METHODS FOR TREATING NEUROMUSCULAR JUNCTION-RELATED DISEASES

Abstract

The present invention relates to methods for treating neuromuscular junction-related diseases. In particular, the present invention relates to a method of treating a neuromuscular junction-related disease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one inhibitor of glycogen synthase kinase 3 (GSK3).

Claims

1. A method for treating a neuromuscular junction-related disease in a human subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of glycogen synthase kinase 3 (GSK3), wherein said neuromuscular junction-related disease is a myasthenic syndrome and the inhibitor of GSK3 is tideglusib.

2. A method for treating a neuromuscular junction-related disease in a human subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of glycogen synthase kinase 3 (GSK3), wherein said neuromuscular junction-related disease is selected from the group consisting of myasthenia gravis, Lambert-Eaton syndrome, Miller Fischer syndrome, congenital myasthenic syndrome, botulism, and organophosphate poisoning, and the inhibitor of GSK3 is tideglusib.

3. The method of claim 4, wherein the disease is myasthenia gravis.

4. The method of claim 4, wherein the disease is congenital myasthenic syndrome.

5. A method for rescuing neuromuscular junction (NMJ) defects in a human subject affected with an NMJ-related disease, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of glycogen synthase kinase 3 (GSK3), wherein said NMJ-related disease is a myasthenic syndrome and the inhibitor of GSK3 is tideglusib.

6. A method for rescuing neuromuscular junction (NMJ) defects in a human subject affected with an NMJ-related disease, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of glycogen synthase kinase 3 (GSK3), wherein said neuromuscular junction-related disease is selected from the group consisting of myasthenia gravis, Lambert-Eaton syndrome, Miller Fischer syndrome, congenital myasthenic syndrome, botulism, and organophosphate poisoning, and the inhibitor of GSK3 is tideglusib.

7. The method of claim 8, wherein the disease is myasthenia gravis.

8. The method of claim 8, wherein the disease is congenital myasthenic syndrome.

Description

FIGURES

[0038] FIGS. 1A-1K. Generation of MuSKΔCRD transgenic mice.

[0039] (1A) Schematic representation of the wild type (WT), MuSK.sup.flox(CRD), and recombined MuSKΔCRD alleles. Arrowheads, primers for genotyping. (1B) Examples of Neo Southern blot hybridization analysis of the injected ES mutant clones. (1C) Genotyping by PCR. The 234 and 200 bp bands represent WT and MuSKΔCRD alleles, respectively. (1D) Western blot of MuSK or MuSKΔCRD using MuSK antibodies in HEK293T cells transfected with MuSK-HA or MuSKΔCRD-HA or in MuSKΔCRD primary myotubes after MuSK immunoprecipitation. (1E) 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 MuSKACRD-HA was performed in duplicate. (1F) Confocal images of P60 WT or MuSKΔCRD TA muscle cross sections stained with MuSK antibody (red) together with α-BTX (AChR, green). (1G) Quantification of WT and mutated MuSK signal intensities at the synapse. (1H) Koelle's histochemical staining of acetylcholinesterase (AChE) performed on isolated TA muscle fibers from P90 WT and MuSKΔCRD. (1I) Quantification of AChE activity extracted from P90 WT and MuSKΔCRD soleus and diaphragm. (1J) Examples of myotubes isolated from WT or MuSKΔCRD primary cultures, treated or not with recombinant agrin and stained with α-BTX. (1K) Quantitative analysis of the number of AChR clusters in WT and MuSKΔCRD myotubes. Data are shown as mean±SEM. ***p<0.001. ns, non significant. N=3 independent experiments. Scale bar in the merged image in 1E, 20 μm; in 1G, 50 μm.

[0040] FIGS. 2A-2H. Impaired muscle prepatterning in MuSKΔCRD embryos.

[0041] (2A-2B) Confocal images of whole mount left hemidiaphragms from E14 WT and MuSKΔCRD embryos stained with neurofilament (NF, red) and synaptophysin (Syn, red) antibodies (2A-2B) together with α-BTX (AChRs, green, 2B). Arrowheads, AChR clusters. White dashed lines delineate the synaptic endplate band and include most AChR clusters. (2C-2H) Quantitative analysis of the mean neurite length (2C), the endplate band width (2D), the AChR clusters number (2E), volume (2F), intensity (2G) and non innervated AChR clusters (2H). Number of AChR clusters analyzed: 790 in WT and 461 in MuSKΔCRD. Data are shown as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001, N=4 embryos per genotype, Mann-Whitney U test. Scale bar in the merged image in 2A, 300 μm; in 2B, 50 μm.

[0042] FIGS. 3A-3J. Aberrant NMJ formation in E18.5 MuSKΔCRD embryos.

[0043] (3A) Confocal images of whole mount left hemidiaphragms from E18.5 WT and MuSKΔCRD embryos stained with α-BTX to visualize AChR clusters. Right panels show enlarged images of boxed regions in left panel. White dashed lines delineate the synaptic endplate band and include most AChR clusters. Insets in right panels are higher magnification views of AChR clusters. (3B-3E) Quantifications of the endplate band width (3B), the AChR clusters number (3C), volume (3D) and intensity (3E). Numbers of AChR clusters analyzed: 806 in WT and 604 in MuSKΔCRD. (3F) Confocal images of whole mount left hemidiaphragms from E18.5 WT and MuSKΔCRD embryos stained as in FIG. 2B. (3G-3J) Quantitative analysis of the length (3G-3H) and the number (3I-3J) 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 shown as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001, ns: non significant. N=6 embryos per genotype, Mann-Whitney U test. Scale bar in the merged image in 3A, 300 μm; in 3F, 50 μm.

[0044] FIGS. 4A-4E. Diaphragm innervation defects in P5 MuSKΔCRD mice.

[0045] (4A) Confocal images of whole mount P5 WT and MuSKΔCRD left hemidiaphragms stained as FIG. 2B. White dashed lines delineate the synaptic endplate band and include most AChR clusters. (4B-4E) Quantitative analysis of the endplate band width (4B), the AChR clusters number (4C), volume (4D) and intensity (4E). Numbers of AChR clusters tested: 263 in WT and 120 in MuSKΔCRD. Data are shown as mean±SEM. *p<0.05 and ***p<0.001. N=4 embryos per genotype, Mann-Whitney U test. Scale bar, 50 μm.

[0046] FIGS. 5A-5H. Immature and fragmented NMJs in MuSKΔCRD adult mice.

[0047] Whole mount isolated muscle fibers from P20 and P60 WT and MuSKΔCRD TA were stained with neurofilament (NF, red) and synaptophysin (Syn, red) antibodies together with α-BTX (AChRs, green). (5A) 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. (5B-5D) Quantification analysis of the AChR cluster area (5B), the synaptophysin area (5C) and overlap area of pre and postsynaptic elements (5D) in P20 WT and MuSKΔCRD mice. (5E-5H) Quantification analyses of the number of fragments per AChR clusters (5E), the AChR cluster area (5F), the synaptophysin area (5G) and the overlap ratio of pre- and postsynaptic elements (5H) in P60 WT and MuSKΔCRD mice. Data are shown as mean±SEM of at least 50 NMJs. *p<0.05, ***p<0.001, ns: non significant. N=6 animals per genotype, Mann-Whitney U test. Scale bar in the merged image, 10 μm.

[0048] FIGS. 6A-6M. Disorganised NMJ ultrastructures in MuSKΔCRD mice.

[0049] (6A-6G) Representative electron micrographs (EM) of P120 WT and MuSKΔCRD TA NMJs. 6A-6C, examples of WT NMJs. 6D-6G, examples of MuSKΔCRD NMJs. 6B and 6E are higher magnification views of 6A and 6D respectively. (6H-6J) Quantification analyses of the synaptic vesicle density (6H), diameter (6I) and the number of JFs (6J) in MuSKΔCRD compared to WT mice. (6K) Representative EM of P120 WT and MuSKΔCRD TA structure. (6L) Quantification of the distance between Z-lines in MuSKΔCRD and WT mice. (6M) Representative EM of myelin sheath in P120 WT and MuSKΔCRD TA. Data are shown as mean±SEM. ns: non significant. N=4 animals per genotype, Mann-Whitney U test. N, nerve; MF, muscle fiber; SVs, synaptic vesicles; JFs, junctional folds; m, mitochondria; SBL, synaptic basal lamina; arrow, presynaptic membrane; arrowhead, postsynaptic membrane; white arrow, Z-line; star, M-line. Scale bar in 6A-6G, 500 nm; in 6H and 6J, 1 μm.

[0050] FIGS. 7A-7L. MuSKΔCRD mice progressively develop muscle weakness, fatigability and decreased muscle contraction.

[0051] (7A) Micro-Computed tomography scans of P90 WT and MuSKΔCRD mice. (7B) P120 WT and MuSKΔCRD kyphotic index. (7C) Latency to fall quantifications during a rail-grip test at various time-points (P20, P40, P60 and P90). (7D-7E) Quantification of fore limb (7D) and hind limb (7E) grip strengths in WT and MuSKΔCRD mice. (7F) 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. (7G-7H) Peak amplitudes of nerve-evoked single twitch and tetanic stimulations in WT and MuSKΔCRD mice. mN: millinewton; g: gram. (7I) 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. (7J) Quantification of the fatigability in WT and MuSKΔCRD. (7K) Example of spontaneous twitch induced by unique stimulation observed in MuSKΔCRD muscles. The bottom line corresponds to the stimulator. Calibration scales in WT apply to MuSKΔCRD. (7L) Confocal images of whole mount P90 WT and MuSKΔCRD left hemidiaphragms stained as in FIG. 2B. Arrow, loss of postsynapse. Arrowhead, denervated postsynapse. Star, fragmented NMJ. Data are shown as mean±SEM. *p<0.05. N=3 animals per genotype in 7A-7B and 7L, N=6 animals per genotype in 7C-7E, N=5 animals per genotype in 7F-7K. Mann-Whitney U test. Scale bar in 7L, 50 μm.

[0052] FIGS. 8A-8L. LiCl treatment rescues NMJ defects in MuSKΔCRD embryos.

[0053] (8A-8B) Quantitative analysis of the number of AChR clusters in myotubes isolated from WT or MuSKΔCRD primary cultures and treated or not with Wnt11 (8A) or LiCl (8B). (8C) Examples of Wnt11-treated WT, Wnt11-treated MuSKΔCRD and LiCl-treated MuSKΔCRD primary myotubes stained with β□catenin together with Dapi to visualize catenin translocation to nuclei (arrowheads). (8D) Confocal images of whole mount E18.5 WT, NaCl-treated MuSKΔCRD and LiCl-treated MuSKΔCRD left hemidiaphragms stained as in FIG. 2B. White dashed lines delineate the synaptic endplate band and include most AChR clusters. (8E-8L) Quantification of the endplate band width (8E), AChR clusters number (8F), volume (8G) and intensity (8H). Number of AChR clusters tested: 2235 in WT, 846 in NaCl-treated MuSKΔCRD embryos and 1714 in LiCl-treated MuSKΔCRD embryos. (8F-8I) Quantitative analyses of the length of primary and secondary nerve branches (8I-8J), the number and the length of bypassing neurites (8K-8L). At least 300-400 primary and secondary nerve branches were analyzed per condition. Data are shown as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001. ns: non significant. N=6 embryos per genotype, two-way ANOVA or Mann-Whitney U test. Scale bar in the merged image in 8C, 20 μm; in 8D, 50 μm.

[0054] FIGS. 9A-9H. LiCl treatment restores NMJ morphological defects and motor function in adult MuSKΔCRD mice.

[0055] (9A) 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). (9B) Confocal images of synapses from P40 WT, NaCl-treated or LiCl-treated MuSKΔCRD whole mount isolated TA muscle fibers stained with neurofilament (NF, red) and synaptophysin (Syn, red) antibodies together with α-BTX (AChRs, green). (9C-9E) Quantification analyses of the AChR cluster area (9C), the synaptophysin area (9D) and the number of fragments per AChR clusters (9E). (9F) Latency to fall quantifications during a rail-grip test at various time-points (P20, P40, P60 and P90). (9G and 9H) Quantification of fore (9G) and hind limb (9H) grip strength in WT, NaCl-treated or LiCl-treated MuSKΔCRD mice. Data are shown as mean±SEM of at least 50 NMJs. *p<0.05, **p<0.01, and ***p<0.001; ns: non significant. N=6 animals per genotype, two-way ANOVA. Scale bar in the merged image in 9A and 9B, 10 μm.

EXAMPLE

[0056] Material & Methods

[0057] Animals

[0058] All experiments on mice were performed in accordance with European Community guidelines legislation and reviewed by the local ethical committee of the Paris Descartes University (No CEEA34.LS.030.12). The investigators had valid licenses (No A-75-1970) to perform experiments on live vertebrates delivered by the Direction des Services Veterinaires (Prefecture de Police, Paris, France). The animal house and the experimental room of Paris Descartes University had received the agreement of the same authority (No B75-06-07). Experimental procedures were performed on C57BL/6 male mice and mutant mice were always compared to Wt littermates.

[0059] Generation of MuSKΔCRD mutant mice and genotyping.

[0060] The MuSK.sup.ΔCRD/ΔCRD mutant mouse line, lacking MuSK 315-478 amino acids corresponding to the CRD was established at the Mouse Clinical Institute (MCI/ICS) using proprietary vector containing floxed Neomycin resistance cassette and Protamine-Cre cassette (Illkirch, France; http://www-mci.u-strasbg.fr). The use of protamine cassette in the construction vector offers an efficient solution for auto-excision of the floxed region when chimaera mice were bred with Cre-expressing mice. The targeting vector was constructed by successive cloning of PCR products and contained a 5.5 kb fragment (corresponding to the 5′ homology arm), a 4 kb floxed fragment including Protamine Cre and Neomycin selection cassettes, and a 5 kb fragment (corresponding to the 3′ homology arms). Two LoxP sequences delimitating the floxed fragment were located upstream of exon 9 and downstream of exon 11. The linearized construct was electroporated in Balb/CN mouse embryonic stem (ES) cells. Targeted ES clones were screened by 5′ external and Protamine Cre (inside the targeting vector) LongRange PCR and by Neo Southern blot. Two positive ES clones were injected into C57BL/6N blastocysts, and derived male chimaeras gave germline transmission. The resulting lines were crossed with a Cre deleter mouse generated on a pure inbred C57BL/6N background, in which the CRE gene is driven by the chicken (3 actin promoter, and showing high and stable recombination efficiency to induce deletion of the floxed region (Birling et al., 2012). Forward (Ef) and reverse (Wr and Lxr) primers were used for genotyping for the WT allele and for the MuSKΔCRD allele. The WT amplified sequence was 234 bp-long while the knock-out amplification was 200 bp-long.

[0061] Antibodies

[0062] The following antibodies were used: polyclonal and monoclonal Alexa Fluor® 488 conjugated (Life Technologies, 1/1000), polyclonal Cy™ 3-conjugated (Jackson immunoresearch, 1/1000), monoclonal and polyclonal Peroxidase conjugated (Amersham, 1/10000), rabbit monoclonal anti-synaptophysin (Life Technologies, 1/5), polyclonal anti-neurofilament 68 kDa (Chemicon, 1/1000), polyclonal anti-neurofilament 165 kDa (DSHB, Iowa, USA, 1/750), polyclonal anti-HA (1/2500; Abcam), monoclonal anti-beta catenin (Life Technologies, 1/500). Polyclonal anti-MuSK (Abcam, 1/200), monoclonal anti-transferrin receptor (TfR, Invitrogen, 1/500) and anti-α-tubulin (Sigma-Aldrich, 1/6000) were used for western blot. Polyclonal anti-MuSK (1/500) used for immunohistochemistry is a gift from M. Ruegg (Germany) Dapi (1/20000) and α-bungarotoxin (α-BTX) Alexa Fluor® 488 conjugate (1/1000) were purchased from Euromedex and Life Technologies, respectively.

[0063] Plasmids

[0064] The rat MuSK-HA and MuSKΔCRD cDNA plasmids have been previously described (Cartaud et al., 2004; Strochlic et al., 2012).

[0065] Biotinylation of Cell Surface MuSK and MuSKΔCRD and Western Blot Analyses

[0066] HEK293T cells (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine and 2% penicillin/streptomycin (500 U) at 37° C. in 5% CO.sub.2. Cells were grown to 70% confluence and transfected (2 to 7 μg of plasmids) using Fugen (Promega) transfection technique. 48 h after transfection, cells were washed with cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 and incubated with 0.5 mg/ml EZ-link NHS-SS-biotin (Thermo Scientific Pierce) in the same buffer at 4° C. for 30 min. The labeling reaction was quenched by incubation with 50 mM glycine and 0.5% BSA for 5 mM. Cells were then rinsed and harvested in lysis buffer (Tris 50 mM, NaCl 150 mM, EDTA 3 mM, Triton X100 1%) containing a cocktail of protease inhibitors (Roche). Lysates were centrifuged at 20000 g for 15 mM and supernatants were pre-cleared with Sepharose beads. Biotin-labeled proteins were recovered by incubation with BSA-treated streptavidin-agarose beads for 3 h at 4° C. (Thermo Scientific Pierce). Bound proteins were resolved by a 7% NuPAGE Novex Tris-acetate gel and detected by Western blot using HA antibodies. The membrane transferrin receptor (TfR) was used as a loading control to normalize the results. Relative signal intensity of total and cell surface MuSK or MuSK□CRD proteins was measured using ImageJ software. The levels of MuSK and MuSKΔCRD in total extracts were normalized to tubulin-alpha signals.

[0067] Muscle Primary Cultures.

[0068] Muscle cells were isolated from P7-P10 hind limb Tibialis Anterior (TA) and Gastrocnemius muscles from MuSKΔCRD or WT mice. Briefly, muscle tissues were excised, separated from connective tissue, minced in dissecting medium (DMEM-F12 medium containing 2 mM glutamine, 2% penicillin/streptomycin (500 U), 2% Fungizone) and dissociated in dissecting medium containing 0.2% type I collagenase (Gibco) for 90 min at 37° C. in water bath. Cells were centrifugated, filtered and resuspended in proliferating medium (dissecting medium supplemented with 20% horse serum and 2% Ultroser G, Pall). After overnight pre-plating, cells were expanded in Matrigel (Corning) coated dishes for 3 to 5 days and differentiated in differentiating medium (dissecting medium supplemented with 2% horse serum) for 5 days. When indicated, myotubes were treated with recombinant Agrin (0.4 μg/ml, R&D system), Wnt11 (10 ng/ml, R&D system) or LiCl (2.5 mM, Sigma) for 16 h.

[0069] Computed Tomography Scan Analysis and Measurement of the Kyphotic Index.

[0070] Micro Computed Tomography (micro-CT) scan analysis was performed in collaboration with the imaging platform PIPA installed in the imaging laboratory of EA 2496 (Montrouge). P90 WT and MuSKΔCRD mice were sedated using 1.5% isoflurane in air (TEC 3 Anestéo France). Entire body in dorsal and ventral decubitus of each animal was scanned by Quantum FX Perkin Elmer micro-CT device (Caliper Rikagu) in dynamic mode. A tube voltage of 90 kV and a tube current of 160 μA were selected. Total scan time was 2×17 seconds per total animal scan. The scan field of view was 2×60 mm with a spatial resolution of 118 micros (voxel size). Each scan was achieved by optimizing the resolution/dose ratio. Therefore, for the resolution selected, each animal was exposed to a low dose of 26 mGy. Image reconstructions and measurements were performed by the Osirix software v.5.6. The kyphotic index (KI) was determined from the direct multiplanar reconstructions as previously described (Laws and Hoey, 2004). Briefly, the distance (D1) from the seventh cervical vertebra (C7) to the sixth lumbar vertebra (L6) and then the perpendicular distance (D2) from D1 to the point of maximum vertebra curvature were measured. KI corresponds to the ratio D1/D2. KI is inversely proportional to kyphosis.

[0071] Grip Strength Measurement

[0072] Animals were allowed to grasp a metal rail suspended in midair and their latency to release the rail was recorded. Each mouse (N=6 for each genotype) was subjected to five trials with at least 10 min rest period between tests.

[0073] The grip strength was measured using a grip force tensiometer (Bioseb) according to the TREAT-NMD guidelines. Forelimbs and hindlimbs traction strength were recorded according to the manufacter's instructions. Three measurements were performed per animal.

[0074] Ex Vivo Isometric Tension Analyses

[0075] P120 Wt and MuSKΔCRD mice were euthanized by dislocation of the cervical vertebrae followed by immediate exsanguination. Left hemidiaphragm muscles with their respective associated phrenic nerves, were mounted in a silicone-lined bath filled with Krebs—Ringer solution of the following composition: 154 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose and 5 mM HEPES (buffered at pH 7.4 with NaOH), continuously perfused with 02 at 23.1+/−0.4° C. One of the hemidiaphragm tendons (at the rib side) was securely anchored onto the silicone-coated bath via stainless steel pins, while the other tendon was tied with silk thread, via an adjustable stainless steel hook, to an FT03 isometric force transducer (Grass Instruments, West Warwick, R.I., USA). Muscle twitches and tetanic contractions were evoked by stimulating the motor nerve via a suction microelectrode adapted to the diameter of the nerve, with supramaximal current pulses of 0.15 ms duration, at frequencies indicated in the text. For each preparation investigated, the resting tension was adjusted at the beginning of the experiment (to obtain maximal contractile responses), and was monitored during the whole duration of the experiment. Signals from the isometric transducer were amplified, collected and digitized with the aid of a computer equipped with an analogue to digital interface board (Digidata 1200, Axon Instruments, Union City, Calif., USA) using Axoscope 9 software (Axon Instruments).

[0076] Electron Microscopy

[0077] P120 Wt and MuSKΔCRD mice were sedated using 1.5% isoflurane in air (Minerve Equipement vétérinaire). Tibialis anterior were then dissected and immediately fixed in 2% glutaraldehyde and 2% paraformaldehyde in PBS for 1 h at room temperature and overnight at 4° C. TA muscle were rinsed in PBS and AChE staining following Koelle's protocol was performed. The endplate-containing tissue blocks were cut in small pieces. Subsequently, tissue samples were washed three times in 0.1M sodium phosphate buffer, incubated 30 min at 4° C. in 1% osmic acid in sodium phosphate buffer and dehydrated in graded ethanol solutions (2×10 min in 70%, 2×10 mM in 90% and 2×10 mM in 100% ethanol). Samples were then incubated twice in propylene for 1 mM, 10 mM in 50% 50% Epon-50% propylene oxide, then embedded in Epon and incubated for polymerisation 24 h at 60° C. Finally, 90 nm sections were cut on a Reichert Ultracut S and layed on a grid for staining 10 mM with 2% uranyl acetate and 4 min with lead citrate at RT. The observations were performed with a JEOL 1011 transmission electron microscope and the images recorded at 80 kv with a GATAN Erlangshen 1000 camera.

[0078] Whole Mount Staining of Diaphragms

[0079] Diaphragm muscles were dissected and fixed (4% paraformaldehyde in PBS) for 1 h at room temperature and further fixed (1% formaldehyde in PBS) overnight at 4° C. Muscles were washed three times for 15 minutes in PBS, incubated for 15 minutes with 100 mM glycine in PBS and rinsed in PBS. Muscles were permeabilized (0.5% Triton X-100 in PBS) for 1 h and blocked for 4 hours in blocking buffer (3% BSA, 5% goat serum and 0.5% Triton X-100 in PBS). Muscles were incubated overnight at 4° C. with rabbit polyclonal antibodies against neurofilament and synaptophysin in blocking solution. After three 1-hour washes in PBS, muscles were incubated overnight at 4° C. with Alexa-488 goat anti-rabbit IgG and Alexa-594-conjugated-α-bungarotoxin (α-BGT) in blocking solution. After three 1-hour washes in PBS, muscles were flat-mounted in Vectashield (Vector Labs) mounting medium.

[0080] Immunohistochemistry (Tissue Sections and Isolated Muscle Fibers)

[0081] For tissue sections analyses, dissected Soleus and Tibialis Anterior muscles from P90 adult mice were fixed (1% paraformaldehyde in PBS) for 1 hour at 4° C., rinsed twice at 4° C. in PBS, cryoprotected (30% sucrose in PBS) overnight at 4° C., embedded in TissueTek (Sakura) and quickly froze in isopentane cooled in liquid nitrogen. Cryostat cross sections (12 μm) were permeabilized with 0.5% Triton X-100 in PBS for 10 min and labeled with various antibodies as for whole mount immunostaining. Incubation times were the following: 1 hour for blocking, overnight at 4° C. for incubation with primary antibodies and 1 hour for incubation with secondary antibodies.

[0082] For isolated muscle fibers analyses, dissected and isolated Soleus, Extensor Digitorum Longus (EDL) and Tibialis Anterior muscles fibers from P20 and P60 adult mice were fixed (4% paraformaldehyde in PBS) for 30 mM at 4° C. and rinsed with PBS at RT. Isolated muscle fibers were labeled with antibodies as for whole-mount immunostaining Incubation times were the following: 3 hours for blocking, overnight at 4° C. for incubation with primary or secondary antibodies.

[0083] Images Acquisition and Processing

[0084] All images were collected on a microscope (model BX61; Olympus) equipped with a Fast 1394 Digital CCD FireWire camera (model Retiga 2000R; Qimaging) and a 20× objective or on a confocal laser scanning microscope (Zeiss LSM-710) equipped with a 20× objective and 63× oil objective. Collected Z-stacks confocal images (5 to 20 stacks with 1 to 1.5 μm (20×) z-steps) and image capture were made using LSM Image Browser. The same laser power and parameter setting were applied to ensure fair comparison between WT and MuSKΔCRD muscles. Multiple Tile Scanned images were taken to cover the size of the whole diaphragm. Confocal images presented are single-projected image derived from overlaying each set of stacks. For quantification of the AChR clusters number, volume and intensity, image stacks were quantified using the ImageJ (version 1.46m) plugin “3D object counter” (Bolte and Cordeliéres, 2006). The threshold intensity was set by visual inspection of AChR clusters, being the same between WT and MuSKΔCRD images. The endplate band width was defined by the distance between the two farthest AChR clusters from the main nerve trunk. Around 100 measurements regularly spaced and covering the entire diaphragm were taken. At least 4 diaphragms or 50 isolated muscle fibers of each genotype were analyzed and quantified. To evaluate β-catenin translocation to subsynaptic nuclei in isolated muscle fibers, image stacks corresponding to nuclei were used for quantification using the ImageJ intensity plot profile measuring the intensity of β-catenin and Dapi fluorescence within the segmented line.

[0085] Neonatal and Pregnant Mice Intra-Peritoneal Injections.

[0086] E12 pregnant mice or P10 adult mice were intraperitoneally injected with LiCl (Sigma, 600 mM-10 μl/g body weight) or placebo NaCl solution (0,0009%-10 μl/g body weight). Daily injections were made from E12 to E18 or from P10 to P60. After embryos genotyping, E18.5 or P60 diaphragms of LiCl-treated MuSKΔCRD mice were analyzed and compared to WT or NaCl-treated MuSKΔCRD.

[0087] Statistical Analysis

[0088] All data were expressed as means±SEM. Statistical analyses and graphs were performed with Prism 6.0 (Graphpad) software. All data were analyzed using the Mann-Whitney U-test or two-way ANOVA, wherever appropriate (P<0.05 considered significant). Each experiment was conducted a minimum of three times.

[0089] Results

[0090] Generation of MuSKΔCRD Transgenic Mice.

[0091] The inventors generated a mouse line deleted from the MuSK CRD by homologous recombination (FIG. 1A). To construct the MuSK-CRD floxed allele, we created a targeting vector consisting of the MuSK gene flanked by two loxP sites upstream of exon 9 and downstream of exon 11. The construct was electroporated in Balb/CN mouse embryonic stem (ES) cells and the injected clones were controlled by Neo Southern blot performed on two distinct digests using a Neo probe to verify the 5′ arm integration by homologous recombination (Apa L1 and Eco R1) and the 3′ arm integration (Drd 1 and Xcm 1, FIG. 1B). For all digests, a single band of the expected size was obtained indicating correct integration and absence of second random integration. Two positive and independent ES cell clones were injected into C57BL/6N blastocysts to generate two independent MuSK.sup.flox(CRD) mice. These mice were then crossed with Cre deletes mice in order to generate offspring with MuSK CRD deletion (see Material and Methods, FIG. 1C). Moreover, a single band corresponding to MuSK deleted from its CRD was detected in MuSKΔCRD primary myotubes after MuSK immunoprecipitation confirming that the CRD deletion occurred in the mutant mice (FIG. 1D). Both heterozygous MuSK.sup.+/ΔCRD and homozygous MuSK.sup.ΔCRD/ΔCRD (MuSKΔCRD) mutant mice were indistinguishable from wild-type (WT) mice. Although 5% of the MuSKΔCRD mice died few days after birth being smaller in weight and size, exhibiting respiratory failure as well as limb motor deficiency, most of them were viable, able to suck milk and developed up to adulthood with normal fertility. In addition, no difference in the weight curve between MuSKΔCRD and WT mice could be detected.

[0092] Given that deletion of MuSK CRD could lead to a deficit of the mutated MuSK expression at the cell membrane, we quantified the level of membrane WT or mutated MuSK using in vitro biotinylation experiments and tissue immunohistochemistry. Labeling of surface proteins by biotinylation in HEK293T cells expressing MuSK-HA or MuSKΔCRD-HA showed similar levels of MuSK and MuSKΔCRD at the plasma membrane (FIG. 1E). Moreover, both WT and mutated MuSK colocalized with membrane AChRs clusters labeled with α-bungarotoxin (BTX) in P60 WT and MuSKΔCRD tibialis anterior (TA), indicating that deletion of MuSK CRD does not disrupt the mutated MuSK membrane expression at the NMJ (FIG. 1F). Quantification of the level of WT and mutated MuSK signal intensity at the synapse, showed an increase of 70% in MuSKΔCRD compared to WT mice (FIG. 1G). In addition, since MuSK localization at the synapse is required for anchoring acetylcholinesterase (AChE) in the postsynaptic membrane (Cartaud et al., 2004), we asked whether AChE localization and activity was disturbed in MuSKΔCRD muscles. Histochemical staining revealed that AChE was accumulated in the postsynaptic membrane of both WT and MuSKΔCRD P90 TA muscles (FIG. 1H). No difference in AChE activity could be detected between WT and MuSKΔCRD muscles (FIG. 1I).

[0093] To test whether deletion of MuSK CRD could disturb agrin-Lrp4-MuSK signaling, we quantified agrin-induced AChR clusters in WT and MuSKΔCRD primary muscle cultures. No difference in the number of AChR clusters following agrin treatment was detected in MuSKΔCRD compared to WT primary myotubes, indicating that MuSK CRD deletion does not perturb agrin-induced AChR clustering. These data suggest that agrin is able to interact with Lrp4 in the Lrp4/MuSKΔCRD complex and that MuSKΔCRD/Lrp4 interaction can transduce downstream signaling (FIGS. 1J and 1K).

[0094] Deletion of MuSK CRD Affects NMJ Formation.

[0095] Given that Wnts are known to play a role during the early steps of NMJ formation through binding to MuSK CRD (Jing et al., 2009; Strochlic et al., 2012), we tested whether deletion of MuSK CRD could lead to NMJ defects during development. First, we compared NMJ phenotype in E14 WT and MuSKΔCRD embryos (FIG. 2). Whole-mount diaphragms were stained with α-BTX to detect AChR clusters and with a mixture of antibodies against neurofilament (NF) and synaptophysin (Syn) to label axonal branches and nerve terminals respectively. Both dorsal and ventral portions of each hemidiaphragm were innervated indicating that axonal extension is fully developed in MuSKΔCRD embryos (FIG. 2A). Nonetheless, the neurites were increased in length by 82% (FIGS. 2B and 2C). In addition to this presynaptic defect, AChR clustering was also affected in MuSKΔCRD embryos. In WT embryos, AChR clusters were concentrated in the central zone of the muscle as expected during prepatterning. In contrast, in mutant embryos, AChR clusters were almost undetectable and were distributed in a 2-fold wider muscle area (FIGS. 2B and 2D, arrowheads). Quantitative analysis revealed a mean 64% decrease in the number of MuSKΔCRD AChR clusters (FIG. 2E). Moreover, MuSKΔCRD AChR clusters volume and intensity were respectively reduced by 70% and 65% and non innervated AChR clusters were increased by 30% in MuSKΔCRD compared to WT embryos (FIGS. 2F-2H). These results suggest that deletion of MuSK CRD leads to an early developmental defect of the NMJ with a severely reduced AChR prepatterning.

[0096] We further analyzed NMJ morphology later during development in E18.5 MuSKΔCRD diaphragms (FIG. 3). Whereas AChR clusters were tightly restricted to a thin endplate band in WT hemidiaphragms, the endplate band width was 2-fold larger in MuSKΔCRD (FIGS. 3A and 3B). Furthermore, AChR clusters were reduced in number by 40% (FIG. 3C), volume by 25% (FIG. 3D), and intensity by 14% (FIG. 3E). All AChRs clusters were innervated in WT and MuSKΔCRD, however, MuSKΔCRD embryos showed aberrant extension of motor axons, bypassing AChR clusters and growing excessively toward the periphery of the muscle (FIG. 3F). Although the number of primary and secondary branches was not significantly affected, the length of primary as well as secondary branches was increased by 75% and 46% respectively in MuSKΔCRD mutants compared to WT embryos (FIGS. 3G-J).

[0097] Taken together, these results indicate that MuSK CRD deletion drastically perturbs NMJ formation as exemplified by reduced prepatterned and neural AChR clusters as well as exuberant neurite outgrowth.

[0098] MuSKΔCRD Adult Mice Exhibit Immature and Fragmented NMJs.

[0099] As previously mentioned, MuSKΔCRD mutant mice are viable at birth. Therefore, we wondered whether NMJ defects observed during development would be also detected in newborn and adult mutant mice. NMJ phenotype analyzed in P5 WT and MuSKΔCRD whole-mount preparations of diaphragm revealed AChR clusters deficit and neurite outgrowth defects in MuSKΔCRD similar to those observed in MuSKΔCRD embryos (FIG. 4A-E). To assess whether the pre- and postsynaptic counterparts were still affected in MuSKΔCRD adult mice, we analyzed NMJ morphology on isolated muscle fibers from WT and MuSKΔCRD TA muscles at P20 and P60 (FIG. 5). At birth, the shape of the endplates is ovoid and as NMJs mature, the endplates begin to acquire their branched postnatal topology (Kummer et al., 2006; Marques et al., 2000). In P20 MuSKΔCRD mice, most of the endplates analyzed were ovoid and compact compared to the perforated WT ones suggesting that NMJs are immature (FIG. 5A, left panel). Both analyzed AChR endplates of WT and MuSKΔCRD mice were innervated as confirmed by nerve terminals staining which colocalized with AChR clusters (FIG. 5A, left panel). However, quantitative analysis revealed that the number of AChR clusters as well as nerve terminal area per NMJ were significantly reduced by 49% and 58% respectively in MuSKΔCRD mice compared to WT mice without affecting the final overlap area between pre and postsynaptic elements (FIG. 5B-D).

[0100] At P60, WT NMJs formed a continuous branched postnatal topology and exhibited a typical “pretzel-like” structure (FIG. 5A, right panel). At this stage, 10% of the analyzed NMJs in MuSKΔCRD were similar in shape to WT ones. However, the structure of most MuSKΔCRD synapses (90% of the analyzed NMJs) was severely altered with the following characteristics: the postsynaptic network was discontinuous and isolated AChR clusters were frequently observed, suggesting fragmentation of the NMJs. Indeed, the number of AChRs fragments per NMJ was increased by 4-fold (FIG. 5E). Moreover, the total occupied AChR clusters area per NMJ was significantly reduced by 40%, suggesting a loss of AChR-rich domains (FIG. 5F). Axonal branches appeared discontinuous and fragmented in correlation with the postsynaptic apparatus parceling. The nerve terminal area was reduced by 39% without affecting the overlap area between pre and postsynaptic elements (FIGS. 5G and 5H). Similar NMJ defects were observed in other muscles types including fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus.

[0101] Taken together, our findings indicate that the CRD deletion of MuSK perturbs NMJs maturation that finally leads to a severe dismantlement of the post- and presynaptic apparatus in adult mice.

[0102] MuSKΔCRD mice display altered NMJ ultrastructures.

[0103] To analyze the morphological alterations of muscle and NMJs at the ultrastructural level in MuSKΔCRD mice, we performed electron microscopic analysis on TA muscle of P120 WT and MuSKΔCRD mice (FIG. 6A-M). Although presynaptic specialization of nerve terminals appeared normal in MuSKΔCRD mice with abundant mitochondria in the axoplasm, quantification analyses revealed a 67% decrease in MuSKΔCRD synaptic vesicle density compared to WT mice without affecting the mean synaptic vesicle diameter (FIGS. 6H and 6I). Moreover, on the postsynaptic side, about 30% of MuSKΔCRD NMJs exhibited highly disorganized or very few junctional folds (JFs) compared to WT mice (78% decrease of JFs in MuSKΔCRD, FIGS. 6A-F and 6J). In some case, compared to WT, the distance between JFs and muscle fibers was increased and abundant accumulation of mitochondria beneath the postsynaptic membrane was observed in MuSKΔCRD mice (FIG. 6G). However, no morphological difference between WT and MuSKΔCRD overall muscle structure could be detected (FIG. 6K). Indeed, the sarcomeric organization and the distance between Z-lines were similar between WT and MuSKΔCRD mice (FIG. 6L). In addition, MuSKΔCRD mice did not show any alteration in the myelin sheath diameter (FIG. 6M).

[0104] Collectively, these results indicate that MuSK CRD deletion alters both pre- and postsynaptic ultrastructures in MuSKΔCRD NMJs, which may result in a motor activity deficit.

[0105] MuSKΔCRD Mice Progressively Develop Muscle Weakness, Fatigue and Motor Defects.

[0106] During the first two weeks after birth, MuSKΔCRD mice developed normally without any gross physical signs of muscle weakness compared to WT mice. After this period, changes in the trunk region became progressively evident by the abnormal spine curvature caused by the shrinkage of the pelvic and scapular region. The computed tomography scan analysis performed on P90 WT and MuSKΔCRD mice illustrates the kyphosis developed by MuSKΔCRD mice (FIG. 7A). The kyphotic index (KI, see Material and Methods) used to appreciate the kyphotic severity degree, was significantly reduced in MuSKΔCRD compared to WT confirming the presence of a severe spine deformation in mutant mice (FIG. 7B, Laws and Hoey, 2004). To assess whether motor function was altered in MuSKΔCRD mice, we performed a grip test assay on young and adult mice. Whereas the latency to fall from the rail increased with age in WT mice, MuSKΔCRD mice exhibited poor motor performance from P20 to P90 as determined by a reduced latency to fall (latency decrease: P20, 62%; P40, 33%; P60, 64%; P90, 72%; FIG. 7C). To further investigate the origin of motor defect in MuSKΔCRD mice, we measured the grip strength of the forelimbs (FIG. 7D) and the hind limbs (FIG. 7E). In MuSKΔCRD as in WT mice, the forelimb and the hind limb grip strength increased with age. However, the grip strength of MuSKΔCRD mice was reduced at all time-points compared to WT mice indicating a muscle weakness (forelimb grip strength decrease: P20, 26%; P40, 23%; P60, 20%; P90, 30%; hindlimb grip strength decrease: P20, 37%; P40, 18%; P60, 23%; P90, 21%; FIGS. 7D and 7E).

[0107] To confirm the occurrence of muscle weakness in MuSKΔCRD, we analyzed the ability of P120 WT and MuSKΔCRD left hemidiaphragms to evoke twitches and tetanic contractions in response to phrenic nerve stimulation at different frequencies ex vivo. As shown in FIG. 7F, WT as MuSKΔCRD muscles developed and maintained tetanic contractions, in a wide range of nerve-stimulation frequencies. However, MuSKΔCRD muscles developed less force than WT ones. Indeed, in MuSKΔCRD mice, the strength of muscle twitch upon nerve stimulation was significantly reduced compared to WT mice, both upon single and tetanic stimulations (strength decrease of muscle twitch: single twitch, 59%; T40 Hz, 54%; T60 Hz, 47%; T80 Hz, 44%; T100 Hz, 42%; FIG. 7G). The developed muscle specific force, defined as the muscle strength (mN) normalized to the muscle weight (g) was significantly reduced in MuSKΔCRD compared to WT muscles, both upon single and tetanic (T100 Hz) stimulations respectively by 52% and 24% (FIG. 7H). One expected pathophysiological consequence of NMJ structural changes is fatigable muscle weakness as observed in myasthenia (Hantaï et al., 2013). We therefore evaluated the muscle fatigue strength after a train of tetanic nerve stimulations (T60 Hz) and found that MuSKΔCRD muscles exhibited a degree of fatigue more pronounced than WT muscles (fatigability increase: 30%, FIGS. 7I and 7J). Interestingly, we also observed spontaneous twitches after one twitch induced by unique stimulation in MuSKΔCRD mice, suggesting the presence of muscle denervation processes (FIG. 7K, Heckmann and Ludin, 1982). To further confirm this issue, non innervated AChR clusters could be detected in P90 MuSKΔCRD whole-mount diaphragm preparations (FIG. 7L).

[0108] Our data demonstrate that MuSK CRD deletion compromises motor performance, affects muscle strength and lead to increased muscle fatigability. This is in agreement with clinical symptoms generally observed in patients suffering from CMS.

[0109] Lithium Chloride Rescues NMJ Phenotype of MuSKΔCRD Mice.

[0110] Deletion of MuSK CRD, impairing Wnt/MuSK interaction is likely to perturb Wnt signaling at the NMJ. Indeed, treatment of WT primary myotubes with Wnt11, a member of the Wnt family known to interact with MuSK CRD and required for AChR clustering (Jing et al., 2009; Zhang et al., 2012) induced a 4.5-fold increase in the number of AChR clusters that was fully abolished (80% decrease) in Wnt11-treated MuSKΔCRD primary myotubes, demonstrating that deletion of MuSK CRD alters Wnt-induced AChR clustering (FIG. 8A). In addition, β-catenin translocation to the nucleus was strongly reduced in Wnt11-treated MuSKΔCRD compared to Wnt11-treated WT primary myotubes, indicating that Wnt activation of the canonical pathway is affected in MuSKΔCRD muscle cells (FIG. 8C). Since previous reports suggest that the Wnt canonical pathway is involved in neuromuscular synapse formation (Li et al., 2008; Liu et al., 2012; Wu et al., 2012), we then reasoned that forced activation of the Wnt beta-catenin signaling pathway during development could thwart impaired NMJ formation and compensate at least partially MuSKΔCRD NMJ phenotype. To test this hypothesis, we set up a pharmacological approach using LiCl, a well-known reversible inhibitor of the Gsk3 and activator of Wnt/β-catenin signaling (Klein and Melton, 1996; Stambolic et al., 1996; Wada, 2009). LiCl treatment of MuSKΔCRD primary myotubes resulted in a 6-fold increase in the number of AChR clusters and increased β-catenin translocation to the nucleus compared to Wnt11-treated MuSKΔCRD myotubes suggesting that LiCl is able to rescue AChR clustering and Wnt canonical signaling in MuSKΔCRD myotubes (FIGS. 8B and 8C). We then tested the effect of LiCl treatment on NMJ formation in vivo in MuSKΔCRD mice. Repeated intraperitoneal injections of LiCl or placebo (NaCl) from E12 to E18.5 in pregnant mice were performed and the phenotype of E18.5 LiCl-treated MuSKΔCRD NMJs was compared to NaCl-treated MuSKΔCRD and WT NMJs (FIG. 8D). Remarkably, LiCl treatment almost fully rescued the postsynaptic phenotype in E18.5 MuSKΔCRD embryos (FIG. 8D). The endplate band width in LiCl-treated MuSKΔCRD embryos was reduced by 23% compared to NaCl-treated mutant embryos and was close to the endplate band of WT embryos (FIG. 8E). Moreover, LiCl-treated MuSKΔCRD embryos significantly gained AChR clusters in number (by 103%), volume (by 186%) and intensity (by 20%) and were almost indistinguishable from WT embryos (FIG. 8F-H). In addition, presynaptic defects were improved in LiCl-treated MuSKΔCRD. The increased length of primary and secondary branches observed in MuSKΔCRD was reduced respectively by 35% and 28% in LiCl-treated MuSKΔCRD embryos, being almost similar to WT embryos (FIGS. 81 and 8J). In addition, the number and length of bypassing neurites were reduced by 33% and 138% respectively in LiCl-treated MuSKΔCRD embryos compared to NaCl-treated ones (FIGS. 8K and 8L). Taken together, these results indicate that LiCl treatment almost fully rescued both pre- and postsynaptic defects of MuSKΔCRD mutants, with NMJs being phenotypically indistinguishable from WT NMJs.

[0111] To further investigate the beneficial effect of LiCl treatment on NMJ maintenance in adulthood, MuSKΔCRD mice were injected with LiCl or placebo (NaCl) from P10 to P60 and NMJ morphology and motor functions as well as beta-catenin translocation to subsynaptic nuclei were analyzed (FIG. 9). LiCl treatment resulted in a strong increase of β-catenin translocation to subsynaptic nuclei in LiCl-treated MuSKΔCRD compared to NaCl-treated MuSKΔCRD isolated P40 TA muscle fibers as shown in the intensity plot profiles measuring the fluorescence intensity of beta-catenin and Dapi along the segmented lines (FIG. 9A). In addition, we found that NMJ structures of P40 TA isolated muscle fibers from LiCl-treated MuSKΔCRD were increased in size compared to NaCl-treated MuSKΔCRD NMJ (FIG. 9B). The AChR cluster and synaptophysin area in LiCl-treated mutants were increased by 40% and 105%, respectively compared to NaCl-treated mice (FIGS. 9C and 9D). Importantly, the number of AChRs fragments per NMJ in LiCl-treated mutants was decreased by 44% compared to NaCl-treated mice (FIG. 9E). Moreover, LiCl treatment significantly improved MuSKΔCRD mice latencies to fall from the rail grip as well as fore and hind limb strength compared to NaCl-treated MuSKΔCRD mice (FIG. 9F-H). Taken together these data demonstrate that LiCl treatment to postnatal MuSKΔCRD mice improves the NMJ morphological defects, muscle strength and restores beta-catenin translocation to synaptic nuclei suggesting that MuSK CRD plays a role during NMJ maintenance in adulthood likely in part via activation of the Wnt β-catenin signaling pathway

DISCUSSION

[0112] Here, we have investigated the functional role of the MuSK-Wnt binding domain (CRD) during NMJ formation and maintenance in vivo. To this end, we generated mutant mice deficient for MuSK CRD. Deletion of MuSK CRD leads to severe alteration of both pre- and postsynaptic elements during early muscle prepatterning (E14) and NMJ differentiation (E18.5) mainly characterized by (i) a drastic deficit in AChR clusters and (ii) exuberant axonal growth bypassing AChR clusters. Moreover, MuSK CRD deletion is pathogenic in adult mice, inducing CMS-like symptoms including kyphosis, NMJs dismantlement, muscle weakness and fatigability as previously observed in other mice models of CMS (Bogdanik and Burgess, 2011; Chevessier et al., 2008, 2012; Gomez et al., 1997; Webster et al., 2013). We also report that NMJ innervation defects in MuSKΔCRD mice can be rescued in vivo by LiCl treatment. Taken together, our data uncover a critical role for MuSK CRD in NMJ formation and function in adulthood.

[0113] Wnts proteins are known to be involved in muscle prepatterning early during NMJ formation (Wu et al., 2010). Moreover, in zebrafish, Wnt11 induced aneural AChRs clustering requires the CRD of Unpplugged/MuSKD(Jing et al., 2009). However, zebrafish lacking muscle prepatterning are able to form NMJ and are fully motile leaving open the question of the exact role of the prepatterning in NMJ functioning (Jing et al., 2009; Gordon et al., 2012). Here, we demonstrate that deletion of MuSK CRD in mammals severely impairs muscle prepatterning since the number of AChR clusters is drastically reduced (63%) and non innervated AChR clusters are strongly increased (30%) in E14 MuSKΔCRD embryos. Why deletion of MuSK CRD does not fully abolished muscle prepatterning remains unclear. Three hypothesis could explain this observation: first, it has been shown in vitro that deletion of MuSK CRD reduces but does not fully inhibit the binding activity of Wnt proteins to MuSK (Bank et al., 2014; Zhang et al., 2012). Thus, we cannot exclude that Wnts elicited muscle prepatterning requires other domains in MuSK. Second, Frizzled (Fzd) receptors are expressed in skeletal muscle cells and could mediate Wnt signaling to contribute to muscle prepatterning (Avilés et al., 2014; Strochlic et al., 2012). Finally, it has been suggested that MuSK and Lrp4 expression in early fused myofibers is sufficient to auto-activate MuSK and initiate muscle prepatterning (Burden et al., 2013; Kim and Burden, 2008). Wnts binding to MuSK CRD could therefore maintain or reinforce MuSK activation to amplify muscle prepatterning.

[0114] Our results demonstrate that MuSKΔCRD expression at the NMJ is increased by 70% in mutant compared to WT mice. In zebrafish, Wnt proteins have been shown to regulate the level of MuSK expression at the plasma membrane via activation of Wnt-induced MuSK endocytosis (Gordon et al., 2012). Therefore, deletion of MuSK CRD could disturb MuSK translocation from the plasma membrane to intracellular compartment leading to MuSK membrane accumulation and reduce Wnt/MuSK downstream signaling. Accordingly, we show that Wnt 11-induced AChR clustering as well as β-catenin translocation to the nucleus are impaired in MuSKΔCRD primary myotubes suggesting that deletion of MuSK CRD alters downstream Wnt canonical signaling during NMJ formation.

[0115] With innervation, nerve terminals release agrin that binds to Lrp4 and subsequently increases MuSK phosphorylation further enhancing AChR clustering (Kim et al., 2008; Zhang et al., 2008, 2011). Among the key molecules involved in NMJ formation, MuSK, Lrp4, Rapsyn and Dok7 knock-out mice lack both aneural and agrin induced AChRs clusters (DeChiara et al., 1996; Gautam et al., 1999; Okada et al., 2006; Weatherbee et al., 2006). In contrast, although muscle prepatterning is severely affected in MuSKΔCRD embryos, our results show that at E18.5, upon innervation, NMJs are able to form but are abnormal with reduced AChR clusters number (30%), volume and density suggesting that (i) prepatterning is not indispensable but necessary for normal progression of NMJ differentiation, (ii) innervation only partially compensates early AChR clusters deficit. Since we show that MuSK CRD deletion does not affect agrin-induced AChR clustering in primary myotubes, Wnt proteins may bind to other receptors at the postsynaptic membrane including Fzd, thus activating signaling mechanisms leading to inhibition of agrin-induced AChR clustering. Consistent with this, it has been recently shown that Fzd9 is highly expressed in skeletal muscle when NMJs form and its overexpression in culture myotubes impairs agrin-induced AChR clustering (Avilés et al., 2014).

[0116] In MuSK null mutant mice, motor axons grow excessively throughout the muscle (DeChiara et al., 1996). Similarly, both during muscle prepatterning and later during NMJ formation, MuSKΔCRD motor axons overshoot AChR clusters and grow aberrantly all over the muscle. This result underlines the importance of the MuSK CRD in regulating a muscle retrograde stop signal for motor axons. In support of this hypothesis, studies of conditional invalidation or overexpression of muscle key components of Wnt canonical signaling including Lrp4 and ß-catenin in vivo in mice suggest a role for Wnt canonical signaling to direct a retrograde signaling required for presynaptic differentiation (Li et al., 2008; Liu et al., 2012; Wu et al., 2012a, 2012b).

[0117] Intriguingly, despite severe NMJ formation defects, MuSKΔCRD mice are viable at birth and reach adulthood without any obvious abnormal phenotype during the first two weeks. In contrast, mice deficient for MuSK fail to form NMJs and die at birth due to respiratory failure (DeChiara et al., 1996). These data suggest that the remaining activity of MuSK deleted from its CRD is sufficient to prevent mutant mice from lethality. However, two weeks after birth, MuSKΔCRD mice start to develop CMS-like symptoms. Morphological analysis of adult MuSKΔCRD NMJs reveals abnormal endplates architecture, with an immature phenotype at P20 followed by a severe dismantlement at P60. This has been observed in all muscles analyzed including diaphragm, TA, soleus and EDL suggesting that MuSK CRD is necessary for all striated muscles to guarantee NMJ integrity. This abnormal NMJ phenotype is likely to be the consequence of early NMJ defects during development since NMJ defects similar to those observed in mutant embryos are detected in P5 MuSKΔCRD diaphragms. In this hypothesis, beside its role for synapse positioning early during NMJ formation, muscle prepatterning would also have a so far unsuspected role in NMJ functioning in adult mice. However, we cannot exclude a specific MuSK CRD-dependent role of Wnts during NMJ maintenance in adulthood. Further investigations are required to discriminate the role of MuSK CRD during NMJ formation in embryos and maintenance in adulthood.

[0118] NMJ fragmentation is often associated with muscle weakness as it has been described in CMS patients (Slater et al., 2006). Indeed, MuSKΔCRD adult mice develop fatigable muscle weakness highlighted by abnormal performance in the grip test assay, ex vivo isometric diaphragm contraction and fatigability measurement in response to nerve stimulation. Interestingly, one mutant mouse over five mice tested for isometric diaphragm contraction exhibits spontaneous twitches after a unique stimulation, a phenomenon often caused by muscle denervation (Heckmann and Ludin, 1982). In line with this observation, analysis of the NMJ innervation pattern in P90 MuSKΔCRD mice reveals the presence of non innervated AChR clusters suggesting a denervation-like process.

[0119] Electron microscopy analyses of adult MuSKΔCRD NMJs reveal defects in presynaptic vesicle density and postsynaptic folds structure associated with either few or highly disorganized junctional folds (JFs). AChR clusters and JFs are required for the genesis of an efficient endplate potential leading to the activation of the voltage-gated sodium channels concentrated in the depth of the JFs (Engel and Fumagalli, 1982; Marques et al., 2000). Thus, reduced presynaptic vesicle density, fragmented endplates as well as disorganized or fewer JFs are most probably responsible for the fatigable muscle weakness observed in adult MuSKΔCRD mice. Our investigation of the muscle pattern does not reveal major changes except mild muscle atrophy with reduced fiber size (data not shown). This muscle atrophy may participate to the muscle weakness but it may also result from defective NMJ maintenance since muscle activity is required for muscle trophicity (Schiaffino et al.,

[0120] LiCl is currently used as a pharmacological reagent to treat bipolar, Parkinson's and Hungtinton's diseases (Klein and Melton, 1996; Schou, 2001; Chiu et al., 2011; Yong et al., 2011). Remarkably, LiCl treatment rescues beta-catenin signaling and improves the impaired NMJ defects in both MuSKΔCRD embryos and adult mice suggesting that the defects observed during NMJ formation and maintenance in MuSKΔCRD mice are in part due to inhibition of the Wnt canonical signaling pathway. However, given that Gsk3 is a pivotal kinase interacting with multiple signaling pathways including PI3K-PTEN-Akt-mTOR or Ras-Raf-MEK-ERK, we cannot rule out the possibility that LiCl regulates other signaling pathways involved in NMJ development (McCubrey et al., 2014). Interestingly, LiCl was shown to be efficient in the treatment of occulopharyngeal muscular dystrophy through activation of the Wnt canonical pathway and to improve skeletal muscle strength in mouse models of myotonic dystrophy (Abu-Baker et al., 2013; Jones et al., 2012). Our results provide the first evidence that LiCl or other Gsk3 inhibitors can also be used as therapeutic reagents or in complement to the treatment currently available of neuromuscular disorders associated to Wnt-MuSK deficiency.

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