Sheytanova, Ludmila Martinova; (2018) Development of a human cell model of amyloid β seeding and aggregation to investigate Alzheimer's Disease pathology. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Alzheimer’s disease (AD) is characterized by extracellular plaques of amyloid β (Aβ) and intracellular tangles of microtubular tau proteins. Aβ is produced through sequential cleavage of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. Seeded aggregation of oligomeric Aβ (AβO) contributes to disease progression as demonstrated by intracerebral inoculation of transgenic mice with human-derived AD brain homogenates. To date, however, there is no high-throughput cell model for Aβ seeding and the present project investigated an approach to address this gap. Eleven human neuroblastoma lines were evaluated for their endogenous APP, BACE1, γ-secretase, and Aβ levels. Wild-type or mutant (Swedish, Iberian, or NL-F) APP695 was cloned with BACE1 into a retroviral vector and was stably overexpressed in two cell lines with opposite levels of APP and BACE1 expression, SK-N-BE(2) and GI-ME-N. The Aβ peptides secreted by each mutant were evaluated via mass spectrometry and relative amounts of Aβ1-38, 1-40, and 1-42, were quantified with a highly sensitive enzyme-linked immunosorbent assay (ELISA). Aβ levels were compared to those produced by 7PA2 cells, a wellcharacterized model of APP processing. APP-overexpressing SK-N-BE(2) cells secreted equivalent or higher Aβ amounts; the NL-F line had the highest levels of Aβ1-42, which is particularly prone to oligomerization. This line was inoculated with diluted homogenate from human AD brain with proven seeding ability, in parallel to 7PA2 and native GI-ME-N cells, in which Aβ was not detected. The lines were grown for several splits post-seeding. Cell supernatant from each split was evaluated for sustained AβO secretion post-seeding with an AβO-specific ELISA. Seed uptake and propagation was quantified at each split by immunocytochemistry. No AβOs were detected in cell supernatants due to assay sensitivity limitations and intracellular uptake was too variable. Hence, pilot experiments to explore seeded aggregation were not conclusive and further exploration of this system is needed.

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