Figure 7.

Differential degradation of tau in cytosol versus vesicles. Native U2OS cells were exposed to tau fibrils overnight, then treated with trypsin, washed, and incubated for indicated times. Cells were then subjected to biochemical fractionation to isolate tau in the cytosol versus organelle (vesicle) fractions. For organelle fractions, seeds were extracted by immunoprecipitation to avoid cytotoxicity of vesicle contents. The respective fractions were transduced with Lipofectamine 2000 into V2L biosensor cells. A and B, time course of tau seeding activity present in cytosol (A) versus organelle (B) fractions revealed degradation in both. A t test was performed to compare samples with the corresponding untreated control (0 h). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. C and D, bafilomycin (Baf; 200 nM) was added to block lysosomal acidification. This slowed loss of tau seeding activity in the organelle fraction but had no effect on seeding within the cytosol. A t test was performed to compare samples with the corresponding untreated control (0 h). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. E and F, MG132 (10 μM) was added to block proteasome activity. E, MG132 had no effect on seeding in the organelle fraction, (F) but slowed cytoplasm tau degradation. Assays were performed in technical triplicate. Data are representative of three similar experiments for MG132 and bafilomycin treatment for cytosolic fractions and two replicates for organelle fractions. Error bars represent SEM. A t test was performed to compare samples with the corresponding untreated control (0 h). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.