Tau seeds translocate across the cell membrane to initiate aggregation

Neurodegenerative tauopathies, including Alzheimers disease and related disorders, are caused by intracellular aggregation of tau protein in ordered assemblies. Experimental evidence suggests that tau assemblies propagate pathology across brain networks. Tau seeds enter cells through endocytosis but must access the cytoplasm to serve as templates for their own replication. The mechanism by which this occurs is unknown. To study tau uptake, we began with a whole-genome CRISPR knockout screen, which indicated a requirement vacuolar H+ ATPase (v-ATPase) components. Treatment with Bafilomycin A1, an inhibitor of the v-ATPase, also reduced tau entry. We next tested direct modifiers of endolysosomal trafficking. Dominant-negative Rab5a expression uniquely decreased tau uptake, as did temporary cold temperature during tau exposure, consistent with a primary role of endocytosis in tau uptake. However, despite reducing tau uptake, these interventions all paradoxically increased intracellular seeding. Consequently, we generated giant plasma membrane vesicles (GPMVs), which cannot undergo endocytosis, and observed that tau fibrils and monomer translocated into the vesicles, in addition to TAT peptide, whereas transferrin and albumin did not. In every case, tau required binding to heparan sulfate proteoglycans (HSPGs) for cell uptake, seeding, or GPMV entry. These findings are most consistent with direct translocation of tau seeds across the lipid bilayer, a novel mechanism of entry into the cytoplasm.