Abstract
We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2−/−) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2−/− mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.
Highlights
The release of the neurotransmitter acetylcholine (ACh) from the axon terminal of the spinal motor neuron (SMN) activates the postsynaptic acetylcholine receptor (AChR) and elicits an action potential in the target muscle fiber[1,2,3]
To understand the origin of Rspo[2] that promotes AChR clustering at the neuromuscular junction (NMJ), transgenic mice expressing Rspo[2] in skeletal muscle or SMNs were generated according to the conventional transgenic mice strategy[32]
To confirm expression of FLAG-tagged Rspo[2] protein, frozen sections of the tibialis anterior (TA) muscle and the spinal cord from muscle creatine kinase (MCK)-RSPO2 and vesicular acetylcholine transporter (VAChT)-RSPO2 mice were stained with anti-FLAG antibody
Summary
The release of the neurotransmitter acetylcholine (ACh) from the axon terminal of the spinal motor neuron (SMN) activates the postsynaptic acetylcholine receptor (AChR) and elicits an action potential in the target muscle fiber[1,2,3]. Neuronal agrin released from the nerve terminal[7] binds to the low-density lipoprotein receptor-related protein 4 (LRP4) on the motor endplate and promotes phosphorylation of the muscle-specific kinase (MuSK) to induce AChR clustering[8,9]. Muscle-derived molecules including laminins, FGFs, collagens, BDNF, GDNF, Wnts, and TGF-β4 are implicated in orchestrating pre- and postsynaptic NMJ formation. In cultured myogenic C2C12 cells, Rspo[2] is able to promote muscle differentiation through Wnt/β-catenin signaling[30]. To dissect the differential roles of SMN-derived Rspo[2] and muscle-derived Rspo[2] on AChR clustering and NMJ formation in mouse embryos, we generated transgenic mice expressing Rspo[2] in SMNs or in skeletal muscle on the background of Rspo2−/−
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