Fig 1: Ttbk1 ASOs selectively suppress Ttbk1 expression in the temporal cortex of PS19. A The scheme of the experimental design for ASO-mouse Ttbk1 injection in PS19 mice. PS19 mice were injected with ASO-Ttbk1 #1 or #2, or ASO-control at 6 month of age. Mice are euthanized 8 weeks after the injection for qPCR, ELISA, and immunohistochemistry. B Body weight measurement at the end point. C, D qPCR analysis of mRNA level of temporal brain tissue for Ttbk1 (C) and Ttbk2 (D). Reduced mRNA level of Ttbk1 but not Ttbk2 was observed by ASO-Ttbk1 treatment. **** denotes < 0.0001 and *** denotes < 0.001 compared to ASO-control treated group as determined by one-way ANOVA and Dunnett’s multiple comparison (WT saline treated n = 6, ASO-control n = 7; ASO-Ttbk1 #1 n = 6; ASO-Ttbk1 #2 n = 8 mice)
Fig 2: Phosphorylated tau level in PS19 mice treated with ASO targeting Ttbk1. Protein expression levels determined by ELISA for p-tau for epitope pS396 (A, B), pT231 (C, D) and pT181 (E, F), and total tau in sarkosyl soluble (S2; A, C, E) and insoluble (P2; B, D, F) fractions isolated from the hippocampal brain tissue in PS19 mice after ASO treatment. * denotes < 0.05, ** < 0.01, *** < 0.00, **** < 0.0005 compared to ASO-control treated group as determined by one-way ANOVA and Dunnett’s multiple comparison (ASO-control n = 7; ASO-Ttbk1 #1 n = 6; ASO-Ttbk1 #2 n = 8 mice)
Fig 3: RNAseq analysis of the temporal cortex from PS19 mice treated with ASO-Ttbk1. A, B Volcano plot for the comparison between ASO-control and ASO-Ttbk1 treated group. The horizontal axis shows log2 (fold change) and the vertical axis means log10 (adjusted p-value). Cutoff values were set at ± 1.5 for FC and 0.05 for adj. p value. C Genes significantly altered by ASO-Ttbk1 treatment compared to ASO-control in the venn diagram. Cutoff values were set at ± 1.5 for FC and 0.05 for adj. p value. D List of 11 genes commonly and significantly altered by ASO-Ttbk1#1 or #2 treatment. The numbers represent the Log2 (Fold change). E Results of pathway enrichment analysis by Reactome for genes significantly altered by ASO-Ttbk1#1 treatment. F Top list of gene set enrichment analysis (GSEA) by ASO-Ttbk1#1 and #2 treatment. NES; Normalized enrichment Score. All Adj. p values were below 0.01. G Fold change in genes involved in antigen presentation by ASO-Ttbk1#1 and #2 treatment
Fig 4: Ttbk1 ASOs suppresses pS422 tau level in PS19 mice. A Protein extraction scheme. B Western blot results against pS422 and total tau (tau5) antibodies with soluble fraction S1 isolated from the hippocampal tissue 8 weeks after the injection of ASO-Ttbk1 or -control. C: WT mouse sample H: WT mouse samples under hypothermia to induce tau phosphorylation as a positive control, M: Marker, 1–8: Samples from PS19 mice injected with ASO-control, ASO-Ttbk1#1, or ASO-Ttbk1#2. C Protein expression levels of pS422 normalized by total tau. All membranes were always loaded with a constant amount of the same positive control (PC) at the same time, and all bands of pS422 and tau5 were quantified as a ratio to PC. One-way ANOVA and Dunnett’s multiple comparison. * denotes < 0.05. D Association analysis of pS422 level normalized by total tau and TTBK1 protein level in S1 fraction of the hippocampal tissues measured by ELISA. The GraphPad was used to confirm the Gaussian distribution, and then simple liner regression analysis was performed. E Representative immunofluorescence image of phosphorylated tau (pS422) in the hippocampal region in PS19 mice injected with ASO-control, ASO-Ttbk1#1, or ASO-Ttbk1#2 at 8 weeks post injection. pS422 (red) and DAPI (blue) immunostaining in the CA1 and mossy fiber. Scale bar = 100 μm. F Mean intensity measurement of pS422 in the mossy fiber region. **p < 0.01 compared to ASO-control treatment group as determined by One-way ANOVA and Dunnett’s multiple comparison. G Quantification of pS422 positive neuronal volume normalized by total CA1 volume (ASO-control n = 7; ASO-Ttbk1 #1 n = 6; ASO- Ttbk1 #2 n = 8 mice)
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