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INTRODUCTION |
ATP-binding cassette transporter A1
(ABCA1)1 is a member of a
large superfamily of evolutionarily conserved transmembrane proteins that transport lipids, proteins, and drugs across cellular membranes (1). Mutations in the ABCA1 gene cause high density lipoprotein (HDL)
deficiency syndromes, such as Tangier disease, which are characterized by the virtual absence of HDL and apolipoprotein A-I
(apoA-I), cholesterol deposition in tissue macrophages, and prevalent
atherosclerosis (2, 3). ABCA1 mediates cholesterol efflux and secretion
of excess cholesterol from cells to lipid-free apolipoproteins and is a
major determinant of plasma HDL concentration (4). Thus, ABCA1 controls
reverse cholesterol transport, a metabolic pathway whereby excess
cholesterol in peripheral tissues is removed and transported to the
liver (5). Because of its ability to deplete macrophages of cholesterol
and to raise plasma HDL levels, ABCA1 has been studied mainly for its
role in the pathogenesis of atherosclerosis (5, 6). Nonetheless, ABCA1 mRNA and protein are widely distributed among multiple tissues including brain (7, 8), suggesting a more generalized role for ABCA1.
Data regarding the possible physiological function of ABCA1 in the
central nervous system, however, are limited. ABCA1 expression and
ABCA1-mediated lipid secretory pathway are regulated by hydroxysterols
and 9-cis-retinoic acid, ligands for nuclear hormone liver X
receptors (LXRs) and retinoid X receptors (RXRs), respectively (9). The
LXRs function as heterodimers with RXRs, and these dimers can be
activated by ligands for either receptor (10). In conditions that
result in high cholesterol levels, LXR activation increases the
mRNA levels of several target genes primarily involved in lipid
metabolism including ATP-binding transporters. Nuclear receptors LXR
and RXR are also expressed in neurons and glia (7, 11).
Cholesterol plays major structural and functional role in the central
nervous system. Although the central nervous system comprises less than
10% of the total body mass, it contains approximately one quarter of
all of the unesterified cholesterol (12). Almost all of the central
nervous system cholesterol is derived from in situ
biosynthesis and is transported by lipoproteins similar to plasma HDL
(13). The main apolipoproteins are apoE, produced by astrocytes (14)
and microglia (15), and apoA-I from the systemic circulation or from
brain endothelial cells (16). Mutations that affect the synthesis and
intracellular traffic of cholesterol in neurons lead to
neurodegeneration like that is seen in Smith-Lemli-Opitz syndrome (17)
and Niemann-Pick type C disease (18), and disturbances in brain
cholesterol metabolism may contribute to the pathogenesis of
Alzheimer's disease (AD) (19). AD is a senile dementia, characterized by extracellular deposits of amyloid
(A
) peptide, derived from the amyloid precursor protein (APP) cleavage (20). To generate A
, a
small percentage of APP is cleaved by two enzymes:
-secretase-1 (21)
and a still unidentified
-secretase, producing two secreted products, A
and soluble APP
(sAPP
). Most of the APP is cut by
a proteinase named
-secretase-generating soluble APP
(sAPP
). The residual C-terminal APP fragment (CTF-
) remains within the plasma membrane and can be cleaved by
-secretase activity yielding p3 fragment and thus precluding A
production. Previous in
vitro studies have shown that cellular cholesterol modulates APP
processing in cell lines and primary neurons. A
production and
secretion are reduced dramatically when cellular cholesterol levels are decreased by inhibiting de novo synthesis with
hydroxymethylglutaryl-CoA reductase inhibitors (22) alone or in
combination with the cholesterol-extracting agent
methyl-[
]-cyclodextrin and statins (23). Furthermore, elevated
cellular cholesterol levels decrease
-secretase activity (24)
and increase
- and
-secretase activity (25, 26).
To clarify further the central nervous system function of ABCA1, we
first examined ABCA1 expression and transcriptional regulation in brain
cells. We found high expression of ABCA1 in neurons in the cortex,
basal forebrain, and hippocampus of the rat brain. We then demonstrated
ABCA1 transcriptional up-regulation by established ligands for the
LXRs/RXRs and a functional increase in apolipoprotein-mediated cholesterol efflux in primary neuronal and glial cultures. More importantly, we showed that in non-neuronal and neuronal cells overexpressing a human Swedish variant of APP (APPsw), ligand-activated LXR/RXR, alone or in combination with ABCA1-mediated cholesterol depletion, caused substantial reduction in the stability of APP C-terminal fragments and decreased A
production.
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MATERIALS AND METHODS |
Chemicals
The following were purchased from Sigma and used in all
experiments: delipidated calf serum,
-mercaptoethanol,
L-glutamine, 22R-hydroxycholesterol
(22R), 9-cis-retinoic acid (RA),
22S-OH cholesterol (22S), leupeptin, aprotinin,
and AEBSF. We obtained 1,2-[3H]cholesterol (specific
activity 45 Ci/µmol) from PerkinElmer Life Sciences. Tissue culture
flasks and plates were from Corning (Corning, NY) and Falcon (Lincoln,
NJ). 22R, RA, and 22S were dissolved in ethanol.
Antibodies
Microtubule-associated protein 2 (MAP-2) and rat integrin
(CD11b) mouse antibodies (Abs) were from Chemicon International (Temecula, CA). Anti-glial fibrillary acidic protein Ab was from Sigma.
Rabbit polyclonal anti-ABCA1 was from Novus (Littleton, CO). Goat
anti-ABCA1 was from Santa Cruz Biotechnology (Santa Cruz, CA). TAU-5 Ab
was from NeoMarkers (Fremont, CA). Mouse anti-
-tubulin Ab was
from Santa Cruz Biotechnology. The 6E10 monoclonal Ab (Signet, Dedham, MA) recognizes the first 17 amino acids of the A
peptide. 6E10 Ab was used for Western blotting to detect full-length
APP (APPfl) and sAPP
and for immunoprecipitation of total Ab. Rabbit C8 polyclonal Ab (generous gift from Dr. Denis Selkoe, Harvard University) was used to detect CTF resulting from
- or
-secretase cleavages. Secondary Abs conjugated to horseradish peroxidase and to
alkaline phosphatase were from Jackson ImmunoResearch (West Grove, PA).
Alexa-labeled secondary Abs and DiL-Ac-LDL were from Molecular Probes
(Eugene, OR).
Cell Culture
Primary Neuronal Cultures--
Primary neuronal cultures were
made from dissociated cortices and hippocampi of 17-19-day-old
Sprague-Dawley rat embryos as described previously (27). Briefly,
cortices and hippocampi were dissected and incubated with 1×
trypsin/EDTA (Invitrogen) for 10 min at room temperature. The trypsin
was inactivated with complete defined neurobasal medium supplemented
with B27, GlutaMax II, 10% horse serum, 5% fetal bovine serum, and
antibiotics (medium and additives were from Invitrogen). A single cell
suspension was obtained after filtering through a 70-µm Falcon cell
strainer (BD Biosciences, Franklin Lakes, NJ). For all experiments
except immunostaining, neurons were plated at a high density (2 × 106/ml, 2 ml/well) on 100 µg/ml
poly-D-lysine-coated six-well Costar plates (Stony Brook,
NY). For immunostaining neurons were plated at low density (2 × 105/ml, 0.250 ml/well) on 200 µg/ml
poly-D-lysine-coated Nunc Permanox chamber slides (Nalge,
Naperville, IL). One h after plating the medium was changed with
complete neurobasal medium as above, without serum. At day in
vitro 2 the neuronal cultures were treated with cytosine-
-D-arabinoside (4 µM final
concentration) to suppress proliferation of non-neuronal dividing cells.
Mixed Glial Cultures--
Mixed glial cultures were isolated
from the cerebral cortices of 21-day-old Sprague-Dawley rat embryos or
newborn pups as described previously (28). The cortices were minced and
dissociated by trituration in 0.25% trypsin and 0.01% DNase-I. After
the addition of Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and antibiotics (growth medium), the supernatant was
passed through a 70-µm cell strainer, resuspended in growth medium,
and plated in poly-L-lysine-coated T75 Falcon flasks at a
density of 1.5-2.0 × 107 cells/flask. The mixed
glial cultures were grown for 1 week before collecting the microglia
and astrocytes. To isolate microglia, we cultured the cells for 7 days
and then vigorously agitated the confluent cultures on a rotary shaker
for 15 h (37 °C, 180 rpm). The resulting cell suspension, which
was rich in microglia and oligodendroglia, was placed in T75 plastic
flasks (1 × 105/ml, total volume of 10 ml) and
allowed to adhere at 37 °C. After a 1-3-h adhering interval,
loosely adhering cells (most of which were oligodendroglia) were
removed by gently shaking the flasks at room temperature. The strongly
adhering microglia were then released by vigorous shaking in defined
medium with 0.2% trypsin, resuspended, and 15% fetal bovine serum was
added. The purity of microglial cultures was assessed by the uptake of
DiL-Ac-LDL according to the original protocol (28) and the
manufacturer's instructions. More than 95% of the cells harvested
from the medium after shaking took up DiL-Ac-LDL. To isolate astrocytes
from the mixed glial culture, we harvested the adherent astroglial
cells, which were detached by trypsin/EDTA treatment. These cells were then collected, pelleted by centrifugation, and resuspended in fresh
growth medium. Antibodies to the following markers were used to
identify the specific cell types: MAP-2 for neurons, glial fibrillary
acidic protein for astrocytes, and CD11b for microglia. The purity of
these enriched neuronal, astroglial, and microglial cultures as
determined by the specific staining of the markers was greater than
95%. All cells were incubated at 37 °C, 5% CO2 and
95% air. Cells were seeded in six-well plates or T25 Corning flasks,
and then astrocytes and microglia were grown to 80-90% confluence
before use. Astrocytes were used up to six passages.
CHOAPPsw Cells--
CHOAPPsw cells (a generous gift from
Dr. Ruth Perez, University of Pittsburgh) were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
500 µg/ml Geneticin (Invitrogen) as described before (29).
H4 (Human Neuroglioma) Cells--
H4 cells were obtained from
ATCC and transfected with pcDNA3.1HygroAPPsw. A stable cell line
was established after a 1-month selection with 250 µg/ml
hygromycin (Invitrogen) (33).
RNA Isolation and RT-PCR
To determine the expression of ABCA1 in neurons and glia, we
used reverse transcription followed by PCR amplification (RT-PCR). Total RNA from enriched primary embryonic rat neuronal, astrocytic, and
microglial cell cultures was isolated using RNeasy Kit (Qiagen, Valencia, CA). RNA concentrations were determined
spectrophotometrically using a DU640 spectrophotometer
(Beckman). Mouse forward ABCA1-341F (5'-TCCCGGCGAGGCTCCCGGTGT-3') and reverse ABCA1-899R
(5'-CAGCTCTTGGGCCAGGCCCCC-3') primers (NCBI accession NM_013454)
were used for semiquantitative RT-PCR. The primers for rodent GAPDH
(Clontech, Palo Alto, CA) were used to amplify an
internal control. Semiquantitative RT-PCR for rat ABCA1 levels was
performed using 0.5 µg of total RNA and two-step RT-PCR. The reverse
transcription reaction and synthesis of the first-strand cDNA were
run at 42 °C for 60 min using Advantage RT-For-PCR
(Clontech) and random primer hexamer. The PCR was
carried out for 40 cycles (denaturation at 95 °C for 15 s,
annealing and extension at 60 °C for 1 min) using a PCR kit
(Promega) and the above ABCA1 primers. Data quantification and
analysis were calculated relative to the level of the control. Each
sample was assayed in triplicate during two independent experiments.
Northern Blotting
Northern blotting was performed as reported previously (30) with
the following modifications. Total RNA (~5 µg) was separated on 1%
denaturing agarose gel containing 2.2 M formaldehyde
(Ambion, Austin, TX), transferred to NytranSuperCharge membrane
(Schleicher & Schuell), UV cross-linked, and processed for detection of
mRNA using NorthernMaxTM system (Ambion). A 518-bp
digoxigenin-labeled antisense single stranded DNA probe was
generated by asymmetric PCR amplification using a DIG Probe Synthesis
kit (Roche Molecular Biochemicals), ABCA1-879R primer and 2 µl of
the first strand cDNA reaction as generated for the RT-PCR.
Overnight hybridization of the probe to the immobilized RNA was carried
out in ULTRAhybTM Ultrasensitive Hybridization Buffer (Ambion), and the
membrane was processed in digoxigenin Wash and Block Buffer set
(Roche). To verify equal loading of RNA, we stripped the ABCA1 probe
from the membranes and rehybridized them with a rodent GAPDH control probe. The hybridized probe/anti-IG-alkaline phosphatase complex was
visualized on x-ray film (Kodak) after incubation of the membrane with
CDP-Star ultrasensitive chemiluminescent substrate for alkaline phosphatase (Roche). The relative intensities of the hybridization signals were determined by densitometry (Molecular Dynamics, model 300A) and quantified.
Radioactive Labeling and LXR/RXR Ligand
Stimulation
For cholesterol efflux studies, monolayers of cells were washed
and then incubated for 24 h in complete culture medium containing 2 µCi/ml 1,2-[3H]cholesterol, antibiotics, and 10%
fetal bovine serum, as described previously (30, 31). Parallel cultures
incubated with nonradioactive cholesterol were used to measure and
compare the amount of ABCA1 protein and its mRNA levels. After the
24-h labeling period, cells were washed and then incubated with medium
plus 1% delipidated calf serum (Sigma) plus or minus the indicated
ligands (10 µM 22R and 10 µM
RA). After this 8-h incubation, some of the wells were washed with
phosphate-buffered saline (PBS) and the cells lysed in 0.5 N NaOH. These wells provided a base line (time zero = T0) value for total 1,2-[3H]cholesterol content for time
course experiments. Unlabeled monolayers were harvested for total RNA
isolation or protein analysis at different time points.
Measurement of Cholesterol Efflux
Cells containing 1,2-[3H]cholesterol were treated
with LXR/RXR ligands 22R (10 µM) and RA (10 µM). These cells and vehicle control cells were washed
with medium and incubated for 24 h or in the time course
experiments for 4-24 h in the presence or absence of cholesterol
acceptors (apoA-I or apoE). ApoA-I from human plasma and recombinant
apoE3 (Sigma) were used at concentrations of 15-30 µg/ml. After
incubation, the medium was centrifuged to remove any cells. The cells
were washed and lysed in NaOH. Aliquots of medium and cell lysates were
assayed by liquid scintillation counting. The percent efflux was
calculated by dividing the radioactivity in the medium by the sum of
the radioactivity in the medium and cell lysate.
Apolipoprotein-specific [3H]cholesterol efflux was
measured as the fraction of total radiolabeled cholesterol (cells and
medium) appearing in the medium in the presence of apolipoproteins
after subtraction of values for apolipoprotein-free medium. For time
course experiments cholesterol efflux was quantified by removing
replicate 150-µl aliquots of the incubation medium containing the
indicated acceptor at different time points and the cholesterol efflux
determined as above. The radioactivity of the cell lysate at the
starting point were considered as total cholesterol.
Measurement of Cholesterol Content
Cells were washed in PBS and divided in half for determination
of cholesterol content and for protein extraction. Cholesterol mass was
determined as described previously (32) with a slight modification.
Extraction was performed with chloroform:isopropyl alcohol (3:1), and
after centrifugation clear supernatant was decanted. The solvent was
evaporated under nitrogen, the residue dissolved, and total cholesterol
(free cholesterol and cholesteryl esters) concentration determined by
enzymatic assay (Infinity cholesterol reagent, Sigma) using a standard
curve with cholesterol standards from Sigma. The cholesterol mass was
normalized to the total protein (µg of cholesterol/mg of protein).
Protein Isolation and Western Blotting Analysis
For Western blot analysis cellular extracts were prepared from
primary neurons, microglia, and astrocytes. Cells were washed and
scraped in PBS and lysed in 10 mM Tris-HCl, pH 7.3, 1 mM MgCl2, and 0.25% SDS, 1% Triton X-100 in
the presence of protease inhibitors (10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 10 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride). Cellular extracts were centrifuged to remove debris.
For ABCA1 Western blot analysis, extracts containing 20-50 µg of
total protein were reduced with 2-mercaptoethanol in
NuPAGETM loading buffer (without boiling), loaded,
electrophoresed on 3-8% NuPAGETM Tris acetate gels
(Novex, San Diego), and transferred to nitrocellulose membranes. ABCA1
was detected with a rabbit primary Ab raised against human ABCA1
protein. For Western blot analyses of APPfl, CTF, sAPPa, and A
, we
used cellular extracts and conditioned media and 10-20% Tricine gels.
Membranes were then incubated with a goat anti-rabbit IgG conjugated to
horseradish peroxidase and processed for visualization by enhanced
chemiluminescence ECL PlusTM (Amersham Biosciences) according to the
manufacturer's protocol. Western blotting with anti-
-tubulin A
was used as an internal standard. The relative intensities of the bands
were quantified by densitometry (Molecular Dynamics).
Immunoprecipitation and ELISA for A
Immunoprecipitation and ELISA for A
were performed
essentially as before (33, 34). Briefly, A
was immunoprecipitated from the conditioned medium and immunoblotted using the 6E10 Ab. ELISA
for A
was performed using the same 6E10 as the capture Ab and
anti-Ab40 and anti-Ab42 polyclonal Abs (BioSource International, Camarillo, CA) to detect Ab1-40 and Ab1-42,
respectively. The amount of A
was normalized either to the total
protein or to the expression of APPfl as measured by Western blotting.
Tissue Preparation for Immunohistochemistry
The study fully conformed to the guidelines outlined in the
Guide for the Care and Use of Laboratory Animals from the U.S. Department of Health and Human Services and was approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Young mid-age (250-275 g) Harlan Sprague-Dawley rats were deeply anesthetized with pentobarbital (Nembutal, 80-100 mg/kg; Abbott Laboratories, North Chicago, IL) and perfused transcardially with 100 ml 0.1 M PBS (pH 7.4) followed by 500 ml of 4%
paraformaldehyde with 15% saturated picric acid in 0.1 M
phosphate buffer (pH 7.4). After perfusion, the brain was removed and
placed into the same fixative for 30 min, then immersed in 4%
paraformaldehyde in neutral 0.1 M phosphate buffer at
4 °C overnight. The brain was transferred to 15% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h, then
to 30% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4 °C until it sank. The cryoprotected brain was frozen and cut in
coronal plane on a cryostat (Jung CM 1800; Brodersen Instrument, Valencia, PA) at a 40-µm thickness.
ABCA1 Immunohistochemistry
Immunohistochemistry for ABCA1 was conducted using the free
floating technique as described previously (35). Sections were preblocked with 10% normal goat serum and 0.1% Triton X-100 in 0.1 M PBS and incubated overnight at 4 °C with a rabbit
anti-ABCA1 polyclonal Ab (1:750) in 0.1 M PBS with 5%
normal serum and 0.1% Triton X-100. Affinity-purified, biotinylated
goat anti-rabbit IgG (1:50; Jackson ImmunoResearch
Laboratories) was incubated as secondary Ab with 5% normal serum and
0.1% Triton X-100 in 0.1 M PBS at 4 °C for 2 h at
room temperature. Between all steps, the tissue was rinsed three times
with 0.1% Triton X-100 in 0.1 M PBS, for 10 min each time.
The peroxidase reaction was developed by a DAB Substrate Kit (Vector,
Burlingame, CA) with the addition of 2.5% nickel as a reaction
intensifier. Sections were rinsed several times in 0.1 M
Tris-buffered saline, mounted on gelatinized slides, dehydrated in
alcohols, defatted in xylenes, and placed on cover slips for analysis
with an Olympus Vanox AH-2 microscope. Three sections of brain tissue
from three animals were processed for immunoreactivity. To control for
nonspecific immunostaining, we incubated additional sections in the
absence of primary Ab or with an irrelevant secondary Ab. In both
control paradigms, there was no detectable immunostaining of tissue sections.
ABCA1 Immunostaining of Primary Neuronal Cultures
Cells were plated on four-well poly-D-lysine-coated
Permanox slide chambers. Primary neuronal cultures, plated at low
density (5 × 104 cells), were fixed between day
in vitro 2 and day in vitro 7. The cells were
fixed with 4% paraformaldehyde at room temperature for 30 min,
permeabilized with 0.2% Triton X-100 for 10 min, and blocked for
1 h in PBS containing 5% bovine serum albumin, 0.2% Tween 20, and 5% goat serum. After blocking the cells were incubated overnight
with primary Abs followed by a 1-h incubation with a secondary Ab. Two
different anti-ABCA1 Abs were used to detect ABCA1: goat anti-ABCA1
primary Ab (Santa Cruz) and rabbit anti-ABCA1 (Novus). Secondary Abs
for detection of anti-ABCA1 primary Abs were donkey anti-goat and goat
anti-rabbit Alexa488-labeled secondary Abs. For MAP-2 and TAU-5 we used
anti-mouse Alexa546-labeled secondary Ab. The slides were examined with
a fluorescent microscope (Olympus IX-70), and images taken through a
SPOT-2 CCD camera were processed using Image software (National
Institutes of Health). The following negative controls were used to
prove the specificity of ABCA1 immunostaining: (a) staining
with primary anti-ABCA1 Ab preincubated with the blocking peptide
(Santa Cruz); and (b) staining only with secondary Abs.
There was no staining detected in any of the negative control experiments.
Statistical Analysis
Results are reported as the mean ± S.E. Statistical
significance was determined by two-tailed Student's t test
(GraphPad Prism version 3.0, GraphPad Software, San Diego).
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RESULTS |
Distribution and Localization of ABCA1 in Rat Brain--
ABCA1
immunostaining was detected throughout the adult rat brain. In the
cerebral cortex, the Ab exclusively labeled neuronal cells.
ABCA1-positive neurons were observed in all examined cortical layers
(Fig. 1A). In these cells,
localization of the immunoreaction product was restricted to the cell
membrane (Fig. 1B). In subcortical regions, ABCA1
immunoreactive cells were found in the hypothalamus, thalamus,
amygdala, and cholinergic basal forebrain (Fig. 1, C-F). Among thalamic nuclei, the reticulate nucleus displayed the most abundant cellular ABCA1 immunostaining. Large neurons of the
cholinergic nucleus basalis were one of the most abundantly
immunolabeled neurons. In addition, both neurophil and neuronal
staining was particularly intense in the hippocampus. Dentate granule
cells as well as hilar neurons showed strong ABCA1 immunoreactivity that was restricted to the cell periphery (Fig.
2, A and B),
similar to the staining pattern observed in cortical neurons. The CA1 and CA3 pyramidal cells displayed even more intense immunoreaction, with some immunoreactive apical dendrites. Stratum oriens of both fields showed a dense neurophil staining (Fig. 2, C and
D). These results showed that ABCA1 is expressed
predominantly in the neuronal cells of adult rat brain.

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Fig. 1.
Brain distribution of ABCA1. Low
(A, C, and E) and high power
(B, D, and F) photomicrographs
illustrating immunohistochemical localization of ABCA1 Ab in coronal
tissue sections from adult rat brain are shown. Neuronal cells in all
cortical layers show ABCA1-positive staining restricted to the cell
membrane (A and B). Intense cell labeling was
also found in the hypothalamus and amygdala (C), thalamic
reticulate nucleus (D), and in the nucleus basalis
(E and F). Amy, amygdala;
Ht, hypothalamus; NB, nucleus basalis;
ot, optic tract; Ret. N., reticulate nucleus of
thalamus. Scale bar = 200 µm (A,
C, and E); 50 µm (B, D,
and F).
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Fig. 2.
ABCA1 immunostaining of adult rat
hippocampus. Granule cells within dentate granular layers and
hilar neurons show strong ABCA1 immunoreactivity that is localized
mainly in the periphery of cell bodies (A and B).
In the CA1, neuronal staining is strong in the pyramidal cell layer
(C). Although some immunoreactive dendrites are seen in
stratum radiatum, stratum oriens displays a dense neuropil staining.
The CA3 pyramidal cells are less intensely stained, which is contrasted
by dark labeling of few displaced pyramidal neurons (D,
arrow). Dgl, dentate gyrus granular layer;
CA1, CA3, hippocampal CA fields; so,
stratum oriens; sp, stratum pyramidale; sr,
stratum radiatum. Scale bar = 200 µm (A),
100 µm (B), and 50 µm (C and
D).
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ABCA1 Expression in Primary Neurons--
To determine whether
ABCA1 was transcribed in neurons, we performed Northern blotting on
total RNA extracted from embryonic rat neurons. Fig.
3A (lanes 1 and
2) illustrates a band of ~8 kb corresponding to the
predicted size of ABCA1 mRNA. To examine whether ligands for the
nuclear receptors LXR and RXR activate ABCA1 transcription in neurons
similar to what has been shown in macrophages, we treated primary
neurons for 24 h with 22R and RA, which are ligands for
LXR and RXR, respectively. Fig. 3A (lanes 3 and
4) showed a substantial increase in ABCA1 mRNA with this combination, which was accompanied by a marked increase in ABCA1 protein (Fig. 3B). It should be noted that we and others
(30, 36) detected several bands by Western blotting, suggesting the possibility of post-translational modification with lower molecular mass species migrating at ~210 kDa.

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Fig. 3.
ABCA1 expression and cholesterol efflux in
primary neurons. A, Northern blotting for ABCA1.
Primary neurons were treated for 24 h with vehicle (C)
or 22R + RA, and total RNA was isolated. Northern blotting
was performed using a single stranded antisense DIG-labeled ABCA1
probe. Membranes were probed with a GAPDH cDNA probe to ensure
equivalent RNA loading and its integrity. Lanes 1 and 2, control; lanes 3 and 4, 22R and RA treatments.
B, neurons were treated as in A, lysed in lysis
buffer, and Western blot analysis was performed using anti-ABCA1
polyclonal Ab. Western blotting with -tubulin Ab was used as a
loading control. Lane 1, control; lane 2,
22R and RA. Quantification of ABCA1 protein level,
normalized for -tubulin expression, is presented below. Data are the
mean ± S.E., n = 3. C and
D, ABCA1 mediates cholesterol efflux in primary neurons.
Cells were labeled with [3H]cholesterol, and cholesterol
efflux was determined after a 24-h incubation with 30 µg/ml apoA-I
(C) or 15 and 30 µg/ml apoE3 (D).
22R + RA was present during the incubation with
apolipoproteins. Apolipoprotein-specific efflux was determined as
indicated under "Materials and Methods." Data (mean ± S.E.)
are the result of two (in C) or one (in D)
experiment(s) in triplicate. ***, p < 0.001; **,
p < 0.002; and *, p < 0.01 compared
with control nontreated with LXR/RXR ligands. E, 24 h
treatment with 22R + RA and 20 µg/ml apoA-I decreased
cholesterol content in neurons. Cellular lipids were extracted with
chloroform/methanol, and the total cholesterol (free cholesterol and
cholesteryl esters) concentration was determined by enzymatic assay as
described under "Materials and Methods." The cholesterol mass was
normalized to the total protein (µg of cholesterol/mg of protein) and
presented as a -fold of control. n = 5, *,
p < 0.05.
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We found that application of 10 µM 25-hydroxycholesterol,
which occurs naturally in the brain (37), also increased ABCA1 mRNA
and protein levels (data not shown). Previously, it was shown in
macrophages and fibroblasts that increased cholesterol efflux to
lipid-free apolipoproteins correlated with increased levels of ABCA1
expression. To examine the potential connection between ABCA1
expression and function in neurons, we labeled the cells with
radioactive cholesterol for 24 h and induced the expression of
ABCA1 for additional 8 h. Cholesterol efflux was activated with
the addition of apoA-I and measured 24 h later. Because ABCA1 was
shown to mediate cholesterol transport not only to apoA-I but also to
other exchangeable apolipoproteins in non-neuronal cells (38), we
compared apoE- and apoA-I-specific cholesterol efflux after treatment
with ligands for LXR/RXR (Fig. 3D). ApoE was chosen because
it is the main apolipoprotein in the central nervous system and the
ApoE3 isoform, in particular, because it has the highest allelic
frequency in the population. The increase in ABCA1 protein level was
concomitant with the marked up-regulation in
apolipoprotein-dependent cholesterol efflux, which was
consistent with increased ABCA1 functionality. The treatment with
ligands for LXR/RXR increased cholesterol efflux to lipid-free apoA-I and apoE3 in a similar fashion, with apoA-I showing slightly higher efflux than apoE3 (Fig. 3, C and D). As expected,
the cholesterol efflux was dependent on the exogenous apolipoprotein
concentration (Fig. 3D).
Next we examined whether the increased cholesterol efflux after
treatment with LXR/RXR ligands affected the cholesterol content in
neurons. Fig. 3E shows that treatment with 22R + RA, and apoA-I, resulted in a small but significant decrease in
cholesterol content compared with the control cells (no ligands, no
apoA-I). It should be noted that ABCA1-mediated cholesterol efflux
requires the addition of lipid-free/lipid-poor apolipoproteins (4), and
therefore, in this experiment we compared ABCA1-dependent
(cells treated with ligands and apoA-I) versus
ABCA1-independent (control cells, no ligands, no apoA-I) reduction in
cholesterol content. Therefore, we concluded that ABCA1 was expressed
in neurons, and its expression and function were regulated by LXR/RXR
ligands. We demonstrated also that stimulation of
apolipoprotein-specific cholesterol efflux decreased cholesterol
concentration in neurons.
Time Course of 22R and RA Up-regulation of ABCA1 mRNA, Protein
Expression, and Cholesterol Efflux--
We examined further the
kinetics of ABCA1 mRNA expression, protein synthesis, and
cholesterol efflux (Fig. 4). A 5-fold
increase in ABCA1 mRNA was detected within the first 4 h after
treatment with 22R and RA. There was no increase in ABCA1
mRNA after treatment with 22R or RA alone at this time
point. The ABCA1 mRNA level continued to increase steadily after
22R and RA treatment, and at the 24 h time point it was
more than 30-fold greater compared with the control. A 24-h treatment
with any of the ligands alone resulted in a significant but lower
increase in ABCA1 mRNA, consistent with previous results in
non-neuronal cells, indicating that ligands for LXR/RXR act
synergistically to increase ABCA1 expression (39, 40).

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Fig. 4.
Time course of ABCA1 expression and
apolipoprotein-dependent cholesterol efflux in
neurons. Cells were treated with 22R or RA alone or
combination of 22R and RA, and total RNA and protein
extracts were prepared at 4-h intervals. Northern blot (A)
and Western blot (B) and quantification of ABCA1 mRNA
and protein levels were performed as in Fig. 3, A and
B. Data for Northern blotting are the result of one
experiment in triplicate and for Western blotting, a single experiment.
C, time course of apolipoprotein-dependent
cholesterol efflux in primary neurons treated with LXR/RXR ligands.
Cells were labeled for 24 h with [3H]cholesterol,
incubated for 8 h with 22R + RA and then with apoA-I
(30 µg/ml) plus 22R and RA. Aliquots of the efflux medium
were taken at 4-h intervals, and apoA-I-dependent
cholesterol efflux was determined as indicated under "Materials and
Methods." Data are presented as a percentage (mean ± S.E.) of
the total radioactivity in cells and medium; each point is an average
of triplicate experiments. *, p < 0.05 compared with
control nontreated with LXR/RXR ligands.
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Immunolocalization of ABCA1 in Neurons--
To characterize
further ABCA1 localization in neurons, we used primary cultures of
cortical and hippocampal neurons that showed high levels of ABCA1
expression. To determine in which of the nerve processes ABCA1 was
localized, we costained neurons with anti-MAP-2 and anti-tau Abs. In
neurons, MAP-2 was present in soma and dendrites with little or no
immunostaining detectable in axons (41). Fig.
5, A and B, shows
that ABCA1 and MAP-2 were expressed in the same cells, although the
distribution of these two proteins differed to some extent. ABCA1 and
MAP-2 colocalized in the cell body and in some of the neurites, whereas
ABCA1 staining was extended to processes not immunostained for MAP-2
(Fig. 5C). To determine whether ABCA1 was present
specifically in axons, we costained neuronal cultures with the anti-tau
Ab TAU-5, which recognizes both the phosphorylated and unphosphorylated
forms of tau.

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Fig. 5.
ABCA1 localization in primary
neurons. Six-day-old neurons were immunostained with goat
anti-ABCA1 (A) and with mouse anti-MAP-2 (B)
primary Abs. C represents merged images. Three-day-old
neurons were stained with goat anti-ABCA1 (D) and TAU-5
(E) Abs. The inset shows the presence of ABCA1
and tau in the growth cones of developing axons. Secondary Abs were
Alexa488-labeled for ABCA1 and Cy3-labeled for MAP-2 and TAU-5.
Scale bar = 40 µm. F shows that ABCA1 was
localized to the neuronal membrane and is a higher magnification of
6-day-old neurons immunostained with rabbit anti-ABCA1 primary Ab and
anti-rabbit Alexa488-labeled secondary Ab. Arrow, neuronal
membrane. Scale bar = 20 µm.
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Microtubule-associated protein tau plays an important role in axonal
morphology, growth, and polarity and, in contrast to MAP-2, is
localized to axons (41). As visible from Fig. 5, D and
E, in day in vitro 3 neurons, ABCA1 and tau
colocalized to axons and specifically to the growth cones of developing
axons (Fig. 5E, inset). There was no staining
with primary anti-ABCA1 Ab preincubated with the blocking peptide (data
not shown). Fig. 5F is a higher magnification of 6-day-old
neurons and demonstrates that ABCA1 also localized to the plasma
membrane (Fig. 5F, arrow) and in intracellular
compartments. Similar patterns of distribution were observed previously
in HeLa cells (42). From these experiments we concluded that in neurons
ABCA1 was localized to cell body, dendrites, and axons.
ABCA1 Expression in Primary Astrocytes--
Immunohistochemistry
experiments showed very little ABCA1 immunostaining of glial cells in
the white matter and cortex in agreement with a previous report (8).
Considering the important role of astrocytes in the formation of brain
lipoprotein complexes and transport of cholesterol (43), we next
examined ABCA1 expression in these cells using a semiquantitative
RT-PCR method. Astrocytic cultures were prepared from 21-day-old
embryos or newborn pups and used after several passages to ensure the
absence of surviving neurons. Fig.
6A, lane 1, shows a
584-bp ABCA1 product generated by the two-step RT-PCR approach,
confirming astrocytic ABCA1 expression. RT-PCR using GAPDH-specific
primers generated a 960-bp product (lower panel) and showed
preserved RNA integrity and equal RNA load in all samples.

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Fig. 6.
ABCA1 expression in astrocytes.
Astrocytes were treated for 24 h with vehicle (lane 1),
22R + RA (lane 2), or 22R alone
(lane 3). Total RNA was isolated, and RT-PCR (A)
and Northern blotting (B) were performed as indicated under
"Materials and Methods" and in Fig. 3A. Quantification
of the ABCA1 mRNA level, normalized for GAPDH expression, is
presented below. C, ABCA1 protein expression in astrocytes.
Cells were treated as in A, and Western blotting was
performed as in Fig. 3B. Quantification of the ABCA1 protein
level, normalized for -tubulin expression, is presented below.
Lane 1, control; lane 2, 22S;
lane 3, 22R + RA; lane 4,
22R. D, ABCA1-mediated cholesterol efflux in
primary astrocytes. Cells were labeled for 24 h with
[3H]cholesterol and incubated for 8 h with
22R ± RA and for 24 h with 30 µg/ml apoA-I plus
the indicated ligands. Apolipoprotein-specific efflux was determined as
indicated under "Materials and Methods." Data are presented as a
percentage (mean ± S.E.) of the total radioactivity in cells and
medium; each point was derived from triplicate determinations from two
independent experiments. ***, p < 0.001 compared with
control nontreated with LXR/RXR ligands.
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Treatment with 22R (Fig. 6A, lane 3)
or the combination of 22R and RA (lane 2) for
24 h resulted in a significant increase of ABCA1 mRNA. To
supplement the semiquantitative RT-PCR analysis, we used Northern
blotting to quantify better the transcriptional regulation of ABCA1 by
oxysterols. We found a 30-fold increase in ABCA1 expression in
astrocytes (Fig. 6B, lane 2) after treatment of
astrocytes with LXR/RXR ligands. ABCA1 protein was also increased more
than 7-fold after 22R alone (Fig. 6C, lane
4) or after a combination of 22R and RA (lane
3).
ABCA1 Expression in Primary Microglia--
Finally, we determined
ABCA1 expression in microglial cells, which express markers usually
found in tissue macrophages and activated monocytes and, thus, may
assume macrophage functionality in the central nervous system. Because
ABCA1 is highly expressed in macrophages, we were interested in whether
microglia also expressed ABCA1. Like astrocytes, microglia participate
in the formation of central nervous system lipoproteins but to a lesser
extent. RT-PCR using total RNA from primary microglial cells confirmed a low expression level of ABCA1 (Fig.
7A, lane 1). ABCA1
expression was also detected by Northern blotting, although the basal
expression of ABCA1 was very low (Fig. 7B, lane
1). The expression of ABCA1 mRNA and protein was increased
considerably after treatment with 22R (Fig. 7, B
and C, lanes 3). The combination of
22R and RA resulted in a 2-fold increase of ABCA1 mRNA
and protein over a single treatment with 22R, replicating
the findings in neurons and astrocytes (Fig. 7, B and
C, lanes 2). The increased ABCA1 expression in
microglial cells correlated with the increase in apolipoprotein-specific efflux (Fig. 7D). The -fold increase
after LXR/RXR ligand treatment was highest in the microglia (Fig.
7D, ~5.5-fold).

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Fig. 7.
ABCA1 expression in microglia. Primary
microglia were treated for 24 h with vehicle (lane 1),
22R + RA (lane 2), or 22R alone
(lane 3). Total RNA and protein were isolated, and RT-PCR
(A), Northern blot (B), and Western blot
(C) were performed as in Fig. 6. D,
ABCA1-mediated cholesterol efflux in primary microglia. Cells were
labeled for 24 h with [3H]cholesterol and incubated
for 8 h with 22R ± RA and for 4 h with 30 µg/ml apoA-I plus the indicated ligands. Apolipoprotein-specific
efflux was determined as indicated under "Materials and Methods."
Data are presented as a percentage (mean ± S.E.) of the total
radioactivity in cells and medium and are an average of one experiment
in triplicate. **, p < 0.01 compared with control
without LXR/RXR ligands.
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A
Secretion in CHOAPPsw Cells Treated with 22R and
RA--
Previous studies suggest that decreasing the amount of
cholesterol in the cells shifts APP processing toward the
-secretase pathway (24) and reduces A
production in neurons (23). In contrast,
high cholesterol concentration in the medium of cultured cells inhibits
the secretion of sAPP
(44, 45). To determine whether the
LXR/RXR-mediated increase in ABCA1 expression affects cholesterol
concentration and consequently influences APP processing we analyzed
CHO cells stably expressing the Swedish variant of APP695 (CHOAPPsw).
Treatment of CHOAPPsw cells with 22R + RA resulted in
significant increase in ABCA1 protein (Fig.
8A, upper panel) and did not change the steady-state level of APPfl (Fig. 8A,
lower panel). To induce cholesterol efflux, 100 µg/ml
apoA-I was added to the cells stimulated with LXR/RXR ligands, but not
to control cells. After 24 h, culture medium was collected for
measuring A
and sAPP
. Cells were washed in PBS and divided for
cholesterol content measurement and protein extraction. Cholesterol
mass was determined as described above and normalized to the total
protein. The cholesterol content in cells treated with 22R + RA but not in control cells decreased after the addition of apoA-I
(Fig. 8B, 22R + RA + apo column)
and doubling the 22R + RA concentration did not produce any
further decrease in the amount of cellular cholesterol (Fig.
8B, 2 X 22R + RA + apo column).

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Fig. 8.
ABCA1 expression, cholesterol content, and
A secretion in CHOAPPsw cells after LXR/RXR
ligand treatment. A, Western blotting for ABCA1 and APPfl
after treatment with 20 µM 22R + RA. APPfl
protein expression was determined by immunoblotting using 6E10 Ab.
B and C, cells were treated with 22R + RA, and cholesterol efflux was initiated with 100 µg/ml apoA-I.
Control cells (C-apo) did not receive apoA-I. After 24 h, medium was collected and used for determination of A . Cells were
washed in PBS and divided by half for determination of cholesterol
content and for protein extraction. B, total cholesterol
content in CHOAPPsw cells after treatment with 22R + RA.
Total cholesterol was determined as in Fig. 3E and
normalized to the total protein (TP). C, ELISA
for A 1-40 was performed as explained under "Materials and Methods." The amount of
A was normalized to the amount of total protein and presented as
pg/ml/mg total protein. *, p < 0.05, **,
p < 0.005, ***, p < 0.001 versus control. ++, p < 0.005 versus 22R + RA. 22R + RA is 10 µM 22R and RA, and 2 X 22R is 20 µM 22R and RA. D, total A was
immunoprecipitated from the medium using the 6E10 Ab and the samples
immunoblotted with the 6E10 Ab as described under "Materials and
Methods." Below is the quantification of the band intensity presented
as a -fold of control after normalization to APPfl. Data (mean ± S.E.) are the results of triplicate determinations from one
experiment.
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To determine the effect of 22R and RA on APP processing, we
measured A
1-40 secretion by sandwich ELISA. Conditioned media from CHOAPPsw cells used to determine intracellular cholesterol content were centrifuged and aliquots utilized for A
measurement. Fig. 8C shows that there was more than a 2-fold reduction in
the secretion of A
in cells treated with LXR/RXR ligands and apoA-I compared with the control. The decrease in A
1-40
secretion correlated with the decreased intracellular cholesterol
level. In addition, a doubling in the 22R and RA
concentration produced an even more pronounced decrease in
A
1-40 secretion (Fig. 8C, 2 X 22R + RA + apo), suggesting that 22R + RA treatment affected A
1-40 secretion in a
concentration-dependent manner. To confirm the effect of
activated LXR/RXR on A
secretion, we immunoprecipitated total A
(including A
1-40 and A
1-42) using 6E10 Ab, which recognizes all A
species. A concentration of 20 µM 22R + RA and apoA-I decreased total A
secretion more than 5-fold (Fig. 8D). There was no
significant change in the secreted sAPP
(data not shown). These
results clearly showed that in non-neuronal cells 22R and
RA, ligands for LXRs/RXRs induced ABCA1 expression, reduced cholesterol
content, and in the presence of apoA-I decreased A
secretion.
Role of 22R and RA on APP Processing and A
Secretion in Neuronal
Cells--
To examine whether the ligands for LXR/RXR have the same
effect on A
secretion in neuronal cells, we used H4 human
neuroglioma cells stably expressing APPsw (H4APPsw). We found that
treatment of H4APPsw cells with 10 µM 22R + RA
increased ABCA1 protein expression (Fig.
9A) in a manner similar to
that seen with CHO cells. The increase in ABCA1 expression was
accompanied by more than a 2-fold increase in cholesterol efflux to 50 µg/ml apoA-I (Fig. 9B, 22R + RA + apo
column). Treatment with 22R + RA alone resulted
in a small but significant increase in cholesterol efflux compared with
control cells not treated with ligands and apolipoproteins (Fig.
9B, 22R + RA-apo versus C-apo,
p < 0.05). As expected, apoA-I alone did not change
ABCA1 expression and caused a small but statistically significant
increase in cholesterol efflux over control cells without apoA-I (Fig.
9, A and B, compare C
apo with C + apo). Incubation of H4APPsw cells with LXR/RXR ligands did not change the APP
precursor level either with or without apoA-I (Fig. 9C). In
contrast, 22R + RA considerably decreased the level of
intracellular APP-CTF, which is a product of
- and
-secretase
cleavage (total APP-CTF), regardless of the presence or absence of
apoA-I (Fig. 9D). To examine the effect of LXR/RXR on A
secretion, we immunoprecipitated and immunoblotted total A
with 6E10
Ab. Fig. 10A (22R + RA + apo lane) shows that 10 µM
22R + RA and apoA-I decreased total A
secretion more than
2-fold. 22R + RA treatment alone resulted in a small but
significant decrease in total A
secretion (~35%, 22R + RA lane), and apoA-I had no statistically significant effect (C + apo lane). The decrease in total A
secretion caused
by 22R + RA and apoA-I was accompanied by small but
significant increase in the secretion of sAPP
(Fig. 10B,
22R + RA + apo lane), which was confirmed in
multiple independent experiments. Surprisingly, 22R + RA
alone increased sAPP
secretion even more profoundly (Fig.
10B, 22R + RA lane) compared with the
22R + RA treatment with the addition of apoA-I.

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Fig. 9.
ABCA1 expression, cholesterol efflux, and CTF
content in H4APPsw cells after LXR/RXR ligand
treatment. H4 cells were stably transfected with
APP695sw and treated with 10 µM
22R + RA and 50 µg/ml apoA-I. A, ABCA1 protein
expression was determined by Western blotting as in Fig. 3.
B, cells were labeled with [3H]cholesterol,
and cholesterol efflux was determined after a 24-h incubation with 10 µM 22R + RA and 50 µg/ml apoA-I. Data
(mean ± S.E.) are the result of triplicate determinations from
one experiment. C and D, LXR/RXR ligands
decreased CTF. Cells were treated as in A and B,
and APPfl (C) and total CTF (D) were detected by
Western blotting using the C8 polyclonal Ab that recognized full-length
APPfl and its C-terminal domain. D shows the quantification
for total CTF band intensity presented as a -fold of control after
normalization to APPfl. Values are the mean ± S.E.,
n = 6. *, p < 0.05, **,
p < 0.005 ***, p < 0.001 versus C-apo. +, p < 0.05 and ++,
p < 0.01 versus 22R + RA + apo.
C apo is a control without apoA-I; C + apo is a control plus
apoA-I.
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Fig. 10.
A secretion and
sAPP production in H4APPsw cells treated with
22R and RA. Cells were treated with 10 or 20 µM 22R + RA, and cholesterol efflux was
initiated with 50 µg/ml apoA-I. A, total A was immunoprecipitated from the medium using the 6E10 Ab and
the samples immunoblotted with the 6E10 Ab as in Fig. 8D.
Below is the quantification of the band intensity presented as a -fold
of control after normalization to APPfl. Data (mean ± S.E.) are
the result of triplicate determinations from one experiment. *,
p < 0.05; ***, p < 0.001 versus control, apoA-I. B, sAPP was detected
by Western immunoblotting using 6E10 Ab. ***, p < 0.001 versus control. Data are the result of at least three
experiments in triplicate. C and D, ELISA for
A 1-40 and A 1-42 was performed as
explained under "Materials and Methods," normalized to the
APPfl and presented as a -fold of control without apoA-I. Values are
the mean ± S.E. n = 6 for A 1-40
and n = 3 for A 1-42. *,
p < 0.05; **, p < 0.01 and; ***,
p < 0.001 versus C-apo; +,
p < 0.05 versus 22R + RA + apo.
22R + RA is 10 µM and RA, and 2X is
20 µM 22R and RA.
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To determine the effect of LXR/RXR treatment specifically on the
secretion of A
1-40 and A
1-42, we
applied sandwich ELISA. The levels for A
1-40 and
A
1-42 were normalized to the level of APPfl and
presented as a -fold of the control not treated with ligands and apoA-I
(C
apo). Fig. 10, C and D, shows that treatment
with 22R + RA and apoA-I produced a statistically significant decrease in A
1-40 and
A
1-42, respectively (compare C
apo and
22R + RA + apoA-I columns). A doubling in the
22R + RA concentration (2X + apo column) reduced
A
1-40 secretion more than 3-fold (Fig. 10C)
and A
1-42 more than 2-fold (Fig. 10D), thus
demonstrating a concentration-dependent effect. The results
from these experiments provide important evidence that ligand
activation status of LXR/RXR influences APP processing by possibly two
distinct mechanisms: one related to ABCA1-mediated cholesterol efflux
and depletion of cellular cholesterol content, and another independent
of changes in cholesterol concentration.
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DISCUSSION |
ABCA1 acts as a major regulator of peripheral cholesterol efflux
and plasma HDL metabolism. Because of its ability to deplete macrophages of cholesterol and to raise plasma HDL levels, ABCA1 has
been studied mainly for its role in the pathogenesis of
atherosclerosis. Interestingly, approximately half of Tangier disease
patients exhibit a nonspecific peripheral neuropathy that may be motor, sensory, or mixed in type (46). Morphologically, the neuropathy is
characterized by lipid inclusion in Schwann cells, neuronal loss,
axonal degeneration, and demyelination of peripheral nerves. Thus, we
believe it is reasonable to hypothesize that ABCA1 could have an
important role in neuronal functionality and viability.
ABCA1 mRNA is widely distributed among multiple tissues including
brain (7). In this study, we have found ABCA1 mRNA and protein
expression in neurons, astrocytes, and microglial cells from embryonic
rat brain. In adult rat brain we found high expression of ABCA1 in
neurons from different brain regions. Large neurons of the cholinergic
nucleus basalis together with CA1 and CA3 pyramidal neurons were among
the most abundantly immunolabeled neurons. In all cell types examined
ABCA1 expression was increased by LXR/RXR ligands. Here we also
demonstrated that these ligands increased apolipoprotein-specific
cholesterol efflux in neurons, astrocytes, and microglia in parallel
with the increased ABCA1 expression. In a previous study, Whitney
et al. (7) reported that LXR/RXR ligands increased the
expression of ABCA1 mRNA in neurons and astrocytes
prepared from embryonic mouse brain. In contrast to our observations,
however, they found that increased ABCA1 expression correlated with the
increased cholesterol efflux in astrocytes but not in neurons. An
important difference between our and their studies, however, is that we
measured apolipoprotein-specific, cholesterol efflux rather than efflux
without the addition of apolipoproteins. According to the current
model, the first step of ABCA1-regulated cholesterol efflux depends on
the association of lipid-free/lipid-poor apolipoproteins with the cell
surface (47, 48). Apolipoproteins bind either directly to ABCA1 or to
other receptors on the plasma membrane (49) to activate ABCA1-mediated lipid secretion. Thus, ABCA1 is unique among ABC transporters in that
it requires cellular apolipoprotein interaction before lipids are
translocated to its site in the membrane (4). In astrocytes the
addition of apolipoproteins may be less critical for
ABCA1-dependent cholesterol efflux because they secrete
apoE, and LXR/RXR ligands were shown to increase the expression of apoE (50). As mentioned above, we found that values for cholesterol efflux
in astrocytes and microglia were much higher than in neurons, consistent with their role in the secretion of cholesterol and apolipoproteins and brain cholesterol transport.
Our observations suggest a possible important role for ABCA1 in the
efflux, transport, and redistribution of cholesterol in the central
nervous system. Cholesterol in nerve cells comes mainly from de
novo synthesis. Recent data, however, suggest that the ability of
central nervous system neurons to form synapses is limited by the
availability of exogenous cholesterol (51). Mauch et al.
(51) showed that neurons produce enough cholesterol to survive, but
massive synaptogenesis requires large amounts of cholesterol and,
therefore, is dependent on cholesterol production by astrocytes. In
contrast to glia, neurons do not engage in secretion of brain
apolipoproteins and formation of lipoprotein complexes. Instead, they
express numerous low density lipoprotein receptors to acquire
lipoproteins (52). Furthermore, the cholesterol produced by glia and
delivered via apoE/apoA-I-containing lipoproteins to neurons may be of
particular importance during neuronal injury and remodeling (43). In
fact, a recent study demonstrated that ABCA1 mRNA was dramatically
up-regulated in neurons and glia in areas of damage by hippocampal AMPA
lesion (53). The regulation of ABCA1 expression by oxysterols through
LXRs is physiologically relevant in vivo because the brain
has a mechanism for the production of oxysterols, and LXR/RXRs are
expressed in neurons and glia (7, 11). Moreover, a recent study
demonstrated that LXRs have an important function in lipid homeostasis
in the brain and that loss of these receptors results in severe brain
abnormalities and neurodegeneration (11).
Cholesterol may also contribute to the pathogenesis of AD (54-56).
Cholesterol is an important lipid that controls membrane fluidity in
neurons, and its distribution throughout the membrane is not uniform:
some patches of membrane termed lipid rafts contain high densities of
cholesterol and are characterized by low membrane fluidity (57). There
is an evidence that A
production is associated with lipid rafts and
the probable explanations are that
-secretase-1 and
-secretase
activities are localized in rafts or that a fraction of APP is sorted
to the axonal membrane of neurons and in particular to lipid rafts (25,
26). Reducing the amount of cholesterol in the membrane increases
fluidity of the membrane and may shift APP processing toward the
-secretase pathway thus reducing A
production in neurons (22,
24). Recent genetic data have linked AD to ABCA1; carriers of a common
genetic isoform of ABCA1 associated with increased HDL-cholesterol
levels showed delayed age at onset of AD (58, 59). Our results reveal
that treatment of both non-neuronal and neuronal cells with
22R and RA induced ABCA1 expression and increased
apoA-I-mediated cholesterol efflux, consequently decreasing cellular
cholesterol content. Importantly, A
1-40 and
A
1-42 were also decreased. In contrast, treatment with ligands without apolipoproteins induced only a small increase in the
cholesterol efflux. Surprisingly, in both treatments the steady-state
level of CTF was reduced considerably, whereas the secretion of sAPP
increased. These results suggest that the effects of 22R and
RA on APP processing might depend not only on ABCA1-mediated cholesterol efflux but on additional factors. Oxysterols regulate other
genes affecting cholesterol homeostasis, including ABCG1, sterol
regulatory element-binding protein processing, and
hydroxymethylglutaryl-CoA reductase (60, 61). Furthermore, oxysterols
and cholesterol decrease the expression of low density lipoprotein
receptors including low density receptor-related protein (LRP) (62). It
is important that LRP has been implicated in the pathogenesis of AD,
and the absence of LRP affects APP processing (63).
It is interesting to contrast our results with those recently published
by Fukumoto et al. (53), who demonstrated a modest increase in the secretion of A
1-40 and
A
1-42 peptides derived from proteolytic processing of
endogenous mouse wild type APP. It is possible that the species used in
the two studies are important in the 22R + RA effects
on A
secretion: we used CHO and human cells and Fukumoto et
al. (53) used mouse cells. In addition, we
examined the processing of the human Swedish mutant form and not the
endogenous murine APP. Perhaps most important, we studied A
processing in the presence of the physiologically relevant ApoA-1. We,
therefore, suggest that the role of the extracellular environment might
be critical for LXR/RXR ligand effects.
In conclusion, the results presented here support the hypothesis that
ABCA1 regulates cholesterol efflux in brain cells and the formation of
brain lipoproteins. Accordingly, ABCA1 may prevent the accumulation of
excess cholesterol in neurons by increasing the internal cycling of
brain cholesterol among glial cells and neurons or by stimulating
cholesterol flux out of central nervous system. Thus, ABCA1 may affect
APP processing, decrease A
secretion, and consequently decrease
amyloid burden in the brain.