Research Topics

NEUROMUSCULAR SYNAPSE FORMATION

Our laboratory uses mouse molecular genetics and molecular biological approaches to study the signaling mechanisms that regulate the formation of neuromuscular synapses. The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. As a consequence, acetylcholine receptor (AChRs) become highly concentrated in the postsynaptic membrane and arranged in perfect register with active zones in the presynaptic nerve terminal, insuring for fast, robust and reliable synaptic transmission. The signals and mechanisms responsible for this complex differentiation program are poorly understood but require the neurally derived ligand, Agrin, and the receptor tyrosine kinase, MuSK, which are critical to form and maintain synapses. Defects in this signaling pathway, which lead to aberrant presynaptic and postsynaptic differentiation and a reduced number of AChRs at synapses, are responsible for a variety of congenital neuromuscular disorders.

Muscle prepatterning
Recent studies have demonstrated that muscle is pre-specialized in the central, prospective synaptic region prior to and independent of innervation. Thus, key features of postsynaptic differentiation, including AChR expression, are established in a muscle-autonomous manner, and motor neurons refine and sharpen, rather than induce this prepattern of postsynaptic differentiation. These findings have led to a revised model for the steps and mechanisms that regulate neuromuscular synapse formation and indicate that motor axons approach muscles that are regionally-specialized, or prepatterned, prior to innervation, in a manner that preconfigures the prospective zone of innervation.

Genetic studies in mice have shown that muscle prepatterning requires MuSK, which is itself prepatterned in the central region of muscle independent of innervation. Our studies indicate that this regionally-confined pattern of MuSK expression arises from stochastic expression of MuSK in developing myotubes, the subsequent and symmetric pattern of muscle growth and positive feedback loops that stabilize and enhance the early pattern of MuSK expression. Muscle prepatterning has a critical role in defining where synapses will form in muscle, as ectopic MuSK expression disrupts muscle prepatterning, leads to ectopic axon growth and synapse formation and causes defects in muscle use, leading to kyphosis, impaired mobility and labored breathing. We are currently studying how developing motor axons recognize and respond to this MuSK-dependent muscle prepattern.

Agrin/MuSK signaling

Following contact with the growth cone of a developing motor neuron, developing muscle fibers undergo a still further complex differentiation program in the synaptic region, which is dependent upon motor neuron-derived signals. Arrival of the nerve, and its attendant signals, converts the AChR prepattern into the more refined pattern of AChR transcription and AChR clustering characteristic of mature synapses. This modification and sharpening is dependent upon two separable nerve-dependent programs: one program utilizes neurally derived Agrin to maintain AChR expression at nascent synaptic sites by activating MuSK, and a second program, triggered by acetylcholine (ACh), extinguishes AChR clustering at sites that are not stabilized by Agrin/MuSK signaling. These two programs thus ensure the stable expression of AChR clusters selectively at nascent synapses.

Upstream of MuSK
Although Agrin stimulates the rapid tyrosine phosphorylation of MuSK in cultured myotubes, Agrin fails to stimulate MuSK tyrosine phosphorylation in myoblasts or fibroblasts transfected with a MuSK expression vector. These findings have suggested that additional myotube-specific activities are required for Agrin to stimulate MuSK phosphorylation. Because purified, recombinant Agrin fails to bind MuSK, at least one of the missing components may be a receptor, or co-receptor for Agrin.

Lrp4, a member of the low-density lipoprotein (LDL) receptor-related protein (LRP) family, is required for neuromuscular synapse formation, and the defects in presynaptic and postsynaptic differentiation are virtually identical in lrp4 and MuSK mutant mice. These and additional data suggest that Lrp4 and MuSK function in the same pathway at synapses and raise the possibility that Lrp4 may function as a co-receptor with MuSK and confer responsiveness to Agrin, ideas that we are currently testing. We are currently using biochemical and genetic methods to study how Lrp4 regulates the formation of neuromuscular synapses.

Downstream from MuSK
How does MuSK activation lead to presynaptic and postsynaptic differentiation? Agrin stimulates the rapid tyrosine phosphorylation of MuSK, and the kinase activity of MuSK is essential for Agrin to stimulate clustering and tyrosine phosphorylation of AChRs. Signaling downstream from MuSK depends upon phosphorylation of a tyrosine residue (Y553) in the juxtamembrane region of MuSK. Phosphorylation of this tyrosine leads to recruitment and tyrosine phosphorylation of Dok-7, a PTB domain-containing protein that engages additional, but unknown signaling pathways essential for synapse formation. Mice lacking Dok-7 have defects identical to MuSK mutant mice, and mutations in human Dok-7 are a major cause of congenital myasthenic syndromes with defects in neuromuscular synapses. We are currently using biochemical methods and mouse molecular genetics to study the signaling pathways downstream from MuSK/Dok-7.

Since Dok-7 binds selectively to tyrosine phosphorylated MuSK, Dok-7 acts downstream of MuSK. Nonethleless, Dok-7 also regulates MuSK phosphorylation, since a failure to recruit Dok-7 leads to substantially reduced levels of MuSK tyrosine phosphorylation. Consistent with these data, phosphorylation of MuSK Y553 is required to fully activate MuSK kinase activity. Taken together, these data indicate an unusual role for an adapter protein, since Dok-7 acts upstream from MuSK to stimulate/maintain MuSK phosphorylation and downstream from MuSK to couple MuSK phosphorylation to AChR clustering and postsynaptic differentiation. Together with the Hubbard lab at the Skirball Institute, we are using biochemical and structural methods to study how Dok-7 stimulates MuSK tyrosine phosphorylation.