Wade A. Grow

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 β-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 β-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 α-dystroglycan.

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 β 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, β‐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.

Reduced Glycosaminoglycan Sulfation Diminishes the Agrin Signal Transduction Pathway

Proteoglycans consist of a protein core complexed to glycosaminoglycan (GAG) side chains, are abundant in skeletal muscle cell membranes and basal lamina, and have important functions in neuromuscular synapse development. Treatment with chlorate results in the undersulfation of heparan sulfate and chondroitin sulfate GAGs in cell culture. In addition, chlorate treatment decreases the frequency of spontaneous acetylcholine receptor (AChR) clustering in skeletal muscle cell culture. AChRs and other molecules cluster to form the postsynaptic component of neuromuscular synapses. Chlorate treatment is shown here to decrease the frequency of agrin-induced AChR clustering and agrin-induced tyrosine phosphorylation of the AChR β-subunit. These data suggest that reduced GAG chain sulfation decreases the frequency of AChR clustering by diminishing the agrin signal transduction pathway.

Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture

We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10mM, 25mM, and 100mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering.

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.

Dietary Genistein Influences Number of Acetylcholine Receptors in Female Diabetic Jejunum

Background Intestinal dysfunction in the ob/ob mouse model of diabetes mimics that seen clinically. Methods We determined the effects of a 4-week genistein diet (600 mg genistein/kg food) on intestinal function (contractility, morphology, AChR, and motility) in female ob/ob and lean mice. Results Contractility of the jejunum in response to incrementally increasing concentrations of KCl was comparable in ob/ob females and lean controls regardless of a genistein-diet. There were no changes in the wall thickness measured. We assessed the number of clusters of AChR in the jejunum wall; AChR were decreased by 48% in ob/ob mice versus leans, and the genistein diet reversed this. In utilizing a video-imaging system to evaluate gastrointestinal motility, we determined that the distance between consecutive contractile events was significantly increased by 1.87-fold in ob/ob mice versus leans, and the genistein diet was without effect. Conclusions These data suggest that slowed intestinal transit in the diabetic ob/ob mouse may be due in part to decreased AChR and decreased contraction events occurring per unit time. A genistein diet rescues the number of AChR to levels of leans yet did not change the number of contractile events. Feeding ob/ob mice a genistein-rich diet has potential therapeutic benefits towards improving the debilitating diabetes-related gastrointestinal dysfunction.