Structural characterization of tau aggregation variability and maturity in isolated cell types of the brain

摘要

Abstract Recent structural and biochemical work reveals the microtubule-associated protein tau (MAPT/tau) adopts different toxic filament conformations or strains that are specific to different tauopathy diseases. Assembly of these toxic tau conformations is thought to occur through a prion-type mechanism in which tau forms cross-β sheet amyloid aggregates that template and catalyze the conversion of soluble tau. Cryo-electron microscopy (cryo-EM) has proven indispensable for determining high-resolution structures of these conformations from ADRD brain-derived tissue, establishing a critical platform for identifying the disease-relevant targets for drug design. Further, recent cryo-EM efforts have revealed distinct, disease-specific conformations of ADRD tau filaments, and raised the hope that the newly solved structures could be useful in more precise drug design. However, it has been difficult to approach the flat, repetitive surfaces of amyloids and the discovery of amyloid- binding diagnostic compounds has been limited to random screening. Moreover, existing structures are derived from bulk tissue preparations from late stage, primarily sporadic samples. Thus, we hypothesize that therapeutically relevant states of tau are not fully understood, including states that arise in different brain cell types or during initial stages of disease. Importantly, tau filament structures have not been determined from a pathologically relevant ADRD animal model, hindering development of therapies that target tau disease conformations. We hypothesize this is due to low abundance of structurally tractable tau fibrils from existing mouse models. These challenging questions prompted us to use combined imaging approaches involving novel, structurally sensitive tau-binding small molecule dyes and cryo-EM. We plan to revolutionize ex vivo characterization of tau brain deposits through the advancement of our methods to separate and isolate glial and neuronal cell types from human brain tissue and characterization of our novel rat model for pathogenic tau structures. These goals are based on our strong preliminary work in which we have determined the first high- resolution structure of a medically-relevant small molecule bound site-specifically to disease-relevant tau filaments, and developed structurally-sensitive dye imaging methods that reveal distinct states of tau in different cell types and diseases. Furthermore, we developed custom antibody-functionalized EM grids for purification of biochemically-relevant ADRD tau filaments from small volumes of bulk tissue that will: 1) enable cryo-EM studies on precious early stage brain regions with sparse tau deposits, 2) purify tau filaments based on known PTM markers of tau maturity, and 3) reduce the need for amplification methods prone to structural infidelity. This innovative proposal is built on the established collaboration between Southworth and Condello, whose labs will leverage combined strengths in cryo-EM structural, cell biological and histological approaches for predicting, prioritizing and determining structures of disease-, cell type-, and age-specific tau filaments and ligand-bound co-complexes at a sufficient resolution for ligand modeling and structure-guided design.