The patterning and formation of synapses is fundamental to nervous system function. The long-term objective of our research is to understand the cellular and molecular mechanisms underlying the formation of synapse, using the vertebrate neuromuscular junction (NMJ) as our model. During the initial stages of neuromuscular synaptogenesis, nascent AChRs are clustered preferentially in the central region of the muscle. The presence of these AChR clusters coincides with the arrival of the nerves, but the majority of these clusters are not directly contacted by the ingrowing nerves;subsequent development leads to Formation of juxtaposed apposition between the pre-synaptic nerve terminal and the post-synaptic AChR. These nerve-independent initial AChRs are termed muscle prepatterning. A critical question that remains unanswered is whether the prepatterning of postsynaptic proteins such as AChRs contributes in any way to the formation of the NMJs. We hypothesize that these fetal AChRs are essential for defining the normal innervation boundaries critical for the establishment of initial neuromuscular synaptic pattern. This hypothesis will be tested by two independent, but complementary, approaches: 1) in mutant mice deficient in the AChR y-subunit gene, and 2) in mouse embryos treated with conotoxins that specifically recognize and block fetal AChRs. We will examine the formation of synaptic patterns in the absence of the initial AChR clustering to determine the extent that fetal AChRs contribute to pre-synaptic differentiation and pattern formation. Furthermore, using electrophysiology, we will characterize synaptic transmission in the developing NMJ in the absence of the y-subunit. Using conotoxins to block the fetal AChRs, we will determine whether the function of postsynaptic AChRs is essential for the formation of the NMJ. This proposed project will provide a better understanding of the mechanisms underlying synapse formation. In addition, since AChR deficiency is implicated in neuromuscular disorders such as myasthenia gravis (MG) and congenital myasthenic syndromes (CMSs), this proposed project will also provide a basis for better understanding the pathogeneses of these neuromuscular diseases. Furthermore, increasing evidence shows that distal axonal or neuromuscular synaptic dysfunction occurs prior to the degeneration of motoneurons in diseases such as amyotrophic lateral sclerosis (ALS). Therefore, elucidating mechanisms underlying the structural and functional changes of the pre-synaptic nerves at the NMJ will also provide insight towards a better understanding of motoneuron diseases and may provide new targets for therapeutic intervention for motoneuron diseases.
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