The deposition of abnormal tau hyperphosphorylated on several residues is a typical feature of several tauopathies. The phosphorylation pattern of tau was therefore assessed in the brain homogenates obtained from the mice at different ages. For quantitative determination of tau phosphorylation at specific sites associated with tauopathies29, several additional AlphaLisa assays were established. As before, we used the HT7 or TAU-13 capture antibodies coupled to the donor beads, whereas detection was performed with monoclonal antibodies specific to different phosphorylated tau residues coupled to the acceptor beads. For each sample, the abundance of the phosphorylated epitopes was normalized to the total amount of human tau, in order to also take into account the different levels of WT and P301S tau as a function of age. Overall, the data demonstrated tau phosphorylation as early as 1.5 months after vector injection in both the AAV-WT and AAV-P301S groups. However, phosphorylation extent varied for most residues, as a function of age and presence of the P301S mutation (Fig. 5a).Figure 5Human 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.