formation of toxic fibrils of alzheimer’s amyloid β-protein-(1–40) by monosialoganglioside gm1,...

doi:10.1016/j.jmb.2007.05.069 J. Mol. Biol. (2007) 371, 481–489

Formation of Toxic Fibrils of Alzheimer’s Amyloidβ-Protein-(1–40) by Monosialoganglioside GM1,a Neuronal Membrane Component

Takuma Okada, Masaki Wakabayashi, Keisuke Ikedaand Katsumi Matsuzaki⁎

Graduate School ofPharmaceutical Sciences,Kyoto University, Sakyo-ku,Kyoto 606-8501, Japan

Abbreviations used: AD, Alzheimamyloid β-protein; GM1, monosialoDMEM, Dubecco's modified Eagle'sethidium homodimer-1; CD, circulathioflavin-T; SEC, size-exclusion chrfluorescence intensity; NGF, nerve gtransmission electron microscopy.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2007 E

A pathological hallmark of Alzheimer's disease (AD) is the deposition ofamyloid β-protein (Aβ) in fibrillar form on neuronal cells. However, the roleof Aβ fibrils in neuronal dysfunction is highly controversial. This studydemonstrates that monosialoganglioside GM1 (GM1) released fromdamaged neurons catalyzes the formation of Aβ fibrils, the toxicity andthe cell affinity of which are much stronger than those of Aβ fibrils formedin phosphate-buffered saline. Aβ-(1–40) was incubated with equimolarGM1 at 37 °C. After a lag period of 6–12 h, amyloid fibrils were formed, asconfirmed by circular dichroism, thioflavin-T fluorescence, size-exclusionchromatography, and transmission electron microscopy. The fibrils showedsignificant cytotoxicity against PC12 cells differentiated with nerve growthfactor. Trisialoganglioside GT1b also facilitated the fibrillization, althoughthe effect was weaker than that of GM1. Our study suggests an exacerbationmechanism of AD and an importance of polymorphisms in Aβ fibrilsduring the pathogenesis of the disease.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: Alzheimer's disease; amyloid β-protein; monosialogangliosideGM1; toxicity; polymorphism
*Corresponding author
Introduction

One of the pathological hallmarks of Alzheimer'sdisease (AD), a progressive neurodegenerative di-sorder, is the deposition of senile plaques composedmainly of aggregated (fibrillar) amyloid β protein(Aβ) in the brain.1–3 Accordingly, the aggregationand deposition of Aβ on neuronal cells is the keystep in the onset of AD. The lack of a strongcorrelation between the severity of AD and thenumber of Aβ fibrils in the AD brain2 has promptedextensive efforts to identify toxic Aβ aggregates.There is a growing body of evidence to suggest that

er's disease; Aβ,ganglioside GM1;medium; EthiD-1,

r dichroism; Th-T,omatography; FI,rowth factor; TEM,

ng author:

lsevier Ltd. All rights reserve

rather than fibrils, soluble oligomers such as Aβ-derived diffusible ligands formed with clusterin orin cold medium,4–7 Aβ oligomers prepared in acidicsolution,8–10 amylospheroids prepared by rotatingan Aβ solution,11 Aβ globulomer incubated in thepresence of SDS or fatty acid,12 Aβ*56 extractedfrom APP-transgenic mouse brains,13 or Aβ oligo-mers naturally secreted from cells,14 show neuro-toxicity. On the other hand, Aβ fibrils were alsoreported to induce functional deficits in neuronalcells and cell death.9,15–23 Notably, Aβ fibrils wererecently reported to show polymorphisms.24 Theextent of mechanical stress during the preparation offibrils affects their structure and their toxicity.On the other hand, the concentration of mono-

sialoganglioside GM1 (GM1), a neuronal membranecomponent, in the cerebrospinal fluid was found tobe significantly higher in patients with AD (∼30 nM)compared with age-matched controls (∼10 nM)because GM1 is released from damaged neurons.25GM1 exists as micellar aggregates in these con-centrations.26 Gangliosides including GM1 and tri-sialoganglioside GT1b (GT1b) have been suggestedto play a pivotal role in the pathogenesis of AD.27

d.

mailto:[email protected]

482 Formation of Toxic Aβ-1–40 Fibrils Mediated by GM1

In this study, we investigated the effects of GM1and GT1b on the aggregation and cytotoxicity ofAβ-(1–40) and found that gangliosides, especiallyGM1, catalyze the formation of neurotoxic fibrils.

Results

Secondary structure of Aβ-(1–40) in thepresence of GM1

Circular dichroism (CD) measurements werecarried out to estimate the secondary structure ofAβ-(1–40) in the presence of GM1 at various GM1-to-Aβ molar ratios (Figure 1(a)). In PBS, thespectrum of 50 μM Aβ-(1–40) was characteristic ofa random coil conformation. In the presence of500 μM GM1, the protein assumed an α-helix-richconformation, as judged from double minimaaround 208 nm and 222 nm. CD spectra at higherconcentrations of GM1 could not be obtainedbecause of strong light absorption by GM1 itself.The presence of an isodichroic point at 202 nmindicated that Aβ is in a two-state equilibriumbetween an α-helix structure and the random coil(Figure 1(a)).

Aggregation of Aβ-(1–40)

Aβ-(1–40) (50 μM) was incubated in the presenceor in the absence of 50 μM GM1 in PBS at 37 °Cwithout any agitation. Aggregation was monitoredby CD, fluorescence of the amyloid specific dyethioflavine-T (Th-T), and size-exclusion chromato-graphy (SEC).Just after the mixing of Aβ-(1–40) with 50 μM

GM1, most of the protein molecules were in an un-ordered (Figure 1(b)), Th-T negative (Figure 1(d)),and monomeric (elution volume, 17.8 ml) (Figure1(e) 1) state similar to that in the absence of GM1(Figure 1(c)–(e) 3). Note that the population of GM1-bound Aβ-(1–40) was too small to be detected underthese conditions. After incubation for 6 h, thesecondary structure and aggregational state didnot change, whereas a conformational transitionwas observed after 12–24 h. CD spectra changedfrom a helical spectrum at 12 h to a β-sheet spectrumas characterized by a minimum at 220 nm at 24 h(Figure 1(b), blue and green). The fuorescenceintensity (FI) of Th-T also increased (Figure 1(d)),indicating the aggregation of Aβ. The supernatantafter a mild centrifugation (10,000g for 10 min)contained only a small amount of monomeric Aβ-(1–40) (Figure 1(e), 2), and soluble oligomers werenot detected.In the absence of GM1, Aβ-(1–40) remained in a

monomeric state with an unordered structure evenafter incubation for 24 h (Figure 1(c)–(e) 4). In thepresence of a lower concentration of GM1 (25 μM),Aβ-(1–40) aggregated much more slowly (Figure1(d)), indicating that a slight increase in the con-centration of GM1 significantly facilitates the aggre-gation of Aβ.

Cytotoxicity of incubated Aβ-(1–40)

PC12 cells were differentiated into neuron-likecellswithnervegrowth factor (NGF)and treatedwithvarious Aβ preparations for 24 h, and cytotoxicitywas evaluated by staining live and dead cells withcalcein-AM and ethidium homodimer-1 (EthiD-1),respectively. No remarkable morphological changewas observed when cells were treated with PBS(Figure 2(a) and (b)), monomeric Aβ-(1–40) (Figure2(c) and (d)), or GM1 (Figure 2(e) and (f)). In contrast,a 12 h-preincubated Aβ-(1–40)–GM1 mixture (1: 1)(Figure 2(g) and (h)) exhibited significant toxicity.About half of the cells were dead, and neuriteretraction was observed even in live cells. A 24 hpreincubated mixture showed stronger cytotoxicity.Most cells were detached from the bottom of thedishes (data not shown). Because of the detachment,a quantitative estimation of cell viability was per-formed by measuring the FI of calcein-AM in a 96-well plate (Figure 2(i)). Neither Aβ-(1–40) nor GM1was cytotoxic. The cyotoxicity of the Aβ-GM1mixture was dependent on the preincubation period.Non-incubated or preincubated for 6 h mixtureswere not cytotoxic, whereas the toxicity of mixturespreincubated for 12 h and 24 h was significant, inaccordance with changes in the aggregational state,as detected by CD, Th-T assay, and SEC (Figure 1(b),(d) and (e)).To identify cytotoxic species, a 24 h preincubated

equimolar mixture of Aβ-(1–40) and GM1 waswashed three times by centrifugation (200,000g for30 min). The final pellet showed cytotoxicity similarto that of the original 24 h preincubated mixture(Figure 3(a)). The cell viability of the pellet fractionestimated by the EthiD-1 assay was 51.2(±10.8)%(±S.E), indicating that the percentage cell viabilityvalue was not dependent on the assay method. Incontrast, the supernatant of the first centrifugationwas non-toxic (Figure 3(a)). The protein concentra-tion of the supernatant was under the detection limit(1.0 μM). We succeeded in isolating a toxic fractionof the incubated Aβ-(1–40)–GM1mixtures, thereforethe pellets were used in the following assays.The cytotoxicity of Aβ-(1–40) aggregates formed

in the presence of GM1 was compared with that ofAβ-(1–40) aggregates formed in the absence of GM1.In the latter case, a trace amount (1.0 μM) of pre-formed fibrils had been added as a seed.28 Althoughthe predominant morphology of Aβ-(1–40) aggre-gates formed in the presence of GM1 were typicalnon-branched amyloid fibrils (11.8(±0.8) nm, width)(Figure 4(a)), that of Aβ-(1–40) aggregates formedin the absence of GM1 apparently consists of shorterfibrils and associate laterally with each other(14.5(±0.9) nm, width) (Figure 4(b)). The cytotoxicityof Aβ-(1–40) aggregates formed in the absence ofGM1 was much weaker (almost non-toxic) (Figure3(b) 2) than that of the aggregates whose formationwas mediated by GM1 (Figure 3(b) 3).Transmission electron microscopy (TEM) images

of the toxic fraction used in the cytotoxicity assay(Figure 4(a)) show the presence of small particles

Figure 1. GM1-induced conformational changes and aggregation of Aβ-(1–40). (a) CD spectra of 50 μMAβ at 37 °C inthe presence of 0 μM (black), 50 μM (orange), 100 μM (blue), 250 μM (green), and 500 μM (red) GM1, respectively. CDspectra of 50 μMAβ-(1–40) incubated at 37 °C for 0 h (black), 6 h (red), 12 h (blue), and 24 h (green) (b) in the presence of50 μMGM1 or (c) in the absence of GM1. (d) The formation of amyloid fibrils (50 μMAβ-(1–40)) was monitored by the Th-T assay in the presence of 0 μM (▴), 25 μM (○), and 50 μM (▪) GM1 (mean±S.D, n=3, *pb0.01, ** pb0.001). (e) The SECprofiles of the supernatants after centrifugation (10,000g, 10 min) of Aβ-(1–40) (50 μM) prior to (1, 3) and after incubationfor 24 h (2, 4), with (1, 2) or without 50 μM GM1 (3, 4). (black arrow, void volume; red arrowhead, 21,200 Da; bluearrowhead, 4400 Da).

483Formation of Toxic Aβ-1–40 Fibrils Mediated by GM1

(∼10 nm in diameter), which are considered to beGM1 micelles.29 To confirm that the cytotoxicity ofthe mixture originates from amyloid fibrils, not the

particles, the following two experiments werecarried out. First, daughter fibrils were preparedby incubating 50 μM monomeric Aβ with the soni-


cated pellet fraction as a seed (1.0 μM) for 1 h at37 °C and the cell viability assay was performed.Daughter fibrils were as toxic as parent fibrils(Figure 3(b) 4). Second, the resuspended pellet wasextruded with a 100 nm pore polycarbonate mem-brane (Nuclepore, WA), and the protein concentra-

tion and the cytotoxicity of the filtrate were esti-mated. The protein concentration was under thedetection limit (1.0 μM) and no cytotoxicity wasobserved (Figure 3(b) 5). These lines of evidenceindicated strongly that the origin of the cytotoxicityin the preincubated Aβ-(1–40)–GM1 mixture isamyloid fibrils.Neuronal cells contain gangliosides other than

GM1. To determine if the formation of toxic Aβ-(1–40)aggregates is specific to GM1, the cytotoxicity ofAβ-(1–40) pellets mediated by GT1b was examined.The cytotoxicity of Aβ-(1–40) incubated in the pre-sence of equimolar GT1b for 24 h was significantlyweaker than that of Aβ-(1–40) incubated in thepresence of GM1 (Figure 3(b) 6).The Th-T FI of theformer Aβ aggregates (63.6±13.0 (S.D.), n=3)was significantly lower than that of the latter (100±2.9 (S.D.), n=3).

Binding of Aβ aggregates to cells

To understand the mechanism of different toxicityof the Aβ fibrils, the binding of the fibrils to cells wasexamined by the Congo red assay (Figure 5). Aβ-(1–40) fibrils formed in the presence of GM1 showedsignificantly stronger binding to cell membranesthan Aβ-(1-40) fibrils formed in the absence of GM1.

Discussion

The concentration of Aβ in the brain is too low(∼nM) for the protein to aggregate spontaneously,suggesting that the aggregation is triggered byenvironmental factors specific to the brain's patho-logical state. Proteins such as apo J,4,30 and metalions, e.g. Zn2+,31 have been reported to facilitate theaggregation of Aβ. In addition to soluble factors, thecomponents of membranes also promote the aggre-gation. Yanagisawa et al. discovered that GM1–bound Aβ (GM1-Aβ) in human brains exhibitedearly pathological changes associated with AD,32

GM1-Aβ was shown to serve as a seed for theformation of amyloid fibrils.33,34

Figure 2. Cytotoxicity of Aβ-(1–40) co-incubated withGM1. (a), (c), (e) and (g), Confocal laser scanningmicrographs of differentiated PC12 cells double-stainedwith calcein-AM (live cells, green) and ethidium homo-dimer-1 (dead cells, red) and (b), (d), (f) and (h)corresponding differential interference contrast images.Cells were treated for 24 h at 37 °C with (a) and (b) PBS, (c)and (d) 50 μMAβ-(1–40), (e) and (f) 50 μMGM1, or (g) and(h) a mixture of 50 μM Aβ-(1–40) and 50 μM GM1. Allsamples had been incubated for 12 h at 37 °C and mixedwith the same volume of cell medium containing NGFbefore the cytotoxicity assay. (i) The cell viability wasdetermined as a function of the preincubation time basedon the FI of calcein-AM (mean±S.E.; n=6; *pb0.001against a positive control (PBS treatment) ). Symbols:(▴), Aβ (50 μM) alone; (□), GM1 (50 μM) alone; (▪), amixture of 50 μM Aβ and 50 μM GM1 were preincubatedat 37 °C for various lengths of time.

Figure 3. Identification of toxic species in a preincubated Aβ-(1–40)–GM1 mixture. (a) Aβ-(1–40) (50 μM) wasincubated with 50 μM GM1 for 24 h at 37 °C. The cytotoxicity of the whole mixture (Total), the supernatant aftercentrifugation (200,000g, 30 min) (Sup), or the resuspended pellet after three washing procedures (Pellet). (b) Comparisonof the toxicity of various forms of Aβ-(1–40) (final, 50 μM). Monomer (1), fibrils polymerized in the absence of GM1 (2),fibrils formed by a 24 h incubation with equimolar GM1 (3), and daughter fibrils prepared from sample 3 (4), the filtrate(pore radius; 100 nm) of sample 3 ([Aβ]b1 μM) (5) and fibrils formed by a 24 h incubation with equimolar GT1b (6).(mean±S.E.; n=6 ; *pb0.001, against a positive control treated with PBS).


GM1 is one of the glycosphingolipids composingthe lipid raft microdomains in neuronal cell mem-branes.35,36 Previous studies demonstrated that theproduction of Aβ occurs, at least partly, in lipid raftsand is regulated by GM1,37 and other raft com-ponents,38,39 and vice versa. Aβ modulates the me-tabolism of raft components,18,40 implying that lipidrafts play an important role in the pathogenesisof AD. Recently, a significant increase in GM1 was

Figure 4. TEM images of Aβ-(1–40) fibrils. Aβ-(1–40)fibrils were formed by coincubation for 24 h (a) withequimolar GM1 and (b) in the absence of GM1. The scalesbars represent 100 nm.

reported in Aβ-positive nerve terminals from ADcortex.41

Our group proposed a molecular mechanism forthe GM1-mediated aggregation of Aβ.34,42 Unor-dered soluble Aβ recognizes and binds to GM1clusters, which are formed in raft-like membranesin a cholesterol-dependent manner, changing itsconformation from an α-helix-rich to a β-sheet-richstructure as the molar ratio of Aβ-to-GM1increases. The β-sheet-rich Aβ serves as a seedfor the elongation of amyloid fibrils. Our CD(Figure 1(b)), Th-T (Figure 1(d)), SEC (Figure 1(e)), and TEM (Figure 4(a)) data show that Aβfibrils rich in β-sheets are also formed by thecatalysis of micellar GM1 with a lag time of 6–12 h.Micelles of synthetic detergents, such as SDS, arealso reported to facilitate the fibrillization of Aβ-(1–40), although toxicity was not determined.43 Incontrast, polyunsaturated fatty acids at micellarconcentrations stabilize soluble Aβ-(1–42) protofi-brils, thereby hindering their conversion to fibrils.44

Gangliosides other than GM1 also accelerate thefibrillization of Aβ in raft-like membranes,although their effects are weaker than that ofGM1.34 The same is true in the case of micellargangliosides (Figure 3).We previously reported that the accumulation of

Aβ at ganglioiside-rich domains of cell membranesleads to cytotoxicity.45 Recently, toxic globular Aβoligomers have been shown to be generated in thepresence of raft-like liposomes with limited amountsof GM1.46 Thus, GM1 mediates the formation ofdifferent types of toxic Aβ aggregates.The current study clearly indicated that Aβ-(1–

40), which shows lower toxicity than Aβ-(1–42), canform toxic fibrils in the presence of GM1 (Figures 2and 3), suggesting that the released GM1 byneuronal damage may exacerbate neurodegenera-tion in AD. In striking contrast, Aβ fibrils formed inthe absence of GM1 are much less toxic (Figure 3)

Figure 5. Binding of Aβ aggregates to cells. Confocal laser scanning micrographs of Aβ aggregates bound to (a)–(c)(red) NGF-differentiated PC12 cells stained with Congo red and (d) FI of Congo red per cell. Cells were incubated for30 min with Aβ-(1–40) fibrils formed by incubation for 24 h with equimolar GM1 ((a) and (d) 1), Aβ-(1–40) fibrilspolymerized in the absence of GM1 ((b) and (d) 2), and Aβ-(1–40) monomer ((c) and (d) 3) and subsequently stained with20 μM Congo red. The final concentration of Aβ was 25 μM.


because of weaker binding to cell membranes(Figure 5). We are currently investigating the struc-tural differences between the two kinds of fibrils indetail. Various types of amyloid fibrils with differentstructures and toxicity as well as soluble oligomersappear to be involved in the pathogenesis of AD in acomplicated way.

Experimental Procedures

Materials

Dulbecco's modified Eagle's medium (DMEM), horseserum, bovine serum, penicillin, streptomycin, and EthD-1 were purchased from Invitrogen (Carlsbad, CA). Aβ-(1–40) was obtained from Peptide Institute (Minou, Japan).Calcein-AM, GM1 and GT1b were purchased from DojinLaboratories (Kumamoto, Japan), Wako (Tokyo, Japan),and Sigma (St louis, MO), respectively. All otherchemicals were obtained from Nacalai Tesque (Kyoto,Japan).

Protein solution

Aβ-(1–40) was dissolved in 0.02% (v/v) ammonia onice, and any large aggregates that might act as a seed foraggregation were removed by ultracentrifugation in500 μl polyallomer tubes at 540,000g at 4 °C for 3 h.

The protein concentration of the supernatant wasdetermined in triplicate by Micro BCA protein assay(Pierce, Rockford, IL). The supernatant was collectedand stored at −80 °C. Just before the experiment, apotion of the stock solution was mixed with an equalvolume of double-concentrated PBS (NaCl 16.0 g/l, KCl0.40 g/l, Na2HPO4 2.30 g/l, and KH2PO4 0.40 g/l, pH7.4).

Ganglioside solution

Powdered bovine brain GM1 or GT1b was dissolvedin a chloroform/methanol 1:1 (v/v) mixture and storedat −80 °C. The solvent was removed by evaporation in arotary evaporator. The residual lipid film, after dryingunder vacuum overnight, was hydrated with PBS (NaCl8.00 g/l, KCl 0.20 g/l, Na2HPO4 1.15 g/l, and KH2PO40.2 g/l, pH 7.4) and vortex-mixed to produce the GM1solution or GT1b solution. The concentration of gang-lioside was quantified in triplicate by the resorcinol-hydrochloric acid method.47

CD measurements

CD spectra were measured at 37 °C on a Jasco J-820apparatus, using a 1.0 mm or 0.1 mm pathlength quartzcell to minimize the absorbance due to buffer compo-nents. Eight scans were averaged for each sample. Theaveraged blank spectra (GM1 solution or buffer) weresubtracted.


Aggregation assay

Aβ-(1–40) (50 μM) was incubated with GM1 in PBS at37 °C for 24 h without agitation and the aggregation wasmonitored by the Th-T assay and SEC. The Th-T assay wascarried out as follows.48,49 The sample (final Aβ concen-tration, 0.5 μM) was added to a 5 μM Th-T solution in50 mM glycine buffer (pH 8.5). Fluorescence at 490 nmwas measured at an excitation wavelength of 446 nm on aShimadzu RF-5300 spectrometer with a cuvette holderthermostatically controlled at 25 °C. The statisticalanalysis was performed using ANOVA (n=3).The protein was fractionated with a Sephacryl S-300

column with an exclusion limit of 150,000 Da (GEHealthcare Bio-Science Corp., NJ), which had beenpretreated with an excess of BSA to block non-specificbinding of proteins. The column was eluted with PBS at aflow rate of 0.5 ml/min and the protein was detected bymeasuring UV absorbance at 220 nm. Each sample wascentrifuged at 10,000g for 10 min to remove insolublecomponents and the supernatant (25 μl) was injectedonto the column. Molecular mass was estimated withFITC-dextrans (4400 Da, 21,200 Da, 42,000 Da, 70,000 Da,and 150,000 Da) as standards.50 The blank chromato-graphs (GM1 solution or buffer) were subtracted.

Cell culture

Rat pheochromocytoma PC12 cells were cultured inDMEM containing 5% (v/v) horse serum, 10% (v/v)bovine serum, 100 units/ml of penicillin, and 0.1 mg/mlof streptomycin at 37 °C with 5% (v/v) CO2. After beingplated at a density of 5000 cells/well onto a Biocoat poly-d-lysine-coated 96-well Black/Clear plate (Becton Dick-inson, England) for a viability assay and at a density of100,000 cells/ dish onto a poly-d-lysine-coated 35 mmglass-bottomed dish for confocal laser scan microscopy,cells were incubated for 24 h at 37 °C with 5% CO2 anddifferentiated with 50 ng/ml of NGF in serum-freemedium for five to seven days.

Viability assay and cell observation

A mixture of 50 μM Aβ-(1–40) and 50 μM GM1 thathad been incubated for various lengths of time wasdiluted with the same volume of NGF-containing non-serum medium. A 100 μl portion of the solution wasapplied to each well, and incubated for 24 h at 37 °C with5% CO2. The sample was removed and 100 μl of 1 μMcalcein-AM in PBS was applied gently and incubated for1 h at 37 °C for the live cell assay, and 50 μl of 3 μMEthiD-1 in PBS was added to each well (final concentra-tion 1 μM) without removing the sample solution for thedead cell assay. Calcein-AM is a membrane-permeabledye that is cleaved by intracellular esterases in living cellsto produce the fluorophore calcein. EthiD-1 can penetratethe plasma membranes of only dead cells. The linearitybetween the number of living or dead cells and FI ofcalcein (excitation, 495 nm; emission, 535 nm), or FI ofEthiD-1 (excitation, 530 nm; emission, 620 nm) wasconfirmed. In the live cell assay, the FI of cells treatedwith only vehicle and the background FI of each wellwere defined as a positive control (viability;100%) and asa negative control (viability; 0%), respectively. In the deadcell assay, the FI of cells treated with only vehicle and theFI of cells treated with 70% (v/v) methanol for 30 minwere defined as a positive control (viability; 100%) and as

a negative control (viability; 0%), respectively, accordingto the manufacturer's instructions. Confocal laser scan-ning microscopy was used to observe cells. Cells in aglass-bottomed dish were stained with 0.2 μM calcein-AM (excitation, 488 nm; emission, BP 505–530 nm) and0.2 μM EthiD-1 (excitation, 543 nm; emission, LP 560 nm)for 30 min and washed twice. The cells were visualizedusing the 20× C-Apochromat objective of a Zeiss LSM 510confocal laser scanning microscope. The statistical analy-sis was performed using ANOVA (n=6) and thesignificance was assessed against a positive control.

Preparation of amyloid fibrils in the absence ofGM1

Amyloid fibrils in solution were prepared asdescribed.28 For the preparation of seeds, 200 μM Aβ-(1–40) was incubated at 37 °C for three days in PBS (pH 7.4)and the formation of fibrils was confirmed by the Th-Tassay. The fibril suspension was diluted with ultrapurewater to 7 μM. The solution was sonicated on ice with 20intermittent pulses (pulse, 0.6 s; interval, 0.4 s; output level2) using an ultrasonic disruptor (UD-201, Tomy, Tokyo,Japan) equipped with a microtip TP-030. Seed fibrils(1 μM) were mixed with monomeric 50 μM Aβ-(1–40) inPBS (pH 7.4) and incubated at 37 °C for 12 h withoutagitation. The formation of amyloid fibrils was confirmedby the Th-T assay and TEM. The TEM experiments werecarried out by the Ultrastructure Research Institute,Hanaichi Co. Ltd (Okazaki, Japan). Samples were spreadon carbon-coated grids, negatively stained with uraniumacetate, and examined under a JOEL 1200EX electronmicroscope with an acceleration voltage of 100 kV.

Congo red assay

NGF-differentiated PC12 cells on a glass-bottomed dishwere incubated with each Aβ sample (final concentration,25 μM) for 30 min, washed twice, stained with 20 μMCongo red for 30 min and washed twice. The cells werevisualized using the 20× C-Apochromat objective of aZeiss LSM 510 confocal laser scanning microscope(excitation, 543 nm; emission, LP 560 nm). For quantitativeestimation, the FI of Congo red (expressed in 256 tones)multiplied by the pixel number was summed andnormalized to the cell number examined (∼100 cells)after the background fluorescence was subtracted.

Acknowledgements

This work was supported by the Takeda ScienceFoundation and the ONO Medical Research Foun-dation. We thank Professor Kazuhisa Nakayama foraccess to the confocal laser scanning microscopysystem.

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Edited by J. Weissman

(Received 20 February 2007; received in revised form 8 May 2007; accepted 21 May 2007)Available online 31 May 2007

formation of toxic fibrils of alzheimer’s amyloid β-protein-(1–40) by monosialoganglioside gm1,...

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