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INTRODUCTION |
Formation of senile plaques composed of amyloid
-protein
(A
)1 is one of the
hallmarks of Alzheimer's disease (AD) (1). A
, a small protein of
Mr ~4,000, is cleaved out sequentially with
- and
-secretases from a large membrane protein called
-amyloid precursor protein (APP), and secreted into the
extracellular space. There are two major species of A
that are
defined by their C-terminal lengths; A
40 terminates at Val-40, and
A
42 terminates at Ala-42. The latter has a much stronger tendency to
aggregate into fibrils and accounts for less than 10% of secreted
A
. However, A
42 is the most predominant form found in senile
plaques and is considered the initial species to be deposited in aged
and AD brains (2, 3).
Three causative genes, APP, presenilin 1 (PS1),
and PS2 have been identified in early onset familial AD (1),
and their mutations appear to converge on an increased production of
A
42. However, the pathological mechanisms of sporadic AD, explaining more than 90% of AD patients, remain unknown. Interestingly, there is
growing evidence for a significant linkage between A
and cholesterol metabolism. First, cholesterol loading or depletion affects A
generation both in vitro (4, 5) and in vivo (6).
Second, the levels of total cholesterol and LDL-cholesterol, but not
those of HDL-cholesterol, in the serum correlate with the amount of A
42 in AD brains (7). Third, aggregated A
preferentially binds to
cholesterol in vitro (8). Fourth, a substantial fraction of
intracellular A
is localized in the detergent-insoluble membrane domain that is rich in glycosphingolipid and cholesterol (9, 10).
Fifth, the presence of an E4 isoform of apolipoprotein E, an essential
molecule for cholesterol metabolism, is a strong risk factor for
developing AD (11). Sixth, high dietary cholesterol accelerates
AD-related pathologies, including A
deposition, in a transgenic
mouse model (12). However, no detailed mechanisms for clarifying
A
-cholesterol interacting sites are known. We hypothesized that both
cholesterol and A
metabolisms are linked intracellularly, and to
examine this possibility, we focused on Niemann-Pick type C (NPC) disease.
NPC is an autosomal recessive neurovisceral storage disease (13),
characterized by the presence of numerous foam cells in the bone marrow
and visceral organs. Its hallmark is an intracellular accumulation of
unesterified cholesterol and other lipids, especially sphingolipids, in
vacuoles resembling late endosomes (14). A major gene (NPC1)
responsible for NPC has recently been identified by positional cloning
(15, 16). The cDNA sequence predicts a 1,278-residue polytopic
integral membrane protein (NPC1) containing a putative
cholesterol-sensing domain. The main features of the NPC phenotype can
be mimicked by cultured cells exposed to a variety of reagents called
class 2 amphiphiles, such as U18666A (17). It is now believed that
these reagents act directly on the NPC1 protein (18). Based on these
facts, we hypothesized that intracellular A
metabolism might be
changed in these cholesterol-perturbed cells.
In this study, we investigated the intracellular A
in two cell
lines. These were NPC model cells treated with a class 2 amphiphile, and a Chinese hamster ovary (CHO) cell mutant line that is deficient in
NPC1 protein due to premature termination of its translation (19). We
found that intracellular cholesterol levels strongly affect A
42
accumulation and aggregation in late endosomes and that this A
42
accumulation was also observed in NPC mouse brains.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Transfection--
CHO cells, stably transfected
with wild-type (7WD10) and C-terminally truncated (
C) APP751
cDNAs (20), human embryonic kidney 293 cells transfected with
APP695 cDNA (K295) (21), and CHO mutants 25RA and CT43 (19) were
grown and maintained as described previously. For cholesterol depletion
studies, monolayers of 7WD10 cells were washed twice with
phosphate-buffered saline and cultured in cholesterol starvation medium
(F-12 medium; Life Technologies, Inc.) with 10% delipidated fetal calf
serum, 35 µM oleic acid (Sigma), 50 µM
mevinolin, and 230 µM mevalonate (Sigma). Mevinolin, a
competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase, blocks the synthesis of steroidal and nonsteroidal isoprenoids (22). Delipidated fetal calf serum was prepared according
to a procedure modified by Cadigan et al. (22).
Water-soluble cholesterol and methyl-
-cyclodextrin were purchased
from Sigma. For transient transfection, APP695 cDNA was cloned into
the vector pCXN (23) and transfected with LipofectAMINE (Life Technologies).
Subcellular Fractionation--
To avoid contamination of the
cell lysate with the cell surface-adsorbed A
, cells were harvested
by incubation with trypsin at 37 °C. A 1% Triton X-100
(Triton)-insoluble fraction of whole cell homogenates was prepared as
described previously (10). Percoll (Amersham Pharmacia Biotech)
gradient centrifugation was performed according to Lange et
al. (24). Late endosomes were isolated according to van der Goot
(25) using sucrose gradients containing 3 mM imidazole (pH
7.4). Three interfaces, at 8.5/25% (interface 1), 25/35%
(interface 2), and 35/40.6% (interface 3) were carefully aspirated
(800 µl each) and pelleted.
Western Blotting--
Each fraction obtained by subcellular
fractionation was centrifuged at 540,000 × g for 20 min in a TLX ultracentrifuge (Beckman). The resulting pellets were
extracted with 70% formic acid at room temperature for 30 min, and the
extract was dried by Speed Vac (Savant Instruments). The proteins
solubilized with the sample buffer were subjected to Western blotting
as described previously (26). Cellular APP was extracted with 1%
Triton in Tris saline (TS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) and was subjected to Western blotting. In the
present study, the samples were adjusted by their volumes and loaded on
gels, except for that in Fig. 6A. The A
antibodies used
were BAN50 (epitope: A
1-10), 6E10 (A
1-17: Senetek PLC), BA27
(specific for A
40), and BC05 (specific for A
42) (27). Monoclonal
antibodies (5A3/1G7) recognize the midportion of APP (20). Antibodies
to Rab7 and Rab5a were purchased from CytoSignal and Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), respectively. The antibodies to
caveolin 1 and BiP were from Transduction Laboratories and StressGen
Biotechnologies Corp., respectively. The 6C4 anti-lysobisphosphatidic
acid monoclonal antibody was a generous gift from Dr. J. Gruenberg
(University of Geneva) (28).
Enzyme-linked Immunosorbent Assay (ELISA)--
ELISA for A
in
the culture medium (29) and lysobisphosphatidic acid among fractions
(30) was performed as described previously. BALB/c npcnih
and wild-type BALB/c mice were obtained from the Jackson Laboratory. ELISAs for A
in mouse brains were performed by a procedure described elsewhere (31). The data were statistically analyzed using Student t test.
Flow Cytometric Analysis and Sorting--
For flow cytometry,
samples from interface 1 were incubated with 50 µg/ml filipin (Sigma)
for 30 min at 4 °C and immediately analyzed using a FACSVantage flow
cytometer (Beckton Dickinson Immunocytometry systems). The flow rate
for analysis and sorting was about 2,000 events/s. The nozzle tip was
50 µm in diameter.
Determination of the Cholesterol Content--
Lipids were
extracted from the samples with hexane/isopropyl alcohol (3:2,
v/v), and evaporated under nitrogen flow. The cholesterol content of
each sample was determined using a cholesterol determination kit (Kyowa
Medix, Tokyo).
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RESULTS |
A
Accumulates within the Cells Treated with U18666A--
We
first assessed the A
levels within NPC model cells. CHO cells,
stably expressing APP751 (7WD10), were treated with 3 µg/ml U18666A
for 24 h (14). After harvesting by incubation with trypsin, the
cells were homogenized with 1% Triton in TS, and the homogenates were
centrifuged at 540,000 × g for 20 min. The resulting
Triton-insoluble pellet was then extracted with 70% formic acid, and
the A
levels in the extract were assessed by Western blotting (Fig.
1A). The U18666A treatment
remarkably enhanced A
immunoreactivity in the Triton-insoluble
fraction, whereas A
was barely detected in the same fraction of
nontreated cells. Increased signal intensities of A
monomers at ~4
kDa and dimers at ~5-6 kDa on the blot were apparent (26) (Fig.
1A). Those signals in the high molecular mass range should
represent nonspecific reactivities, because the secondary antibody
alone labeled most of these bands. Compared with the BA27
immunoreactivity, there was an enormous increase in the BC05
immunoreactivity, indicating that the major A
species that
accumulates following U18666A treatment is A
42.

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Fig. 1.
A accumulation in
NPC model cells. A, CHO cells (7WD10), stably
transfected with wild-type APP751 cDNA, were incubated with or
without 3 µg/ml U18666A for 24 h. After homogenization, 1%
Triton-insoluble fractions were pelleted and extracted with 70% formic
acid, and A in the extract was probed with various A antibodies,
as described under "Experimental Procedures." The
immunoreactivities for all A antibodies were greatly enhanced by the
U18666A treatment. An increase in the level of A 42 is apparently
much greater than that of A 40. Synthetic A 42 (50 pg) is loaded in
the right-most lane. B, a 1%
Triton-soluble fraction of homogenates was subjected to Western
blotting. The levels of Triton-soluble A 42 also increased by the
U18666A treatment, whereas that of A 40 did not. Truncated A
species (asterisk) is weakly detected with BA27, and its
level is not changed by the treatment. C, the U18666A
treatment did not alter the expression levels of APP, as shown by
Western blotting with 5A3/1G7 antibodies. Similar results were obtained
in three independent experiments.
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When Triton-soluble fractions were similarly analyzed, the signal for
BC05 immunoreactivity was also increased by the treatment, whereas the
intensities of BA27-immunoreactive bands at 4 and ~3 kDa (presumably
representing a truncated species of A
) were unchanged (Fig.
1B). Interestingly, the A
dimer, that was prominent in
the Triton-insoluble fraction, was undetectable in the Triton-soluble fraction. We found that the Triton-soluble fraction consistently contained a greater amount of A
than did the Triton-insoluble fraction (data not shown). The U18666A treatment did not alter the
expression level of APP (Fig. 1C); thus, an increase of A
was not a result of overproduction of its precursor.
To examine whether these phenomena are independent of the cell type, we
applied the same experimental conditions to K295 cells and obtained the
same results (data not shown).
A
Accumulates in Cholesterol-rich Compartments--
Next, we
determined the particular cellular compartment in which A
accumulates. Since NPC cells accumulate mainly unesterified cholesterol, we simply speculated that A
resides in cholesterol-rich compartments. Thus, we fractionated cell homogenates on Percoll gradients according to an established protocol widely used for separating cholesterol-rich compartments (24). A
42 was recovered only in fraction 1 in which free (unesterified) cholesterol was accumulated after the U18666A treatment (Fig.
2, A and B).
However, due to the fractionation process, endosomal and lysosomal
markers were distributed over the fractions (data not shown), and thus this protocol was judged not to be useful for determining the compartment in which A
accumulates.

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Fig. 2.
A 42 accumulation in
cholesterol-rich fractions. A, the homogenate of 7WD10
cells was fractionated on Percoll gradients, and the cholesterol
content of each fraction was determined. Treatment of the cells with
U18666A for 24 h shifted the distribution of free cholesterol to
the buoyant region (fraction 1). This is a representative of three
independent experiments. B, the homogenate of
U18666A-treated cells was fractionated on Percoll gradients, and each
fraction was centrifuged. The resulting pellets were extracted with
70% formic acid, and subjected to Western blotting using BC05.
Immunoreactivity for BC05 was detected only in fraction 1. C, to isolate late endosomes, 7WD10 cells were homogenized,
and the homogenates were centrifuged to equilibrium on sucrose
gradients. Interfaces 1, 2, and 3 correspond to the 8.5/25,
25/35, and 35/40.6% sucrose interfaces, respectively (see
"Experimental Procedures"). U18666A treatment induced cholesterol
accumulation exclusively at interface 1. D and E,
distribution of the markers specific for subcellular organelles: 6C4
antigen for late endosomes as shown by an ELISA assay (D),
Rab7 for late endosomes, Rab 5a for early endosomes, caveolin for
caveolae, and BiP for ER as shown by Western blotting (E).
Interface 1 produces a late endosome-enriched fraction. After U18666A
treatment, immunoreactivity for BC05, but not for BA27 was detected at
interface 1, suggesting that the treatment induces A 42 accumulation
in late endosomes. Similar results were obtained in four independent
experiments.
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Because free cholesterol accumulates in late endosomes of NPC cells
(14), we isolated late endosomes directly, using sucrose density
gradient centrifugation. This protocol enabled us to fractionate late
endosomes into interface 1 and early endosomes and plasma membrane into
interfaces 2 and 3 (Fig. 2C) (25). As shown in Fig.
2C, a U18666A-induced increase of free cholesterol was
observed in interface 1. To confirm the validity of this fractionation, each fraction was probed for specific markers including the 6C4 antigen
(for late endosomes) (Fig. 2D), Rab7 (for late endosomes), and Rab 5a (for early endosomes) (Fig. 2E). The obtained
results clearly showed that interface 1 indeed represents the late
endosome-rich fraction, in which a remarkable increase in the level of
A
42 was observed by the U18666A treatment (Fig. 2E).
These findings indicate that the U18666A treatment induces an
accumulation of both A
42 and free cholesterol in late endosomes.
A
40 was not always detectable in any of the three interfaces, even
in the presence of U18666A; therefore, the intracellular A
levels
were routinely assessed only with BC05.
Recently, we and others have reported that a significant fraction of
intracellular A
resides in the detergent-insoluble membrane domain
(9, 10). Caveolae are examples of such domains, but caveolin 1, a
caveolae-resident protein, was located at interface 3 (Fig.
2E). Endoplasmic reticulum (ER) is assumed to be a site for
A
42 generation (4, 32). However, ER markers, BiP (Fig. 2E) and calnexin (data not shown), were undetectable in
interface 1. Thus, caveolae and ER are not the sites in which the
A
42 accumulates following U18666A treatment.
The U18666A treatment itself did not alter the locations of several
marker proteins examined (data not shown). All of the observations
described above that were made in 7WD10 cells were reproduced in K295
cells (data not shown).
A
42 Accumulates in Filipin-positive Granules--
To further
confirm the colocalization of A
42 with free cholesterol, we isolated
cholesterol-rich vesicles from 7WD10 cells using a
fluorescence-activated cell sorter (33). Because free cholesterol can
be specifically and sensitively labeled with filipin, a fluorescent
probe (34), interface 1 prepared from U18666A-treated cells was
incubated with filipin, and the suspension was immediately analyzed
using a fluorescence-activated cell sorter with a UV filter setting.
There were two fluorescence peaks in filipin-labeled samples (Fig.
3A). The first peak (R1) was
also present in the unlabeled samples; thus, R1 should represent
autofluorescence from unlabeled membranes. The second peak (R2)
consisted of a population of highly fluorescent vesicles. Sorted
materials from the two peaks were separately pelleted, and each pellet
was extracted with 70% formic acid and subjected to Western blotting.
As shown in Fig. 3B, a BC05-positive signal, most intense at
the A
dimer, was detected only in the R2 fraction, demonstrating
that at least a part of the accumulated A
42 indeed localizes in
cholesterol-residing compartments.

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Fig. 3.
Western blotting of sorted filipin-labeled
vesicles. Late endosome-rich membranous organelles (interface 1)
were labeled with filipin and sorted by flow cytometry. R1 consisted of
the fluorescence from membranous organelles that were not labeled with
filipin (autofluorescence), while R2 represented a homogenous
population of filipin-labeled highly fluorescent vesicles (see
"Results"). Each fraction was pelleted and extracted with
70% formic acid and subjected to Western blotting using BC05. A 42
monomers and dimers were detected only in the R2 fraction.
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Intracellular A
Accumulation Does Not Affect A
Secretion--
We next examined whether the U18666A treatment causes
any detectable alterations in constitutive A
secretion. 7WD10 cells were cultured with or without 3 µg/ml U18666A for 24 h. After a
brief wash, the cells were maintained in culture for another 6 h
with or without U18666A, and the levels of A
in the conditioned medium were quantified by ELISA. As shown in Table
I, no statistical difference in the
levels of secreted A
40 and A
42 between the treated and untreated
cells was found. The ratio of A
42 to the total A
was also not
altered (Table I). These results indicate that A
42 in late endosomes
is not the source of secreted A
42.
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Table I
Effect of U18666A on A secretion
7WD10 cells were cultured confluent with or without 3 µg/ml U18666A
for 24 h. After washing, cells were continuously cultured in fresh
medium with or without U18666A for another 6 h. The amount of A
in the conditioned medium was measured by ELISA. Average A 42 ratios
are shown in the right-most column.
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Accumulated A
42 Disappears upon Withdrawal of
U18666A--
Since U18666A treatment has reversible effects on
cholesterol transport (35, 36), we examined whether withdrawal of
U18666A had an effect on A
accumulation and A
secretion. As shown
in Fig. 4A, the accumulated
A
42 became completely undetectable 24 h after the withdrawal of
U18666A. Any trace amount of A
was not detected in other interfaces
(data not shown), indicating that accumulated A
42 disappeared from
cellular membranous compartments.

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Fig. 4.
Further characterization of
A 42 that accumulated in late endosomes.
A, after incubation with U18666A for 24 h, 7WD10 cells
were washed and further cultured in the absence of the reagent for 0, 24, and 48 h. Twenty-four hours after depletion of the reagent,
the level of cholesterol (µg/mg protein) at interface 1 was
dramatically reduced, and the immunoreactivity for A 42 disappeared.
B, interface 1 was carefully sampled and divided into halves
and separately pelleted. One pellet was extracted with 2% SDS, while
the other with 70% formic acid. The final volumes of both samples were
adjusted, and the samples were subjected to Western blotting using
BC05. While the SDS extract exhibited no A -derived signals, the
formic acid extract (FA) contained A monomers, dimers,
and higher order A oligomers. C, CHO cells stably
expressing C-APP lacking the internalization signal were treated
with or without 3 µg/ml U18666A for 24 h. After fractionation,
interface 1 was taken and subjected to Western blotting using BC05.
Even in this cell line, the treatment induced an intracellular
accumulation of A 42, indicating that the internalization of the cell
surface APP is not required for A accumulation in late
endosomes.
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We then measured A
levels by ELISA in the cultured medium after
withdrawal of the reagent. After being exposed to U18666A for 24 h, 7WD10 cells were briefly washed and cultured for another 3 or 6 h in the regular medium. The A
content in the conditioned medium was
compared with that of sister cultures incubated without U18666A (Table
II). We did not observe any
detectable change in the A
secretion level after the removal of
U18666A.
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Table II
A secretion after U18666A treatment
After a 24-h treatment with U18666A (+), cells were washed and
maintained in culture for the indicated chase time in the absence of
the reagent. The amount of A in conditioned media was measured by
ELISA. For controls, sister cultures were incubated without U18666A
( ) for 24 h. Average A 42 ratios are shown in the right-most
column.
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A
42 That Accumulates in Late Endosomes Is Aggregated--
Since
the presence of intracellular, highly insoluble A
has been reported
(37), we examined the insolubility characteristics of the A
that
accumulated in late endosomes. Interface 1, prepared from
U18666A-treated cells, was divided into halves and separately pelleted.
One pellet was extracted with the 2% SDS sample buffer, while the
other was extracted with 70% formic acid. As shown in Fig.
4B, accumulated A
was extracted only with formic acid,
but not with SDS, although similar amounts of proteins other than A
were extracted with SDS and formic acid, as judged by Coomassie staining (data not shown). Thus, the A
42 accumulated in late endosomes is most likely aggregated into an SDS-insoluble form.
Internalization of Cell Surface APP Is Not Required for
U18666A-induced A
Accumulation--
Production and release of A
by 7WD10 cells depend primarily on internalization of APP from the cell
surface, and its endocytosis signal is located in the cytoplasmic
domain (20, 38). To determine whether this APP internalization is
essential for the accumulation of A
42 in late endosomes, CHO cells
stably expressing APP751 lacking nearly the entire cytoplasmic domain
(
C), were exposed to U18666A. The
C cells exhibited significant
reductions in the APP internalization and in the production and
secretion of A
(20, 38). As shown in Fig. 4C, remarkably
increased levels of A
42 were again observed in interface 1 following
the U18666A treatment. These results may indicate that internalization
of cell surface APP is not required for U18666A-induced A
accumulation in late endosomes.
Intracellular Cholesterol Levels Affect A
42 Accumulation in Late
Endosomes--
The above results strongly suggest a tight correlation
between A
42 accumulation and cellular cholesterol levels. To examine this possibility, 7WD10 cells were cultured in cholesterol starvation medium for 2 days. The cells were then further cultured in the cholesterol starvation medium with or without 3 µg/ml U18666A for
24 h. Although cholesterol starvation for 3 days resulted in an
average 80% decrease in the total cellular cholesterol levels, the
U18666A treatment somewhat increased the cholesterol level in interface
1 compared with that of untreated cells (Fig.
5). As shown in Fig. 5, under the
cholesterol-depleted conditions, U18666A again induced A
42
accumulation in interface 1. When the cells were cultured with 10 µg/ml exogenous water-soluble cholesterol (cholesterol and
methyl-
-cyclodextrin complex) for 24 h in the presence of
U18666A, apparently greater amounts of A
42 monomers and dimers were
observed at interface 1. Methyl-
-cyclodextrin alone (180 µg/ml)
together with U18666A did not increase the accumulation as much. These
data indicate that the A
42 accumulation in late endosomes is under
the influence of cellular cholesterol levels.

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Fig. 5.
Cellular cholesterol levels regulate
A accumulation in late endosomes. 7WD10
cells were cultured in the cholesterol starvation medium for 2 days and
further cultured in the presence of indicated reagents for 24 h.
After fractionation, interface 1 of each sample was prepared and
subjected to Western blotting using BC05. Even under
cholesterol-starved conditions, U18666A (3 µg/ml) induces A
accumulation in late endosomes. However, exogenous cholesterol
apparently enhances the accumulation. Appreciable cholesterol levels at
interface 1 are indicated. DLFC, delipidated calf serum;
CD, methyl- -cyclodextrin. Similar results were obtained
in three independent experiments.
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NPC1-deficient CHO Cells Accumulate A
42--
Although it is
most likely that U18666A acts directly on the NPC1 protein, we examined
whether a mutant cell that lacks the NPC1 gene can also
accumulate A
. Mutant CT43 is a cholesterol-trafficking CHO cell
mutant that carries a mutation in the NPC1 gene, resulting in production of a nonfunctional NPC1 protein (19). The parental cell
25RA has a gain-of-function mutation in the sterol regulatory element-binding protein and cleavage-activating protein. The line 25RA
shows normal intracellular cholesterol trafficking and thus was used as
a control. Both cell lines were transiently transfected with APP695
cDNA, and the levels of expressed APP were assessed by Western
blotting followed by densitometric quantification. The Triton-insoluble
fractions of the cell lysate, after normalization to the expression
levels of APP, were assessed by Western blotting. We found that CT43
contained higher levels of A
42 within the cell lysate than did 25RA
(Fig. 6A). Thus, we concluded
that both the cell lines deficient in NPC1 gene and the NPC
model cells accumulated A
, and especially A
42, in a similar
manner within the cells.

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Fig. 6.
A accumulates in
NPC1-disrupted CHO mutant and in
NPC1-deficient mouse brain. A, NPC1
mutant (CT43) and the parental (25RA) CHO cells were transiently
transfected with APP695 cDNA. Two days later, cells were
homogenized in 1% Triton in TS and spun down. The volumes of
Triton-insoluble samples were adjusted to the expression levels of APP
and loaded on the gel. A greater amount of A 42 monomers and dimers
are detected in CT43 cells. This is a representative of four
independent experiments. B and C, brains of
6-week-old BALB/c npcnih (Homozygote (Homo)) and
wild-type BALB/c mice (WT) (n = 3) were
homogenized in TS. After centrifugation, TS-insoluble pellets were
extracted with 6 M guanidine HCl and subjected to ELISA.
The results were analyzed by Student's t test. The A 42
(p = 0.033) and A 40 levels (p = 0.049) are significantly increased in NPC1-deficient
mice.
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A
Accumulates in NPC Mouse Brains--
Finally, we examined
whether A
42 accumulates in vivo (i.e. in NPC
mouse brains). We used a well characterized mutant mouse on a BALB/c
strain, named BALB/c npcnih, in which the NPC1
gene is disrupted (16). This mouse exhibits progressive
neurodegeneration and dies around 10 weeks of age. Brains from three
wild-type BALB/c and three homozygote BALB/c npcnih mice,
at the age of 6 weeks, were homogenized in TS. After centrifugation, TS-insoluble A
was extracted with 6 M guanidine HCl in
50 mM Tris-HCl, pH 7.6, and subjected to ELISA. As shown in
Fig. 6, B and C, the A
40 and A
42 levels are
significantly increased in NPC1-deficient mice compared with
those in wild-type mice.
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DISCUSSION |
Several lines of evidence suggest a linkage between cholesterol
metabolism and susceptibility to AD. Further, many reports indicate the
correlation of cholesterol metabolism with A
secretion levels. Here,
we have demonstrated the intracellular accumulation of A
, especially
A
42, along with free cholesterol in late endosomes in
cholesterol-perturbed NPC cells. This cholesterol accumulation does not
affect A
secretion levels, but the extent of A
accumulation in
late endosomes is strongly influenced by their cholesterol levels.
Importantly, this accumulated A
42 in late endosomes appeared to be
in an SDS-insoluble aggregate form. Thus, late endosomes of NPC cells
are a novel pool of intracellular aggregated A
.
The mechanism of interaction between aggregated A
and cholesterol
remains to be clarified. One possible explanation is that the observed
A
42 accumulation in late endosomes results from altered membrane
trafficking induced by an abnormal lipid content in NPC cells. Cellular
cholesterol levels (39) and lysobisphosphatidic acid, a late endosomal
lipid, (28), are known to modulate endosomal membrane trafficking. In
NPC cells, intracellular trafficking of lipids other than sterol is
also disrupted. Thus, abnormal lipid accumulation, not only for
cholesterol, may possibly affect intracellular membrane and A
/APP
trafficking and may induce A
42 aggregation in late endosomes.
However, we prefer a more straightforward explanation; free cholesterol
(together with other molecules) directly or indirectly (via other
molecules) interacts with A
(especially an aggregated form of
A
42), and wherever free cholesterol accumulates within the cell,
aggregated A
42 also accumulates. Earlier studies have shown that the
rate of degradation of 125I-labeled low density lipoprotein
was not impaired in normal CHO cells treated with U18666A (36) or in
mutant CHO cells defective in the NPC1 gene (19, 22).
These results rule out the remote possibility that the massive
accumulation of A
in NPC cells might be due to the capacity of the
endosomes for protein degradation being compromised by the massive
accumulation of cholesterol and/or other lipid in this compartment. It
is notable that, even under cholesterol-depleted conditions, U18666A
induced mobilization of remaining free cholesterol to late endosomes,
which apparently accompany A
42. One may argue that this assumption
is contradictory to an in vivo observation; significant
A
42 accumulation occurs in NPC mouse brains where free cholesterol
is reported not to be accumulated (40). However, it is quite possible
that, even if free cholesterol levels are not increased in the brain,
those in late endosomes may be significantly increased and coupled with A
42 accumulation.
The effects of U18666A on cholesterol trafficking can be completely
reversed when the reagent is withdrawn (35, 36). Free cholesterol that
accumulates in late endosomes is sorted either to the plasma
membrane/ER (19) or secreted into the culture medium (41). We took
advantage of this property of U18666A to examine the relocation of the
A
accumulated in late endosomes. Within 24 h after withdrawal
of U18666A, the accumulated A
42 disappeared completely from any of
the membranous compartments. One may speculate that the accumulated
A
is sorted to lysosomes for rapid degradation. Alternatively, the
A
42 may migrate to the plasma membrane/ER or be secreted into the
culture medium, through interaction with cholesterol and/or other
lipids. Because A
was observed neither in interface 2 nor in
interface 3, where the plasma membrane and ER are localized (data not
shown), it is possible that the accumulated A
is released into the
culture medium. However, no increased secretion of A
after
withdrawal of U18666A was detected by ELISA. There could be two
explanations for this observation, which are not mutually exclusive.
First, a change in the A
levels in the medium is so small that ELISA fails to detect it. Second, our ELISA system may exclusively detect A
monomer, but not A
dimers or oligomers (26). Thus, if the A
42 that accumulates in late endosomes is secreted in an aggregated form, the ELISA cannot detect it.
As seen in Figs. 1A and 5, A
monomer and dimer appeared
even in the absence of U18666A treatment. Thus, indiscernible amounts of A
must accumulate and aggregate even in non-NPC cells. Because cholesterol trafficking is normal in these cells, the aggregated form
of A
might be shed in a constitutive manner into the extracellular space, along with cholesterol. These released A
aggregates may act
as a seed for A
fibrillation, thereby leading to A
deposition in
the brain (42). In fact, a recent report by Walsh et al. (43) supports this view, showing that intracellular A
aggregation is
essential for the extracellular A
oligomerization. Conversely, in
the NPC brain, intracellularly accumulated A
42 cannot be efficiently secreted and accumulated within cells as seen in Fig. 6, B
and C. This may possibly explain why no A
deposition is
observed in the NPC brain.
Most interestingly, abundant neurofibrillary tangles, the other
hallmark of AD, are observed in the brains of NPC patients with a
slowly progressive clinical course (44). Those neurofibrillary tangles
in the NPC brain are morphologically and biochemically indistinguishable from those in the AD brain (44). Thus, in addition to
aberrant cholesterol trafficking, long term intracellular A
accumulation might accelerate neuronal degeneration, which eventually
leads to neurofibrillary tangle formation in the NPC brain.