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Isolation and characterization of distinct populations of amyloid β aggregates using size exclusion chromatography and structure-specific antibody Shyu, Chih Chieh

Abstract

One of the most significant challenges in the study of Alzheimer’s disease (AD) is the elucidation of causative agents. AD belongs to a family of diseases characterized by proteinopathy—misfolded amyloidogenic proteins forming aggregates. Pathogenic amyloid aggregates are capable of prion-like propagation, serving to seed template misfolding and propagate aggregation. Literature strongly implicates amyloid-β aggregates (Aβ) as causative agent in AD, impairing synaptic transmission, causing cell death, and serving to propagate cytotoxic insults. Canonical protein aggregation proceeds in three distinct stages: with misfolding of initial monomers, followed by spontaneous formation of oligomers, and ending with deposition of insoluble fibrils as AD plaques in the brain. Aβ oligomers (AβO) have been shown to exert the most potent toxicity and propagation of aggregation both in vitro and in vivo, with monomers and fibrils being orders of magnitude weaker. As literature suggests the structure of amyloidogenic proteins confer toxicity; great efforts are being made to understand their structural and functional biology, including Aβ. However, Aβ aggregation is complex and non-linear, and the transience of oligomeric populations in solution hamper efforts to identify specific disease-causing isoforms. Literature also shows significant differences in toxicity and aggregation kinetics between synthetic and biologically-derived AβOs. As AβO causes AD, great efforts are being made to neutralize aggregate activity, with therapeutic antibodies at the forefront. However, all antibodies that underwent clinical trials up to date have failed to meet their efficacy endpoints, despite showing plaque clearance. These antibodies targeted regions of Aβ sequence, which led to broad spectrum reactivity to Aβ monomers and aggregates. This observation emphasizes the importance of elucidating AβO-specific epitopes for targeted therapy. It is thus imperative to identify and isolate individual populations of AβOs from both synthetic preparations and biological sources for target determination. In my project, size exclusion chromatography (SEC) was used to separate and collect Aβ aggregates by size, and the seeding ability of various AβOs was examined using Thioflavin-T assay. Finally, surface plasmon resonance (SPR), a state-of-the-art label-free technology that monitors protein-protein interactions in real-time, was employed to determine the specificity of antibodies developed in-house targeting structural features unique to AβO.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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