Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.
Increasing evidence supports the idea that the initial events of A beta oligomerization and cytotoxicity in Alzheimer's disease involve the interaction of amyloid A beta-derived diffusible ligands (ADDLs) with the cell membrane. This also indicates lipid rafts, ordered membrane microdomains enriched in cholesterol, sphingolipids and gangliosides, as likely primary interaction sites of ADDLs. To shed further light on the relation between ADDL-cell membrane interaction and oligomer cytotoxicity, we investigated the dependence of ADDLs binding to lipid rafts on membrane cholesterol content in human SH-SY5Y neuroblastoma cells. Confocal laser microscopy showed that A beta 1-42 oligomers markedly interact with membrane rafts and that a moderate enrichment of membrane cholesterol prevents their association with the monosialoganglioside GM1. Moreover, anisotropy fluorescence measurements of flotillin-1-positive rafts purified by sucrose density gradient suggested that the content of membrane cholesterol and membrane perturbation by ADDLs are inversely correlated. Finally, contact mode atomic force microscope images of lipid rafts in liquid showed that ADDLs induce changes in raft morphology with the appearance of large cavities whose size and depth were significantly reduced in similarly treated cholesterol-enriched rafts. Our data suggest that cholesterol reduces amyloid-induced membrane modifications at the lipid raft level by altering raft physicochemical features
Several lines of evidence suggest that the initial events of amyloid-p peptide (Ap) oligomerization and deposition in Alzheimer's disease (AD) involve the interaction of soluble oligomers with neuronal membranes. In this study, we show that Ap42 oligomers are recruited to lipid rafts, which are ordered membrane microdomains rich in cholesterol and gangliosides, resulting in lipid peroxidation, Caz* dyshomeostasis and membrane permeabilization in primary fibroblasts from familial AD patients (FAD) bearing APPValTlTIle, PS-1Leu392Val or PS-1Metl46Leu gene mutations. Moreover, the presence of significant$ higher levels of lipid peroxidation correlated with greater structural modification in detergent resistant domains (DRMs) isolated from APP and PS-l fibroblasts, compared to WT fibroblasts from healthy subjects.Modulation of raft GMl, including modest depletion of GMI content and interference with GMt exposure or negative chaqge, precluded the interaction of amyloid aggregates with the plasma membrane and the resulting cell damage in FAD fibroblasts and rat brains cortical neurons. These findìngs suggest a specific role for raft domains as primary mediators of amyloid toxicity in AD neurons.
The authors describe the interaction of biological nanostructures formed by β(2) -microglobulin amyloid fibrils with three-dimensional silicon microstructures consisting in periodic arrays of vertical silicon walls (≈3 μm-thick) separated by 50 μm-deep air gaps (≈5 μm-wide). These structures are of great interest from a biological point of view since they well mimic the interstitial environment typical of amyloid deposition in vivo. Moreover, they behave as hybrid photonic crystals, potentially applicable as optical transducers for label-free detection of the kinetics of amyloid fibrils formation. Fluorescence and atomic force microscopy (AFM) show that a uniform distribution of amyloid fibrils is achieved when fibrillogenesis occurs directly on silicon. The high resolution AFM images also demonstrate that amyloid fibrils grown on silicon are characterized by the same fine structure typically ensured by fibrillogenesis in solution.
We studied the effect of the model amyloid‐forming protein, HypF‐N (the N‐terminal domain of the prokaryotic hydrogenase maturation factor HypF), in its native and aggregated form on the integrity of model lipid membranes, including those more representative of natural membranes, using AFM and surface pressure measurements. Using fluorescence spectroscopy, we studied the effect of lipids on the structure of HypF‐N. We find that native protein has a larger effect on model membranes than aggregated protein. Model membranes composed of lipid raft forming mixtures (phosphatidylcholine: sphingomyelin: cholesterol, 1:1:1) were much more resistant to insertion of protein and membrane disruption than those composed of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylserine (PS). Addition of cholesterol to the latter mixture made the membranes less resistant to protein insertion. Liposomes containing the negatively charged lipid PS induced a strong quenching in fluorescence of HypF‐N (indicating a change in the environment of tryptophan and tyrosine residues) while those containing only PC had little effect. Preliminary denaturation experiments indicate relatively strong binding of HypF‐N to PS:PC liposomes.Research was supported by Saint Lawrence University, University of Genoa and University of Florence.
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