Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Area of Science:

• Neuroscience
• Biochemistry
• Cell Biology

Background:

• Neurodegenerative diseases, including Alzheimer's and tauopathies, are linked to the spread of toxic protein aggregates called proteopathic seeds.
• Tau seeds play a critical role in disease progression, necessitating sensitive detection methods in biological samples.

Purpose of the Study:

• To engineer an ultra-sensitive assay for detecting tau seeding activity.
• To enable the study of transcellular propagation of proteopathic seeds in neurodegenerative diseases.

Main Methods:

• Developed a FRET-based assay using monoclonal HEK 293T cells expressing tau repeat domains fused to CFP/YFP.
• Utilized flow cytometry to quantify aggregation-induced FRET upon cellular uptake of proteopathic tau seeds.
• Validated the assay with recombinant tau seeds and brain homogenates from tauopathy models and human subjects.

Main Results:

• The assay detects femtomolar concentrations of recombinant tau seeds with a three-log dynamic range.
• Demonstrated compatibility with biological samples, including human and mouse brain homogenates.
• Showed potential for detecting other proteopathic seeds like alpha-synuclein and use in primary neuronal cultures.

Conclusions:

• The FRET flow cytometry assay offers high sensitivity and broad applicability for studying tauopathies and other protein aggregation disorders.
• This tool facilitates research into the mechanisms of neurodegeneration and the development of diagnostics and therapeutics.