Erythrocytes as a complex membrane system to elucidate plasma membrane perturbations associated with amyloid-related disorders

Mathew Sebastiao,^1,2 Mathilde Fortier,^1,2 Noe Quittot,^1,2 Isabelle Marcotte,^1,2 Steve Bourgault^1,2

¹Université du Québec à Montreal, Département de chimie
²Québec Network on Protein Function, Engineering, and Applications (PROTEO), Canada

The formation of amyloid fibrils and their tissue deposition are associated with numerous diseases, known as amyloidoses. Historically, amyloid fibrils were presumed to be the toxic agents responsible for these pathologies. However, recent studies have indicated that pre-fibrillar species are more cytotoxic than fully formed fibrils. These oligomeric proteospecies are suspected to disrupt the plasma membrane (PM), initiating pathways of cell death. Most biophysical characterizations of the interactions between amyloidogenic proteins and PM have used simplified models such as unilamellar vesicles. To better represent the complexity and heterogeneous nature of the PM, erythrocyte membranes were used instead of traditional lipid bilayers. As an amyloidogenic protein, the human islet amyloid polypeptide (IAPP) was used due to its potent cytotoxicity, high aggregation propensity, and the large library of analogs available among our team. Monomeric IAPP exhibited a significant hemolytic activity in erythrocytes indicating membrane perturbation, which was not observed for pre-assembled IAPP fibrils and for a non-aggregating peptide analog (rat IAPP). Nonetheless, the lipid bilayer of the PM remained intact even at elevated monomeric IAPP concentrations, despite the clear presence of amyloid. These findings were confirmed by thioflavin-T fluorescence, Congo red binding, atomic force microscopy, transmission electron microscopy, and solid-state NMR spectroscopy. Confocal microscopy and FRET experiments indicated that intact membranes were clustered by aggregated protein and potentially exchanged lipids between vesicles. This study supports a better understanding of the fundamental cytotoxic pathways of amyloids, and underlines the importance of developing new strategies to study amyloid-related pathologies using biologically relevant models.