N-Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission
N-Linked glycosylation of Tau is implicated in Alzheimers disease, yet how it influences Tau propagation and neuronal uptake remains unclear. Here, we use chemically defined N-glycoforms of 2N4R Tau and the K18 repeat domain to map how glycan site and structure reprogram Tau-polyanion binding, seeding, and entry into human neurons. Site-specific GlcNAc or high-mannose glycans at Asn359 or Asn410 remodel heparin and RNA binding kinetics in biolayer interferometry assays, revealing avidity-dominated, slow-off heparin interactions that are dampened by N-glycans and faster RNA interactions that can be potentiated by small GlcNAc modifications. Cross-seeding experiments show that N-glycosylation selectively tunes nucleation compatibility between Tau proteoforms; GlcNAc at Asn410 in full-length Tau and small glycans at Asn359 in K18 most strongly accelerate aggregation. In HEK293T FRET biosensor cells and iPSC-derived cortical neurons, N-glycans enhance or suppress seeding and pHrodo-reported uptake in a site- and glycan-dependent manner, with Asn410 glycosylation markedly potentiating cellular seeding. Pharmacological inhibition with atropine, dynasore, and cytochalasin D further indicates that N-glycans rebalance muscarinic, dynamin-dependent, and actin-dependent uptake routes. Together, these data establish N-linked glycosylation as a tunable regulator of Tau trafficking and prion-like spread, coupling extracellular receptor usage to intracellular cofactor engagement, and reveals druggable nodes in uptake.