Monica Pallitto, PhD 2001

Thesis Title: Kinetic characterization and modulation of beta-amyloid aggregation and toxicity

β-amyloid (Aβ), the primary protein component of Alzheimer's plaques, is neurotoxic after aggregation into fibrils. We devised a modular strategy for generating compounds that inhibit Aβ toxicity, based on linking a recognition element for Aβ to a disrupting element designed to interfere with Aβ aggregation. The hybrid peptide KLVFF-KKKKKK provided significantly improved protection against Aβ toxicity compared to the recognition peptide alone. None of the cytoprotective peptides prevented Aβ aggregation; rather, they increased aggregate size and altered aggregate morphology. A desire to further understand Aβ aggregation leads to the primary goal of this work: the development of an experimental and mathematical representation of the kinetics of Aβ aggregation. This model could be used to help elucidate differences in aggregates exhibiting varying levels of neuronal toxicity, and aid in design and analysis of therapeutic molecules. In order to create such a kinetic model, reproducible and well-characterized initial conditions were selected. Aβ dissolved in 8 M urea is monomeric and lacks secondary structure. Upon dilution into physiological buffer, Aβ aggregates into fibrils. The self-assembly process was followed by monitoring both the monomer/oligomer distribution using size exclusion chromatography, and average size using light scattering. A significant amount of Aβ remained as monomers and dimers, even in the presence of very large aggregates. High molecular weight species formed very rapidly, within minutes of dilution. Interestingly, the initial aggregate size was greater at lower concentrations. Fibril linear density increased with concentration and time. We used this experimental data to develop a quantitative mathematical model describing the kinetics of growth of Aβ aggregates. A highly reactive 'intermediate' species is formed that nucleates and adds to Aβ filaments. Filaments laterally associate to form fibrils. These fibrils continue to grow by end-to-end association. We further examined the relationship between aggregation and toxicity using our model to aid interpretation. Aggregation conditions that produced predominantly long filaments were significantly more toxic in vitro than our other samples. We hypothesize that filaments are very important in Aβ-mediated toxicity and that our inhibitors work by driving the process of lateral association, thus reducing the number of filaments available to interact with cells.