Figure 1.

CMT2D neuropathy is influenced by the VD pathway. (A) Experimental diagram showing the deprivation of VD from the diet. VD⁻ diet, VD-deficient diet. (B and C) VD deficiency worsened motor defects of CMT2D mice in the hindlimb extension test and rotarod test (n ≥ 11 for each group). (D) Representative images showing NMJs in the gastrocnemius muscles with motor nerve terminals (labelled in cyan) and acetylcholine receptors on the muscle (labelled in magenta). Scale bar, 20 μm. (E) Quantification of NMJ denervation (n = 3 for each group). (F) Representative images of sciatic nerve sections. Scale bar, 5 μm. (G) The percentage of axons with a diameter >2 μm (n = 3 for each group). (H) Experimental diagram of Cal administration. (I and J) Cal treatment improved the motor performance of CMT2D mice in the hindlimb extension test and rotarod test (n ≥ 10 for each group). (K) Cal treatment significantly attenuated NMJ denervation in CMT2D mice (n = 3 for each group). (L) Cal treatment significantly increased the percentage of large-diameter axons (with a diameter >2 μm) in the sciatic nerves of CMT2D mice (n = 3 for each group). (M–P) RT-qPCR analysis of VEGF (VEGF-A₁₆₄ isoform) mRNA expression levels. (M) VD deficiency significantly downregulate VEGF-A₁₆₄ expression level in the gastrocnemius muscles of 7-week-old mice (n = 6 for each group). (N) VEGF-A₁₆₄ expression level was significantly decreased in the gastrocnemius muscles of adult VDR KO mice (n ≥ 3 for each group). (O) VEGF-A₁₆₄ expression level was upregulated in WT mouse gastrocnemius muscles after a single injection of 60 μg/kg Cal (n = 4 for each timepoint). (P) Cal (10 nM) treatment induced a rapid (<1 h) increase in VEGF-A₁₆₄ expression level in C2C12 cells (n = 3 independent experiments). Data are presented as mean ± SEM. Statistical analysis was performed with two-way ANOVA (B, C, E, G, and I–L), Student's t-test (M, N, and P), or one-way ANOVA (O). n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.