Fig. 4: Increased Sirt2 activity in AD impairs Fzds transcription.

A WB analyses of total and pSIRT2 levels in hippocampal nuclear extracts of human control/BI-III subjects showing decreased levels of pSIRT2.2 at early AD. B ChIP-qPCR analyses of FoxO1 in WT and NLGF hippocampal samples showing increased FoxO1 levels at Fzd1 and Fzd7 promoters in AD. No differences are observed at Fzd5 or Fzd9 promoters. C Scheme representing Sanguinarine (SAN) and AS1842856 FoxO1 inhibitor (Fox1Oi) treatment in the in vitro AD organotypic model for 7 days and 72 h respectively. D Scheme representing the levels of Sirt2 at Fzd1 and Fzd7 and upon FoxO1 inhibition in AD. E ChIP-qPCR showing that FoxO1i treatment reduces Sirt2 levels at Fzd1 and Fzd7 promoters in hippocampal organotypic cultures of NLGF while not changing the levels of Sirt2 in WT or at Fzd5 or Fzd9 promoter, suggesting FoxO1 recruits Sirt2 to Fzd1 and Fzd7 promoters in AD. F qPCR analyses of Fzds expression upon FoxO1 inhibition in vehicle (Veh) and Aβo treated neurons, showing that FoxO1i prevents Fzd1 downregulation without modulating Fzd5 or Fzd9 mRNA levels. FoxO1 inhibition downregulates Fzd7 expression per se and fails to prevent its downregulation in Aβo treated neurons. G WB analyses of PP2Cα in hippocampal nuclear extracts of human control/BI-III subjects, sowing increased levels of PP2Cα in human BI–III group. H qPCR analyses of total mRNA levels from WT and NLGF hippocampal organotypic cultures treated with vehicle or SAN. Our results show that SAN treatment rescues Fzd1 and Fzd7 mRNA levels and does not show any effect on Fzd5 or Fzd9 mRNA levels. I Scheme representing increased nuclear levels of the phosphatase PP2C in AD and how PP2C inhibition in AD rescues H4K16ac levels at Fzd1 and Fzd7 promoters and their transcription. J ChIP-qPCR showing that SAN treatment rescues the levels of H4K16ac at Fzd1 and Fzd7 promoters in AD while no changes are observed in WT or at Fzd5 and Fzd9 promoter. Data are represented as mean + SEM. Statistical analyses by t-Test in A for total SIRT2.2 and pSIRT2.1 and pSIRT2.2, and by Mann-Whitney for total SIRT2.1; in B in by t-Test for all genes analysed; E Two-way ANOVA followed by Tukey’s post hoc for all genes analysed; in F by Two-way ANOVA followed by Games-Howell post hoc for all genes analysed; in G by t-Test; in H Two-way ANOVA followed by Tukey’s post hoc for Fzd1, Fzd7 and Fzd9 and Kruskal-Wallis followed by Dunn’s multiple comparison for Fzd5; in J Two-way ANOVA followed by Tukey’s post hoc for Fzd7 and Fzd9 and Kruskal-Wallis followed by Dunn’s multiple comparison for Fzd1 and Fzd5. N is indicated in each bar by the number of symbols. Asterisks indicate *p < 0.05; **p < 0.01; ***p < 0.005.