Since PIKfyve inhibition did not reduce the neuronal internalization of K18 PFFs, we hypothesized that the reduction in seeded tau aggregation might be caused either by a change in the fate of the seeds with consequent reduced release into the cytoplasm or by increased degradation. We started by evaluating how YM-201636 could affect the subcellular distribution of K18 PFFs in neurons. We treated primary hippocampal neurons with either 1% DMSO or 1 μM YM-201636 followed by incubation with AF488-labeled K18 PFFs for 16 h to allow internalization and transport into EDITORS' PICK: PIKfyve mediates tau aggregates transport and its seeding lysosomes. As mentioned before, LAMP1-positive organelles in neurons treated with YM-201636 appeared enlarged and reduced in number when compared with DMSO control (Fig. S8B). While there were no changes in the amount of K18 PFFs being internalized by neurons, the percentage of both K18 and FL Tau PFFs colocalizing with LAMP1 vesicles was reduced by 50% after YM-201636 treatment (Fig. 6, A and B). XYZ projection imaging shows that the majority of K18 PFFs can be seen close to LAMP1-positive vesicles, but not inside (Fig. S8A). On the other hand, K18 PFFs are slightly increased in number in EEA1-positive vesicles as well, thus pointing to a partial trapping or delayed transit of labeled K18 PFFs in earlier endosomal compartments. Because an absence of K18 PFFs in lysosomes could potentially be explained by an increased degradative capacity, we investigated lysosomal proteolytic activity. We used DQ-BSA, a self-quenchable substrate for hydrolase activity that, following endocytic uptake and delivery to degradative compartments, produces fluorescence upon cleavage. The intensity of DQ-BSA positive vesicles was decreased by 27% when primary hippocampal cultures were treated with 1 μM YM-201636, thus indicating reduced transport of cargo into proteolytic vesicles and consequently lowered degradation of substrates and excluding increased hydrolytic activity (Fig. 6G). YM-201636 treatment also reduced the number of cathepsin B-and D-positive vesicles, corroborating the DQ-BSA **data** and the idea of impaired degradation capacity in neurons (Fig. 6, H and I).