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