Article pubs.acs.org/Langmuir

Cholesterol Drives Aβ(1−42) Interaction with Lipid Rafts in Model Membranes Silvia Seghezza, Alberto Diaspro, Claudio Canale,* and Silvia Dante Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy S Supporting Information *

ABSTRACT: The molecular mechanism at the basis of the neurodegenerative process related to Alzheimer’s disease (AD) is triggered by the local composition of the neural plasma membrane. The role of cholesterol is controversial. In this investigation the interaction of the AD peptide amyloid-beta (1− 42) with model membranes containing lipid rafts has been investigated by atomic force microscopy techniques. Supported lipid membranes made of phospholipids/sphingomyelin/cholesterol have been investigated as a function of the molar content of cholesterol, in a range spanning the phase diagram of the lipid system. The administration of amyloid-beta induced a phase reorganization of the lipid domains, when the cholesterol molar fraction was below 5%. At the same time, a mechanical destabilization and an appreciable thinning of the membrane induced by the peptide were detected. The major interaction was observed in the presence of the gel phase Lβ, and was enhanced by a low cholesterol amount. With the appearance of the liquid ordered phase Lo, the effect was hindered. At high cholesterol content (20% mol), no detectable effects in the bilayer morphology or in its mechanical stability were recorded. These findings give new insights on the molecular mechanism of the amyloid/membrane interaction, highlighting the peculiar role of cholesterol.

1. INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia in the elderly population. With the exception of sporadic cases of early onset AD, it affects people over age 65 and its incidence increases dramatically with aging. It is characterized by progressive memory loss and neuronal degeneration and death. Decades of research in the field have allowed formulating different hypotheses, supported by the observation of distinctive hallmarks, about the molecular mechanisms that can cooperatively lead to neurodegeneration. Unfortunately, many details still need to be clarified and an effective therapeutic strategy is still missing. Among these theories, the amyloid cascade hypothesis1−3 suggests that the neurodegenerative effect is mainly due to the action of small peptides consisting of 39−42 amino acidic residues, the amyloid-beta (Aβ) peptides, which tend to self-aggregate into insoluble fibrils constituting the extracellular neuritic plaques observed in the brains of AD patients. Neurodegeneration is thought to start much before plaques formation, probably caused by the interaction of small membrane-soluble Aβ aggregates with plasma membrane.4,5 The most abundant Aβ species in senile plaques are the ones with 40 and 42 residues; in particular, Aβ(1−42), is thought to be the main one responsible for neurotoxicity in AD. Aβ peptides are produced through the cleavage of a transmembrane protein, the amyloid precursor protein (APP), by two enzymatic complexes, γ- and β-secretase, operating in specific membrane lipid domains known as detergent-resistant microdomains or rafts.6,7 These liquid ordered lipid domains, enriched in cholesterol, © 2014 American Chemical Society

sphingolipid, and proteins, are thought to have an important role in many cellular processes such as cell signaling and protein trafficking8−10 and might be a preferential target for pathogenic agents.11,12 In this scenario, the role of cholesterol appears to be crucial, both for its contribution in lateral domain organization13−15 and for its capability of modulating important membrane properties such as fluidity.16,17 Recent studies have proved a direct connection between content and distribution of cholesterol inside the membrane and Aβ, showing that cholesterol can both modulate amyloidogenesis18−21 and trigger or prevent specific types of membrane-peptide interactions. The results are extremely controversial and it has still to be demonstrated whether high cholesterol content may increase neurotoxicity or, on the contrary, has a protective role against neurodegeneration. Part of the literature asserts that an increase in cholesterol can facilitate Aβ penetration inside the membrane,22−24 trigger peptide aggregation,25,26 and lead to the formation of pores,27,28 which can alter the ionic balance. On the other side, mechanical simulations29 and in vitro tests on cells30−32 and model membranes33,34 showed that a high cholesterol content can prevent membrane disruption, reduce fibrillization, protect against peptide insertion, and decrease Aβ cytotoxic effect. It is also important to notice that cholesterol distribution between different domains, rather than Received: July 25, 2014 Revised: October 31, 2014 Published: October 31, 2014 13934

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means of a two component epoxy glue. Silicon surface was cleaned with 20 mM sodium dodecyl sulfate (SDS) solution under sonication for 30 min, rinsed with Milli-Q water (Millipore, resistivity 18.2 MΩ cm) several times, and put in an UV/ozone chamber (BioForce Nanosciences, Ames, IA) for at least 30 min to remove any organic contamination. In order to get uniform bilayer coverage, overnight incubation at room temperature was required, keeping the samples in a closed chamber with 100% relative humidity (rh). Before AFM measurements, SLBs were gently rinsed three times with Milli-Q water to remove vesicles in excess from the liquid subphase in contact with the bilayer. Aβ Peptide Preparation and Administration. Aβ(1−42), purchased from Bachem (Germany), was pretreated with trifluoroacetic acid (TFA, Sigma-Aldrich) at concentration 1 mg/mL55 to destroy seeds and aggregates. A few microliters of this solution was dried under nitrogen flux, redissolved in Milli-Q water, sonicated for 5 min, and centrifuged for 10 min at 1000 rpm to separate possible aggregates. Aβ(1−42) monomer solution was then directly administered to the sample under the atomic force microscope head with a 1 μM final concentration and incubated for 10 min before further measurements. Before injection, the AFM tip was lifted up 100 μm from the sample. AFM Measurements. All AFM measurements were performed by using a Nanowizard III (JPK Instruments, Germany) mounted on an Axio Observer D1 (Carl Zeiss, Germany) inverted optical microscope. V-Shaped DNP silicon nitride cantilevers (Bruker, Billerica, MA), with a nominal spring constant ranging from 0.12 to 0.48 N/m, with a resonance frequency in air ranging from 40 to 75 kHz, and a tip with typical curvature radius of 20−60 nm were used. The actual spring constant of each cantilever was determined in situ, using the thermal noise method.56 AFM Imaging. SLBs were imaged in intermittent contact mode in liquid with an oscillating frequency of 10−15 kHz. Images of 5 μm × 5 μm and 2 μm × 2 μm were collected in different areas of the sample before and after protein administration. In order to reduce possible damage to the sample, the image set point was kept above 75% of free oscillation amplitude and tip lateral velocity was in the range 2−4 μm/ s. An analysis of height distribution was performed to quantify the variation in coverage of different phase domains. Quantitative imaging mode (QI, JPK Instruments), which represents the topography related to tip position at a specific force load, was used during a preliminary analysis on different lipid mixtures in order to qualitatively evaluate tip penetration inside the sample after bilayer rupture: QI images deriving from 256 × 256 force−distance curves were acquired on different samples, with maximum force load either below (0.8 nN) or above Fb (18 nN). For each curve, the tip speed was 30 μm/s and the curve length was 60 nm (maximum force below Fb) or 210 nm (maximum force above Fb). Breakthrough Force Measurement. At least two maps of force− distance curves were acquired on each sample before and after peptide administration, with each map consisting of 400 curves collected in a 10 μm × 10 μm area. Force load was in the range 12−16 nN, above the maximum expected breakthrough force, in order to detect the breakthrough event for each curve. Curve length (300 nm) and tip vertical velocity (1 μm/s) were maintained constant. Force−distance curve data sets were analyzed with a home-built Matlab (MathWorks, Natick, MA) algorithm which allows Fb detection for each curve. Variation in Fb distributions before and after protein administration has been considered for each measured sample. Differences in the distance covered by the tip during bilayer penetration, which is directly related to bilayer thickness, were also taken into account.

overall brain cholesterol content, is thought to be altered in AD patients.35,36 Planar membranes, such as supported lipid bilayers (SLBs),37−39 are extremely suited for atomic force microscopy (AFM) investigation. The high lateral and vertical resolution of AFM easily allows distinguishing between different lipid phases and detecting small features such as pores or peptide aggregates on the membrane surface.40−47 Important parameters related to the mechanical stability of the membrane are available. In this work we investigate the breakthrough force (Fb), that is, the force required by the tip to penetrate the bilayer;43,48−52 in particular, we study the fine modifications induced by Aβ(1− 42) monomers on model membranes containing different percentages of cholesterol after short incubation time. We chose a lipid mixture containing monounsaturated phospholipids and sphingomyelin in the ratio 2:1, in order to reproduce a small raft domains organization. Phospholipid headgroup composition, consisting of 90% zwitterionic choline and 10% negative serine, aims to mimic the lipid charge distribution of the neural membrane. The cholesterol content was varied in the range 0−20% to compare with the results of a previous study, in which the interaction of Aβ(25−35) with lipid membranes made of a binary mixture of phospholipids and cholesterol was reported.34 Interestingly, at the 2:1 phospholipid/SM molar ratio (i.e., the ratio employed in this investigation), the introduction of different percentages of cholesterol leads the lipid system through different phases and phase coexistence.53,54 Low molar concentration of the peptide, initial step of the aggregation kinetics, and short incubation time have been chosen as conditions to investigate the molecular mechanisms that trigger the membrane−peptide interaction. In the analysis, we take into account both topographical domain reorganization and variation in Fb distributions before and after peptide administration. The study is carried out by conventional AFM imaging techniques and force−distance curve acquisition, supplemented by information obtained employing a recently developed force−distance curve based imaging mode.

2. EXPERIMENTAL SECTION Supported Lipid Bilayers. For vesicles preparation, we used 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and sphingomyelin (SM) (brain, porcine), all purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol (Chol) from Sigma-Aldrich (St. Louis, MO). Four different lipid mixtures were prepared, containing phospholipids and SM in a 2:1 mol/mol ratio plus a variable molar percentage of Chol: 0%, 1%, 5%, and 20%. Phospholipid composition was POPC/POPS 9:1 mol/mol. Lipid powders were dissolved in chloroform/methanol (Sigma-Aldrich) 2:1, mixed according to the chosen compositions and gently dried under a nitrogen flux. Aliquots were then incubated overnight under vacuum, in order to remove any solvent trace, and resuspended in phosphate buffer saline (PBS 1×) in a concentration of 1 mg/mL to form multilamellar vesicles (MLVs). Each MLV suspension was presonicated for 1 h at 60 °C and subsequently extruded 11 times through a polycarbonate membrane with 100 nm pores using a commercial extruder (Avanti Polar Lipids). Extrusion was performed on a hot plate that was kept at 60 °C. After cooling at room temperature, the obtained large unilamellar vesicle (LUV) suspensions were diluted 10-fold in PBS 1× and then 50 μL of each suspension was administrated to the substrate to allow vesicle fusion and bilayer formation. Silicon (1,0,0) N-doped substrates (1 cm × 1 cm, Siltronic AG, Germany) were used. Native oxide was not removed. Silicon was used as a substrate, since it is a common material used in many biophysical techniques and often employed in the biosensors field. The substrates were mounted on microscope slides by

3. RESULTS AFM Imaging. Before examining the effect of Aβ peptide on SLBs, a preliminary AFM study on phospholipid/SM/Chol bilayer formation on SiO2 surface was performed (Figure S1, Supporting Information). Samples without cholesterol or containing a small percentage of cholesterol (up to 5%) were characterized by a lateral organization presenting raftlike 13935

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Figure 1. AFM topographies of SLBs acquired before (images on the left of the panels) and after (images on the right of the panels) Aβ(1−42) administration. Histograms below each image represent the corresponding height distribution: Gaussian fittings have been performed to estimate the variation in phase coverage (horizontal axis, relative height [nm]; vertical axis, counts [#]). Each panel represents one exemplifying result for each lipid mixture. SLB composition is POPC/POPS/SM 9:1:5 mol/mol/mol + X% mol Chol (panel A, X = 0; panel B, X = 1; panel C, X = 5; panel D, X = 20). Images have been collected in the same area before and after peptide injection. Exact overlapping was not achievable, likely due to sample drift induced by thermal effects. Blue star markers identify corresponding points in each topography couple. Images were acquired in intermittent contact mode in liquid. Scale bars, 1 μm; Z-range, 2 nm.

domains, protruding 0.8 ± 0.1 nm with respect to the rest of the membrane. These small circular ordered lipid domains, sizing from tens to few hundreds of nanometers, were distributed all over the sample, separated by disordered lipid regions or, in some cases, clustered together. According to literature,13,14 we assume that SM and Chol were located in the thicker ordered phase, while the disordered one was mainly composed of phospholipids. Samples with high cholesterol percentage (20% or more) presented a rough surface on which it was not possible to distinguish between different lipid phases. Time required for complete bilayer formation could vary from 3 to 12 h, depending on uncontrolled environmental conditions such as temperature and rh. We observed that overnight incubation allowed achieving complete coverage for all the samples, which appeared stable and reproducible. For this reason, we adopted this solution for sample preparation, maintaining a high rh in order to avoid evaporation. For Aβ(1−42) experiments, we selected four lipid mixtures containing POPC/POPS/SM in a molar ratio 9:1:5 and different molar percentage of cholesterol: 0%, 1%, 5%, and 20%. Exemplifying bilayer topographies per each composition are shown in Figure 1. Three samples per mixture have been examined before and after peptide administration. Only the

samples that did not present evident anomalies in terms of homogeneity of the SLB and presence of defect in the force− distance curve were employed. As first, SLB starting topography was inspected by AFM and force−distance curves were acquired. Then Aβ solution was directly injected on the sample under AFM head and incubated for 10 min unperturbed, before measuring again. The first image was generally acquired 15−20 min after peptide injection; subsequent observations proved that Aβ induced modification did not change significantly over time. Since the SLB was not moved during incubation, we obtained a direct comparison of the same areas before and after the action of the peptide. A reorganization of phase domains on samples with low (1−5%) or none cholesterol content (Figure 1A−C) was the most relevant effect. After peptide administration, the area occupied by ordered lipid phase became larger and domains clustering increased significantly. In samples containing 20% Chol, instead, topography was unaltered (Figure 1D). Since SLBs were completely covering SiO2 surface, it was not possible to measure the actual thickness of lipid phases through image analysis. The height difference between lipid domains was not affected by Aβ. Samples displaying an ordered phase coverage above 80% after peptide administration were not considered in this analysis since tip size 13936

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Figure 2. Left graph: force−distance curve acquired on a SLB. Right graph: force vs tip-sample separation representation of the trace signal of the curve on the left. Tip speed, 1 μm/s; force set point, 18 nN. Discontinuity representing tip penetration inside the SLB is highlighted (Breakthrough Event). The maximum force before rupture (Breakthrough Force, Fb) and the distance covered by the tip during penetration (Breakthrough Length, Lb) can be directly measured from the discontinuity.

effects57 could affect the measurement when AFM probe must follow the profile of very sharp and narrow pits. In order to quantify the change in phase coverage, an analysis of height distribution was performed for each couple of corresponding images (histograms in Figure 1): samples containing 0%, 1%, and 5% cholesterol displayed a bimodal distribution that could be fitted with two Gaussians; the coverage of the two phases was derived from the measurement of the areas below the two curves. Considering all the possible couples of corresponding images, we observed an increase in the area occupied by the ordered phase after peptide administration. The variation was 28.2% ± 4.8% for the 0% Chol samples, 31.4% ± 2.1% for 1% Chol, and 26.1% ± 2.8% for 5% Chol. Breakthrough Force Measurement. A typical force− distance curve on SLBs is shown in Figure 2: after the contact point, the measured force increases almost linearly with a local slope that depends both on SLB deformation and on elastic cantilever deflection; this last contribution is generally subtracted before the analysis so that only the relative distance between tip and SLB surface is considered in Z-direction (Figure 2, graph on the right). If the applied force load is sufficiently high, a discontinuity is observed in the trace part of the force−distance curve, indicating local bilayer failure and tip penetration inside the SLB. After this point, the tip is pushed on the rigid substrate and its relative distance from the sample remains constant. The critical force at which bilayer failure occurs could be taken into account to characterize the mechanical stability of the considered SLB. We examined Fb variation to understand how Aβ(1−42) affects the mechanical stability of specific model membranes. Force−distance curves, 20 × 20, have been collected on at least two different 100 μm2 areas per sample before and after Aβ(1−42) administration. Figure 3 represents normalized Fb distributions before (blue, left box plot) and after peptide (pink, right box plot) acquired on the samples shown in Figure 1. Percentage variations of mean Fb values of all the measured samples are summarized in Table 1. The overall trend consisted in a shift of Fb distributions toward lower force values. The highest variation was detected for samples containing 1% Chol;

Figure 3. Distributions of Fb measured on SLBs before and after Aβ(1−42) administration. Each couple of box plots represents an exemplifying sample for each mixture: the blue box plot on the left refers to Fb distribution before Aβ; the pink one on the right illustrates Fb distribution after Aβ action. Connection lines between the two mean Fb values, before and after the peptide, are represented as a guide to the eye. Data normalization has been done independently for each sample, considering the percentage variations in respect to the median of Fb distribution before Aβ. For each distribution, N > 800.

samples with 0% and 5% Chol (samples S2 and S3) presented an intermediate behavior, while only a small and not significant decrease was measured in the case of 20% Chol mixture (samples S2 and S3). Another value that can be extracted from the breakthrough discontinuity is the distance covered by the tip during bilayer penetration (Figure 2 right graph), which we termed breakthrough length (Lb). If we assume that, after bilayer rupture, the tip reaches the substrate below, Lb represents SLB thickness after its maximum deformation. Variations in Lb distributions before and after Aβ are summarized in Figure 4 and in Table 1. Box plots referring to absolute values of Lb for each mixture are shown in Figure S3 in the Supporting Information. A significant Lb decrease is observed for samples containing 0% and 1% Chol; in particular, for samples without cholesterol, the variation of the mean values is close to −20%. 13937

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Table 1. Percentage Variations of Fb and Lb Mean Values Induced by Aβ(1−42) Administration to SLBsa lipid mixture POPC/POPS/SM 9:1:5 + 0% Chol mean Fb variation mean Lb variation a

POPC/POPS/SM 9:1:5 + 1% Chol

POPC/POPS/SM 9:1:5 + 5% Chol

POPC/POPS/SM 9:1:5 + 20% Chol

S1

S2

S3

S1

S2

S3

S1

S2

S3

S1

S2

S3

−6.1% −19.1%

−6.2% −17.0%

−6.3% −19.4%

−17.3% −7.4%

−17.3% −8.8%

−17.2% −12.7%

−21.3% −10.7%

−7.8% −2.1%

−7.1% +0.0%

−10.7% −2.9%

−2.1% −1.5%

−3.5% −2.2%

Sample ID: S1−S3. For each sample before and after peptide, N > 800.

cholesterol molar fraction higher than 20% were unperturbed in the presence of the peptide. These facts seem to support the theories that identify membrane cholesterol as a protective agent against amyloid mechanisms of neurodegeneration.29−33 In the samples with lower cholesterol content, on the contrary, we observed an effect consisting in an increase in ordered phase coverage and a decrease in Fb distribution, probably associated with membrane destabilization. The results of the mechanical analysis are in agreement with previous findings by our group43 studying lipid bilayers supported on polymer cushion. Assuming that cholesterol may inhibit the interaction of Aβ(1−42) peptide with plasma membrane, we could expect that this effect is less relevant with the increase of cholesterol content and should be enhanced in its absence. Experimental data, instead, show that the greatest modification is observed for 1% Chol samples. In order to explain this nonlinear behavior, we analyzed the characteristics of the observed lipid phase domains. The phase diagram described in Marsh’s review54 refers to the ternary mixture POPC/SM/Chol; since phospholipids phase mainly depends on length and unsaturation content of the two hydrocarbon chains, we think it can be representative also for our mixture containing POPC/POPS 9:1 in place of POPC alone. The diagram identifies three possible lipid phases: a fluid phase mainly composed of phospholipids, generally termed liquid disordered (Lα); a gel or solid ordered phase consisting of SM and phospholipids (Lβ); a fluid phase containing SM, phospholipids, and cholesterol, termed liquid ordered (Lo). According to the diagram, our mixtures containing 0% and 1% cholesterol are located in the region of coexistence of liquid disordered and gel phases (Lα + Lβ), clearly distinguishable in the acquired images; the one with 5% cholesterol is in the three-phase coexistence area (Lα + Lo + Lβ), and the 20% one is in the phases coexistence region of liquid disordered and liquid ordered phase (Lα + Lo). Considering this domain organization, our results suggest that Aβ(1−42) peptide mainly destabilizes the gel phase and does not interact significantly with the Lα and Lo phases. The increased fluidity of Lβ phase causes an increase in the area per molecule and triggers a recruitment of part of phospholipids present in the disordered phase, causing the enlargement of the ordered area observed in Lβ containing samples. The damaging effect on Lβ phase is more relevant if a small percentage of cholesterol is present (1% Chol). Interestingly, recent literature reports that a small amount of cholesterol causes a change in packaging and orientation of saturated lipids in the gel phase, transforming a tilted gel phase to a more disordered untilted solid state.58 Assuming that such a change occurs in our system as well, this could explain the difference that we observed between 0% and 1% Chol samples, both belonging to the same region of the phase diagram. If cholesterol content further increases (5% Chol), small Lo domains, not clearly detectable on our samples through AFM imaging, start forming on the Lβ phase and, as a

Figure 4. Distributions of Lb measured on SLBs before and after Aβ(1−42) administration. Each couple of box plots represents an exemplifying sample for each mixture: the blue box plot on the left refers to Lb distribution before Aβ; the pink one on the right illustrates Lb distribution after Aβ action. Connection lines between the two mean Lb values, before and after the peptide, are represented as a guide to the eye. Data normalization has been done independently for each sample, considering the percentage variations in respect to the median of Lb distribution before Aβ. For each distribution, N > 800.

The other lipid mixtures, instead, do not display significant variations in Lb distributions.

4. DISCUSSION The effect of Aβ(1−42) small soluble species, present at the very initial step of aggregation kinetics, has been investigated considering possible topographical reorganization and change in the mechanical stability of model lipid membranes, evaluated through the measurement of Fb. In order to overcome the effect of inherent sources of variability (sample variability, AFM tip variability), statistical parameters before and after the peptide are expressed in terms of percentage variations in respect to the median of initial Fb distributions (the typical variability of Fb absolute values, as derived from the analysis performed on four exemplifying samples for each mixture are shown in Figure S2 in the Supporting Information). Results are reproducible and allow identifying a trend for each lipid mixture, with the exception of the few cases in which starting Fb values are much higher than the ones generally observed (Figure S2, the first 5% Chol and the first 20% Chol samples are the ones that displayed the anomalous behavior indicated in Table 1). The first evidence emerging from the results is that samples containing 20% of cholesterol are almost not affected by the action of the peptide: this result is in agreement with previous findings on a similar system obtained by neutron diffraction.34 In that case, the short fragment Aβ(25−35) was found to interact with fluid phospholipid membranes with 0% or 1% molar cholesterol content, whereas the membranes containing a 13938

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cholesterol against membrane destabilization induced by Aβ(1−42) is strongly related to lipid phase organization. In particular, membrane destabilization, here characterized via Fb variation, seems to be mainly caused by Aβ(1−42) interaction with lipid gel phase. Thus, the presence of cholesterol has a protective effect by triggering the transition from Lβ into Lo phase, the latter, according to our findings, being not affected by the peptide. A preventive role against bilayer thinning, independent of phase organization, was also observed. All these findings provide deeper insight on the molecular mechanisms that regulate the interaction of Aβ(1−42) with lipid membranes.

consequence, Aβ-induced destabilization decreases. When cholesterol percentage is sufficiently high (20% Chol), only small Lα and Lo domains are present, mixed together to form a rough surface; the absence of Lβ phase, thus, seems to be protective against peptide-induced membrane destabilization. The preferential interaction of Aβ(1−42) with gel phase domains of lipid bilayers was already reported in phase separated DOPC/DPPC lipid bilayers, using a combination of AFM and fluorescence microscopy.41 Recently, Dies et al.59 using X-ray diffraction showed that Aβ(25-35) and Aβ(1−42) interact and embed in the gel phase of anionic lipid membranes of DMPC/DMPS; in that case, the membrane was made of saturated phospholipids and a high content of cholesterol favored the interaction with the peptide. In contrast to our findings, several investigations in the literature report about an enhanced interaction of Aβ species with membranes, in the presence of high cholesterol levels. For instance, Fantini et al.23 using the Langmuir technique reported that cholesterol accelerates the interaction of Aβ with a membrane containing gangliosides GM1. As a main difference with respect to our system, the negative charge of the membrane in that investigation was carried by GM1, instead than by POPS, i.e., it was specifically located in the lipid rafts; additionally, the peptides investigated were Aβ(1-40) and a short fragment Aβ(5-16). Similarly, using the monolayer approach, Ji et al.24 showed that spontaneous insertion of Aβ(1-40) in monolayers at the air/water interface is enhanced in the presence of cholesterol; saturated phospholipids (DPPC, DPPS and DPPG) were used in that investigation as well. Taking into account all this information, interaction of Aβ seems therefore to be strictly correlated with membrane fluidity, beside Aβ species and molecular components of the membrane itself. The other value we derived from force−distance curve analysis was the length associated with tip penetration inside the bilayer (Lb). We observed a significant decrease of Lb values in the case of samples without cholesterol. The effect is halved with the addition of 1% cholesterol and becomes negligible in the other cases. A set of QI images acquired with high force set point (Figure S4, Supporting Information) proved that the tip does not completely penetrate inside the lipid ordered regions. This phenomenon does not depend on the presence of cholesterol and is thus associated with SM. This fact implies that, even if Lb is directly proportional to bilayer thickness, it does not represent its actual measurement. Nevertheless, since further investigation demonstrated that Aβ peptide does not significantly affect the phenomenon (Figure S5, Supporting Information), we think that Lb decrease is mainly due to a bilayer thinning effect that can be prevented by the presence of cholesterol inside the membrane. Thinning of the membrane upon action of Aβ peptides has been often reported in the literature, especially in relationship to ion leakage induced by small oligomeric Aβ species.1,60

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ASSOCIATED CONTENT

S Supporting Information *

AFM topographies of POPC/POPS/SM 9:1:5 mol/mol/mol SLBs containing 0%, 1%, 5%, 20%, and 40% molar cholesterol (Figure S1). Distributions of breakthrough force Fb measured on POPC/POPS/SM 9:1:5 mol/mol/mol SLBs containing 0%, 1%, 5%, and 20% molar cholesterol (Figure S2). Distributions of breakthrough length Lb measured on POPC/POPS/SM 9:1:5 mol/mol/mol SLBs containing 0%, 1%, 5%, 20% molar cholesterol (Figure S3). QI images collected in liquid on POPC/POPS/SM 9:1:5 mol/mol/mol SLBs containing different molar percentage of cholesterol, with force set point below and above Fb (Figure S4). QI images collected in liquid on POPC/POPS/SM 9:1:5 mol/mol/mol SLBs containing different molar percentage of cholesterol after Aβ(1−42) administration, with force set point below and above Fb (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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5. CONCLUSIONS In this work, we employed AFM techniques to characterize the interaction between Aβ(1−42) peptides and multiphase lipid membranes. In particular, we studied the changes induced by Aβ(1−42) on the morphology and on the mechanical stability of phospholipids/SM/Chol membranes at different concentrations of cholesterol. This study indicates that at high concentration of cholesterol (20%) the membrane structure is substantially unaltered, suggesting that the role played by 13939

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