Figure 2.

Aggregated tau directly inhibits 26S proteasomes and associates with proteasomes in brains of mice with tauopathy. (a) Immunoblot analysis for tau and phosphorylated (pS396 and pS404) tau epitopes from soluble and insoluble fractions in HEK293 cells nontransfected (NT) or stably transfected with WT human tau (hTau) or double-mutant P301L and V337M tau. (b) Native PAGE assay (top) and quantification (bottom) of 26S proteasome activity and levels (assessed by immunoblotting for β5 subunit) in NT HEK293 cells and HEK293 cells transfected with WT hTau or P301L and V337M tau. The densitometric quantification of 26S proteasome activity normalized to 26S proteasome levels (c) Immunoprecipitation with antibody to human tau of cortical brain lysates from Mapt−/− (negative control) and rTg4510 mice at indicated ages and immunoblot analysis of Rpt6, Rpt5, Rpn5 and 20S α-subunits 1–7 (α1–7) expression and a reciprocal experiment using anti-Rpt6 for immunoprecipitation and anti-hTau for immunoblotting. The total extracts (input) for tau and Rpt6 are shown as loading controls. (d,e) Rate of succinyl-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc) hydrolysis by 26S proteasomes, incubated with tau monomers, LMW aggregates and fibrils generated from recombinant tau (d), or incubated with tau monomers and LMW aggregates generated by incubating recombinant tau at indicated time points (e). Untreated WT 26S proteasomes were used as a control for d and e. At least three biological replicates were performed with stably transfected clones (a,b). For immunoprecipitation (c), two animals per experiment were analyzed in three biological replicates (n = 6 mice). Purified proteasomes from n = 6 brains from 3-month-old WT were used for d and e, and at least three independent experiments were performed (d,e). Error bars, means ± s.e.m. *P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey’s multiple comparison post hoc test).