The dynamic nature of amyloid-beta protein aggregation and its association to Alzheimer’s disease

Abstract

[cat] La proteïna beta amiloide (Aß) es troba estretament lligada a la malaltia d’Alzheimer (MA). Tot i el seu rol central en la malaltia, Aß es produeix de forma regular en humans sans. És el processament aberrant de la proteïna que en determina la seva acumulació i agregació, primer en intermedis oligomèrics transitoris que evolucionen cap a les estructures fibril·lars que composen les plaques amiloides dipositades al cervell dels malalts de la MA. La neurotoxicitat associada a la malaltia s’atribueix a les espècies intermèdies, per bé que se’n desconeix l’estequiometria i l’estructura. En la present tesi doctoral, hem determinat l’estequiometria i l’estructura dels oligòmers d’Aß formats en els estadiatges inicials de l’agregació. Hem provat que aquests oligòmers mostren neurotoxicitat en cultius neuronals primaris. Addicionalment, hem demostrat que el clàssic anàlisi per electroforesi en gel proporciona resultats confusos a l’hora de determinar l’estequiometria dels oligòmers. Els resultats presentats en la tesi doctoral també mostren evidències de que les fibres amiloides d’Aß es troben en equilibri amb monòmers i oligòmers de baix pes molecular. Aquest equilibri ha demostrat ser dependent de les propietats fisicoquímiques de les pròpies fibres d’Aß.

[eng] 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. In 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. The 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.