Development of sensitive fluorescent and spectroscopic assays to study tau assemblies implicated in neurodegenerative diseases

Abstract

Protein misfolding and fibrillization are core pathological hallmarks of neurodegenerative diseases. Abherrent forms of the tau protein are implicated in the pathology of diseases including Alzheimer’s disease, Pick’s disease, and Frontotemporal dementia; where it assembles into filamentous inclusions within cells in the form of neurofibrillary tangles. In the last twenty years, tauopathy research has had several pivotal findings, including the identification of the neurotoxic effects of the small oligomeric forms of the tau protein, the structural heterogeneity of fibrillar tau, and the capability of tau to form biomolecular condensates. These new insights have highlighted the need for more sensitive methods to characterize the early stages of the fibrillization process, observing initial protein conformational changes, and subsequent events. The main aim of this work is to develop methods that report on processes implicated near the nucleation and elongation phases, by 1.) characterizing the liquid-liquid phase separation processes of the protein and 2.) developing affinity-based detection assays to provide kinetic data for the oligomerization process. In the first objective, intrinsic and extrinsic fluorescence-based methods are introduced as techniques to determine temperature-dependent phase transitions of proteins and labeled biomolecules. These methods allow low volume and low concentration measurements of LLPS as an alternative to absorbance turbidity measurements. Furthermore, supplementary methods of data analysis using dimension reduction techniques such as principal component analysis greatly improve the scope of the technique by allowing trends in high throughput screens to be easily identified. The second part of this work focuses on the development of aptamer-based fluorescence spectral shift assays which quantify the change in the tau monomer concentration in solution. This assay is optimized to perform measurements at pathologically relevant concentrations to produce real-time information on the formation of small oligomers - a process missed by Thioflavin T assays. Finally, signal amplification is explored in the development of spectral shift in-solution plasmonic assays. The oligonucleotide - gold nanorod conjugates described in the work produce a highly sensitive detection tool, with improved signal-to-noise ratios. As such, each of the methods developed in this thesis aims to challenge the state of the art of biophysical characterization of the various assembly states of tau.