Figure 6.
Accumulation of insoluble tau and induction of pathological tau conformation after targeted tau expression. A, Tau solubility profile from lysates of infected hippocampal slices. The profile was generated using buffers of increasing stringency: high salt, 1% Triton, RIPA buffer, 2% SDS, and 70% formic acid. In wt tau (441 and 352 isoform)-infected slices, the majority of tau was soluble and present in the high salt extract with almost no insoluble tau. In contrast, a significant portion of PHP tau isoforms was insoluble. No tau was detected in the 70% formic acid fraction. An equal amount of the material from each extraction step was loaded, and Tau-5 antibody was used for immunoblot analysis. B, Schematic representation that indicates the epitopes of the conformational-dependent antibodies Alz50 and MC1. C, Immunoblot of lysates of infected hippocampal slices. The PHP tau proteins showed a strong increase in immunoreactivity for Alz50 and MC1 antibodies compared with the respective wt proteins. D, E, Quantitation of the immunoblots. Conformational alteration for PHP tau was increased for both adult and fetal tau isoforms at different times after infection (day 3 and day 4). To determine quantitatively the alterations in EGFP–tau conformation, all data obtained with conformation-dependent antibodies were normalized to total tau (Tau-5) levels in the samples, and then the difference in conformation between EGFP–PHP tau and EGFP–wt tau was determined. Data for EGFP–PHP tau are expressed as percentage of the value for EGFP–wt tau (wt tau set as 100%). All values are shown as the mean ± SEM from three independent experiments. Data were analyzed using Student's t test and considered to be significantly different from EGFP-wt tau at ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. F, Double immunofluorescence staining of all infected neurons stained with the MC1 antibody showed a subcellular distribution closely reflecting the EGFP fluorescence. Scale bar, 100 μm.