Cellular models for the investigation of the structural dynamics and activity of human tau protein aggregate formation

Evidence for the significant role of tau protein in Alzheimer's disease (AD) highlights the need for deep investigation of intracellular tau aggregation pathways and formation of aberrant tau polymorphs within cellular models. Intrinsically disordered proteins with low sequence complexity like hyperphosphorylated tau protein can form condensed folded structures that contribute to neurodegeneration. Candidates for aberrant intracellular tau folding and aggregation have been identified including liquid-liquid phase separation (LLPS) and we will investigate if nanocrystal growth at ribosomes could be observed. Recent research discovered that the differences in tau folding at the core of pathological tau aggregates can differentiate AD from other tauopathies. It is hypothesized that disease specific changes in the cellular environment surrounding tau, such as patterns of hyperphosphorylation, could lead to differential core structures. Combining advanced structural determination methods including cryoEM, cryoFIB, and cryo-electron tomography, we are working on direct imaging of intracellular tau aggregates in cells as well as autopsy tissue derived from human brain. The goal is to develop new cellular models of human tau aggregation using both mammalian and insect cell lines which we employ to evaluate intracellular forces necessary to create unique pathological tau structures beginning with patterns of hyperphosphorylation. We overexpressed the six human tau isoforms in both Sf9 insect cells and SH-SY5Y mammalian cells and induced this intracellular tau to form aggregates by incubating the cells with a small molecule tau aggregation inducer Congo Red. Using cryo-focused ion beam, we can mill thin lamellae through vitrified cells used for future cryo-tomography imaging and 3D reconstruction. Using advanced structural methods, we can compare our aggregates with authentic human brain-derived AD tau aggregates as well as visualize intracellular interactions.