UBC Theses and Dissertations
Characterization of the mechanisms underlying Alzheimer's disease and multiple sclerosis Herculano, Bruno de Araujo
Deposition of the amyloid β protein (Aβ) into neuritic plaques is the neuropathological hallmark of Alzheimer's Disease (AD). Aβ is generated through the cleavage of the Amyloid Precursor Protein (APP) by β-secretase and γ-secretase. Currently, the evaluation of APP cleavage by β-secretase in experimental settings has largely depended on models that do not replicate the physiological conditions of this process. We have developed a chimeric protein construct, ASGβ, incorporating the β-site cleavage sequence of APP targeted by β-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by β-secretase, ASGβ releases a phosphatase-containing portion that can be measured in the culture medium, and an intracellular fraction that can be detected through western blot. Subsequently, we have generated a cell line stably expressing ASGβ that can be utilized to assay β-secretase in real time. Our findings suggest this system could be a high-throughput tool to screen compounds that aim to modulate β-secretase activity and Aβ production under physiological conditions, as well as evaluating factors that regulate this cleavage. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described and more recently we have identified novel mutations in 2 genes, NLRP12 and NCOA3 in familial forms of the disease. Our findings show that one of the discovered mutations on NLRP12 affects the maturation of Caspase-1, and that the mutation discovered on NCOA3 leads to a higher basal level of inducible Nitric Oxide Synthase (iNOS). These findings show novel mechanisms causing the disruption of immune and inflammatory responses that could in turn lead to the symptoms observed in MS patients. Our study suggests that further characterization of the pathways affected by the NLRP12 and NCOA3 could lead to a better understanding of the phenomena causing MS, and the development of novel, more effective treatments for the treatment of symptoms.
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