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

Area of Science:

• Neuroscience
• Stem Cell Biology
• Genetics

Background:

• Pathological accumulation of four-repeat (4R) tau protein is a hallmark of frontotemporal dementia (FTD).
• Existing human neuronal models for studying 4R tauopathy and screening therapies are limited.
• Developing robust models is crucial for advancing FTD therapeutic strategies.

Purpose of the Study:

• To create a human neuronal model for high-throughput screening of therapies targeting 4R tau accumulation in FTD.
• To engineer induced pluripotent stem cell-derived neurons (i³Neuron) that accurately recapitulate key features of 4R tauopathy.
• To validate the utility of this model for drug discovery and development.

Main Methods:

• Developed induced pluripotent stem cell (iPSC)-derived i³Neuron lines expressing high levels of 4R tau, driven by FTD-associated mutations (S305N or S305N/IVS10+3).
• Utilized CRISPR-Cas9 gene editing to introduce an HiBiT luminescence tag at the endogenous MAPT locus for precise tau quantification.
• Cultured i³Neuron lines for 28 days to observe the development of hyperphosphorylated, mislocalized, and seed-competent tau assemblies.

Main Results:

• The engineered i³Neuron lines expressed >75% 4R tau and exhibited hyperphosphorylation and somatodendritic mislocalization.
• Neurons developed endogenous seed-competent tau and pentameric formyl thiophene acetic acid (pFTAA)-positive tau assemblies.
• The CRISPR-engineered HiBiT-tagged i³N model demonstrated predictable responses to compounds targeting tau clearance, confirming its suitability for drug screening.

Conclusions:

• The developed i³Neuron platform effectively models key pathological features of 4R tauopathy.
• This robust human neuronal system provides a valuable tool for identifying therapeutic modulators of pathological tau.
• The model facilitates scalable screening for novel FTD therapies.