Figure 2. Disease-associated tau seeds preferentially accumulate in autophagic vesicles.

(A) Primary neurons were treated with WT or P301L tau seeds for 2 days and fractionated into vesicular and cytosolic fractions. The presence of WT and P301L seeds within the biochemical fractions was evaluated by immunoblotting with site-specific tau antibodies and markers of vesicular and cytosolic fractions, including EEA1, LC3, and GAPDH. The tau MTBR fragment is labeled as tauRD, which migrates as monomers and dimers, whereas endogenous full-length mouse tau is labeled as mouse tau.

(B) Quantification of tau seeds (monomer and dimer) present in the vesicular fraction was performed with site-specific or total tau antibodies. The extent of tau modifications was normalized to total tau levels. Error bars indicate SEM; n = 3 biologically independent experiments. p value was determined by unpaired t test. *p < 0.05.

(C) Primary neurons were treated with myc-tagged WT or P301L tau seeds and analyzed by double-labeling with the myc antibody (red) to mark tau seeds in combination with LC3 antibody (green). The arrows in the inset highlight regions of co-localization. Scale bar, 50 mm.

(D) Quantification of co-localization between tau seeds and vesicular markers was determined as a ratio of the number of co-localized tau seeds per total number of tau seeds. Error bars indicate SEM; n = 6 biologically independent experiments. p value was determined by unpaired t test. n.s. p > 0.05, ***p < 0.001.

(E) Primary neurons were treated with WT or P301L tau seeds for 2 days in the presence or absence of 3MA or MG-132 to impair autophagic or proteasome-mediated degradation, followed by immunoblotting with ac-K280 and total tau antibodies.

(F) Quantification of tau seed acetylation normalized to total tau seed levels. Error bars indicate SEM; n = 3 biologically independent experiments. p value was determined by one-way ANOVA with Tukey’s test for multiple comparisons among groups. n.s. p > 0.05, **p < 0.01.