Amyloid-β fibrils assembled on ganglioside-enriched membranes contain both parallel β-sheets and turns

Some protein and peptide aggregates, such as those of amyloid-β protein (Aβ), are neurotoxic and have been implicated in several neurodegenerative diseases. Aβ accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer's disease. Although the presence of Aβ deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between Aβ secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that Aβ40 fibrils can be obtained by incubating seed-free Aβ40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection–absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel β-sheets, whereas self-assembled Aβ40 molecules formed antiparallel β-sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn Aβ40. Our findings indicate that Aβ generally self-assembles into antiparallel β-structures but can also form protofibrils with parallel β-sheets by interacting with ganglioside-bound Aβ. We concluded that by promoting the formation of parallel β-sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of Aβ fibrils. These results advance our understanding of ganglioside-induced Aβ fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer's disease.

Oligomerization and fibril formation are generally spontaneous (15) but are enhanced by numerous factors including metal ions (16,17) and gangliosides (18). The latter, which abundant in the nervous system, are often visualized by cholera toxin B subunit, which binds GM1, Gal␤1-3GalNAc␤1-4(Neu5Ac␣2-3)Gal␤1-4Glc1-1Ј-Cer (19,20). Levels of gangliosides including GM1 are within 1-2% in the extracellular leaflet of the plasma membrane in the nervous system (21). However, gangliosides actually exist with high density in lipid rafts comprising sphingomyelin and cholesterol (22). Ganglioside-enriched microdomains at neuronal membranes are considered among the key sites for the onset of Alzheimer's disease. A␤ assembly at neuronal membranes (23,24), in which a ganglioside-bound A␤ (GA␤) complex acts as an endogenous seed, was first reported by Yanagisawa et al. (18) and was eventually demonstrated on ganglioside-containing liposomes using a thioflavin T assay, EM, and antibody assays (25,26). The toxicity of the A␤ assembly resulting from the GA␤ complex has been assessed using rat PC12 pheochromocytoma cells (27) and human neuroblastoma SH-SY5Y cells (28). These cells express gangliosides, including GM1, and nerve growth factor receptor-mediated neuronal cell death or cellular damage has been indicated. More recently, atomic force microscopy (AFM) of a reconstituted lipid bilayer containing mouse synaptosomal lipids has suggested that A␤-sensitive ganglioside nanoclusters promote A␤ 40 assembly (29, 30). In addition, the chain length of ganglioside GD1b was found to influence A␤ 42 assembly at the neuronal membrane in human precuneus with amyloids (31).
Structural studies of A␤ polymerized at ganglioside-containing membranes are limited and contradictory. For example, Matsuzaki and Horikiri (32) found by CD that A␤ 40 forms ␤-sheets at liposomes containing GM1, e.g. liposomes of sphingomyelin/cholesterol/GM1 (5:2:3). Similarly, FTIR attenuated total reflection spectroscopy indicated that A␤ 40 forms antiparallel ␤-sheets at dry-cast films of egg yolk L-␣-phosphatidylcholine/GM1/A␤ 40 (40:10:1) (32) or at liposomes of GM1/cho-lesterol/sphingomyelin (4:3:3) (33). On the other hand, NMR data collected by Utsumi et al. (34 -36) suggest that A␤ 40 forms ␣-helices at membranes containing GM1 because of the hydrophobic environment. Recently, Hu et al. (37) also showed by Raman spectroscopy that ␣-helices and ␤-sheets of A␤ 40 were eventually observed with a planar lipid bilayer composed of GM1/sphingomyelin/cholesterol (5:55:40 and 20:40:40), although the topography of the fibrils was not investigated. Among these studies, spectroscopic evidence of gangliosideinduced fibrillar A␤ with parallel ␤-sheets has not yet been reported. In light of these results, a unifying model of A␤ assembly is needed to explain most of the structural data.
Previously, we found that A␤-sensitive ganglioside nanoclusters in neuronal membranes induce a conformational change in A␤ 40 (29, 38). This and other characteristic properties of A␤-sensitive ganglioside nanoclusters are mimicked by ganglioside-enriched planar membranes composed of ganglioside (GM1, GM2, GD1a, GD1b, or GT1b)/sphingomyelin/cholesterol (10:45:45) (30). These membranes are constructed by depositing a monolayer composed of ganglioside, sphingomyelin, and cholesterol with a lateral plasma membrane pressure (30 mN m Ϫ1 ) on phospholipid-coated mica. This type of membrane is more stable than conventional liposomes (39) and supported lipid bilayers (37) with the same lipid composition, and AFM images can be obtained in a few days. In addition, various lipid compositions of the membrane are acceptable even if there are lipids incapable of forming liposomes.
Using FTIR reflection-absorption spectroscopy, we have now determined the secondary structure of A␤ 40 fibrils obtained in 15 min to 72 h on membranes containing GM1 at a 20% molar ratio (GM1/sphingomyelin/cholesterol, 20:40:40). The data indicate that the fibrils form turns and parallel ␤-sheets within 48 h. On the other hand, A␤ 40 assembled on membranes with 20% glucosylceramide (GlcCer), as well as self-assembled A␤ 40 , forms antiparallel ␤-sheets. Solid-state NMR analyses by Tycko and co-workers (10,40,41) indicate that A␤ 40 predominantly forms cross-␤-structures based on parallel ␤-sheets stabilized by intermolecular hydrogen bonds. Our results imply that by promoting the formation of parallel ␤-sheets, highly ganglioside-enriched nanoclusters also accelerate the elongation of A␤ fibrils.

Formation of A␤ fibrils on GM1-enriched membranes
As described previously (30), A␤ 40 fibrils were formed at ganglioside-enriched, planar, bilayer membranes, which were prepared by depositing 20:40:40 monolayers of GM1, sphingomyelin, and cholesterol onto 1-palmitoyl-2-oleoyl-sn-glycelo-3-phosphocholine (POPC)-coated mica (GM1-enriched membrane (Fig. 1A)). This composition mimics that of GM1enriched microdomains and has frequently been used for ganglioside-induced A␤ assembly (39,42). To confirm fibril formation, membranes were imaged by AFM in water after incubation with A␤ 40 . GM1-enriched microdomains and A␤ assemblies were then visualized as areas higher than 4 nm on binarized AFM images. GM1-enriched microdomains before incubation with A␤ 40 had a diameter of 30 -300 nm (Ͼ700 domains in a 5 m ϫ 5 m area), and the apparent size of the domains increased after incubation with A␤ 40 for 15 min (60 -500 nm, 280 domains) (Fig. S1). This topological change suggests that A␤ molecules are deposited on the GM1-enriched membrane to yield an A␤ layer (30). After 48 h, over a dozen A␤ 40 fibrils Ͼ1-m long were clearly observed in a 5 ϫ 5-m area. These A␤ fibrils accumulated on round-shaped GM1-enriched microdomains, as reported previously (30).
To measure FTIR in air, after the formation of A␤ 40 fibrils, membranes were dried overnight and imaged in air (Fig. 1B,  upper panel). The binalized AFM images of GM1-enriched membranes after incubation with A␤ 40 for 15 min, 48 h, and 72 h are shown in Fig. 1B (lower panel) at a height threshold of 2.0 -3.0 nm. Drying slightly altered the shape of GM1-enriched microdomains, but A␤ 40 fibrils were still identifiable, and more A␤ 40 fibrils were observed after 72 h than after 48 h. On the other hand, one long fibril (Ͼ3 m) and several short fibrils were observed on GlcCer-enriched membranes after 72 h (Fig.  2). These results indicate that GM1 generates and elongates A␤ fibrils more effectively than GlcCer.

Generation of parallel ␤-sheets of A␤ on ganglioside cluster Immobilization of GM1-enriched membranes on gold-coated glass
FTIR reflection-absorption spectra were collected to investigate the formation of lipid bilayers on gold-coated glass. Seven characteristic peaks in a monolayer of POPC were assigned according to the literature to CH 3 , CH 2 , PO 2 Ϫ , and ester C-O stretching vibration ( Fig. 3A and Table 1) (43,44). A strong peak corresponding to CϭO stretching vibration ((CϭO), 1742 cm Ϫ1 ) was also observed. On the other hand, amide I (1660 cm Ϫ1 ) and amide II (1547 cm Ϫ1 ) were observed in monolayers containing GM1 or GlcCer at a 20% molar ratio, as both lipids contain ceramide (44,45). Two peaks, at 3342 and 1379 cm Ϫ1 , in these membranes were assigned to N-H stretching and -CH 3 scissoring vibration, respectively. The spectrum of a lipid bilayer composed of POPC as the first layer and GM1/sphingomyelin/cholesterol as the second layer is simply a superposition of the spectrum of each, clearly implying that a lipid bilayer with GM1 was formed on gold-coated glass.

A␤ deposition on GM1-and GlcCer-enriched membranes
To investigate the interaction between A␤ and a GM1-enriched membrane, FTIR reflection-absorption spectra were collected after 15 min, 24 h, 48 h, and 72 h. Incubation with A␤ 40 shifted amide I and II peaks to 1663 and 1541 cm Ϫ1 , respectively (Fig. 3B, upper panel, and Table S1). In addition, the height of these peaks significantly increased with time, as plotted in Fig. 4 along with peak shifts, indicating A␤ 40 accumulation. The absorbance continued to increase even at 72 h, although peak shifts had nearly stabilized by that point.
The amide I and II bands in GlcCer-enriched membranes were at positions similar to those in GM1-enriched membranes (Fig. 3B, lower panel, and Fig. 4), implying comparable molecular structures in both membranes in light of the surface selection rule of reflection-absorption spectroscopy. In addition, the peaks were of similar relative intensity at 15 min and 72 h, implying comparable A␤ 40 accumulation on both membranes, despite drastic differences in their ability to form fibrils (Figs. 1B and 2).
The strongest peak of the amide I region from 15 min to 72 h was shifted from 1676 to 1663 cm Ϫ1 observed in the raw spectrum of GM1-enriched membranes (Fig. 3B, upper panel, Fig.  S2, and Table S1). The second-derivative reflection spectra indicated that two peaks (1661-1662 and 1691-1695 cm Ϫ1 )   Table S1.

Generation of parallel ␤-sheets of A␤ on ganglioside cluster
corresponding to the turns were appeared after 48 and 72 h ( On the other hand, a strong peak at 1668 cm Ϫ1 with two shoulders was observed in the raw spectrum of GlcCer-enriched membranes after 72 h (Figs. 3B, lower panel, and 5B). The second-derivative spectra indicate that the two shoulders segregate into two peaks at 1631 and 1697 cm Ϫ1 . Because the peak of GlcCer-enriched membranes after 72 h at 1631 cm Ϫ1 was distinguishable from that of GM1-enriched membranes at 1635 cm Ϫ1 , these peaks at 1631 and 1635 cm Ϫ1 are assigned to antiparallel and parallel ␤-sheets, respectively (51). Two peaks, at 1631 and 1676 cm Ϫ1 , of GlcCer-enriched membranes were assigned to a pair of peaks for antiparallel ␤-sheets. From these results, we concluded that GM1-enriched membranes induced A␤ 40 fibrils with parallel ␤sheets, in contrast to GlcCer-enriched membranes with antiparallel ␤-sheets ( Fig. 5C and Table 2).

Secondary structure of self-assembled A␤ based on attenuated total reflection spectra
Seed-free A␤ 40 was self-assembled for 15 min and 48 h (6), dropped on a suitable plate, and dried under nitrogen gas for 50 min. Three peaks at around 1630, 1670, and 1697 cm Ϫ1 in the attenuated total reflection spectrum at 15 min were again observed in the spectrum at 48 h (Fig. 6A). The pair of peaks at 1630 and 1666 -1670 cm Ϫ1 in the second-derivative spectra are characteristic of antiparallel ␤-sheets because the major component at 1612-1640 cm Ϫ1 was accompanied by a minor component at 1670 -1690 cm Ϫ1 (Table 2) (50). The peak at 1697 cm Ϫ1 is attributed to a turn structure.

Structural features of residual A␤ in the supernatant incubated with GM1-enriched membranes
Although A␤ 40 fibrils formed on GM1-enriched membranes contain turns and parallel ␤-sheets (Fig. 5A), raw and secondderivative attenuated total reflection spectra (Fig. 6B) indicate that residual A␤ 40 in the supernatant is structurally similar to self-assembled A␤ 40 , with major and minor components at 1630 and 1679 cm Ϫ1 corresponding to antiparallel ␤-sheets (Table 2). An AFM image of the residual A␤ 40 in the supernatant after a 48-h incubation with a GM1-enriched membrane supports the FTIR spectra, short fibrils, 150 nm or less in length, were observed (Fig. 6C).

Discussion
The objective of this study was to investigate the secondary structure of A␤ assembled on GM1-enriched membranes with a view to clarify the mechanism of assembly. Often, A␤ assembly on ganglioside-containing membranes is investigated in solution by fluorescence thioflavin T assay and CD, because such membranes are often prepared as liposomes. However, seemingly contradictory results have been reported, preventing the formulation of a unified mechanism of ganglioside-induced A␤ assembly. For example, Fukunaga et al. (33) reported that GM1-induced A␤ 40 contains antiparallel ␤-sheets based on FTIR but did not observe the ␣-helices detected on NMR (36). Recently, ␣-helices and ␤-sheets were detected by Raman spectroscopy of A␤ 40 deposited for 24 h on supported lipid bilayers composed of GM1/sphingomyelin/cholesterol (5:55:40 and 20:40:40) (37). There is no evidence of ganglioside-induced fibrillar A␤ 40 with parallel ␤-sheets that implies cross-␤ structures (10,40,41). Most unfortunately, however, the topography of A␤ assemblies are not always investigated when the secondary structure of A␤ assemblies is assessed.
We attempted to image A␤ 40 assemblies directly with time via AFM using membranes containing GM1/sphingomyelin/  We have now determined from the secondary structures of A␤ 40 assemblies from second-derivative reflection-absorption spectra (Fig. 5A) that A␤ 40 fibrils deposited on GM1-enriched membranes for 48 -72 h consist of turns (1661-1662 cm Ϫ1 and 1691-1695 cm Ϫ1 ) and parallel ␤-sheets (1635-1637 cm Ϫ1 ). The parallel ␤-sheets are clearly distinguishable from antiparallel ␤-sheets, typified by two characteristic peaks at 1631 and 1676 cm Ϫ1 , formed on GlcCer-enriched membranes (Fig. 5B). In this case, the difference in secondary structure correlates with the AFM data ( Figs. 1 and 2). In light of our results, a model of ganglioside-induced A␤ assembly was proposed (Fig. 7) in which monomeric A␤ forms an initial layer not only on GM1-enriched nanoclusters but also on sphingomyelin/cholesterol area (step a) (29, 30). NMR (36) and Raman spectroscopic studies (37) indicated that A␤ at ganglioside nanoclusters then forms helices (step b, GA␤ formation); however, a prominent helical peak at 1650 -1657 cm Ϫ1 was not observed (see Fig. 5A). Subsequently, A␤ molecules in contact with ganglioside-bound helical A␤ formed turn and antiparallel ␤-sheets and transited to parallel ␤-sheets (Fig. 7, steps c and d) (14). Previous FTIR findings of GM1-induced A␤ with antiparallel ␤-sheets, reported by Matsuzaki and Horikiri (32) and Fukunaga et al. (33), seem to resemble the present oligomeric A␤ assembly (Fig. 7, steps c and f). Finally, protofibrils with parallel ␤-sheets were formed and extended (Fig. 7, step e) as observed on reflection-absorption spectra at 48 -72 h (Fig. 5A). The parallel ␤-sheet structure is stabilized by intermolecular interactions between A␤ molecules and ␤-sheet side chains to form cross-␤ units (9). Multiple molecular dynamics simulations support the binding of A␤ to ganglioside clusters through the combination of a CH-/OH-interaction, a Lys 28 -Neu5Ac interaction, and hydrophobic interactions at the C terminus and also support the involvement of two or three A␤ molecules of the GA␤ complex in the formation of the parallel ␤-sheet (52).

Generation of parallel ␤-sheets of A␤ on ganglioside cluster
Based on attenuated total reflection spectra, the secondary structure of residual A␤ 40 in the supernatant of the solution on GM1-enriched membranes is similar to that of A␤ 40 deposited on GlcCer-rich membranes, with a major (1631 cm Ϫ1 ) and a minor peak (1676 cm Ϫ1 ) attributable to antiparallel ␤-sheets (Fig. 6B), which were also detected in A␤ 40 self-assembled for 48 h (Fig. 6A). Indeed, most of the residual A␤ 40 molecules in the supernatant may not interact at all with the GM1-enriched membrane and therefore will self-assemble in the same way as seed-free A␤ 40 (Fig. 6C). A␤ 40 self-oligomerized into antiparallel ␤-sheets (Fig. 7, step g), as described previously by Stroud et al. (13) and Fu et al. (14) and confirmed by reflectionabsorption spectroscopy of self-assembled A␤ 40 and residual A␤ 40 in the supernatant of the solution on GM1-enriched membranes after 48 h (Fig. 6). That A␤ 40 deposited on GlcCer/ sphingomyelin/cholesterol also formed antiparallel ␤-sheets (Fig. 5B), confirming that a GM1-enriched nanocluster is required to form fibrils with parallel ␤-sheets.
We noted that a mixture of GM1/sphingomyelin/cholesterol (20:40:40) formed a lipid bilayer with POPC on gold-coated glass as well as on mica (Fig. 1A) (30), with characteristic peaks of POPC ((CϭO), 1742 cm Ϫ1 ) and GM1 (amide I and II, 1660 and 1547 cm Ϫ1 , respectively) observable on reflectionabsorption spectra (Fig. 3A and Table 1). The transfer ratio of the 20% GM1 monolayer onto the POPC-coated slide was almost 1.0 (see "Experimental Procedures"), implying the formation of a suitable bilayer of 20% GM1 and POPC. The intensity (absorbance) of amides I and II in GM1-and GlcCer-containing membranes increased with time (Fig. 3), suggesting an accumulation of A␤. This result also suggests a comparable accumulation of an A␤ layer on both membranes, although AFM indicated that A␤ fibrils were selectively deposited on GM1-enriched membranes only (Figs. 1B and 2), as reported previously (30).
In conclusion, the data indicate that A␤ generally self-assembles into antiparallel ␤-structures but is competent to form protofibrils with parallel ␤-sheets, in this case by interaction with GA␤. This model is based on data from AFM and FTIR of a ganglioside-enriched planar membrane. The A␤ 40 topography obtained by AFM was eventually linked to secondary structures obtained by FTIR. In addition, an initial A␤ layer was also detected by both methods, suggesting that the formation of this layer may explain the seemingly contradictory data in the literature. Our data also highlight the growing significance of molecular dynamics simulation in investigating the interaction between A␤ and neural membranes. Finally, these data advance our understanding of ganglioside-induced A␤ fibril formation on neuronal membranes, which may accelerate the development of novel therapies against Alzheimer's disease.

Lipids
Monosialoganglioside GM1 from bovine brain and GlcCer from human (Gaucher disease) spleen were purchased from Sigma or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sphingomyelin from bovine brain and synthetic POPC were from Matreya LLC (State College, PA) and Sigma, respectively.

Preparation of GM1-enriched membranes and AFM
GM1-enriched lipid bilayers were prepared on mica as described previously (29,30). Briefly, a POPC monolayer was prepared at 25°C at the air-water interface of a Langmuir-Blodgett trough (FSD-220, USI Corp., Fukuoka, Japan), with Generation of parallel ␤-sheets of A␤ on ganglioside cluster water as the subphase, and deposited horizontally on freshly cut 1 ϫ 1-cm mica at a surface pressure of 35 mN m Ϫ1 (Fig. 1A, POPC-coated mica). To form the bilayer, a second monolayer consisting of GM1/sphingomyelin/cholesterol (20:40:40, molar ratio) at a surface pressure of 30 mN m Ϫ1 was loaded horizontally onto POPC-coated mica by deposition.
The GM1-enriched membrane was incubated with 10 M seed-free soluble A␤ 40 in PBS for 15 min to 72 h at 37°C (30,31). After washing three times with PBS, the membrane was imaged at 25°C in water using an SPM-9600 atomic force microscope (Shimadzu Corp., Kyoto, Japan) and a 38-m soft cantilever (BL-AC40TS-C2, Olympus, Tokyo, Japan) with integrated pyramidal silicon nitride tips with spring constant 0.1 N m Ϫ1 . Multiple topographic images (2 ϫ 2 m, n Ն 3) were acquired in dynamic mode at 1-2 Hz, and representative images were used in further analyses.
To estimate the A␤-coated areas, AFM images were binarized based on height, and pixels were counted using the GNU Image Manipulation Program. In particular, areas higher than around 4 nm on a binarized image were considered A␤-coated layers. Fibril length and domain size were measured in ImageJ (National Institutes of Health, Bethesda, MD) using a line drawn along a fibril. We estimated the lengths of the long and short axes of an A␤ assembly and defined them as fibrils when the long/short axis aspect ratio was Ͼ3.

Immobilization of GM1-enriched membranes onto gold-coated glass
Glass slides (40 ϫ 20 ϫ 1.1 mm) coated with an evaporated gold layer 300 nm thick and a stabilizing chromium layer 50 nm thick were purchased from Geomatec (Yokohama, Japan) and treated for 10 min with a UV/ozone cleaner (Procleaner TM Plus, BioForce Nanosciences) to remove organic impurities. The slide was then coated by vertical deposition at a surface pressure of 35 mN m Ϫ1 with a POPC monolayer prepared at 25°C at the air-water interface of a Langmuir minitrough (Minitrough System 2, KSV Instruments Ltd., Helsinki, Finland) with water as the subphase. After drying overnight, a monolayer consisting of GM1/sphingomyelin/cholesterol (20: 40:40) or GlcCer/sphingomyelin/cholesterol (20:40:40) was deposited horizontally at a surface pressure of 30 mN m Ϫ1 . The transfer ratio of lipid to the slide was 1.0 Ϯ 0.2 as calculated from changes in the lipid area and the area of the slide that thickened. Finally, the GM1-enriched lipid bilayer was incubated for 15 min to 72 h at 37°C with 10 M seed-free soluble A␤ 40 in PBS. After careful washing with water three times, the membrane was dried overnight for FTIR reflection-absorption spectroscopy.

Reflection-absorption spectroscopy
IR spectra were recorded on a Magna 550 FTIR spectrometer (Thermo Fisher Scientific) equipped with a VR1-NIC variableangle reflection accessory (Harrick Scientific Products, Inc., Pleasantville, NY) and an Hg-Cd-Te detector cooled with liquid nitrogen (53). A p-polarized IR ray was obtained using a wire grid polarizer (PWG-U1R, Harrick Scientific Products, Inc.). Data were collected at a modulation frequency 60 kHz, with angle of incidence 80°from the surface normal and number of accumulations 1000.

Attenuated total reflection spectroscopy
Attenuated total reflection spectra were collected as described previously (53). Briefly, 84 M seed-free soluble A␤ 40 in MilliQ water was incubated at 25°C for 15 min or 48 h to induce self-assembly. About 15 l of the resulting solution was dropped on a germanium plate and dried under nitrogen gas for 50 min. Spectra were collected using a single-reflection accessory (Spectra-Tech Foundation Performer, Thermo Fisher Scientific) and a germanium prism, with number of accumulations 1000. To compare these with the reflection-absorption spectra, attenuated total reflection spectra were transformed into absorbance (␣) spectra according to ␣ ϭ 4k/, where k and are the imaginary parts of the complex refractive index (1.5) and the wavelength, respectively.

Second-derivative analysis of FTIR spectra
FTIR spectra were analyzed in OMNIC, version 7.3. The second derivative (54) of each spectrum was calculated by the Savitzky-Golay method (55).