Abstract

The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding.

Highlights

  • Unlike dioleoyl phosphatidylcholine (DOPC) and POPC, dilauroyl phosphatidylcholine (DLPC) Liposomes Inhibit A␤ Fibrillation—Lipid bilayers can modulate the rate of amyloid formation by A␤, the extent of modulation varies depending on the bilayer composition, incubation conditions, and detection method [23,24,25]. thioflavin-T (ThT), a dye that fluoresces in the presence of amyloid fibrils, can probe for the formation of fibrillar aggregates and is useful in delineating mechanistic deviations in amyloid aggregation (26 –28)

  • ThT was used to initially probe the aggregation propensity of A␤ in the presence of large unilamellar vesicles (LUVs) of DOPC, POPC, and DLPC and characterize kinetic differences that result from different interactions caused by variation in hydrophobic thickness

  • End state aggregates formed in the absence and presence of either DOPC or POPC were examined by CD spectroscopy and transmission electron microscopy (TEM) (Fig. 2)

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Summary

The abbreviations used are

A␤, amyloid-␤; AD, Alzheimer’s disease; DLPC, dilauroyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; ThT, thioflavin-T; LUV, large unilamellar vesicle; TEM, transmission electron microscopy; CMC, critical micelle concentration; AFM, atomic force microscopy; SLB, smooth supported lipid bilayer; GM1, Gal␤1,3GalNAc␤1,4(NeuAc␣2,3)-Gal␤1,4Glc-ceramide. It is more likely that the hydrophobic thickness of lipid bilayers influences the generation and/or stabilization of higher order membrane-associated species because of the ability of these A␤ aggregates to insert themselves within the bilayer [15] Both DOPC and POPC have been previously applied to examine the interactions between lipid bilayers and amyloidogenic proteins (8, 16 –19). It was observed that DLPC bilayers are capable of remodeling mature A␤ fibrils into a semi-unfolded intermediate, a feat previously observed only after treatment with high concentrations of denaturants or exposure to high frequency sonication [22] These results show that thin bilayers are able to dramatically alter the aggregation of A␤ compared with conventional lipid bilayers, and this redirected misfolding can stabilize distinct, membrane-associated species not observed previously

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