Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with ‘dying-back’ neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.
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The data that support the findings of this study and biological materials used are available from the corresponding authors upon request.
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The authors thank L. Schaeffer (University of Lyon, France), A. Verger (Université Lille Nord de France, France), J. Weishaupt (Ulm University, Germany) and R. Perlingeiro (University of Minnesota, USA) for the gift of plasmids, and N. Charlet Berguerand for advice on siRNA treatment. They would also like to thank the General Instruments Facility of the Faculty of Science of Radboud University and I. Alexopoulos in particular for advice on image acquisition and analyses. The authors would like to thank S. Seltenheim, L. Dietz and R. Zienecker for excellent technical support, A. Knehr (Department of Neurology, Ulm University) for collecting human material, and B. Mayer (Institute for Epidemiology and Medical Biometrics, Ulm University) for advice on statistical analyses. They are grateful to all the participants for willingly providing them with hair and muscle samples. This work was directly supported by the following grants: ALS Association Investigator Initiated Awards (grants 2235, 3209 and 8075 to L.D. and C.L.-T.); the Frick Foundation (award 2013 to L.D. and C.L.-T); Association Française contre les Myopathies (grant 18280 to L.D., C.L.-T. and E.S.); Virtual Helmholtz Institute ‘RNA dysmetabolism in ALS and FTD’ (WP2 to L.D., A.L. and T.M.B.); the DZNE (Ulm site) and agence nationale de la recherche (ToFU, EpiFUS to L.D.); the Bundesministerium für Bildung und Forschung (BMBF01EK1611C to T.M.B. and M.D.); the Max Planck Society (to E.S.); and the Donders Center for Neuroscience (to E.S.). Work in the authors’ laboratories is supported by ARSla (call 2014 and 2016 to L.D.), the fondation ‘recherche sur le cerveau’ (call 2015 to L.D.), Axa Banque Patrimoniale (Bourse recherche maladies rares to L.D.), Fondation pour la recherche médicale (Equipe FRM to L.D.), the Muscular Dystrophy Association (MDA479773 to E.S.), the EU Joint Programme—Neurodegenerative Disease Research (JPND; grant numbers ZonMW 733051075 (TransNeuro) and ZonMW 733501073 (LocalNMD) to E.S.) and an ERC consolidator grant (ERC-2017-COG 770244 to E.S).
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks Thomas Lloyd and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 NMJ morphology defects in the gastrocnemius of newborn FusΔNLS/ΔNLS and Fus−/− mice.
a-f Dot plots showing the percentage (%) of innervated endplates (a, d), endplate surface area as % of Fus+/+ littermate controls (b, e) and the total number of endplates in the gastrocnemius (c, f) of newborn FusΔNLS/ΔNLS (a-c, closed circles) and Fus−/− (d-f, closed circles) mice, as compared to Fus+/+ mice (open circles). Two-tailed unpaired t-test; **p<0.01, ***p=0.0005; N= 12 versus 11 (a), 17 versus 14 (b), 4 versus 5 (c), 8 versus 8 (d), 15 versus 13 (e), 9 versus 10 (f) mice. Average ± SEM. g-k, Quantification of ultrastructural NMJ defects in gastrocnemius of E18.5 Fus+/+, Fus−/− and FusΔNLS/ΔNLS mice. g, The neuromuscular contact was scored as intact (1) when pre- and postsynaptic membranes were closely apposed and the synaptic cleft was filled with electron dense material. A widened synaptic cleft was scored as 0, and intermediate morphology as 0.5. h, The presence or absence of postsynaptic membrane foldings was scored as 1 or 0, respectively. i,j, Presynaptic (i) and postsynaptic (j) membrane integrity was scored as intact (1), thin (0.5) or disrupted (0). k, The abundance of synaptic vesicles was scored as abundant (1), rare (0.5) or absent (0). For g-k: Kruskal-Wallis test with Dunn’s multiple comparisons test; *p<0.05, **p<0.01; N= 5 Fus+/+, 4 Fus−/− and 5 FusΔNLS/ΔNLS mice; for each parameter, the average value per animal (based on 4-14 NMJs analyzed per animal) was used as a data point. Average ± SEM.
a,b, Dystrophin staining to visualize individual muscle fibers in extensor digitorum longus (EDL) muscle of newborn Fus+/+ versus FusΔNLS/ΔNLS mice. Scale bar: 100µm. c,d, Dot plots showing the total number of muscle fibers in EDL muscle of newborn Fus+/+ (open circles) versus FusΔNLS/ΔNLS (c, closed circles) or Fus−/− (d, closed circles) mice. p=0.52 (c) by two-tailed unpaired t-test; N= 6 versus 5 (c) and 2 versus 2 (d) mice. Average ± SEM. e, Dot plot showing the total number of muscle fibers in TA of newborn Fus+/+ (open circles) versus Fus−/− (closed circles) mice. p=0.054 by two-tailed unpaired t-test; N= 7 versus 4 mice. Average ± SEM. f, Dot plot showing the total number of endplates in TA of 1-month-old male Fus+/+ versus FusΔNLS/+ mice. p=0.59 by two-tailed unpaired t-test; N= 3 versus 7 mice. Average ± SEM.
a, Schematic illustrating the working principle of the ROSAmT/mG reporter mice. The chicken β-actin core promoter with a CMV enhancer (pCA) drives expression of the loxP-flanked (triangles) coding sequence of membrane-targeted tandem dimer Tomato (mT). After Cre-mediated excision of the mT sequence, the pCA promoter drives expression of membrane-targeted EGFP (mG). pA: polyadenylation sequence. b, Transverse sections through the TA of adult ROSAmT/mG/+ (control, upper panels) and MyoDiCre/+; ROSAmT/mG/+ (experimental, lower panels) mice were labeled with phalloidin (to visualize muscle fibers), antibodies against tdTomato and GFP, and DAPI. Scale bar: 20µm. c, Bar graph showing the percentage (%) or GFP-positive muscle fibers in ROSAmT/mG/+ and MyoDiCre/+; ROSAmT/mG/+ mice. One-sample Wilcoxon signed rank test (mT/mG; MyoD-iCre versus 0); *p=0.031; N= 6 versus 6 mice. Average ± SEM.
a,b, Relative mRNA levels of Chrn genes, including Chrna1, Chrnb1, Chrng, Chrnd, and Chrne, in gastrocnemius muscle of E18.5 Fus+/+ versus FusΔNLS/ΔNLS mice (a) and TA muscle of 1-month-old male Fus+/+ versus FusΔNLS/+ mice (b). Note that Chrne is not expressed at E18.5, and Chrng is not expressed at 1 month of age. Two-tailed unpaired t-test; *p<0.05, **p=0.0055; N= 4 Fus+/+ versus 5 FusΔNLS/ΔNLS (a) and 4 Fus+/+ versus 6 FusΔNLS/+ mice (b). Average ± SEM.
Supplementary Figure 5 R495X mutant FUS mislocalizes to the cytoplasm and induces cytoplasmic mislocalization and granular nuclear staining of wild type FUS in C2C12 cells.
a, Representative images of C2C12 cells co-transfected with plasmids expressing N-terminal GFP-tagged wild type human FUS (FUS_WT::GFP) and N-terminal Myc-tagged human FUS, either wild type (FUS_WT::Myc) or R495X mutant (FUS_R495X::Myc). 24 h later, cells were immunostained for Myc and Hoechst was used to label nuclei. Scale bar: 25 µm. b,c, Quantification of the percentage of cytoplasmic relative to total (nuclear + cytoplasmic) FUS_WT::GFP (b) and the percentage of granular relative to total nuclear FUS_WT::GFP (c) in C2C12 cells co-transfected with either FUS_WT::Myc (open circles) or FUS_R495X::Myc (closed circles). Nested one sample Wilcoxon-test (b); ***p=0.0005; Nested t-test (c); *p=0.024; N= 3 transfections, with for each 11-12 images analyzed and 10-50 transfected cells per image. Average ± SEM.
Supplementary Figure 6 FUS and ERM collaborate to promote transcription of Chrn genes in subsynaptic nuclei.
a, FUS is required for Agrin induced transcriptional activation of the Chrne promoter. Luminescence intensity (in RLU) measured in C2C12 cells 24 h after transfection with siCt (25 nM) or siFus (25 nM) and a luciferase reporter plasmid carrying the promoter of Chrne gene, and further treated for 24 h with agrin (10 ng/mL) or its vehicle. Data of 10 independent transfections are shown. Each transfection included 3 to 6 technical replicates per condition. For each transfection, luminescence intensity was normalized to siCt without agrin treatment after removal of statistical outliers, and the median value of the technical replicates was determined per condition, plotted in the graph and used for statistical analysis. Lines and error bars indicate median ± interquartile range. Two-way ANOVA with Tukey post-hoc test for multiple comparisons; **p=0.0041; ***p<0.001; ns: not significant. b, HEK293 cells were transfected with FLAG-ERM and Myc-Fus expression plasmids and lysates were immunoblotted before (input) and after (IP) immunoprecipitation with anti-Myc, in the presence or absence of RNase. c, Endogenous FUS was immunoprecipitated from HEK293 cells using a specific antibody (IP) or beads only, in the presence or absence of RNase. Native lysates (input) and immunoprecipitates were analyzed by Western blotting with anti-FUS or anti-ERM antibodies. Co-IP experiments (b, c) were independently repeated 5 (b) or 6 (c) times with similar results. Blot images in panels b and c were cropped. Supplementary Fig. 10 shows uncropped blots.
a, Time course of the spheres-based protocol for myogenic differentiation, including progenitor and final myotube differentiation steps. Myogenic cells were grown in spheres for 6 weeks and then plated to form multinucleated myotubes. b, Relative mRNA levels of the myogenic markers MYOG, PAX7 and CHRNA1 as determined by quantitative real time PCR (qPCR) on hiPSC-derived myocytes from two control and two FUS-ALS patients at different time points during differentiation (4, 5, 6, 7 and 8 weeks). The HMBS gene (encoding hydroxymethylbilane synthase) was used as housekeeping gene for normalization. Transcript levels of CHRNA1 were significantly reduced in FUS-ALS myotubes after 8 weeks of differentiation. ***p<0.001 by two-way ANOVA with Bonferroni post-test. N= 3 independent experiments per genotype, with each experiment representing the average of two technical replicates. Average ± SEM. c, Differentiated muscle cells stained positive for the muscle-specific intermediate filament desmin, the contractile filament myosin heavy chain (MHC), MYOG, the skeletal muscle ryanodine receptor isoform RyR1 and the voltage-dependent dihydropyridine receptor alpha subunit (DHPRα). The latter two are intracellular calcium channels that mediate calcium release from the sarcoplasmic reticulum, an essential step in muscle contraction. Staining for FUS and DAPI revealed FUS localization in nuclei. Differentiation was repeated three times with similar results. Scale bar: 10µm. d, hiPSC-derived myotubes were labeled with BTX (AChR, yellow), FUS (green), actinin (red) and DAPI (blue). FUS predominantly localized to nuclei in both CNTL and FUS-ALS myotubes. The microfilament protein α-actinin, which is necessary for attachment of actin filaments to the sarcomeric Z-disks, was correctly localized to Z-disks. Differentiation was repeated three times with similar results. Scale bar: 10 µm.
a,b, Size of single BTX positive puncta within endplates in motor neuron-myotube co-cultures. Compared to CNTL-CNTL co-cultures, combinations containing FUS-ALS myotubes showed reduced single particle size. Panel a shows co-cultures derived from CNTL1 and FUS1 or FUS2, panel b shows co-cultures derived from FUS1 and its isogenic control CNTL3. **p<0.01; ***p<0.001 by Kruskal-Wallis test with Dunn’s test for multiple comparisons. N= 759 (CNTL1 MT + CNTL1 MN), 846 (CNTL1 MT + FUS1 MN), 3467 (FUS1 MT + CNTL1 MN), 1872 (FUS1 MT + FUS1 MN), 307 (CNTL1 MT + FUS2 MN), 364 (FUS2 MT + CNTL1 MN), 841 (FUS2 MT + FUS2 MN), 978 (CNTL3 MT + CNTL3 MN), 1842 (CNTL3 MT + FUS1MN), 1799 (FUS1 MT + CNTL3 MN), 1116 (FUS1 MT + FUS1 MN). N= 3 co-cultures per combination. Average ± SEM. c, Motor neuron-myotube co-cultures were labeled for FUS (green), AChR (BTX, yellow), neurofilament heavy chain (NF-H, red, labels axons) and DAPI (blue). FUS localized to nuclei of both CNTL and FUS myotubes (white arrowheads), but in FUS-mutant motor neurons, FUS localized to both nucleus and cytoplasm, in contrast to nuclear FUS localization in CNTL motor neurons (red arrowheads). Scale bar: 10µm. d, Relative mRNA levels of myogenic differentiation markers (PAX7, MYOD, MYOG) and AChR subunits (CHRNA1, CHRNG, CHRNE) in motor neuron-myotube co-cultures as determined by qPCR. As myotube differentiation was induced by PAX7 overexpression, it was expected that no significant differences in PAX7 expression were found across the different genotypic combinations. The HMBS gene was used as housekeeping gene for normalization. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Bonferroni post-test. N= 3 independent co-cultures per genotype, with each representing the average of two technical replicates. Average ± SEM.
a, Distribution of muscle fiber size in muscle biopsies from control (gray bars) and FUS-ALS patients (green bars). Muscle fibers were assorted in the indicated size categories based on their surface area (in μm2) and the percentage of fibers in each category is shown in the bar graph. b, Density of nuclei in muscle biopsies from control (gray circles) and FUS-ALS patients (green circles) as a proxy for muscle atrophy. The number of nuclei per field of 13,547 μm2 is shown in the dot plot. One-way ANOVA with Tukey’s multiple comparisons test; **p<0.01, ***p<0.001; N= 4 fields. Average ± SEM. c, Dot plot showing cytoplasmic FUS staining intensity in muscle biopsies from control (gray circles) and FUS-ALS patients (green circles). Kruskal-Wallis test with Dunn’s multiple comparisons test; *p<0.05, ***p<0.0001. Number of fields in which the average cytoplasmic FUS staining intensity is measured=3 (CNTL4), 5 (CNTL5), 2 (CNTL6), 4 (FUS1), 5 (FUS4), 10 (FUS5). Average ± SEM. d, Dot plot showing BTX-positive endplate area in muscle biopsies from control (gray circles) and FUS-ALS patients (green circles). One-way ANOVA with Tukey’s multiple comparisons test; *p<0.05, ***p<0.0001. Number of endplates=3 (CNTL4), 6 (CNTL5), 4 (CNTL6), 5 (FUS1), 7 (FUS4), 24 (FUS5). Average ± SEM.
Supplementary Figure 10 Working model illustrating how FUS may regulate transcription of Chrn genes in subsynaptic myonuclei.
Agrin secreted from motor nerve endings binds to LRP4 on postsynaptic membranes, resulting in MuSK activation and subsequent activation of ERM through Rac1 and JNK pathways. FUS is enriched in subsynaptic nuclei, interacts with ERM, binds to AChR gene promoters and cooperates with ERM to stimulate transcription of Chrn genes.