Fig. 9

SD-induced increase in mRNA levels of APP and β-secretase expression, and decrease in APP O-GlcNAcylation. Zebrafish brains were analyzed after exposure to SD or RSD, with or without a recovery period, as well as after RSD without recovery (RSD-R). In some conditions, intraperitoneal GlcN (20 μg/g) was administered. After the induction of SD, SD(R), RSD-R, or RSD, the zebrafish brains were sacrificed. A, B mRNA expression levels of pooled brain samples (n = 3/group) were measured using quantitative real-time PCR with specific primers. Values are presented as the mean ± SEM for each target gene expression, normalized to β-actin levels using the delta–delta Ct method. The experiments were independently replicated three times. For statistical analysis, the Mann–Whitney test was used for the two-group comparisons, and the Kruskal–Wallis test with the original FDR of Benjamini and Hochberg was used for comparisons involving more than three groups (*p < 0.05 versus Con). C Representative confocal images (40x) of DAPI (blue), APP (C, green), BACE-1 (D, green), and merged immunofluorescence staining in the Dl region of zebrafish brain are displayed. Enlarged images are presented in the white boxes. The graphs represent the quantitative results for each antibody with normalization to the DAPI levels (n = 3 ~ 4/group). For statistical analysis, the Mann–Whitney test was performed the two-group comparisons, while the Kruskal–Wallis test with the original Benjamini and Hochberg false FDR was used for comparisons involving more than three groups (**p < 0.01 versus Con). D WGA pull-down assays were performed using total protein extracted from whole brains of zebrafish (3 brain pools per group). The total cell lysates were incubated with WGA agarose, and the proteins bound to WGA were analyzed using Western blot assay with specific antibodies. The graphs represent the quantitative results for the level of WGA-bound APP, normalized to the total APP levels. The experiments were independently replicated three times. For statistical analysis, the Mann–Whitney test was performed (*p < 0.05, **p < 0.01 versus Con, ^#p < 0.05 versus SD). E β-Secretase activities were measured in the whole brain of zebrafish (n = 4 ~ 5/group). Values are presented as the mean ± SEM. For statistical analysis, the Mann–Whitney test was performed for the two-group comparisons, and the Kruskal–Wallis test with the original FDR of Benjamini and Hochberg was performed for comparisons involving more than three groups (*p < 0.05, **p < 0.01 versus Con, #p < 0.05 versus SD or RSD-R)