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

Area of Science:

• Neuroscience
• Biochemistry
• Drug Discovery

Background:

• Tau protein aggregation is a key feature of neurodegenerative diseases, such as Alzheimer's disease.
• Developing effective anti-aggregation therapeutics is crucial but challenged by a lack of suitable screening methods.
• Existing methods often fail to accurately replicate cellular conditions for drug screening.

Purpose of the Study:

• To develop a high-throughput, cell-based assay for monitoring tau aggregation in living cells.
• To create a physiologically relevant platform for identifying novel anti-tau aggregation therapeutics.
• To enable efficient screening of potential drug candidates.

Main Methods:

• Utilized split GFP technology in suspension-adapted HEK293 cells for real-time tau aggregation monitoring.
• Co-transfected cells with tau proteins fused to complementary GFP fragments, generating fluorescence upon aggregation.
• Employed flow cytometry for rapid, quantitative analysis and simultaneous assessment of compound efficacy and cytotoxicity.

Main Results:

• The assay successfully detected tau aggregation in cells without external inducers, attributed to enhanced protein expression.
• A known urea-based tau aggregation inhibitor demonstrated a dose-dependent reduction in fluorescence, confirming assay sensitivity.
• The system proved compatible with flow cytometry for high-throughput screening.

Conclusions:

• The developed split GFP assay provides a robust and efficient platform for studying tau aggregation.
• This method significantly advances drug discovery for neurodegenerative diseases by offering a physiologically relevant screening tool.
• It facilitates the identification of novel therapeutics targeting tau aggregation.