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 terminals. 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 activity. 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.

Figure 1. Lrp4-expressing non-muscle cells induce clustering of Synapsin in motor axons

a, Explants from the ventral neural tube of HB9::GFP transgenic mice, containing GFP-expressing motor neurons (MN), were co-cultured with primary muscle cells or non-muscle cells. b, Synapsin (blue) accumulates in motor axons (green) in apposition to clusters of AChRs (red) that form in muscle, marking synaptic sites. c, Synapsin is homogenously distributed in motor neurons that are co-cultured with control 3T3 cells, expressing mCherry alone, but clustered (arrows) in motor axons that contact 3T3 cells expressing Flag-Lrp4-mCherry (Lrp4-3T3). d, The number and size of Synapsin puncta are five-folder greater in axons contacting 293 cells expressing Flag-Lrp4 than control cells (m ± s.e.m., n=3). The bar = 10 μm.

Figure 2. Lrp4, attached to polystyrene beads, induces presynaptic differentiation in motor neurons

a, Lrp4-LDLa-Fc, attached to polystyrene beads, induces clustering of Synapsin (red) in motor axons (green). Many Synapsin clusters are in close apposition with Lrp4-beads (inset). b, Lrp4 specifically induces clustering of Bassoon (blue) as well as Synapsin (red). c, Lrp4-beads induce a ~12-fold increase in the number of Synapsin puncta (m ± s.e.m., n=3). d, Lrp4-beads induce synaptic puncta, marked by Synapsin and Bassoon, in agrin mutant motor neurons. The response of agrin mutant motor neurons is not significantly different (106%) from wild-type motor neurons. e,f, Depolarization stimulates uptake (Stimulate) and release (Restimulate) of FM 4-64FX in motor axons at contact sites with Lrp4-LDLa beads, visualized by bright field (BF) microscopy (m ± s.e.m., n=3). The cartoon shows the LDLa repeats and β-propeller domains in Lrp4. The bar = 50 μm in a, 10 μm in b and d and 5 μm in e.

Figure 3. Lrp4 binds to motor axons

a, AP-ecto-Lrp4 binds to motor axons. b, AP-ecto-Lrp4-LDLa binds preferentially to distal (D) rather than proximal (P) segments of motor axons. c, AP-ecto-Lrp4 binds to distal segments of agrin mutant motor axons.

Figure 4. Lrp4 is essential for presynaptic differentiation independent of MuSK activation

a–i, In agrin mutant mice, a three-fold increase in MuSK expression, conferred by the MuSK-L transgene, restores AChR clusters and presynaptic differentiation . j–o, In lrp4 mutant mice, MuSK-L restores AChR clusters but not nerve terminal differentiation; instead, motor axons continue to grow beyond the prepatterned zone and fail to contact AChR clusters. p, In agrin mutant mice carrying MuSK-L, ~90% of AChR clusters are contacted by motor axons. In lrp4 mutant mice that carry MuSK-L, ~15% of AChR clusters are contacted by motor axons; these contacts may be incidental, as motor axons grow and branch extensively throughout muscle of lrp4 mutant mice, inevitably placing axons in the vicinity of AChR clusters (m ± s.e.m., n=3).