Nucleation Dependent Polymerization of Amyloid-ß (Aß) as an Important Determinant of Aß Amyloid Formation and Neurotoxicity : Implications for the Pathogenesis of Alzheimer's Disease and Design of Therapies

Aggregation and fibril formation of amyloid-β (Aβ) peptides play a pivotal role in the pathogenesis of Alzheimer's disease (AD). Aβ peptides, principally comprising of 40 or 42 amino acid residues (Aβ40 and Aβ42), are produced by proteolytic processing of the amyloid precursor protein by β and γ-secretase activity. A vast number of academic and biomedical industry projects are devoted to the discovery and development of therapeutic agents which inhibit the process of Aβ aggregation, and associated neurotoxicity, as potential disease modifying therapies for AD. Despite decades of research, the mechanism of Aβ neurotoxicity leading to development of AD and the identity of toxic Aβ species remain debatable. Nevertheless, independent lines of evidence implicate Aβ oligomers, which are precursors to fibrils, as putative toxic species and promising therapeutic targets. Aβ oligomers are characterized by heterogeneity in size and morphology distribution and have been shown to alter normal neuronal physiology in a manner that implicates their causal association with AD pathogenesis. The primary objective of this thesis work was to elucidate the structural determinants of Aβ neurotoxicity, the relationship of fibril formation process with neurotoxicity and identify neurotoxic Aβ species. Towards achieving these goals, we first developed and optimized reproducible protocols for generating various Aβ species, including monomers, protofibrils and fibrils, and established methods for detailed characterization of their structural properties. To better understand the relationship between Aβ aggregation and neurotoxicity, cell culture assays using primary neurons and neuronal cell lines were developed and used to assess the toxicity of various Aβ aggregates. Using these assays and biochemical tools, we sought to answer some of the key outstanding questions in the field concerning the role of Aβ amyloid formation process in neurodegeneration in AD. What is the nature of neurotoxic Aβ species and how does the process of its formation relate to neurodegeneration in AD? The identification of toxic Aβ species and/or process of its formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in AD, and development of effective diagnostic tools and therapeutic interventions. To achieve this, we successfully isolated protofibrils of distinct sizes and morphology distribution and compared their fibrillization and neurotoxicity to monomeric and fibrillar forms of Aβ. The results from our studies implicate an ongoing Aβ polymerization process, rather than distinct Aβ aggregate states (fibrillar or non-fibrillar), as primary determinant of Aβ neurotoxicity. We showed that crude Aβ42 preparations containing mixtures of monomers and heterogeneous oligomers were significantly more toxic than purified Aβ42 protofibril preparations. Subfractionation of protofibril preparations, to separate different protofibril species, resulted in further attenuation of their toxi