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Home , Dystrobrevin, Neuromuscular junction

SnapShot: Steven J. Burden Skirball Institute of Biomolecular Medicine, New York University Medical School, New York, NY 10016, USA

Motor approach muscles that are prepatterned

NEUROMUSCULAR JUNCTION Muscle

Bone Muscle fibers Motor terminal

Motor axons Motor axons Muscle membrane Nucleus AChR transcription AChR cluster MuSK Lrp4

Innervation Synaptic cleft

MUSCLE

MOTOR

NMJ ACh CONGENITAL MYASTHENIA

Agrin Hypomorphic in these Lrp4 impair postsynaptic MuSK differentiation. The number of Dok-7 AChRs at is reduced, Rapsyn which compromises the reliability of synaptic transmission and leads to muscle and fatigue. MuSK Antagonizes Mutations in these genes impair differentiation synaptic transmission, leading to AChR and fatigue. AChE ChAT encodes the enzyme for Stabilizes synthesizing ACh, and Nav1.4 differentiation ChAT Nav1.4 encodes a voltage-activated expressed in AChR .

MYASTHENIA GRAVIS

Cause accelerated degradation Auto- of AChRs and structural to AChRs disorganization of the , P leading to

P P Auto-antibodies Cause structural disorganization Dok-7 Rapsyn to MuSK of the synapse, leading to Myasthenia Gravis.

P Crk/ Crk-L P P

AChR Erm/GABP/ERG MuSK Rho GTPases Cortactin, α-actinin JNK Dvl, Pak, Tid1 Lrp4 Abl1/2 ACF7, , AChE ETS

826 Cell 144, March 4, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.02.037 See online version for legend and references. SnapShot: Neuromuscular Junction Steven J. Burden Skirball Institute of Biomolecular Medicine, New York University Medical School, New York, NY 10016, USA

The neuromuscular synapse—the connection between motor and skeletal muscle—controls our ability to move and breath, and thus, it is essential for survival. terminals release the (ACh), which diffuses across the synaptic cleft and binds to acetylcholine receptors (AChRs) in the muscle membrane. Binding of ACh opens the -gated channel and depolarizes the muscle membrane, which initiates an that stimulates . AChRs must be concentrated in the postsynaptic membrane at extremely high density, greater than 10,000 AChRs/um2, to ensure that ACh elicits a sufficient of the muscle membrane potential to trigger the action potential mechanism. This reliability is achieved by two general mechanisms: (1) anchoring AChRs (through rapsyn) and other critical muscle-derived in the postsynaptic membrane, and (2) selectively increasing expression of these genes in myofiber nuclei positioned near the synaptic site, a process called “synapse-specific transcription.” In addition, robust synaptic transmission relies upon the organization and alignment of active zones, or neurotransmitter release sites, in the nerve terminal directly opposed to AChRs in the muscle. This SnapShot illustrates the molecules and mechanisms that are currently known to lead to this specialized organization of the muscle plasma membrane at the neuromus- cular synapse, a process called “synaptic differentiation.” Defects in these pathways lead to neuromuscular diseases called congenital myasthenia, typified by muscle weakness and fatigue. The molecules and mechanisms that direct the formation and maintenance of neuromuscular synapses are likely similar to those in synapses of the central , where these signaling mechanisms are less well understood. Therefore, understanding how neuromuscular synapses form and work remains a paradigm for elucidating principles that can then be applied to less accessible synapses in the .

Synapse Formation Muscles are prepatterned with regions of increased AChR transcription (blue) and AChR clustering (red) in the central, prospective synaptic region of muscle prior to and inde- pendent of innervation. Muscle prepatterning requires low-density lipoprotein receptor-related 4 (Lrp4) and (muscle-specific kinase) MuSK and specifies the region where motor axons form synapses, marked by nerve terminals juxtaposed to AChRs. Motor nerve terminals focally release agrin. Agrin is a large that stabilizes postsynaptic differentiation at preexisting sites by binding Lrp4 and activating MuSK. Agrin can also induce new postsynaptic specializations. Therefore, the final cohort of synapses on the muscle fiber includes those recognized by motor axons and those induced by motor axons. ACh depolarizes muscle, disperses AChRs that are not juxtaposed to nerve terminals, and antagonizes postsynaptic differentiation by mechanisms that are poorly under- stood. In contrast, Agrin overcomes the extinguishing activity of ACh and selectively stabilizes postsynaptic differentiation at synapses. Binding of Agrin to Lrp4 stimulates association between Lrp4 and MuSK and increases MuSK kinase activity. Phosphorylation of Y553 in the juxtamembrane region of MuSK stimulates recruitment and phosphorylation of Dok-7. This causes Dok-7 to dimerize, stimulate MuSK kinase activity, and then recruit CT10 regulator of kinase (Crk) and Crk-L. Formation of a MuSK/Dok- 7-signaling complex is essential to activate both a Rac/Rho-dependent and a Rapsyn-dependent pathway, which leads to the anchoring and clustering of AChRs. Crk/Crk-L directly activates Rho GTPases, dishevelled (Dvl), kinases (e.g., Pak, Abl1/2), and a cochaperone of the heat shock protein (i.e., Tid1). The pathway that activates transcription specifically at the synapse is not as well understood. However it likely involves activation of the E-twenty six (Ets) family of transcrip- tion factors in a JNK-dependent manner. ETS transcription factors stimulate expression of multiple genes encoding synaptic proteins, including AChR, MuSK, Lrp4, acetylcho- linesterase (AchE), and utrophin. In the SnapShot, fundamental components of neuromuscular synapse formation are large and in bold, whereas if genetic evidence for their suggested role in synapse formation is lacking, the protein name is smaller.

Synapse Maintenance Maintaining neuromuscular synapses requires the continual action of genes that are necessary for synapse formation as well as additional genes essential to stabilize neuromus- cular synapses. These include proteins that regulate postsynaptic maturation, such as Src-family kinases, a-, LL5b, Syne-1, and collagen XIII, as well as proteins that regulate presynaptic differentiation, including fibroblast growth factors (FGFs), collagen IV, beta2-, and CD24.

References

Arber, S., Burden, S.J., and Harris, A.J. (2002). Patterning of skeletal muscle. Curr. Opin. Neurobiol. 12, 100–103.

Engel, A.G., Ohno, K., and Sine, S.M. (2003). Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat. Rev. Neurosci. 4, 339–352.

Farrugia, M.E., and Vincent, A. (2010). Autoimmune mediated neuromuscular junction defects. Curr. Opin. Neurol. 23, 489–495.

Hallock, P.T., Xu, C.F., Park, T.J., Neubert, T.A., Curran, T., and Burden, S.J. (2010). Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L. Genes Dev. 24, 2451–2461.

Kim, N., and Burden, S.J. (2008). MuSK controls where motor axons grow and form synapses. Nat. Neurosci. 11, 19–27.

Kim, N., Stiegler, A.L., Cameron, T.O., Hallock, P.T., Gomez, A.M., Huang, J.H., Hubbard, S.R., Dustin, M.L., and Burden, S.J. (2008). Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135, 334–342.

Kummer, T.T., Misgeld, T., and Sanes, J.R. (2006). Assembly of the postsynaptic membrane at the neuromuscular junction: paradigm lost. Curr. Opin. Neurobiol. 16, 74–82.

Okada, K., Inoue, A., Okada, M., Murata, Y., Kakuta, S., Jigami, T., Kubo, S., Shiraishi, H., Eguchi, K., Motomura, M., et al. (2006). The muscle protein Dok-7 is essential for neuro- muscular . Science 312, 1802–1805.

Sanes, J.R., and Lichtman, J.W. (2001). Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat. Rev. Neurosci. 2, 791–805.

Wu, H., Xiong, W.C., and Mei, L. (2010). To build a synapse: signaling pathways in neuromuscular junction assembly. Development 137, 1017–1033.

826.e1 Cell 144, March 4, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.02.037