environment- and mutation-dependent aggregation behavior of alzheimer amyloid β-protein

Environment- and mutation-dependent aggregation behavior of

Alzheimer amyloid b-protein

Naoki Yamamoto,* Kazuhiro Hasegawa,� Katsumi Matsuzaki,� Hironobu Naiki�and Katsuhiko Yanagisawa*

*Department of Dementia Research, National Institute for Longevity Sciences, Obu, Japan

�Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui,

Fukui, Japan

�Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

Abstract

The deposition of amyloid b-protein in the brain is a funda-

mental process in the development of Alzheimerı́s disease;

however, the mechanism underlying aggregation of amyloid

b-protein remains to be determined. Here, we report that a

membrane-mimicking environment, generated in the pres-

ence of detergents or a ganglioside, is sufficient per se for

amyloid fibril formation from soluble amyloid b-protein. Fur-

thermore, hereditary variants of amyloid b-protein, which are

caused by amyloid precursor protein gene mutations, inclu-

ding the Dutch (E693Q), Flemish (A692G) and Arctic (E693G)

types, show mutually different aggregation behavior in these

environments. Notably, the Arctic-type amyloid b-protein, in

contrast to the wild-type and other variant forms, shows a

markedly rapid and higher level of amyloid fibril formation in

the presence of sodium dodecyl sulfate or GM1 ganglioside.

These results suggest that there are favorable local environ-

ments for fibrillogenesis of amyloid b-protein.

Keywords: Alzheimer’s disease, amyloid, amyloid b-protein,

detergent, ganglioside, membrane.

J. Neurochem. (2004) 90, 62–69.

One of the questions about the molecular pathophysiology

of Alzheimer’s disease (AD) is how the soluble, non-toxic

amyloid b-protein (Ab) is converted to its aggregated, toxicform. In the case of familial AD caused by mutations of the

amyloid precursor protein (APP) gene outside the Absequence (London and Swedish types) and presenilin

(presenilin 1 and presenilin 2) genes, it is likely that Abdeposition in the brain is induced by the accelerated and/or

altered generation of Ab, particularly Ab42, an aggregation-prone form of Ab (Selkoe 1997). However, in the case ofsporadic AD, a major form of the disease, no evidence has

ever been reported to indicate that Ab generation is altered. Itmay therefore be possible to assume that Ab deposition inthe brain is facilitated not only by its accelerated and/or

altered generation but also by as yet unknown aggregation-

promoting local environmental factor(s). This possibility is

supported by the evidence that Ab deposits in preferred areasin an AD brain. The environment-dependent acceleration of

Ab aggregation may be particularly significant for the

development of hereditary cerebral amyloidosis, which is

caused by APP gene mutations within the Ab sequence(Levy et al. 1990; Hendriks et al. 1992; Rozemuller et al.

1993; Nilsberth et al. 2001), for the following reasons: first,

the levels of secreted Ab42 are not increased but ratherdecreased by these mutations, including the Dutch (E693Q),

Italian (E693K) and Arctic (E693G) types but not the

Flemish (A692G) type (Nilsberth et al. 2001); second, the

Dutch-type Ab predominantly deposits in the blood vesselsof the brain (Rozemuller et al. 1993) whereas the Arctic-type

Abwas suggested to deposit preferentially in the parenchymaof the brain (Nilsberth et al. 2001).

The presence of detergents, including octyl-b-glucopyr-anoside (OG) and sodium dodecyl sulfate (SDS), or solvents,

including trifluoroethanol and hexafluoroisopropanol, alters

Received January 9, 2004; accepted February 13, 2004.

Address correspondence and reprint requests to Katsuhiko

Yanagisawa, MD, Department of Dementia Research, National Institute

for Longevity Sciences, 36–3 Gengo, Morioka, Obu, Japan 474–8522.

E-mail: [email protected]

Abbreviations used: Ab, amyloid b-protein; AD, Alzheimer’s disease;APP, amyloid precursor protein; CD, circular dichroism; CMC, critical

micelle concentration; EM, electron microscopy; OG, octyl-b-gluco-pyranoside; SDS, sodium dodecyl sulfate; TBS, Tris-buffered sulfate;

ThT, thioflavin T; ZW, Zwittergent 3–14.

Journal of Neurochemistry, 2004, 90, 62–69 doi:10.1111/j.1471-4159.2004.02459.x

62 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 62–69

the secondary structure of soluble Ab (Hollosi et al. 1989;Barrow and Zagorski 1991; Barrow et al. 1992; Burdick

et al. 1992; Laczko-Hollosi et al. 1992; Otvos et al. 1993;

Shao et al. 1999). Moreover, it was recently reported that

trifluoroethanol could accelerate Ab fibrillogenesis throughits partial unfolding (Fezoui et al. 2002).

The aims of the present study were twofold: first, to

determine whether membrane-mimicking environments are

sufficient per se for amyloid fibril formation from soluble Aband, second, to investigate how hereditary Ab variantsbehave in these environments. The results of the present

study suggest that the interaction of Abwith lipids is crucialfor the initiation of Ab aggregation in the brain and that thereare preferred environments for the aggregation of each

hereditary Ab variant.

Experimental procedures

Materials

OG, SDS, cholesterol and sphingomyelin were purchased from

Sigma-Aldrich (St Louis, MO, USA). Zwittergent 3–14 (n-tetra-

decyl-N,N-dimethyl-3-ammonia-1-propanesulfonate) (ZW) was

obtained from Calbiochem (San Diego, CA, USA). Bovine brain

GM1 ganglioside was obtained from Wako (Osaka, Japan).

Synthetic Ab (wild-type and variant forms of Ab40 and wild-typeAb42) were purchased from the Peptide Institute (Osaka, Japan).

Preparation of ‘de-seed’ Ab solutions

‘De-seed’ Ab solutions were prepared essentially as we previouslyreported (Naiki and Gejyo. 1999). Briefly, synthetic Ab40 (wildtype and variant forms) was dissolved in 0.02% ammonia solution at

500 lM and Ab42 (wild type) at 250 lM, and the solutions werecentrifuged at 540 000 g for 3 h using an Optima TL ultracentrifuge

(Beckman, Fullerton, CA, USA). The supernatant was collected and

stored in aliquots at ) 80�C until use. Immediately before use,aliquots were thawed and diluted with Tris-buffered saline (TBS)

(150 mM NaCl, 10 mM Tris-HCl, pH 7.4).

Determination of critical micelle concentrations (CMCs)

The critical micelle concentrations (CMCs) of detergents (OG, ZW

and SDS) were determined using the fluorescence probe 8-anilino-1-

naphtalene (ANS) as previously reported (Ortner et al. 1979). Briefly,

solutions of detergents were incubated with ANS, and fluorescence

intensity in the incubation mixtures was determined using a

spectrofluorophotometer (RF-5300PC, Shimadzu, Japan); the exci-

tation wavelength was 385 nm with a slit width of 5 nm, and the

emission intensity was recorded at 475 nmwith a slit width of 10 nm.

Thioflavin T (ThT) assay

The ThT assay was performed as described elsewhere (Naiki and

Gejyo. (1999), using a spectrofluorophotometer (RF-5300PC). The

Ab (Ab40 and Ab42) solution described above was incubated at37�C at a concentration of 50 lM. Optimum fluorescence intensitymeasurements of amyloid fibrils were obtained at excitation and

emission wavelengths of 446 nm and 490 nm respectively, with

the reaction mixture (1.0 mL) containing 5 lM ThT and 50 mM

glycine-NaOH buffer, pH 8.5. Fluorescence intensity was measured

immediately after preparing the mixture.

Circular dichroism (CD) spectroscopy

Ab40 at a concentration of 50 lM in TBS was used in CD

spectroscopy. CD spectra were measured on a Jasco J-720 apparatus

interfaced to an NEC PC9801 microcomputer (Tokyo, Japan), using

a 1-mm path-length quartz cell to minimize the absorbance owing to

buffer components. The instrumental outputs were calibrated with

non-hygroscopic ammonium d-camphor-10-sulfonate as reported

previously (Takakuwa et al. 1985). Four scans were averaged for

each sample. The averaged blank spectra of TBS with or without

detergents (ZW or SDS) were subtracted.

Preparation of liposomes

Cholesterol, sphingomyelin and GM1 ganglioside were dissolved in

chloroform/methanol (1 : 1) at a molar lipid ratio of 40 : 40 : 20,

42.5 : 42.5 : 15, and 45 : 45 : 10 to generate GM1-containing

liposomes. GM1-lacking liposomes were prepared by mixing

cholesterol and sphingomyelin at a molar lipid ratio of 1 : 1. The

mixtures were stored at ) 80�C until use. Immediately before use,the lipids were resuspended in TBS at a GM1 ganglioside

concentration of 2.5 mM, and suspensions were subjected to

freezing and thawing. The lipid suspension was centrifuged once

at 15 000 g for 15 min and the resulting pellet was resuspended in

TBS. Finally, the suspension was subjected to sonication on ice.

Electron microscopy (EM)

Samples (2 lL) were diluted with 38 lL distilled water. These dilutedsamples were spread on carbon-coated grids, allowing the solution to

stand for 1–2 min before removing any excess with filter paper. After

evaporating the residual solution, the grids were negatively stained

with 1% phosphotungstic acid (pH 7.0). Again, the solution on the

grids was removed with filter paper and the residual solution evapor-

ated. These samples were examined under a Hitachi H-7000 electron

microscope (Tokyo, Japan) with an acceleration voltage of 75 kV.

Congo red staining

Congo red staining was performed as described elsewhere (Naiki

and Nakakuki. 1996). Briefly, a part of the sample was centrifuged

at 4�C for 2 h at 20 000 g. Pellets were spread on glass slides, driedovernight, stained with Congo red, and examined under a polarized

light microscope for orange–green birefringence.

Data analysis

Data were expressed as mean ± SD of five independent experi-

ments. Statistical analysis was performed by two-way ANOVA

combined with Scheffe’s test for all paired comparisons. p < 0.05

was interpreted to be statistically significant.

Results

Detergent-induced aggregation of wild-type Ab40 and

Ab42

To perform a kinetic study of the aggregation of synthetic

wild-type and variant forms of Ab40 and wild-type Ab42,we prepared ‘de-seed’ solutions of Ab as reported previously

Ab aggregation in membrane-mimicking environment 63

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 62–69

(Naiki and Gejyo. (1999) to avoid spontaneous Ab aggre-gation in the presence of pre-existent seeds (Walsh et al.

1997; Naiki et al. 1998; Hasegawa et al. 1999). ThT

fluoresence intensity in the ‘de-seed’ Ab solutions at

50 lM did not increase during the incubation period of48 h and 6 h for Ab40 and Ab42 respectively (Figs 1 and 2).We then incubated Ab with detergents at various concentra-tions. ThT fluoresence intensity in Ab40 solution increasedin the presence of the detergents OG, ZW and SDS (Fig. 1).

The increase in ThT fluorescence intensity was dependent on

the detergent concentration in the incubation mixture. We

determined the CMC of each detergent in the solution

conditions used in the present study. The CMCs were 0.5%,

0.005% and 0.0375% for OG, ZW and SDS respectively.

Peak ThT fluorescence intensity in the incubation mixtures

containing Ab40 and detergents was obtained at the CMCsfor OG and SDS and at a higher concentration than the CMC

for ZW (Fig. 1). Interestingly, profiles of the increase in ThT

fluorescence intensity were different from each other; that is,

there was a lag phase of 8 h before ThT fluoresence intensity

started to increase in Ab40 solution in the presence of SDS(Fig. 1c).

We also examined whether ThT fluorescence intensity in

Ab42 solutions was increased in the presence of detergents atconcentrations that provided peak ThT fluorescence intensity

in the Ab40 solutions. ThT fluorescence intensity in Ab42solution was increased in the presence of detergents. Peak

ThT fluorescence intensity in Ab42 solutions in the presenceof OG or ZW was essentially similar to that in Ab40solutions. However, ThT fluorescence intensity in Ab42solution in the presence of SDS reached a plateau at a much

lower level than those obtained in the the presence of OG and

ZW (Fig. 2a).

We then examined the possibility that Ab42 aggregationwas induced by SDS at lower concentrations. Surprisingly, in

contrast to the incubation of Ab40, Ab42 aggregation wasmarkedly accelerated in the presence of SDS below the CMC

without a lag phase (Fig. 2b).

Aggregation behavior of Ab variants in the presence

of detergents

We then investigated how hereditary Ab variants, includingthe Dutch-, Flemish- and Arctic-type Ab (Ab40), behave in

Fig. 1 Kinetics of Ab fibrillogenesis in the presence of detergents.

Soluble Ab40 was incubated in the presence of OG (a), ZW (b) and

SDS (c) at various concentrations indicated in the figures, or in the

absence of detergent (control). Fluorescence intensity of ThT was

obtained by excluding background activity in the absence of Ab40 and

detergent. Values are representative of three experiments.

Fig. 2 Kinetics of Ab fibrillogenesis in the presence of detergents. (a)

Soluble Ab42 was incubated in the presence of OG, ZW and SDS at

concentrations that provided peak values of ThT fluorescence intensity

in Ab40 solutions, or in the absence of detergent (control). (b) Soluble

Ab42 was incubated in the presence of SDS at various concentrations,

or in the absence of SDS (control). Fluorescence intensity of ThT was

obtained by excluding background activity in the absence of Ab42 and

detergent. Values are representative of three experiments.

64 N. Yamamoto et al.


membrane-mimicking environments. In the presence of OG

or ZW the Dutch-type Ab solution showed a more rapidincrease in ThT fluorescence intensity, with a higher peak,

than the Flemish- and Arctic-type Abs (Figs 3a and b). Incontrast, in the presence of SDS, the solution of Arctic-type

Ab, but not those of Dutch- and Flemish-type Abs, showed asteep increase in ThT fluorescence intensity without a lag

phase (Fig. 3c).

To morphologically characterize aggregates of Ab variantsformed in the presence of detergents, we employed EM. In

the presence of ZW, the wild-type Ab formed fibrils with adiameter of 8–10 nm and helical structures, whereas the

Dutch- and Flemish-type Abs formed rather thin fibers with adiameter of 6–7 nm (Fig. 4a). In contrast, Arctic-type Abformed short and curved thin fibers with a diameter of

6–7 nm, which can be defined as protofibrils (Walsh et al.

1997) (Fig. 4a). In the presence of SDS, the wild-,

Dutch- and Flemish-type Abs formed fibrils with similarcharacteristics to those formed in the presence of ZW.

However, surprisingly, the Arctic-type Ab formed fibrilsdistinct from those formed in the presence of ZW. These

fibrils were indistinguishable from those of wild-type Ab(Fig. 4b). We attempted to perform EM of fibrils formed in

the presence of OG; however, we failed to fix Ab aggregateson carbon-coated grids for unknown reasons (data not

shown).

To further characterize the Arctic-type Ab aggregation inthe presence of ZW or SDS, we performed CD spectroscopy.

In the experiment with wild-type Ab, an immediate transitionfrom a random coil to a b-sheet was observed in the presenceof SDS; however, it was incomplete in the presence of ZW

(Fig. 5a). A typical curve for a b-sheet was obtained with Absolution preincubated for 24 h in the presence of ZW before

CD measurements (data not shown). The Arctic-type Abmade the transition from random coil to b-sheet both in the

Fig. 3 Kinetics of Ab fibrillogenesis. Different types of soluble Ab40

(wild type, d; Dutch type, j; Flemish type, m; Arctic type, r) were

incubated in the presence of 0.5% OG (a), 0.02% ZW (b), 0.0375%

SDS (c) or in the absence of detergents (respective open symbol for

each Ab). Fluorescence intensity of ThT was obtained by excluding

background activity in the absence of Ab40 and detergent. Values are

representative of three experiments.

Fig. 4 Electron micrographs of the solutions of Ab (Ab40), including

wild-, Dutch-, Flemish- and Arctic-type Abs, incubated for 24 h in the

presence of 0.02% ZW (a) or 0.0375% SDS (b). Bar, 50 nm.



presence of SDS and ZW immediately after preparation of

solutions (Fig. 5b). A previous study showed that protofi-

brils have a secondary structure characteristic of amyloid

fibrils (Walsh et al. 1999); this result also suggests that the

Arctic-type Ab fibrils formed in the presence of ZW are

protofibrils.

Aggregation behavior of Ab variants in the presence

of GM1 ganglioside

The results of the present study (Figs 3 and 4) led us to

examine whether the aggregation of Ab variants is acceler-ated in the presence of GM1 ganglioside because we

previously identified a GM1 ganglioside-bound Ab in ADbrains; on the basis of its unique molecular characteristics,

we hypothesized that ganglioside-bound Ab accelerates the

aggregation of soluble Ab by acting as a seed (Yanagisawaet al. 1995, 1997; Yanagisawa and Ihara. 1998). Indeed,

results of previous in vitro studies support this possibility

(Choo-Smith et al. 1997; McLaurin et al. 1998; Kakio et al.

2001, 2002). In our present experiment, the ThT fluorescence

intensity was highest in a mixture of Arctic-type Ab andGM1 ganglioside-containing liposomes (Fig. 6a). Notably,

the difference in increase in ThT fluorescence intensity

between the Arctic-type and other Abs was more marked inthe mixture of Ab and liposomes with a lower concentrationof GM1 ganglioside (Fig. 6a). To confirm that the increase in

ThT fluorescence intensity in the Arctic-type Ab solution in

the presence of GM1 ganglioside was caused by amyloid

fibril formation, we performed EM and also Congo red

staining of precipitates obtained from the incubation mix-

tures. Fibrils with typical morphological features of amyloid

were observed by EM (Fig. 6b). The fibrils also showed

typical birefringency under polarized microscopy (Fig. 6b,

inset).

Discussion

In the present study, we report that membrane-mimicking

environments, generated in the presence of detergents or

GM1 ganglioside, are sufficient per se for amyloid fibril

formation from soluble Ab. The effect of detergents on theinduction of Ab aggregation was dependent on detergent

Fig. 5 Secondary structures of wild-type (a) and Arctic-type (b) Ab40.

CD spectra of the Ab40 solutions were measured in the absence of

detergents (control) or in the presence of 0.02% ZW or 0.0375% SDS.

WildDutchFlemishArctic

a

a

bc

c

0

50

100

150

50/50/0 45/45/10 42.5/42.5/15 40/40/20

CH/SM/GM1

fluor

esce

nce

(arb

itrar

y un

it)

Fig. 6 Ab (Ab40) fibrillogenesis in the presence of GM1 ganglioside.

(a) Fluorescence intensity of ThT in mixtures of Ab, including wild-,

Dutch-, Flemish- and Arctic-type Abs, incubated for 24 h in the pres-

ence of liposomes with various concentrations of GM1 ganglioside.

Molar ratios of cholesterol (CH), sphingomyelin (SM) and GM1 gan-

glioside (GM1) are indicated in the figure. Values in each column are

mean ± SD of five values. ap < 0.01, bp < 0.02, cp < 0.001 (two-way

ANOVA combined with Scheffe’s test). (b) Electron micrograph of the

Arctic-type Ab solution incubated for 24 h in the presence of liposomes

containing cholesterol, sphingomyelin and GM1 ganglioside at a lipid

molar ratio of 40 : 40 : 20. Bar, 50 nm. Inset, birefringency of fibrils

observed under a polarized microscope.



concentration. In general, the detergents were most potent

either at or above their CMCs, suggesting that the interface

between water and the surface of detergent micelles is a

preferred space for the initiation of Ab aggregation. Thispossibility is supported by recent studies using insulin and

tau (Sharp et al. 2002; Chirita et al. 2003). Thus, one of the

possible mechanisms underlying the aggregation of soluble

proteins, including Ab, in vivo is the adsorption of a given

protein to the lipid membrane surface, followed by the

unfolding of the protein to induce the subsequent aggregation

of soluble proteins. At this point, it remains to be elucidated

why the effects on Ab aggregation induction differ fromdetergent to detergent. It might be assumed that the presence

or absence of a negative charge (e.g. SDS) and sugar

molecule (e.g. OG) modulates the hydrophobic micro-

environment on the surface of detergent micelles (Sundari

and Balasubramanian 1997; Kuroda et al. 2003).

Another question that should be clarified in future studies

is why Ab40 and Ab42 showed different features ofaggregation in the presence of SDS. Although further studies

are needed, the preference of adsorption to the membrane

surface and unfolding on the membrane surface may be

different between Ab40 and Ab42 in a given environment;for example, Ab42 may adopt a stable a-helical structure thatprevents fibril formation on SDS micelles, as recently

demonstrated in a study using a peptide from human

complement receptor 1 (Pertinhez et al. 2002).

Several mutations inside the Ab sequence have beenidentified as being responsible for hereditary Ab amyloido-sis, including AD and cerebral hemorrhage (Levy et al.

1990; Hendriks et al. 1992; Nilsberth et al. 2001). Unlike

APP gene mutations outside the Ab sequence, the pheno-types of these mutations inside the Ab sequence are ratherdiverse but not uniform. Interestingly, the Dutch- and Arctic-

type mutations, which involve the substitution of a different

amino acid at the same position, show distinct phenotypes of

Ab amyloidosis (Rozemuller et al. 1993; Nilsberth et al.

2001). To date, many efforts have been made to elucidate the

pathological effects of the mutations inside the Ab sequenceon the induction of Ab deposition in the brain. We cannotexclude a possibility that these mutations accelerate Abdeposition through alteration of APP processing; however,

previous studies demonstrated a high aggregation rate of Abfor the Dutch- and Iowa-type mutations (Wisniewski et al.

1991; Van Nostrand et al. 2001; Fraser et al. 1992; Clements

et al. 1993), the formation of more toxic oligomeric and

fibrillar species of Ab by Dutch- and Arctic-type mutations(Dahlgren et al. 2002), and alteration in sensitivities to

peptidase degradation for these mutations (Morelli et al.

2003; Tsubuki et al. 2003).

In the present study, Ab variants, including the Dutch-,Flemish- and Arctic-type Abs, behaved differently in a givenmembrane-mimicking environment; the Dutch-type Abshowed the highest aggregation rate in the presence OG and

ZW, whereas the Arctic-type Ab had the highest aggregationrate in the presence of SDS. Consistent with previous studies

(Clements et al. 1993; Van Nostrand et al. 2001; Murakami

et al. 2002), the Flemish-type Ab showed the lowest

tendency to aggregate under any conditions. Furthermore,

we report, for the first time, that the Arctic-type Ab shows amarkedly accelerated aggregation in the presence of GM1

ganglioside. Although ZW is an ionic detergent, the total

charge of ZW is neutral because it has both positive and

negative charges at the head group. In contrast, SDS and GM1

ganglioside have only a negative charge at the head group.

Thus, negative charges on the surface of micelles or

liposomes are favorable for the Arctic-type Ab, but not forother variant forms of Ab, to initiate aggregation.Recently, it has been reported that the Arctic-type Ab

potentially forms protofibrils, in contrast to the wild-type Ab.It was suggested that the accelerated formation of protofibrils

is related to neurotoxicity of the Arctic-type Ab (Nilsberthet al. 2001). In agreement with this previous finding, we

found in the present study that the Arctic-type Ab indeedformed fibrils, of a type that can be defined as protofibrils, in

the presence of ZW. Interestingly, however, the Arctic-type

Ab formed fibrils with morphological features of amyloidfibrils in the presence of SDS or GM1 ganglioside. Notably,

the aggregation of Arctic-type Ab to form amyloid fibrils inthese environments occurred at a higher rate than that of the

wild-type Ab. Because affected subjects with the Arctic-typemutation have clinical features of early-onset AD (Nilsberth

et al. 2001), it is intriguing to speculate that the Arctic-type

Ab has a greater tendency to aggregate than the wild-type Abin an environment in which the wild-type Ab also formsfibrils. This possibility may be supported by the result of the

present study; that is, the Arctic-type Ab has a potency toaggregate even in the presence of liposomes with a lower

concentration of GM1 ganglioside. Taken together, these data

suggest that the negatively charged membrane surface is

likely to be a preferred environment for the Arctic-type Abaggregation.

The preferred environment for Dutch-type Ab aggrega-tion may be different from those of the wild- and Arctic-

type Abs. This possibility is supported by the results of thepresent study and also by evidence that clinical and

pathological features of affected individuals with the

Dutch-type mutation contrast with those of sporadic and

Arctic-type AD. In this context, we have to pay particular

attention to previous reports indicating that the Dutch-type

Ab rapidly aggregates in cultures of smooth muscle cellsprepared from human leptomeningeal blood vessels (Davis

and Van Nostrand 1996; Van Nostrand et al. 1998),

suggesting that blood vessels, but not cerebral parenchyma,

provide a particularly favorable environment for Dutch-type

Ab aggregation.In conclusion, the results of the present study suggest that

local environments are important for Ab aggregation in the



brain. Indeed, a recent transplantation study using a mouse

model for AD has highlighted the significance of local

environments for extracellular amyloid formation (Meyer-

Luehmann et al. 2003). A challenge for future studies is to

elucidate whether local environments, including lipid com-

position and/or distribution in neuronal membranes, can be

altered with risk factors for the development of AD.

Acknowledgements

We thank Dr W. E. Van Nostrand and Ms Y. Hanai for critical

reading and preparation of the manuscript. This work was supported

by a Grant-in-Aid for Scientific Research on Priority Area (C) from

the Ministry of Education, Culture, Sports, Science and Technology,

Japan and a grant from the Organization for Pharmaceutical Safety

and Research of Japan.

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environment- and mutation-dependent aggregation behavior of alzheimer amyloid β-protein

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