Search for molecules involved in the formation of the nerve-muscle synapse

Efficient synaptic transmission requires a high local specialization of pre- and postsynaptic cells: the presynaptic nerve terminal must be competent to secrete neurotransmitter substance in response to an invading action potential, and the postsynaptic cell must express in the synaptic portion of its cell membrane a high density of receptor molecules for neurotransmitter which is required for signal transduction into the postsynaptic cell. The reciprocal signalling mechanisms that regulate and coordinate pre- and postsynaptic differentiation during synapse formation are only poorly understood. At one synapse in the peripheral nervous system, the neuromuscular junction (NMJ), some of the signalling cascades involved are known. Specifcally, Agrin, a heparansulfate proteoglycan that is secreted by motor neurons and interacts with a muscle specific receptor tyrosine kinase (MuSK) has been shown to trigger the differentiation of a postsynaptic membrane in the muscle fiber in the absence of a nerve.
However, experiments in our laboratory have shown that the secretion of Agrin by motor neurons and the expression of its receptor MuSK in the fiber surface are not sufficient to induce motor neurons to make synapses on muscle fibers. Furthermore, it is known from classical experiments that muscle fibers must be denervated, i.e. electrically inactive, to be susceptible to motor innervation. Therefore, we hypothesized that denervated muscle fibers secrete factors or express molecules on their surface that promote neuromuscular synapse formation.
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In an attempt to identify such molecules, we performed differential display of mRNAs expressed in innervated and in denervated muscle fibers. In addition to many different genes whose mRNAs were up- or down-regulated by denervation and which are known to be involved in protein turnover, cytoskeletal rearrangements and energy metabolism, we also observed that the mRNA for a putative surface molecule of the immunoglobulin superfamily (IgSF), Embigin, was highly upregulated in denervated muscle. Quantitative Real-Time PCR and Northern blot analysis showed that the level of Embigin mRNA was increased between 50 and 150 fold in denervated mouse and rat muscles. Based on the homology of its extracellular domain with SynCAM an IgSF member recently shown to drive synaptic assembly in the central nervous system, we hypothesized that Embigin is involved in denervation-induced neuromuscular synapse formation.
The rat and mouse orthologs of Embigin were cloned, and the developmental expression pattern of Embigin mRNA in muscle was analyzed by Northern blot analysis and by quantitative RT-PCR. Importantly, Embigin mRNA increased upon denervation 1-2 days prior to ectopic endplate formation, a time course consistent with a role in neuromuscular synapse development. Furthermore, exogenous stimulation of denervated muscle repressed Embigin mRNA, again consistent with such a role. Embigin mRNA expression is also induced during differentiation of the mouse myogenic cell line C2C12 which are known to form synapses. Cell biological experiments using HEK293 cells transfected with an Embigin full length clone suggested, however, that unlike other IgSF members such as NgCAM, Embigin does not mediate adhesion via homophilic interactions. Finally, we tested whether Embigin expressed in surface of COS-7 cells induced the differentiation of presynaptic terminals by co-cultured chicken ciliary ganglion neurons. However, unlike with cells expressing a transmembrane isoform of Agrin, no effect could be seen. It is hypothesized that Embigin could make the motor neuron growth cone approaching the muscle fiber adhere transiently to the muscle fiber which would allow Agrin to be deposited locally and in sufficient quantities to activate MuSK, thus initiating the formation of a neuromuscular synapse.