Báo cáo khoa học: Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide doc

Bio-chemical analysis of the Ab peptides isolated from AD Keywords Alzheimer’s disease; amyloid beta peptide; fibril formation; lysophosphatidylcholine; phospholipid–Ab interaction Corre

amyloid beta peptide

Abdullah Md Sheikh and Atsushi Nagai

Department of Laboratory Medicine, Shimane University School of Medicine, Izumo, Japan

Introduction

Alzheimer’s disease (AD) is a neurodegenerative

disorder that is manifested clinically as progressive

dementia Histopathologically, it is characterized by

degenerative changes in the neurons, together with

intra-neuronal deposition of hyperphosphorylated Tau

and extracellular accumulation of peptides comprising 39–43 amino acids, called amyloid beta (Ab) peptides, which are generated by secretase-mediated cleavage of transmembrane amyloid precursor protein [1] Bio-chemical analysis of the Ab peptides isolated from AD

Keywords

Alzheimer’s disease; amyloid beta peptide;

fibril formation; lysophosphatidylcholine;

phospholipid–Ab interaction

Correspondence

A Nagai, Department of Laboratory

Medicine, Shimane University Faculty of

Medicine, 89-1 Enya-cho, Izumo 693-8501,

Japan

Tel ⁄ Fax: +81 853 20 2312

E-mail: anagai@med.shimane-u.ac.jp

(Received 14 July 2010, revised 29

Novem-ber 2010, accepted 6 DecemNovem-ber 2010)

doi:10.1111/j.1742-4658.2010.07984.x

Phospholipids are known to influence fibril formation of amyloid beta (Ab) peptide Here, we show that lysophosphatidylcholine (LPC), a polar phos-pholipid, enhances Ab(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate Conversely, LPC did not have an enhancing effect on Ab(1-40) fibril formation, and appeared to be inhibi-tory Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Ab(1-40) and Ab(1-42) in opposite ways Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Ab(1-42), but not of Ab(1-40) Tris-tricine gradient SDS⁄ PAGE revealed that LPC increased the formation of higher-molecular-weight species of Ab(1-42), including trimers and tetramers LPC had no such effect on Ab(1-40), and thus may specifi-cally influence the oligomerization and nucleation processes of Ab(1-42) in

a manner dependent on its native structure Dot-blot assays confirmed that LPC induced Ab(1-42) oligomer formation at an early time point Thus our results indicate that LPC specifically enhances the formation of Ab(1-42) fibrils, the main component of senile plaques in Alzheimer’s disease patients, and may be involved in Alzheimer’s disease pathology

Structured digital abstract

(uni-protkb: P05067 ) bind ( MI:0407 ) by fluorescence technology ( MI:0051 )

(uni-protkb: P05067 ) and A beta (1-40) (uniprotkb: P05067 ) bind ( MI:0407 ) by comigration in sds

Abbreviations

Ab, amyloid beta peptide; AD, Alzheimer’s disease; LPC, lysophosphatidylcholine; ThT, thioflavin T.

brain indicated that an Ab peptide consisting of 42

res-idues, Ab(1–42), is the principal species associated with

senile plaques in AD, while an Ab peptide consisting

of 40 residues, Ab(1–40), is more abundant in

cerebro-vascular amyloid deposits and cerebrospinal fluid [2]

Genetic analysis of familial AD patients, as well as

animal studies, showed that genetic alterations found

in familial AD, such as amyloid precursor protein or

pre-senilin mutations, increase the production and

deposition of Ab in the brain [1,3–7] This in turn

initi-ates a cascade of events leading to AD-related

neuro-toxicity and the appearance of AD plaques [8]

Moreover, the Ab is deposited mainly in fibrillary

form, and the fibrils have been shown to be intimately

associated with dystrophic neurons and activated glial

cells [8] Therefore, the Ab fibril formation process is

considered to have a central role in AD pathology

Lysophosphatidylcholine (LPC) is a bioactive polar

phospholipid that is produced by phospholipase

A2-mediated hydrolysis of phosphatidylcholine [9]

Studies in our laboratory and others have established

the neuroinflammatory and neurodegenerative

proper-ties of LPC [10–12] Neuroinflammatory processes

have an essential role in AD pathology [13]

Phospho-lipase A2 activity was reported to be specifically

increased in astrocytes in the cortical area of AD

patients where neurodegeneration was evident [14],

indicating that lipid metabolism, including that of

LPC, may be changed in the brain of AD patients

Indeed, the concentration of LPC is increased in the

white matter of aged human brains exhibiting senile

atrophy of the Alzheimer type; in addition, the LPC to

phosphatidylcholine ratio is decreased in the

cerebrospinal fluid of AD patients [15,16] Moreover,

LPC increases Ab-induced neuronal apoptosis [17]

Taken together, these reports suggest that LPC may

have an important role in AD pathology

There have been many studies on the interaction of

Ab and phospholipids in relation to AD pathology,

including factors such as membrane disruption and

neurotoxicity, conformational changes and Ab fibril

formation process [18–21] The Ab-interacting

phos-pholipids are mainly acidic, including phosphatidic

acid, phosphatidylserine, phosphatidylinositol,

cardioli-pin and phosphatidylethanolamine [20] Recently, it

has been shown that neutral zwitterionic phospholipid,

such as phosphatidylcholine, can also interact with Ab

peptide and affect its conformation and fibril

forma-tion [19] However, the effects of LPC on Ab fibril

for-mation have not been investigated in detail In this

study, we used an in vitro system to examine the

mech-anisms by which LPC influences fibril formation of

Ab(1-40) and Ab(1-42)

Results

Effects of LPC on Ab(1-42) fibril formation

To investigate the effects of LPC on Ab(1-42) fibril formation, we incubated increasing concentrations (starting from 250 nm) of Ab(1-42) in a fibril-forming buffer with or without 20 lm LPC for 8 h No fibrils were detectable at concentrations of Ab(1-42) of up to

10 lm, as revealed by thioflavin T (ThT) fluorescence assay However, addition of LPC (20 lm) induced the fibril formation process at 5 lm Ab(1-42) (Fig 1A) Subsequent fibril formation increased linearly with respect to Ab(1-42) concentration (r2> 0.94) in the presence or absence of LPC, although the slopes were significantly different (1.1 without LPC versus 1.89 with LPC, P < 0.001) To investigate the dose-depen-dent effect, we added increasing concentrations of LPC

to 50 lm Ab(1-42), and fibril formation was allowed to proceed for 30 min at 37C We observed a linear increase of ThT fluorescence with increasing LPC con-centration (r2= 0.98) (Fig 1B) The effects of LPC

on fibril formation became apparent at 5 lm (mean ThT fluorescence 5.7; arbitrary units), and reached a plateau at 120 lm LPC (mean ThT fluorescence 37.9) However, transmission electron microscopy showed that LPC did not change the overall morphology of Ab(1-42) fibrils (Fig 1G)

Previous reports have shown that peptide concen-tration affects the fibril formation process [22] Our preliminary experiments also showed that the lag phase was decreased at a high Ab(1-42) concentra-tion, causing difficulties in the analysis of fibril formation kinetics (data not shown) Therefore, in order to investigate the effects of LPC on Ab(1-42) fibril formation kinetics, we choose a peptide concen-tration of 12.5 lm At this concenconcen-tration, Ab(1-42) fibril formation showed typical sigmoid kinetics, with

a lag phase of between 4 and 8 h (Fig 1C), and reached a plateau at 16 h When 20 lm LPC was added, the lag phase became as short as 15 min, and the ThT fluorescence reached a plateau within 2 h (Fig 1D)

Next we investigated the effects of the vesicular form

of LPC on the Ab(1-42) fibril formation process For this purpose, an increasing concentration of LPC lipo-somes was added to 50 lm Ab(1-42), and fibril forma-tion was allowed to proceed for 30 min As in the case

of non-vesicular LPC, a linear increase of fibril forma-tion was observed with increasing LPC liposome concentration (r2< 0.93) (Fig 1E) However, the rate

of fibril formation was higher (slope 2.2 for LPC liposome versus slope 0.4 for non-vesicular LPC,

P< 0.001) LPC liposomes affected the fibril

forma-tion process from 5 lm LPC (mean ThT fluorescence

4.8), and the effect reached a plateau at 40 lm LPC

(mean ThT fluorescence 84.1) Like non-vesicular LPC,

LPC liposomes greatly decreased the lag phage to less

than 30 min, and the fibril formation process reached

a plateau within 2 h (Fig 1F)

Effects of LPC on Ab(1-40) fibril formation

Next, we investigated the fibrillogenic properties of

Ab(1-40) Significant fibril formation was observed at

20 lm Ab(1-40) after 8 h incubation, suggesting that

the critical micelle concentrations (CMC) was between

10 and 20 lm (Fig 2A) under these conditions

How-ever, LPC (20 lm) increased the CMC to between 20

and 50 lm (Fig 2A) Similarly, a kinetic study showed

that the lag phase of Ab(1-40) fibril formation at

50 lm was between 4 and 6 h (Fig 2B) LPC (20 lm)

increased the lag period to between 6 to 8 h, with a corresponding delay in reaching the plateau (Fig 2B)

Effects of LPC on the change of intrinsic tyrosine fluorescence during Ab fibril assembly

Next we investigated the effect of LPC on tyrosine (Tyr) fluorescence of Ab during fibril assembly Upon excitation at 277 nm, the emission maximum of Tyr fluorescence is approximately 304 nm [23] Fibril formation of Ab peptides in the absence or presence of LPC did not produce any shift of the Tyr fluorescence maximum Incubation of Ab(1-40) or Ab(1-42) in fibril-forming buffer caused a time-dependent decrease

of Tyr fluorescence intensity (Fig 3A–D), although the change was extremely small in the case of Ab(1-42) Incubation of Ab(1-40) for 2 h in the presence of LPC increased the Tyr fluorescence compared with 0 h incu-bated Ab(1-40) alone or Ab(1-40) plus LPC (Fig 3A);

120

A

40

80

ThT fluorescence ThT fluorescence

0

Aββ (1-42) µ M :

8

12

C

Aβ (1-42) 12.5 µ M

0

4

Time (h):

40

60

80

100

Aβ (1-42) 50 µ M

0

20

LPC liposome (µ M )

a

40

B

20 30

Aβ (1-42) 50 µ M

0 10

D

E

G

F

LPC (µ M ):

8 12 16

0 4

Time (h):

Aβ (1-42) 12.5 µ M

LPC lipo 20 µ M

10 20 30

0

Aβ (1-42) 12.5 µ M

LPC lipo 20 µ M

Time (h):

b

Fig 1 Effect of LPC on Ab (1-42) fibril formation (A) Various concentrations of Ab(1-42) peptide were incubated in fibril-forming buffer with no LPC (open circles) or with 20 l M LPC (closed circles) at 37 C for

8 h (B) Ab(1-42) (50 l M ) was incubated with increasing concentrations of LPC for 30 min

at 37 C (C,D) Ab(1-42) (12.5 l M ) was allowed to form fibrils in the absence (C) or presence (D) of 20 l M LPC for the indicated times (E) Dose-dependent effect of LPC liposomes on Ab(1-42) fibril formation LPC liposomes were prepared as described in Experimental procedures Various concentra-tions of LPC liposomes were added to

50 l M Ab(1-42) peptide, and fibril formation was allowed to proceed for 30 min, with monitoring by ThT fluorescence measure-ment (F) Fibril formation kinetics of Ab(1-42) in the presence of LPC liposomes Ab(1-42) (12.5 l M ) was allowed to form fibrils in the presence of 20 l M LPC liposomes for the indicated times For (A–F), fibril formation was monitored by the ThT fluorescence assay as described in Experimental procedures, and expressed in arbitrary ThT fluorescence units (G) Ab(1-42) (50 l M ) was allowed to form fibrils for 24 h in the absence (a) or presence (b)

of 20 l M LPC, and fibril morphology was investigated by electron microscopy.

thereafter a time-dependent decrease of the

fluores-cence was observed (Fig 3B) Addition of LPC

decreased the Tyr fluorescence of Ab(1-42) after 2 h

of incubation compared to that of Ab(1-42) alone

(Fig 3C,D), but thereafter the fluorescence remained

unchanged up to 24 h (Fig 3D)

Surface hydrophobicity of Ab aggregates

The fluorescent dye 8-anilino-1-naphthalene sulfonic

acid (ANS), which is widely used in protein-folding

studies, was used to investigate the structural features

of Ab aggregates When ANS binds to solvent-exposed

hydrophobic regions on protein surfaces, an increase

in the fluorescence intensity and a blue shift of the

emission maximum are observed [24] We found that

incubation of Ab peptides in fibril-forming buffer

caused an increase of fluorescence with a blue shift

(Fig 3E–H) When Ab(1-42) was incubated in the

presence of LPC, ANS fluorescence was significantly

increased compared to Ab(1-42) alone (Fig 3G,H),

suggesting an increase of hydrophobicity In the case

of Ab(1-40), LPC did not cause an increase of ANS fluorescence (Fig 3E,F)

SDS⁄ PAGE analysis of Ab peptide during fibril assembly

Next we investigated the Ab species that were gener-ated during fibril formation, and the effects of LPC on them Ab(1-40) or Ab(1-42) (50 lm) were allowed to form fibrils in the presence of 0 or 20 lm LPC for 0,

1, 4, 8 and 24 h, and the products were separated by SDS⁄ PAGE using a 10–20% gradient tris-tricine gel system Both Ab(1-40) and Ab(1-42) produced dimeric and tetrameric species in fibril-forming buffer, although they mostly remained in monomeric form (Fig 4A,B) When Ab(1-42) was further incubated in fibril-forming buffer, the amount of monomeric species decreased time-dependently, and an initial increase in tri- and tetrameric species was observed, followed by a time-dependent reduction (Fig 4B) Addition of LPC affected the mono-, tri- and tetrameric species of Ab(1-42), decreasing the amount of monomer, and increas-ing the amounts of tri- and tetrameric species, com-pared to the peptide alone at the same time point (Fig 4B) However, no significant effect of either LPC

or incubation time was apparent with regard to dimeric species of Ab(1-40) or Ab(1-42) (Fig 4A, B) Conversely, LPC did not have any significant effect on the concentration of Ab(1-40) monomer (Fig 4A)

Dot-blot immunoassay of Ab oligomer Next we examined the oligomeric species formed during fibril formation of Ab(1-42) peptide, using an oligomer-specific antibody [25] Our dot-blot immuno-assay showed that, in the case of Ab(1-42) alone, oligomer was detectable after 8 h incubation in fibril-forming buffer (Fig 4C) However, when LPC was added, oligomer formation was enhanced and became detectable as early as 1 h after the start of incubation (Fig 4C)

Effects of LPC on the rate of Ab fibril elongation

To further examine the effect of LPC on Ab fibril for-mation, we investigated whether LPC influenced the elongation phase To eliminate the nucleation process (lag phase) and focus on Ab elongation, we monitored fibril formation for Ab(1-40) and Ab(1-42) in the pres-ence of pre-formed sonicated fibrils In a preliminary experiment, pre-formed sonicated fibrils were incu-bated in fibril-forming buffer for up to 48 h, and no increase in ThT fluorescence was observed during that

12

15

18

A

B

0

3

6

9

*

*

0.25 0.5 1 5 10 20 50

A β (1-40) µ M :

25

30

35

5

10

15

20

A β (1-40) 50 µ M

Time (h): 0 0 5 10 15 20 25 30

Fig 2 Effect of LPC on Ab(1-40) fibril formation (A) Various

concentrations of Ab(1-40) peptide were incubated in fibril-forming

buffer with 20 l M LPC (closed squares), or without LPC (open

squares) at 37 C for 8 h (B) Fibril formation kinetics of Ab(1-40).

Ab(1-40) (50 l M ) was incubated in fibril-forming buffer at 37 C for

the indicated times in the absence (open circles) or in the presence

of 20 l M LPC (closed circles) For (A) and (B), the fibril formation

was monitored by ThT fluorescence assay as described in

Experi-mental procedures, and expressed in arbitrary ThT fluorescence

units *P < 0.001 versus Ab(1-40) alone at the same time point.

time (data not shown) Addition of pre-formed

soni-cated fibrils effectively eliminated the lag phase, and a

linear increase in fibril formation was observed

(r2> 0.8) (Fig 5) In the case of Ab(1-42), addition of

LPC significantly increased the rate of elongation

(slope 0.89 versus 0.2, P < 0.001) (Fig 5B) However,

LPC had no effect on the rate of elongation of

Ab(1-40) (slope 0.8 versus 0.75, P = 0.74) (Fig 5A)

Discussion

Our key observations are that LPC increased

fibrillo-genesis of Ab(1-42) by decreasing both the lag phase

and the critical peptide concentration required for fibril formation This fibril formation-enhancing char-acteristic of LPC is specific for Ab(1-42) In the case

of Ab(1-40), LPC (20 lm) actually increased the lag period and critical peptide concentration for fibril formation, suggesting that it may have an inhibitory effect on Ab(1-40) fibrillogenesis This differential effect of LPC on Ab(1-40) and Ab(1-42) fibrillogenesis was also supported by the findings that the phospho-lipid differentially regulates the micro-environment during fibril formation of these two peptides However,

as we did not investigate Ab(1-40) fibrillogenesis in the presence of higher concentrations of non-vesicular and vesicular LPC, a fibril formation-enhancing effect at higher concentration cannot be ruled out

Lipids are known to influence several fibrillogenic processes For example, negatively charged phospho-lipids, such as lysophosphatidic acid and lysophosphat-idylglycerol, increase fibrillogenesis of b2-microglobulin [26] Ab has affinity for negatively charged lipids, such

as phosphatidylinositol and ganglioside, and peptides bound to negatively charged lipid membranes can self-associate into b-sheets [20,27,28] However, a recent study showed that zwitterionic phospholipid vesicles, such as phosphatidylcholine liposomes, can also inter-act with Ab(1-40), possibly through the phosphocho-line head group, and a-helix or b-sheet formation is promoted depending on the salt concentration, lipid:peptide ratio and temperature [19] Although we used LPC in both vesicular and non-vesicular form, non-vesicular LPC showed a dose-dependent effect on

Ab fibril formation, starting at a low LPC:peptide

35

Aββ 1-40

30

35

Aβ 1-40 25

20 25 15

2 h

15 5

C

10

D

20

25

Aβ 1-42

10

5

Tyrosine fluorescence Tyrosine fluorescence

Tyrosine fluorescence Tyrosine fluorescence

2 h

10

280 300 320 340

0

h

h

0

E

Aβ 1-40

Aβ 1-40

F

40

60

Aβ 1-42 Aβ 1-42

Aβ – 0 h

Aβ + LPC – 0 h

Aβ – 2 h, 24 h

Aβ + LPC – 2 h, 24 h

20

2 h

Aβ 1-42

0

40 60

20

0

Wavelength (nm)

Wavelength (nm)

Wavelength (nm)

280 300 320 340

Wavelength (nm)

Aβ 1-42 + LPC

Fig 3 Effect of LPC on Ab peptide fibril-forming micro-environ-ment Ab peptide (50 l M ) was incubated in fibril-forming buffer in the absence or presence of 20 l M LPC for the indicated times Ab fibril samples (20 lL) were added to glycine buffer (pH 8.5, 50 m M final concentration) to make a total volume of 200 lL Tyrosine (Tyr) fluorescence was analyzed using a spectrofluorimeter with excitation at 277 nm and emission in the range of 280–350 nm as described in Experimental procedures (A,C) Normalized Tyr fluores-cence spectra of Ab(1-40) (A) or Ab(1-42) (C) alone or in the pres-ence of LPC, incubated for 0 or 2 h (B,D) Time-dependent changes

in the Tyr fluorescence maxima for Ab(1-40) (B) and Ab(1-42) (D) (E,F) ANS emission spectra (E) and time-dependent change of the fluorescence maximum (F), of Ab(1-40) are shown (G,H) ANS emission spectra (G) and time-dependent fluorescence maximum (H) of Ab(1-42) are shown For ANS fluorescence analysis, 20 lL of

Ab fibril samples and ANS (10 l M final concentration) were added

to glycine buffer (pH 8.5, 50 m M final concentration) to make a total volume of 200 lL, and ANS fluorescence was analyzed using a spectrofluorimeter with excitation at 360 nm and emission in the range of 400–600 nm as described in Experimental procedures.

*P < 0.05 versus Ab peptide alone at the same time point.

ratio (1 : 2) and low LPC concentration (as low as

5 lm) However, vesicular LPC showed greater ability

to enhance fibril formation than non-vesicular LPC,

suggesting that the polar phosphate head group of

LPC may play a critical role in interaction with

Ab(1-42) during fibril formation

The intrinsic Tyr fluorescence of Ab peptide is

not highly sensitive to the local micro-environment,

displaying modest decreases in fluorescence intensity during fibril formation [23] On the other hand, the quantum yield of ANS fluorescence is greatly increased after binding to hydrophobic patches during the fibril formation process of Ab peptide, suggesting that it is

an excellent probe to monitor the fibril formation micro-environment [29] Our intrinsic Tyr fluorescence experiments demonstrated that LPC modulates the fluorescence of Ab(1-40) and Ab(1-42) in an opposite manner, albeit modestly Also, ANS experiments showed that LPC exposes the hydrophobic patches of Ab(1-42) peptide only The change in Tyr and ANS fluorescence induced by LPC is indicative of potential LPC–peptide interaction and differential changes in the micro-environment during the 40) and Ab(1-42) fibril formation processes [29,30]

The observations that LPC almost abolished the lag phase and decreased the critical concentration of Ab(1-42) aggregation suggest that LPC may act as seeds in the fibril formation process However, as LPC had no

40

60

A

B

A ββ 1-40

20

0

40

50

A β 1-42

20 30

0 10

Time (min)

Time (min)

Fig 5 Effect of LPC on the rate of Ab(1-40) and Ab(1-42) fibril elongation (A,B) Ab(1-40) (A) or Ab(1-42) (B) (11 lg, 50 l M ) were allowed to form fibrils in the presence of 0.2 lg of pre-formed sonicated fibrils in the absence (open circles) or presence (closed circles) of 20 l M LPC, for the indicated times Fibril formation was monitored by ThT fluorescence assay, and expressed in arbitrary fluorescence units.

A ββ 1-40 198.5

kDa

A

B

C

116.2

84.8

53.9

37.4

29

19.8

6.8

A β (50 µ M ):

LPC (20 µ M ):

A β (50 µ M ):

LPC (20 µ M ):

A β 1-42

A β 1-40

198.5

kDa

116.2

84.8

53.9

37.4

29

19.8

6.8

LPC

(–)

(+)

0 h 1 h 2 h h 4 h 8 h 24 h

Fig 4 Effect of LPC on Ab peptide oligomerization (A,B) Ab

pep-tide (50 l M ) was incubated in fibril-forming buffer in the absence or

presence of 20 l M LPC for the indicated times After fibril

forma-tion, 2.5 lg of Ab(1-40) (A) or Ab(1-42) (B) were separated by

10–20% gradient Tris ⁄ tricine SDS ⁄ PAGE, and bands were stained

with Coomassie blue as described in Experimental procedures.

(C) For dot-blot immunoassay, aliquots of 10 lL of Ab fibrils were

spotted on a poly(vinylidene difluoride) membrane, and Ab(1-42)

oligomers were detected using an oligomer-specific antibody as

described in Experimental procedures.

such effect on Ab(1-40) fibril formation, it may not act

as seeds, but rather may specifically influence the

olig-omerization and nucleation processes of Ab peptides,

depending on their native structure This is consistent

with our finding that LPC exclusively increased the

tri-meric and tetratri-meric species of Ab(1-42) but not those

of Ab(1-40) Indeed, Ab(1-42) oligomer was detectable

as early as 1 h after the start of the fibril formation

process, supporting the idea that LPC affects the

oligo-merization process of Ab(1-42)

Fibril formation of both Ab(1-40) and Ab(1-42) is

nucleation-dependent, and both peptides have been

shown to have surfactant properties due to the

pres-ence of hydrophobic amino acids at the C-terminus, a

region that is critical for nucleation and fibril

forma-tion [31] The presence of two more hydrophobic

amino acids at the C-terminus causes Ab(1-42) to

oligomerize much faster than Ab(1-40) does [32], and

it was proposed that this difference in fibril formation

kinetics is due to conformational differences between

the peptides [33] These findings imply that

hydropho-bicity is a determinant of Ab oligomerization and fibril

formation processes Our ANS experiments showed

that LPC significantly increased the hydrophobicity of

Ab(1-42) only, so this increased hydrophobicity may

be critical for the enhanced nucleation and fibril

formation of Ab(1-42)

In conclusion, our findings show that LPC increases

fibrillogenesis of Ab(1-42), the major component of

Alzheimer’s disease plaque, and thus LPC may play a

role in the pathology of Alzheimer’s disease

Experimental procedures

Materials

Lysophosphatidylcholine (LPC) was purchased from Avanti

Polar Lipids (Alabaster, AL, USA) and dissolved in water

To prepare unilamellar LPC vesicles, 25 mg of lyophilized

LPC was hydrated with 10 mL of water, followed by

soni-cation at room temperature for 30 min in a bath sonicator

The Ab peptides Ab(1-40) and Ab(1-42) (Peptide Institute,

Osaka, Japan) were each dissolved in 0.1% NH3at a

con-centration of 250 lm, aliquoted immediately (in order to

avoid the need for repeated freeze-thaw cycles), and stored

at )70 C, according to the manufacturer’s instructions

Chromatographic data provided by the manufacturer

con-firmed the monomeric purity of the peptides Thioflavin T

(ThT) was obtained from Wako Pure Chemicals

(Rich-mond, VA, USA), and deionized and filter sterile water was

purchased from Sigma-Aldrich (St Louis, MO, USA)

Pre-stained protein size markers were purchased from Bio-Rad

(Hercules, CA, USA)

Ab peptide fibril formation

For fibril formation, a solution of synthetic Ab peptide in fibril formation buffer (50 mm phosphate buffer pH 7.5 and 100 mm NaCl) was prepared with or without LPC or LPC liposomes at the concentrations indicated The reac-tion mixture was incubated at 37C without agitation for the indicated times, and then the fibril formation reaction was terminated by quickly freezing the samples

Assessment of Ab fibril formation on the basis of ThT fluorescence

The presence of b-sheet structures and the kinetics of fibril formation were monitored by means of ThT fluorescence spectroscopy Samples were diluted tenfold with glycine (pH 8.5, 50 mm final concentration) and ThT (5 lm final concen-tration) ThT fluorescence was measured using a fluorescence

Tokyo, Japan), with excitation and emission wavelengths of

446 and 490 nm, respectively [34] The normalized florescence intensity of fibrillary Ab was obtained by subtracting the flo-rescence intensity of buffer alone from that of the sample

Electron microscopy

Electron microscopy was performed as described previously [35] In brief, after Ab fibril formation, 10 lL of sample was applied to a carbon-coated Formvar grid (Nisshin EM, Tokyo, Japan) and incubated for 1 min The droplet was then displaced with an equal volume of 0.5% v⁄ v glutaral-dehyde solution and incubated for an additional 1 min The grid was washed with a few drops of water and dried Finally, the peptide was stained with 10 lL of 2% w⁄ v ura-nyl acetate solution for 2 min This solution was soaked off, and the grid was air-dried and examined under an elec-tron microscope (EM-002B, Topcon, Tokyo, Japan)

Tyrosine fluorescence spectroscopy

Tyrosine fluorescence of Ab peptide was measured using a Hitachi F2500 spectrofluorimeter, with excitation at 277 nm

280–350 nm, at a scan rate of 300 nmÆmin)1 Slit widths for excitation and emission were 5 nm The fluorescence emission spectrum of buffer only (background intensity) was subtracted from the emission spectrum of the samples The emission max-imum data is presented as the mean of three independent experiments, and is expressed in arbitrary fluorescence units

ANS fluorescence spectroscopy

The fluorescence intensity change of 8-anilino-1-naphtha-lene sulfonic acid (ANS) was used to evaluate the relative

exposure levels of hydrophobic surfaces of Ab aggregates

[30] Fluorescence intensity measurements were obtained

using a Hitachi F2500 spectrofluorimeter, with excitation

at 360 nm The emission spectra were read from 380 to

for excitation and emission were 5 nm The data of

emis-sion maximum is presented as the mean of three

fluorescence units

Gel electrophoresis and staining

tris-tricine gel system (Invitrogen, Carlsbad, CA, USA) After

fibril formation, 2.5 lg of Ab peptide was mixed with 2·

SDS non-reducing sample buffer (Invitrogen) making a

total volume of 20 lL, incubated at 85C for 2 min, and

separated by electrophoresis The gel was washed briefly

with water, fixed in fixation buffer (40% methanol and

10% acetic acid) for 30 min, and stained with Coomassie

Blue G250 (Biosafe Coomassie; Bio-Rad) for 1 h The

stained gel was washed with water overnight and scanned

using a gel scanner (Bio-Rad)

Dot-blot immunoassay

After fibril formation, an aliquot (10 lL) of Ab(1-42)

pep-tide was applied to poly(vinylidene difluoride) membrane

using a manifold Then the membrane was immunoblotted

with an oligomer-specific antibody (A11, Invitrogen) This

oligomer-specific antibody reacts specifically to a variety of

soluble oligomeric protein⁄ peptide aggregates regardless of

their amino acid sequence, and does not react with either

monomer species or insoluble fibrils; it reacts only with Ab

oligomer species of at least octamer [25] Immunoreactive

oligomer was detected using horseradish

peroxidase-conju-gated anti-rabbit IgG and an enhanced chemiluminescence

kit (Amersham, Little Chalfont, UK), according to the

manufacturer’s instructions

Elongation assay

The elongation assay was performed as described

previ-ously [34] In brief, 50 lm Ab(1-40) or Ab(1-42) peptide

monomer in fibril formation buffer was incubated at 37C

for 48 h to prepare Ab fibrils Then the whole reaction

mixture was sonicated for 10 min

To eliminate the lag phase, and for analysis of the

elon-gation phase, 11 lg of synthetic Ab monomer was

incu-bated in fibril formation buffer in the presence of 0.2 lg of

sonicated Ab fibrils in a total volume of 50 lL at 37C for

the indicated times Then fibril formation was assayed by

measuring ThT fluorescence

Statistical analysis

The results are expressed as mean ± SEM of at least three independent experiments Statistical analysis for comparing mean values was performed using one-way ANOVA, followed by Scheffe´’s post hoc test Linear regression and tests of whether the slopes are significantly different were performed using graphpad prism software (GraphPad Software, La Jolla, CA, USA) The fibril formation kinetics were analyzed using sigmaplot software (Systat Software Inc, San Jose, CA, USA) P values < 0.05 indicate statisti-cal significance

References

1 Selkoe DJ (1996) Amyloid b-protein and the genetics of Alzheimer’s disease J Biol Chem 271, 18295–18298

2 Barrow CJ & Zagorski MG (1991) Solution structures

of beta peptide and its constituent fragments: relation

to amyloid deposition Science 253, 179–182

3 Rumble B, Retallack R, Hilbich C, Simms G, Multhaup G, Martins R, Hockey A, Montgomery P, Beyreuther K & Masters CL (1989) Amyloid A4 protein and its precursor in Down’s syndrome and Alzheimer’s disease N Engl J Med 320, 1446–1452

4 Citron M, Oltersdorf T, Haass C, McConlogue L, Hung AY, Seubert P, Vigo-Pelfrey C, Lieberburg I & Selkoe DJ (1992) Mutation of the b-amyloid precursor protein in familial Alzheimer’s disease increases b-protein production Nature 360, 672–674

5 Cai XD, Golde TE & Younkin SG (1993) Release of excess amyloid beta protein from a mutant amyloid beta protein precursor Science 259, 514–516

6 Suzuki N, Cheung TT, Cai XD, Odaka A, Otvos L Jr, Eckman C, Golde TE & Younkin SG (1994) An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants Science 264, 1336–1340

7 Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Hardy J, Hutton M, Kukull W

et al.(1996) Secreted amyloid b-protein similar to that

in the senile plaques of Alzheimer’s disease is increased

in vivoby the presenilin 1 and 2 and APP mutations linked to familial Alzheimer’s disease Nat Med 2, 864–870

8 Hardy J & Selkoe DJ (2002) The amyloid hypothesis

of Alzheimer’s disease: progress and problems on the road to therapeutics Science 297, 353–356

9 Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL

& Steinberg D (1984) Modification of low density lipo-protein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospho-lipids Proc Natl Acad Sci USA 81, 3883–3887

10 Ousman SS & David S (2000) Lysophosphatidylcholine

induces rapid recruitment and activation of macrophages

in the adult mouse spinal cord Glia 30, 92–104

11 Ousman SS & David S (2001) MIP-1a, MCP-1,

GM-CSF, and TNFa control the immune cell response that

mediates rapid phagocytosis of myelin from the adult

mouse spinal cord J Neurosci 21, 4649–4656

12 Sheikh AM, Nagai A, Ryu JK, McLarnon JG, Kim SU

& Masuda J (2009) Lysophosphatidylcholine induces

glial cell activation: role of rho kinase Glia 57, 898–

907

13 Salminen A, Ojala J, Kauppinen A, Kaarniranta K &

Suuronen T (2009) Inflammation in Alzheimer’s disease:

amyloid-b oligomers trigger innate immunity defense

via pattern recognition receptors Prog Neurobiol 87,

181–194

14 Stephenson D, Rash K, Smalstig B, Roberts E,

John-stone E, Sharp J, Panetta J, Little S, Kramer R &

Clemens J (1999) Cytosolic phospholipase A2is induced

in reactive glia following different forms of

neurodegen-eration Glia 27, 110–128

15 Wender M, Adamczewska-Goncerzewicz Z, Szczech J &

Godlewski A (1988) Myelin lipids in aging human

brain Neurochem Pathol 8, 121–130

16 Mulder C, Wahlund L, Teerlink T, Blomberg M,

Veerhuis R, van Kamp GJ, Scheltens P & Scheffer PG

(2003) Decreased lysophosphatidylcholine⁄

phosphatidyl-choline ratio in cerebrospinal fluid in Alzheimer’s

disease J Neural Transm 110, 949–955

17 Qin ZX, Zhu HY & Hu YH (2009) Effects of

lysophosphatidylcholine on b-amyloid-induced neuronal

apoptosis Acta Pharmacol Sin 30, 388–395

18 McLaurin J & Chakrabartty A (1996) Membrane

disruption by Alzheimer b-amyloid peptides mediated

through specific binding to either phospholipids or

gangliosides Implications for neurotoxicity J Biol

Chem 271, 26482–26489

19 Yoda M, Miura T & Takeuchi H (2008)

Non-electro-static binding and self-association of amyloid b-peptide

on the surface of tightly packed phosphatidylcholine

membranes Biochem Biophy Res Commun 376, 56–59

20 Chauhan A, Ray I & Chauhan VPS (2000) Interaction

of amyloid beta-protein with anionic phospholipids:

possible involvement of Lys28 and C-terminus aliphatic

amino acids Neurochem Res 25, 423–429

21 Nagarajan S, Ramalingam K, Reddy PN, Cereghetti DM,

Malar EJP & Rajadas J (2008) Lipid-induced

confor-mational transition of the amyloid core fragment

Ab(28–35) and its A30G and A30I mutants FEBS J

275, 2415–2427

22 Burdick D, Soreghan B, Kwon M, Kosmoski J,

Knauer M, Henschen A, Yates J, Cotman C & Glabe

C (1992) Assembly and aggregation properties of

synthetic Alzheimer’s A4⁄ b amyloid peptide analogs

J Biol Chem 267, 546–554

23 Maji SK, Amsden JJ, Rothschild KJ, Condron MM & Teplow DB (2005) Conformational dynamics of amyloid b-protein assembly probed using intrinsic fluorescence Biochemistry 44, 13365–13376

24 Hawe A, Sutter M & Jiskoot W (2008) Extrinsic fluo-rescent dyes as tools for protein characterization Pharm Res 25, 1487–1499

25 Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW & Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis Science 300, 486–489

26 Ookoshi T, Hasegawa K, Ohhashi Y, Kimura H, Takahashi N, Yoshida H, Miyazaki R, Goto Y & Naiki H (2008) Lysophospholipids induce the nucleation and extension of b2-microglobulin-related amyloid fibrils

at a neutral pH Nephrol Dial Transplant 23, 3247–3255

27 McLaurin J, Franklin T, Chakrabartty A & Fraser PE (1998) Phosphatidylinositol and inositol involvement in Alzheimer amyloid-b fibril growth and arrest J Mol Biol 278, 183–194

28 Choo-Smith LP & Surewicz WK (1997) The interaction between Alzheimer amyloid b(1-40) peptide and gangli-oside GM1-containing membranes FEBS Lett 402, 95– 98

29 Kremer JJ, Pallitto MM, Sklansky DJ & Murphy RM (2000) Correlation of b-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes Biochemistry 39, 10309–10318

30 Lindgren M, Sorgjerd K & Hammarstrom P (2005) Detection and characterization of aggregates, prefibril-lar amyloidogenicoligomers, and protofibrils using fluo-rescence spectroscopy Biophys J 88, 4200–4212

31 Soreghan B, Kosmoski J & Glabe JC (1994) Surfactant properties of Alzheimer’s Ab peptides and the mecha-nism of amyloid aggregation J Biol Chem 269, 28551– 28554

32 El-Agnaf OM, Mahil DS, Patel BP & Austen BM (2000) Oligomerization and toxicity of b-amyloid-42 implicated in Alzheimer’s disease Biochem Biophys Res Commum 273, 1003–1007

33 Shen L, Ji HF & Zhang HY (2008) Why is the C-termi-nus of Ab(1-42) more unfolded than that of Ab(1-40)? Clues from hydrophobic interaction J Phys Chem B

112, 3164–3167

34 Naiki H & Nakakuki K (1996) First-order kinetic model of Alzheimer’s amyloid fibril extension in vitro Lab Invest 74, 374–383

35 Walsh DM, Lomakin A, Benedek GB & Condron MM (1997) Amyloid b-protein fibrillogenesis Detection of a protofibrillar intermediate J Biol Chem 272, 22364– 22372