Yeast as a platform for identification of chemo-protectors of Alzheimer's disease
Creators: name: Porzoor, Afsaneh
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310102
subject: Cell development, proliferation and death
classificationCode: 310113
subject: Synthetic biology

description: The complexity of cellular pathways in neurodegenerative disease has limited the understanding of the molecular mechanisms underlying Alzheimer’s disease (AD). Therefore, to gain insight into AD and to devise potential therapeutic approaches, simple models are employed. Saccharomyces cerevisiae is currently utilised for analysing cellular toxicity and protein aggregation in neurodegenerative diseases including AD. This study has therefore focused on improvements and validation of the yeast screening model and assays to generate a more suitable disease model that can be used successfully for designing therapeutic strategies and interventions which correlate with in vitro and mammalian testing systems.<br><br>This thesis investigates and shows for the first time that the pretreatment method of synthetic Aβ42 peptide determines its activity and ultimately how it interacts with yeast cells. The effects of Aβ42 appear to be limited to the yeast cell wall and interactions with the amyloidogenic cell wall proteins. S. cerevisiae was found to be more resistant than Candida glabrata to the effect of Aβ42 peptide. It was identified that conformational changes in the peptide due to preparation methods, determine its fate on toxicity and proliferation. Hexafluoroisopropanol pretreated Aβ42 had a greater tendency to aggregate on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Quiescent cells were found to be more resistant to the toxicity of Aβ42 peptide than non-quiescent cells. Also, a preparation which results in toxicity to PC12 neuronal cells caused proliferation of S. cerevisiae and C. glabrata cells. This further indicated that extracellular toxicity assay in yeast using chemically-synthesised Aβ42 peptide results in a different outcome.<br><br>My study shows for the first time that exogenous folate (folic acid or folinic acid) causes C. glabrata cells suspended in water to undergo two cycles of cell division and to form multiple buds. This effect was limited to cells in stationary phase and was more profound in quiescent cells. This study further exploited the cellular uptake of folate by utilising a putative folate transporter (YJL163C) and examined its role. Folate uptake appeared normal in YJL163C overexpressed yeast cells and a deletant mutant strain. Furthermore, the YJL163C deletion did not abolish folate-stimulated cell division. However, YJL163C overexpression in a diploid resulted in meiotic cell divisions. The Aβ42 was toxic to the YJL163C deletion stain suggesting that the YJL163C putative transporter was not a unique receptor for Aβ42.<br><br>Another novel outcome of this study was the identification of the most suitable isomers of bio-inspired compounds with anti-amyloidogenic properties using both in vivo assays and in vitro assays. The positions of the hydroxyl moieties on the aromatic rings were found to be the major determinant of their potency rather than number of hydroxyl groups. This data implied that any yeast screening assays should be complemented with conventional in vitro assays to ensure their validity prior to high-throughput analyses. Another major development was the identification that the GFP-Aβ42 fusion system, which was utilised as a screening platform for identification of anti-oligomerisation of compounds, could only be used when expressed in AHP1 deletant mutant strain while no effect was observed in the wild-type strain. The data from this study highlights the pitfalls that exist in current yeast model systems and how they should be improved for future investigations.