Human WT tau is more subjected to phosphorylation in the mouse forebrain than P301S and 2P tau. (a) AlphaLisa assays are performed on forebrain protein homogenates to determine the levels of human tau phosphorylation at the indicated residues, as function of age (1.5, 3 and 7 months after injection) and tau variant (AAV-WT or AAV-P301S). Data represent relative levels of phosphorylated tau, normalized to the total amount of human tau determined in a parallel AlphaLisa. Note that WT tau is more heavily phosphorylated than P301S tau at residues Ser396, Ser 202/Thr 205, Thr 181 and Thr 231. For all conditions: n = 6. Statistical analysis: two-way ANOVA followed by Tukey’s post-hoc test, *p < 0.05; **p < 0.01; ***p < 0.001. (b) Representative images of phosphorylated tau deposition, 7 months after injection of AAV-WT and AAV-P301S. Immunolabeling with anti-phospho tau antibodies AT8 (Ser202/Thr205) in the cortex (left panels) and with PHF1 (Ser396) in the hippocampus (right panels) are shown. Note the localization of P301S tau in the neuronal soma and dendrites. Scale bar: 100 μm. (c) AlphaLisa quantification of human tau phosphorylation at residues Ser 202/Thr 205, Thr 231, Ser 396 and Thr 181 in mouse forebrain homogenates, 7 months after injection of either AAV-WT or AAV-2P. The graphs represent the levels of phosphorylated tau relative to total human tau; n = 8 mice per group. Statistical analysis: two-tailed Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001. (d) Representative immunodetection of phosphorylated tau using the AT8 (Ser 202/Thr 205) and PHF1 (Ser 396/Ser 404) antibodies in sagittal sections of the mouse cortex 7 months after injection of either AAV-WT or AAV-2P. Note the decreased immunoreactivity for phosphorylated tau in AAV-2P-injected mice. Scale bar: 100 μm.