LRP4 signaling in neuromuscular junction formation

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between motoneurons and muscle fibers, has contributed greatly to understanding of the general principles of synaptogenesis, as well as of neuromuscular disorders. NMJ formation requires intimate interaction between motoneurons and muscle fibers. For example, in antegrade signaling, motoneurons release agrin that binds LRP4, a member of the LDL receptor family, in muscle cells to activate the receptor tyrosine kinase MuSK, both of which are required for NMJ formation. Downstream of MuSK was not well understood, except that AChR concentration absolutely requires the adapter protein rapsyn. However, exactly how signals are transduced from MuSK activation to AChR concentration is not well understood. On the other hand, skeletal muscles are known to be critical to the development of axon terminals of motoneurons. In contrast to antegrade regulation, much less is understood about molecular mechanisms of retrograde regulation of presynaptic differentiation by muscle fibers. In the past few years, we have begun to address these critical issues. In preliminary results, first, we discovered that the classic adaptor protein rapsyn is an E3 ligase. Knockin mice carrying the mutation of a single residue necessary for the enzymatic activity are unable to form the NMJ. Mechanistic studies suggest that rapsyn promotes the conjugation of nedd8, a small ubiquitin-like protein, to AChR -subunit. These observations identify a previously unappreciated enzymatic activity of rapsyn and a role of neddylation in synapse formation. In addition, we found that muscle-specific LRP4 mutant mice display profound deficits in motoneuron terminals. In particular, LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of co-cultured neurons, indicating that the extracellular domain of LRP4 (ecto-LRP4) is synaptogenic. These observations corroborate that muscle LRP4 may have a novel function – as a retrograde signal for presynaptic development, in addition to the well-stablished role as agrin’s receptor. Based on these exciting preliminary results, we propose two hypotheses. First, MuSK increases rapsyn enzyme activity to promote neddylation that is necessary for clustering. Second, muscle LRP4 regulates presynaptic differentiation by interacting with a yet unidentified protein. To test these hypotheses, we will investigate how rapsyn regulates postsynaptic assembly and to identify mechanisms by which muscle LRP4 controls presynaptic differentiation. Results will provide a better understanding of mammalian NMJ formation. Pathogenesis of neuromuscular disorders is known to involve abnormal synaptic structure and mutations of proteins essential for NMJ formation. In fact, mutations and/or autoimmunization of agrin/LRP4/MuSK signaling proteins have been implicated in congenital myasthenic syndrome. Recent evidence from various laboratories including ours indicates that patients with myasthenia gravis (MG) develop antibodies against agrin and LRP4. Therefore, our research will contribute to a better understanding of pathogenic mechanisms of these neuromuscular disorders.