Differential detection of tau aggregation species using a small molecule probe for cell-based drug screening

Tau aggregation into polymorphic species, including oligomers, fibrils, and neurofibrillary tangles (NFTs), is central to the pathology of Alzheimer’s disease and related tauopathies. As an intrinsically disordered protein, tau undergoes liquid-liquid phase separation (LLPS), a metastable state that regulates its aggregation pathways. Detecting and understanding these mechanisms are essential for developing tau-targeted therapies, yet current tools for differentiating LLPS and polymorphic tau species remain limited. Here, we introduce TP-11, a small-molecule fluorescent probe that detects distinct stages of tau aggregation. TP-11 exhibits unique fluorescence intensity, quantum yield, and lifetime signatures in response to tau LLPS, oligomers, and fibrils. Using fluorescence intensity measurements, fluorescence lifetime imaging microscopy (FLIM), and single-particle total internal reflection fluorescence (TIRF) imaging, TP-11 enables highly specific detection of tau species. In addition, TP-11 supports cell-based drug screening assays, facilitating the identification of tau aggregation modulators and mechanisms through high-content imaging with potential for high-throughput applications. TP-11 thus provides a powerful tool for probing tau aggregation across experimental conditions and advancing therapeutic development, with strong potential for quantifying neurotoxic tau species in vitro and in cells.