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The Journal of Neuroscience, September 15, 2001, 21(18):7226-7235
A Novel Action of Alzheimer's Amyloid 
-Protein (A):
Oligomeric A Promotes Lipid Release
Makoto
Michikawa,
Jian-Sheng
Gong,
Qi-Wen
Fan,
Naoya
Sawamura, and
Katsuhiko
Yanagisawa
Department of Dementia Research, National Institute for Longevity
Sciences, Obu, Aichi 474-8522, Japan
 |
ABSTRACT |
Interactions between amyloid 
-protein (A) and lipids have
been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these
interactions has not been fully understood. We examined the effect of
A on lipid metabolism in cultured neurons and astrocytes and found
that oligomeric A, but not monomeric or fibrillar A, promoted
lipid release from both types of cells in a dose- and time-dependent
manner. The main components of lipids released after the addition of
A were cholesterol, phospholipids, and monosialoganglioside (GM1).
Density-gradient and electron microscopic analyses of the conditioned
media demonstrated that these A and lipids formed particles and were
recovered from the fractions at densities of ~1.08-1.18 g/ml, which
were similar to those of high-density lipoprotein (HDL) generated by
apolipoproteins. The lipid release mediated by A was abolished by
concomitant treatment with Congo red and the PKC inhibitor, H7, whereas
it was not inhibited with
N-acetyl-L-cysteine. These A-lipid
particles were not internalized into neurons, whereas HDL-like
particles produced by apolipoprotein E were internalized. Our findings
indicate that oligomeric A promotes lipid release from neuronal
membrane, which may lead to the disruption of neuronal lipid
homeostasis and the loss of neuronal function.
Key words:
amyloid -protein; cholesterol release; phospholipid; high-density lipoprotein; cultured neurons; Alzheimer's disease
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INTRODUCTION |
The mechanism underlying the
initiation of the clinicopathological process in Alzheimer's disease
(AD) is assumed to be the age-related aggregation of amyloid
-protein (A) (Selkoe, 1994
; Esiri et al., 1997). This assumption
has been supported in part by the findings that highly aggregated A
fibrils, but not A monomers, induce neurodegeneration (Mattson et
al., 1993; Pike et al., 1993; Lorenzo and Yankner, 1994). This
assumption has also been challenged by recent evidence indicating that
A oligomers also play an important role in AD pathogenesis (Walsh et
al., 1997; Hartley et al., 1999) and that neurodegeneration is induced in mouse brain without amyloid plaque formation (Chui et al., 1999;
Hsia et al., 1999).
The role of lipid metabolism in the pathogenesis of AD has been
highlighted by the finding that apolipoprotein E (apoE) epsilon 4 is a
strong risk factor for the development of AD (Corder et al., 1993;
Saunders et al., 1993; Strittmatter et al., 1993). The findings that
monosialoganglioside (GM1)-bound A is the initially deposited A
species in AD brain (Yanagisawa et al., 1995), and that amyloid fibril
formation is induced by A binding to membrane vesicles containing
ganglioside (Choo-Smith et al., 1997) and phospholipids (Terzi et al.,
1995), suggest that interactions of A with lipids play a crucial
role in the pathogenesis of AD. In addition, it has been reported that
A modulates cholesterol metabolism in the plasma membrane (Liu et
al., 1998) and membrane functions by altering the physicochemical
properties of membrane constituents including lipids (Muller et al.,
1995; Mason et al., 1996; McLaurin and Chakrabartty, 1996; Avdulov et
al., 1997). Previous studies have shown that the properties of this
interaction of A with lipids are dependent on the aggregation state
of A (Avdulov et al., 1997; Mason et al., 1999). These lines of
evidence indicate that A interacts with neuronal membranes,
disrupting its lipid environment. The interactions of A with lipids
have also been demonstrated in physiological conditions; A has been shown to be associated with high-density lipoproteins (HDLs) in the
cerebrospinal fluid (Koudinov et al., 1996; Fagan et al., 2000) and
human plasma (Koudinov et al., 1994; Biere et al., 1996; Matsubara et
al., 1999). However, the mechanism underlying the formation of these
A-lipid complexes is poorly understood, and their significance in
cholesterol metabolism in the CNS or in the pathogenesis of AD remains
to be elucidated.
We have recently shown that apoE modulates cellular cholesterol
metabolism in an isoform-specific manner (Michikawa et al., 2000), and
tau phosphorylation is enhanced in cholesterol-deficient neurons (Fan
et al., 2001) and in the brains of Niemann-Pick disease type C mice
(Sawamura et al., 2001), suggesting that cholesterol is the key
molecule in the pathogenesis of tauopathy. These lines of evidence have
led us to the question of whether A affects cholesterol homeostasis
in neurons, leading to the abnormal phosphorylation of tau. In this
study, we conducted experiments to examine the effect of A on
cholesterol metabolism in cultured neurons.
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MATERIALS AND METHODS |
Cell culture. All experiments were performed in
compliance with existing laws and institutional guidelines. Neuron-rich
cultures were prepared from cerebral cortices as described previously
(Michikawa and Yanagisawa, 1998), with some modifications. In brief,
uteri of gravid rats at embryonic days (E) 17-18 were removed under anesthesia. Cerebral cortices from fetal rat brains were dissected, freed of meninges, and diced into small pieces; the cortical fragments were incubated in 0.25% trypsin and 20 mg/ml DNase I in PBS (8.1 mM
Na2HPO4, 1.5 mM
KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH
7.4) at 37°C for 20 min. The fragments were then dissociated into
single cells by pipetting. The cells were suspended in the feeding
medium and plated onto poly-D-lysine-coated
24-well plates at a cell density of 2 × 104/cm2. The
feeding medium consisted of DMEM nutrient mixture (DMEM/F12, 50:50) and N2 supplements. More than 99% of the
cultured cells were identified as neurons by immunocytochemical
analysis using monoclonal antibody against microtubule-associated
protein 2, a neuron-specific marker, at 3 d in culture. For
astrocyte-rich cultures, mixed glial cells were prepared according to a
previously described method (Isobe et al., 1999). In brief, dissociated
cells were prepared from E17-18 rat cerebral cortices as described
above and seeded in 75 cm2 flasks at a
cell density of 1 × 107 in DMEM
containing 10% FBS. After 2 weeks of incubation in vitro, the cells in the astrocytic monolayer were removed by vigorously shaking the flasks. The medium with floating cells was removed, and the
remaining monolayer cells were trypsinized (0.1%) and reseeded onto
12-well plates.
Preparation of oligomeric A. Synthetic A1-40 (TFA
salt) was purchased from Peptide Institute (Osaka, Japan; lot numbers 490703, 491131, 500324, 500520, 500701, and 501001), Bachem (Bubendorf, Switzerland; lot numbers 518765 and 519600), and Sigma (St. Louis, MO;
lot number 38H49581). A was dissolved in dimethyl sulfoxide (DMSO)
at 13.3 M and diluted with PBS to obtain a 350 µM stock solution. A solution was then
incubated for 24 hr at 37°C (iA). After incubation, A solution
was filtered with a 0.45 µM Millipore filter
(Millipore, Bedford, MA). Peptide concentrations of both nonfiltered and filtered A were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The aggregation state of A in both solutions was monitored by measuring the
intensity of thioflavin-T fluorescence. Fresh A1-40 was dissolved
in the same manner to obtain 350 µM solution
and used for experiments immediately after determination of its peptide
concentration. For electron microscopic analysis, each sample was
centrifuged in PBS in a SW 41-Ti swing rotor (at 4°C) for 48 hr at
34,200 rpm using a Beckman TL-70. After centrifugation, electron
microscopic analysis of the lower part of each solution containing
resuspended pellet, if any, was performed.
Quantification of released and intracellular cholesterol and
phosphatidylcholine. Neurons in 6- or 12-well plates were labeled in DMEM supplemented with N2 supplements
containing 37 Bq/ml of [14C]acetate
(DuPont NEN) for 48 hr, the time period necessary to achieve isotopic
steady state in these cells, as has been described previously
(Michikawa et al., 2000). Astrocytes in 12-well plates were labeled in
DMEM with 5% FBS containing 37 Bq/ml of
[14C]acetate (DuPont NEN) for 48 hr. The
labeled neurons and astrocytes were rinsed three times with fresh DMEM
and treated with the reagents that were examined. Aliquots of
1.0 ml of each conditioned medium were filtered with a 0.45 µm
Millipore filter and then transferred into clean glass tubes containing
4.0 ml of chloroform/methanol (2:1 v/v). The organic phase was
separated from the aqueous phase, washed twice by vigorous shaking with
3 ml of chloroform/water (1:1 v/v), separated from the aqueous phase by
centrifugation, and dried under N2 gas. For
extraction of intracellular lipids, dried cells were incubated in
hexane/isopropanol (3:2 v/v) for 1 hr at room temperature. The solvent
from each plate was removed and dried under N2
gas. The organic phases were redissolved in 50 µl of chloroform, and
10 µl of each sample was spotted on activated silica gel
high-performance thin-layer chromatography (HPTLC) plates (Merck,
Darmstadt, Germany). The lipids were separated by sequential
one-dimensional chromatography using the chloroform/methanol/acetic acid/water (25:15:4:2, v/v/v/v) solvent system, followed by another run
in hexane/diethyl ether/acetic acid (80:30:1).
[14C]cholesterol,
[14C]sphingomyelin, and
[14C]phosphatidylcholine were used as
standards. The chromatography plates were exposed to radiosensitive
films, and each lipid was visualized and quantified with BAS2500 (Fuji
Film, Tokyo, Japan). The amount of lipid release was calculated as the
percentage of released lipid relative to the total lipid content
(released plus intracellular lipid).
Density gradient ultracentrifugation. After incubation with
iA at 8 µg/ml for 24 or 48 hr, the neuronal or astrocyte
culture medium was filtered with a 0.45 µm Millipore filter. A
discontinuous KBr gradient was prepared in a 14 × 89 mm
ultracentrifuge tube (Ultraclear, Beckman) from the bottom to the top
with 2 ml of sample at a density of 1.30 gm/ml, 3 ml at 1.21 gm/ml, 2 ml at 1.063 gm/ml, 2 ml at 1.19 gm/ml, and 4 ml at 1.006 gm/ml KBr
solution (all salt solutions contained 0.1% disodium-EDTA and 0.002%
sodium azide, pH 7.4). The sample in the KBr gradient was centrifuged using a Beckman TL-70 ultracentrifuge in a SW 41-Ti swing rotor (at
4°C) for 48 hr at 34,200 rpm. After density gradient centrifugation, 12 fractions (1.0 ml each) were collected from the top of the gradient
using a micropipette. The densities of the fractions were determined by
measuring the weight of each 100 µl fraction collected. The last
fraction was stirred to dissolve the pellet. The cholesterol and
phospholipid contents were determined in each fraction. Five
milliliters of chloroform/methanol (2:1 v/v) solvent were added to 1 ml
of each fraction, and the mixture was stirred vigorously. After
centrifugation, the organic phase was removed from each fraction and
dried under N2 gas. The organic residue was
dissolved in a small volume of chloroform, and the total cholesterol and phospholipid contents were determined using cholesterol and phospholipid determination kits (Kyowa Medix, Tokyo, Japan and Wako,
Osaka, Japan, respectively).
Viability assay. The release of the cytoplasmic enzyme,
lactate dehydrogenase (LDH), into culture medium was determined for the
quantification of cell death. Fifty microliters of culture medium
were transferred to a fresh 96-well flat-bottomed plate and a
colorimetric LDH-release assay was performed according to the
instructions of the manufacturer (Promega, Madison, WI); absorbances were read at 490 nm immediately thereafter. For determination of total
LDH, the neuronal cultures were incubated with 100 mM H2O2 for 10 min at room
temperature and released LDH was determined, and the percentage of
released LDH per total LDH in each culture was calculated.
Measurement of thioflavin-T binding to aggregated A.
Determination of the aggregated A state in solution was performed on the basis of a previously established method (LeVine, 1995; LeVine, 1999). A 350 µM stock solution of
A1-40 was prepared as described above and incubated for 24 hr at
37°C. The solution was then diluted to threefold with PBS. One-half
the amount of the solution was filtered with a 0.45 µM pore-sized Millipore filter. The protein concentration of nonfiltered and filtered solution was determined using
a bicinchoninic acid protein assay kit. Freshly dissolved A was
prepared as described above and diluted to threefold with PBS, and its
protein concentration was determined. The concentration of each
solution was then adjusted to 50 µM with PBS.
Steady-state fluorescence measurements for A were performed with a
multiplate reader (Fluoroskan Ascent, Labsystems Inc., Franklin, MA)
(excitation 446 nm, emission 490 nm) in 48-well plates. Each well
contained 10 µl of 50 µM A in 1000 µl/well of 5 µM thioflavin-T in 50 mM glycine-NaOH, pH 8.5.
Immunoblot analysis of A, apoE, apoJ, and GM1
ganglioside. For immunoblot analysis of A, samples of each
fraction isolated by density gradient ultracentrifugation were
dissolved in sample solubilizing buffer consisting of 50 mM Tris-HCl, pH 6.8, 10% glycerol, 4% SDS, 10%
mercaptoethanol, 8 M urea, and 0.01% bromophenol blue. For analysis of apoE and GM1 ganglioside, samples of each fraction were dissolved in equal volumes of Laemmli buffer. They were
then subjected to 4-20% gradient Tris/tricine SDS-PAGE
(Dai-ichi Pure Chemical Co., Tokyo, Japan). The separated proteins were transferred onto an immobilon or polyvinylidene difluoride membrane (Millipore) with a semidry electrophoretic transfer apparatus (Nihon
Eido, Tokyo, Japan) using a transfer buffer (0.1 M Tris, 0.192 M glycine,
and 20% methanol). The blots were blocked with 100% Block Ace
(Dainippon Pharmaceutical Co., Osaka, Japan) for 1 hr and incubated
with primary antibodies overnight at 4°C. The primary antibodies used
were monoclonal antibodies specific for the A1-40 ending site, BA27
(Suzuki et al., 1994) (at a final concentration of 3.1 µg/ml),
specific for human A1-17, 6E10 (Kim et al., 1990) (Senetek, St.
Louis, MO) (at a final concentration of 1 µg/ml), polyclonal
anti-apoE antibody, AB947 (Chemicon, Temecula, CA) (1:1,000), and
anti-apoJ antibody (Rockland, Gilbertsville, PA) (at a final
concentration of 0.2 µg/ml). The blots were washed four times with
PBS-T (PBS containing 0.05% Tween 20) within a period of 60 min and
then incubated with secondary antibodies (horseradish
peroxidase-conjugated anti-goat or anti-mouse antibodies, used at a
final concentration of 0.4 µg/ml) for 1 hr. For the detection of GM1
ganglioside, the membrane was probed with horseradish peroxidase-conjugated cholera toxin B (Sigma) (final concentration at
42 ng/ml) overnight at 4°C. In between steps, the blots were washed
four times with PBS-T for 15 min. Bound antibodies or cholera toxin was
detected using ECL (Amersham Pharmacia Biotechnology).
Immunoprecipitation of lipids in association with A
and apoE. The neurons and astrocytes were labeled with 37 Bq/ml of [14C]acetate (DuPont NEN) for
48 hr, followed by three washes in DMEM, and treated with 8 µM iA. The conditioned media, in which neurons and astrocytes were cultured in the presence of iA for 24-48 hr, were filtered. The filtered conditioned media were then incubated with 1 µl of mouse monoclonal antibody, 6E10, or goat polyclonal antibodies, AB947, and anti-apoJ antibody and mouse normal
mouse IgG, together with 100 µl of 20% protein G-Sepharose (Amersham
Pharmacia Biotechnology) slurry under rotation at 4°C overnight. The
immunoprecipitated lipids associated with protein G-Sepharose were
washed in PBS-T three times and solubilized in a solution of
chloroform/methanol (2:1 v/v), and the solution was evaporated by
N2 gas. The organic phases were redissolved in 20 µl of chloroform, and all samples were spotted on activated silica
gel HPTLC plates; the lipids were separated by sequential one-dimensional chromatography using the chloroform/methanol/acetic acid/water (25:15:4:2) solvent system, followed by another run in
hexane/diethyl ether/acetic acid (80:30:1). The chromatography plates
were exposed to radiosensitive films, and each lipid was visualized and
quantified with BAS2500. The amount of immunoprecipitated lipid was
calculated as the percentage of lipid relative to the total lipid in
the media.
Immunoelectron microscopy. The solutions containing
A-lipid particles were obtained by density gradient
ultracentrifugation, diluted with H2O to a
density of 1.006, and centrifuged again at 46,000 rpm in a SW 50.1-Ti
rotor for 24 hr at 4°C. The 400 µl portion at the bottom was used
for electron microscopic study. The solutions were placed on a
carbon-coated electron microscopy grid. Nonspecific binding was blocked
by incubation in PBS with 1% bovine serum albumin (BSA) for 10 min.
The grids were then placed on a droplet of either the A-specific
antibody, 6E10 (at final concentration of 5 µg/ml), or normal mouse
IgG for 60 min (both diluted in PBS, 0.1% BSA), and passed over seven
droplets of washing solution (PBS) for 1 min each. The grids were
placed on a droplet of anti-mouse IgG conjugated to 5 nm colloidal gold particles for 60 min (Sigma; diluted 1:20 in PBS, 0.1% BSA), passed over seven droplets of washing solution (PBS), and passed over another
seven droplets of distilled water. The specimens were then negatively
stained with 2% sodium phosphotungstate.
Statistical analysis. Statistical analysis was performed
using StatView computer software (Macintosh), and multiple pairwise comparisons among the sets of data were performed using ANOVA and the
Bonferroni t test.
| |
RESULTS |
Characterization of A used in this study
We studied the effect of A1-40 on lipid metabolism from the
point of view of lipid release from neurons and astrocytes in culture.
Because A1-42 is known to be highly amyloidgenic and assumed to
play a critical role in the pathogenesis of AD, the effect of A1-42
on cellular lipid metabolism is also an important issue that needs to
be addressed. However, the fact that synthetic A1-42 is very
difficult to handle and that oligomerized A1-40 as well as
A1-42 can be associated with lipids led us to use A1-40 in the
present study. To characterize A used in this study, A1-40
incubated for 24 hr at 37°C at 350 µM
(iA-nonfiltered), A1-40 incubated in the same way followed by
filtration through a 0.45 µM Millipore filter
(iA-filtered), and freshly dissolved A (fresh A) were
subjected to thioflavin-T assay, Western blot analysis, and electron
microscopy. Determination of A peptide concentration in each sample
was performed using a bicinchoninic acid protein assay kit (Pierce,
Rockford, IL). The concentration of A in each solution was then
adjusted to 100 µM using PBS, and the solutions were used
for the experiments. As we reported previously (Isobe et al., 2000),
the intensity curve of thioflavin-T reaction with A, which was
incubated at 350 µM at 37°C, was saturated at 24 hr of
incubation. The fluorescence intensity of iA-filtered was similar to
that of A-nonfiltered, whereas that of fresh A was as low as
background levels of PBS (Fig.
1a). Immunoblot analysis showed that tetrameric, trimeric, dimeric, and monomeric A were found in the samples of iA-nonfiltered and iA-filtered, whereas only dimeric and monomeric A were found in fresh A (Fig.
1b). Electron microscopy showed that fibrils were formed in
the samples from iA-nonfiltered, 7.9 ± 0.5 nm in diameter and
118 ± 14 nm in length for measurable fibrils (Fig.
1c), although the length was difficult to determine because
of the twisted configuration. The morphological characteristics of
these fibrils are identical to those of protofibrils with curvilinear
structures of 4-11 nm in diameter and <200 nm in length as has been
reported previously (Walsh et al., 1997). In contrast, electron
microscopy of iA-filtered and fresh A did not reveal any
structures such as protofibrils or A-derived diffusible ligands
(ADDLs) (Fig. 1d,e).
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Figure 1.
Characterization of A1-40. A1-40 was
prepared as described in Materials and Methods. a, The
aliquots of iA-nonfiltered,
iA-filtered, fresh
A, and PBS were subjected to thioflavin-T
assays as described in Materials and Methods. Three independent
experiments were performed, and similar results were obtained.
b, The equal volume of 2× sample solubilizing buffer
was added to each A solution, of which the concentration was
normalized with PBS. The samples were then subjected to 4-20%
Tris/tricine SDS-PAGE, followed by Western blot analysis.
c, Electron micrograph of each sample is shown. The
samples were centrifuged at 34,500 rpm for 48 hr using a SW 41-Ti
rotor. Electron microscopic analysis of the lower part of each solution
containing the resuspended pellet was performed. Results of negative
staining show that fibrillar structures are found in the sample of
iA-nonfiltered
(c); however, no fibril is detected in the
samples of iA-filtered
(d) or fresh A
(e). Scale bar, 50 nm.
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Oligomeric A promotes lipids release from neurons
When we analyzed lipids released from the cells, all the
conditioned media of the cultures treated with iA were examined after filtration with a 0.45 µm Millipore filter. Electron
microscopic study did not reveal any structures such as fibrils,
protofibrils, or ADDLs in the conditioned media of the neuronal
cultures incubated with iA-nonfiltered or iA-filtered for 24 hr
(data not shown). The dose-response curves for the release of
cholesterol and phosphatidylcholine from neurons at 4 hr of incubation
with iA-filtered are shown in Figure
2a. Incubation with iA
promoted the release of cholesterol (essentially all unesterified) and
phosphatidylcholine from neurons in a dose-dependent manner. Saturation
of A-mediated lipid release was observed at A concentrations
higher than 1 µM, which can be expected to be
present in local extracellular spaces in vivo. The time
course of cholesterol and phosphatidylcholine release from cultured
neurons into the medium in the presence of 8 µM A is shown in Figure 2b. The cholesterol and
phosphatidylcholine release mediated by A increased in a
time-dependent manner. We used six batches of A1-40 obtained from
Peptide Institute, one batch of A1-40 from Sigma, and two batches
of A1-40 from Bachem. The iA prepared from these batches
promoted lipid release from neurons. Because among these A peptides
tested, A peptide from Peptide Institute has the strongest ability
to promote lipid release (data not shown), we used A peptide
obtained from Peptide Institute.
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Figure 2.
Effect of iA on cholesterol and
phosphatidylcholine release from neurons in culture. Neuron-rich
cultures were labeled with [14C]acetate for 48 hr
as described in Materials and Methods. Cells were then washed three
times with DMEM and incubated with iA at various concentrations for
4 hr. Synthetic A1-40 dissolved at high concentration and incubated
at 37°C for 24 hr, followed by filtration, was used. The released
lipids in the media and the cellular lipids were extracted and analyzed
as described in Materials and Methods. The iA-mediated release of
cholesterol and phosphatidylcholine (PC)
(a) was significantly increased in a
dose-dependent manner. Each data point represents mean ± SE for
three samples. For the time course study of iA-mediated lipid
release from neurons, cultured neurons were labeled with
[14C]acetate for 48 hr and then washed three times
with DMEM and incubated with iA at a final concentration of 8 µM. The iA-mediated release of cholesterol and PC
(b) increased with incubation time. Each data
point represents mean ± SE for three samples. Effect of Congo red
on iA-mediated lipid release and effect of fresh A on lipid
release from neurons were investigated using labeled neurons with
[14C]acetate for 48 hr. c, Cells
were washed three times with DMEM and then incubated with iA (10 µM), iA (10 µM) with Congo red
(CR) (10 µM), CR alone (10 µM), freshly dissolved A
(frA) (10 µM), and frA plus
CR (10 µM) in serum-free N2 medium for 24 hr.
The release of cholesterol and PC in iA-treated culture medium was
abolished by concurrent treatment with Congo red. Freshly dissolved
A1-40 did not promote lipid release from these cells. Each
data point represents mean ± SE for four samples.
*p < 0.005 versus CONT, iA + CR, frA, and
frA + CR. CONT, Control cultures;
iA, incubated A1-40; CR, Congo
red; frA, fresh A1-40. d, Cells
were washed three times with DMEM and then incubated with none
(CONT), iA (5 µM), iA (5 µM) + NAC (1 mM), NAC (1 mM),
H2O2 (2 mM), and
H2O2 (2 mM) + NAC (1 mM). *p < 0.001 versus CONT and NAC;
**p < 0.0001 versus H2O2 + NAC; #p < 0.06 versus CONT and NAC.
NAC, N-acetyl-L-cysteine.
e, The cultures were washed three times with DMEM and
then incubated with none (CONT), iA (5 µM), and iA (5 µM) + H7 (30 nM) for 16 hr at 37°C, and the lipids in the medium and
the cells were quantified as described in Materials and Methods.
*p < 0.004 versus CONT and iA + H7.
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Because Congo red is known to inhibit oligomerization of A by
stabilizing A monomer (Podlisny et al., 1995, 1998), we next examined whether A-mediated lipid release is inhibited after concurrent treatment with Congo red. A was incubated at high concentration for 24 hr at 37°C, filtered, and added into neuronal cultures. As shown in Figure 2c, iA promoted lipid
release from neurons, whereas A incubated with 10 µM Congo red for 24 hr at 37°C lost its
function as a lipid acceptor. In addition, freshly dissolved A did
not induce lipid release from neurons (Fig. 2c), suggesting
that the aggregated form of A is necessary for acquiring a lipid
acceptor function.
Because the concentrations of A used in this study were high, they
may have induced oxidation of cell membranes and thereby could be
cytotoxic (Schubert et al., 1995; Mark et al., 1996), leading to lipid
leakage from cells damaged by A. Therefore, we next performed
experiments to determine whether free radicals are involved in
iA-mediated lipid release in the neuronal cultures. The neuronal
cultures were subjected to the following treatments: none, iA, iA + N-acetyl-L-cysteine (NAC), NAC,
H2O2, and
H2O2 + NAC for 8 hr. The
amount of lipids released into the culture media in each culture was
determined. NAC at a concentration of 1 mM had no
effect on iA-mediated lipid release, whereas lipid leakage caused by
H2O2 was significantly
inhibited by 1 mM NAC (Fig. 2d).
It is known that lipid release is an active cellular process and that
intracellular signaling molecules such as PKC are involved in cellular
cholesterol release (Theret et al., 1990; Mendez et al., 1991; Li and
Yokoyama, 1995; Mendez, 1997). To confirm that the iA-mediated lipid
release is an active cellular process and not a nonspecific
physicochemical phenomenon, we next examined the effect of a PKC
inhibitor, H7, on the iA-mediated lipid release from neurons. As
shown in the Figure 2e, H7 completely inhibited lipid
release mediated by iA.
Density gradient analysis of lipid particles produced by neurons in
the presence of iA
The characteristics of the released cholesterol and phospholipids
and the localization of iA added exogenously to the serum-free media
in cultured neurons were examined. The conditioned media of the
neuronal cultures treated with iA (10 µM) were
filtered with a 0.45 µM Millipore filter and centrifuged
at 34,500 rpm for 48 hr at 4°C in a tube using a Beckman SW 41-Ti
rotor. Figure 3, a and
b, show the results of density gradient ultracentrifugation of the conditioned medium containing iA. They show that most of the
cholesterol and phospholipids are distributed similarly across the
gradient, with both having densities of ~1.08-1.18 gm/ml (fractions
5, 6, 7, and 8), which correspond to the densities of HDL (Fig.
3a). The cholesterol to phospholipid ratio (w/w) at peak
density was 0.35. Western blotting analysis using anti-A antibody,
BA27, and anti-apoE antibody, AB947, was performed with the samples
from the fractions. Exogenously added A was recovered mainly from
fractions 4-8 (Fig. 3b). A in these fractions was detected as monomers and dimers under denatured conditions. Endogenous GM1 ganglioside was identified in fractions 5-8 (Fig. 3b).
However, apoE was not detected in these fractions, and apoJ, which is
secreted by neurons, was localized in fractions 10-12 (Fig.
3b). GM1 is known to have a strong positive
curvature-imposing potency, which is required for the formation of
small particles such as HDL (Epand, 1998), suggesting that GM1 may
contribute to the formation of A-lipid particles by bending of lipid
membranes in a convex manner.
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Figure 3.
Density gradient ultracentrifugation
analysis of the culture medium of neurons in the presence of iA or
H2O2. Neuronal cultures plated in six-well
plastic plates were incubated with A1-40 (10 µM) in
serum-free N2 medium for 24 hr. The culture medium was
collected, filtered through a 0.45 µm filter, and subjected to an
initial discontinuous density gradient prepared using KBr solution as
described in Materials and Methods. a, After
ultracentrifugation, fractions were obtained, and the density,
cholesterol, and phospholipids content in each fraction were
determined. b, Aliquots of 10 µl from each fraction
were mixed with the same volume of SDS buffer, subjected to SDS gel
electrophoresis, and immunoblotted with antibodies against A (BA27),
apoE (AB947), and apoJ. GM1 ganglioside in each fraction was detected
with HRP-conjugated chorea toxin-B. c, Forty-eight hours
after plating in serum-free N2 medium, the neuronal
cultures plated in six-well plastic plates were washed in DMEM and
incubated with A1-40 (10 µM) or 5 mM
H2O2 in serum-free N2 medium for
indicated periods. The percentage of LDH released from the cultures was
determined as described in Materials and Methods. The data are
mean ± SE of triplicate. d, Forty-eight hours
after plating in serum-free N2 medium, the neuronal
cultures plated in six-well plastic plates were washed in DMEM and
incubated with 5 mM H2O2 in
serum-free N2 medium for 24 hr. The culture medium was
collected, filtered through a 0.45 µm filter, and subjected to an
initial discontinuous density gradient prepared using KBr solution as
described in Materials and Methods. After ultracentrifugation,
fractions were obtained, and the density and concentrations of
cholesterol and phospholipids were determined.
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These results indicate that iA-mediated lipid release results
in the formation of A-lipid particles. To confirm that
iA-mediated lipid release is not the nonspecific lipid leakage from
neuronal cultures by free radicals generated by iA, we determined
whether HDL-like particles were generated in the conditioned medium of the neuronal cultures damaged by
H2O2. As shown in Figure
3c, iA at 20 µM has no toxic
effect on neuronal cultures until 144 hr of treatment, whereas
H2O2 at 5 mM exhibits a toxic effect on neuronal cultures
at 24 hr of treatment assayed by LDH release (Fig. 3c). The
conditioned media of these cultures were collected, and lipids, the
release of which from neurons was caused by
H2O2, were analyzed by
density-gradient ultracentrifugation. As shown in Figure 3d,
the distribution pattern of lipids shows no peak of HDL and was quite
different from that mediated by iA (Fig. 3a). These lines
of evidence together with the finding shown in Figure 2d
clearly indicate that lipid release mediated by iA is not
nonspecific lipid leakage from damaged cells by a cytotoxic effect of
iA.
Oligomeric A promotes lipid release from astrocytes
We further examined the effects of iA on the release of
cholesterol and phosphatidylcholine from cultured astrocytes. The astrocyte cultures were labeled with
[14C]acetate for 48 hr, washed in DMEM,
and incubated with iA at various concentrations for 4 hr. Lipid
concentration released into the media and cellular lipid content were
determined. The dose-response curves for the release of cholesterol
and phosphatidylcholine at 4 hr of incubation with iA are shown in
Figure 4, a and b, respectively. Incubation with iA promoted the release of cholesterol and phosphatidylcholine from astrocytes in a dose-dependent manner. Saturation of iA-mediated release of these lipids was observed at
iA concentrations higher than 1 µM. In
contrast to iA, freshly solubilized A at 10 and 30 µM did not promote lipid release from astrocytes (Fig. 4, a and b, respectively).
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Figure 4.
iA promotes lipid release from
astrocytes in culture. Astrocyte-rich cultures were labeled with
[14C]acetate for 48 hr as described in Materials
and Methods. Cells were then washed three times with DMEM and incubated
with iA1-40 or fresh A at various concentrations for 4 hr. The conditioned media were collected and then filtered. The lipids
that were released into the medium and the intracellular lipids were
extracted and analyzed as described in Materials and Methods. iA
( ) promoted the release of cholesterol (a) and
phosphatidylcholine (PC) (b) in a
dose-dependent manner; fresh A ( ) did not. Data are mean ± SE for four samples. The scale bars are smaller than the symbol size at
0 µM (a and b). Density
gradient ultracentrifugation analysis was performed with the
conditioned medium of astrocytes in the presence of iA. Astrocytes
plated in six-well plastic plates were incubated with A1-40 (10 µM) in DMEM for 24 hr. The culture medium was collected,
filtered through a 0.45 µm filter, and subjected to an initial
discontinuous density gradient prepared using KBr solution as described
in Materials and Methods. After ultracentrifugation, the solution
was fractionated. The density and cholesterol and phospholipid content
in each fraction were determined (c). Aliquots of
10 µl from each fraction were mixed with the same volume of SDS
buffer, subjected to SDS gel electrophoresis, and immunoblotted with
antibodies against A (BA27), apoE (AB947), and apoJ. GM1 ganglioside
in each fraction was detected with HRP-conjugated chorea toxin-B
(d).
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Density gradient analysis of lipid particles produced by astrocytes
in the presence of iA
The conditioned media of the astrocyte cultures treated with iA
(10 µM) were filtered with a 0.45 µM
Millipore filter and centrifuged at 34,500 rpm for 48 hr at 4°C in a
tube using a Beckman SW 41-Ti rotor. Density gradient analysis of lipid
released from astrocytes showed that a major part of cholesterol and
phospholipids distributed similarly across the gradient, with both
having densities at ~1.08-1.17 gm/ml (fractions 5, 6, 7, and 8),
which corresponded to the densities of HDL (Fig. 4c). The
cholesterol to phospholipids ratio (w/w) at peak density was 2.22. Western blotting analysis using anti-A antibody, BA27, and anti-apoE
antibody, AB947, was performed with the samples from the fractions.
Exogenously added A was recovered mainly in the fractions between 4 and 8 (Fig. 4d). A was detected as monomer and dimer
under denaturing conditions. Endogenous GM1 ganglioside and apoE were
identified in the fractions between 5 and 8 (Fig. 4d).
However, apoJ, which is secreted from neurons, was localized in
fractions 10, 11, and 12 (Fig. 4d).
Morphological and biochemical analysis of lipid particles
associated with A
To confirm the conformation of A-lipid particles isolated by
density gradient analysis of filtered culture medium of neurons in the
presence of iA, fractions containing A-lipid particles were
diluted with distilled water to a density of 1.006 gm/ml and
centrifuged at 34,500 rpm for 48 hr at 4°C in a tube using a Beckman
SW 41-Ti rotor. The conditioned media of the neuronal cultures in the
presence of 0.25 µM of apolipoprotein E3 (apoE3) were
also analyzed. The lower part of each solution was subjected to
immunoelectron microscopic study. Analysis of samples from neuronal
conditioned media in the presence of iA revealed that lipoprotein
particles were spherical, with a mean diameter of 29.4 ± 1.1 nm,
similar to the appearance of, but larger than, HDL-like particles
formed via the apoE-mediated manner, the mean diameter of which is
11.4 ± 0.5 nm (Fig.
5a,b). In addition,
A was found to be associated with lipid particles as demonstrated by
the immunoreactivity of lipid particles against the anti-A antibody,
6E10, using electron microscopy (Fig. 5c). In contrast, A
immunoreactive lipid particles were not detected in negative control
samples (Fig. 5d), suggesting that iA is directly
associated with lipid particles.
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Figure 5.
Electron micrographs of lipid particles associated
with iA and apoE3. Neuronal cultures were incubated with 10 µM iA or 0.25 µM apoE3 for 24 hr. The
conditioned media of these cultures were collected, filtered with a
0.45 µm Millipore filter, subjected to an initial discontinuous
density gradient prepared using KBr solution, and centrifuged at 34,500 rpm for 48 hr using a SW 41-Ti rotor. After centrifugation, 12 fractions were isolated, and lipid concentration and the density in
each fraction were determined. HDL fractions were then diluted with
10-fold volumes of distilled water, followed by centrifugation at
34,500 rpm for 48 hr using a SW 41-Ti rotor. Electron microscopic
examination of the lower part of each solution was performed. Negative
staining of electron micrographs of lipid particles in the presence of
apoE and iA is shown (a and b,
respectively). Results of immunoelectron microscopy show that
exogenously added iA1-40 forms complexes with lipid particles as
demonstrated using the antibody directed against human A1-17, 6E10
(Kim et al., 1990). c, Gold labeling is considered to be
associated with lipid particles. d, In contrast, lipid
particles were not labeled with gold without 6E10. Scale bar, 50 nm.
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We further examined whether A-lipid particles contain other
acceptors such as apoE and apoJ. The filtered conditioned medium of
neuronal cultures was subjected to immunoprecipitation using antibodies
including anti-apoE antibody, AB947, anti-A antibodies, 6E10,
anti-apoJ antibody, or normal mouse IgG as a negative control. The
ratio of cholesterol and phospholipids associated with protein G-Sepharose to those in the conditioned medium is shown in Figure 6. Approximately 12% of total
cholesterol and phospholipids in the conditioned medium was
immunoprecipitated with 6E10, whereas the percentages of both lipids
immunoprecipitated with AB947, anti-apoJ antibody, and normal mouse IgG
were significantly lower than those with 6E10 in neuronal culture (Fig.
6Aa,b). In the conditioned medium of
astrocytes incubated with 10 µM iA, the percentages of cholesterol and phospholipids immunoprecipitated with
6E10 were 4.7 and 3.1%, respectively, of those in the conditioned medium, and those immunoprecipitated with AB947 were 4.7 and 3.2% of
those in the conditioned medium, respectively (Fig.
6Ba,b). However, as seen in neuronal
cultures, the percentages of either lipid immunoprecipitated with
anti-apoJ antibody and normal mouse IgG were significantly lower than
those with 6E10 or AB947 in astrocyte cultures.
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Figure 6.
Binding of A by immobilized anti-A antibody,
6E10, results in capture of lipids. Filtered conditioned medium of
neuronal and astrocyte cultures incubated with iA for 48 hr was
subjected to immunoprecipitation using anti-A antibody (6E10),
anti-apoE antibody (AB947), anti-apoJ antibody, and normal mouse IgG.
A, The protein-G-Sepharose-associated lipids were
determined using the kits described in Materials and Methods. The
quantity of cholesterol (a) and phospholipids
(b) immunoprecipitated with 6E10 from the
conditioned medium of neuronal culture incubated with iA was ~12%
of the total cholesterol and phospholipids in the initial conditioned
medium. However, those immunoprecipitated with anti-apoE antibody,
anti-apoJ antibody, or normal mouse IgG were significantly low.
B, In astrocyte culture medium, the quantity of
cholesterol (a) and phospholipids
(b) immunoprecipitated with 6E10 and AB947 from
the conditioned medium incubated with iA was significantly higher
than those with anti-apoJ antibody or normal mouse IgG. Western blot
analysis using anti-apoE antibody (AB947) and anti-A antibody (BA27)
was performed with the immunoprecipitates. The bands corresponding to
A monomers and dimers is labeled only in immunoprecipitates by 6E10,
whereas no band or a faint band was detected in those by AB947,
anti-apoJ antibody, and normal mouse IgG in neurons
(Ac). The bands corresponding to A monomers and
dimers and apoE are labeled in both immunoprecipitates by 6E10 and
AB947, respectively, whereas no band or a faint band was detected in
those by anti-apoJ and normal mouse IgG for astrocytes
(Bc). Data are mean ± SE for four samples.
*p < 0.01 versus 6E10, anti-apoJ, and normal IgG
(Ac); *p < 0.01 versus anti-apoJ
and normal IgG (Bc).
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Western blotting analysis using anti-A antibody, BA27, and anti-apoE
antibody, AB947, was performed with the immunoprecipitates. The pellet
fractions from the immunoprecipitation of neuronal conditioned medium
with the anti-A antibody contained exogenously added A, being
compatible with lipid content in association with immunoprecipitates (Fig. 6Ac). In contrast, those
from the immunoprecipitation of astrocyte-conditioned medium with the
anti-A antibody contained both exogenously added A and endogenous
apoE (Fig. 6Bc). In addition, those of
astrocyte-conditioned medium with the anti-apoE antibody contained both
exogenously added A and endogenous apoE (Fig. 6Bc).
Binding and internalization of A-lipid particles
into neurons
Because iA1-40 was found to be an acceptor of lipids from
neurons and astrocytes to form A-lipid particles, it was considered appropriate to perform further study on binding and internalization of
A-lipid particles produced by iA. The conditioned media of [14C]acetate-labeled astrocytes in the
presence of iA at 10 µM or apoE3 at 0.25 µM in DMEM after 24 hr incubation were collected. A-lipid particles were recovered from HDL fractions by density gradient centrifugation and dialyzed. The fractions containing A-lipid particles were collected and dialyzed, and the radioactivity contained was normalized by a scintillation counter. Neuronal cultures
were then incubated with the equal amount of
[14C]labeled-A-lipid particles in
DMEM for 20 min at 4 or 37°C. The cultures were washed with cold PBS
three times and dried under the fresh airflow. The lipids were
extracted with hexane/isopropanol (3:2 v/v), separated by HPTLC, and
quantified with BAS2500. Figure 7
demonstrates the binding activity and internalization efficacy of
A-lipid particles and HDL-like particles formed by exogenously added
iA and apoE3, respectively. The ratio of the labeled-cholesterol activity associated with neurons per total cholesterol activity in the
added medium was significantly lower at both 4 and 37°C in the
cultures incubated with conditioned medium treated with iA1-40
(Fig. 7a). In contrast, it was significantly higher both at
4 and 37°C in the cultures incubated with apoE. Similar results were
observed for the ratio of the labeled phosphatidylcholine in
association with the cells (Fig. 7b).
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Figure 7.
Binding affinity and internalized efficacy of
A-lipid particles into neurons. Astrocyte-rich cultures were labeled
with [14C]acetate for 48 hr as described in
Materials and Methods. Cells were then washed three times with DMEM and
incubated with 10 µM iA 1-40 or 0.25 µM
human recombinant apoE3 for 24 hr. The conditioned media were obtained,
filtered using a 0.45 µm filter, and subjected to density-gradient
ultracentrifugation at 34,000 rpm for 48 hr in a Beckman SW 41-Ti
rotor. The HDL fractions were then collected and dialyzed. The
radioactivity in each sample was determined by a scintillation counter
and normalized with DMEM. The normalized conditioned medium containing
iA or apoE was added to neuronal cultures at 4 or 37°C. Twenty
minutes after the addition, the cultures were washed three times with
cold PBS and dried under air flow at room temperature. The lipids in
each culture were extracted by incubation with hexane/isopropanol (3:2
v/v) solution for 1 hr. Then the solution was moved into tubes and
dried under N2 gas. The extracted lipids were then
dissolved in chloroform, developed in HPTLC, and quantified by BAS2500
(Fuji Film, Tokyo, Japan). a, The ratio of the
labeled cholesterol associated with neurons was significantly lower at
both 4 and 37°C in the cultures incubated with conditioned medium
treated with iA1-40. However, it was significantly higher at both 4 and 37°C in the cultures incubated with apoE. Similar results were
observed for the ratio of the labeled phosphatidylcholine in
association with the cells (b). Data are
mean ± SE for six samples. *p < 0.003.
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DISCUSSION |
In the present study, we found a novel action of A: oligomeric
A can promote lipid release from astrocytes and neurons to form
A-lipid particles consisting of cholesterol, phospholipids, GM1
ganglioside, and A. A-lipid particles produced by oligomeric A
have very low binding affinity to neurons and therefore are not
internalized into neurons, suggesting that oligomeric A may affect
intracellular lipid metabolism. Because high concentrations of A are
known to induce oxidation and can be cytotoxic (Schubert et al., 1995;
Mark et al., 1996), we have examined the toxicity of A used in this
study and found that iA has no cytotoxic effect on neurons until 144 hr of treatment, as demonstrated by LDH assay. We have also found that
NAC, a potent antioxidant molecule, has no effect on iA-mediated
lipid release, and lipids released from the cells after the addition of
H2O2 do not form lipid
particles, which were recovered in HDL fractions. These lines of
evidence clearly indicate that lipid release mediated by iA is not
nonspecific lipid leakage from damaged cells by cytotoxic effect of
iA. Because Congo red is a well known dye that not only binds to
A fibrils and A oligomers to inhibit fibril formation but also
inhibits A oligomerization by stabilizing A monomer (Podlisny et
al., 1995, 1998), we examined the effect of Congo red on iA-mediated lipid release. We also performed experiments to determine whether freshly dissolved A can serve as a lipid acceptor. We found that neither freshly prepared A nor iA incubated with 10 µM Congo red removes lipids from neurons. These findings
suggest that oligomerized A is required for acquiring the ability to
promote lipid release from the cells. In addition, iA-filtered does
not contain any fibrils or ADDLs (Fig. 1c). Moreover,
all conditioned media of cultures treated with iA were examined
after filtration, and electron microscopic study did not detect any
fibrils in the samples after 24 hr incubation (data not shown). These
lines of evidence exclude the possibility that A fibrils, but not
A oligomers, act as lipid acceptors. Taken together, oligomerization
to dimers, trimers, tetramers, and possibly larger assemblies is
indispensable for A to acquire the ability to promote lipid release.
The molecular mechanism by which oligomeric A, but not monomeric
A, promotes cholesterol and phospholipid release remains obscure.
However, one can explain the mechanism based on the differential binding affinity of A to lipids. It has been reported that the binding efficacy of lipids, including cholesterol, phosphatidylcholine, and free fatty acids, to A is increased when the added A forms polymers (Avdulov et al., 1997). Aggregated A exhibits strong electrostatic interactions with the surface of model membranes, which
appear to mediate its neurotoxicity (Hertel et al., 1997). In addition,
a synthetic peptide in an amphipathic -sheet structure was found to
associate with lipids, including cholesterol and phosphatidylcholine,
and is being proposed as a model for apolipoprotein B (Osterman et al.,
1984), suggesting that cholesterol and other lipids bind to hydrophobic
areas of amphipathic -sheet. These lines of evidence suggest
that cholesterol and other lipids may bind to hydrophobic areas of
aggregated A to form A-lipid particles.
Another interesting point is that A-lipid particles generated in the
cultured astrocytes are cholesterol rich compared with HDL-like
particles produced by apolipoproteins, such as apoE or apoAI (Ito et
al., 1999; Michikawa et al., 2000). In addition, our finding that the
average size of A-lipid particles produced by iA (29.4 ± 1.1 nm) is larger than that of HDL-like particles produced by apoE
(11.4 ± 0.5 nm) in neuronal cultures suggests that there could be
two species of lipid particles, one produced by iA and the other
produced by apoE. The size of HDL-like particles produced by apoE is
consistent with that previously reported in astrocyte conditioned
medium or in CSF (LaDu et al., 1998; Fagan et al., 1999). The
mechanisms underlying these cell type- and acceptor type-specific
discrepancies are not clear, and further studies are needed to address
these issues.
Interestingly, A-lipid particles produced by iA cannot bind to
neurons and thus are not internalized into the cells, whereas HDl-like
particles produced by apoE can do so. This may be because neurons have
no receptors to bind A. It has been shown that under physiological
conditions, a significant amount of soluble A is associated with
apoE-containing HDL particles in CSF, AD brain, and culture medium
(Koudinov et al., 1994, 1996; Biere et al., 1996). In contrast to
oligomeric A, when monomeric A forms complexes with such
particles, apoE receptors bind and internalize these lipid complexes
into the cells, thereby modulating the amount of A in the brain
parenchyma through cellular clearance mechanisms (Holtzman et al.,
1999). This may be because the protein component of HDL under
physiological conditions is predominantly apoE and not A, and thus
apoE can function as a ligand to its receptors. These lines of evidence
raise the question of whether A directly binds to apoE and not to
lipids, or directly binds to lipids to form apoE-lipid-A complexes.
It is difficult to answer this question using astrocyte cultures,
because astrocytes synthesize and secrete both apoE and A (Figs.
4d, 6Bc). However, our data on
iA-mediated lipid release from neurons can exclude the former
possibility, because lipid particles associated with iA contained
neither apoE nor apoJ (Figs. 3b, 6Ac).
These data indicate that iA interacts directly with lipid particles.
Previous studies have shown that A oligomers (dimers, trimers,
tetramers, and possibly larger assemblies) are formed in the
conditioned media of certain cell lines that constitutively secrete
A and that endogenous and synthetic A can assemble into stable
oligomers at physiological concentrations in culture (Podlisny et al.,
1995, 1998; Xia et al., 1997). Recently, A oligomers have been
identified in CSF of AD patients (Pitschke et al., 1998). These lines
of evidence together with our present results may allow us to assume
that oligomeric A accumulates extracellularly under
pathophysiological conditions such as the AD brain, and this oligomeric
A, in turn, may stimulate lipid release from neurons, leading to
disruption of cholesterol homeostasis in the CNS.
We have recently found that the deficiency in intracellular cholesterol
content causes tau phosphorylation (Fan et al., 2001). We have also
demonstrated that tau is hyperphosphorylated in brains of mice with
Niemann-Pick disease type C (Sawamura et al., 2001), which is known as
a cholesterol storage disease involving late endosomes and lysosomes
with defective intracellular sterol trafficking (Scriver et al., 1995).
On the basis of these lines of evidence, together with the findings
reported here, we hypothesize that oligomeric A promotes lipid
release, which in turn may reduce cellular cholesterol levels, thereby
promoting tau phosphorylation and neurodegeneration, as observed in AD
brain. Our observations in the present study also provide new insight
into the central issue concerning the pathogenesis of AD, that is, the
relationship between amyloid plaque formation and the development of
neurofibrillary tangles in neurons.
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FOOTNOTES |
Received March 19, 2001; revised June 5, 2001; accepted July 6, 2001.
We are grateful to K. Matsuzaki and S. Yokoyama for helpful comments
and suggestions. This study was supported by a research grant for
Longevity Sciences (H11-001), Research on Brain Science from Ministry
of Health and Welfare, by CREST (Core Research for Evolutional Sciences
and Technology), Japan, and by Ono Research Foundation and Life Science
Foundation of Japan.
Correspondence should be addressed to Dr. Makoto Michikawa, National
Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi
474-8522, Japan. E-mail: michi{at}nils.go.jp.
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Lipid binding to amyloid
TOP
ABSTRACT
INTRODUCTION
