On the aberrant nature of tau protein: biophysical approaches to characterise and target aggregation in neurodegeneration

Abstract

Tau is a multifaceted and dynamic protein vital to a number of physiological processes, notably in the architecture of the cytoskeleton. Its key role in the assembly and stabilisation of microtubules, which are critical for molecular shuttling and axonal growth, underscores the significance of tau in cellular health. Although the protein is central to many fundamental processes, tau is also heavily implicated in the onset and spread of neurodegenerative conditions, collectively known as tauopathies. As a result, this added complexity muddles our complete understanding of the protein in physiology and pathology, making the treatment of dementia all the more difficult. These disorders are hallmarked by inclusions of neurofibrillary tangles (NFTs) principally comprised of the microtubule-associated protein. Interestingly, these aggregates are abnormally phosphorylated and acetylated, suggesting a regulatory mechanism of posttranslational modifications (PTMs). Nevertheless, how tau transitions from monomer to disordered, unstable oligomeric intermediates, and, ultimately, highly structure, β-sheet rich fibrils, remains elusive. In this thesis, we seek to answer this very question, with the ultimate aim of designing therapeutics that can modify the course of the disease. To achieve this, we have combined the advantages of metadynamic structural biology and bioorthogonal chemistry to investigate the relative contributions of site-specific PTMs to the stability of tau-microtubule interactions. Our experiments, validated by in silico predictions, revealed a strong correlation between structural heterogeneity and the number of contacts made by tau within the structural ensemble. Furthermore, the magnitude of destabilisation largely depends on the which residue is modified. Our results were corroborated concomitantly by proteomic analysis serendipitously conducted independently of our study. Together, these findings indicate that certain PTMs, above others, are fundamental to initiating the amyloid cascade of tau protein. With this understanding, we next utilised a chemical kinetics approach to divulge microscopic processes from macroscopic measurements of tau aggregation. Doing so elucidated a more holistic model for the amyloid pathway. As such, we have determined that the aggregation of tau in Alzheimer’s and Pick’s diseases is comprised of a series of elementary steps. Although the rates at which these two disease-specific fragments of tau aggregate substantially differ, we have identified secondary pathways, such as fragmentation and surface-catalysed secondary nucleation, as key contributors to the formation of toxic misfolded oligomers of tau. This finding serves as the basis for our drug discovery platform. Finally, building on this kinetic framework, we developed a drug screening assay to identify therapeutics capable of inhibiting tau aggregation. Our screening strategy encompassed a variety of small molecules originating from various sources, namely, natural products, a hypothesis-free epidemiological meta-analysis for drug repurposing, molecular docking simulations, and a machine learning derivatisation. Some of these candidates, despite presenting therapeutical potential in epidemiological and neuropathological data, did not inhibit aggregation. In contrast, other small molecules demonstrated a capacity to bind the fibril surface and inhibit the deposition of tau protein when in the presence of pre-formed aggregates. This gives reason to conclude that inhibitors which bind fibrils may do well to significantly reduce the total population of tau oligomers and, consequently, reduce toxicity and mitigate disease propagation. Taken together, the results of this thesis carve out one possible pathway for the aberrant self-assembly of tau, from monomer to fibril. We have determined that certain PTMs greatly reduce the affinity of tau for the microtubule surface and that these sites correlate with the average number of contacts within the tau-microtubule complex. Accordingly, these molecular events subsequently serve to increase the likelihood of stochastic, primary nucleation events, oligomeric conversion, and amyloidogenesis. Moreover, the application of chemical kinetics can provide fundamental information that further quantifies the inhibitory potential of therapeutics for a given amyloid system. This serves as an additional tool to be exploited in the early-stage drug discovery process for the ultimate treatment of misfolding protein diseases.