Study of the effects of aminosterols and berberine on the structural and dynamical properties of biological membranes and quantification of their protective role against misfolded protein oligomers

Many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, are associated with the self-assembly of peptides and proteins into fibrillar aggregates. Soluble misfolded oligomers formed during the aggregation process, or released by mature fibrils, play a relevant role in neurodegenerative processes through their interactions with neuronal membranes. Natural aminosterols are considered promising drug candidates against neurodegeneration, and one relevant protective mechanism occurs via their binding to biological membranes and inhibiting the binding of amyloidogenic proteins and their cytotoxic oligomers. Another natural compound with a potential therapeutic effect is the plant alkaloid berberine, which has been reported to possess antioxidant and anti-inflammatory activities and to protect neuronal cells from death induced by oxidative stress, but its mechanism of action is not yet clear, and there is no evidence whether it exerts its protective effects through the interaction with biological membranes. In the first part of this thesis, we obtained a quantitative measurement of the affinity of a pair of toxic/nontoxic oligomers of the model protein HypF-N for the lipid membranes of liposomes in the form of large unilamellar vesicles (LUVs) by using fluorescence quenching experiments with probes embedded in the polar and apolar regions of the LUV bilayer and toxic/nontoxic oligomers, and another oligomer membrane binding assay using fluorescently labelled oligomers and unlabeled LUVs. With both approaches, we found that toxic oligomers have a membrane affinity 20- 25 times higher than nontoxic oligomers. Circular dichroism spectroscopy, intrinsic fluorescence measurements and fluorescence resonance energy transfer (FRET) indicated that neither oligomer type changes its structure upon membrane interaction. LUVs enriched with trodusquemine had a significantly decreased affinity for these toxic species. The affinity of the oligomers for the lipid membranes increased and slightly decreased with GM1 ganglioside and cholesterol content, respectively, indicating that physico-chemical properties of lipid membranes modulate their affinity for misfolded oligomers. In the second part of this thesis, we compared three natural chemically different aminosterols, namely squalamine, trodusquemine and the newly discovered 2 aminosterol ENT-03. Using unlabelled LUVs and fluorescently labelled aminosterols and an aminosterol-membrane binding assay we quantified the different binding affinities of the three aminosterols for the membrane. Moreover, the three aminosterols caused different (i) LUV charge neutralization, as observed with ζ potential measurements; (ii) LUV bilayer mechanical reinforcement, as observed with breakthrough force measurements on supported lipid bilayers (SLBs) with atomic force microscopy (AFM); and (iii) redistributions of key lipids within membranes of LUVs, as inspected with lipid-lipid FRET. Following a dose-dependent quantification of their different abilities in protecting cultured cells against Ca2+ influx induced by amyloid-β oligomers, a global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane perturbations, and furthermore correlated aminosterol-mediated protection with well-defined chemical moieties, linking quantitatively their chemistry to their protective effects on biological membranes. In the third part of this thesis, we focused on the plant alkaloid berberine, and we demonstrated its ability to interact with the membrane of LUVs, with a slightly higher affinity for the hydrophobic core of the lipid bilayer, as investigated using berberine-induced fluorescence quenching of LUVs containing fluorescent probes, light scattering and equilibrium dialysis experiments with unlabeleld LUVs and berberine. Concentration- and temperature-dependent fluorescence anisotropy measurements indicated that the interaction between berberine and LUVs induced an overall stiffening of the membrane. Moreover, berberine decreased the affinity of toxic oligomers for the membrane, as observed with fluorescence quenching experiments with fluorescent probes embedded in LUVs bilayer and toxic oligomers. An ANS binding assay and circular dichroism spectroscopy in the absence of LUVs highlighted the ability of this alkaloid to shield the exposure of hydrophobic clusters to the solvent of toxic oligomers, without significantly altering their secondary structure, suggesting an additional therapeutic potential of this molecule against misfolded oligomers based on its direct effect on oligomers, in addition to its protective binding to the membranes.