Figure 4. Salicylate Inhibits p300 and Reduces Ac-Tau (K174) In Cultured Neurons and PS19 mice.
(a–c) Genetic deletion of p300 lowers levels of ac-K174, p-tau and total tau. (a) Lysates from primary mouse neurons that were derived from p300F/F pups, and infected with lenti-WT tau and lenti-control or lenti-cre virus were immunoblotted with anti-p300, ac-tau (AC312), p-tau (AT8), or t-tau (Tau5) antibody. (b) Quantification of levels of total tau (t-tau) (left), ac-K174 (middle), and AT8-positive p-tau (right). (c) Quantification of the levels of ac-K174 (left) or p-tau (right) relative to t-tau. Levels in non-treated cells were set as 1. n=11 from three independent experiments, *** p < 0.001, ** p < 0.01, unpaired student t-test (ac-tau), Kruskal-Wallis test (p-tau). (d) Rat primary cortical neurons infected with lenti-WT tau were treatment with 0, 5, 10 mM salicylate for 24 hr (DIV 12). Representative immunoblot of p300, ac-tau, p-tau and t-tau. Quantification of the levels of p300 (left), ac-tau (middle) or AT8-positive p-tau (right) relative to t-tau, in primary rat neurons treated with salicylate. Levels in non-treated cells were set as 1. n = 4 from 4 independent experiments.. *, p < 0.05, **, p < 0.01, one –way ANOVA, Tukey-Kramer post-hoc analyses. (e) Representative immunoblot and quantification of ac-H3K18 and H3 in cortical histone extract from 10–11-month-old NTG and PS19 littermates. Levels in NTG group were set as 1. n=9 mice/group, ** p < 0.01, unpaired student t-test. (f–h) Oral gavage of SSA (225 mg/kg) inhibits p300 activity in the brain. Representative immunoblot and quantification of ac-H3K18 and H3 (f, male, n=11/group), ac-H2AK5 and H2A (g, female, n=8/group) or ac-H2BK12K15 and H2B (h, male, n=11/group). Levels in vehicle-treated group were set as 1. * p < 0.05,unpaired student t-test. (i) SSA treatment lowers levels of total hTau protein, but not mRNA. Levels of total hTau protein were measured by ELISA (left). Levels of hTau mRNA were measured by qRT-pCR (right). Levels in vehicle-treated group were set as 1. n=11 mice/condition, 10–11 months old male, * p < 0.05, unpaired student t-test. ns=not significant. (j) SSA treatment lowers levels of ac-tau relative to total tau. Representative immunoblot of ac-tau (AC312), p-tau (AT8), and t-tau (Tau5). Vehicle-treated non-transgenic NTG and tau KO mice were included as controls. Quantification of ac-tau (left) or AT8-positive p-tau relative to t-tau (middle) or GAPDH (right), in the soluble hippocampal lysates from PS19 mice treated with vehicle or SSA. Levels in vehicle-treated group were set as 1. n=8 mice/condition, 10–11 months old female, ** p < 0.01, *, p < 0.05, unpaired student t-test. Values are mean ± SEM (b–j). WT, wild-type. NTG, non-transgenic.