Endolysosomal inhibition uncouples tau uptake from intracellular seeding.

Neurodegenerative tauopathies, including Alzheimer's disease, appear to be driven by propagation of tau assemblies, which must access the cytoplasm to recruit monomer and self-replicate, a process termed "seeding." The prevailing model holds that tau seeds enter cells via macropinocytosis and reach the cytosol through lysosomal rupture or micro-perforation. Our findings revise this model by revealing that endocytosis is not required for seeding. Using genome-scale CRISPR screening, we identified multiple v-ATPase components whose loss reduced tau uptake (measured by flow cytometry) yet paradoxically increased seeding (measured by FRET biosensors). Acute v-ATPase inhibition with bafilomycin A1 produced the same effect in v2L tau biosensors and iPSC-derived neurons. Among regulators of endosome maturation, dominant-negative Rab5a decreased internalization while enhancing cytoplasmic templating. Cholesterol depletion produced identical results. Strikingly, transient hypothermia eliminated virtually all detectable tau uptake and dramatically increased seeding, without affecting subsequent tau monomer or aggregate degradation. We conclude that efficient endolysosomal trafficking does not appear to be required for cytoplasmic seeding under the conditions studied here. Across diverse perturbations, reduced endolysosomal flux consistently enhanced tau seeding, consistent with prior work indicating that most internalized aggregates are routed toward degradation rather than amplification. To seed effectively, tau must cross the plasma or vesicular membranes into the cytoplasm. We have found that proper endolysosomal trafficking suppresses cytoplasmic tau seeding, as all perturbations augmented this process. These findings reframe the role of the endolysosomal system in tau seeding and identify membrane transit rather than macropinocytosis itself as a critical gateway to cytoplasmic tau amplification.