Scalable human neuronal models of tauopathy producing endogenous seed-competent 4R tau
Pathological accumulation of four-repeat (4R) tau is central to several frontotemporal dementia (FTD) subtypes but human neuronal models amenable to high-throughput screening of 4R tau-targeting therapies remain limited. To address this gap, we developed iPSC-derived i3Neuron (i3N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (S305N or S305N/IVS10+3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mis-localisation. Unlike any other stem cell model of 4R tauopathy, these i3N neurons develop endogenous seed-competent tau and present pFTAA-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered a HiBiT luminescence tag at the endogenous MAPT locus into the S305N/IVS10+3 iPSC line, enabling precise quantification of tau levels and pharmacological responses. The model responded predictably to compounds affecting tau clearance, demonstrating its suitability for drug discovery. Overall, this i3N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.