From the Department of Dementia Research, National
Institute for Longevity Sciences, Gengo 36-3, Morioka, Obu, 474-8522, Japan, § Protein Research Foundation, Peptide Institute,
Inc., 4-1-2 Ina, Minoh, Osaka 562, Japan, ¶ Department of
Pathology, Fukui Medical University, Fukui 910-1193, Japan,
Laboratory for Mass Spectrometry, Rockefeller University, New
York, New York 10021-6399, and ** Department of Molecular Biology, J5,
Central Institute for Mental Health, 68159 Mannheim, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Deposition of aggregated amyloid A We have recently reported the detection of a novel A Regarding the involvement of cellular cholesterol in the generation of
the pathogenic protein, it must be noted that cholesterol-rich lipid
microdomains within cells, called caveolae-like domains, have been
reported to be the likely sites of the conversion of the normal
cellular form of prion protein (PrPc) to its pathogenic
form (PrPsc) (8-11). Thus, it would be of great interest
to investigate whether the novel A MDCK Cell Culture--
Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum was
used as the culture medium. We plated 2.5-3.0 × 105
MDCK cells transfected with human APP695 cDNA, with full-length APP
(wild-type MDCK cell) or APP with a truncated cytoplasmic domain ( Thioflavin T Assay and Congo Red Assay of Fibril Formation of
Synthetic A Electron Microscopy--
Samples were spread on carbon-coated
grids, negatively stained with 1% phosphotungstic acid, pH 7.0, then
examined under a Hitachi H-7000 electron microscope with an
acceleration voltage of 75 kV.
Enzyme Immunoassay--
The enzyme immunoassay (EIA) was
performed essentially as described previously (3, 15). Briefly, 100 µl of the media of the MDCK cell cultures were diluted with 400 µl
of buffer C (20 mM phosphate buffer (pH 7.0), 0.4 M NaCl, 2 mM EDTA, 10% Block Ace (Dai-nippon,
Tokyo, Japan), 0.2% bovine serum albumin, and 0.05%
NaN3), and 100 µl of the mixture were subjected to the
multiwell plates coated with 4G8, a monoclonal antibody specific for
A Effect of Addition of the A Inhibition of de Novo Cholesterol Synthesis--
To inhibit
de novo cholesterol synthesis, MDCK cells were incubated for
90 min with compactin (Sigma), a potent competitive inhibitor of
3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, at
concentrations of 0.5 and 1.0 µM, before changing the
media to fresh ones. The cells were further cultured for 3 days with compactin, and then 1.5-ml aliquots of the medium from the apical and
basolateral compartments of the culture were used for immunoprecipitation.
Treatment with Filipin--
MDCK cells were cultured in medium
containing filipin (Sigma), a polyene antibiotic that specifically
binds to cholesterol, at concentrations of 0.1 and 0.3 µg/ml for 90 min before changing the media to fresh ones. The cells were further
cultured for 3 days with filipin, and then 1.5-ml aliquots of
the medium from the apical and basolateral compartments of
the culture were used for immunoprecipitation.
Addition of Exogenous Cholesterol--
To confirm that the
seeding ability of the apical A Determination of the Total Cellular Level of Cholesterol and the
Level of de Novo Cholesterol Synthesis--
To determine the total
level of cellular cholesterol in MDCK cells, cultures treated with
compactin (1 µM) or filipin (0.3 µg/ml) for 2 days were
washed 3 times in PBS and dried at room temperature. The samples were
extracted with hexane/isopropanol (3:2 v/v) and dried with nitrogen.
The total cholesterol levels in the samples were determined using a
cholesterol determination kit (Kyowa Medical Co. Ltd., Tokyo, Japan).
Protein concentration was determined using the BCA protein assay kit
(Pierce), with bovine serum albumin as the standard. To determine the
level of de novo cholesterol synthesis, MDCK cells were
pretreated with compactin (1 µM) and filipin (0.3 µg/ml) in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum for 2 h. 2 µCi/ml [14C]acetate was
added into the cultures treated with the compounds. After 3 h of
incubation, the cultures were washed three times in PBS and dried at
room temperature. The samples were then extracted with
hexane/isopropanol (3:2 v/v) and dried with nitrogen. The samples were
quantitatively spotted on thin-layer chromatography plates and
developed in a solvent system of hexane/ethyl ether/acetic acid
(80:30:1). The radioactivities of the spots were detected and
quantified by the Bio-imaging Analyzer System-2500 Mac (Fuji Film Co.
Ltd., Tokyo, Japan).
Mass Spectrometry--
Mass spectrometric analysis was performed
essentially as described elsewhere (17). Aliquots of medium (5 ml) from
the apical and basolateral compartments were incubated with 4G8 (5 µg) at 4 °C overnight for immunoprecipitation of A MDCK cells were cultured as described previously (6). Stable
transfection of these cells with APP cDNA was performed as described elsewhere (12). To investigate whether the novel A-protein
(A
), a proteolytic cleavage product of the amyloid precursor protein
(1), is a critical step in the development of Alzheimer's disease (2). However, we are far from understanding the molecular mechanisms underlying the initiation of A
polymerization in vivo.
Here, we report that a seeding A
, which catalyzes the
fibrillogenesis of soluble A
, is generated from the apically
missorted amyloid precursor protein in cultured epithelial cells.
Furthermore, the generation of this A
depends exclusively on the
presence of cholesterol in the cells. Taken together with mass
spectrometric analysis of this novel A
and our recent study (3), it
is suggested that a conformationally altered form of A
, which acts
as a "seed" for amyloid fibril formation, is generated in
intracellular cholesterol-rich microdomains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 is physiologically
secreted into the extracellular space; however, why and how soluble
A
aggregates and forms amyloid fibrils remains to be elucidated. A
great deal of effort has been made to clarify this issue, using mainly
in vitro systems. In most such experiments, it has been
found that A
at much higher concentrations than those prevailing in
biological fluids is needed for A
aggregation. Thus, it has been
hypothesized that aggregation of soluble A
involves seeded
polymerization (4, 5), although this assumption has not yet been proved
in vivo.
in the apical
compartment of cultures of MDCK cells that had been stably transfected
with APP cDNA (
C MDCK cell) with a truncated cytoplasmic domain
(
C APP) (3). This A
species possesses unique molecular characteristics including its appearance as a smear on immunoblots and
altered immunoreactivity. Significantly, these molecular
characteristics disappeared dramatically following treatment of the
cells with compactin or filipin, an inhibitor of de novo
cholesterol synthesis and a cholesterol-binding drug, respectively.
Based on previously reported evidence for
C APP being missorted to
the apical surface (6) and the cholesterol concentrations of the apical
plasma membrane and apical transport vesicles being higher than those in other cellular membranes (7), we concluded that a novel A
is
generated from apically missorted APP in a
cholesterol-dependent manner.
detected in our recent study (3),
the generation of which is exclusively dependent on the presence of
cholesterol in the cell, has the potential to act as a seed for
fibrillogenesis of soluble A
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C
MDCK cell) (12) onto 24-mm Transwell filters (Costar) and cultured
these cells on the filters for 3 days. To determine the integrity of
the cell monolayers that grew on the filters, measurement of the
electric resistance between the apical and basolateral compartments of
the MDCK cell culture was performed by immersing electrodes into each
of the compartments. The MDCK cells culture media were changed 3 days
after plating, and the cells were cultured 24 h longer.
--
Thioflavin T assay was performed as described
elsewhere (13), on a spectrofluorophotometer (RF-5300PC, Shimadzu,
Kyoto, Japan). Optimum fluorescence measurements of amyloid fibrils
were obtained at the excitation and emission wavelength of 446 nm and 490 nm, respectively, with the reaction mixture (1.0 ml) containing 5 µM thioflavin T (Nakalai tesque, Inc., Kyoto, Japan) and
50 mM of glycine-NaOH buffer, pH 8.5. Fluorescence was
measured immediately after making the mixture and averaged for an
initial 5 s. Synthetic A
(A
1-40, Bachem Switzerland) was
initially dissolved in ice-cold distilled water at a concentration of
100, 200, and 300 µM, and then diluted with 9 volumes of
PBS. Aliquots of the A
solutions were incubated in Eppendorf tubes
at 37 °C. Every hour after the start of incubation, 10 µl of the
solution of synthetic A
was taken and mixed with 990 µl of the
reaction mixture. The lot number of the A
used in the experiment of
Fig. 1 was 518765 and that of the A
used in the other experiments
was 510313. Peak fluorescence was dependent on the concentration of
A
. We used 20 µM A
peptide for this study. Congo
red assay was performed as described elsewhere (14).
17-24 (16). Appropriate amounts of synthetic A
40 (A
1-40,
Bachem Switzerland) were applied to the multiwell plates for the
construction of a standard curve. The plates were incubated at 4 °C
overnight. After rinsing with PBS, loaded wells were reacted with
appropriately diluted horseradish peroxidase-conjugated BA27, a
monoclonal antibody specific for A
40, at 4 °C overnight. Bound
enzyme activities were measured using the TMB Microwell peroxidase
substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Immunoprecipitated from the Medium
of the MDCK Cells on the Fibril Formation of Synthetic
A
--
Aliquots (1.5 ml) of the medium from the transfected MDCK
cell cultures were incubated with 4G8 (1.5 µg) at 4 °C overnight for A
immunoprecipitation 3 days after changing the medium. The mixtures were then incubated with protein G-Sepharose at 4 °C for
3 h and centrifuged. The pellets were washed thoroughly in RIPA
buffer once, and then in Tris-saline buffer four times. The A
in the
immunoprecipitates was extracted in 25 µl of a buffer containing 2%
SDS by boiling. Following dilution with 975 µl of Tris-saline buffer,
5 µl of the solution (containing 1 pmol of the immunoprecipitated
A
) was mixed with 10 µl of the synthetic A
solution (containing
2 nmol of A
1-40) and 85 µl of PBS buffer. The level of the
immunoprecipitated A
was determined by enzyme immunoassay (data not
shown), and the molecular ratio between synthetic A
and the
immunoprecipitated A
was approximately 2,000:1. Amyloid fibril
formation of synthetic A
was quantitatively determined by measuring
fluorescence intensity at 2 and 6 h after starting the incubation
as described before. The background fluorescence intensity was
determined using an extract of protein G-Sepharose that was incubated
with 4G8 in fresh medium.
depends on the presence of
cholesterol, we investigated whether the effect of compactin and
filipin was reversed by the addition of exogenous free cholesterol.
. The
mixtures were then incubated with protein G-Sepharose at 4 °C for
3 h and centrifuged. The pellets were washed thoroughly with RIPA
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl,
0.1% SDS, 0.5% deoxycholic acid, 1.0% Nonidet P-40) once and then
with distilled water four times. The immunoprecipitated A
was
extracted with 10 µl of trifluoroacetic acid. 1 µl of the extracted
solution was mixed with 10 µl of UV-laser desorption matrix
(trifluoroacetic acid/water/acetonitrile (1:20:20, v/v/v), containing
saturated
-cyano-4-hydroxycinnamic acid; 0.5 µl of this
mixture was loaded onto the mass spectrometer sample probe and dried at
room temperature. Mass spectra were measured using a UV-laser
desorption/ionization time-of-flight mass spectrometer (Voyager Elite,
PerSeptive Biosystem).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
in the
medium of the apical compartment of the
C MDCK cell cultures, referred to as apical A
, potentially accelerates amyloid fibril formation of synthetic A
, we performed a thioflavin T assay as described previously (13), using the A
peptide immunoprecipitated from the conditioned media. As shown in Fig.
1a, when synthetic A
(A
1-40) was incubated with the apical A
derived from the
C
MDCK cells, the fluorescence increased without a lag phase and
proceeded to equilibrium hyperbolically. This time-course curve
suggests that the apical A
acts as a seed in this experiment. A
perfect linear semilogarithmic plot (r = 0.998) shown
in Fig. 1b, indicates that F(t)
satisfies a differential equation: F'(t) = B
CF(t), where B
and C are constants (18). Based on this differential
equation, amyloid fibril formation from synthetic A
incubated with
the apical A
can be explained by a first-order kinetic model;
i.e. the extention of amyloid fibrils may proceed via the
consecutive association of synthetic A
first onto the apical A
then onto the ends of growing fibrils (18).
View larger version (58K):
[in a new window]
Fig. 1.
Seeding ability of the apical
A on the amyloid fibril formation. Panel
a, effects of the immunoprecipitated A
on the kinetics of
amyloid fibril formation in vitro. 50 µM
synthetic A
1-40 was incubated at a ratio of 2,000:1 with A
immunoprecipitated from the media of the apical (
) and basolateral
(
) compartment of
C MDCK cells, and the apical (
) and
basolateral (
) compartment of wild-type MDCK cells. Amyloid fibril
formed were quantified using the thioflavin T assay. Note that when
synthetic A
was incubated with the apical A
derived from the
C
MDCK cells (
), the fluorescence increased without a lag phase and
proceeded to equilibrium hyperbolically. Panel b, the
semilogarithmical plot of the difference:
A-F(t) versus incubation
time. F(t) is the fluorescence as a function of
time in the case of synthetic A
incubated with the apical A
derived from the
C MDCK cells, and A is the tentatively
determined F(
) shown as a broken line in
panel a. Linear regression and correlation coefficient were
calculated (r = 0.998). Note that
F(t) is described by a differential equation:
F'(t) = B
CF(t), that is, it follows a first-order kinetic
model (see text). Panel c, electron micrographs of the
mixture incubated for 3 h at 37OC containing 50 µM of synthetic A
plus A
immunoprecipitated from
the media of the apical (left panel) or basolateral
(right panel) compartment of
C MDCK cells. Note that
typical amyloid fibrils were formed from synthetic A
plus the apical
A
(left panel), whereas no fibrillar structures were
formed from synthetic A
plus the basolateral A
(right
panel). The bar indicates a length of 100 nm.
Electron microscopic analysis showed the formation of typical amyloid
fibrils with a diameter of approximately 10 nm and helical structure
(Fig. 1c, left panel). Similar helical filament
structure was observed when 400 µM of synthetic A
(A
1-40) was incubated at pH 7.5, 37 °C for 3 days (18). When
synthetic A
was incubated with other types of immunoprecipitated
A
, the increase in the fluorescence was small and no fibrillar
structures were observed with electron microscopic analysis (Fig. 1,
a and c, right panel).
Accelerated fibrillogenesis upon addition of the apical A was
further confirmed using Congo red assay (data not shown). The extent of
enhancement of fibrillogenesis by the apical A
was statistically
significant (Fig. 2a). We
excluded the possibility that these results were caused by alteration
in the amount of A
secreted from the cells by determining the level
of A
by enzyme immunoassay (Fig. 2b).
|
To investigate the molecular mechanism of generation of the apical A
with seeding ability, we first asked whether the generation is
dependent on the cellular cholesterol because the level of cholesterol
in the apical plasma membrane is higher than that of basolateral plasma
membrane (7). When we incubated the cells with compactin or filipin,
acceleration of fibrillogenesis upon addition of the apical A
was
substantially inhibited in a dose-dependent manner as shown
in Fig. 3a. Again, we excluded
the possibility that these results were caused by alteration in the
amount of A
secreted from the cells by determining the level of A
by enzyme immunoassay (Fig. 3b). To further confirm that the
seeding ability of the apical A
depended on the presence of
cholesterol, we performed an experiment to see if the effect of
compactin and filipin was reversed by the addition of exogenous
cholesterol. As shown in Fig. 3c, the inhibition of the
apical A
-induced acceleration of fibrillogenesis of synthetic A
following treatment with compactin and filipin, was dramatically
reduced by the addition of exogenous cholesterol. Total cellular level
of cholesterol was not dramatically decreased in cultures treated with
compactin or altered at all in those treated with filipin, whereas the
de novo cholesterol synthesis was markedly suppressed in
cultures treated with compactin (Fig. 3d). These results
suggest that generation of the seeding A
requires the presence of
cholesterol in specific microdomains and does not depend on the total
cellular level of cholesterol. The altered A
species was also
generated in cultures that were grown in media not containing fetal
bovine serum or supplemented with lipoprotein-deprived serum (data not
shown), indicating that its generation does not depend on the presence
of lipoprotein(s) or other factors in the serum.
|
We then attempted to characterize the novel SDS-stable A species. It
was previously reported that A
can form SDS-stable oligomers (19);
thus, we attempted to determine its molecular mass using
matrix-assisted laser desorption/ionization mass spectrometry. The
molecular mass of the major peak for the apical A
was identical to
that of human A
6-40 peptide (Fig.
4a), whereas that of the A
immunoprecipitated from the media of the basolateral compartment was
identical to the molecular mass of A
5-40 (data not shown), which is
consistent with a previous report (12). Notably, the molecular mass of
the apical A
did not change following treatment of the cells with
compactin (Fig. 4b), whereas its smearing on the immunoblot
was lost following compactin treatment (data not shown) as described
previously (3). To exclude the possibility of a different A
species
being extracted in the experiment of mass spectrometry because of a
difference in the used extracting buffer, we performed Western blotting
analysis of the immunoprecipitates extracted with trifluoroacetic acid,
which was used for the mass spectrometric study, but the smear
appearance persisted (data not shown). This result indicates that the
acquisition of these unique molecular characteristics by the apical
A
is not due to the association of the A
with other molecules,
but rather because of some conformational alteration. This assumption
is also supported by our previous finding that the altered
immunoreactivity of this novel A
was restored by treatment of the
cells with compactin (3). Furthermore, formic acid treatment of the
apical A
abolished its ability to act as a seed (data not
shown).
|
Here we report, for the first time, that a seeding A, which
catalyzes the fibrillogenesis of soluble A
, is endogenously generated in cell culture. A noteworthy finding in this study is that
generation of the seeding A
depends exclusively on the presence of
cholesterol as shown in Fig. 3, a and c.
Determination of the intracellular site of the generation of the
seeding A
remains to be determined; however, lipid microdomains,
called rafts (20), sharing a high content of cholesterol and
glycosphingolipid with caveolae or caveolae-like domains are likely to
be the best candidate for the following reasons: first, the axonally
sorted APP, analogous to apically sorted APP in epithelial cells, is conveyed via caveolae-like domains in neurons (21); second, localization of APP in caveolae has recently been reported (22); third,
further evidence to support the generation of A
in the cholesterol-rich microdomains is accumulating (23, 24).
At this point, it is extremely difficult to elucidate the molecular
mechanism of acquisition by the apical A of its unique molecular
characteristics, including its seeding ability; however, it may be
reasonable to assume that the apical A
adopts an altered conformation based on the following experimental results obtained from
this and previous studies (3): first, the immunoreactivity of the
apical A
to BAN50, in addition to its smearing behavior on gel
electrophoresis, changed following treatment of the cells with
compactin without any alteration in its mass number (Ref. 3 and Fig.
4); second, the BAN50 immunoreactivity for the apical A
recovered
following treatment of the A
with formic acid (3). In the putative
conformational alteration of the apical A
, auxiliary factors
localized in the lipid microdomains may be involved, as is suggested in
the conversion of the normal cellular form of prion protein
(PrPc) to its pathogenic form (PrPsc) (9).
Among the candidates for such factors, we prefer to consider GM1
ganglioside for the following reasons: first, GM1 ganglioside is one of
the main resident molecules in the microdomains (25); second, we have
previously found GM1 ganglioside-bound A
in human brains in the
early stages of Alzheimer's disease (26); third, A
undergoes
alteration of its secondary structure via interaction with GM1
ganglioside (27, 28); and fourth, it has recently been reported that
amyloid fibril formation of A
is drastically accelerated in the
presence of GM1 ganglioside (14). Thus, it is intriguing to speculate
that the A
generated from the apically missorted APP undergoes
conformational alteration via association with GM1 ganglioside in the
lipid microdomains and then acts as a template for the consecutive
conversion of a nascent soluble A
into a seeding A
.
Recently, much attention has been focused on the conformational
alteration of constitutive proteins in the brain in various neurodegenerative diseases (4, 5, 29). In such processes, a
constitutive protein in the brain undergoes minor perturbations of
structure, leading to an increase in -sheet content; it has been
proposed that these disease processes be grouped into one new category,
the conformational diseases (29). Conformational conversion
of the prion protein with resultant aggregation of its pathogenic form
is a well-known example belonging to this category (30). Although
Alzheimer's disease could also be included in the conformational
diseases group (4, 5, 29), to date, no study has ever shown the
generation of a conformationally altered isoform of A
with seeding
ability. In this regard, our results present, for the first time,
evidence for the generation of a seeding A
in cell culture, and
furthermore, may be used to explain the molecular mechanism underlying
initiation of amyloid fibril formation in vivo.
Finally, from the results of this and other (31) studies, one can
consider the possibility that missorting of APP or altered intracellular trafficking of A plays a role in the pathogenesis of
Alzheimer's disease. Further studies, using polarized differentiated neurons should be carried out to investigate the consequences of
generation of a seeding A
from axonal or presynaptic membranes.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Y. Hanai for preparing this manuscript and I. Yamaguchi for technical support in the electron microscopic analysis.
![]() |
FOOTNOTES |
---|
* This study was supported by a research grant for Longevity Sciences (8A-1) and Brain Research Science from the Ministry of Health and Welfare and by Core Research for Evolutional Science and Technology, Japan.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.: 81-562-44-5651 (ext. 834); Fax: 81-562-44-6594; E-mail: katuhiko{at}nils.go.jp.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
-protein;
APP, amyloid precursor protein;
MDCK, Madin-Darby canine
kidney cell;
PBS, phosphate-buffered saline;
EIA, enzyme
immunoassay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|