Figure 2.

Synaptic activation induced by Bic/4-AP triggers translocation of tau into dendritic spines of primary cortical neuron cultures. A, Confocal imaging of cortical neurons (14 DIV) cotransfected with EGFP-Tau and LifeAct-RFP. Left, Spatial repartition of EGF-Tau (green) and LifeAct-RFP (red) in nonactivated neuron. The merged image displays only minor colocalization (yellow). Scale bar, 5 μm. Right, Higher magnification of EGFP-Tau, LifeAct in dendritic spines (designated box in the left) of a neuron after synaptic activation triggered by 15 and 30 min of Bic/4AP exposure. The merged image shows the activity driven colocalization of tau/LifeAct (yellow) in the activated dendritic spine after 15 and 30 min Bic/4-AP exposure. Scale bar, 1 μm. B, Quantification of fluorescence intensity mediated by EGFP-Tau in the head of spines during synaptic activation (Bic/4-AP treatment). The graph represents the evolution of ΔF/F₀ generated by EGFP-Tau in neurons subjected to synaptic activation or not. Statistical analysis was performed by two-way ANOVA followed by Bonferroni's posttest (mean ± SEM, ***p < 0.0001, control n = 40 spines on 3 independent cultures; Bic/4-AP n = 162 spines on 5 independent cultures). C, Representative Western blot of endogenous tau expression in a whole lysate extract of cortical neurons (14 DIV) exposed to Bic/4-AP for 15 and 30 min. Quantification of tau expression normalized to actin levels does not display any change. D, Representative Western blots of protein expression from primary cortical cultures after fractionation. Presynaptic protein (synaptophysin), postsynaptic proteins (PSD-95, GluA1, GluN2A, GluN2B, Fyn), cytoskeletal protein (actin), and tau in non-PSD fraction (Triton-soluble fraction) and PSD-fraction (Triton-insoluble fraction) on primary cortical neurons treated 15 min with Bic/4-AP. E, Quantitative analysis of PSD-95, GluA1, GluN2B, GluA1, Fyn, actin, and tau in the PSD fraction under control and Bic/4-AP (15 min) treated conditions is shown in graphs (mean ± SEM, 2-tailed Student's t test,***p < 0.001, **p = 0.0026, *p = 0.0101, N ≥ 7 independent cultures).