Zach W. Hall

Synaptic structure and development: The neuromuscular junction

Synaptic transmission--the process by which signals are transferred from a neuron to its target--is a fundamental function of neurons. Most neurobiologists look to the synapse to find the patterns of connectivity that account for neural specificity, the information processing that underlies behavior, and the plasticity responsible for learning. Accordingly, it is no surpris e that we are keenly interested in how synapses are formed. What is surprising is that virtually all of our current understanding of synaptogenesis derives from the study of just one synapse, the vertebrate skeletal neuromuscular junction. This synapse lies outside of the brain and does not even have a neuron as its postsynaptic element. It is the only synapse in vertebrates or invertebrates, however, whose structure and function are sufficiently well understood that the mechanisms regulating its development can be analyzed. The features of simplicity and accessibility that have enabled investigation of the mature synapse facilitate this analysis. In addition, unlike central neurons, muscles are readily reinnervated following nerve damage, allowing synaptogenesis to be studied in the adult, uncomplicated by processes such as neurogenesis that occur only in the embryo. Finally, although interneuronal synapses differ from neuromuscular junctions in important ways, our fragmentary knowledge to date encourages the belief that similar principles govern the development of both. For all of these reasons, this review is devoted to the neuromuscular junction. We begin by describing the cytological and molecular architecture of this synapse, making three main points: that chemical synapses are designed for rapid, focal transmission of information; that this task is performed by highly specialized preand postsynaptic domains that lie in precise juxtaposition across the synaptic cleft; and that many of the components forming these domains have now been isolated and characterized. We then examine the complex series of inductive interactions between nerve and muscle that regulate synaptic development. These interactions are mediated by membrane, extracellular matrix, and soluble molecules that are only now being identified. A major point is that nerve and muscle can each synthesize and assemble synaptic components on its own. In vivo, however, they do so in coordination and only at points of juxtaposition. Thus, intercellular interactions during synaptogenesis localize and refine the synaptic functions of each cell. The result is a process of synaptic maturation that proceeds to completion in a series of overlapping steps over a prolonged interval. As the signaling molecules are identified and the regulatory circuits that define these steps are unraveled, the classic histological and physiological descriptions of synaptogenesis are being reformulated in molecular and mechanistic terms.

The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans

Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the y site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.

Dystroglycan binds nerve and muscle agrin

Neurally released agrin is thought to cluster acetylcholine receptors (AChRs) and other synaptic proteins in the postsynaptic membrane during synaptogenesis at the neuromuscular junction. We have examined the binding of nerve and muscle agrins, which have dramatically different abilities to cluster AChRs, to the membrane proteins of Torpedo electric organ and C2 myotubes. Both bound with approximately nanomolar affinity to a single component identified as alpha-dystroglycan: agrin binding was blocked by antibodies to alpha-dystroglycan, and agrin bound to purified alpha-dystroglycan. Dystroglycan was altered in two genetic variants of C2 muscle cells that fail to form spontaneous clusters of AChRs and that show a diminished response to agrin. Antibodies that blocked alpha-dystroglycan binding, however, failed to block the clustering of AChRs by neural agrin. Although alpha-dystroglycan is the major agrin-binding protein in Torpedo and myotube membranes, its physiological role is unclear.

RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Nuclear domains in muscle cells

Article de synthese sur les interactions des noyaux cellulaires presents en grande quantite dans les fibres musculaires des vertebres

Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle

We used specific antibodies to gamma, delta, and epsilon subunits to characterize acetylcholine receptor (AChR) in extracts and at endplates of developing, adult, and denervated rat muscle. The AChRs in normal adult muscle were immunoprecipitated by anti-epsilon and anti-delta, but not by anti-gamma antibodies, whereas AChRs in denervated and embryonic muscles were precipitated by anti-gamma and anti-delta, but showed little or no reactivity to anti-epsilon antibodies. In immunofluorescence experiments, AChRs at neonatal endplates bound antibodies to gamma or delta, but not epsilon, subunit, whereas those in adult muscles bound antibodies to epsilon or delta, but not gamma, subunit. AChRs at denervated endplates and at developing endplates between postnatal days 9 and 16 bound all three antibodies. We conclude that the distribution of gamma and epsilon subunits of the AChR parallels the distribution of AChRs with embryonic and adult channel properties, respectively.

Developmental regulation of highly active alternatively spliced forms of agrin

Agrin is an extracellular matrix protein involved in clustering acetylcholine receptors during development of the neuromuscular junction. We have previously shown that alternative splicing at three sites generates multiple forms of rat agrin and that a novel 8 amino acid insert is the most important in determining biological activity. In the present study we have examined the expression of agrin during development with particular emphasis on determining the tissue distribution of the splicing variants at each site. Our principal observation is that the variants containing the sequence most responsible for biological activity are expressed exclusively in neural tissue and that their expression is highly regulated during development. We also show that muscle expresses less active forms and that agrin immunoreactivity during synaptogenesis is initially not limited to synaptic sites, but becomes progressively restricted to the synapse as development proceeds.

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin‐treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR‐associated MuSK, as demonstrated by MuSK phosphorylation. In agrin‐treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the β subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin‐induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

Building synapses: agrin and dystroglycan stick together

A major effort of the past decade for those studying synaptic development has been to identify the molecular signals whose carefully choreographed exchange between pre- and postsynaptic cells regulates the local differentiation of each cell to form the mature synapse. Now that several of these factors [agrin, ACh-receptor inducing activity (ARIA) and calcitonin gene-related peptide] have been identified and isolated, efforts have moved toward understanding their receptors and the intracellular signaling pathways by which the factors achieve their effects. One of the most intensively studied of the synaptic signaling molecules is agrin, a large protein synthesized and released by motor neurons that induces ACh receptors and other synaptic molecules in muscle cells to accumulate at the sites of nerve contact. Recent efforts to discover the agrin receptor have led to a surprising conclusion: the only agrin-binding component so far detected in muscle cells is dystroglycan, an extracellular protein that is part of the complex of proteins associated with dystrophin, and its homologue, utrophin. Because dystroglycan binds laminin, and dystrophin binds actin, the complex containing these two proteins is thought to link the extracellular matrix to the cytoskeleton. Those interested in synapses are now pondering whether dystroglycan has a new and unexpected role as a signaling receptor for agrin-induced ACh-receptor clustering, whether it serves as an auxiliary for another receptor, or whether it serves as a receptor for an entirely different agrin-mediated function.