Sialic acid inhibits agrin signaling in C2 myotubes.

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.

Acetylcholine receptors are required for postsynaptic aggregation driven by the agrin signalling pathway.

To investigate the role of acetylcholine receptors (AChRs) in the aggregation of postsynaptic molecules on muscle cells, we utilized the 1R- genetic variant of C2 muscle cells which has very little expression of AChRs in its cell membrane. On C2 myotubes, AChRs cluster spontaneously, with the frequency of clustering greatly enhanced by motor neuron-derived agrin. Signal transduction events driven by agrin, including the tyrosine phosphorylation of muscle-specific kinase (MuSK) and the AChR beta subunit, have been implicated as requirements of postsynaptic scaffold assembly. We show here that some molecules of the postsynaptic scaffold spontaneously aggregate and colocalize on 1R- myotubes at very low frequency, including an as yet unidentified agrin binding molecule, beta-dystroglycan and MuSK. Agrin is unable to increase the frequency of these aggregations, but does cause tyrosine phosphorylation of MuSK. We conclude that free molecules can associate into aggregates independently of AChRs, but AChRs are required for high-frequency molecular aggregation driven by the agrin signalling pathway. MuSK tyrosine phosphorylation appears to precede a requisite event involving AChRs that aggregates postsynaptic molecules.

Agrin-independent activation of the agrin signal transduction pathway.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.

A mechanism for acetylcholine receptor clustering distinct from agrin signaling.

Acetylcholine receptors (AChRs) and other postsynaptic molecules cluster spontaneously on cultured C2 myotubes. The frequency of clustering is enhanced by neural agrin, neuraminidase, or calcium through a signaling pathway which includes tyrosine phosphorylation of a muscle-specific kinase (MuSK) and the AChR beta-subunit. Vicia villosa agglutinin (VVA) lectin, previously shown to potentiate agrin-induced clustering on C2 myotubes, is shown here to also potentiate neuraminidase- and calcium-induced clustering of AChRs, while having no effect on the level of tyrosine phosphorylation of MuSK or the AChR beta-subunit. We propose that VVA lectin increases the frequency of AChR clustering through a mechanism that is distinct from agrin signaling, and that may involve alpha-dystroglycan.

Muscle fiber type correlates with innervation topography in the rat serratus anterior muscle.

Previous studies have reported that motoneurons from the sixth spinal nerve (C6) innervate the majority of muscle fibers in the rat serratus anterior (SA) muscle. The seventh spinal nerve (C7) innervates a limited number of SA fibers, increasing caudally. This topographic map is partially reestablished following denervation. In the present study, muscle fibers of the SA were stained with monoclonal antibodies for the muscle-specific fast myosin heavy chain (F-MHC) and slow myosin heavy chain (S-MHC) proteins. We found that the majority of fibers in the SA muscle stained for F-MHC antibody, and the percentage of muscle fibers staining for S-MHC antibody increased caudally. When newborn SA muscles were denervated and then reinnervated by the entire long thoracic (LT) nerve or only the C6 branch to the LT nerve, the reinnervated muscle had the normal proportion of muscle fibers expressing S-MHC protein. However, if the LT nerve was crushed and only C7 motoneurons allowed to reinnervate the SA muscle, a greater percentage of muscle fibers stained for S-MHC antibody than normal. We conclude that there is a correlation between muscle fiber type and innervation topography in the SA muscle of the rat.

Differential delay of reinnervating axons alters specificity in the rat serratus anterior muscle.

Previous studies have shown remarkable rostrocaudal selectivity by regenerating motoneurons to the rat serratus anterior (SA) muscle after freezing, crushing, or sectioning the long thoracic (LT) nerve. The LT nerve contains motoneurons from both the sixth and seventh cervical spinal nerves (C6 and C7), with C6 motoneurons as the major source of innervation throughout the muscle, and with C7 motoneurons innervating a larger percentage of muscle fibers caudally than rostrally. To determine if synaptic competition can play a role in neuromuscular topography, both the LT nerve and the branch carrying C6 (rostral) motoneurons to the LT nerve were crushed in newborn rats. This approach provides a temporal advantage to regenerating C7 (caudal) motoneurons. After an initial period during which C7 motoneurons reinnervated a larger proportion of muscle fibers than normal in all SA muscle sectors, C6 motoneurons regained their original proportion of rostral muscle fibers. Caudally, however, C7 motoneurons maintained an expanded territory. With this two-site crush method, the number of C6 motoneurons that reinnervate the SA muscle was significantly decreased from normal, whereas the number of C7 motoneurons remained the same. It is concluded that when C7 motoneurons are given a temporal advantage, synaptic specificity can be altered transiently in rostral muscle sectors and permanently in caudal sectors, and this is correlated with a disproportionate loss of C6 motoneurons. Moreover, this may be an important model for studies of synaptic competition, where terminals destined to be eliminated can be identified beforehand.