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

Lysophosphatidylcholine modulates fibril formation of
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:
(Received 14 July 2010, revised 29 Novem-
ber 2010, accepted 6 December 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 A b(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
l
MINT-8077403: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uniprotkb:P05067)
bind (
MI:0407)byelectron microscopy (MI:0040)
l
MINT-8077463: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uniprotkb:P05067)
bind (
MI:0407)byfilter binding (MI:0049)
l
MINT-8077369, MINT-8077387: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uni-
protkb:
P05067) bind (MI:0407)byfluorescence technology (MI:0051)

l
MINT-8077417, MINT-8077428, MINT-8077436, MINT-8077448: A beta (1-40) (uni-
protkb:
P05067) and A beta (1-40) (uniprotkb:P05067) bind (MI:0407)bycomigration in sds
page (
MI:0808)
Abbreviations
Ab, amyloid beta peptide; AD, Alzheimer’s disease; LPC, lysophosphatidylcholine; ThT, thioflavin T.
634 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS
brain indicated that an A b 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
A
2

-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 A
2
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)ofAb(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 (r
2
> 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 37 °C. We observed a linear
increase of ThT fluorescence with increasing LPC con-
centration (r
2
= 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 concentration, 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 (r
2

< 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,
A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation
FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 635
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 fluorescenceThT fluorescence
ThT fluorescence
ThT fluorescenceThT fluorescence
0204060
0
Aβ
β
(1-42) µM:
8
12
C
A
β
(1-42) 12.5 µM
0102030
0

4
Time (h):
40
60
80
100
A
β
(1-42) 50 µM
020406080100
0
20
LPC liposome (µM)
a
40
B
20
30
A
β
(1-42) 50 µM
0 20406080100
0
10
D
E
G
F
LPC (µM):
8

12
16
0246810
0
4
Time (h):
A
β
(1-42) 12.5 µM
LPC lipo 20 µM
10
20
30
0102030
0
A
β
(1-42) 12.5 µM
LPC lipo 20 µM
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.
LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai
636 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS
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. A b(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 A b(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) µM:
25
30
35

ThT fluorescence ThT fluorescence
5
10
15
20
A
β
(1-40) 50 µM
Time (h):
0 5 10 15 20 25 30
0
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.
A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation
FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 637
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
(r

2
> 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 b
2
-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
A
B
35
Aβ
β
1-40
30
35
A
β
1-40
25
20
25
15
2 h
15

5
C
10
D
20
25
A
β
1-42
30
A
β
1-42
10
15
20
5
Tyrosine fluorescence
Tyrosine fluorescence
Tyrosine fluorescence
Tyrosine fluorescence
2 h
10
280 300 320 340
0
h
024824
h
024824
0

E
A
β
1-40
30
30
A
β
1-40
F
GH
20
20
10
10
0
24 h
0
40
60
A
β
1-42
A
β
1-42
A
β
– 0 h
A

β
+ LPC – 0 h
A
β
– 2 h, 24 h
A
β
+ LPC – 2 h, 24 h
20
ANS fluorescence ANS fluorescence
ANS fluorescence ANS fluorescence
2 h
A
β
1-42
400 500 600
0
40
60
20
0
024824
h
024824
h
Wavelength (nm)
400 500 600
Wavelength (nm)
Wavelength (nm)
280 300 320 340

Wavelength (nm)
A
β
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 lLof
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.

LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai
638 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS
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 Ab(1-40) 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
Aβ
β
1-40
20
0
40
50
A
β
1-42
20
30
ThT fluorescence ThT fluorescence
0 1020304050
0
10
Time (min)
0 1020304050
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.
Aβ
β
1-40
198.5
kDa
A
B
C
116.2
84.8
53.9
37.4
29
19.8
6.8
A
β
(50 µM):
LPC (20 µ
M):
A
β
(50 µM):
LPC (20 µ
M):
0 h
1 h
A

β
1-42
A
β
1-40
198.5
kDa
116.2
84.8
53.9
37.4
29
19.8
6.8
0 h
1 h
LPC
(–)
(+)
++ +++ +++++
++–– +++–––
4 h 8 h
24 h
++++++++++
++–– +++–––
4
h8h
24 h
0 h
1 h 2 hh4 h 8h24 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 lgofAb(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 lLofAb 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.
A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation
FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 639
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 tetrameric 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 A b(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% NH
3
at 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 37 °C 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
spectrophotometer (F2500 spectrofluorimeter, Hitachi,
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 lLof2%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.
Fluorescence emission was scanned in the range of
280–350 nm, at a scan rate of 300 nmÆmin
)1
.Slitwidthsfor
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
LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai
640 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS
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
600 nm, at a scan rate of 300 nmÆmin

)1
. Slit widths
for excitation and emission were 5 nm. The data of emis-
sion maximum is presented as the mean of three indepen-
dent experiments, and is expressed in arbitrary
fluorescence units.
Gel electrophoresis and staining
SDS ⁄ PAGE was performed using a 10–20% gradient tris-
tricine gel system (Invitrogen, Carlsbad, CA, USA). After
fibril formation, 2.5 lgofAb peptide was mixed with 2·
SDS non-reducing sample buffer (Invitrogen) making a
total volume of 20 lL, incubated at 85 °C 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 37 °C
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 l gof
sonicated Ab fibrils in a total volume of 50 lLat37°C 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 vivo by 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, W itztum 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.
A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation
FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 641
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 A
2
is 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.
LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai
642 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS