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J Biol Chem, Vol. 274, Issue 26, 18801-18807, June 25, 1999
From the The effects of soluble and aggregated amyloid
Alzheimer's disease
(AD)1 is a progressive
neurodegenerative disorder characterized by the accumulation of
neuritic plaques composed of amyloid It has been postulated that the biological activity of A In this study, the effects of aggregated and soluble A Chemicals--
A Synaptic Plasma Membrane Isolation--
Synaptic plasma
membranes (SPM) were isolated from F344 male rats (3 months old) using
discontinuous Ficoll-sucrose gradients as described previously (14,
18-24). Rat cortex was homogenized in a sucrose buffer (0.32 M sucrose and 5 mM HEPES, pH 7.4) containing 0.5 mM EDTA at 4 °C. The homogenate was centrifuged at
578 × g for 8 min, and the supernatant was removed and
centrifuged at 17,300 × g for 10 min. The resulting
pellet (P2) was suspended in sucrose buffer and then layered over 7.5 and 13% Ficoll solutions (w/v Ficoll/sucrose buffer) containing 0.5 mM EDTA. The gradients were centrifuged in a Beckman SW 28 rotor at 80,000 × g for 30 min. The material at the
7.5 and 13% interfaces was carefully removed, and sucrose buffer was
added and centrifuged at 17,300 × g for 15 min. The
pellet enriched in synaptosomes was resuspended in sucrose buffer and
centrifuged at 12,000 × g for 10 min. SPM were
prepared by lysing synaptosomes in 5 mM Tris-HCl, pH 8.5. The synaptosomal suspension was kept on ice (4 °C) and vortexed every 20 min for 1 h. The suspension was then centrifuged at
41,000 × g for 20 min. The pellet was resuspended in
15 ml of cold distilled water and underlayered with 15 ml of 0.75 M sucrose buffer containing 1.5 mM Tris, 3 mM HEPES, 0.25 mM EDTA, pH 7.4, and centrifuged at 41,000 × g for 30 min. SPM at the interface were
removed and pelleted at 41,000 × g for 20 min. The SPM
pellet was resuspended in phosphate-buffered saline, pH 7.4.
Gel Electrophoresis of A Preparation of Reconstituted Brain Membrane Multilamellar
Vesicles--
Porcine brain phosphatidylcholine lipids dissolved in
chloroform (1.0 mg/ml) were dried down under a stream of nitrogen gas to a thin film in a test tube while vortexing. Residual solvent was
removed by drying for 3 h under a vacuum. A 1.0-ml aliquot of
buffer (0.5 mM HEPES, 154.0 mM NaCl, pH 7.3)
containing a specified concentration of A Small Angle X-ray Diffraction--
SPM or reconstituted brain
multilamellar vesicles prepared in the presence and absence of A Fluorescence Spectroscopy--
An SLM 8100 fluorimeter
(Spectronics Inc., Rochester, NY) was used to determine SPM annular and
bulk fluidity using procedures previously reported by our laboratory
(14, 18, 19, 24). The pyrene excimer/monomer fluorescence intensities
ratio when pyrene was excited through energy transfer from tryptophans
on SPM proteins (excitation = 286 nm) and when pyrene was excited at
its own excitation wavelength (excitation = 334 nm) were used to
calculate annular fluidity and bulk fluidity, respectively. Fluorimeter
cuvette temperature was maintained at 36.5 °C with a circulating
water bath. Band pass slits were 8 nm on excitation and 4 nm on
emission. Pyrene emission spectra were recorded in a 350-500-nm
interval. SPM were suspended in PBS (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4,
1.5 mM KH2PO4, 20 mM
HEPES, pH adjusted to 7.4 with Tris base) that served as the control
buffer or PBS containing A SDS-Polyacrylamide Gel Electrophoresis of
A Effects of Soluble and Aggregated A
Direct subtraction of the normalized SPM membrane electron density
profiles demonstrated pronounced changes in structure following the
addition of soluble versus aggregated A
The addition of aggregated A Effects of Soluble and Aggregated A Effects of Soluble and Aggregated A
When pyrene that had partitioned into SPM was excited at 334 nm, the
fluorescence intensity of the maximas was increased as compared with
pyrene excited through energy transfer (Figs. 6 and
7). This large difference in fluorescence
intensity indicated that the fraction of pyrene in close proximity to
SPM proteins containing tryptophan contributed very little to the
estimation of bulk fluidity. The ratio of intensity of pyrene excimer
fluorescence intensity at 480 nm to fluorescence intensity of pyrene
monomer at 373 nm was used for the estimation of bulk fluidity when
pyrene was excited at 334 nm. When soluble A Alzheimer's disease neuropathology is characterized by insoluble
A The relative contribution of A One-dimensional electron density profiles generated from the x-ray
diffraction data indicated a membrane structure with unit cell
periodicities that ranged from 75.1 to 85.2 Å, depending on the
presence of A The fluorescence anisotropy experiments indicated that both soluble and
aggregated A The increase in SPM fluidity observed in the presence of
A There is increasing evidence that A The mechanism underlying the neurotoxicity of A *
This work was supported by a Nathan Shock Pilot Grant (to
R. P. M.), the Medical Research Program of the Department of
Veterans Affairs (to W. G. W.), and National Institutes of
Health Grants AG11056 (to W.G.W.) and AA10806 (to N. A. A.).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: Allegheny University
Hospital, 320 E. North Ave., 15ST, Pittsburgh, PA 15212-4772. Tel.:
412-359-4815; Fax: 412-359-6390.
The abbreviations used are:
AD, Alzheimer's
disease;
A
Distribution and Fluidizing Action of Soluble and Aggregated
Amyloid 
-Peptide in Rat Synaptic Plasma Membranes*
§,,,, Membrane Biophysics Laboratory, Departments
of Medicine and Biochemistry, MCP Hahnemann University School of
Medicine, Allegheny Campus, Pittsburgh, Pennsylvania 15212-4772 and the
¶ Geriatrics Research, Education and Clinical Center, Veterans
Affairs Medical Center and the Department of Pharmacology, University
of Minnesota School of Medicine, Minneapolis, Minnesota 55417
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptide (A) on cortical synaptic plasma membrane (SPM) structure
were examined using small angle x-ray diffraction and fluorescence
spectroscopy approaches. Electron density profiles generated from the
x-ray diffraction data demonstrated that soluble and aggregated
A1-40 peptides associated with distinct regions
of the SPM. The width of the SPM samples, including surface hydration,
was 84 Å at 10 °C. Following addition of soluble
A1-40, there was a broad increase in electron density
in the SPM hydrocarbon core ±0-15 Å from the membrane center, and a
reduction in hydrocarbon core width by 6 Å. By contrast, aggregated
A1-40 contributed electron density to the phospholipid
headgroup/hydrated surface of the SPM ±24-37 Å from the membrane
center, concomitant with an increase in molecular volume in the
hydrocarbon core. The SPM interactions observed for
A1-40 were reproduced in a brain lipid membrane system.
In contrast to A1-40, aggregated A1-42
intercalated into the lipid bilayer hydrocarbon core ±0-12 Å from
the membrane center. Fluorescence experiments showed that both soluble
and aggregated A1-40 significantly increased SPM bulk
and protein annular fluidity. Physico-chemical interactions of A
with the neuronal membrane may contribute to mechanisms of
neurotoxicity, independent of specific receptor binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptide (A) variants,
extracellular matrix components, and apolipoproteins (1, 2). A is an
amphipathic, 39-42-residue peptide that is derived by proteolytic
cleavage of the transmembrane glycoprotein, the amyloid precursor
protein; the A domain is composed of 28 extracellular and 12-14
transmembrane amino acid residues of amyloid precursor protein (3). An
increase in the production and abnormal accumulation of A in the
brain has been implicated in the etiology of AD. Several studies have shown that A analogs can directly disrupt neuronal function, contributing to cell death associated with the development of AD
(4-8).
is related
to its ability to form insoluble aggregates in solution (9-11),
although the cellular mechanism of action is not well understood. A
recent study from Cotman and co-workers (12) showed that A
neurotoxicity is independent of stereoisomer-specific ligand-receptor
interaction because both all-D- and
all-L-stereoisomers of A25-35 and
A1-40 had similar neurotoxic activity. This finding
suggests that A modulates membrane function by a nonreceptor-mediated mechanism, potentially as a result of altering the
physico-chemical properties of membrane constituents, including lipids
and proteins (13-16). Indeed, previous membrane equilibrium binding
experiments have demonstrated that the A25-35 fragment is highly lipophilic (KP > 102);
the peptide intercalates deep into the membrane bilayer hydrocarbon core, as determined by small angle x-ray diffraction approaches (15).
In addition, aggregated A1-40 has strong electrostatic interactions with the surface of model membranes that appear to mediate
its neurotoxicity (17). It was also reported that soluble A1-40 has pronounced effects on the molecular
anisotropy properties of rat SPM samples, consistent with
peptide-induced changes in the conformation of membrane lipid
constituents (14).
on membrane
structure and lipid dynamics were evaluated using a combination of
small angle x-ray diffraction and fluorescence spectroscopy approaches.
X-ray diffraction analyses were utilized to compare the molecular
structures of reconstituted and intact neuronal membranes following the
addition of soluble versus aggregated A
(A1-40 and A1-42). In parallel
experiments, fluorescence spectroscopy techniques were carried out
using pyrene as a fluorescent probe to quantify the effects of A on
both bulk and annular lipid fluidity. The results of these experiments
provide new insights into the physico-chemical interactions of A
with neuronal membranes as a function of the peptide aggregation state.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-40 (lot ZN571) and
A1-42 (lot ZN327) were purchased from Bachem California
(Torrance, CA). The silver staining kit was from Bio-Rad. Benzo
[def]phenanthrene (pyrene; P-2146) and all other chemicals
were purchased from Sigma. Porcine brain phosphatidylcholine was
purchased from Avanti Polar Lipids (Alabaster, AL). The fatty acid
composition of the brain phosphatidylcholine lipids included: 16:0
(30%), 18:1 (30%), 18:0 (14%), 18:2 (9%), 20:4 (6%), and 22:6
(3%), as determined by gas-liquid chromatographic analysis. The
overall ratio of saturated to unsaturated fatty acids was 0.8:1.
1-40 and
A1-42--
A1-40 and
A1-42 in 1.0 ml of distilled water were individually
incubated for 0 and 48 h in darkness with continuous stirring at
37 °C. The peptide aggregates formed were mixed with glycerol and
separated with 11.5% nondenaturing gels. The following protein
reference standards were used: aprotinin, 
-lactalbumin, trypsin
inhibitor, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, egg albumin, and bovine albumin. The gels were run at 70 min at constant amperage (60 mA/gel) using Bio-Rad Powerpac 200. Bands
were visualized using silver staining. Optical density and molecular
weights were determined using Eagle Eye II video system and EagleSight
software (version 3.2).
1-40 or
A1-42 was added to the dried lipid to yield a final
protein:lipid mass ratio of 1:10.
for
x-ray diffraction analysis were oriented by centrifugation. The
membrane suspensions were centrifuged in a Sorvall AH-629 swinging
bucket ultracentrifuge rotor (DuPont) at 35,000 × g
for 90 min at 5 °C in Lucite sedimentation cells, each containing an
aluminum foil substrate (25). For these experiments, 250 µg of
phospholipid was used for each sample. The mole ratio of
A1-40 to phospholipid in the samples was 1:23 for the
SPM samples and 1:55 for the reconstituted brain membrane samples.
These ratios were selected based on previous analyses with shorter A
fragments (15) and designed to produce detectable differences in
electron density. Following centrifugation, the supernatants were
removed, and each membrane multilayer pellet was mounted on a curved
glass support and suspended overnight in a humidity chamber containing
a saturated salt solution. The oriented membrane samples were then
placed in sealed brass canisters with thin aluminum foil windows in
which temperature and relative humidity were controlled (as described
above). The oriented SPM samples were aligned at near-grazing incidence
with respect to a collimated x-ray beam. The radiation source was a
monochromatic x-ray (CuK
x-ray, 
= 1.54 Å) from
a Rigaku RU-200 high brilliance rotating anode x-ray generator (Rigaku
USA, Danvers, MA). The diffraction data were collected on both a
one-dimensional position-sensitive electronic detector (Innovative
Technologies, Inc., Newburyport, MA) and two-dimensional PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) plate. The sample-to-detector
distance was 150 mm. Each individual diffraction peak used for the
x-ray diffraction analysis was background-corrected using a linear
subtraction routine that averaged the noise. The intensities used for
these analyses were at least 3 orders of magnitude above background
noise. At least six separate x-ray diffraction experiments were
conducted for membranes prepared in the absence and presence of soluble
versus aggregated A analogs. The lamellar intensity
functions from the oriented membrane samples were corrected by a factor
of s = 2sin
/, the Lorentz correction, in which
is the wavelength of the x-ray radiation (1.54 Å), and is the
Bragg angle equal to one-half of the angle between the incident beam
and the scattered beam. A swelling analysis was used to assign
unambiguous phases to the experimental structure factors (26).
1-40 that had been incubated
for 0 or 48 h. SPM (50 µg of protein) were added to PBS control
buffer or PBS containing A1-40 and were incubated for
30 min in a thermostated water bath at 36.5 °C with continuous
shaking in darkness. Samples were then transferred to a 1.0-ml quartz
cuvette and placed in a thermostated cuvette chamber. Pyrene
(10
5 M in 1.0 µl of 102
M solution in dimethylformamide) was added to the sample at
the rate of 1.0 µl/min with constant stirring. Pyrene was then
excited 1 min later through energy transfer from tryptophan (excitation = 286 nm), and fluorescence emission spectra of pyrene were recorded. Considering that the Forster radius (the energy transfer-limiting distance) for the tryptophan-pyrene donor-acceptor pair is 3 nm (27),
only pyrene located in the annular lipid (adjacent to proteins) was
excited, and the fluidity of the annular lipid was considered
proportional to the ratio
Fe/Fm, where
Fe and Fm are the
fluorescence intensities of pyrene eximer (emission = 480 nm) and
monomer (emission = 373 nm), respectively. Pyrene was then excited
at 334 nm, and the bulk fluidity was considered to be proportional to
the ratio Fe/Fm obtained
with the 334-nm excitation wavelength.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-40--
The effects of A on membrane structure
were evaluated following the addition of freshly solubilized and
preincubated (48 h) peptide. Fig. 1 shows
that incubation of A1-42 (Panel A) and
A1-40 (Panel B) for a 48-h period produced
peptide aggregation, as determined by gel electrophoresis. However, the distribution of the molecular weights of A1-40 and
A1-42 were quite different. Dimers, trimers, and
polymers were observed for A1-40 that had been
incubated for 48 h (Fig. 1B). Approximately 44% of
A1-40 that had been incubated for 48 h was in a dimeric form. Dimers of A1-40 (10%) were also noted
immediately following peptide solubilization, but trimers and polymers
were not detected. Distribution of A1-42 revealed
monomeric and tetrameric forms (Fig. 1A); a 48 h
incubation of A1-42 resulted in 74% of the peptide as
a tetramer, whereas the 0-h incubation showed only 11% of the peptide
as a tetramer.
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Fig. 1.
Aggregation of
A 1-42 and
A1-40. A peptides were
incubated in 1.0 ml of distilled water for 0 or 48 h in darkness
with continuous shaking at 37 °C. The samples were examined by
electrophoresis on 11.5% nondenaturing gels as described under
"Experimental Procedures." Panel A is
A1-42 at 0 (lane 1) and 48 h
(lane 2) incubation. Panel B is
A1-40 at 0 (lane 3) and 48 h
(lane 4) incubation. Standards (std) were
aprotinin, -lactalbumin, trypsin inhibitor, carbonic anhydrase,
glyceraldehyde-3-phosphate dehydrogenase, egg albumin, and bovine
albumin.
1-40 on SPM
Structure--
Small angle x-ray diffraction data from oriented SPM
samples produced five strong, reproducible diffraction orders at
10 °C (Fig. 2A). The unit
cell periodicity, or d space (the distance from the center
of one membrane to the next, including surface hydration), for the
control was 85.2 ± 0.3 Å. Following addition of soluble
A1-40 or aggregated A1-40 at a peptide to phospholipid mole ratio of 1:23, the d space values were
reduced to 77.9 ± 0.3 and 75.1 ± 0.3 Å, respectively.
One-dimensional electron density profiles generated from the
diffraction data by Fourier analysis indicated a centrosymmetric
membrane bilayer structure. The primary reason for a centrosymmetric
structure is that the stacking of the membranes results in a random
orientation for the SPM with respect to the multilayer direction. The
SPM electron density profiles were normalized to the phospholipid headgroup region, a region of relatively high electron density, after
applying identical correction factors to the raw diffraction data (28,
29).
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Fig. 2.
Representative x-ray diffraction patterns
from oriented rat cortical SPM samples (A) and
reconstituted porcine brain phospholipid bilayers (B)
at 10 °C and 74% relative humidity. The diffraction data were
collected on a one-dimensional position-sensitive electronic detector,
as described under "Experimental Procedures." A maximum of either
five or four diffraction orders were collected for the SPM and
reconstituted membrane samples, respectively.
1-40
(Figs. 3 and
4). The addition of soluble
A1-40 produced a broad increase in electron density
±0-16 Å from the center of the membrane concomitant with a 5 Å reduction in the intrabilayer headgroup separation from 51 Å to 46 Å.
The large increase in electron density associated with the center of
the membrane suggests that soluble A1-40 intercalates
deep into the membrane hydrocarbon core, a region containing
hydrophobic, phospholipid acyl chains. As a result of disrupting the
intermolecular packing of membrane phospholipid acyl chains, the
overall membrane width was effectively reduced by
A1-40. Similar changes in membrane width have been
observed as a function of disordering the membrane hydrocarbon core by
either reducing membrane cholesterol content or increasing the sample
thermal energy (30, 31). The addition of soluble A1-40
also produced a reduction in peak width at half-maximum corresponding
to the phospholipid headgroup region by 2 Å. This finding indicates
that the motion of the phospholipid headgroup is more restricted in the
presence of soluble A1-40, as compared with control SPM
samples.
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Fig. 3.
A, superimposed one-dimensional electron
density profiles for SPM samples in the absence (solid line)
and presence (dashed line) of soluble
A 1-40. The d space values for the samples
were 85.2 Å (control) and 77.9 Å (soluble A) at 10 °C. The mole
ratio of A1-40 to phospholipid was 1:23. B,
superimposed one-dimensional electron density profiles for SPM samples
in the absence (solid line) and presence (dashed
line) of aggregated A1-40. The d space
values for the samples were 85.2 Å (control) and 75.1 Å (aggregated
A1-40) at 10 °C. The mole ratio of
A1-40 to phospholipid was 1:23.
View larger version (17K):
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Fig. 4.
A, superimposed one-dimensional electron
density profiles for reconstituted porcine brain phospholipid bilayer
samples in the absence (solid line) and presence
(dashed line) of soluble A 1-40.
The d space values for the samples were 52.8 Å (control)
and 53.6 Å (soluble A1-40) at 10 °C. The mole ratio
of A1-42 to phospholipid was 1:55. B,
superimposed one-dimensional electron density profiles for
reconstituted porcine brain phospholipid bilayer samples in the absence
(solid line) and presence (dashed line) of
aggregated A1-40. The d space values for the
samples were 52.8 Å (control) and 55.5 Å (aggregated
A1-40) at 10 °C. The mole ratio of
A1-40 to phospholipid was 1:55.
1-40 to SPM samples
produced changes in membrane structure that were distinct from those observed with soluble A1-40. Instead of an increase in electron density associated with the membrane hydrocarbon core, a
marked decrease in electron density was observed throughout this region of the membrane following the addition of aggregated A1-40. Aggregated A1-40 also produced a
broad increase in electron density associated with the SPM phospholipid
headgroup and hydrated surface ±25-36 Å from the center of the
membrane. These differences in electron density suggest that aggregated A1-40 is associated primarily with the hydrated surface of the SPM samples, as opposed to the phospholipid acyl chains. Moreover, the presence of A1-40 in this region of the
membrane can effect a reorganization in the hydrocarbon core acyl
chains, resulting in increased molecular volume, as evidenced by the
decrease in hydrocarbon core electron density. These changes in
membrane structure associated with the addition of either soluble or
aggregated A1-40 were observed over a broad range of
temperature (10-40 °C) and relative humidity levels (72-93%).
1-40 and
A1-42 on Reconstituted Brain Membrane
Structure--
Small angle x-ray diffraction data from oriented SPM
samples produced four strong, reproducible diffraction orders (Fig.
2B). The interactions of soluble and aggregated A analogs
with lipid vesicles reconstituted from brain phosphatidylcholine were
determined by using small angle x-ray diffraction approaches. Despite
the fact that protein was not present in these vesicles, the molecular locations of soluble and aggregated A1-40 peptide were in large part similar to that observed for the intact SPM preparation (Figs. 5 and
6). The unit cell periodicity, or
d space, for the control brain lipid bilayer was 52.8 ± 0.3 Å at 20 °C. Following addition of soluble or aggregated
A1-40 at a peptide to phospholipid mole ratio of 1:55,
the d space values were 53.6 ± 0.3 and 55.5 ± 0.3 Å, respectively. One-dimensional electron density profiles
generated from the diffraction data indicated a centrosymmetric
membrane structure, as predicted for a model lipid bilayer preparation.
In the presence of soluble A1-40, there was a broad
increase in electron density associated with the hydrocarbon core
±0-18 Å from the center of the membrane. The addition of aggregated
A1-40 to the reconstituted brain membranes produced
distinct changes in electron density. Specifically, the addition of
soluble A1-40 produced an increase in electron density
associated with the glycerol backbone/phospholipid head group region of
the lipid bilayer, extending ±11-27 Å from the center of the
membrane. The addition of aggregated A1-40 effected a
broad reduction in electron density associated with the
membrane hydrocarbon core (±0-10 Å), similar to that observed with
the SPM samples. By contrast, aggregated A1-42 had different interactions with the membrane lipid vesicles when compared with A1-40. Both soluble and aggregated forms of
A1-42 contributed electron density only to the membrane
hydrocarbon core over a broad region extending ±0-12 Å from the
membrane center (Fig. 5) and had no effect on overall membrane width or
d space. The effect of A on membrane structure was
dose-dependent.
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Fig. 5.
A, superimposed one-dimensional electron
density profiles for reconstituted porcine brain phospholipid bilayer
samples in the absence (solid line) and presence
(dashed line) of soluble A 1-42.
The d space values for the samples were 54.8 Å (control)
and 54.8 Å (soluble A1-42) at 10 °C. The mole ratio
of A1-42 to phospholipid was 1:59. B,
superimposed one-dimensional electron density profiles for
reconstituted porcine brain phospholipid bilayer samples in the absence
(solid line) and presence (dashed line) of
aggregated A1-42. The d space value for the
sample containing aggregated A1-42 was 54.8 Å at
10 °C. The mole ratio of A1-42 to phospholipid was
1:59.
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Fig. 6.
Effects of soluble
A 1-40 on tryptophan and
pyrene fluorescence in SPM. Lines a and b,
SPM endogenous tryptophan in the absence or presence of
106 M A1-40. Line
c, SPM endogenous tryptophans and pyrene in the absence of
A1-40. Line d, SPM endogenous tryptophans
and pyrene in the presence of 106 M
A1-40. The eximer/monomer fluorescence ratio
(Fc/Fm) for pyrene when
the probe was excited through energy transfer from SPM tryptophan was
used as an indicator of annular fluidity.
1-40 on Annular
and Bulk Fluidity--
Annular fluidity and bulk membrane fluidity
were determined by using the excimer/monomer fluorescence intensity
ratio of the fluorescent probe pyrene. Annular fluidity was measured
when pyrene was excited through radiationless energy transfer from SPM
proteins containing tryptophan (excitation = 286 nm). Bulk
fluidity was determined when pyrene was excited at 334 nm. The
fluorescence intensity of SPM endogenous tryptophans is shown in Fig. 6
(line a); this fluorescence intensity was markedly decreased
when pyrene was added (Fig. 6, line b). The observed
reduction in fluorescence intensity of tryptophan in the 330-350 nm
wavelength interval was due to energy transfer from tryptophan to
pyrene (excitation = 334 nm). The result of energy transfer was
the observation of maximas of pyrene monomers in the wavelength
interval 370-400 nm and a hill-shaped portion of the spectra with a
maxima typical for the pyrene excimer at 480 nm. The ratio of
intensities associated with the pyrene excimer at 480 nm and pyrene
monomer at 373 nm was used to calculate annular fluidity when pyrene
was excited through energy transfer from SPM tryptophans (Fig. 6).
There was a large increase in the pyrene excimer fluorescence intensity at 480 nm with little if any effect on the fluorescence intensity of
pyrene monomer at 373 nm when A1-40 was added to the SPM samples (Fig. 6, line c). The means ± S.E.
(Fe/Fm) of annular fluidity of the control SPM and SPM incubated with soluble
A1-40 were 0.435 ± 0.012 and 0.474 ± 0.014, respectively, and were significantly different
(p 
0.01). A1-40 that had been
incubated for 48 h prior to being added to SPM also significantly
(p 0.01) increased annular fluidity
(Fe/Fm = 0.467 ± 0.009).
1-40 was
added to SPM, there was a marked increase in pyrene excimer
fluorescence intensity at 480 nm with no effect on the fluorescence
intensity of pyrene monomer at 373 nm (Fig. 7). The means ± S.E.
of bulk fluidity of the control SPM and SPM + A1-40
were 0.448 ± 0.009 and 0.477 ± 0.008, respectively, and
were significantly different (p 0.01). Bulk fluidity
of SPM was also significantly (p 0.01) increased by
A1-40 that had been incubated for 48 h prior to
being added to SPM (Fe/Fm = 0.469 ± 0.006).
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Fig. 7.
Effects of soluble
A 1-40 on pyrene
fluorescence in SPM. Pyrene was excited at 334 nm. Maximas at 373 and 480 nm represent monomer and excimer intensities, respectively. The
ratio of eximer and monomer intensities was used as an indicator of
bulk fluidity. Line a, SPM control, no
A1-40. Line b, SPM incubated with
106 M A1-40. An increase in
pyrene excimer fluorescence intensity was observed in the presence of
A1-40 as compared with control SPM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
that accumulates progressively in the limbic and cerebral cortices. A peptides have been shown to modify various cellular functions, leading to increased neuronal vulnerability. It has been
recently reported that the effects of A on neuronal viability were
not mediated by specific receptor interactions (12) but potentially by
changes in the structure and dynamics of membrane lipid constituents
(17). Therefore, the purpose of the present experiments was to examine
the effects of both soluble and aggregated A on membrane structure
using a combination of x-ray diffraction and fluorescence spectroscopy approaches.
1-42 and
A1-40 to AD pathogenesis has been the subject of
numerous studies. There is evidence that A1-42 is
important in the early development of AD pathology because of its
association with diffuse plaques, loosely aggregated deposits of
amyloid protein (32-34). In addition, as compared with
A1-40, A1-42 has been shown to have a
greater aggregation potential (35, 36) and is produced in relatively
higher amounts in familial forms of the disease, including presenilin
and amyloid precursor genetic alterations (for review see Refs. 32 and
37). However, immunohistochemical studies have shown that
A1-40 is the predominant form in neuritic plaques, a
lesion that develops later in the disease process (34, 38, 39). Studies
by Shin et al. (40) suggest that A1-42 is
essential to the early development of AD pathology but not sufficient
to promote the formation of mature, neuritic plaques unless succeeded
by A1-40 deposition. These findings support a
"seeding" hypothesis that aggregates of A1-42 act
as the initiation factors for early plaque formation followed by
progressive accumulation of A1-40 in the AD brain (35,
36). These previous studies suggest that A1-42 and
A1-40 likely have distinct roles in neuritic formation
and justify the need to examine their separate effects on membrane
structure and neuronal behavior.
1-40 peptide. The dimensions of the intact
SPM sample were significantly larger than those observed for
reconstituted brain lipid bilayers (52.8 Å). Differences in the
dimensions of the intact versus reconstituted brain lipid bilayer are attributed to the presence of integral and surface membrane
proteins. Direct subtraction of electron density profiles showed that
soluble and aggregated A1-40 interacted with different
regions of the SPM sample. Soluble A1-40 was located in
the hydrophobic core region of the SPM, whereas aggregated A1-40 was associated with the phospholipid headgroup or hydrophilic area of SPM, as illustrated in Fig.
8. The distinct membrane interactions
observed following the addition of soluble and aggregated
A1-40 were reproduced in a reconstituted porcine brain
lipid bilayer system consisting of phosphatidylcholine with
heterogeneous acyl chains. However, the pronounced effects of
A1-40 on the overall dimensions of the SPM preparation were not reproduced in the model membrane preparation. The different effects of A1-40 on SPM structure may be attributed to other constituents in the SPM system, such as protein and other lipid
molecules. Interestingly, both soluble and aggregated
A1-42 interacted only with the membrane lipid bilayer
hydrocarbon core, as evidenced by a broad increase in electron density.
This finding suggests that the longer A derivative has stronger
hydrophobic properties that would result in van der Waals'
interactions with the phospholipid acyl chains. The additional two
nonpolar amino acids (isoleucine and alanine) associated with the
carboxyl terminus of A1-42 may also contribute to its
greater aggregation rate in solution, as compared with
A1-40 (36, 41-43).
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Fig. 8.
A schematic representation of the proposed
molecular membrane interactions of soluble versus
aggregated A 1-40 with
synaptic plasma membranes, based on the results of this study.
Soluble A1-40 intercalates deep into the plasma
membrane hydrocarbon core, whereas aggregated A1-40
interacts with the membrane bilayer at the headgroup/water interface,
resulting in a marked reorganization of the lipid bilayer, including
increased trans-gauche isomerizations.
1-40 increase the fluidity of SPM samples.
Unlike the x-ray diffraction findings, the fluorescence anisotropy
measurements could not distinguish differences between soluble
versus aggregated A1-40. Effects of soluble
A1-40 on SPM fluidity are consistent with findings
reported earlier (14). Another study, however, found that
A1-40 reduced fluidity of a brain homogenate
preparation (13). In both our previous study and the current study, we
used isolated SPM. In addition, fluidity in SPM was determined using
the pyrene probe, whereas polarization of the fluorescent probe
diphenylhexatriene was used in the brain homogenate preparation. A
appears to behave differently in a membrane preparation, as compared
with a heterogenous brain homogenate system (13).
1-40 may explain the reported effects of A on
permeability of neurons to Ca2+, Na+, and
K+ and the increase in KCl-induced neuronal
Ca2+ in brain neurons and lymphocytes (5, 44). It has been
previously reported that changes in the fluidity of the membrane-lipid
environment can alter the function of ion channels (45). Ethanol, for
example, also increases membrane fluidity, enhancing Ca2+
flux into neurons (46). The combined physico-chemical effects of A
on bulk and protein annular fluidity may contribute to mechanisms of
neurotoxicity (17). A-induced membrane structure changes may also
lead to further increases in A production by increasing the access
of certain proteases to abnormal, membrane-associated cleavage sites on
amyloid precursor protein following disruptions in the organization of
the lipid bilayer.
interacts with lipids in a
multifaceted manner. Differences in the location of soluble and
aggregated A1-40 in SPM observed in the present study may have resulted from interactions of soluble versus
aggregated A1-40 with distinct regions of membrane
lipids. It has been demonstrated that amyloid fibril formation can be
induced by A1-40 binding to membrane vesicles
containing gangliosides (47) and vesicles reconstituted from acidic and
negatively charged phospholipids (48, 49). We recently reported that
A1-40 aggregates bind fluorescent-labeled cholesterol,
phosphatidylcholine, and stearic acid (50). The affinity of cholesterol
for aggregated A1-40 was significantly higher as
compared with other lipids that were examined. Thus, membrane
cholesterol may act as an anchor for polymers of A1-40.
It was observed that significant binding of cholesterol to
A1-40 occurred after polymers were formed and that
membranes enriched in cholesterol may be more susceptible to deposition
of A1-40 (50). For example, the exofacial or outer
leaflet of SPM from aged mice contains more than twice the amount of
cholesterol present in the exofacial leaflet of SPM from young mice
(20). This large difference in the transbilayer or asymmetric
distribution of cholesterol could hinder either the uptake into or
efflux of A1-40 from membranes, resulting in the
accumulation and deposition of A in membranes.
has not yet been
elucidated. A alters several different cell functions, which would
argue against a specific cellular target, such as a receptor. The
results of this study support a growing body of data suggesting that
A has strong physico-chemical interactions with membranes, leading
to alterations in the lipid bilayer environment and loss of neuronal
membrane function.
FOOTNOTES
ABBREVIATIONS
, amyloid -peptide;
SPM, synaptic plasma membranes;
PBS, phosphate-buffered saline.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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