Fig 1: (A) High-dose treatment of D1-NBP alleviates mitochondrial accumulation by promoting mitochondrial dynamics. (A–D) Representative kymograph (A) and quantitative analysis of the percentage of stationary, anterograde, and retrograde mitochondria in the axons (B), mitochondrial load per 50 μm (C), and mitochondrial size (D) in cerebellum slices subjected to Ctrl+ phosphate buffered saline (PBS), Ctrl+NBP, Ctrl+SNPH OE, Ctrl+SNPH OE+H-NBP, low glucose and low oxygen (LGLO)+PBS, LGLO+NBP, LGLO+SNPH OE, LGLO+SNPH OE+H-NBP. The high-dose treatment of D1-NBP alleviated the LGLO-induced impairment of retrograde and anterograde mitochondrial transport. A one-way ANOVA with Tukey's correction. For the percentage stationary, anterograde, and retrograde mitochondria analysis, n = 60 mitochondria per group. For mitochondrial load per 50 μm analysis, n = 20 axons per group. For mitochondrial size analysis, n = 400 per group. Scale bar, 50 μm and 5 min, respectively. Data are represented as means ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant differences).
Fig 2: (A) High-dose treatment of D1-NBP mitigated demyelination and cognitive impairment by rescuing mitochondrial dynamic in vivo. (A–E) Representative TEM images depicting myelination status (Upper panel) and mitochondrial load in the axons (A) and quantitative analysis of the G-ratio (B), mitochondrial load per area (C), mitochondrial length (D), and mitochondrial diameter (E) in BCAS+Placebo, BCAS+H-NBP, BCAS+SNPH OE, and BCAS+SNPH OE+H-NBP groups. The therapeutic effects of H-NBP on mitigating demyelination and cognitive impairment were dependent on mitochondrial dynamics. Scale bar, 2 μm. A one-way ANOVA with Tukey's correction. For G-ratio, n = 80 myelinated axons. For mitochondrial load per area analysis, n = 20 visual fields (4 visual fields per mouse, 5 mice per group). For mitochondrial length analysis, n = 50 (10 mitochondria per mouse, 5 mice per group). For mitochondrial diameter analysis, n = 50 (10 mitochondria per mouse, 5 mice per group). (F–H) Representative silver staining images. (F) Quantitative analysis of the number of spines per 10 μm. (G) Different kinds of spine morphology in different groups (H). A one-way ANOVA with Tukey's correction. For the number of spines per 10 μm analysis, n = 10 axons per mouse, 5 mice per group. For different kinds of spine morphology analysis, n = 10 axons per mouse, 5 mice per group. Scale bar, 10 μm. (I–K) Working and reference memory were assessed by the eight-arm radial maze. The improvement of working memory by H-NBP in BCAS mice was abolished by SNPH OE, as evidenced by the increased revisiting errors. (I) Less different choices (J) in the BCAS+H-NBP+SNPH OE group. There is no significant difference in the spatial reference memory between different groups (K). A two-way ANOVA with the Dunnett's post-hoc test, n = 11 mice in each group. Data are represented as means ± SD (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant differences).
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