From the Neuroscience Research Institute, University
of California, Santa Barbara, California 93106 and the
§ Biochemistry Department, Yeungnam University, Kyongsan
712-749 Korea
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ABSTRACT |
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Amyloid protein (A
P) forms senile plaques
in the brain of the patients with Alzheimer's disease. The early-onset
AD has been correlated with an increased level of 42-residue A
P
(A
P1-42). However, very little is known about the
role of A
P1-42 in such pathology. We have examined the
activity of A
P1-42 reconstituted in phospholipid
vesicles. Vesicles reconstituted with A
P show strong
immunofluorescence labeling with an antibody raised against an
extracellular domain of A
P suggesting the incorporation of A
P
peptide in the vesicular membrane. Vesicles reconstituted with A
P
showed a significant level of 45Ca2+ uptake.
The 45Ca2+ uptake was inhibited by (i) a
monoclonal antibody raised against the N-terminal region of A
P, (ii)
Tris, and (iii) Zn2+. However, reducing agents Trolox and
dithiothreitol did not inhibit the 45Ca2+
uptake, indicating that the oxidation of A
P or its surrounding lipid
molecules is not directly involved in the A
P-mediated
Ca2+ uptake. An atomic force microscope was used to image
the structure and physical properties of these vesicles. Vesicles
ranged from 0.5 to 1 µm in diameter. The stiffness of the
A
P-containing vesicles was significantly higher in the presence of
calcium. The stiffness change was prevented in the presence of zinc,
Tris, and anti-A
P antibody but not in the presence of Trolox and
dithiothreitol. Thus the stiffness change is consistent with the
vesicular uptake of Ca2+. These findings provide
biochemical and structural evidence that A
P1-42 forms
calcium-permeable channels and thus may induce cellular toxicity by
regulating the calcium homeostasis in Alzheimer's disease.
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INTRODUCTION |
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A pathological hallmark in brain tissue from patients with
Alzheimer's disease (AD)1 is
the accumulation of amyloid protein (A
P), a 39-43-amino acid-long polypeptide, as morphologically heterogeneous neuritic plaques and cerebrovascular deposits (1, 2). A
P is derived primarily
from a proteolytic cleavage of the
-amyloid precursor protein
(A
PP), a highly conserved and widely expressed integral membrane
protein with a single membrane-spanning polypeptide. The amount and the
nature of polypeptides vary considerably among various forms of ADs:
A
P1-40 and A
P1-42 are differentially accumulated in sporadic Alzheimer's disease and non-demented brain samples (3) and a mutation in presenilins is linked with an increased
ratio of A
P1-42/A
P1-40 in familial
Alzheimer's disease (4-7). The early-onset familial AD has been
correlated with an increased level of A
P1-42. However,
very little is known about the role of A
P1-42 in such
pathology and about the mechanism(s) of its action.
Accumulating evidence suggests an early and causative role
of APs in the pathogenic cascade (8-11). Postulated mechanisms of
A
P toxicity include, by its interaction with the tachykinin neuropeptide system, a surface membrane effect (12); by changing cellular ionic concentration via formation of plasma membrane channels
(13-15); and by activating oxidative pathways and making cells more
responsive to oxidative stress (for review see Refs. 16 and 17).
Reactive oxygen species and the antioxidant defenses work probably by
altering the lipid peroxidation and membrane composition. However,
A
P polypeptides associated with the reactive oxygen hypothesis have
produced conflicting effects on cytoskeletal organization and cell
lysis (18-23).
The commonly observed change in the cellular ion concentration involves
increased calcium level (24-26) either indirectly via modulating the
existing Ca2+ channel or directly via cation-selective
channels formed by APs. Support for the cation-selective A
P
channels are accumulating. Arispe and his collaborators (13-15, 27)
have reported cation-selective channels formed by
A
P1-40 when reconstituted into lipid bilayers and in
the membrane patches excised from hypothalamic gonadotropin-releasing
hormone neurons. Kagan and his collaborators (28) have also recorded
channel-like activity when A
P25-35 was reconstituted in
lipid bilayers as well as for both A
P1-40 and
A
P1-42 reconstituted in lipid
bilayer,2 though, with less
reliability and reproducibility than the A
P25-35 current (28). Whether A
P1-42 toxicity is also mediated via A
P1-42 forming calcium-permeable ion channel is
unclear.
The molecular structure of AP oligomers, especially as an ion
channel, is unknown. Durell et al. (29) have developed
theoretical models for the structure of ion channel formed by the
membrane-bound A
P1-40. However, no direct structural
data from EM, NMR, x-ray diffraction, or other microscopic techniques
are available to support the presence of the A
P channel.
We have used an atomic force microscope (AFM) (30) integrated with a
light and fluorescence microscope (31) to examine the mechanism(s) of
AP1-42 toxicity. A
P1-42 was reconstituted in phospholipid vesicles and were imaged in buffer to
reveal the A
P-membrane complex and a channel-like structure. Consistent with the possibility of fresh A
Ps forming ion-permeable channels (i) fluorescently labeled anti-A
P antibodies were localized in A
P-reconstituted vesicles, (ii) vesicles reconstituted with A
P
show a significant level of 45Ca2+ uptake which
was blocked by anti-A
P-antibody, Zn2+, and tromethamine,
but not blocked by antioxidants, and (iii) A
P-reconstituted vesicles
have considerably higher stiffness in the presence of calcium.
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EXPERIMENTAL PROCEDURES |
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Reconstitution of AP1-42 into
Liposomes--
Human A
P1-42 and phospholipids were
purchased from Bachem (Torrance, CA) and Avanti Polar (Birmingham, AL),
respectively. Liposomes were prepared from both
palmitoyloleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylserine. 10 µl of phospholipids (10 mg/ml palmitoyloleoylphosphatidylethanolamine:
palmitoyloleoylphosphatidylserine::1:1) in chloroform was
dried under argon gas, and then 2 ml of buffer (10 mM
HEPES, pH 7.4) was added. The mixture was then bath sonicated for 20 min. For the incorporation of A
P1-42 into liposomes, the phospholipids were first dried under argon gas, then 1.5 ml of
buffer was added, followed by 20 min of bath sonication. The A
P
stock solution (5 mg/ml) was added, and the mixture was sonicated for
another 20 min. The final concentration of phospholipids and A
P is 1 mg/ml.
Immunolabeling with Anti-AP Antibody--
A mouse monoclonal
antibody raised against the N' terminus of A
P (named 3D6 antibody
[anti-A
P 1-5 (DAEFR)]) was obtained from Dr. Russell Rydel at
Athena Neurosciences (San Francisco, CA). Goat anti-mouse IgG
conjugated with Cy3 (1 mg/ml) was purchased from Chemicon International
(Temecula, CA). Liposomes were adsorbed on glass coverslips and fixed
with 4% paraformaldehyde for 10 min, then washed with
phosphate-buffered saline (PBS), and then blocked with PBS containing
3% BSA and 1% goat serum to minimize nonspecific binding. Primary
antibody (diluted 500-fold) was added in the presence of 3% BSA and
1% goat serum and incubated for 1 h at room temperature. After
washing with PBS, the sample was incubated with secondary antibody
(500-fold dilution) at the same condition as primary incubation.
Fluorescent images were obtained using 40× high numerical aperture
objective lens with an inverted Olympus inverted fluorescence
microscope.
Measurement of 45Ca2+ Uptake into
Liposomes--
45Ca2+ uptake was measured by
the modified method of Nakade et al. (32). 25 µl of
liposomes reconstituted with/without AP1-42 was
incubated with 75 µl of HEPES buffer (10 mM, pH 7.4)
containing 2 µCi of 45Ca2+. Calcium influx
was measured separately for each perturbation: anti-A
P-antibody,
Zn2+, Tris, Trolox, and DTT, respectively. After incubation
for 1 min at 30 °C, in the presence of a perturbation, the calcium
influx reaction was stopped with a blocking solution containing 300 µl HEPES buffer, 0.3 mM CaCl2, 5 mM ZnCl2, 10 mM Tris, and 15 µg/ml 3D6 antibody. The 400-µl mixture of liposomes and the
blocking solution was immediately loaded onto a Chelex 100 column (bead volume: 3 ml) (Bio-Rad) which was pre-equilibrated with a buffer containing 200 mM sucrose, 20 mM Tris-HCl (pH
7.4 at room temperature), and 0.3% BSA. The column was then washed
immediately with 5 ml of a buffer containing 200 mM sucrose
and 20 mM Tris-HCl (pH 7.4) to take the liposomes. The
45Ca2+ content of the liposomes was measured
with a Beckman liquid scintillation counter.
Imaging Liposomes with Atomic Force Microscopy--
AFM images
were obtained as described
(33),3 using a prototype of
Bioscope AFM and a Multimode AFM (Digital Instruments, Santa Barbara,
CA). Contact mode AFM was used for most of the images. Oxide-sharpened
silicon nitride tips with a nominal spring constant of about 0.06 newton/m (Digital Instruments) were used for most experiments. All
imaging was conducted on wet and hydrated liposomes. For liposomes
reconstituted with/without AP1-42, 20-50 µl of
sample was deposited on a clean glass Petri dish and left for 30 min.
The surface of the Petri dish was then rinsed with a buffer (10 mM HEPES, pH 7.4) and imaged in the buffer. The imaging
force was regularly monitored and kept to a minimum. The imaging force
varied from subnanonewton to tens of nanonewtons. All imaging was
performed at room temperature (22-24 °C).
Measuring Viscoelastic Properties--
We measured stiffness of
AP vesicles by the AFM force-mapping technique as
described (34) using the Nanoscope III software (Version 4.23R2;
Digital Instruments, Santa Barbara). Force maps were taken by raster
scanning the tip over the sample with 64 × 64 measuring points
with a pixel resolution of 95 nm. Each force map thus consists of a
topographical image of 64 × 64 pixels, and a force curve is
stored for every pixel. Force maps were obtained alternately with the
regular height and error modes of imaging the surface topography, using
the same cantilever.
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RESULTS AND DISCUSSION |
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Immunolocalization of AP1-42 on the Reconstituted
Liposomes--
Liposomes reconstituted with fresh
A
P1-42 show strong immunolabeling with an anti-A
P
antibody. Fig. 1D shows a
fluorescence labeling image of liposomes reconstituted with
A
P1-42. For comparison, no immunofluorescence labeling
was observed in the liposomes prepared without A
P1-42
(Fig. 1B). Also, there was no immunofluorescence labeling
observed in vesicles reconstituted with aged A
P1-42
(A
P stored for 24 h or longer). All immunolabeled liposomes
have a well defined vesicular structure as revealed by AFM imaging
(Fig. 1, A and C). Vesicles with strong
immunofluorescence signals appear to have double-layered (two
membranes) disc-shaped structures in AFM images. Vesicle size ranged
from 0.5 to 1 µm. Vesicles without A
Ps are on average half in
diameter but more spherical compared with the vesicles with A
Ps. The
average height of vesicles without A
P is 40 nm and 31 nm for
vesicles with A
P. The vesicular flattening and larger size are
probably due to the protein-lipid interactions (37, 38) as is the case
with other membrane proteins such as gap junctions and acetylcholine
receptors.
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AP Channel-specific Calcium Uptake--
Liposomes reconstituted
with A
P1-42 show a significantly larger (>4-fold)
increase in the influx of 45Ca2+ compared with
the liposomes without A
P1-42 (compare A and
B in the top panel of Fig.
2). In the presence of an antibody raised
against the amino-terminal domain of A
P, there was no significant
influx of 45Ca2+ in the liposomes reconstituted
with A
P (compare A, B, and C in the
top panel of Fig. 2). Such inhibition of calcium
uptake by the anti-A
P antibody shows the specificity of
A
P1-42-induced calcium uptake. The level of inhibition
by anti-A
P antibody would have been greater if all epitopes of the
reconstituted A
Ps were oriented outside of the liposomes.
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Calcium Uptake Induced Change in Vesicular Elasticity--
In
cells, calcium uptake would lead to altered metabolic load and an
imbalance in the ionic homeostasis. In reconstituted vesicles, calcium
uptake should change the physicochemical properties, especially vesicle
stiffness and charge-charge interactions. We examined the change in
vesicle stiffness using the force-mapping feature of the AFM
imaging on vesicles reconstituted with/without AP1-42
(Fig. 3) with a well defined morphology
as revealed in AFM images. The shape and size of the vesicles appear to
vary, but the apparent height of the unilammellar vesicles was
similar.
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ACKNOWLEDGEMENTS |
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We thank Drs. Bruce Kagan and Dennis Clegg for useful advice and critical evaluation of the manuscript, Dr. Russell Rydel of Athena Neurosciences for kindly providing us the antibodies used in our study, and Dr. Paul Hansma for encouragement with the AFM study of Alzheimer's disease.
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FOOTNOTES |
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* This work was supported by grants from the State of California, Department of Health Services, Alzheimer's Disease Program, 95-23336, and Yeungnam University in Korea. Portions of this work have been published as an abstract from the Biophysical Society annual meeting, 1998.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 805-893-2350; Fax : 805-893-2005; E-mail: rlal{at}physics.ucsb.edu.
1
The abbreviations used are: AD, Alzheimer's
disease; AP, amyloid
protein; APP, amyloid precursor protein;
AFM, atomic force microscope; PBS, phosphate-buffered saline; BSA,
bovine serum albumin; DTT, dithiothreitol.
2 T. Mirzabekov, M. C. Lin, W. L. Yuan, P. J. Marshall, M. Carman, K. Tomaselli, I. Lieverburg, and B. L. Kagan, personal communication.
3 Y. J. Zhu, Y. Zhang, and R. Lal, submitted for publication.
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REFERENCES |
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