs and many other components at the NMJ.

The N-terminal of the agrin molecule contains matrix-binding sites, while the C-terminal contains membrane-binding sites and is necessary for AChR-clustering. Localization studies have shown that both muscle and motor neurons produce agrin, that motor neurons transport agrin to the nerve terminus, and that agrin from both sources is localized at the NMJ. A neuron-specific form of agrin has also been identified that it is about 1000-fold more active in inducing AChR clustering than agrin expressed by muscle.

A major component of the agrin receptor complex is a muscle-specific receptor tyrosine kinase, called MuSK (Muscle Specific Kinase), which is expressed at low levels in myoblasts and is then upregulated with the onset of myoblast fusion and differentiation. While the overall level of this receptor declines in mature muscle, the receptors become concentrated in postsynaptic membranes at the NMJ. Knockout mice which lacked MuSK showed a phenotype similar to the agrin knockout. Finally, MuSK activity is stimulated specifically by the neuronal isoform of agrin. Nonetheless, agrin does not bind directly to the extracellular domain of MuSK. Thus additional myotube components or myotube-specific modification of MuSK is necessary to form an active agrin receptor.

Analysis of rapsyn, a protein identified in biochemical studies as co-purifying with the AChR, showed that it aggregates AChR in gain-of-function studies. While AChRs are diffusely expressed along the surface of non-muscle cells transfected with AChR subunit genes, co-transfection with rapsyn promoted receptor clustering. On the contrary, rapsyn knock-out mice did not aggregate receptors.

Recent studies provide converging evidence for a pivotal role of Wnt signalling in NMJ development. Wnts are a family of secreted glycoproteins that regulate diverse cellular processes, including cell proliferation and fate determination. The role of Wnt signalling in the regulation of synaptogenesis was first discovered in the developing rodent cerebellum, where Wnt7a is used by granule cells as a retrograde signal for axon and growth cone remodelling. At the vertebrate NMJ, Wnt signalling regulates the prepatterning of AChR receptors before the arrival of axons and later by collaborating with Agrin. Before the arrival of motor axons aneural AChRs clusters form in the central domain of the muscle. Although MuSK expressed in the muscle is crucial in this process, Agrin is not required. Thus, MuSK appears to be activated by an alternative ligand. In zebrafish embryos, knockdown of Wnt11r, expressed in tissues surrounding the spinal cord, results in severe defects in the clustering of aneural AChRs. Wnt11r binds to Unplugged/MuSK receptors and requires unplugged for its function. Thus, binding of Wnt11r to MuSK activates a signalling cascade that stimulates the clustering of aneural AChRs in the central region of the muscle before the arrival of the motor axons.

Wnt signalling also contributes to the assembly of NMJs by collaborating with Agrin. Gain and loss of function studies reveal that Wnt promotes the formation of AChR clusters during NMJ development in the chick limb. Moreover, Dvl1 knockout mice exhibit defects in AChRs clustering. In cultured myotubes, Wnt3, expressed by motoneurons, increases the formation of small but unstable AChR clusters. These clusters become stable and larger in the presence of Agrin. Thus, Wnt3 collaborates with Agrin by increasing the formation of microclusters, which can be converted into stable large AChR clusters. Agrin induces the formation of large AChR clusters through the activation of Rac and Rho and requires Dvl. Interestingly, Wnt3 alone activates Rac1 whereas blockade of Rac suppresses the effect of Wnt3 microcluster formation. These studies show that Wnt signalling collaborates with Agrin to regulate the assembly of the vertebrate NMJ.

More recent experiments have indicated that Schwann cells also play an active role in NMJ formation. Schwann cells synthesize and secrete trophic factors, such as neuregulin and nerve growth factor, which promote motorneuron survival and growth, and Schwann cell process extension. Studies in which Schwann cell progenitor cells were destroyed by genetic manipulation of neuregulin signalling prior to their migration into the periphery showed that, in embryos lacking Schwann cells, axons grow toward muscles and form neuromuscular contacts; however, such contacts quickly disappear, the motor neurons die, and the animals are nonviable at birth or shortly after. Terminal Schwann cells have a function at the time of nerve-terminal injury; that is, after damage to the nerve terminal, Schwann cells may become phagocytic and remove nerve-terminal debris. During the process of debris removal, Schwann cells invade the space between the muscle fiber and the degenerating nerve terminal and, as the nerve grows to re-innervate the muscle fiber, Schwann cells form paths for axonal growth.

Through all these studies the following pathway for formation of the NMJ can be established: Agrin is released from motor neuron growth cone. MuSK activates rapsyn-dependent-AChR aggregation. Many muscle components, including the receptors for ARIA, are promoted to aggregate at the synapse. ARIA released from motor neurons promotes myotube nuclei directly below the receptor to up-regulate transcription of synapse-specific components. Transcription of AChR genes and other postsynaptic components is inhibited in nuclei in regions away from the synapse. Depolarization (Ca2+ influx) of the muscle membrane by activation of AChR by presynaptic release of acetylcholine from motor nerve terminals is necessary for repression.

Although much remains to be learned about synapse formation, the study of the structure and function of the vertebrate skeletal NMJ, with its seductive simplicity, has provided valuable insights and probes for investigating the apparently more complex synapses occurring between neurons. The experimental results described in this essay are of more than developmental interest. Many of the processes involved in synapse formation likely remain active at adult synapses, where they mediate changes in synaptic efficacy that underlie neuronal plasticity. The next challenge will be to not only identify the full cohort of synaptic organizers but to gain insight into how they constructively signal to, induce and maintain synapses. Ultimately this knowledge will advance our understanding of the basics of neural activities.