Abstract 2445 Elucidating Tau Fibril Formation using Correlative Cryo-CLEM in situ

Accurate modeling of tau protein aggregation in diverse cell systems, such as fluorescently tagged tau-expressing cell lines or 'biosensor' cells, is crucial for advancing tauopathy research. This encompasses understanding the early stages of tau fibril formation and localization within these cells, a vital but underexplored aspect in studying these neurodegenerative diseases. Our aim was to elucidate the structure and formation of tau fibrils in fluorescently tagged tau-expressing cell lines and iPSC-derived human neurons, assessing their resemblance to amyloid structures in tauopathies and exploring early stages of fibril formation. We employed a suite of advanced imaging techniques, including cryo-confocal microscopy, cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and cryo-focused ion beam (cryo-FIB) milling, to analyze tau fibrils in these cell models seeded with brain extracts from tauopathy cases and recombinant tau. Our findings demonstrate that these cell lines can form amyloid structures similar to neurofibrillary tangles found in tauopathies. Cryo-EM and cryo-ET analyses revealed cross-β sheets and protein chains spaced 4.7Å apart. Our preliminary data using cryo-FIB and cryo-ET provide insights into the early stages of fibril formation and localization within the cells. These findings support the use of fluorescently tagged tau-expressing cell lines as valid models for studying tau fibril formation in tauopathies and lay a foundation for further understanding the early development of these conditions in cell models. This research was generously supported by the National Institutes of Health (NIH), National Institute on Aging (NIA) and the National Institute of Neurological Disorders and Stroke (NINDS).