Understanding Amyloid β Aggregation and the Interaction with Ions via Supercritical Angle Microspectroscopy

For decades, Alzheimer's disease (AD) poses a severe threat to human lives by being one of the primary cause of neurodegeneration worldwide. With the growing incidence, pathological diagnosis is time-consuming and also lags in detecting plaques at their early stages of formation. The proper understanding of Amyloid β (Aβ) aggregation which commonly associated with AD is the state-of-the-art for the development of therapeutics and diagnostics. The supercritical angle optical technique takes the advantage of evanescent wave phenomena, offers excellent surface sensitivity and can be used for the simultaneous measurement of the bulk solution and the surface of a lipid membrane. In this doctoral thesis, the interaction of Aβ proteins with CaCl2 and the role of other metal ions on the aggregation process are studied in vitro using supercritical angle fluorescence (SAF) and Raman (SAR) microscopy and spectroscopy. The thesis project work focus on three themes. In the first part, the Aβ aggregation during different times is studied. The formation of protein aggregation follows long incubation times, involving adsorbing and resorbing from the lipid membrane. The Aβ1-42 is known to be more cytotoxic than Aβ1-40, therefore studying both pathway and comparing the aggregation mechanism is important. A long term study over three days is performed, where measurements occurs at different aggregation times. To observe, if the lipids have an effect on incubation, the proteins are incubated for 1 and 72 hours before adding to the lipid membrane. The results of the first experiment shows that a long incubation time of 70-77 hours is needed for aggregation growth. In general, the aggregates in the SAF channel have a smaller size, due to the fact, that aggregation growth needs longer time. For aggregation at the surface, small aggregation seeds are needed, which are formed at the surface and once anchored into the membrane, they can start to grow to larger aggregates. It is shown that the small aggregates start to grow over a time period of 70 hours. The incubation after 1 hour brings small aggregates in the SAF and large in the bulk. Large aggregates from the bulk do not attach to the membrane, however, the aggregates grow directly on the lipid membrane via the seeding mechanism. In addition, similar experimental conditions with the same protein concentration are carried out for the Raman measurements for direct comparison of the results. The aggregation is enhanced at higher protein concentration. Only Aβ in high concentration after a comparably long incubation time has the ability to form filaments. In the second part, the Aβ aggregation in the presence of calcium ions is analyzed using the same methods for aggregation and structure characterization as described in part 1. A structural change of the peptides towards weaker bonds of the aggregates when aggregated in presence of calcium ions, which are larger in size is confirmed. It is shown that calcium ions form a protecting layer at the lipid membrane, which hinders protein insertion. This part of the thesis will pave a new pathway for better understanding of Aβ aggregation in the context of calcium homeostasis. In the third part, the role of other ions such as ZnBr2, ZnCl2, CuBr2 and CuCl2 and effects of ions in modulating aggregation behavior is studied. The results showed that chloride ions, due to their smaller size and lower polarizability compared to bromide, enhance the redox activity of Cu²⁺, leading to stronger Aβ aggregation. In contrast, Zn²⁺ stabilizes Aβ at lower concentrations but promotes aggregation at higher concentrations due to electrostatic interactions and ion-bridging effects. Finally, the principal component analysis (PCA) of the Raman data is performed for further statistical analysis of the results and in particularly fundamental understanding of Aβ protein aggregations.