AbstractAlzheimer’s disease (AD) is the most common form of dementia with around 520,000 individuals currently diagnosed in the UK. Despite the devastating impact of AD there are currently no successful therapeutic approaches to treat the condition. AD is characterised by the misfolding and aggregation of the Amyloid-β (Aβ) peptide. Whilst pinpointing the exact species of Aβ that exerts toxicity is difficult, it is generally accepted that oligomeric forms of the peptide confer toxicity. Various oligomer sizes and conformers impart varying mechanisms of toxicity but ultimately, soluble oligomers of Aβ initiate a cascade of downstream toxic events that eventually lead to the neuronal cell death responsible for AD. Recent application of cryogenic electron microscopy (cryo-EM) and solid-state NMR (ssNMR) techniques have revealed high-resolution structural information regarding the architecture of Aβ within fibres.
Here, a semi-rational approach has been applied to design peptide libraries utilising one of these recent high-resolution structures as a template. In particular, we focus on two key regions, an outer β-sheet strand and the central dimeric interface, which are instrumental in formation of the Greek-key motif common to most amyloid folds. The intracellular Protein-fragment complementation assay (PCA) was employed, and a further novel transcription block survival (TBS) assay developed, along with successful proof-of-concept experiments, and applied to successfully identify peptide inhibitors of Aβ1-42. The study utilised Thioflavin-T (ThT) fluorescence aggregation assays along with Circular Dichroism (CD), Transmission Electron Microscopy (TEM) and photoinduced crosslinking experiments to demonstrate the ability of the selected peptides to impact upon Aβ aggregation. The study next sought to explore how the altered aggregation of Aβ would translate within a cell-based assay. Therefore, an Aβ-induced toxicity assay was optimised within a differentiated SH-SY5Y cell line applying low concentrations of Aβ. Upon application of the identified peptide hits it was observed both could partially rescue Aβ-induced toxicity within the human derived neuronal-like cell line as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) viability assays. The peptide sequences identified herein present exciting targets for potential therapeutic candidates to target Aβ-induced toxicity in AD.
Overall, this study demonstrates the successful application of the PCA assay and the development and application of the novel, intracellular TBS screening platform to identify peptide hit sequences able to alter Aβ-induced aggregation. Optimisation of an Aβ-induced toxicity assay within a relevant human-derived cell line also provides a platform to assess the potential of future drug candidates and reveals the potential for the peptide sequences identified to partially rescue Aβ-induced toxicity.
|Date of Award
|16 Nov 2022
|Jody Mason (Supervisor) & Robert Williams (Supervisor)