The dynamic nature of amyloid-beta protein aggregation and its association to Alzheimer’s disease
Year: 2015
Type: dissertation
Abstract: Amyloid-beta protein (Aß) is strongly linked to the aetiology of Alzheimer’s disease (AD). Even though Aß has a central role in AD, this protein is normally produced in healthy humans. It is the aberrant processing of Aß that determines its accumulation and aggregation into large oligomer species that evolve into the fibrillar structures deposited in amyloid plaques in the brain of AD patients. The neurotoxicity observed in AD has been attributed to the oligomeric intermediates, although their stoichiometry and structure still remain unknown. This is the reason why no AD therapeutic approaches tackling Aß aggregation have arrived to the market yet. \nIn the present thesis, we have determined the stoichiometry and the structure of the oligomers formed in the early stages of Aß aggregation. Due to their highly dynamic nature, we have first obtained their covalent counterparts through the use of a photo-induced crosslinking methodology. Successful isolation of cross-linked Aß dimers and trimers has been achieved by means of a disaggregating treatment coupled to size exclusion chromatography. The combined study of these isolated cross-linked species through ion mobility coupled to electrospray ionization mass spectrometry, circular dichroism and molecular dynamics simulations, has shown that Aß dimers and trimers possess a globular shape without defined secondary structure. Moreover, we have proved that these cross-linked oligomers induce calcium influx, an intracellular marker for neurotoxicity, in primary neuroglial cultures, and that these cross-linked species effectively modulate Aß aggregation. Additionally, we have demonstrated that classical SDS-PAGE analysis provides misleading results when determining oligomer stoichiometry. \nThe present thesis was also aimed at providing insights into the role of Aß amyloid fibrils in AD. Aß fibrils had been previously shown to be in dynamic equilibrium with soluble Aß species. The objective was to find evidences on the nature of these soluble species in equilibrium with Aß fibrils. Determination of their stoichiometry has been achieved by incubating Aß fibrils with insulin degrading enzyme, an enzyme which we have demonstrated to specifically proteolyze monomeric Aß. Subsequent analysis of Aß fibrils by hydrogen/deuterium exchange monitored by mass spectrometry has revealed that Aß fibrils are in equilibrium with monomers and low molecular weight oligomers. This equilibrium has been proved to be highly dependent on the physicochemical properties of Aß fibrils.
Author: Sílvia Vilaprinyó Pascual
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1195043082