We semiquantitatively assessed ThioS staining using a blinded rater who gave a score from 1 (no staining) to 5 (maximum staining) in all control and anti-tau antibody-treated mice. By semiquantitative assessment, HJ8.5 treatment significantly reduced ThioS staining compared to PBS and HJ3.4 (Figures 6A and 6B). We also stained mice treated with PBS, HJ8.5, and HJ9.3 (n = 6 from each group) with PHF1 monoclonal antibody, which recognizes tau phospho-residues Ser396 and Ser404 (Otvos et al., 1994). AT8 and PHF1 staining significantly correlated (r = 0.630, p = 0.005) (Figure S7A), showing that two anti-phospho tau antibodies to different tau epitopes give similar
results. Many neurodegenerative diseases, including tauopathies, exhibit microglial activation in areas of the brain surrounding protein
aggregation and cell injury. We assessed microglial activation in the treatment groups using anti-CD68 antibody ERK inhibitor mw (Macauley et al., 2011) (Figures S6C–S6G). HJ8.5 and HJ9.3 treatment reduced microglial activation in piriform cortex, entorhinal cortex, and amygdala compared to controls (Figures S6C–S6G). HJ9.4 had a weaker effect in the piriform cortex compared to HJ8.5 and HJ9.3 (Figures S6E–S6G), consistent GW-572016 in vivo with the AT8 staining results (Figure 4A). Microglial activation strongly correlated with AT8 staining in all samples (r = 0.511, p = 0.0038) (Figure S7B). To determine the level of soluble and insoluble tau in the cortex, we performed sequential biochemical extraction with RAB (aqueous buffer), radio immunoprecipitation assay (RIPA) (detergent buffer), and 70% formic acid (FA) to solubilize the final pellet. We quantified total tau by ELISA with anti-tau antibody HJ8.7, which detects both human and mouse tau with the same
KD (0.34 pM). We excluded the possibility that the treatment antibodies would interfere with the ELISA by spiking positive control samples with these antibodies prior to analysis and observing no interference (data not shown). We analyzed all mice that were assessed by pathological analysis in Figure 5. Total tau levels in the RAB (Figure 6A)- or RIPA (Figure 6B)-soluble fractions were similar among all groups. We analyzed the detergent-insoluble/70% until FA-soluble fractions by neutralizing the samples prior to ELISA and western blot. We analyzed every animal studied and found that HJ8.5 and HJ9.3 decreased detergent-insoluble tau by >50% versus controls (Figure 6C). Representative samples (n = 4 from each group) illustrate by western blot decreased levels of insoluble tau in mice treated with HJ8.5 and HJ9.3 (Figure S7C). Insoluble tau levels were no different in HJ9.4-treated groups versus PBS or HJ3.4. We also assessed human and mouse tau specifically in the detergent-insoluble/70% FA-soluble fractions in n = 6 mice per group in which the mean AT8 staining reflected the mean values of results in Figure 4.