MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance

Abstract

The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain (cysteine-rich domain [CRD]). Dysfunction of MuSK CRD in patients has been recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSKΔCRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSKΔCRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.

Keywords: MuSK; Wnt; congenital myasthenic syndrome; lithium chloride; neuromuscular junction; synaptogenesis.

Figure 1.

Generation of MuSKΔCRD transgenic mice. A, Schematic representation of the KO strategy. First panel, Genomic structure of the MuSK gene encompassing MuSK CRD. Second panel, Targeting vector. Third panel, MuSK-targeted allele (MuSK^flox(CRD)). Fourth panel, Recombinant MuSK allele (MuSK^ΔCRD). Ef, Wr, and Lxr (black arrowheads), primers for PCR genotyping; Efl, Nr, Cref, and Er, primers for PCR validation of the ES clones (blue arrowheads for 5′ external and violet arrowheads for protamine Cre PCR). Pink line indicates 3′ external Southern blot probe. Green line indicates neo internal Southern blot probe. A, ApaL1; D, Drd1; X, Xcm1. B, Examples of 5′ external and protamine Cre PCR validation and internal and external Southern blot hybridization analyses of two injected ES mutant clones (lanes 1 and 2). Amplified bands of the expected size for PCR (8.2 and 6 kb) and for 5′ internal (ApaL1, targeted band, 11.1 kb), 3′ internal (Drd1, targeted band, 9.5 kb), and 3′ external (Drd1; WT band, 7.4 kb; targeted band, 9.5 kb) Southern blots were obtained. C, Genotyping by PCR using a three primer set. The 234 and 200 bp bands represent WT and MuSKΔCRD alleles, respectively. D, Western blot of MuSK or MuSKΔCRD using MuSK antibodies in E18.5 MuSK^+/ΔCRD or MuSKΔCRD hindlimb muscle after MuSK immunoprecipitation. E, Western blot of cell surface and total MuSK-HA and MuSKΔCRD-HA in HEK293T cells transfected with MuSK-HA or MuSKΔCRD-HA. Transferrin receptor (Tfr) and α-tubulin were used as a loading control for biotinylated proteins and input, respectively. Transfection of MuSK-WT-HA or MuSKΔCRD-HA was performed in duplicate. F, Confocal images of P60 WT or MuSKΔCRD TA muscle cross sections stained with MuSK antibody (red) together with α-BTX (AChR, green). G, Quantification of WT and mutated MuSK signal intensities at the synapse. H, Koelle's histochemical staining of AChE performed on isolated TA muscle fibers from P90 WT and MuSKΔCRD. I, Quantification of AChE activity extracted from P90 WT and MuSKΔCRD soleus and diaphragm. J, Examples of myotubes isolated from WT or MuSKΔCRD primary cultures, treated or not with recombinant agrin and stained with α-BTX. K, Quantitative analysis of the number of AChR clusters in WT and MuSKΔCRD myotubes. Data are mean ± SEM. ***p < 0.001. ns, Not significant. N = 3 independent experiments. Scale bar: F (merged image), J, 20 μm; H, 50 μm.

Figure 2.

Impaired muscle prepatterning in MuSKΔCRD embryos. A, B, Confocal images of whole-mount left hemidiaphragms from E14 WT and MuSKΔCRD embryos stained with NF (red) and Syn (red) antibodies (A, B) together with α-BTX (AChRs, green, B). Arrowheads indicate AChR clusters. White dashed lines indicate the synaptic endplate band and include most AChR clusters. C–H, Quantitative analysis of the mean neurite length (C), the endplate band width (D), the AChR cluster number (E), volume (F), intensity (G), and non-innervated AChR clusters (H). Number of AChR clusters analyzed: 790 in WT and 461 in MuSKΔCRD. Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test). **p < 0.01 (Mann–Whitney U test). ***p < 0.001 (Mann–Whitney U test). N = 4 embryos per genotype. Scale bar: A, 300 μm; B (merged image), 50 μm.

Figure 3.

Aberrant NMJ formation in E18.5 MuSKΔCRD embryos. A, Confocal images of whole-mount left hemidiaphragms from E18.5 WT and MuSKΔCRD embryos stained with α-BTX to visualize AChR clusters. Right panels, Enlarged images of boxed regions in left panel. White dashed lines indicate the synaptic endplate band and include most AChR clusters. Right panels, Insets, Higher-magnification views of AChR clusters. B–E, Quantifications of the endplate band width (B), the AChR cluster number (C), volume (D), and intensity (E). Numbers of AChR clusters analyzed: 806 in WT and 604 in MuSKΔCRD. F, Confocal images of whole-mount left hemidiaphragms from E18.5 WT and MuSKΔCRD embryos stained as in Figure 2B. G–J, Quantitative analysis of the length (G, H) and the number (I, J) of primary and secondary nerve branches. Number of primary branches analyzed: 273 in WT and 309 in MuSKΔCRD; secondary branches: 266 in WT and 310 in MuSKΔCRD. Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test). **p < 0.01 (Mann–Whitney U test). ***p < 0.001 (Mann–Whitney U test). ns, Not significant. N = 6 embryos per genotype. Scale bar, A, 300 μm; F (merged image), 50 μm.

Figure 4.

Diaphragm innervation defects in P5 MuSKΔCRD mice. A, Confocal images of whole-mount P5 WT and MuSKΔCRD left hemidiaphragms stained as Figure 2B. White dashed lines indicate the synaptic endplate band and include most AChR clusters. B–E, Quantitative analysis of the endplate band width (B), the AChR cluster number (C), volume (D), and intensity (E). Numbers of AChR clusters tested: 263 in WT and 120 in MuSKΔCRD. Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test). ***p < 0.001 (Mann–Whitney U test). N = 4 embryos per genotype. Scale bar, 50 μm.

Figure 5.

Immature and fragmented NMJs in MuSKΔCRD adult mice. Whole-mount isolated muscle fibers from P20 and P60 WT and MuSKΔCRD TA were stained with NF (red) and Syn (red) antibodies together with α-BTX (AChRs, green). A, Confocal images of synapses from P20 and P60 WT and MuSKΔCRD mice. For P60 NMJs, top views of the reconstructed image are represented on the right side. B–D, Quantification analysis of the AChR cluster area (B), the Syn area (C), and overlap area of presynaptic and postsynaptic elements (D) in P20 WT and MuSKΔCRD mice. E–H, Quantification analyses of the number of fragments per AChR clusters (E), the AChR cluster area (F), the Syn area (G), and the overlap ratio of presynaptic and postsynaptic elements (H) in P60 WT and MuSKΔCRD mice. Data are mean ± SEM of at least 50 NMJs. *p < 0.05 (Mann–Whitney U test). **p < 0.01; ***p < 0.001 (Mann–Whitney U test). ns, Not significant. N = 6 animals per genotype. Scale bar (merged image), 10 μm.

Figure 6.

Disorganized NMJ ultrastructures in MuSKΔCRD mice. A–G, Representative electron micrographs of P120 WT and MuSKΔCRD TA NMJs. A–C, Examples of WT NMJs. D–G, Examples of MuSKΔCRD NMJs. B, E, Higher-magnification views of A and D, respectively. H–J, Quantification analyses of the synaptic vesicle density (H), diameter (I), and the number of JFs (J) in MuSKΔCRD compared to WT mice. K, Representative electron micrograph of P120 WT and MuSKΔCRD TA structure. L, Quantification of the distance between Z-lines in MuSKΔCRD and WT mice. M, Representative electron micrograph of myelin sheath in P120 WT and MuSKΔCRD TA. Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test). N = 4 animals per genotype. ns, Not significant; N, nerve; MF, muscle fiber; SVs, synaptic vesicles; m, mitochondria; SBL, synaptic basal lamina. Black arrows indicate presynaptic membrane. Black arrowheads indicate postsynaptic membrane. White arrows indicate Z-line. Stars indicate M-line. Scale bars: A–G, 500 nm; H, J, 1 μm.

Figure 7.

MuSKΔCRD mice progressively develop muscle weakness, fatigability, and decreased muscle contraction. A, Micro-CT scans of P90 WT and MuSKΔCRD mice. B, P120 WT and MuSKΔCRD KI. C, Latency to fall quantifications during a rail-grip test at various time points (P20, P40, P60, and P90). D, E, Quantification of forelimb (D) and hindlimb (E) grip strengths in WT and MuSKΔCRD mice. F, Representative examples of twitch and tetanic contractions evoked by stimulation of the motor nerve in WT and MuSKΔCRD P120 isolated mouse hemidiaphragms. The phrenic nerve was stimulated either with single or tetanic stimuli (600 ms duration) at 20, 40, 60, 80, and 100 Hz. G, H, Peak amplitudes of nerve-evoked single twitch and tetanic stimulations in WT and MuSKΔCRD mice. I, Example of repeated tetanic nerve stimulation (60 Hz, 600 ms duration at 1 Hz) that induced a degree of fatigue more pronounced in MuSKΔCRD than in WT. J, Quantification of the fatigability in WT and MuSKΔCRD. K, Example of spontaneous twitch induced by unique stimulation observed in MuSKΔCRD muscles. Bottom line indicates the stimulator. Calibration scales in WT apply to MuSKΔCRD. L, Confocal images of whole-mount P90 WT and MuSKΔCRD left hemidiaphragms stained as in Figure 2B. White arrows indicates loss of postsynapse. White arrowheads indicates denervated postsynapses. White stars indicate fragmented NMJ. Data are mean ± SEM. *p < 0.05 (Mann–Whitney U test). **p < 0.01 (Mann–Whitney U test). N = 3 animals per genotype in A, B, L. N = 6 animals per genotype in C–E. N = 5 animals per genotype in F–K. Scale bar: L, 50 μm.

Figure 8.

LiCl treatment rescues NMJ defects in MuSKΔCRD embryos. A, B, Quantitative analysis of the number of AChR clusters in myotubes isolated from WT or MuSKΔCRD primary cultures and treated or not with Wnt11 (A) or LiCl (B). C, Examples of Wnt11-treated WT, Wntt11-treated MuSKΔCRD, and LiCl-treated MuSKΔCRD primary myotubes stained with β-catenin together with DAPI to visualize β-catenin translocation to nuclei (white arrowheads). D, Confocal images of whole-mount E18.5 WT, NaCl-treated MuSKΔCRD, and LiCl-treated MuSKΔCRD left hemidiaphragms stained as in Figure 2B. White dashed lines indicate the synaptic endplate band and include most AChR clusters. E–L, Quantification of the endplate band width (E), AChR cluster number (F), volume (G), and intensity (H). Number of AChR clusters tested: 2235 in WT, 846 in NaCl-treated MuSKΔCRD embryos, and 1714 in LiCl-treated MuSKΔCRD embryos. F–I, Quantitative analyses of the length of primary and secondary nerve branches (I, J), the number and the length of bypassing neurites (K, L). At least 300–400 primary and secondary nerve branches were analyzed per condition. Data are mean ± SEM. *p < 0.05 (two-way ANOVA or Mann–Whitney U test). **p < 0.01 (two-way ANOVA or Mann–Whitney U test). ***p < 0.001 (two-way ANOVA or Mann–Whitney U test). ns, Not significant. N = 6 embryos per genotype. Scale bar: C (merged image), 20 μm; D, 50 μm.

Figure 9.

LiCl treatment restores NMJ morphological defects and motor function in adult MuSKΔCRD mice. A, Confocal images of synapses from P40 NaCl-treated or LiCl-treated MuSKΔCRD whole-mount isolated TA muscle fibers stained with β-catenin (red) antibody together with α-BTX (AChRs, green) and DAPI (blue). Examples of intensity plot profiles measuring the fluorescence intensity of β-catenin and DAPI along the segmented lines corresponding to a subsynaptic nucleus are represented on the right side: (a), NaCl-treated MuSKΔCRD; (b), LiCl-treated MuSKΔCRD. B, Confocal images of synapses from P40 WT, NaCl-treated, or LiCl-treated MuSKΔCRD whole-mount isolated TA muscle fibers stained with NF (red) and Syn (red) antibodies together with α-BTX (AChRs, green). C–E, Quantification analyses of the AChR cluster area (C), the Syn area (D), and the number of fragments per AChR clusters (E). F, Latency to fall quantifications during a rail-grip test at various time points (P20, P40, P60, and P90). G, H, Quantification of fore (G) and hindlimb (H) grip strength in WT, NaCl-treated, or LiCl-treated MuSKΔCRD mice. Data are mean ± SEM of at least 50 NMJs. *p < 0.05 (two-way ANOVA). **p < 0.01 (two-way ANOVA). ***p < 0.001 (two-way ANOVA). ns, Not significant. N = 6 animals per genotype. Scale bar: A, B (merged image), 10 μm.