The development of peptide-based inhibitors for Tau aggregation as a potential therapeutic for Alzheimer’s disease

Aggidis, Anthony and Allsop, David (2019) The development of peptide-based inhibitors for Tau aggregation as a potential therapeutic for Alzheimer’s disease. PhD thesis, Lancaster University.

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Abstract

There are currently approximately 50 million individuals worldwide with dementia resulting in predicted global societal costs of up to US $1 trillion. Approximately 60-70% of these individuals have Alzheimer’s disease, which results in a chronic and insidious decline in memory. One of the main proteins that misfolds in this disease is Tau protein, which aggregates into toxic oligomers and neurofibrillary tangles. It is these aggregates, which cause damage to the brain resulting in dementia. As a result, it is imperative to be able to prevent or suppress the pathogenic aggregation of this protein, so the onset of dementia is halted or delayed, improving quality of life. Certain amino acid sequences in Tau such as VQIINK and VQIVYK play important roles in aggregation. Targeting these sequences can potentially prevent aggregation. This project aims to produce effective peptide inhibitors based on the human Tau peptide sequences VQIINK and VQIVYK, to specifically target pathogenic Tau aggregation. Using molecular docking softrware ‘ICM-Pro’ the potential binding locations of a variety of peptide candidates were computationally investigated to determine which will be most successful in a laboratory setting. Recombinant TauΔ1-250 was incubated in the prescense of heparin and subsequently aggregated to display highly ordered parallel, in-register β-strand structures; including fibrils and paired helical filaments presenting the characteristic twist under transmission electron microscope. This aggregation was achieved using of 20μM Tau at pH 7.4 in the presence of 20mM Tris buffer, 1mM DTT, 5μM Heparin, and 15uM ThT and incubated at 37 °C for 48 hours. The first generation of peptides AG01, AG02, AG02, AG02R4, AG02R5, AGR502, AG02PR5, AG02R6, AG02R9, AG02TAT, AG02ΔI, AG02ΔV inhibited approximately 50% of Tau aggregation determined by Thioflavin-T (ThT) fluorescence assay. The next generation, AG03 was slightly more effective, however when retroinverted (RI-AG03) inhibited over 90% of Tau aggregation, confirmed by Thioflavin-T fluorescence assay, transmission electron microscopy, circular dichroism and Congo red birefringence. RI-AG03 was determined to be stable in cells at therapeutic concentrations. After determining stability of RI-AG03 using SDS-PAGE, thermal circular dichroism and mass spectrometry, it was tested in vivo in rough eye Drosophila model. Results suggested that RI-AG03 partially rescued the rough eye phenotype in this model. This research demonstrates that retro-inverted peptide RI-AG03 is a potent inhibitor of Tau aggregation and can be further developed as a novel therapeutic for Tauopathies like Alzhimer’s Disease.