Fig. 4
Pseudo-phosphorylated tau relocates the AIS down the axon in a process mediated by microtubules. Representative photomicrograph of A14- (top) and E14-tau-EGFP (lower) transfected hippocampal neurons stained for ankyrin G a 24 h and b 48 h after transfection. Arrows indicate the start, middle and end of the AIS as determined by quantitative analysis of the c, d axonal fluorescence profile. The dashed line indicates the normalized detection threshold. Quantification of ankyrin G labeling in neurons transfected with E14- (blue) and A14-tau (black) for e 24 h (start p = 0.48, middle p = 0.94, end p = 0.54; E14 n = 61, A14 n = 46) and f 48 h (start p = 0.0001, middle p = 0.0027, end p = 0.0017; E14 n = 47, A14 n = 62). AIS location of E14- and A14-tau-transfected neurons labeled for g βIV spectrin (start p = 0.0041, middle p = 0.0024, end p = 0.0084; E14 n = 55, A14 n = 52) and h NaV1.6 (start p = 0.0042, middle p = 0.0045, end p = 0.0316; E14 n = 53, A14 n = 53). Using ankyrin G staining, the role of site-specific phosphorylation in AIS relocation was determined: i AT180 (start p = 0.0138, middle p = 0.44, end p = 0.76; AT180E n = 53, AT180A n = 52), j 12E8 (start p = 0.0183, middle p = 0.0352, end p = 0.09; 12E8E n = 54, 12E8A n = 51), and k PFH1 (start p = 0.34, middle p = 0.56, end p = 0.75; PHF1E n = 50, PHF1A n = 38). l Treatment with 0.1 µM taxol (magenta) prevents AIS relocation (start, ANOVA, F = 8.05, p = 0.0005; middle, ANOVA, F = 4.057, p = 0.0191; end, ANOVA, F = 6.614, p = 0.0017; E14 n = 57, A14 n = 54, E14+ taxol n = 51). m Microtubule destabilization with 0.05 µM nocodazole (green) relocates the AIS (gray, control, start: p = 0.0169, middle: p = 0.0297, end: p = 0.0856; nocodazole n = 54, control n = 55). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p ≤ 0.0001. Data presented as mean ± SEM. Scale bar 5 µm. Statistical comparisons were made using an unpaired two-tailed Student’s t test or a one-way ANOVA with a Sidak’s post hoc test