Studying Models of the Amyloid Cascade Hypothesis Using Single-Molecule Imaging

Abstract

Observation of beta-amyloid (Aβ) plaques in the brains of Alzheimer’s disease (AD) patients led to the development of the amyloid cascade hypothesis (ACH), which postulates the accumulation of Aβ as the initiator of AD. Aβ can be found in multiple forms in the AD brain; while the insoluble plaques are the most studied species, the smaller, soluble aggregates may be more toxic, leading to neuroinflammation, oxidative stress, and tau phosphorylation. However, their small size and low abundance makes it challenging to study the soluble Aβ aggregates. Our group has been developing single-molecule and super- resolution microscopy techniques to characterise these aggregates. While these methods were previously applied to human samples, they have not been used to study disease models. Cellular and animal models have numerous advantages over human samples, primarily less intra-group variation and the ability to manipulate individual pathways. Thus, these models enable researchers to gain insight over disease mechanisms in a controlled system and test hypothesis such as ACH. Throughout my PhD, I have developed and applied single- molecule detection techniques to induced pluripotent stem cells and organoids, as well as transgenic murine models to test the predictions of the ACH. This thesis provides data on AD-like pathology caused by elevated Aβ aggregation, which can be accelerated by immune challenges and rescued by reducing Aβ levels, the role of glia in the pathogenesis of AD and their relationship to Aβ aggregation, peripheral Aβ aggregates in a mouse model, and the impact of tissue processing on the harvested aggregates. Moreover, novel methodologies to normalise conditioned media samples and study Aβ aggregates in the synapses have been described, and therapeutics against Aβ has been characterised. Overall, this thesis investigated the ACH in a range of cellular and murine models, providing supporting data for the hypothesis and revealing new details of the molecular processes that initiate AD.