Fig 1: Motor neuron degeneration and gliosis. This figure is supported by Extended Data Figure 3-1. A, Representative images of ChAT-stained motor neurons in the lumbar spinal cord of mice at 20 w. GFAP-immunostained (B) and Iba1-immunostained (C) images of the lumbar spinal cord. Scale bar: 200 μm. D, Counts of ChAT-positive neurons in hemi sections of the lumbar spinal cord (WT; n = 4, SOD1G93A; n = 7, Crmp1−/−/SOD1G93A; n = 8, Crmp1ki/ki/SOD1G93A; n = 7). Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test as follows: WT versus SOD1G93A; p = 0.003, WT versus Crmp1−/−/SOD1G93A; p < 0.001, SOD1G93A versus Crmp1−/−/SOD1G93A; p = 0.871, WT versus Crmp1ki/ki/SOD1G93A; p = 0.496, SOD1G93A versus Crmp1ki/ki/SOD1G93A; p = 0.0253. Percentage of GFAP-positive (E) and Iba1-positive (F) area within the ventral horn area (WT; n = 5, SOD1G93A; n = 5, Crmp1−/−/SOD1G93A; n = 5, Crmp1ki/ki/SOD1G93A; n = 5). Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test as follows: (E) SOD1G93A versus Crmp1−/−/SOD1G93A; p = 0.723, SOD1G93A versus Crmp1ki/ki/SOD1G93A; p = 0.292, (F) SOD1G93A versus Crmp1−/−/SOD1G93A; p = 0.457, SOD1G93A versus Crmp1ki/ki/SOD1G93A; p = 0.999. Values are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, N.S. = not significant as determined by one-way ANOVA.
Fig 2: Phosphoproteomic analysis of spinal cords from WT and SOD1G93A mice at 20 weeks of age. A, Top 10 canonical pathways identified based on the molecules that were differentially expressed (max fold change >1.5, ANOVA p < 0.05) between WT and SOD1G93A mice in phosphoproteomics. B, A list of phosphoproteins in semaphorin neuronal repulsive signaling pathway with significant expression changes in the lumbar spinal cords of SOD1G93A mice compared with WT mice (max fold change 1.5; p < 0.05). Phosphorylation sites, fold-change levels, and p-value are also shown. C, Protein–protein interaction (PPI) network of differentially upregulated proteins in SOD1G93A mice visualized by STRING: https://string-db.org/. Red nodes are phosphoproteins associated with axon guidance in reactome pathway. The red arrow indicates Crmp1.
Fig 3: Phenotypic comparisons of SOD1G93A, Crmp1−/−/SOD1G93A, and Crmp1ki/ki/SOD1G93A mice. This figure is supported by Extended Data Figures 2-1, 2-2. A, Kaplan–Meier survival curves for SOD1G93A (black), Crmp1−/−/SOD1G93A (blue), and Crmp1ki/ki/SOD1G93A (red) mice. Median survival duration in SOD1G93A, Crmp1−/−/SOD1G93A, and Crmp1ki/ki/SOD1G93A mice was 167, 173, and 180 d, respectively (the log-rank test, p = 0.044). B, The comparative survival durations for three groups (two-way ANOVA, *p < 0.05). C, Kaplan–Meier curves for disease onset. D, Median disease onset in SOD1G93A, Crmp1−/−/SOD1G93A, and Crmp1ki/ki/SOD1G93A mice was 112, 102, and 112 d, respectively, and the differences were not significant (the log-rank test, p = 0.797). E, Mean disease duration (days from onset to end stage). Mean disease duration in SOD1G93A, Crmp1−/−/SOD1G93A, and Crmp1ki/ki/SOD1G93A mice was 54, 63, and 65 d, respectively. Moreover, the duration was longer in Crmp1ki/ki/SOD1G93A mice than in SOD1G93A mice (one-way ANOVA with Fisher’s LSD test, p = 0.039). F, Rotarod test. Crmp1ki/ki/SOD1G93A mice exhibited a significant improvement in motor function at the late stage (21–24 w) compared with SOD1G93A mice, while Crmp1−/−/SOD1G93A showed shorter latency to fall during the rotarod test at 18 w. Values are means ± SD (SOD1G93A, n = 20; Crmp1−/−/SOD1G93A, n = 20; Crmp1ki/ki/SOD1G93A, n = 20). *,†p < 0.05, **p < 0.01 (two-way ANOVA with Fisher’s LSD test). G, Body weight. Significant differences were not observed between the three lines of model mice. N.S. = not significant.
Fig 4: Blocking Crmp1 phosphorylation at S522 delays denervation at NMJs. A, Representative photomicrographs of NMJs in fixed TA muscles. Nerve axons (green) are stained with 2H3 (NF) plus SV2 (synaptic vesicles) and postsynaptic acetylcholine receptors (red) are stained with α-BTX. Scale bar: 20 μm. B, Crmp1 S522A expression increases endplate occupancy in TA muscle of SOD1G93A mice. The figure shows the percentage of fully innervated (fully occupied), partially denervated (partially occupied), and denervated endplates of the axon terminals in TA muscles from mice with the indicated genotypes. Statistical significance was determined as follows: innervated: WT versus SOD1G93A; p < 0.001, SOD1G93A versus Crmp1−/−/SOD1G93A; p = 0.999, SOD1G93A versus Crmp1ki/ki/SOD1G93A; p = 0.038, denervated: WT versus SOD1G93A; p < 0.001, SOD1G93A versus Crmp1−/−/SOD1G93A; p = 0.955, SOD1G93A versus Crmp1ki/ki/SOD1G93A; p = 0.01, by one-way ANOVA with Dunnett’s multiple comparisons test. Values are means ± SEM (n = 3); *,†p < 0.05, ††p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 5: MAR-ASD autoantibody production in rat dams.A Autoantibody production is induced in rat dams through injection with autism-specific autoantigen peptides. Antibody can then transfer to offspring and influence developmental outcomes. B Levels of serum IgG Ab reactive to MAR-ASD proteins (LDHA/B, STIP1, CRMP1) in rat dams compared between aAb and control animals. Values expressed as fold-change over baseline (week 0) following 4 weeks of injections. C Longitudinal characterization of MAR-ASD IgG persistence in serum from treated dams at 5 weeks and 60 weeks following initial immunization. ELISA data representative of single experiments. D Serum cytokine levels in MAR-ASD and control dams taken 1 week prior to breeding. Data expressed as fold change over baseline (week 0). N = 3 per condition for each experiment, data are expressed as mean ± SEM. Analysis conducted by two-way ANOVA, representative of a single experiment.
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