Figure 9. The dynamics of tau in spines changes is distinct between translocation inducing synaptic activation from Aβ exposure and could be phosphorylation dependent. A, Time lapse of a spine pretreated with Bic/4-AP containing EGFP-tau and showing FRAP. B, Representative FRAP curves (%FRAP normalized to the prebleached intensity of the spine of EGFP-Tau-transfected neurons) of Tau WT (EGFP-Tau 2N4R) after 45 min of Bic/4AP (green, n = 21) or 45 min after Aβo application (red, n = 24) and TauT205A after 45 min of Bic/4AP (blue, n = 21). C, Percentage of EGFP-tau fluorescence or EGFP-tauT205A that was stable (white), dynamic (gray), or unbleached (black). Statistical analysis was performed by 2-tailed t test, ***p < 0.0001. D, Representative Western blot of Tau phospho-Threonine 205 (p-Thr205), phospho-Serine 404 (p-Ser 404), and tau total from PSD-enriched fractions for control, Bic/4-AP (30 min), Aβ (30 min), and Aβ (15 min)+Bic/4-AP (15 min) conditions. E, Quantifications of p-Thr 205 and p-Ser 404 under control, Bic/4-AP, Aβ, or Aβ + Bic/4-AP conditions (mean ± SEM, **,*relative to control; # # #relative to Bic/4-AP, +relative to Aβ, p > 0.05, 1-way ANOVA with Bonferroni's posttest, N = 5 independent cultures). F, Confocal imaging of cortical neurons (14 DIV) cotransfected with EGFP-Tau S404A and LifeAct-RFP. Left, Spatial repartition of EGFP-TauS404A (green) and LifeAct-RFP (red) in untreated neurons. Scale bar, 5 μm. Right, Higher magnification of EGFP-Tau S404A and LifeAct-RFP in dendritic spine neuron after Aβ treatment (15 and 30 min, designated box in the left) by 15 and 30 min of Aβo 100 nm treatment. The merged image shows no colocalization of F-actin and tau. Scale bar, 1 μm. G, Quantification of the fluorescence intensity of EGFP-TauS404A in the head of spines during the Aβo treatment. The graph represents the evolution of ΔF/F0 generated by EGFP-Tau under control conditions (black), EGFP-Tau (red), and EGFP-TauS404A (pink) induced by Aβo 100 nm treatment. Statistical analysis was performed by 2-way ANOVA followed by Bonferroni's posttest (mean ± SEM, ***p < 0.001, control n = 40, N = 3 independent cultures; Tau-WT Aβo 100 nm n = 79, N = 5; Tau S404A n = 98, N = 3 independent cultures).