Fig 2: Area of myelination of spared spinal cord tissue 8 weeks after injury.Box and whiskers graphs showing measurements (in μm2) of MBP immunostaining in transverse cord sections at the epicenter, and 1.5 mm cranial and caudal to it. SI, injuries of severe intensity (n = 7); MI, injuries of moderate intensity (n = 9); SSAD, small surgical approach to debridement. Statistical analysis: Student’s t test was performed separately at each cord level, for each injury intensity. *, p = 0.03; **, p = 0.009.

Fig 3: White matter and cerebral cortex are reduced in adult mTrappc9 deficient mice. (A-B), NFH (axonal tracts) and MBP (myelin) immunostaining of the corpus callosum sections. Compared with WT (C57BL/6 wildtype) and mTrappc9+/+, the corpus callosum (highlighted with white dotted lines) of mTrappc9m/m mice was significantly thinner. Representative images with indicated thickness from n=3 mice/group (A), and quantitative analysis of n=5 sections (B). (C-D), MBP immunostaining of the cerebral cortex sections, with the white line arrows defining the representative thickness of the cerebral cortex (C, n=3 mice). Quantification shows the cortex is significantly thinner in mTrappc9 m/m mice than the other two groups (D, n=6 sections). (E-F), MBP immunostaining of the striatum. The white dotted lines demarcate the striatum area (E, n=3 mice) for quantification of the coverage area of the nerve bundles in the striatum (F, n=6 sections), which shows that nerve bundles are significantly reduced in mTrappc9 m/m mice compared to WT and mTrappc9+/+ mice. (G-I), NFH immunostaining of spinal cord sections. In mTrappc9 m/m mice, there was no significant difference in gray matter (inside the white dotted line), but the area of white matter (outside the white dotted line) was reduced significantly. Representative images from n=3 mice/group (G); n=7 sections (H, I). (J-K), Western blot analysis of MBP and NFH protein expression in cerebral cortex normalized with GAPDH. Both showed a significant decrease in mTrappc9 m/m mice. n=3 mice/group. Data are means ± SEM; One-way ANOVA (B, D, F, H, I); Two-way ANOVA (K); ****P=0.0001; ns, not significant (P>0.05). Scale bars: 50 µm (A); 150 µm (C, E, G).

Fig 4: Myelin sheath in early developing mice and zebrafish is reduced after Trappc9 deficiency, but the myelin sheath formation was not affected. (A-B), RNA in situ hybridization and RT-qPCR analysis of mbpb expression in 72 hpf zebrafish embryos. The expression of mbpb in mutant (zTrappc9m/m) and morphant (MO) zebrafish at 72 hpf was significantly reduced. Representative images from n=20-26 embryos/group in 7 experiments (A); n=3 experiments (B). (C-D), PLP immunostaining of 72 hpf zebrafish embryos. The area percentage of PLP fluorescence in zebrafish hindbrain (red arrowheads) was significantly decreased in MO and zTrappc9m/m zebrafish embryos. Representative images from n=10 embryos/group (C); n=8 sections (D). (E-F), MBP immunostaining of P11 mouse cerebral cortex, showing the area percentage of MBP fluorescence in the corpus callosum was significantly decreased in mTrappc9m/m mice. Representative images from n=3 mice/group I; n=6 sections (F). (G-I), Transmission electron microscope (TEM) micrographs of adult mouse corpus callosum showing no significant difference in the axon perimeter (H) and G-ratio (I) in mTrappc9m/m mice. Representative images from n=13 sections/group (G); n=200-220 myelinated axons (H, I). (J-L), MBP and OLIG2 immunostaining of primary cultured oligodendrocytes, which were differentiated from brain-derived oligodendrocyte precursor cells (OPCs) of P8 mice. The morphology (sholl analysis, K) and branch numbers (L) of oligodendrocytes were not affected by mTrappc9 deficiency. Representative images from n=3 coverslips/group (J); n=6 neurons (K); n=12 neurons (L). Data are means ± SEM; t-tests (L); One-way ANOVA (B, D, F, H, I); Two-way ANOVA (K); ****P≤0.0001; **P≤0.01; ns, not significant (P>0.05). Scale bars: 2 μm (G); 10 μm (J); 50 μm (A, C, E).