Elucidating mechanisms of protein aggregation in Alzheimer’s Disease using antibody-based strategies.

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder. There are two characteristic histopathological hallmarks in the brain: senile plaques and neurofibrillary tangles, composed of insoluble aggregates of the amyloids Amyloid-β (Aβ) and tau protein, respectively. These diagnostic markers, though distinctive, are not apparent effectors of AD pathology. Evidence has mounted suggesting smaller soluble aggregates (oligomers) of Aβ or tau are the true drivers of disease progression. This dissertation presents several amyloid biophysics projects. Aggregate biophysical parameters such as weight, shape, and conformation were measured using a range of methodologies, including Multiangle Light Scattering, Dynamic Light Scattering, UV-Circular Dichroism, UV-Fluorescence, Scanning Electron Microscopy and immunochemistry. In the first, the behavior of 2N3R tau protein was examined in a seeded oligomerization paradigm, where preformed oligomer may act as a template for monomer aggregation. In comparison to unseeded controls an elution time shift in the seeded solution was noted, suggesting conformational change due to seeding. Interestingly, when the seeded solutions were probed with T22, a tau oligomer-specific antibody raised against 2N4R oligomers, no binding was detected, suggesting the seeded 2N3R oligomer conformation is not the same as the seeded 2N4R oligomer. The second project examined protofibril formation in Aβ42/Aβ40 monomer mixtures with variable isoform ratios. Novel methods were developed to generate pre-fibrillar soluble species from monomeric Aβ solutions, thus minimizing the influence of preformed aggregates on the monomer aggregation pathway. Mixed-monomer solutions displayed ratio-dependent aggregation profile changes. Biochemical analysis demonstrated a higher inclusion rate of Aβ42 inclusion into protofibrils. Furthermore, relative protofibril yield and β-sheet secondary structure were both reduced with decreased Aβ42/Aβ40 monomer ratio, which suggests an additional inhibitory effect from Aβ40. The final project presented is characterization of a novel conformation-specific anti-serum, AbSL, developed by the Nichols Lab against prefibrillar Aβ species. Testing demonstrated specificity for Aβ42 protofibrils over Aβ42 monomer or fibril, as well as over Aβ40 protofibrils. The AbSL specificity epitope was probed, and indicated to incorporate part of the N-terminal 1-16 region