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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 36, Issue of September 7, pp.

33561–33568, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Cellular Membrane Composition Defines A␤-Lipid Interactions*


Received for publication, April 23, 2001, and in revised form, June 19, 2001
Published, JBC Papers in Press, July 3, 2001, DOI 10.1074/jbc.M103598200

Stephen A. Waschuk‡, Elyssa A. Elton‡, Audrey A. Darabie‡, Paul E. Fraser‡§, and JoAnne
McLaurin‡¶储
From the ‡Centre for Research in Neurodegenerative Diseases, Departments of §Medical Biophysics and ¶Laboratory
Medicine and Pathology, University of Toronto, Toronto, Ontario M5S 3H2, Canada

Alzheimer’s disease pathology has demonstrated ation with membrane structures. Recent studies have demon-
amyloid plaque formation associated with plasma strated that plaque formation may be initiated in a plasma
membranes and the presence of intracellular amyloid-␤ membrane form (3, 4) and that A␤ deposition in aged dogs is
(A␤) accumulation in specific vesicular compartments. associated with the extracellular leaflet of the plasma mem-
This suggests that lipid composition in different compa- brane (5). Furthermore, the intracellular accumulation of A␤ in
rtments may play a role in A␤ aggregation. To test this lysosomal or late endosomal vesicles in vitro suggest that these
hypothesis, we have isolated cellular membranes from compartments may be involved in neurotoxicity (6 –9).
human brain to evaluate A␤40/42-lipid interactions. Pla- A␤ is generated from the proteolytic cleavage of the amyloid
sma, endosomal, lysosomal, and Golgi membranes were precursor protein in the endoplasmic reticulum to generate
isolated using sucrose gradients. Electron microscopy A␤42 and the trans-Golgi network to generate A␤40 (10 –14). It
demonstrated that A␤ fibrillogenesis is accelerated in

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has also been suggested that A␤40/42 may also be generated at
the presence of plasma and endosomal and lysosomal
the plasma membrane surface. The presence of A␤ in distinct
membranes with plasma membranes inducing an enha-
compartments and the proposal that lipid association is impor-
nced surface organization. Alternatively, interaction of
A␤ with Golgi membranes fails to progress to fibril tant for both neurotoxicity and fibrillogenesis suggest that the
formation, suggesting that A␤-Golgi head group intera- lipid composition and characteristics of these compartments
ction stabilizes A␤. Fluorescence spectroscopy using the may play vital roles in the disease process. Previous studies
environment-sensitive probes 1,6-diphenyl-1,3,5-hexat- (15, 16) have demonstrated accumulation of A␤42 in lysosomal
riene, laurdan, N-⑀-dansyl-L-lysine, and merocyanine 540 compartments that results in membrane damage as shown by
demonstrated variations in the inherent lipid propert- release of lysosomal hydrolases and the lysosomal specific dye
ies at the level of the fatty acyl chains, glycerol bac- acridine orange. Furthermore, A␤-synaptic plasma membrane
kbone, and head groups, respectively. Addition of interactions demonstrate that A␤ has a fluidizing effect on
A␤40/42 to the plasma and endosomal and lysosomal membrane structure as a result of A␤ insertion into the fatty
membranes decreases the fluidity not only of the fatty acyl chain region of the bilayer (17). The role of proteins asso-
acyl chains but also the head group space, consistent ciated with synaptic plasma membranes could not be distin-
with A␤ insertion into the bilayer. In contrast, the Golgi guished from A␤-lipid interactions alone in this study.
bilayer fluidity is increased by A␤40/42 binding which Therefore, we undertook the examination of A␤40 and A␤42
appears to result from lipid head group interactions and in the presence of bilayers formed from lipids isolated from
the production of interfacial packing defects. post-mortem human cortical gray matter. We chose to evaluate
the membranes involved in both the production of A␤, Golgi
and endosomal, and A␤ pathology, plasma and lysosomal mem-
Alzheimer’s disease is an age-related disorder that is char- branes. In order to distinguish between A␤ lipid and A␤ protein
acterized by progressive cognitive decline and neurodegenera- interactions in these compartments, we extracted the lipid
tion (1, 2). Pathological examinations have demonstrated that component and used this as our model membranes. The effects
one of the key features is the presence of amyloid plaques of A␤ were examined as a consequence of sequence, structure,
associated with neuritic degeneration. Senile plaques are com- and concentration, all of which are factors affecting A␤ assem-
posed predominantly of a 40 – 42-residue peptide, amyloid-␤ bly and neurotoxicity. In order to address potential mecha-
(A␤40/42). The development of Alzheimer’s disease pathology nisms to help explain the pathological findings, we examined
has been proposed to be the result of A␤1 deposition in associ- the ability of these membranes to facilitate A␤40/42 assembly
into amyloid fibers by electron microscopy. Changes in the
* This work was supported in part by the Medical Research Council of membrane physical characteristics as a result of A␤ interac-
Canada (to J. M.), the Natural Sciences and Engineering Research tions were followed by fluorescence spectroscopy using environ-
Council of Canada (to J. M.), the Ontario Mental Health Foundation (to ment-specific probes.
P. E. F.), the Scottish Rite Charitable Foundation (to P. E. F.),and the
Ontario Alzheimer’s Association. The costs of publication of this article MATERIALS AND METHODS
were defrayed in part by the payment of page charges. This article must Peptides—A␤40/42, A␤-(1–28) were synthesized by solid phase Fmoc
therefore be hereby marked “advertisement” in accordance with 18
(N-(9-fluorenyl)methoxycarbonyl) chemistry by the Hospital for Sick
U.S.C. Section 1734 solely to indicate this fact.
储 Supported by the Bickell Foundation, the Canadian Foundation for Children’s Biotechnology Center (Toronto, Ontario, Canada). They were
Innovation, and Ontario Innovation Trust. Recipient of the Year 2000 purified by reverse phase high pressure liquid chromatography on a
Young Investigator Fund Scholarship. To whom correspondence should C18 ␮Bondapak column. A␤ peptides were initially dissolved in 0.5 ml
be addressed: Centre for Research in Neurodegenerative Diseases, of 100% trifluoroacetic acid (Aldrich), to ensure that the peptide re-
Tanz Neuroscience Bldg., 6 Queen’s Park Crescent West, Toronto, On-
tario M5S 3H2, Canada. Tel.: 416-978-1035; Fax: 416-978-1878; E-mail:
J.mclaurin@utoronto.ca. lysine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; DPH, 1,6-di-
1
The abbreviations used are: A␤, amyloid-␤; DL, N-⑀-dansyl-L- phenyl-1,3,5-hexatriene.

This paper is available on line at http://www.jbc.org 33561


33562 Cellular Membrane-associated A␤ Fibrillogenesis
mained monomeric and free of fibril seeds, diluted in distilled H2O, and
immediately lyophilized (18). A␤ peptides were then dissolved at 1
mg/ml in 40% trifluoroethanol (Aldrich) in distilled H2O and stored at
⫺20 °C until use. Bee venom mellitin was used as a control peptide
(Sigma).
Cellular Membrane Isolation—All cellular membranes were isolated
from post-mortem human gray matter of five male control subjects with
post-mortem intervals of less than 15 h. The male subjects ranged in
age from 76 to 80 years without documented signs of clinical dementia.
The cause of death in all cases was heart failure. Plasma membranes
were isolated using the method of Hubbard et al. (19), endosomes using
the method of Gorvel et al. (20), and Golgi membranes isolated using the
method of Duden et al. (21), all of which rely upon the separation of the
specific fraction by differential migration in sucrose density gradients.
Lysosomal membranes were isolated using the procedure of Storrie and
Madden (22) using flotation on a metrizamide density gradient. Lipids
were extracted from each membrane fraction using chloroform:metha-
nol (2:1) extraction and subsequent concentration under a stream of N2.
The samples were stored at ⫺20 °C until use. Phospholipid concentra-
tion in all samples was determined using the Bartlett assay (23), and
cholesterol concentration was determined using the Amplex red assay FIG. 1. Negative stain electron microscopy of A␤42 in the pres-
(Molecular Probes, Eugene, OR). ence of plasma, endosomal, and Golgi membranes. A␤42 incu-
Electron Microscopy—A␤40/42 peptides were incubated in the pres- bated in buffer alone (A) demonstrates many long intertwined fibers.
ence and absence of total brain lipid extract bilayers at a final peptide When incubated in the presence of plasma membrane (B), a similar
concentration of 100 ␮g/ml. The A␤ to lipid ratio was maintained at 1:20 structure of the fibrils to A␤42 alone could be detected but with in-
(by weight). For negative stain electron microscopy, carbon-coated pi- creased organization along the vesicle surface. Only minor lateral ag-
gregation was apparent in the fibrils formed in the presence of lysoso-

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oloform grids were floated on aqueous solutions of peptides. After the
mal membranes (C). In the presence of Golgi membranes only a few
grids were blotted and air-dried, the samples were stained with 1%
protofibrils of A␤42 were detected (D). Scale bar is 50 nm.
(w/v) phosphotungstic acid and examined on a Hitachi 7000 electron
microscope operated at 75 kV (29, 30).
Steady State Fluorescence Anisotropy—Anisotropy experiments were (DL, Molecular Probes) was incorporated into lipid vesicles in the pres-
performed on a PTI fluorimeter equipped with manual polarizers as ence and absence of A␤. The fluorescence spectra of DL were evaluated
described previously (24). Excitation and emission wavelengths were after 30 min of incubation at room temperature with an excitation
set at 360 and 425 nm with a slit width of 1 and 4 nm, respectively. Our wavelength of 335 nm and emission scan monitored between 380 and
system was initially calibrated using 1,6-diphenyl-1,3,5-hexatriene 580 nm inclusive. The DL to lipid ratio was maintained at 1:500 (26).
(DPH; Molecular Probes, Eugene, OR) in mineral oil, which should give Merocyanine 540 Absorption Spectroscopy—Merocyanine 540
an anisotropy equal to 1. The g factor was calculated using horizontally (MC540, Molecular Probes) absorption spectra were obtained at room
polarized excitation and subsequent comparison of the horizontal and temperature on a Beckman spectra DU530. The dye was added to
vertical emissions, which for our machine is 0.883. Lipid vesicles were preformed vesicles at a probe:lipid ratio of 1:500 (27). Final MC540
diluted to 250 ␮g/ml in phosphate-buffered saline, incubated for 20 –30 molar concentration in the cuvette was 21.3 ⫻ 10⫺6 M. Absorption
min in the presence and absence of A␤, and then subsequently incu- spectra were obtained between 400 and 600 nm with 1-nm steps. The
bated for a further 30 min with DPH at a 1:500 probe:lipid ratio. lipid-alone base line in the absence of MC540 was subtracted from all
Fluorescence intensity was measured with the excitation polarizer in spectra, and the corresponding spectra are shown in Equation 4,
the vertical position and the analyzing emission polarizer in the vertical
(IVV) and horizontal (IVH) positions; and anisotropy, r, was calculated A ⫺ 关␧D ⫻ C/2兴 共C ⫺ 关monomer兴兲
using Equation 1, 关monomer兴 ⫽ 关dimer兴 ⫽
␧m ⫺ ␧D/2 2
(Eq. 4)
IVV ⫺ gIVH and were then corrected by referring the absorbances at 600 nm to 0.
r⫽ (Eq. 1)
IVV ⫹ 2gIVH After this correction, the absorbance values at 569 nm were used to
calculate the dimerization constant (Kd(app)) as by Bernik and Disalvo
Lipid vesicles in the absence of DPH were measured in order to evaluate (28), see Equation 5.
the effect of light scattering on our measurements. Poly-L-lysine and
bovine serum albumin were used as negative controls for the anisotropy
studies. 关dimer]
Laurdan Generalized Polarization—Steady state excitation and emis- Kapp ⫽ (Eq. 5)
[monomer]2
sion spectra were collected on the PTI fluorimeter. Laurdan (Molecular
Probes) was added to preformed lipid vesicles in the presence and absence where A is the absorbance at 569 nm, ⑀ is the constant for MC540 dimer
of A␤ at a 500:1 lipid:probe ratio. The laurdan generalized polarization or monomer at the given wavelength, ⑀m ⫽ 1.511 ⫻ 105 and ⑀D ⫽ 5400,
(GP) parameter as developed by Parasassi et al. (25) is calculated as and C is the final MC540 concentration.
follows. The emission GP parameter is given by Equation 2.
RESULTS
A␤ Morphological Characteristics—Lipid bilayers have been
I400nm ⫺ I340nm
GPem ⫽ (Eq. 2) shown to affect the assembly of A␤ peptides into amyloid fibers
I400nm ⫹ I340nm
(29 –31). In order to determine if A␤ interactions with different
where I400 nm and I340 nm are the fluorescence intensities measured at cellular membranes affects fibrillogenesis, we examined A␤
all emission wavelengths within 420 and 520 nm. By using fixed exci- structural characteristics in the presence of vesicles formed
tation wavelength of 400 nm and 340 nm, respectively. The excitation from Golgi, plasma, lysosomal and endosomal lipids by nega-
GP is given by Equation 3,
tive stain electron microscopy. In the absence of lipid, A␤ as-
sembles into long fibers of varying length, 350 – 430 Å, with a
I440nm ⫺ I490nm characteristic helical twist of 100 Å (Fig. 1A). These fibers
GPex ⫽ (Eq. 3)
I440nm ⫹ I490nm demonstrated varying extent of lateral aggregation of fibers
into larger bundles, from 50 Å representing single fibers to
where I440 nm and I490 nm are the fluorescence intensities at each exci-
tation wavelength from 320 to 420 nm, measured at fixed emission 200-Å diameter bundles. In the presence of plasma lipid vesi-
wavelengths of 440 and 490 nm, respectively. cles, A␤ assembled into fiber bundles along the surface of the
N-⑀-Dansyl-L-lysine Fluorescence Spectroscopy—N-⑀-Dansyl-L-lysine bilayer (Fig. 1B). A␤ fibers were not found on the surface of the
Cellular Membrane-associated A␤ Fibrillogenesis 33563
TABLE I
Steady state anisotropy measurements of various cellular membranes in the presence and absence of A␤40/42
Anisotropy was measured using DPH fluorescence at a probe:lipid ratio of 1:500. Peptide was added to lipid vesicles at a 1:20 ratio with a final
peptide concentration of 10 ␮M. ND indicates not determined.
Anisotropy
Sample
Golgi Plasma membrane Endosomal Lysosomal
Control 0.295 ⫾ 0.004 0.221 ⫾ 0.000 0.186 ⫾ 0.002 0.232 ⫾ 0.0002
A␤40 0.284 ⫾ 0.002a 0.259 ⫾ 0.001a 0.196 ⫾ 0.001b 0.268 ⫾ 0.0002a
A␤42 0.284 ⫾ 0.004a 0.263 ⫾ 0.008b 0.196 ⫾ 0.0002 0.267 ⫾ 0.019b
Seeded A␤40 0.314 ⫾ 0.011b 0.273 ⫾ 0.009b ND ND
Seeded A␤42 0.303 ⫾ 0.008b 0.276 ⫾ 0.040b ND ND
A␤-(1–28) 0.297 ⫾ 0.002 0.231 ⫾ 0.004 ND ND
Mellitin 0.294 ⫾ 0.003 0.228 ⫾ 0.008 ND ND
a
p ⬍ 0.001.
b
p ⬍ 0.01 by Student’s t test.

bilayers nor in areas devoid of lipid vesicles, suggesting that A␤ TABLE II


Cholesterol and phospholipid analysis
assembly was driven as a result of interaction with the lipid
Cholesterol content in all membranes was determined using the
surface. These results are similar to those that we have re-
Amplex red assay, and phospholipid content was determined using the
ported previously for A␤ interaction with phosphatidylinositol/ Bartlett assay.
brain phosphatidylcholine and total brain lipid vesicles (32,
Molar ratio cholesterol:phospholipid
33). Both endosomal (data not shown) and lysosomal (Fig. 1C)
Experimental Literature citation
vesicles demonstrated A␤ fibers associated with both the edges values values
and surface of the vesicles. Although no fibers were detected in

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Golgi membrane 0.27 0.45–0.5
the absence of lipids, the level of A␤ lateral aggregation and Plasma membrane 0.49 0.4–1.0
organization was less than that detected for the plasma mem- Endosomal 0.08 0.1–0.2
brane lipids. A␤ incubated in the presence of Golgi lipid vesi- Lysosomal 0.47 0.5
cles was almost devoid of A␤ fibers (Fig. 6D). The odd fiber
could be found across the grid but was not intimately associ-
ated with lipid vesicles, and many areas of lipid vesicles could terol level yet still exhibited a fluid membrane structure.
be found devoid of fibers. The odd fiber detected in the presence Therefore, the properties of the different bilayers represent
of Golgi vesicles had morphological characteristics of A␤ pro- differences in the total lipid composition rather than choles-
tofibrils. The Golgi lipid vesicles are reminiscent of our previ- terol per se.
ous results in which we were unable to identify A␤ fibrils in the Previous reports (17, 34 –36, 39) have shown that the addi-
presence of ganglioside/phosphatidylcholine membranes when tion of A␤40 or A␤25–35 to synthetic membrane preparations
A␤ was added as a randomly structured peptide (29). or membranes isolated from red blood cells results in a reduc-
Fatty Acyl Chain Mobility—In order to characterize differ- tion in membrane fluidity. Many A␤ properties have been
ences in the cellular bilayer properties and determine which is linked to the conformation and aggregation state of the peptide.
most influential in determining A␤ fibrillogenesis, we exam- In order to investigate the interactions of A␤40 and A␤42 with
ined the influence of A␤40/42 on the physical properties of lipid bilayers, we chose to examine initially soluble, random
these bilayers. Many fluorescent dyes are available that pene- structured peptide with bilayers. To ensure that A␤ peptides
trate to varying levels into the lipid bilayer and exhibit fluo- meet these criteria and are free of fibril nucleation seeds,
rescent properties indicative of their local environment. We can A␤40/42 peptides were treated with 100% trifluoroacetic acid
utilize these dyes to address the effects of A␤-lipid interactions followed by lyophilization (18). The lyophilized peptide was
within various cellular membranes. Previous studies using immediately solubilized in 40% trifluoroethanol in order to
synthetic lipid bilayers and synaptic plasma membranes have make a 1 mg/ml stock solution. As reported previously (40),
demonstrated a disordering of the fatty acyl chains after inter- A␤40/42 is partly ␣-helical in 40% trifluoroethanol and upon
action with A␤40 and A␤42 (17, 34 –36). In order to determine dilution into phosphate-buffered saline, pH 7.4, A␤40/42 ini-
the effect of A␤40/42 on the mobility of the fatty acyl chains tially adopted a random structure. Our results demonstrate
within bilayers formed from Golgi, endosomal, lysosomal and that addition of randomly structured A␤40 and A␤42 decreased
plasma membrane lipids, we have examined the steady state the membrane fluidity of the plasma membrane, endosomal
fluorescence anisotropy using the dye, DPH (24). The relative and lysosomal membranes in a concentration-dependent man-
motion of the DPH dye molecule within the lipid bilayer is ner as illustrated by an increase in the anisotropy constant
determined by polarized fluorescence and expressed as r, the (Table I and Fig. 2). We could not detect a significant difference
anisotropy constant. This constant is inversely proportional to in the effect of A␤40 and A␤42 on these membranes. In con-
the degree of membrane fluidity. trast, both A␤40 and A␤42 induced a significant increase in
The relative fluidity of the lipid membranes was found to fluidity of the Golgi lipid bilayers as demonstrated by the
vary considerable with Golgi lipid bilayers having the most decrease in the anisotropy constant (Table I). The disordering
rigid structure, whereas plasma membrane, endosomal, and effect of A␤ on Golgi lipid membranes is enhanced by increas-
lysosomal lipid bilayers were more fluid (Table I). Previous ing A␤ concentration (Fig. 2).
studies have shown that synthetic lipid bilayer fluidity is reg- To determine the specificity of A␤40/42-lipid interactions, we
ulated by the amount of cholesterol, which exhibits a bimodal examined A␤-(1–28), which lacks the N-terminal hydrophobic
effect on fluidity with increasing levels of cholesterol (34, 37– region, and bee venom mellitin, a pore-forming peptide. Nei-
39). The cholesterol to phospholipid ratios of the bilayers were ther A␤-(1–28) nor mellitin altered the fluidity of the Golgi
in the range of that reported previously in the literature (Table membranes, whereas both decreased the fluidity of plasma
II). The differences in fluidity detected in this study are not membranes (Table I). These results suggest that the preferen-
solely related to the cholesterol content of these bilayers as the tial increase in Golgi membrane fluidity as a result of A␤40/42
plasma and lysosomal membranes have a significant choles- interaction is peptide- and sequence-specific. It is not surpris-
33564 Cellular Membrane-associated A␤ Fibrillogenesis

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FIG. 2. The effect of A␤ modulation on membrane fluidity of
cellular lipid bilayers was determined by DPH anisotropy. The
FIG. 3. Laurdan emission and excitation spectra of plasma (A)
addition of A␤40 and A␤42 to plasma (A) and Golgi membrane (B)
and Golgi (B) membranes in the presence of increasing concen-
vesicles resulted in contrasting effects on membrane fluidity. Addition
trations of A␤40. Vesicles alone (solid line) and in the presence of 5
of monomeric, randomly structured A␤ decreased the membrane fluid-
(dotted line) and 10 ␮g (dashed line) of A␤40 demonstrate similar
ity of plasma membranes in a concentration-dependent manner,
overall spectral characteristics. The intensity of the spectral maxima
whereas Golgi membrane fluidity was increased. Data represent the
are affected by the addition of A␤; these results demonstrate the in-
mean of at least three separate experiments and are the mean ⫾ S.D.
crease (Golgi membranes) and decrease (plasma membranes) in polar-
Student’s t test indicates the following: *, p ⬍ 0.01; †, p ⬍ 0.001 when
ity of the head group-fatty acyl chain interface.
compared with lipid alone.

ing that mellitin decreases the fluidity of plasma membranes Golgi lipid bilayers demonstrate the characteristic red excita-
as it inserts into membranes to create pores. tion at 340 nm and blue excitation at 380 nm, whereas the
The structural dependence of A␤ effects on membrane fluid- emission spectra indicates a single maximum at 430 nm indic-
ity of plasma membrane and Golgi lipid bilayers was examined ative of blue emission (Fig. 3). The red excitation band intensity
by comparing the random structured peptide with A␤40 and increases in polar solvents, and in hydrogen-bonding solvents,
A␤42 which exhibit ␤-structure. In contrast to the random the red excitation corresponds to the blue emission population
structured peptides, seeded A␤40 decreased the membrane and is especially intense in gel phase lipid bilayers where little
fluidity of both plasma and Golgi membranes (Table I). Similar relaxation occurs. The addition of A␤ to laurdan containing
results were detected for A␤42. This result suggested to us that membranes does not change the shape of either the excitation
A␤ interactions with lipid bilayers is not only dependent on the or emission spectra but affects the intensity of laurdan fluores-
composition of the lipid bilayer but also on the structural char- cence in both plasma and Golgi membranes (Fig. 3). The ratio
acteristics of the peptide. of the blue to red components in the excitation reflects the
Dynamics of Lipid Head Groups and Interface—Besides the polarity of the probe. Addition of A␤40 to plasma membrane
packing of the lipid acyl chains, the dynamics of the polar head bilayers results in an increase in the blue/red excitation ratio
groups and the polarity of the lipid interface are relevant to the from 1.03 to 2.2, indicating that the environment sensed by
interaction of molecules, i.e. A␤, with the membrane surface. In laurdan becomes more hydrophobic after interaction of A␤ with
order to obtain an insight into these properties, laurdan and membranes (Fig. 2). These results suggest that A␤ produces a
N-⑀-dansyl-L-lysine probes were used. Laurdan naphthalene displacement of water molecules from the hydration shell of the
ring is located at the glycerol backbone and is anchored in the membrane, as a result of the promotion of lateral phase sepa-
bilayer by the lauroyl moiety, thereby imparting fluorescence ration and a higher degree of plasma membrane organization.
characteristics that are dependent on the polarity of its envi- Increasing the concentration of A␤ does not further alter the
ronment (25, 41). The advantages of laurdan are that it is blue/red excitation ratio suggesting that the bilayer has a finite
completely non-fluorescent in aqueous environments, is inde- ability to accommodate A␤. The generalized polarization emis-
pendent of pH between 4 and 10, and independent of lipid sion (GPem) for plasma membranes was calculated to be 0.48,
polar head group; therefore fluorescence readings reflect only which increases to 0.57 in the presence of initially random
the polarity of the probe associated with the bilayer. The structured A␤40 and A␤42. The interaction of seeded A␤40/42
spectral properties of laurdan have been described by the demonstrates the same shift of the GPem to 0.56 and 0.54,
general polarization equation for both excitation and emis- respectively, indicating that in the presence of A␤ the mem-
sion spectra, which render information about the lipid phase, brane becomes more structured at the head group-fatty acyl
polarity, and co-existence of multiple lipid phases within a chain interface.
single bilayer (25, 41). In contrast, both Golgi (Fig. 3B) and endosomal (data not
Laurdan excitation spectra in the presence of plasma and shown) bilayer blue/red excitation intensities do not change
Cellular Membrane-associated A␤ Fibrillogenesis 33565
after addition of A␤ suggesting that A␤ binding does not alter
the phase of these lipids. Increasing concentrations of A␤ still
did not induce a change in the ratio of excitation intensities
confirming that these bilayers do not undergo a concentration-
dependent phase transition. The endosomal lipid bilayer exhib-
its similar fluidity as the plasma membrane as illustrated by
DPH studies, yet has different laurdan fluorescence character-
istics suggesting that bilayer-specific lipid composition may
alter the resultant A␤-lipid interactions. In contrast to the
excitation properties of laurdan, the GPem values of Golgi and
endosomal membranes, 0.49 and 0.54, are only affected by the
addition of high concentrations of A␤ demonstrating a decrease
to 0.42 and 0.44 for A␤40. It is interesting that A␤42 has a
lesser effect on the GPem than A␤40 with only modest alter-
ation of the GPem values to 0.47 and 0.51, respectively. These
results suggest that interaction of A␤42 with both Golgi and
endosomal membranes does not alter the micropolarity or hy-
dration of the interfacial region of the lipid bilayer.
To examine the specificity of the changes in laurdan fluores-
cence properties due to A␤40/42 interactions with Golgi and
plasma membranes, we examined the effects of A␤-(1–28) and
mellitin. Addition of A␤-(1–28) and mellitin to both bilayers
does not alter the blue/red excitation ratio. Furthermore, A␤-

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(1–28) does not shift the GPem of either plasma or Golgi mem-
branes. These combined results suggest that A␤-(1–28) does
not alter the micropolarity of the interface and/or insert into
the bilayer. In contrast, mellitin shifts the GPem for Golgi and
plasma membranes from 0.49 and 0.48 to 0.53 and 0.52, re-
spectively. These results suggest that in contrast to A␤ pep-
tides, mellitin binding and pore formation increases the lipid
order after insertion into the bilayer but does not change the
lipid phase.
In order to examine more closely the phase behavior of the
bilayers, the wavelength dependence of both the excitation and FIG. 4. The wavelength dependence of the excitation and emis-
sion generalized polarization of laurdan in plasma (A), endoso-
emission spectra was examined (Fig. 4). Lipid bilayers in a pure mal (B), and Golgi (C) membranes was determined. GP values
gel phase show an independence of generalized polarization were calculated from excitation and emission scans before (solid line)
values as a function of wavelength, whereas liquid crystalline and after addition of A␤42 and 5 (dotted line) and 10 ␮g (dashed line).
bilayers exhibit a dependence on the excitation wavelength Plasma membrane demonstrates a shift in the phase of the lipid bilayer,
whereas endosomal and Golgi membrane phases are unaffected by
(42). Plasma membrane bilayers exhibit a decrease in the GPex addition of A␤42. The laurdan:membrane lipid ratios were 1:500.
and increase in GPem toward shorter wavelengths, an indica-
tion of the co-existence of lipid phases (Fig. 4A). The addition of red shift in the fluorescence maxima after addition of A␤. This
A␤ results in an increase in the slope of the GPem suggesting result suggests that A␤ creates more space between the lipid
that the lipid phase in the bilayer is further altered as a result head groups or causes an increase in the packing defects of
of A␤ binding. Alternatively, the GPex and GPem of Golgi lipid Golgi lipid bilayers.
bilayers is independent of wavelength in the presence and Lipid Head Group Packing and Surface Properties—In order
absence of A␤, confirming that A␤ binding does not alter the to examine the lipid head group spacing and surface properties
lipid phase (Fig. 4B). These results suggest that binding of A␤ of these bilayers, merocyanine 540 absorbance spectral proper-
to Golgi and endosomal membranes can be easily accommo- ties were examined. The spectral characteristics of MC540
dated within the lipid structure, whereas plasma membrane result from binding of monomeric MC540 and subsequent
bilayers undergo a reorganization. dimerization, and both steps are dependent on the packing
The polarity of the lipid interface can be examined using the properties of the lipid head groups (27, 28). MC540 spectra in
fluorescence of N-⑀-dansyl-L-lysine (26, 43); furthermore, it has the presence of plasma membrane is characteristic of mostly
been suggested that DL inserts into cholesterol-free phospho- gel phase lipid head groups, with the characteristic maxima at
lipid domains (44, 45). Due to its molecular structure and 500 and 530 nm (Fig. 5A). A small shoulder is present at 570
location at the interface, DL fluorescence is most sensitive to nm which is characteristic of a small population of monomeric
the packing constraints and hydration. DL exhibits a strong MC540 insertion into the lipid bilayer. These results suggest
fluorescence maximum at 430 nm, which increases in intensity an ordered head group packing in these bilayers as only a small
as a result of A␤-plasma membrane interactions (data not amount of MC540 is inserted into the head group space. Addi-
shown). These results suggest that A␤ increases the polarity of tion of A␤40 to the plasma lipid bilayers decreased the inten-
the interface which is independent of concentration and struc- sity of the MC540 maxima and percent of monomeric MC540
ture. In contrast, both endosomal and Golgi lipid bilayers ex- insertion (Fig. 5B). No difference could be detected between
hibit DL maxima at 430 and 540 and 520 nm, respectively. The random and ␤-structured A␤40 suggesting similar effects on
addition of A␤40 and A␤42 to endosomal bilayers results in a lipid head group rearrangement. A␤42 did not change the
blue shift in the DL maxima; an indication of increased lipid absorbance spectra of MC540, suggesting that A␤42 interac-
packing and was independent of peptide structure (data not tion does not affect the head group packing of the plasma
shown). DL associated with Golgi membranes demonstrated a membrane. On the other hand, similar MC540 spectra results
33566 Cellular Membrane-associated A␤ Fibrillogenesis
FIG. 5. The interaction of A␤40 and
A␤42 with the lipid head groups of
the various cellular membranes was
examined using MC540 absorbance
spectroscopy. MC540 spectra demon-
strate the rigid packing of the plasma
membrane head groups (A, dashed line),
whereas the Golgi membrane head
groups are more fluid (A, solid line). Ad-
dition of A␤40 (dashed line) and A␤42
(dotted line) to Golgi membranes (C) re-
sulted in an increase in the intensity of
the MC540 spectra indicative of A␤-head
group interactions. In contrast, A␤42 had
little effect on the plasma membrane (B)
as indicated by a lack of shift in the spec-
tra, whereas A␤40 decreased the inten-
sity of the spectra indicating increased
packing of the head groups. In contrast,
mellitin (D, dashed line) in the presence
of plasma membrane (D, solid line)
shifted the shape of the MC540 spectra to
one that is indistinguishable from Golgi
membranes, suggesting creation of lipid
head group packing defects or altered
spacing.

TABLE III

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were obtained for endosomal lipid bilayers in the presence of Effect of A␤ on the apparent dimerization constant (Kd(app)) of
A␤ (data not shown). The MC540 spectra in the presence of merocyanine 540 in cellular membranes
plasma and endosomal membranes are consistent with varying Peptide was added to lipid vesicles at a 1:20 ratio with a final peptide
concentration of 10 ␮M.
levels of A␤ insertion into the bilayers.
These results are contrasted by the MC540 spectra in the Apparent dimerization constant (Kd(app)
Sample
presence of Golgi lipid bilayers, which demonstrate maxima at Golgi Plasma Endosome
530 and 570 nm (Fig. 5A). These spectra are indicative of a Control 1.1 ⫻ 10 7
9.1 ⫻ 10 8
3.7 ⫻ 1010
more fluid, liquid-crystalline head group packing and an in- A␤40 1.0 ⫻ 107 2.1 ⫻ 109 0
creased surface potential that allow for increased MC540 mo- A␤42 1.7 ⫻ 107 9.1 ⫻ 108 1.1 ⫻ 1011
Seeded A␤40 2.0 ⫻ 107 1.3 ⫻ 109 1.1 ⫻ 1011
nomeric insertion into the head group space. Addition of A␤40
Seeded A␤42 5.8 ⫻ 106 1.0 ⫻ 109 4.2 ⫻ 109
and A␤42 results in an increase in the intensity of both max-
ima, indicating a more fluid environment and increased head
group space or packing defects (Fig. 5C). In contrast, seeded Furthermore, addition of A␤40/42 did not significantly alter the
A␤40 and A␤42 decrease the intensity of the 570 nm maxima Kd(app) suggesting that the membranes can easily accommodate
suggesting that ␤-structured peptide increases the packing of A␤. Our anisotropy studies suggest that both the plasma and
the head groups of Golgi membranes. The MC540 absorption endosomal lipid bilayers are both fluid bilayers, whereas the
spectra are consistent with A␤40/42-Golgi interactions occur- dimerization constant suggests that the endosomal head group
ring predominantly at the head group space. packing is more rigid than the plasma membrane bilayers.
To investigate the sequence specificity of A␤40/42 interaction Addition of A␤40/42 as a randomly structured peptide did not
with Golgi and plasma membrane bilayers, we examined the alter the Kd(app), suggesting that A␤ binding does not alter head
interaction of A␤-(1–28) under similar conditions. In contrast group packing. The dimerization of MC540 in endosomal lipid
to A␤40/42, A␤-(1–28) did not affect the shape or intensity of bilayers was decreased by A␤ binding as indicated by a 3-fold
the MC540 absorption spectra of Golgi membranes. These re- increase in the dimerization constant (Table III) after addition
sults suggest that A␤-(1–28) does not affect head group packing of both random and ␤-structured peptides. These results sug-
and confirms the DPH and laurdan fluorescent results, which gest that A␤-endosomal interactions further organize the head
suggest that A␤-(1–28) does not insert into the lipid bilayer group packing and are consistent with our anisotropy studies,
(data not shown). Similar to A␤40/42, mellitin increases the which demonstrate a decrease in the fatty acyl chain fluidity as
intensity but not the shape of the MC540 absorption spectra. a result of A␤ binding to endosomal bilayers.
Furthermore, MC540 spectra of plasma membranes in the
presence of mellitin are indistinguishable from that of Golgi DISCUSSION
membranes (Fig. 5D). These results are consistent with mel- A␤-lipid interactions have implications not only for A␤ pro-
litin insertion into the bilayer and creating increased head duction but also for the induction of neurotoxicity and age-
group space or packing defects. associated pathology. The presence of A␤ aggregates, initiation
The MC540 monomer-dimer equilibrium is relevant to the of plaque formation, and the dependence of toxicity on the
packing properties of the bilayers and can be used as an indi- association with specific lipid compartments suggested that
cation of lipid head group spacing (27, 28). We have calculated vesicular lipid composition might be a factor in these processes.
the apparent dimerization constant for plasma, Golgi, and en- Our fluorescence studies on plasma membrane bilayers suggest
dosomal lipid bilayers in the presence and absence of A␤40/42 that A␤ inserts into the fatty acyl region of the bilayer. This
in both random and ␤-structure (Table III). The most apparent result is supported by the anisotropy studies that demonstrate
observation is that the dimerization constant for the various a dramatic increase in membrane organization as a result of A␤
bilayers differs on the order of 2 magnitudes from each other in interaction, the lipid phase shift associated with A␤ as demon-
the order Golgi ⬍ plasma ⬍ endosomal bilayers. These results strated by the laurdan GP and the lack of head group reorga-
suggest that the head group packing of the Golgi membranes is nization as detected by MC540 absorption spectra. Our results
less constrained and can accommodate the MC540 dimers. are consistent with previous reports (34, 36) that demonstrated
Cellular Membrane-associated A␤ Fibrillogenesis 33567
that addition of A␤ to membranes isolated from cerebellum, various compartments, we have not taken into account the
cortex, hippocampus, and striatum or synthetic lipid vesicles effect of endogenous cellular proteins. These integral mem-
results in a decrease in membrane fluidity. In contrast, Mason brane proteins will also have an effect on A␤-membrane
et al. (17) reported that A␤, both random and aggregated, interactions whether as competitors for A␤ binding, such as
increased synaptic plasma membrane fluidity by insertion of proteoglycans, or as modulators of bilayer properties. Our
random A␤ into the fatty acyl chain and the presence of aggre- results demonstrate differences detected in the A␤-lipid in-
gated A␤ at the lipid head groups. Our results are in partial teractions between the various vesicular compartments,
agreement with these results, as we also propose that A␤ exerts which may play a role in not only normal cellular processing
its bilayer effects by inserting into the fatty acyl chains, but and turnover of A␤ but in the progression of disease processes
differ in that our results demonstrate a rigidizing effect. The in Alzheimer’s disease.
discrepancies between these two studies may be accounted for Acknowledgments—We thank Dr. N. Wang at the Hospital for Sick
by the presence of endogenous synaptic membrane proteins Children’s Biotechnology Center for the synthesis of all peptides used in
that may compete with lipids for A␤ binding. Our results fur- this study, Dr. A. Chakrabartty for use of the PTI fluorescence spec-
ther demonstrate the enhancement and organization of A␤ trometer, the Electron Microscopy Suite at the University of Toronto for
use of Hitachi 7000 electron microscope (CIHR Maintenance Grant),
fibrillogenesis in the presence of plasma membrane vesicles. and the Canadian Brain Tissue Bank.
These structural results are consistent with our previous stud-
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