Research

Understanding how signals are exchanged at neuromuscular synapses is fundamental to understanding the principles that govern the formation and function of synapses in the peripheral and central nervous systems (CNS). The discovery of genes critical for forming and maintaining neuromuscular synapses has not only provided insight into the normal mechanisms for synapse formation but also led to the identification of genes that are responsible for congenital myasthenia and for understanding how mutations in these genes lead to deficits in neuromuscular function. Further, because key molecules that direct the formation and maintenance of neuromuscular synapses are expressed in the CNS, understanding how neuromuscular synapses form and work may serve as a paradigm for understanding synapse formation in the less tractable CNS. Our lab uses multiple approaches, including molecular genetics, biochemistry and structural biology to understand how neuromuscular synapses form during development and how synapses are maintained and stabilized in adults. Moreover, we study the causes for neuromuscular diseases, including congenital myasthenia, myasthenia gravis and ALS, and we are using this knowledge to devise therapeutic strategies for these diseases.

Neuromuscular synapse formation is a multi-step process, requiring coordinated interactions between motor neurons and muscle fibers, which lead to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal (Figure 1). As a consequence of this exchange of signals, acetylcholine receptors (AChRs) become concentrated in the postsynaptic membrane and arranged in perfect register with active zones in the presynaptic nerve terminal, ensuring for rapid, robust and reliable synaptic transmission. The principle signals and mechanisms responsible for this process are becoming understood and require: (1) MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, (2) Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, and (3) Lrp4, the muscle receptor for Agrin, which forms a complex with MuSK (Figure 2). These genes play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in these genes or downstream effectors lead to a reduced number of AChRs at synapses and are a major cause of a group of neuromuscular disorders, termed congenital myasthenia. Moreover, auto-antibodies to MuSK, Lrp4 or AChRs, which perturb the structure and function of the synapse, are responsible for myasthenia gravis.
 
Agrin, released from motor nerve terminals, induces and stabilizes postsynaptic differentiation by binding directly to Lrp4, stimulating association between Lrp4 and MuSK and substantially increasing MuSK phosphorylation. Once tyrosine phosphorylated, MuSK recruits and phosphorylates Dok-7. Dok-7 functions both as an inside-out ligand, which stimulates MuSK phosphorylation, and as an adapter protein that assembles a signaling complex, which includes Crk and Crk-L (Figure 2). Formation of this signaling complex is essential to activate a Rac/Rho- and Rapsyn-dependent pathway for clustering AChRs and additional postsynaptic proteins, including Lrp4 and MuSK. In addition, MuSK signaling stimulates synapse-specific transcription, which does not require Rapsyn but likely involves JNK-dependent activation of ETS family transcription factors. Synapse-specific transcription ensures that expression of synaptic proteins is enriched in the synaptic region so that post-translational mechanisms can act efficiently to further concentrate these proteins into the muscle postsynaptic membrane.

Lrp4 not only has a key role in postsynaptic differentiation but also has a central role in presynaptic differentiation. Lrp4 not only binds Agrin and stimulates MuSK phosphorylation to regulate postsynaptic differentiation, but once clustered as a consequence of MuSK activation, also signals in turn to motor axons, halting their growth and stimulating their differentiation. As such, Lrp4 functions bi-directionally and coordinates synaptic differentiation, using distinct domains to control presynaptic and postsynaptic differentiation. How Lrp4 responds to Agrin to stimulate its association with MuSK and how Lrp4 stimulates presynaptic differentiation are currently major topics of study in the lab.

In addition, we have learned that auto-antibodies to MuSK cause MuSK myasthenia gravis by hindering binding between Lrp4 and MuSK, reducing MuSK phosphorylation and impairing synaptic differentiation. We found that a synthetic agonist antibody to MuSK overcomes this inhibition by patient antibodies, and we are exploring use of these agonist antibodies as a therapy for MuSK myasthenia gravis. We reasoned that these agonist antibodies might enhance retrograde signaling, stabilize synapses and reduce nerve terminal withdrawal and detachment in other neuromuscular diseases. Indeed, we found that these agonist antibodies reduce the extent of denervation, preserving neuromuscular synapses in a mouse model of ALS, and we are exploring the possibility that this strategy may be therapeutic for ALS.


Lab Protocols

Electron microscopy of diaphragm muscle

Generating immortalized muscle cell lines

MuSK phosphorylation

Staining frozen sections

Staining muscle cultures

Staining whole mounts of the diaphragm muscle