Towards the validation of a druggable amyloid-beta oligomer as a target for Alzheimer´s disease = Cap a la validació d’un oligomer de beta-amiloide com a diana en la malaltia d’Alzheimer

Abstract

Amyloid-beta peptide (Aβ) is strongly linked to the aetiology of Alzheimer’s disease (AD). Aβ is the main component of the amyloid plaques found in the brain of AD patients, however, Aβ is also present in the brain of healthy humans. It has been described that for this peptide to be neurotoxic, aggregation is needed. The accumulation of Aβ causes aggregation from low order oligomers through different intermediate species up to the formation of amyloid fibrils. However, is not the presence of the fibrils what correlates the harmfulness of the disease, but the concentration of soluble oligomeric intermediate species. Nowadays, is accepted that the neurotoxicity of these oligomers is produced on the membrane. From this point, the laboratory of Dr. Carulla developed the beta-barrel Pore Forming Oligomer (βPFO). βPFO, was produced with Aβ42, the most neurotoxic version of Aβ. It was the first described example of a stable, well-defined and homogeneous membrane oligomer with the ability to form pores on lipid membranes. In this thesis, we aim to advance in the characterization of this βPFO and the validation of βPFO as a druggable target for AD. First, as βPFO was described using detergents as a biomimetic membrane environment, we aimed to move towards a more native environment using natural lipids. By using lipid-detergent micelles we studied βPFO. In this work we demonstrated that βPFO is not able to reconstitute into the common 1,2-dihexanoyl-sn-glycero-3-phosphocholine - 1,2-dimyristoyl-sn-glycero-phosphatidylcholine (DHPC-DMPC) bicelles. Therefore, we described a new type of bicelles using dodecylphosphocholine (DPC) and DMPC, not described in the literature in these conditions until then. We showed that βPFO was able to reconstitute into DPC-DMPC bicelles preserving its overall structure and pore-forming function. Then, to advance towards βPFO validation, we immunized an alpaca with βPFO in order to generate Nanobodies. A Nanobody is a fragment of the single-chain antibodies produced by camelids, with many different properties from conventional antibodies, their reduced size, their cavity specificity, their ease of modification and production, etc. Upon the Nanobodies generation, we selected the ones specific against βPFO obtaining 11 different Nanobodies. Using enzyme-linked immunosorbent assay (ELISA) we showed that they had both, a high specificity for βPFO compared to monomeric and fibrillar Aβ42, and a high affinity for them. Moreover, we showed that these generated Nanobodies, were binding to βPFO in different manners affecting differently the protection they could cause to proteolysis. Finally, we demonstrated that upon Nanobody binding on membrane-inserted βPFO, some of the Nanobodies did not affect the current pass across the bilayer while others reduced the current pass and two of them completely blocked the pore formed. In the future, these Nanobodies could serve, not only as a tool to validate as a player βPFO in the context of AD, but also as possible therapeutics.