Conformational changes in the tau protein, including transition towards a “paperclip-like” conformation, or towards the formation of β-structures aggregating into PHF, occur in the development of tauopathies26, and can precede the formation of NFTs27. We analyzed the presence of misfolded tau in the brain of AAV-injected mice using conformation-specific tau markers. Immunohistochemistry for MC1, an antibody specific for early changes in tau conformation, revealed the presence of abnormally folded tau in the brain regions overexpressing human tau, including the cortex and hippocampus (Fig. 4a). The staining was observed in the AAV-WT, AAV-P301S and in the AAV-2P groups of mice, 7 months after vector injection.Figure 4Aggregated forms of human tau progressively accumulate in the brain of mice injected with AAV-P301S. (a) Representative images of the hippocampus immunolabeled with the conformational MC1 antibody. Note the presence of MC1-positive neurons and neurites, 7 months after injection of either AAV-WT, AAV-P301S or AAV-2P. Scale bar: 100 μm. (b) Quantification of MC1-positive misfolded tau in forebrain protein homogenates using an HT7/MC1 AlphaLisa at 1.5 and 7 months post-injection. Statistical analysis: Kruskal-Wallis test, H(2, N = 38) = 28.25. (c) HT7/MC1 values are normalized for total human tau determined with a HT7/TAU-13 AlphaLisa. (d) The amount of human tau multimers is determined with a HT7/HT7 single-epitope AlphaLisa and normalized for total human tau. For c and d: data represent relative arbitrary units. Statistical analysis: two-way ANOVA followed by Tukey’s post-hoc test, group × time effect (c) F(2, 36) = 6.55, (d) F(2, 36) = 3.88, *p < 0.05; **p < 0.01; ***p < 0.001. (e) Significant correlation between the amount of MC1-immunoreactive human tau and human tau multimers (Pearson). (b–d) 1.5 and 3 months: AAV-maxFP: n = 2; AAV-WT and AAV-P301S: n = 6. 7 months: AAV-maxFP: n = 6; AAV-WT: n = 8; AAV-P301S: n = 10 mice. (f–h) Representative histological analysis, 7 months after AAV-P301S injection: (f) AT100 immunolabeling of phosphorylated tau in the hippocampus (CA1). Scale bar: 100 µm. (g) Thioflavine-S positive neurofibrillary deposits in cortical neurons. Scale bar: 25 µm. (h) Gallyas silver staining of argyrophilic filamentous inclusions in cortical neurons. Scale bar: 25 μm. (i) Detection of misfolded tau (HT7/MC1 AlphaLisa) in mouse forebrain homogenates, at 3 and 7 months after either AAV-WT or AAV-2P injection. Single mouse values were normalized for total human tau (HT7/TAU-13 AlphaLisa); n = 8 mice per group. (j) Detection of tau multimers (HT7/HT7 single-epitope AlphaLisa); n = 8 mice per group. Statistical analysis: two-tailed Student’s t test.