title: R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5.
abstract: 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.
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.|Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakashima
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Bisei
authors.initials: B
authors.lastname: Ohkawara
authors.affiliation: Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Ishigaki
authors.affiliation: Department of Neurology, Nagasaki Kawatana Medical Center, Nagasaki, Japan.
authors.forename: Takayasu
authors.initials: T
authors.lastname: Fukudome
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.|Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kenyu
authors.initials: K
authors.lastname: Ito
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.|Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Mikito
authors.initials: M
authors.lastname: Tsushima
authors.affiliation: Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Konishi
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Okuno
authors.affiliation: Department of Occupational Therapy, Nagasaki University School of Health Sciences, Nagasaki, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Yoshimura
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Ito
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Sobue
authors.affiliation: Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyama
authors.affiliation: Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ishiguro
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D061346:Laser Capture Microdissection; D008297:Male; D051379:Mice; D009046:Motor Neurons / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000378:metabolism*; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013116:Spinal Cord / Q000378:metabolism; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D000098907:R-Spondins
chemical_list: D020794:Receptor Protein-Tyrosine Kinases; D011950:Receptors, Cholinergic; D043562:Receptors, G-Protein-Coupled; D019699:Thrombospondins; C493597:Lgr5 protein, mouse; C518904:RSPO2 protein, mouse; D000098907:R-Spondins; C511695:MuSK protein, mouse