Membrane Fusogenic Activity of the Alzheimer's Peptide Aβ(1–42) Demonstrated by Small-Angle Neutron Scattering

doi:10.1016/j.jmb.2007.11.076

Journal of Molecular Biology

Volume 376, Issue 2, 15 February 2008, Pages 393-404

https://doi.org/10.1016/j.jmb.2007.11.076 Get rights and content

Abstract

Amyloid-β peptide (Aβ) is considered a triggering agent of Alzheimer's disease. In relation to a therapeutic treatment of the disease, the interaction of Aβ with the cell membrane has to be elucidated at the molecular level to understand its mechanism of action. In previous works, we had ascertained by neutron diffraction on stacked lipid multilayers that a toxic fragment of Aβ is able to penetrate and perturb the lipid bilayer. Here, the influence of Aβ(1–42), the most abundant Aβ form in senile plaques, on unilamellar lipid vesicles of phospholipids is investigated by small-angle neutron scattering. We have used the recently proposed separated form factor method to fit the data and to obtain information about the vesicle diameter and structure of the lipid bilayer and its change upon peptide administration. The lipid membrane parameters were obtained with different models of the bilayer profile. As a result, we obtained an increase in the vesicle radii, indicating vesicle fusion. This effect was particularly enhanced at pH 7.0 and at a high peptide/lipid ratio. At the same time, a thinning of the lipid bilayer occurred. A fusogenic activity of the peptide may have very important consequences and may contribute to cytotoxicity by destabilizing the cell membrane. The perturbation of the bilayer structure suggests a strong interaction and/or insertion of the peptide into the membrane, although its localization remains beyond the limit of the experimental resolution.

Introduction

The emerging trend for the explanation of neurodegeneration in Alzheimer's disease (AD) imputes the cause of neurotoxicity to the interaction of soluble amyloid beta forms with neural cells.¹ Amyloid-β peptides (Aβs) are peptides naturally found in the cerebrospinal liquids, and little is known about their physiological function. They are the product of the enzymatic cleavage of a longer transmembrane protein, the amyloid precursor protein, and they have been identified more than two decades ago as the principal component of the proteinaceous deposits characteristic of brain tissues of patients with AD.² These so-called “senile plaques” primarily contain fibrils of Aβ, and it has been commonly believed that the Aβ fibrils were the toxic form of this peptide.3, 4, 5 More recently, it has been shown that soluble forms of Aβ cause neuronal dysfunction in vivo,⁶ and it was demonstrated that Aβ oligomers are more toxic than fibrils.⁷ In addition, it has been shown that Aβ(1–42) in the soluble form is nondetectable in plaque-free normal brain.⁸ Taken together, these experimental facts tend to identify the soluble and diffusible Aβ forms as the trigger of the neurodegenerative cascade of AD. Nevertheless, the mechanism of action of Aβ continues to remain unknown. The study of the interaction of Aβ with the neuronal membrane is a topic of great interest, in the attempt to identify whether the soluble amyloid beta binds to specific receptors or adsorbs nonspecifically to various receptors and channel proteins. In addition, there is evidence that Aβ and other amyloidogenic proteins can penetrate the membrane, leading to permeabilization and to pore formation.⁹ In our previous investigations, we had applied neutron diffraction and selective deuteration to localize the short peptide Aβ(25–35), a toxic fragment of Aβ, into lipid bilayers of different surface charge and composition.10, 11, 12 In the present study, the interaction of the most abundant Aβ in senile plaques, that is, Aβ(1–42), with unilamellar lipid vesicles (ULVs) is investigated by small-angle neutron scattering (SANS). SANS is a well-established technique for the investigation of lipid vesicles¹³ and the change of the parameters describing their structure in different conditions.14, 15, 16, 17 We have applied the recently proposed separated form factor (SFF) method¹⁸ to extruded unilamellar vesicles, and we have extracted the parameters characterizing the vesicle size, size distribution, and vesicle shell (i.e., lipid bilayer) by using different models to describe the bilayer profiles. The effect of Aβ on these parameters has been investigated as a function of pH and of Aβ concentration. The results clearly show the interaction of Aβ with respect to lipid membranes and allow assumptions about its localization in the bilayer.

Section snippets

Theory

SANS measurements from ULV contain different information about the vesicle structure in different regions of the scattering vector q. The low-q region (q < 0.02 Å^–1) is affected by the vesicle shape and size and allows extracting the vesicle radius R and the polydispersivity. The high-q region (q > 0.1 Å^–1) is modulated by the thickness of the shell d and is barely affected by vesicle radius and polydispersivity.

For monodisperse systems, the scattered intensity I(q) is given by: $I (q) = n F^{2} (q) S (q)$ where

Results

We have investigated by SANS the interaction of monomeric and aged Aβ(1–42) with lipid vesicles.

The small-angle scattering curves obtained from the ULV dispersions at pD 7.0 and pD 6.0 at various Aβ(1–42) contents are shown in Fig. 3a and b. As thoroughly discussed elsewhere,²⁵ the low-q region is influenced by the ULV form factor, while the scattering in the high-angle region is dominated by the form factor of the lipid bilayer. It is evident that the peptide affects the experimental curves

Discussion

In this article, the SFF method used to analyze and interpret small-angle scattering has been applied to curves obtained from ULV of phospholipids in the presence of different amounts of Aβ(1–42).

To our knowledge, although this method has already been proposed to investigate ULV of pure phospholipids, this is the first time that this method is employed to explain the change of the bilayer structure as a function of some physicochemical parameters. We have modified an existing fitting routine to

Sample preparation

POPC and the net negatively charged lipid POPS were purchased from Avanti Polar Lipids (Alabaster, AL) and used as received. The deuterated forms of these products, that is, POPC-D31 and POPS-D31, were also from Avanti Polar Lipids. Aβ(1–42) was a product of Bachem (Germany). D₂O was a product of Sigma-Aldrich, and phosphate-buffered saline (PBS) was purchased from Fluka (Germany). The lipids were solubilized in 1:1 chloroform/methanol; the solvent was evaporated under a gentle stream of

Acknowledgements

The authors acknowledge Dr. Jeremy Pencer for kindly providing the fitting routine of the SANS data. This work was partially supported by the Deutsche Forschungsgemeinschaft (SFB 472).

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Journal of Molecular Biology

Membrane Fusogenic Activity of the Alzheimer's Peptide Aβ(1–42) Demonstrated by Small-Angle Neutron Scattering

Abstract

Introduction

Section snippets

Theory

Results

Discussion

Sample preparation

Acknowledgements

Biochem. Biophys. Res. Commun.

Neuron

Neurobiol. Dis.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biophys. J.

Biophys. J.

Biochim. Biophys. Acta

Biochim. Biophys. Acta, Biomembr.

Curr. Opin. Struct. Biol.

Chem. Phys. Lipids

Peptides

J. Biol. Chem.

J. Mol. Biol.

Physica B

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide

Nat. Rev., Mol. Cell Biol.

Neurotoxicity of beta-amyloid and prion peptides

Curr. Opin. Neurol.

Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red

Proc. Natl Acad. Sci. USA

Zn2+ interaction with Alzheimer amyloid beta protein calcium channels

Proc. Natl Acad. Sci. USA

Zn²⁺ interaction with Alzheimer amyloid beta protein calcium channels