Following injection of the WT tau-encoding vector, microtubule arrangement appeared normal and indistinguishable from the normal situation (Fig. 6a,b). However, in both the P301S and 2P tau groups, local increases in the density of the microtubule network were observed mainly in axons (Fig. 6c,d). This includes the axonal boutons, where the microtubules could also be seen close to the synaptic vesicles, and active zone (Fig. 6d). Microtubules were often organized in bundles with hexagonal symmetry, similar to the tau-induced microtubule fascicles described previously30. To quantify this effect, the number of microtubules was determined in individual axons. In both the P301S and 2P conditions, the number of microtubules per axon was significantly increased compared to either non-injected or AAV-WT injected mice (Fig. 6e). When the number of microtubules was normalized to the cross-sectional area of the axon, we also observed a significant increase in density in the P301S and 2P mice versus the control non-injected mice (Fig. 6f). In contrast, the microtubule density was not changed in the dendrites (Fig. 6g). Therefore, we find that the P301S and 2P variants of tau lead to clear changes in microtubule number and density. These results suggest that these two forms of tau, which have a modified amino-acid sequence in the microtubule-binding domain, are more likely to interact with tubulin than WT tau, which is consistent with the reduced level of tau phosphorylation observed in these conditions.Figure 62P-modified and P301S tau increase microtubule number and density in axonal profiles in the mouse cortex. Representative electron micrographs from layer II/III of mouse neocortex neuropil, 7 months post-vector injection. Images are taken close to the site of injection. (a,b) In the mouse injected with AAV-WT, the neuropil shows normal morphology with microtubules scattered in axon terminals and dendrites, similar to the ultrastructure observed in a non-injected mouse (normal cortex). (c,d) In mice injected with either AAV-P301S or AAV-2P, there is an abundance of cytoskeletal elements, with high densities of aligned microtubules (indicated by white arrowheads) located in axons. These are also seen in the axonal boutons (indicated by *). Scale bars: 500 nm. (e) Quantification of the number of microtubules per axon in each condition. (f,g) Quantification of microtubule density in individual axons. Note the significant increase in microtubule number and density in the AAV-P301S and AAV-2P conditions. Statistical analysis: one-way ANOVA followed by Tukey’s post-hoc test, n = 29–30 axons per condition, *p < 0.05; **p < 0.01; ***p < 0.001. (h) Representative electron micrographs from the neocortex, 7 months post-injection of the AAV-P301S tau vector. Enlarged axons containing high densities of aligned microtubules (indicated by #) are present, as well as axonal swellings containing large accumulations of degenerated organelles including whorls, membrane inclusions and degenerating mitochondrial profiles (white arrows). Scale bars: 1 µm (left panels) and 500 nm (right panel).