In these same samples, we assessed levels of AT8 immunoreactive signal by ELISA. The AT8 signal was lower in the antibody-treated Selleck MDV3100 samples (Figure 6F), similar to what was seen for total tau in this fraction. We hypothesized that a reduction of tau aggregation in brain would correlate with a reduction in seeding activity. Thus, we used the cellular biosensor assay to test for P301S brain seeding
activity in the cortical RAB-soluble fractions from the different treatment groups. Our prior data assessing ISF tau in P301S mice suggested the possible presence of extracellular tau aggregates in equilibrium with both the biochemically soluble and insoluble pools of tau (Yamada et al., 2011). We first assessed intracellular aggregation of RD(ΔK)-CFP/YFP after treating the cells with lysates from mice treated with PBS or HJ3.4. Lysates from these groups strongly induced FRET signal (Figure 7A). We observed markedly less seeding
activity in lysates from the cortical tissue of mice treated with HJ8.5 and HJ9.3 (Figure 7A). ALK inhibitor This was not due to residual antibody in the brain lysates, because immunoprecipitation of the brain lysates followed by elution of seeding activity from the antibody/bead complexes produced the same pattern (Figure 7B). Thus, HJ8.5 and HJ9.3 reduce seeding activity in the P301S tau transgenic mouse brain. HJ9.4 did not significantly reduce seeding activity (Figure 7A). Seeding activity strongly correlated with the amount of detergent-insoluble/formic acid-soluble tau detected by ELISA (Pearson’s r = 0.529, p = 0.0001) (Figure 7C) but did not correlate from with total tau in RAB fractions (Figure 7D). We hypothesized that seeding
activity is due to tau aggregates present in the RAB-soluble fraction. To test for this, we performed SDD-AGE followed by western blot. In addition to tau monomer, we observed higher molecular weight tau species present in 3-month-old P301S mice and a larger amount present in 9-month-old P301S mice (Figure 7E). A component of these higher molecular weight species probably constitutes the seeding activity detected in the FRET assay and may be in equilibrium with the tau present in the detergent-insoluble/formic acid-soluble fraction. In studies of P301S tau transgenic mice at 9 months of age, we compared the control and anti-tau antibody-treated groups in a variety of behaviors. The groups did not differ in locomotor activity, exploration, or measures of sensorimotor function (Figure S8). The ability of the anti-tau antibody treatments to rescue cognitive deficits in P301S mice was evaluated by assessing the performance of the mice on the conditioned fear procedure. On day 1, all four treatment groups of mice exhibited similar levels of baseline freezing during the first 2 min in the training chamber.