R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5

At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.

Total RNA was extracted using Trizol reagent (Invitrogen), and was reverse-transcribed by Oligo (dT) 20 Primer (Life Technologies) using ReverTra Ace reverse-transcriptase (Toyobo). cDNA levels were analyzed by qRT-PCR in triplicate using SYBR Premix Ex Taq (Takara) on LightCycler 480 (Roche). cDNA levels were normalized to Gapdh. The primer sequences are listed in Supplementary   Table S2.

Staining of the spinal cord
The spinal cord of adult or E18.5 mice with vertebral bones were fixed with 4% parafolmaldehyde at 4°C overnight, washed with PBS several times, removed with the bone tissues, and then treated with 10%, 15%, and 30% sucrose in PBS sequentially until the tissues sink to the bottom of a vial. Frozen sections of the spinal cord were fixed with acetone for 5 min on ice, washed with PBS several times, covered with Image-iT FX Signal Enhancer (Cell Signaling, #11932) for 30 min and then covered with PBS containing 5% horse serum for 30 min. For staining, the sections were incubated with rabbit anti-Islet1/2 antibody (1:100, Santa Cruz, sc30200), rabbit polyclonal anti-R-spondin 2 antibody (1:500, Abcam, ab73761), or goat polyclonal anti-choline acetyltransferase (ChAT) antibody (1:100, Millipore, AB143) overnight at 4°C in a humidified chamber. After repeated washes with PBS, sections of the E18.5 spinal cord stained for Islet1/2 were incubated with the goat anti-rabbit Alexa 488 secondary antibody (1:100, Molecular Probes, A21206). Sections of the adult spinal cord double-stained for Rspo2 and ChAT were incubated with biotinylated donkey anti-goat secondary antibody for ChAT (1:300, Vector Laboratory, BA9500) for 30 min, and then treated with a combination of the goat anti-rabbit Alexa 488 secondary antibody for Rspo2 (1:100, Molecular Probes, A21206) and streptavidin-conjugated Alexa 594 (1:1000, Invitrogen, S11227) for detecting ChAT for 1 h. Residual antibodies were removed with repeated washes in PBS-T. Finally, the sections were coverslipped with VectaShield mounting medium containing 1.5 μg/ml 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and were visualized using an IX71 microscope (Olympus) or A1Rsi confocal microscope (Nikon). The Islet1/2-immunopositive SMNs were counted by two blinded observers and averaged.

AChR clustering assays
C2C12 myoblasts were seeded on a plate coated with collagen I (BD Biosciences) and treated with the lentivirus for 12 h. After differentiation, C2C12 myotubes were treated with doxycycline for 2 d to induce shRNA expression and then treated with Rspo2 protein, agrin protein or Rspo2-CM to induce AChR clusters for 12 h. Thirty min before fixation in 2% paraformaldehyde, the cells were incubated with 10 g/ml Alexa594-conjugated -bungarotoxin (Invitrogen) for 30 min to label AChR. Fluorescent images were observed under an Olympus XL71 fluorescence microscope and analyzed with MetaMorph software (Molecular Devices). The lengths of AChR clusters and myotubes were defined as the longest axes of Alexa594 signals and GFP signals, respectively, in the lentivirus-transfected cells. AChR clusters with an axis length of less than 4 µm were excluded from the total count of AChR clusters.

Expression vectors, luciferase reporter vectors, lentiviral vectors, and siRNAs
The full-length human LRP4 cDNA (Open Biosystems) was cloned into the EcoRI site and the full-length human and mouse LGR5 cDNA (Open Biosystems) was cloned into BamHI and NotI sites of the pcDNA3.1 mammalian expression vector (Invitrogen). The mouse Musk cDNA in pExpress-1 was purchased from Open Biosystems. ATF2-Luc to quantify the JNK signaling activity 1 and phRL-TK Renilla luciferase vector (Promega) were used for the luciferase reporter assay. The human full-length MUSK and full-length LRP4 cDNA was subcloned into EcoRI and XbaI sites or After 48 h, we confirmed that more than 90% of cells were positive for GFP signals driven by CMV in pLenti. HEK293 cells were transfected with pEGFP-N1 (Clontech) to make control-CM or Rspo2-mycAP to make Rspo2-CM. The cells were cultured in 10% FBS/DMEM for L cells or HEK293 cells, or in 2% horse serum/DMEM for C2C12 cells. The CM was harvested at 48 and 96 h after the transfection. Recombinant rat C-terminal agrin and recombinant Rspo2 were purchased from R&D systems. 

Luciferase assays
HEK293 cells were transfected with ATF2-Luc and phRL-TK along with the Musk and Lrp4 cDNAs. Cells were cultured for 24 h in the presence or absence of purified Rspo2 (100ng/ml) or/and agrin protein (10 ng/ml) in a 96-well plate. Cells were lysed with the passive lysis buffer (Promega) and assayed for the luciferase activity using the Dual luciferase system (Promega). Each experiment was done in triplicate.

Western blotting
Total or precipitated proteins were dissolved in 1x Laemmli buffer, separated on a 12.5%, 10% or 7.5% SDS-polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane after several washing with ice-cold PBS and the cell lysates were incubated with streptavidin sepharose beads (GE healthcare) to purify the biotinylated cell membrane proteins. Western blotting was performed as described above.

Protein preparation of muscle tissues in mice
The left diaphragm muscle in each mouse was dissected at E18.5 and crushed using the Multi-Beads Shocker (Yasui Kikai Corp.). The Minute Plasma Membrane isolation kit (Invent Biotechnologies) was used to isolate total and plasma membrane protein fractions from the crushed muscles. All procedures were performed on ice and followed the manufacturer's protocols. Western blotting was performed as described above.  Table 1. High magnifications of red boxes are shown in lower panels. Note bigger but fewer synaptic vesicles (red arrows) in Rspo2-/mice. SV, synaptic vesicles.