Amyloid-β (Aβ), a short peptide of 39 to 42 amino acids, is formed by the cleavage of the amyloid precursor protein by proteases in the membrane of neurons, and self-assembles into aggregated species. These aggregates take oligomeric (soluble) and fibrillar (insoluble) forms, which have been proven to be toxic to our brain, playing a key role of the onset of Alzheimer’s disease. In the past few decades, many groups have taken on the task of investigating the aggregation process of Aβ. Previous work in our lab has shown that metal complexes can be used as a new family of photoluminescent probes for the detection of Aβ aggregates. More specifically, we have shown that [Re(CO)3(dppz)(Py)]+ can photooxidize Aβ, providing a new way to investigate the interaction between metal complexes and these species. This thesis covers different topics related to the interaction of Aβ and metal complexes including probing Aβ oligomerization using ruthenium complexes, investigating the interactions between Aβ species and rhenium complexes, and exploring the inhibition effect and degradation of Aβ aggregates. Chapter 1 is a review of the photoluminescent metal complexes that have been developed for detection of Aβ aggregates. In the last few years this area of research has exploded making available photoluminescent metal complexes of ruthenium, iridium, rhenium and platinum for the study of Aβ aggregates. In chapter 2, I reported using the photoluminescence anisotropy of [Ru(bpy)2(dpqp)]2+ for the detection in real time of Aβ oligomers. Aβ oligomers are believed to form immediately following monomers, however they are invisible to fluorescence sensors such as Thioflavin T. Given that photoluminescence anisotropy is sensitive to the rotational correlation time of molecules, it is useful for monitoring the formation of biomolecule aggregates. We found that Aβ oligomers start to form from time zero with a steady increase in anisotropy that plateaus after 48 hours. The real-time monitoring of Aβ oligomers is of great importance for understanding the kinetics of aggregation, the forces that bring peptides together and study their inhibition. The formation of Aβ oligomers was supported by various characterization techniques including Western Blot analysis, SDS-PAGE analysis, dynamic light scattering analysis, transmittance electron microscopy and atomic force microscopy. Chapter 3 details the interactions between Aβ fibrils and [Re(CO)3(dppz)(Py)]+. Job plot and binding assay were used to determine the dissociation constant (Kd) as 4.2 ± 0.6 μM. Molecular dynamics simulations were used to propose a binding site for [Re(CO)3(dppz)(Py)]+ on Aβ fibrils at a hydrophobic cleft between Val18 and Phe20. Due to the fact that Aβ fibrils are oxidized by [Re(CO)3(dppz)(Py)]+ after UV irradiation, the binding site was studied using the oxidation site as a chemical footprint. In addition, the study of the photooxidation of Aβ monomers showed that after UV irradiation His14 is the most likely oxidized residue by [Re(CO)3(dppz)(Py)]+. In order to further study the secondary light-switching behavior of [Re(CO)3(dppz)(Py)]+, functional groups were used to simulate the amino acids of Aβ. We found that the photoluminescence of [Re(CO)3(dppz)(Py)]+ was enhanced in the presence of imidazole and dimethyl sulfide, indicating potential photochemical reaction was occurred . In addition, the quantum yield of singlet oxygen produced by [Re(CO)3(dppz)(Py)]+ upon UV irradiation, power flux of the irradiation source, and the quantum yields of photooxidation were determined. In chapter 4, we used [Ru(bpy)2(dpqp)]2+ in conjunction with time-resolved photoluminescence spectroscopy to assess Aβ aggregation. The added information available in the time-decay curves can be mathematically deconvoluted to obtain specific information about [Ru(bpy)2(dpqp)]2+ bound to Aβ. By considering a two sites non-cooperative binding model, the existence of two different binding sites on Aβ was discovered: one that affects the lifetime of [Ru(bpy)2(dpqp)]2+ and one that shows not affect its lifetime. These binding sites were further studied using MD simulations. The formation of Aβ aggregates was monitored in real-time using time-resolved photoluminescence spectrosocopy and confirmed using AFM.
Chapter 5 investigated the inhibition of Aβ aggregation using a real-time assay. The results indicate that upon UV irradiation of [Re(CO)3(dppz)(Py)]+ with Aβ monomers, fibrillar aggregates are not produced. Further studies indicated that the photooxidized Aβ monomers play an important role for the inhibition effect. More investigations including MD simulation and other characterization are needed to explore the mechanism of this inhibition effect which could provide a unique method for the therapeutics of AD. In addition, we studied the photo-degradation of Aβ fibrils using rhenium complexes upon UV irradiation. The spectroscopic results and AFM images confirmed degradation of fibrillar species into small fragments and oligomers. This project is not yet done and needs more detailed and fundamental studies, but will be extremely helpful for the development of therapeutic strategies of AD.