Single molecule detection of Tau seeds in extracellular vesicles from Alzheimer's disease brains

Extracellular vesicles (EVs) play a critical role in the propagation of Tau pathology in Alzheimer's disease (AD). The selective identification and characterization of Tau seed-carrying EVs would help developing new diagnostic tools and therapeutic strategies. While pathogenic forms of Tau can be detected biochemically in EV-enriched fractions, only super-resolution fluorescence imaging provides both morphological and compositional information at the level of single EVs on account of their small size and the low number of target molecules. This was the object of our study. Post-mortem human brain-derived EVs from AD patients and non-demented control individuals were characterized in depth using biochemistry, transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM). Dual-colour SMLM of immobilized EVs, displaying size distributions that were consistent with TEM, enabled statistically meaningful measurements of thousands of EVs in parallel. Owing to the high spatial resolution and capability of SMLM to detect sparse epitopes, we identified pathological Tau proteins in individual EVs from AD samples. Reconstructed super-resolution images of permeabilized EVs demonstrate that pathological Tau forms are frequent cargo proteins of EVs from AD brains. Our data illustrate the power of SMLM to determine the ultrastructure, molecular composition and topology of single EVs and EV populations.