Understanding Aß Peptide Binding to Lipid Membranes: A Biophysical Perspective.

Aß peptides are known to bind neural plasma membranes in a process leading to the deposit of Aß-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aß plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aß-bilayer binding. Concepts such as Aß peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aß-membrane interaction are separately identified and discussed.

The Influence of Lipid Electric Charge on the Binding of Aß(1-42) Amyloid Peptide to Bilayers in the Liquid-Ordered State.

The amyloidogenic Aß peptides are widely considered as a pathogenic agent in Alzheimer's disease. Aß(1-42) would form aggregates of amyloid fibrils on the neuron plasma membranes, thus perturbing neuronal functionality. Conflicting data are available on the influence of bilayer order on Aß(1-42) binding to membranes. In the present study, a biophysical approach was used in which isothermal calorimetry and surface pressure measurements were applied to explore the interaction of Aß(1-42) in either monomeric, oligomeric, or fibrillar form with model membranes (bilayers or monolayers) in the liquid-ordered state that were either electrically neutral or negatively charged. In the latter case, this contained phosphatidic acid, cardiolipin, or ganglioside. The calorimetric studies showed that Aß(1-42) fibrils, oligomers, and monomers could bind and/or be inserted into bilayers, irrespective of electric charge, in the liquid-ordered state, except that monomers could not interact with electrically neutral bilayers. The monolayer studies in the Langmuir balance demonstrated that Aß(1-42) aggregation hindered peptide insertion into the monolayer, hindered insertion in the decreasing order of monomer > oligomer > fibril, and that lipid composition did not cause large differences in insertion, apart from a slight facilitation of monomer and oligomer insertion by gangliosides.

ß-Amyloid (1-42) peptide adsorbs but does not insert into ganglioside-containing phospholipid membranes in the liquid-disordered state: modelling and experimental studies.

ß-Amyloid (Aß) is a 39-43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aß deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aß42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1-5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aß42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aß binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aß plaque formation.

The Binding of Aß42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation.

The binding of Aß42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aß42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aß42 giving rise to more extended ß-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aß42 to sphingomyelin/cholesterol bilayers.

Liposomes functionalized with acidic lipids rescue Aß-induced toxicity in murine neuroblastoma cells.

The loss of synapses and neurons in Alzheimer's disease (AD) is thought to be at least partly induced by toxic species formed by the amyloid beta (Aß) peptide; therefore, therapeutics aimed at reducing Aß toxicity could be of clinical use for treatment of AD. Liposomes are suitable vehicles for therapeutic agents and imaging probes, and a promising way of targeting the various Aß forms. We tested liposomes functionalized with phosphatidic acid, cardiolipin, or GM1 ganglioside, previously shown to have high Aß-binding capacity. Mimicking Aß-induced toxicity in mouse neuroblastoma cell lines, combined with administration of cell viability-modulating agents, we observed that functionalized liposomes rescued cell viability to different extents. We also detected rescue of the imbalance of GSK-3ß and PP2A activity, and reduction in tau phosphorylation. Thus, these liposomes appear particularly suitable for implementing further therapeutic strategies for AD.

Lipid-based nanoparticles with high binding affinity for amyloid-beta1-42 peptide.

The neurotoxic beta-amyloid peptide (Abeta), formed in anomalous amounts in Alzheimer's disease (AD), is released as monomer and then undergoes aggregation forming oligomers, fibrils and plaques in diseased brains. Abeta aggregates are considered as possible targets for therapy and/or diagnosis of AD. Since nanoparticles (NPs) are promising vehicles for imaging probes and therapeutic agents, we realized and characterized two types of NPs (liposomes and solid lipid nanoparticles, 145 and 76 nm average size, respectively) functionalized to target Abeta(1-42) with high affinity. Preliminary immunostaining studies identified anionic phospholipids [phosphatidic acid (PA) and cardiolipin (CL)] as suitable Abeta(1-42) ligands. PA/CL-functionalized, but not plain, NPs interacted with Abeta(1-42) aggregates as indicated by ultracentrifugation experiments, in which binding reaction occurred in solution, and by Surface Plasmon Resonance (SPR) experiments, in which NPs flowed onto immobilized Abeta(1-42). All these experiments were carried out in buffered saline. SPR studies indicated that, when exposed on NPs surface, PA/CL display very high affinity for Abeta(1-42) fibrils (22-60 nm), likely because of the occurrence of multivalent interactions which markedly decrease the dissociation of PA/CL NPs from Abeta. Noteworthy, PA/CL NPs did not bind to bovine serum albumin. The PA/CL NPs described in this work are endowed with the highest affinity for Abeta so far reported. These characteristics make our NPs a very promising vector for the targeted delivery of potential new diagnostic and therapeutic molecules to be tested in appropriate animal models.