Finally, we investigated the phosphoinositide kinase family, PIP5K (43). Within the PIP5K family, subfamilies can be distinguished, which include PIP5K (isoforms a,b,c) and PIP4K (isoforms a,b,c), which synthetize PI 4,5 P 2 (44), and PIKfyve, responsible for PI 3,5 P 2 formation (45). PI 4,5 P 2 modulates the initial steps of endocytosis through different mechanisms, including the modulation of actin polymerization by sequestering of actin binders (46); recruitment of proteins that mediate membrane curvature (e.g., BIN1) (47), and interaction with specific heparan sulfate proteoglycans (HSPG), glycancoated proteins that are reported to interact with K18 PFFs (17). On the other hand, PI 3,5 P 2 regulates endosomal vesicle maturation, in particular the maturing steps between the multivesicular body (MVB), late endosomes and lysosomes, including associated recycling events (48). We decided to target the different subfamilies of PIP5K using LV-shRNAs and test how these affect the process of K18 PFFs internalization using live-cell imaging. After imaging, samples were lysed and effective knockdown was confirmed through RT-qPCR. We tested the efficacy of three distinct LV-shRNAs in knocking down Pikfyve. LV-shRNA1 and 3 efficiently knocked EDITORS' PICK: PIKfyve mediates tau aggregates transport and its seeding down Pikfyve resulting in a concomitant significant decrease in K18-pHrodo signal in neurons. LV-shRNA2 was less effective in the knockdown of Pikfyve, giving rise to likewise less K18-pHrodo signal. (Fig. 3, B and C). Furthermore, none of the control LV-shRNA used could affect Pikfyve mRNA expression of K18-pHrodo signal. These **data** suggest that PIKfyve function may be an important regulator in K18 PFFs internalization. Data on the effect of other PIP5K family knockdown can be consulted in Figure S5. Because the PIKfyve loss of function elicited the highest reduction in the K18-pHrodo signal, we decided to continue our study focusing on PIKfyve.