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Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses

Abstract

Motor axons receive retrograde signals from skeletal muscle that are essential for the differentiation and stabilization of motor nerve terminals1. Identification of these retrograde signals has proved elusive, but their production by muscle depends on the receptor tyrosine kinase, MuSK (muscle, skeletal receptor tyrosine-protein kinase), and Lrp4 (low-density lipoprotein receptor (LDLR)-related protein 4), an LDLR family member that forms a complex with MuSK, binds neural agrin and stimulates MuSK kinase activity2,3,4,5. Here we show that Lrp4 also functions as a direct muscle-derived retrograde signal for early steps in presynaptic differentiation. We demonstrate that Lrp4 is necessary, independent of MuSK activation, for presynaptic differentiation in vivo, and we show that Lrp4 binds to motor axons and induces clustering of synaptic-vesicle and active-zone proteins. Thus, Lrp4 acts bidirectionally and coordinates synapse formation by binding agrin, activating MuSK and stimulating postsynaptic differentiation, and functioning in turn as a muscle-derived retrograde signal that is necessary and sufficient for presynaptic differentiation.

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Figure 1: Lrp4-expressing non-muscle cells induce clustering of synapsin in motor axons.
Figure 2: Lrp4, attached to polystyrene beads, induces presynaptic differentiation in motor neurons.
Figure 3: Lrp4 binds to motor axons.
Figure 4: Lrp4 is essential for presynaptic differentiation independent of MuSK activation.

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Acknowledgements

We thank L. Landmesser and K. Hata for helping us to establish a motor-neuron and muscle co-culture system, W. Zhang for constructs encoding Fc- and AP-tagged forms of Lrp4, T. Jessell and J. Dasen for HB9::GFP mice, J. Sanes for agrin-mutant mice, K. Anderson and L. Niswander for Lrp4-mutant mice, and D. Littman and R. Lehmann for comments on the manuscript. We are grateful to P. Lopez and M. J. Sunshine and personnel in the transgenic mouse core for their assistance. The flow cytometry and transgenic mouse cores are supported by a New York University Cancer Institute Center Support Grant (National Institutes of Health (NIH)/National Cancer Institute (NCI) 5 P30CA16087-31). This work was supported with funds from the National Institutes of Health (NS36193 to S.J.B.), the Skirball Institute and postdoctoral training support to N.Y. from New York State Stem Cell Science (NYSTEM).

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N.Y. designed and carried out all of the experiments in Figs 1, 2 and 3. N.K. designed and carried out the experiments in Fig. 4. S.J.B. helped to design and interpret experiments. All authors wrote and edited the manuscript.

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Correspondence to Steven J. Burden.

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The authors declare no competing financial interests.

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Yumoto, N., Kim, N. & Burden, S. Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses. Nature 489, 438–442 (2012). https://doi.org/10.1038/nature11348

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