Structure-based Studies on Tau Protein Aggregation and Toxicity.

摘要

Tau protein aggregates represent a type of amyloid that are the pathological hallmark of over 20 neurodegenerative diseases, termed tauopathies. These include Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Given the association between the presence of tau aggregates and pathology, the field has a clear interest in determining whether said aggregates can be prevented, and if doing so would be a viable therapeutic strategy. Yet, much emerging evidence implicates soluble oligomers formed from the same protein to be the toxic species in many amyloid diseases. There is thus also a substantial effort devoted to identifying and characterizing these oligomers.;To see whether a rationally-designed peptidic inhibitor of tau fibril formation works as designed, I adapted and refined a kinetic fluorescence-based assay to test candidate inhibitors in two model systems. Using Nanogold particles, I also developed a new negative-stain electron microscopy-based method to ascertain whether an effective inhibitor actually blocks aggregation as designed, and employed mass-per-unit-length measurements from scanning transmission electron microscopy to support the findings. Using covalently-labeled fibrillogenic peptides, I also tried to create a FRET-based assay to screen for compounds that dissolve pre-formed fibrils. Shifting from working exclusively on insoluble assemblies, I later characterized segments from tau protein that we hypothesized would form toxic oligomers. I did this using a combination of cytotoxicity assays and biochemical methods.;This dissertation describes my findings. I determined that the inhibitors specifically block tau fibril formation, and that they indeed do so by capping and preventing elongation of nascent filaments. I also was able to show that a FRET-based disaggregation assay is viable, although though no effective compounds were identified in a pilot screen. And finally, I identified a segment of tau protein that exhibits characteristics suggesting that it is the region responsible for tau's forming toxic oligomers.