Effect of Cholesterol on Membrane Pore Formation by Amyloid β25-35

Abstract

One of the cytotoxicity mechanisms of amyloid β (Aβ) peptide in Alzheimer's disease is membrane permeabilization, yet the mechanism of pore formation remains unclear. Here, Ca2+-permeable membrane pore formation by a cytotoxic fragment of Aβ (Aβ25-35) has been analyzed by biophysical methods. Quin-2-loaded lipid vesicles composed of 0.3 mole fraction of anionic lipid 1-palmitoyl-2-oleoyl-phosphatidylglycerol, xchol mole fraction of cholesterol, and (0.7 - xchol) mole fraction of zwitterionic lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine were prepared at xchol = 0, 0.05, 0.1, 0.2 and 0.4. CaCl2 was added externally so that Quin-2 was sequestered from Ca2+ ions. Quin-2 fluorescence increased exponentially upon addition of Aβ25-35, which was interpreted as membrane pore formation by the peptide, calcium influx and binding to intravesicular Quin-2. The kinetics and the magnitude of fluorescence enhancement were used to evaluate the second order rate constant of pore formation (ka), the affinity constant between the peptide molecules (Kp), and the number of peptide units in the pore (n). Circular dichroism suggested mostly β-sheet structure for the peptide in the presence of lipid vesicles. Attenuated total reflection Fourier transform infrared experiments were performed on Aβ25-35 embedded in lipid multilayers deposited on a germanium plate. These data suggested α-helical and β-sheet structures for the peptide in a lipid environment. The structure of the peptide, i.e., the α-helix/β-sheet ratio, varied as a function of cholesterol content in the membranes. These conformational transitions correlated with changes in pore formation parameters (Kp, ka, n). The data suggest that cholesterol affects Aβ membrane pore formation by multiple mechanisms. At moderate concentration (xchol< 0.1), cholesterol supports pore formation possibly through cholesterol-peptide interactions. Further increase in xchol causes pore inhibition by a membrane solidification mechanism. At xchol = 0.4, cholesterol causes lipid disorder and membrane destabilization.