# A high-throughput drug screening assay for anti-tau aggregation using split GFP and flow cytometry

## TL;DR

A new cell-based assay using split GFP and flow cytometry enables high-throughput screening of drugs that prevent tau protein aggregation, a key feature in Alzheimer's disease.

## Contribution

The novel method uses suspension-adapted HEK293 cells with split GFP to monitor tau aggregation without external inducers, enabling efficient drug screening.

## Key findings

- The system detects tau aggregation via fluorescent signals from split GFP fragments upon aggregation.
- Validation with a urea-based inhibitor showed dose-dependent reduction in tau aggregation and fluorescence.
- The assay allows rapid, quantitative analysis of drug efficacy and cytotoxicity using flow cytometry.

## Abstract

Tau protein aggregation is a hallmark of neurodegenerative diseases, including Alzheimer’s disease, making the development of anti-aggregation therapeutics a critical area of research. Progress in drug discovery has been hindered by the lack of efficient screening methods that accurately reflect cellular conditions. We present a high-throughput cell-based assay utilizing split GFP technology to monitor tau aggregation in living cells. Our system employs suspension-adapted HEK293 cells co-transfected with tau proteins fused to complementary GFP fragments, producing fluorescent signals upon tau aggregation. Notably, our system demonstrates tau aggregation without external aggregation inducers, likely due to the enhanced protein expression in suspension-adapted cells. Validation with a known urea-based tau aggregation inhibitor showed dose-dependent reduction in fluorescence, corresponding to decreased tau aggregation. The assay’s flow cytometry compatibility enables rapid, quantitative analysis of large sample sets while allowing simultaneous assessment of compound efficacy and cytotoxicity. This method advances tau aggregation monitoring and drug discovery by providing a physiologically relevant platform for identifying novel anti-tau aggregation therapeutics.

The online version contains supplementary material available at 10.1038/s41598-025-21680-5.