From the
Division of Molecular Medicine,
Department of Medicine and the
Taub Institute
for Research on Alzheimer's Disease and the Aging Brain, Department of
Pathology, College of Physicians and Surgeons, Columbia University, New York,
New York 10032
Received for publication, January 23, 2003 , and in revised form, May 15, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular lipid homeostasis is controlled by sterol regulator
element-binding proteins, transcription factors regulating cholesterol and
fatty acid synthesis pathways, and by liver X receptors (LXRs),
oxysterol-activated nuclear receptors that induce a battery of genes involved
in cellular lipid efflux and transport
(6,
7). The two forms of LXR,
and
, are both expressed in the brain. LXR
is broadly
expressed in the developing and adult brain and is present in both neurons and
glial cells (8). Recent studies
show an essential role for LXRs in brain structure and function as aging
LXR
/
knockout mice develop cellular lipid inclusions,
abnormalities of the choroid plexus, and closure of the lateral ventricles
(8). Although this pathology is
different from that of AD, LXRs could potentially have a role in modulating
the course of chronic neurodegenerative diseases.
An important target of LXRs is the ATP-binding cassette transporter A1
(ABCA1) (9). ABCA1, the
defective molecule in Tangier disease, mediates efflux of cellular
phospholipids and cholesterol to lipid-poor apolipoprotein, including
apolipoprotein A-I (apoA-I) and apoE, which are present in the cerebrospinal
fluid (10). Treatment of mice
with LXR agonists resulted in increased expression of LXR target genes in the
brain, especially ABCA1 (8),
and LXR activation induces lipid efflux from glial cells
(11). These observations led
us to hypothesize that activation of LXRs in neurons might induce ABCA1,
resulting in cholesterol efflux and reduced secretion of A.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ImmunoblotsThe cells were lysed in buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, and 2 mM EDTA) supplemented with protease inhibitor mixture (Roche Applied Science). Postnuclear lysates were collected by spinning the lysed cells at 8000 x g for 5 min. Postnuclear lysates were fractionated in 415% SDS-polyacrylamide gel electrophoresis and transferred to 0.2-µm nitrocellulose membranes (Bio-Rad). Polyclonal anti-ABCA1 antibody was purchased from Novus (Littleton, CO). Monoclonal anti-actin antibody was purchased from Sigma. Polyclonal anti-SCD antibodies were raised in rabbits (13). Cellular APP was detected by monoclonal antibody 22C11 (Chemicon).
Detection of Secreted AIn experiments with
LXR activation, the cells were first induced with activators for 24 h in DMEM,
5% lipoprotein-deficient serum. The cells were then incubated in fresh medium
for 4 h for A
measurements. In experiments with ABCA1 transient
transfections, 24 h after transfection, the cells were incubated with DMEM, 1%
lipoprotein-deficient serum with or without apolipoproteins for 6 h for
A
measurements. After the indicated treatments, conditioned medium was
collected on ice and centrifuged at 6000 x g for 10 min to
remove cell debris. Immunoprecipitation of A
or C99 was performed with
monoclonal antibody 4G8 (Signet) and protein A/G-conjugated agarose (Santa
Cruz). A
and C99 were then extracted in NuPAGE sample buffer
(Invitrogen) and fractionated in 412% NuPAGE Bis-Tris Gel (Invitrogen).
Fractionated proteins were then transferred to polyvinylidene difluoride
membrane (Bio-Rad) and blotted with monoclonal antibody 6E10 (Signet) after
boiling. The immunoblots were developed using the ECL system (Pierce) scanned
and quantified by ImageQuant (Molecular dynamics). Quantitation of A
40
and A
42 was performed using a commercial A
enzyme-linked
immunosorbent assay kit (BIOSOURCE).
Cellular Cholesterol EffluxThe efflux experiments were
performed as described (13).
Briefly, Neuro2a cells were labeled with 1 µCi/ml
[1,2-3H(N)]-cholesterol (PerkinElmer Life Sciences) in DMEM
containing 5 mM methyl--cyclodextrin:cholesterol at a molar
ratio of 8:1 for 15 min at 37 °C. After washing, the cells were
equilibrated in DMEM, 0.2% bovine serum albumin for 30 min and then used for
efflux experiments. The cells were incubated with 10 µg/ml purified human
apoA-I or apoEs in DMEM, 1% lipoprotein-deficient serum for 6 h, and the
medium was collected and centrifuged at 6000 x g for 10 min to
remove cell debris and cholesterol crystals. The cells were lysed in 0.1
M sodium hydroxide, 0.1% SDS, and radioactivity was determined by
liquid scintillation counting. Efflux was expressed as the percentage of
radioactivity in the medium relative to the total radioactivity in cells and
medium.
Statistical AnalysisThe significance of differences between groups was assessed by Student's t test
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A is secreted in several different forms. Although A
40 (40
amino acids) is the predominant species, A
42 (42 amino acids) is a minor
but more amyloidogenic form. Whereas A
42 is formed predominantly in the
endoplasmic reticulum and trans-Golgi, A
40 is made in plasma membrane,
endocytic compartments, and trans-Golgi
(16,
17). Measurement of both forms
of A
in medium of Neuro2a cells by enzyme-linked immunosorbent assay
revealed that LXR/RXR activation (1 µM TO, 1 µM
9-cis-RA) decreased the secretion of A
40 by about 70%, whereas
A
42 was more moderately reduced (Fig.
2). Because the concentration of A
40 in medium was
8-fold more than that of A
42, the marked decrease in A
signal
in Fig. 1 primarily reflects a
reduction in A
40.
|
Neuro2a cells treated with LXR/RXR activators (1 µM TO and 1
µM 9-cis-RA) showed a marked induction of ABCA1 protein
(Fig. 3A). To
determine whether induction of ABCA1 might be responsible for decreased
secretion of A, Neuro2a cells were transfected with ABCA1 and incubated
with or without apoA-I. Transfection of ABCA1 resulted in levels of ABCA1
protein comparable with that induced by LXR/RXR activators
(Fig. 3A). Expression
of ABCA1 decreased the formation of A
without affecting cellular APP
levels (Fig. 3B).
Surprisingly, the major effect of ABCA1 expression was observed without the
addition of the extracellular acceptor apoA-I, and there was only a slight
further decrease in A
when apoA-I was added to medium
(Fig. 3B). Measurement
of cellular cholesterol efflux showed a small but significant increase in
efflux (1.0 ± 0.1% of total cellular cholesterol) with expression of
ABCA1 and the addition of apoA-I. Similarly, there was an increase in efflux
of cellular phospholipids (1.9% of total cellular choline-labeled lipids).
However, expression of ABCA1 alone did not increase cholesterol or
phospholipid efflux compared with nontransfected controls. We also measured
cellular cholesterol mass. Under our experimental conditions, there was no
detectable cholesteryl ester in cells, either with or without expression of
ABCA1.
|
As a control for possible nonspecific effects of ABCA1 expression unrelated
to functional activity, we transfected Neuro2a cells with a mutant form of
ABCA1 (Walker motif mutant); this mutant is well expressed on the cell surface
but inactive both in lipid efflux and binding of apoA-I
(18). When expressed at levels
similar to those of wild type ABCA1, the mutant failed to alter A
secretion by Neuro2a cells (Fig.
3C), indicating that the effects of ABCA1 expression are
related to the activity of the transporter, even though they do not require
lipid efflux.
Because apoE is a major apolipoprotein in the central nervous system and
the apoE4 isoform is associated with increased risk of AD
(19,
20), we also examined the
effects of apoE on A formation. Expression of ABCA1 with the addition of
apoE also resulted in a profound decrease in A
formation
(Fig. 4). Again, the major
effect was attributable to ABCA1 expression alone. The addition of apoE2
resulted in a small but significant further decrease in A
formation,
whereas apoE3 and apoE4 did not produce significant further reductions in
A
secretion. The addition of apoE isoforms without ABCA1 expression did
not affect A
secretion (not shown).
|
Both - and
-cleavage of APP are required for the generation of
A
. We next carried out a series of experiments to determine which step
is affected by ABCA1 overexpression. To measure
-cleavage, we
immunoprecipitated the
-secretase cleavage product (C99) from cell
lysates. There was an 85% decrease of cellular C99 in Neuro2a-APPSw
cells transiently transfected with ABCA1 compared with mock transfected
control (Fig. 5A).
-Secretase mediates the final step in A
generation
(1). To assess the direct
effects of ABCA1 expression on
-secretase cleavage of APP, Neuro2a
cells were transfected with constructs encoding C-terminal APP fragments (C99)
containing a Myc epitope tag. ABCA1 expression was found to decrease the
cleavage of C99 peptide, indicating a decrease in
-secretase processing
(Fig. 5B). The
addition of apoA-I did not provide a further significant decrease in A
generation (not shown).
|
In addition to targeting genes involved in cellular cholesterol efflux and
transport, LXRs also activate synthesis of mono-unsaturated fatty acids
(21). SCD is a key LXR target
gene that catalyzes the conversion of stearoylCoA to oleoylCoA and increases
the content of mono-unsaturated fatty acids in cell membrane phospholipids
(22). We have recently shown
that SCD activity decreases the amount of liquid ordered domains in the plasma
membrane (13). There are two
forms of SCD, both LXR targets, with similar catalytic activity and cellular
effects (22). Using an
antibody that recognizes both forms of SCD, we showed that treatment of
Neuro2a cells with LXR activators resulted in a modest 1.6-fold increase in
SCD protein (Fig. 6A).
Transient transfection of SCD in Neuro2a cells resulted in a decrease in
A secretion into medium (Fig.
6B). Because Neuro2a cells have high basal levels of SCD
activity, we also carried out similar experiments in 293 cells that have much
lower basal SCD expression
(13). Transient overexpression
of SCD resulted in an increase in APPs
formation (not shown) and a
profound decrease in A
generation
(Fig. 7A) that was
associated with a marked decrease in
-secretase cleavage of the C99
peptide (Fig. 7B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our studies were undertaken as a test of the hypothesis that LXR activation
would increase lipid efflux and thereby decrease A generation. However,
LXR activators decreased A
formation in the absence of extracellular
acceptors (Fig. 1), and the
addition of apoA-I or any of the three isoforms of apoE resulted in only minor
further changes in A
(Figs.
3 and
4). Serving as a control for
nonspecific effects related to ABCA1 expression, the Walker motif mutant of
ABCA1 failed to decrease A
formation
(Fig. 3C). These
findings indicate that the decrease in A
is related to an intrinsic
cellular activity of ABCA1. ABCA1 probably acts as a lipid translocase at the
plasma membrane (26) and
causes changes in plasma membrane morphology
(27). Recently, Vaughan and
Oram (28) showed that ABCA1
expression increases cell surface cholesterol oxidase-accessible domains,
indicating a redistribution of cholesterol toward the outer membrane,
independent of extracellular lipid acceptors. Decreased A
formation
might result from an ABCA1-induced redistribution of membrane cholesterol
either at the plasma membrane or in the Golgi or endocytic compartments.
Although ABCA1-mediated lipid translocation could affect raft organization,
it seems unlikely that an alteration in plasma membrane rafts fully accounts
for our findings. ABCA1 appears to be localized in and to induce cholesterol
efflux from nonraft membrane regions
(29), and ABCA1 expression did
not alter the distribution or amount of plasma membrane liquid ordered regions
in 293 cells.2 In
contrast, SCD activity decreases membrane liquid ordered regions
(13), and was associated with
increased -cleavage of APP, similar to the effects of cholesterol
depletion, However, changes in SCD protein levels in Neuro2a cells were modest
and unlikely to account for a major part of the effect of LXR activators.
Both ABCA1 and SCD expression were associated with a decrease in
-secretase cleavage of the C-terminal 99 amino acids of APP (Figs.
5B and
7). In the case of
APPSw, ABCA1 decreased
-cleavage by 85%
(Fig. 5A), but the
overall decrease in A
secretion was only around 60%
(Fig. 3B), which is
comparable with the effect of ABCA1 on
-cleavage
(Fig. 5B). This
suggests that under these conditions
-cleavage is the rate-limiting
step in A
secretion. Our findings suggest that this key enzyme of
A
formation is highly sensitive to its membrane lipid environment,
possibly reflecting the fact that
-secretase mediates an
intramembranous cleavage of APP. Alterations in membrane fluidity caused by
lipid translocation or increased content of mono-unsaturated phospholipids
could lead to a decrease in
-secretase activity. Although the cellular
compartmentalization of
-secretase activity is not completely
understood (1), several reports
suggest that A
formation mainly occurs in the endocytic compartments
(30) where components of
-secretase, including presenilins, are shown to be present
(31). An alternative
interpretation of our results is that induction of ABCA1 activity results in
altered cellular trafficking of APP or the C-terminal fragment of APP,
providing less substrate for
-secretase cleavage.
The finding that cellular cholesterol depletion leads to less A
secretion has fostered the idea that statins could be useful in the treatment
of AD. Epidemiological studies have suggested that statin therapy is
associated with decreased prevalence of AD
(32,
33). However, brain
cholesterol is derived by local synthesis (not from plasma low density
lipoprotein), and statins would have to be present in the brain at high levels
to alter neuronal lipid metabolism. Although human studies show an association
between statin treatment and decreased prevalence of AD, such associations can
reflect the influence of confounding factors, as appears to be the case for
statins and bone disease (34).
A recent placebo-controlled prospective trial of statin therapy in the elderly
failed to show any improvements in cognitive function
(35).
Inhibition of cholesterol esterification by acylCoA:cholesterol acyl transferase inhibitors has also been proposed to favorably affect processing of APP (36). Because cholesteryl esters have minor solubility in membranes and are thought to be present in cells as inert lipid droplets, it is unlikely that these effects are related to cholesteryl ester accumulation. One possibility suggested by our studies is that acylCoA:cholesterol acyl transferase inhibition leads to accumulation of cellular free sterol and conversion to LXR ligands via endogenous oxysterol synthesizing enzymes such as 24-cholesterol hydroxylase (37). Cholesteryl esters were not stored in cells in appreciable amounts under the conditions of our experiments, and changes in cellular acylCoA:cholesterol acyl transferase activity are thus unlikely to account for the findings.
Our findings, along with another recent report
(38), suggest that LXR
activators currently being developed for the treatment of atherosclerosis
might have therapeutic efficacy in Alzheimer's disease. LXR/RXR activation
markedly decreased A40 but had less effect on A
42 secretion.
Although A
42 is thought to be more amyloidogenic than A
40, much
more A
40 is secreted by neurons, and both forms are found in amyloid
plaques (2). Therefore,
controlling the predominant A
40 secretion could be beneficial. Our
results differ from another report where a 65% increase in A
42 and no
significant change in A
40 was found in Neuro2a cells treated with TO and
9-cis-retinoic acid (10 µM)
(39). The reasons for this
discrepancy are not clear. However, our findings substantially agree with
another report (38), which
appeared while our paper was under review. Our results extend these latter
studies by the direct demonstration that ABCA1 and SCD expression decreases
A
formation and that these effects are observed without lipid efflux
from cells and involve a decrease in
-secretase cleavage.
In contrast to the more general approach of cholesterol biosynthesis inhibition, LXR activation may directly regulate genes that favorably modulate plasma membrane composition and structure in the brain (8). Tangier disease patients have not been reported with premature dementia, suggesting that ABCA1 may not have an essential role in protecting against AD. However, this does not rule out the possibility that increased expression of ABCA1 has a protective role, just as it does in atherosclerosis (40).
![]() |
FOOTNOTES |
---|
¶ Ellison Medical Foundation New Scholar in Aging.
|| To whom correspondence should be addressed. Tel.: 212-305-9418; Fax: 212-305-5052; E-mail: art1{at}columbia.edu.
1 The abbreviations used are: A, amyloid
peptide; AD,
Alzheimer's disease; APP, amyloid precursor protein; LXR, liver X receptor(s);
ABCA1, ATP-binding cassette transporter A1; apo, apolipoprotein; SCD, stearoyl
CoA desaturase; RXR, retinoid X receptor; C99, 99-residue C-terminal fragment
of APP; 9-cis-RA, 9-cis-retioic acid; TO, TO901317; DMEM,
Dulbecco's modified Eagle's medium.
2 Y. Sun, J. Yao, T.-W. Kim, and A. R. Tall, unpublished data.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|