Scalable human neuronal models of tauopathy producing endogenous seed-competent 4R tau

The accumulation of pathological 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 very limited. To address this, we developed induced pluripotent stem cell (iPSC)-derived i³Neuron (i³N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (Ser³⁰⁵Asn; S305N or S305N/IVS10 + 3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mislocalization. These i³N neurons develop endogenous seed-competent tau and present pentameric formyl thiophene acetic acid-(pFTAA)-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered an 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.