Our failure to understand how Agrin stimulates MuSK tyrosine phosphorylation is perhaps the most fundamental and glaring gap in our understanding of signaling at the neuromuscular synapse. We found that Lrp4, a member of the LDL receptor family, is essential for Agrin to stimulate MuSK tyrosine phosphorylation, suggesting that Lrp4 is a key component of an Agrin receptor complex and potentially the long-sought receptor for Agrin. The experiments described here are designed to determine whether Lrp4 binds Agrin and/or MuSK and how Lrp4 mediates Agrin responsiveness and synaptic differentiation. How MuSK, once activated, stimulates postsynaptic differentiation is similarly poorly understood. Dok-7 has recently been identified as an adaptor protein, which is recruited to tyrosine phosphorylated MuSK and which is essential for synaptic differentiation. Further, mutations in Dok-7 are a major cause of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). The experiments described here are designed to identify and study the proteins that are recruited to Dok-7, which will be critical to understand how Dok-7 regulates synaptic differentiation and how mutations in Dok-7 cause CMS. The proposed studies are clinically relevant for several reasons. First, mutations in Dok-7 cause CMS, so understanding how Dok-7 works will contribute to a better understanding of this disease and may lead to novel therapeutic strategies. Second, mutations in genes that are downstream from Dok-7 may likewise cause CMS, so identifying the pathway downstream from Dok-7 is likewise important. Third, because mutations in other synaptic genes (e.g. AChR subunits, rapsyn, MuSK, AChE) also cause CMS, mutations that interfere with the function of Lrp4 selectively in muscle may also be responsible for CMS. Finally, because 20% of patients with myasthenia gravis are sero- negative for auto-antibodies to AChR or MuSK, these individuals presumably carry auto-antibodies to other synaptic proteins, possibly Lrp4.

Public Health Relevance

The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and developing muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. 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. Defects in this signaling pathway are responsible for a variety of congenital neuromuscular disorders and could underlie or contribute to neurodegenerative diseases such as ALS. The experiments described here are designed to determine how Lrp4 and Dok-7, two newly discovered components of this signaling pathway mediate Agrin responsiveness, lead to MuSK phosphorylation and stimulate synaptic differentiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS036193-15
Application #
8256772
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Porter, John D
Project Start
1998-02-01
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
15
Fiscal Year
2012
Total Cost
$394,511
Indirect Cost
$161,761
Name
New York University
Department
Orthopedics
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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Banerjee, Santanu; Gordon, Laura; Donn, Thomas M et al. (2011) A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration. Development 138:3287-96
Gomez, Andrea M; Burden, Steven J (2011) The extracellular region of Lrp4 is sufficient to mediate neuromuscular synapse formation. Dev Dyn 240:2626-33
Bergamin, Elisa; Hallock, Peter T; Burden, Steven J et al. (2010) The cytoplasmic adaptor protein Dok7 activates the receptor tyrosine kinase MuSK via dimerization. Mol Cell 39:100-9
Ruggiu, Matteo; Herbst, Ruth; Kim, Natalie et al. (2009) Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing. Proc Natl Acad Sci U S A 106:3513-8
Stiegler, Amy L; Burden, Steven J; Hubbard, Stevan R (2009) Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK. J Mol Biol 393:1-9
Borges, Lucia S; Yechikhov, Sergey; Lee, Young I et al. (2008) Identification of a motif in the acetylcholine receptor beta subunit whose phosphorylation regulates rapsyn association and postsynaptic receptor localization. J Neurosci 28:11468-76
Kim, Natalie; Burden, Steven J (2008) MuSK controls where motor axons grow and form synapses. Nat Neurosci 11:19-27
Kim, Natalie; Stiegler, Amy L; Cameron, Thomas O et al. (2008) Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135:334-42
Mansilla, Francisco; Dominguez, Carlota A G; Yeadon, James E et al. (2008) Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein. J Cell Biochem 105:847-58

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