Article Cite This: Langmuir XXXX, XXX, XXX-XXX
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Size and Shape of Amyloid Fibrils Induced by Ganglioside Nanoclusters: Role of Sialyl Oligosaccharide in Fibril Formation Teruhiko Matsubara,† Masaya Nishihara,† Hanaki Yasumori,† Mako Nakai,† Katsuhiko Yanagisawa,‡ and Toshinori Sato*,† †
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama 223-8522, Japan Department of Alzheimer’s Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, 7-430 Morioka, Obu 474-8511, Japan
‡
S Supporting Information *
ABSTRACT: Ganglioside-enriched microdomains in the presynaptic neuronal membrane play a key role in the initiation of amyloid ß-protein (Aß) assembly related to Alzheimer’s disease. We previously isolated lipids from a detergent-resistant membrane microdomain fraction of synaptosomes prepared from aged mouse brain and found that spherical Aß assemblies were formed on Aß-sensitive ganglioside nanoclusters (ASIGN) of reconstituted lipid bilayers in the synaptosomal fraction. In the present study, we investigated the role of oligosaccharides in Aß fibril formation induced by ganglioside-containing mixed lipid membranes that mimic the features of ASIGN. Ganglioside nanoclusters were constructed as ternary mixed lipid bilayers composed of ganglioside (GM1, GM2, GM3, GD1a, or GT1b), sphingomyelin, and cholesterol, and their surface topography was visualized by atomic force microscopy. Aß fibril formation on the nanocluster was strongly induced in the presence of 10 mol % ganglioside, and Aßsensitive features were observed at cholesterol contents of 35−55 mol %. GM1-, GD1a-, and GT1b-containing membranes induced longer fibrils than those containing GD1b and GM2, indicating that the terminal galactose of GM1 along with Nacetylneuraminic acid accelerates protofibril elongation. These results demonstrate that Aß fibril formation is induced by ASIGN that are highly enriched ganglioside nanoclusters with a limited number of components and that the generation and elongation of Aß protofibrils are regulated by the oligosaccharide structure of gangliosides.
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INTRODUCTION Amyloid ß-protein (Aß) is generated from amyloid precursor protein via cleavage by proteases at the cell membrane.1,2 Aß assemblies are neurotoxic, and Aß deposition is also linked to the formation of senile plaques, which are a pathological hallmark of Alzheimer’s disease (AD). The typical length of Aß is around 40 residues;3,4 Aß(1−40) is the most abundant whereas Aß(1−42) is the most toxic form.5 Aß spontaneously assembles into oligomers,6,7 globules (spheroid),8 protofibrils (up to around 200 nm in length),9 and fibrils,10 a process that is accelerated in the presence of metal ions,11,12 hydrophobic substrates,13,14 and gangliosides.15 Aß fibril formation in the brain of AD patients is accelerated by a ganglioside-bound Aß (GAß) complex on neuronal membranes.16,17 GM1 is one of the most important ganglioside components of GAß among several gangliosides that exist in neuronal membranes. GM1 is abundant in the nervous system and can bind cholera toxin B subunit (CTB). We recently reported that the GD1b(d20:1−20:0) to GD1b(d20:1−18:0) ratio also influences the Aß assembly at the neuronal membrane in an amyloid-bearing precuneus.18 We speculated that this was caused by changes in the local environment of gangliosides, including the formation of Aß-sensitive ganglioside nanocluster (ASIGN).19 Generation of sphere-shaped Aß assemblies on © XXXX American Chemical Society
ASIGN was observed by atomic force microscopy (AFM); however, it is not clear that which types of gangliosides and other lipids are responsible for the formation of ASIGN. In a previous study, we identified a GM1-enriched domain as an ASIGN by observing Aß assembly in a membrane composed of lipid extracts from synaptosomes of aged mouse brains.19 Lipids containing GM1 were isolated as the detergent-resistant membrane microdomain (DRM) fraction of the synaptosomes. The GM1 level in the synaptosomal DRM fractions increases with age but does not differ significantly from that in the nonsynaptosomal DRM fraction.20 AFM observations indicated that a monomeric Aß is bound to and forms spherical assemblies on GM1-containing domains of the lipid membrane composed of synaptosomal DRM lipids. The binding of CTB to ASIGN indicated that the latter contains GM1; however, the roles of other gangliosides remain to be determined. To address this issue, the present study investigated the roles of gangliosides GM2, GM3, GD1a, GD1b, GT1b, and GQ1b (Figure 1) in Aß assembly on ASIGN by AFM analysis of the interaction between Aß and ganglioside-containing membranes. Received: June 19, 2017 Revised: October 31, 2017
A
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Pure Chemical Industries, Ltd. (Osaka, Japan). GM3 and GD1a from bovine brain were purchased from HyTest Ltd. (Turku, Finland) and Enzo Life Sciences Inc. (Farmingdale, NY), respectively. Glucosylceramide (GlcCer) from human (Gaucher’s) spleen was purchased from SIGMA or Wako Pure Chemical Industries, Ltd. Lipids, except GT1b and GQ1b, were dissolved in chloroform−methanol (4:1, v/v). GT1b and GQ1b were dissolved in chloroform−methanol (1:1, v/v) and chloroform−methanol−water (65:25:4, v/v/v), respectively. Preparation of Seed-free Aß Solution. A seed-free Aß solution was prepared as previously described.18,19 Synthetic Aß (1−40) (Peptide Institute Co. Ltd., Osaka, Japan) was dissolved in an ice-cold 0.02% ammonia solution. Undissolved peptide aggregates that could potentially serve as seeds were removed by ultracentrifugation at 560 000 × g for 3 h at 4 °C, and the upper third fraction was collected and stored in aliquots at −80 °C until use. The concentration of the Aß solution (200−400 μM) was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA). Immediately before use, aliquots were thawed and diluted with Trisbuffered saline [50 mM Tris, 150 mM NaCl (pH 7.5)]. Preparation of Ganglioside-Containing Membranes and Aß Incubation. To investigate the surface topography of gangliosidecontaining membranes, lipid bilayers were prepared on mica, as previously described.19 Briefly, a POPC lipid monolayer was prepared at the air−water interface of a Langmuir−Blodgett trough at 25 °C with a subphase of water and transferred to freshly cleaved mica (1 × 1 cm) by horizontal deposition at a surface pressure of 35 mN m−1 (POPC-coated mica, Figure 2A). A second lipid monolayer consisting of ganglioside/SM/chol (molar ratio) was loaded onto the POPCcoated mica by horizontal deposition at a surface pressure of 30 mN m−1 to obtain a lipid bilayer (i.e., ganglioside-containing membrane),21 which was incubated with a seed-free Aß solution (1−10 μM) for 15 min−72 h at 37 °C to investigate the interaction with Aß. After washing three times with phosphate-buffered saline, the membrane was analyzed by AFM. AFM Measurements of Ganglioside-Containing Membranes. AFM measurements of ganglioside-containing membranes on mica were carried out in water at 25 °C using an SPM-9600 instrument (Shimadzu Co., Kyoto, Japan).18,19 A 38-μm-long soft cantilever (BLAC40TS-C2; Olympus, Tokyo, Japan) with integrated pyramidal silicon nitride tips and a spring constant of 0.1 N m−1 was used for the measurements. Topographic images (2 × 2 μm) were acquired in the dynamic mode at a scanning rate in the range of 1−2 Hz, and typical multiple images (n ≥ 3) were used for further analyses. To estimate the Aß-bound areas, AFM images were binarized based on membrane height and pixels were counted using Adobe Photoshop
Figure 1. Structure and biosynthetic pathway of ganglio a-series and bseries gangliosides. Glc, glucose; Gal, galactose; GalNAc, Nacetylgalactosamine; Neu5Ac, N-acetylneuraminic acid.
GM1, GD1a, GD1b, and GT1b have been isolated from human amyloid-bearing precuneus.18 To model an ASIGN, a ganglioside-containing membrane was constructed by accumulation of ternary mixed monolayers composed of ganglioside, sphingomyelin (SM), and cholesterol (chol) on phospholipid-coated mica. The highly enriched ganglioside nanocluster exhibited the characteristics of ASIGN, and after incubation with Aß for 24 h, Aß fibrils were observed on a 10 mol % GM1-containing membrane. Aß fibril formation was influenced by the GM1 (≥10 mol %) and chol contents. In addition, five gangliosides (GM1, GM2, GD1a, GD1b, and GT1b) induced fibril formation on ganglioside-containing membranes, in which the Aß fibril length depended on their sialyloligosaccharide structure.
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EXPERIMENTAL SECTION
Lipids. Sphingomyelin (SM) from bovine brain was purchased from SIGMA (St. Louis, MO). Cholesterol (chol) and 1-palmitoyl-2oleoyl-sn-glycelo-3-phosphocholine (POPC) were purchased from Nacalai Tesque Inc. (Kyoto, Japan) and Matreya LLC (State College, PA), respectively. Gangliosides GM1, GM2, GD1b, GT1b, and GQ1b obtained from bovine brain were purchased from SIGMA or Wako
Figure 2. Determination of surface topography of GM1-containing membranes composed of GM1, SM, and chol by AFM. (A) Preparation of GM1containing membranes for AFM measurements. A lipid monolayer of GM1/SM/chol was loaded onto POPC-coated mica to obtain a lipid bilayer (GM1-containing membrane). The height of domain ß (GM1 nanocluster) was higher than that of area α. (B) AFM images of 0−10 mol % GM1containing membranes (left) and section analysis plot (x−y) (right). Molar ratios of GM1/SM/chol were 0:50:50 (SM/chol), 1:49.5:49.5 (1 mol % GM1), 5:47.5:47.5 (5 mol % GM1), and 10:45:45 (10 mol % GM1). B
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Figure 3. Aß deposition on 10 mol % GM1-containing membranes (GM1/SM/chol, 10:45:45). (A) AFM images (original) of GM1/SM/chol (10:45:45) membrane after incubation with 1−5 μM Aß(1−40) at 37 °C for 15 min. To highlight the Aß-bound area, the original AFM image was binarized based on membrane height (binarized; 5 nm threshold). The percentage of white pixels in binarized images (0.4% and 64%) was estimated as the Aß-bound area. (B) Concentration dependence of Aß binding. The Aß-bound area (%) was plotted as a function of Aß concentration. (C) Section analysis plot of the Aß-bound area (x−y). A thin Aß layer (height ∼ 6 nm) and Aß assembly (height > 10 nm) were observed.
Figure 4. Aß protofibril formation on GM1-containing membranes. (A) AFM images of 10 mol % GM1-containing membrane (GM1/SM/chol, 10:45:45) after incubation with 10 μM Aß(1−40) at 37 °C for 12−36 h. Binarized AFM images were obtained by thresholding at 6 nm from the bottom. The section analysis plot (x−y) indicated that Aß protofibrils (height >10 nm) were generated on the Aß layer. (B) AFM images of 1−10 mol % GM1-containing membranes after incubation with 10 μM Aß(1−40) at 37 °C for 48 h. Binarized AFM images (6 nm threshold). The molar ratios of GM1/SM/chol were 1:49.5:49.5 (1 mol % GM1), 5:47.5:47.5 (5 mol % GM1), 7.5:46.25:46.25 (7.5 mol % GM1), and 10:45:45 (10 mol % GM1). (C) Proportion of Aß protofibrils to the Aß-bound area. Aß assemblies were defined as fibrils with long/short axis aspect ratio >3. Aß protofibrils were observed on 7.5 and 10 mol % GM1-containing membranes. N.D., not detected. Elements (Adobe Systems Software, San Jose, CA) or GNU Image Manipulation Program (https://www.gimp.org/) image processing software. For example, the white area of a binarized image by thresholding at 5 nm from the bottom was identified as the Aß-bound area on the ganglioside-containing membrane. We estimated the lengths of the long and short axes of an Aß assembly, which was defined as a fibril when the long/short axis aspect ratio was >3. To measure the length of the Aß fibrils, a line was drawn along a fibril and its length was calculated using ImageJ software (National Institutes of Health, Bethesda, MD). Molecular Modeling. Crystallographic coordinates of the X-ray structures of GM1 pentasaccharide (CTB subunit, 3CHB),22 GT1b heptasaccharide (Hc fragment of tetanus toxin, 1FV2),23 and Aß(1− 40) (2M4J)3 were obtained from the Protein Data Bank (http://www. wwpdb.org/). GD1b saccharide was obtained by a deletion of a Neu5Ac residue from the GT1b saccharide. The molecular model was
visualized with Discovery Studio Viewer Lite software (Accelrys, San Diego, CA).
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RESULTS AFM Analysis of Ganglioside GM1 Nanoclusters. In a previous study, a conformational change in Aß induced by ASIGN in the neuronal membrane was observed.19 GM1 in ASIGN was identified by CTB labeling; we used GM1 as a marker to investigate the Aß assembly induced by gangliosidecontaining membranes. Membrane raftsa type of membrane microdomainare composed of sphingolipids, SM, and chol.24,25 We designed a mixed membrane composed of ternary lipidsi.e., GM1 as well as SM and chol, which were present in equal proportions according to a previous study.26,27 C
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Figure 5. Effect of chol content on Aß fibril generation. (A) 10% GM1-containing membranes composed of 15−75 mol % chol were incubated with 10 μM Aß(1−40) at 37 °C for 15 min and 36 h. Binarized AFM images (6 nm threshold) are shown. Many short Aß protofibrils (3. Protofibril height (width) was typically around 20 nm, with a length of about 100−300 nm.9 Figure 4B shows the surface topographies of 1−10 mol % GM1-containing membranes after incubation with Aß for 48 h. No fibrils were observed on the 1−5 mol % GM1 membrane, but Aß protofibrils coexisted with deposited Aß at 7.5 mol % GM1. The proportion of Aß protofibrils in the Aß-bound area was highest at 10 mol % GM1 (Figure 4C). These results indicate that Aß protofibril formation is strongly induced by membranes highly enriched in GM1 (i.e., containing about 10 mol % GM1). Roles of Chol in Aß Assembly. AFM studies indicated that the GM1 nanocluster is a key lipid domain for Aß assembly into fibrils. To investigate the significance of the chol content in this process, 10 mol % GM1-containing membranes with 15, 35, 55, 65, or 75 mol % chol (GM1/SM/chol) were prepared and Aß deposition was visualized by AFM.32,33 Aß protofibrils were observed on membranes composed of 35−55 mol % chol after 36 h of incubation (Figures 4A and 5A). On the other hand, no protofibrils or large Aß deposits with an island shape (long axis >100 nm) were present on membranes containing 15, 65, or 75 mol % chol (Figure 5A). The Aß-fibril area of membranes containing 45 mol % chol was highest among those containing 15−75 mol % chol, which was consistent with the distribution of the Aß-bound area after 15 min of incubation (Figure 5B). Although domain ß was observed at all GM1/SM/chol ratios (10:75:15, 10:45:45, and 10:15:75) (Figures 2B and S1B), fibril formation was only observed in membranes in which the molar
The GM1-containing membrane was prepared by accumulation of the ternary lipid monolayer composed of GM1/SM/ chol onto 1-palmitoyl-2-oleoyl-sn-glycelo-3-phosphocholine (POPC)-coated mica (Figure 2A), as previously reported.19 The hydrophilic mica surface was coated with POPC to immobilize the monolayer; the surface topography of the GM1containing lipid bilayer was visualized by AFM. When 1−10 mol % GM1 was mixed with SM/chol (1:1), a nanodomain 50−200 nm in size (domain ß) was observed (Figure 2B). Domain ß, which had a height of 3−4 nm, was distinguished from the surrounding phase (area α) (section analysis, Figure 2B, right). The domain was presumed to be the GM1-enriched phase (GM1 nanocluster);27−30 however, its size was not dependent on the GM1 content in the range of 1−10 mol %. In the absence of GM1, the SM/chol lipid monolayer was oriented during the loading step of the monolayer onto POPC-coated mica because of the fluidity of SM/chol (Figure 2B, left). Measurements in AFM images in the present study were made in the dynamic mode to avoid surface topography disruption; i.e., the height of the AFM images was the apparent height and was not comparable to that of AFM images measured in the contact mode, as in our previous study.19 Aß Deposition on GM1-Containing Membranes. To investigate Aß assembly induced by ganglioside-containing membranes, we used a seed-free Aß(1−40). 3,19 After incubation of 1−10 μM Aß solution with a 10 mol % GM1 membrane (GM1/SM/chol, 10:45:45) for 15 min, Aß deposition onto the membrane was visualized by AFM (Figure 3A) and was estimated from an Aß-bound area binarized from an image based on a height with a 5 nm threshold.19 The area was saturated at over 5 μM Aß; therefore, 10 μM was used for subsequent experiments (Figure 3B). Aß was deposited onto the domain ß at 1−2.5 μM. At >5 μM, Aß covered the membrane as a layer with a height of 5 nm. In addition, a thicker Aß assembly (>10 nm in height) was observed on the Aß layer and a shape that was similar to that of the domain ß (Figure 3C). These results suggest that Aß preferentially bound to the domain ß and that the remaining phase (area α) of the membrane was covered with Aß. This observation supports the view that domain ß is the Aß-sensitive GM1 nanocluster (ASIGN). Contrastingly, the generation of the Aß layer is reasonable because Aß is able to bind to SM/ chol membranes at the μM range.31 When 10 μM of Aß solution was incubated with 1 mol % GM1 membrane (GM1/SM/chol, 1:49.5:49.5) for 15 min, D
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Figure 6. Aß fibrils on 10 mol % ganglioside-containing membrane (ganglioside/SM/chol, 10:45:45). (A) AFM images of 10 mol % GM2-, GD1a-, GD1b-, and GT1b-containing membrane were obtained after incubation with 10 μM Aß(1−40) at 37 °C for 48 h. (B) Histograms of Aß fibrils generated on 10 mol % ganglioside-containing membranes. Aß assemblies were defined as fibrils with a long/short axis aspect ratio >3. Fibril length and number were determined from AFM images (2 × 2 μm, n = 3). Statistical comparison of histogram was determined by Kolmogorov−Smirnov test (*, P < 0.05; **, P < 0.01).
longer fibrils (>300 nm) detected (Figure 6B). In contrast, only short protofibrils ( GM1 > GT1b > GD1b > GM2 (Table 1).
ratio of SM to chol was in the 35/55−55/35 range (i.e., 35−55 mol % chol). Aß protofibrils were particularly abundant on the 45 mol % chol membrane (GM1/SM/chol, 10:45:45) (Figure 4). These results indicate that fibril formation is sensitive to the chol content and that Aß tends to form fibrils on the membranes with a SM/chol molar ratio close to 1. Effect of the Glycostructure on Aß Protofibril Formation Induced by Ganglioside-Containing Membranes. To clarify the contribution of glycan structure to Aß protofibril formation, the interaction of Aß with 10 mol % ganglioside (GM2, GM3, GD1a, GD1b, GT1b, or GQ1b)containing membranes was examined by AFM following incubation of ganglioside/SM/chol (10:45:45) with 10 μM Aß solution for 24 and 48 h. After the generation of protofibrils at 24 h was confirmed (Figure S2), fibril length and number at 48 h were determined from the AFM images (Figure 6A). More than 20 protofibrils were observed in GM1, GM2, GD1a, GD1b, and GT1b-containing membranes; 177 ± 21 and 62 ± 17 protofibrils were observed on GD1b- and GT1b-containing membranes, respectively (Table 1). The rank order of
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Table 1. Number and Length of Fibrils Induced by Ganglioside-Containing Membranes (2 × 2 μm, n = 3) glycolipids GlcCer GM3 GM2 GM1 GD1a GD1b GT1b GQ1b
no. of fibrils 4 6 41 28 20 177 62 4
± ± ± ± ± ± ± ±
3 2 4 7 2 21 17 1
total length (μm)
avg of length (μm)
± ± ± ± ± ± ± ±
− − 0.06 0.22 0.26 0.11 0.16 −
0.7 0.7 2.4 6.4 5.2 19.6 9.5 0.5
0.6 0.2 0.5 2.1 1.0 2.0 1.6 0.2
DISCUSSION
In the present study, we investigated key features of ASIGN using ganglioside-containing membranes composed of ternary ganglioside/SM/chol lipids and examined its interaction with Aß by AFM. We previously demonstrated that Aß assemblies in a reconstituted membrane composed of DRM lipids from aged mouse brain are highly enriched in gangliosides including GM1.19 However, the roles of each ganglioside and the minimum lipid composition of ASIGN were unclear. In the present study, we designed a ganglioside-enriched membrane containing ASIGN and used AFM to visualize Aß binding and fibril formation. A planar lipid bilayer was prepared by accumulation of target lipid monolayers on POPC-coated mica, as previously described.18,19 Our method can be used to prepare bilayers with the desired lipid compositions; here, 0− 10 mol % GM1-containing and 15−75 mol % chol-containing membranes were prepared and used to investigate Aß assembly. GM1-enriched membranes composed of GM1/SM/chol induced Aß deposition and fibril formation (Figure 3 and 4). AFM analysis after 15 min of incubation revealed that monomeric Aß bound to the domain ß of GM1-containing membranes at 1 μM Aß; at 5 μM, an Aß layer was observed on the membrane (Figure 3). Although spherical Aß assembly was obviously observed on the membrane of synaptosomal DRM lipids containing GM1 in our previous study,19 Figure 3 shows
protofibril formation frequency was GD1b > GT1b > GM2 > GM1 > GD1a. The GM2 oligosaccharide GalNAcβ1− 3(Neu5Acα2−3)Galβ1−4Glc was required for protofibril formation. Comparison of the total lengths of Aß fibrils indicated that GD1b and GT1b strongly induced Aß fibrils. Histograms for GM1-, GD1a-, and GT1b-containing membranes showed a peak at around 100 nm, with several E
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Figure 7. Proposed model for Aß fibril formation on ganglioside nanoclusters. (A) Schematic illustration of a possible mechanism of ASIGN-induced Aß assembly. (B) Superimposed image of Aß(1−40) on gangliosides. The conformations of Aß(1−40) and ganglioside oligosaccharides were obtained from Protein Data Bank (entries 1FV2 and 2M4J, respectively). Aß(1−40) is shown as a solid ribbon in the molecular surface representation. Oligosaccharides of gangliosides are shown in the stick representation.
on the size and shape of the Aß assemblies, in the present study, we investigated the role of sialyloligosaccharides of the gangliosides on Aß assembly. Surface topographic images of Aß-bound membrane obtained by AFM provided critical information about Aß assembly, including the shape and length distribution (Figure 6). a- and b-Series gangliosides were classified into two groups based on the speed of fibril elongation. Several longer fibrils (>300 nm) were observed on GM1-, GD1a-, and GT1b-containing membranes, on the other hand, whereas GM2 and GD1b induced the formation of many protofibrils that remained short (≤100 nm), even after 48 h, which suggested that their elongation was slow (Figure 7A). The generation and elongation of Aß protofibrils depend on the oligosaccharide structure of the gangliosides, with terminal Gal and Neu5Ac accelerating the elongation process (Figure 7B). Amyloid fibrils contain a cross-ß motif that is a ribbonshaped sheet structure linked by interstrand hydrogen bonds running parallel to the growth direction.3 From the size comparison, an Aß(1−40) monomer is able to be interacted with multiple gangliosides (clustered gangliosides) (Figure 7B). Since GM1, GD1a, GD1b, and GT1b were identified as major gangliosides in the AD brain,18,38 multiple gangliosides are presumed to be involved in Aß assemblies. Two types of bseries ganglioside (GD1b and GT1b) strongly induced Aß fibrils as compared to a-series gangliosides (GM1 and GD1a) (Figure 6); however, a greater decrease in the former was observed in AD patient brains.39 Additionally, in early onset AD patient brains, reduced ganglioside levels accompany the severe loss of neurons.40 These findings indicate that not only the ability of individual gangliosides to induce Aß fibrils but also their composition contributes to Aß fibril formation. Indeed, although only GM3 was generated in the brains of mice with a disrupted GM2 synthase gene (that is, GM1 was lacking in the brain), amyloid deposition was confirmed by biochemical and immunohistochemical analyses.41 In some cases of familial AD, GM3 and GD3 accelerate Aß assembly with Dutch-type (E22Q) and Flemish-type (A21G) mutations, respectively.42 Molecular dynamics simulation of a GM1/SM/chol (20:40:40) lipid bilayer has been useful to estimate GM1 behavior,29 revealing that terminal Gal and Neu5Ac(s) were exposed to water phases and interacted with Aß. In addition, hydrogen bonding between lipids (e.g., GM1-GM1 and GM1chol) might contribute to the formation of the GM1 cluster. Our AFM results demonstrating the formation of ganglioside nanoclusters in the presence of chol and the importance of
that not only Aß assembly with a domain shape but also an Aß layer was generated on the GM1/SM/chol membranes. This difference in the properties of Aß is presumed to be caused by the lipid composition, because the DRM fraction contains many kinds of glycolipids, phospholipids, and other lipids.19 Aß protofibrils were generated on 10 mol % GM1 membranes after 12 h of incubation, coexisting with Aß deposits on the domain ß (Figure 4). These results indicate that monomeric Aß is assembled on GM1/SM/chol membranes containing ASIGN and that GM1 is important for this process. Previously, Aß fibrils were detected on 20 and 50 mol % GM1 membranes (GM1/SM/chol, 20:40:40; GM1/chol, 50:50) that were supported planar lipid bilayers prepared from liposomes, which had similar lipid compositions to those used in the present study.27 For example, fibril formation of Aß at 2 μM on 20 mol % GM1 membrane was observed after 1−12 h incubation, which does not conflict with our results. However, the number of Aß assemblies was not counted, and their morphology was not discussed. Apolipoprotein (apo)E4 knock-in mice showed a twofold increase in chol compared to wild-type mice, which affected chol distribution in the plasma membrane.34 Our AFM results suggest that inheritance of apoE4 is a risk factor for AD development. Aß assemblies depend on the content of chol (Figure 5), which induced Aß fibril formation in the range of 35−55 mol %. This result is accordance with previous findings that the binding of Aß to GM1-containing monolayers and liposomes influences the chol content.26,32,33 Although it did not dictate the topography of the domain ß in GM1/SM/chol membranes (Figure S1B), the chol content contributed to Aß assembly. Aß(1−40) is known to bind to ganglio a-series (GM1, GM2, GM3, and GD1a) and b-series (GD1b and GT1b) gangliosides with binding strengths in the rank order of GT1b > GD1a = GD1b > GM1 > GM2 = GM3, which was determined in a surface plasmon resonance study.35 Circular dichroism (CD) analysis indicated that these gangliosides have the ability to induce the formation of a ß-sheet structure in a similar order (GT1b > GD1a > GD1b > GM1 = GM3).36 However, the strength of fibril formation determined using a thioflavin-T (ThT) assay (GM1 > GD1b > GD1a > GT1b) is not consistent with that of the Aß−ganglioside interaction detected by fluorescence-labeled Aß (GT1b = GD1a > GD1b > GM1).37 Therefore, the contribution of gangliosides to Aß assembly, including fibril formation, should be evaluated carefully. Based F
DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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Langmuir
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ganglioside sugar residues for Aß interaction support this simulation. Potentially, the detailed interactions of oligosaccharides and Aß could be clarified by nuclear magnetic resonance spectroscopy studies.43−45
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CONCLUSION ASIGN-mimic membranes were constructed with the lipid components of 10 mol % GM1 and 35−55 mol % chol. Ganglio b-series gangliosides such as GD1b and GT1b induced Aß fibril formation in addition to a-series gangliosides. AFM analyses indicated that fibril number and length depended on sialyloligosaccharide structure; the GM2 oligosaccharide GalNAcβ1−3(Neu5Acα2−3)Galβ1−4Glc was needed to generate protofibrils, and an additional Gal and Neu5Ac(s) stimulated fibril elongation. We demonstrated that fibril length and shape depended on glycan structures. These findings clarified the mechanisms of ganglioside cluster-induced Aß assembly and provide a basis for the development of therapies to slow AD progression.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02091. Figures including AFM images of ganglioside-containing membranes (ganglioside/SM/chol) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +81-45-566-1771. Fax: +81-45-566-1447. E-mail: sato@ bio.keio.ac.jp. ORCID
Teruhiko Matsubara: 0000-0002-8006-4324 Toshinori Sato: 0000-0002-4429-6101 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by a Japan Society for the Promotion of Science Kakenhi grant (no. 22300118 to T.M. and K.Y.); the Suzuken Memorial Foundation (no. 11-093 to T.M.); Keio Gijuku Academic Development Funds (T.M.); and the Research Funding for Longevity Sciences from the National Center for Geriatrics and Gerontology, Japan (no. 25-19 to T.M. and K.Y.).
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DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.7b02091 Langmuir XXXX, XXX, XXX−XXX
