Overcoming reduced proteostasis in Alzheimer's disease using yeast as model organism

<p>Despite enormous effort to understand the Alzheimer’s disease (AD) and studies to investigate therapeutic strategies, the multifactorial nature of AD and limitations of living human brain for experimental studies have had significant impact in such investigations. The aims of this study are to develop novel yeast based models and bioassays to study the reduced proteostasis in ageing cells. The effects of different compounds including statins, trace amines and bioactive compounds in cellular health were investigated using yeast models of AD.</p> <p>In the first experimental chapter, this study successfully demonstrated the application of yeast models in delineating simvastatin as the most effective statin to act against intracellular amyloid beta 42 (Aβ42). This study revealed that simvastatin’s effect was not replicated by fluconazole treatment suggesting the effect observed after simvastatin treatment is due to some additional effect of inhibiting HMGCR enzyme. During the study, a novel yeast model that expresses human native Aβ42 was designed and evaluated for expression of the human protein. The previously described yeast model expressing green fluorescent protein tagged with Aβ42 (GFP-Aβ42) was also evaluated for its ability to screen statins effect. Most importantly, a matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry based bioassay to quantify the amyloid turnover in the yeast cells was designed and applied to investigate simvastatin’s effect. The results obtained indicated simvastatin effectively reduced both GFP-Aβ42 and native Aβ42 in yeast cells suggesting its potential to restore reduced proteostasis in eukaryotic cells. In summary, from this study a model to express native Aβ42 and bioassays to study amyloid turnover in yeast cell were designed, which was employed to identify the most effective statin that can be beneficial in AD treatment.</p> <p>Considering various indirect evidence from brain microenvironment of AD patients, trace amines are suspected to have some connections in AD pathology. To investigate this connection and to reiterate the importance of yeast AD models in AD research, both the yeast models expressing GFP-Aβ42 and native Aβ42 were evaluated in separate bioassays in the second experimental chapter of this study. A simple culture based method was designed to study the effect of trace amines on cellular mitochondrial health of yeast models expressing GFP-Aβ42. Among the trace amines tested, tyramine was found to significantly exacerbate the respiratory growth of the cell models indicating its combination effect in presence of Aβ42. Tyramine was also found to increase the population of petites in the yeast cells expressing Aβ42. Furthermore, the ability of tyramine to increase Aβ42 induced reactive oxygen species was also evaluated. The results obtained were intriguing as tyramine treated yeast cells produced a high level of ROS as compared to cells treated with tyramine alone suggesting a resultant severe oxidative damage. This study is the first of its kind in identifying that tyramine can exacerbate Aβ42’s effect in cellular health, especially in the mitochondrial health In summary, this study strongly advocated the applications of yeast models and bioassays in finding directions towards possible threats to AD patients and support the quest of finding a perfect strategy to fight against AD.</p> <p>In the third experimental chapter, a set of eleven bioactive compounds were screened for their ability to restore reduced proteostasis in yeast cells expressing GFP-Aβ42. The results obtained from the screen showed most of the compounds significantly reduced GFP-Aβ42 levels. Among the compounds, baicalein and trans-chalcone were found to have the highest effect. Considering the efficacy of baicalein and trans-chalcone, the two compounds were treated in different combinations to evaluate their combination effect. Baicalein and transchalcone synergistically reduced GFP-Aβ42 indicating the combination’s enhanced potential to restore proteostasis in yeast cells. Evaluation of the compounds' effects on growth and redox balance revealed beneficial effects of the individual as well as combination treatments. The combination of the two compounds also showed similar effect in reducing native Aβ42. From these results, it can be concluded that baicalein and trans-chalcone in combination has synergistic ability to restore cellular proteostasis. In conclusion, baicalein and trans-chalcone could be a combination regimen of bioactive compounds that can be tested against AD patients. Once again, the yeast models proved to be highly effective in screening compounds for their anti-AD effects. This study demonstrated an example of how yeast AD models can be useful for studying combination effects of two or more compounds. In summary, it can be confirmed that a combination of rapid yeast assays could provide adequate information on compound screening for drug discovery projects involving evaluation of cellular proteostasis, redox balance and mitochondrial health.</p>