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Originally published In Press as doi:10.1074/jbc.M209085200 on October 15, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48508-48513, December 13, 2002
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Induction of the Cholesterol Transporter ABCA1 in Central Nervous System Cells by Liver X Receptor Agonists Increases Secreted Abeta Levels*

Hiroaki FukumotoDagger , Amy DengDagger , Michael C. IrizarryDagger , Michael L. Fitzgerald§, and G. William RebeckDagger

From the Dagger  Alzheimer Research Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129 and the § Lipid Metabolism Unit, Massachusetts General Hospital, Boston, Massachusetts 02114

Received for publication, September 5, 2002, and in revised form, October 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The expression, function, and regulation of the cholesterol efflux molecule, ABCA1, has been extensively examined in peripheral tissues but only poorly studied in the brain. Brain cholesterol metabolism is of interest because several lines of evidence suggest that elevated cholesterol increases the risk of Alzheimer's disease. We found a largely neuronal expression of ABCA1 in normal rat brain by in situ hybridization. ABCA1 message was dramatically up-regulated in neurons and glia in areas of damage by hippocampal AMPA lesion after 3-7 days. Immunoblot analysis demonstrated ABCA1 protein in cultured neuronal and glial cells, and expression was induced by ligands of the nuclear hormone receptors of the retinoid X receptor and liver X receptor family. ABCA1 was induced by treatment with retinoic acid and several oxysterols, including 22(R)-hydroxycholesterol and 24-hydroxycholesterol. Expression of an ABCA1-green fluorescent protein construct in neuroblastoma cells demonstrated fluorescence in perinuclear compartments and on the plasma membrane. Because the Abeta peptide is important in Alzheimer's disease pathogenesis, we examined whether ABCA1 induction altered Abeta levels. Treatment of neuroblastoma cells with retinoic acid and 22(R)-hydroxycholesterol caused significant increases in secreted Abeta 40 (29%) and Abeta 42 (65%). Treatment with a nonsteroidal liver X receptor ligand, TO-901317, similarly increased levels of secreted Abeta 40 (25%) and Abeta 42 (126%). The increase in secreted Abeta levels was reduced by RNAi blocking of ABCA1 expression. These data suggest that the cholesterol efflux molecule ABCA1 may also be involved in the secretion of the membrane-associated molecule, Abeta .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ABCA1 encodes an ATP-binding cassette protein that promotes efflux of cholesterol and phospholipids from intracellular compartments to high density lipoprotein, lipid-deficient apoAI,1 and other apolipoproteins (1). Disruption of ABCA1 by Tangier disease mutations in humans or by engineered knock-out in mice is associated with a loss of cellular cholesterol efflux, an ablation of circulating high density lipoprotein, and, interestingly, peripheral neuropathies (2-5). Like many genes involved in cholesterol homeostasis, ABCA1 is regulated by the liver X receptors (LXR) (6-8), nuclear receptors activated by oxysterols. ABCA1 is also regulated by peroxisome proliferator- activated receptor delta , which can be activated by fatty acid metabolites (9). Both of these classes of receptors form heterodimers with retinoid X receptors (RXR), which bind retinoic acid; as heterodimers, they alter gene transcription. These systems for ABCA1 induction help decrease cellular cholesterol after cholesterol loading (10).

The importance of cerebral cholesterol metabolism in Alzheimer's disease (AD) risk and pathogenesis is supported by genetic, cell culture, mouse model, and epidemiologic data. ApoE in the central nervous system is implicated in supplying appropriate membrane lipid for development, nerve growth, and responses to injury and repair in the central nervous system (11); allelic polymorphisms in the APOE gene are associated with AD risk (12), and the APOE epsilon 4 allele is associated with increased Abeta deposition in AD brain (13). Abeta formation in cell culture systems is inhibited by cholesterol depletion, beta -hydroxy-beta -methylglutaryl-CoA reductase inhibition, and acyl-CoA:cholesterol O-acyltransferase inhibition (14-16). Amyloid deposition in transgenic mouse models of AD can be accelerated by oral cholesterol loading (17) and reduced by inhibiting cholesterol biosynthesis (18). Finally, hypercholesterolemia has been associated with AD risk (19, 20), and three recent epidemiological studies suggest that cholesterol-lowering drugs can reduce the risk of developing AD (21-23).

There is great need for better understanding of cholesterol transport and processing in the central nervous system. In this work, we found that ABCA1 was expressed in neurons and glia in vivo and in vitro. We found that ABCA1 was induced by ligands of LXR, and this induction lead to increased levels of secreted Abeta . These data suggest that increased cholesterol can alter Abeta levels through its effects on ABCA1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 9-cis-Retinoic acid (R-4643) was purchased from Sigma and dissolved in ethanol at 10 mM. Oxysterols, 22(R)-hydroxycholesterol (H-9384), 25-hydroxycholesterol (H-1015), and 7-ketocholesterol (C-2394) were purchased from Sigma and dissolved in ethanol at 10 mM. 24-hydroxycholesterol was purchased from Medical Isotopes Inc (Pelham, NH) as a mixture of R and S stereoisomers. The LXR agonist, TO-901317, was purchased from Cayman Chemical and dissolved in ethanol. Anti-ABCA1 rabbit polyclonal antibody was generated against human ABCA1 residues 2071-2261 fused to a histidine tag (24). This antibody also recognized rodent ABCA1 (see "Results"). A second anti-ABCA1 polyclonal antibody against a different ABCA1 epitope was obtained from Novus Biologicals.

In Situ Hybridization-- Unilateral hippocampal AMPA lesions were performed in four mice according to published procedures (25), and the mice were sacrificed by cervical dislocation at 1, 3, 7, and 11 days post-surgery. The brains were removed under ether anesthesia and sectioned coronally or sagitally at 16 µm on a cryostat onto sterile Probe-On Plus slides (Fisher Scientific). Two nonlesioned rat brains were also examined. In situ hybridization was performed for ABCA1 expression utilizing a probe against GenBankTM sequence X75926, bases 209-253, according to previously published protocols (26). The sections were fixed for 5 min in ice-cold 4% paraformaldehyde and stored in 95% ethanol at 5 °C. The sections were hybridized overnight with the [35S]adenosine (PerkinElmer Life Sciences) end-labeled 45-mer oligonucleotide probes (10,000 cpm/µl) at 42 °C in sealed chambers humidified with 50% formamide, 0.1% diethylpyrocarbonate water and then washed in 1× standard sodium citrate at 55 °C. The slides were exposed to Amersham Biosciences beta -max autoradiography film for 13 days. The sections were dipped and exposed to Amersham Biosciences LM-1 emulsion for 3 months for cellular resolution.

Cell Culture-- Mouse Neuro2A (neuroblastoma) cells and human IMR-32 (neuroblastoma) cells (American Type Culture Collection) were cultured in Optimem (Invitrogen) supplemented with 10% fetal bovine serum. Mouse BV-2 (microglial) and rat C6 (glioma) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Mouse primary neuronal cultures were generated as described (27). Briefly, cortical neurons were isolated from embryonic day 16 Swiss-Webster mice. Individual cortices was dissociated in calcium-free saline and plated on poly-D-lysine (Sigma)-coated tissue culture dishes at the density of 1.5 × 106 cell/ml. The neurons were grown in neurobasal medium (Invitrogen) plus 10% fetal bovine serum. One hour after plating, medium with serum was replaced with medium containing B-27 supplement (Invitrogen).

Transfections-- Full-length ABCA1 cDNA with a green fluorescent protein tag fused to the second amino acid of ABCA1 was expressed from the pcDNA1 expression vector (24). Neuro2A cells were transiently transfected using FuGENE 6 (Roche Molecular Biochemicals). Green fluorescence was observed 1-2 days later using the Bio-Rad MRC-1024 confocal microscope (excitation at 488 nm, emission at 522 nm).

RNA Inhibition-- Double-stranded RNA (RNAi) was generated homologous to a region of the 5' end of the coding portion of the mouse ABCA1 gene, 5'-AAG TGG CCT GGC CTC TCT TTA-3' (Dharmacon Research Inc., Lafayette, CO). As a negative control, we used a sequence directed against the human Hsp70 gene. Neuro2A cells were transfected with the RNAi with Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. After an overnight exposure, the RNAi complex was removed, the cells were washed one time in growth medium, and the medium was replaced with neurobasal medium with 2% B-27 with or without LXR/RXR agonists for 1 day. Conditioned medium was collected and analyzed for Abeta .

Western Blot-- The cells were lysed in 250 mM sucrose, 10 mM HEPES (pH 7.4), supplemented with complete protease inhibitors (Sigma). The protein levels were quantified by the BCA assay (Pierce), and samples of 50 µg were held in SDS loading buffer with 2.5% beta -mercaptoethanol for 30 min at room temperature but were not boiled prior to loading. The proteins were separated by SDS-6% polyacrylamide gel electrophoresis and transferred to nitrocellulose. The blots were probed with the anti-ABCA1 antibodies (1:1000) and developed with anti-rabbit-linked horseradish peroxidase secondary antibody by chemiluminescence.

Abeta Analyses-- The cells were cultured in neurobasal medium with 2% B-27 for 1 day in the presence or absence of LXR/RXR agonists. Abeta 40 and Abeta 42 levels in the conditioned medium were determined by enzyme-linked immunosorbent assay, with BNT77 as a capture antibody and BA27-horseradish peroxidase for Abeta 40 or BC05-horseradish peroxidase for Abeta 42 as detection antibodies (28).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ABCA1 in Situ Hybridization-- We first wanted to determine whether ABCA1 was expressed in brain. In situ hybridization of rat brain demonstrated widespread expression of ABCA1 (Fig. 1). The highest expression was in the neuronal layers of the cerebellum, followed by the hippocampus and cerebral cortex, with low expression in the white matter tracts (Fig. 1, A and C). As a positive control, the ABCA1 antisense probe bound strongly to sections of mouse liver (Fig. 1B); as a negative control, the ABCA1 sense probe did not bind to brain sections (Fig. 1D).


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  		Fig. 1.   ABCA1 in situ hybridization in rat brain. Rat tissue sections of were probed with a radiolabeled ABCA1 probes. A, brain coronal section, antisense. B, liver, antisense (positive control). C, brain sagittal section, antisense. D, brain sagittal section, sense (negative control).

To investigate the regulation of ABCA1 after acute brain damage, we evaluated ABCA1 expression after excitotoxic lesion of the mouse hippocampus (Fig. 2). Stereotactic AMPA lesioning of the hippocampus resulted in an up-regulation of ABCA1 mRNA beginning gradually at 3 days and continuing to increase through 7 and 11 days post-lesion. No up-regulation was seen at 1 h (Fig. 2B), demonstrating that the increased signal was not an artifact of the lesion procedure. Emulsion-dipped sections demonstrated increased expression in both neuronal and glial elements.


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  		Fig. 2.   Up-regulation of ABCA1 mRNA in mouse brain after AMPA lesion. In situ hybridization for ABCA1 was conducted on coronal sections of mouse brain after a unilateral hippocampal AMPA lesion. A, unlesioned brain. B, 1 h post-lesion. C, 7 days post-lesion. D, 11 days post-lesion.

ABCA1 Immunoblots-- To demonstrate ABCA1 protein expression in neurons, we examined proteins isolated from primary cultures of mouse neurons. Western blot analysis using a polyclonal ABCA1 antibody showed a protein of 220 kDa, the size expected from the published sequence (Fig. 3A). This protein co-migrated with ABCA1 from mouse Neuro2A cells transiently transfected with an ABCA1 expression vector (24).


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  		Fig. 3.   Regulation of ABCA1 expression in neuronal cells. Mouse primary neurons (A) and Neuro2A cells (B and C) were treated in culture with retinoic acid (RA) and oxysterols, which induce gene transcription through RXR and LXR nuclear hormone receptors. Expression of ABCA1 in cell extracts was analyzed by immunoblot analysis. As a control, Neuro2A cells transiently overexpressing ABCA1 from an expression vector demonstrated strong immunoreactivity of ~220 kDa (right lanes). A, primary neurons were treated with 10-7 or 10-5 M (0.1 or 10 µM) RA and 10-7 or 10-5 M 22(R)-hydroxycholesterol (22). B, Neuro2A cells treated with (+) or without (-) 10 µM RA and 22(R)-hydroxycholesterol. C, Neuro2A cells treated with 5 µM RA and 5 µM oxysterols. Lane C, untreated controls; lane 22, 22(R)-hydroxycholesterol; lane 25, 25-hydroxycholesterol; lane 7, 7-ketocholesterol; lane 24, 24-hydroxycholesterol. The location of 250-kDa molecular mass marker is noted at left.

Previous studies demonstrated that ABCA1 was up-regulated by hydroxysterols and retinoic acid (6), which interact with the nuclear receptors LXR and RXR, respectively. To determine whether these agents induce ABCA1 expression in neurons, we treated primary neurons with concentrations of 22(R)-hydroxycholesterol and retinoic acid from 100 nM to 10 µM. One-day treatments with 100 nM of both of these compounds led to an up-regulation of ABCA1 protein compared with untreated cells (Fig. 3A). Increasing concentrations of each compound further increased ABCA1 expression (Fig. 3A).

We also examined whether ABCA1 was expressed in an immortalized cell line derived from mouse neuroblastoma, Neuro2A cells. ABCA1 protein was found at low or undetectable levels in Neuro2A extracts (Fig. 3B). However, when Neuro2A cells were treated with retinoic acid and 22(R)-hydroxycholesterol, expression of ABCA1 was clearly apparent (Fig. 3B). In some experiments, weak up-regulation of ABCA1 was observed in cells treated only with 22(R)-hydroxycholesterol but not in cells treated only with retinoic acid. Similar experiments with human neuroblastoma IMR-32 cells also showed detectable ABCA1 levels only after induction with retinoic acid and 22(R)-hydroxycholesterol.

We examined whether other oxysterols were capable of increasing ABCA1 expression. Neuro-2A cells were treated with retinoic acid and 22(R)-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, or 24-hydroxycholesterol for 1 day (Fig. 3C). ABCA1 immunoblots demonstrated that treatments with 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 24-hydroxycholesterol dramatically increased ABCA1 protein, whereas 7-ketocholesterol was not as efficacious.

Our in situ hybridization data suggested that ABCA1 was also expressed in glial cells after brain lesion. We therefore examined a microglial cell line, BV-2, and a glioma cell line, C-6, for expression of ABCA1 protein. Both glial cell lines expressed detectable levels of ABCA1, and expression was increased after treatment with retinoic acid and 22(R)-hydroxycholesterol (Fig. 4). Expression was also increased after treatment with the nonsteroidal LXR agonist TO-901317 and further increased when treated with TO-901317 in the presence of retinoic acid. The response of ABCA1 in glial cells to LXR ligands was similar to that seen in Neuro2A cells (Fig. 4).


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  		Fig. 4.   Regulation of ABCA1 expression in glial cells. Mouse Neuro2A, mouse BV-2, and rat C6 cells were treated with vehicle (control, lanes 1 and 2), 5 µM retinoic acid and 5 µM 22(R)-hydroxycholesterol (lanes 3 and 4), 1 µM T0-901317 (lanes 5 and 6), or 5 µM retinoic acid and 1 µM TO-901317 (lanes 7 and 8). The cell extracts were collected and analyzed for ABCA1 protein by immunoblot. The molecular mass marker denotes 190 kDa.

Subcellular Localization of ABCA1-- To visualize the subcellular localization of ABCA1 in Neuro2A cells, we transiently transfected these cells with an expression vector of full-length ABCA1 with green fluorescent protein fused to its amino terminus (24). Previous studies with this construct have shown that it is fully competent for cholesterol efflux and that it faithfully co-localizes with untagged ABCA1 at the plasma membrane and in intracellular vesicles (24). Similarly, in Neuro2A cells, confocal microscopic analysis demonstrated prominent expression of ABCA1 in the perinuclear compartments and on the cell surface (Fig. 5). ABCA1-green fluorescent protein was particularly noticeable on processes projecting from the rounded cell bodies (Fig. 5). Thus, ABCA1 is expressed in the plasma membrane of neuronal cells in a manner similar to its expression in non-neuronal cells (29), as expected of a protein whose function is lipid efflux.


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  		Fig. 5.   ABCA1 distribution in Neuro2A cells. Mouse Neuro2A cells were transiently transfected with an ABCA1-green fluorescent protein expression construct. Green fluorescence was visualized by confocal microscopy 1 day after transfection.

Effects of ABCA1 Induction on Secreted Abeta Levels-- Abeta is a 40-42-amino acid, hydrophobic molecule generated by proteolysis of the amyloid precursor protein (APP) (30). We tested whether levels of secreted Abeta were affected by activation of LXR/RXR heterodimers. Neuro2A cells treated with 10 µM 22(R)-hydroxycholesterol and retinoic acid secreted increased levels of both Abeta 40 (29%) and Abeta 42 (65%) (Fig. 6). Similar increases were observed in experiments with 1 or 3 µM 22(R)-hydroxycholesterol. We were concerned that we had altered the cholesterol content of cells in culture with the oxysterol treatments. Therefore, we treated cells with an LXR agonist that was not an oxysterol, TO-901317. Similar increases in secreted Abeta 40 (25%) and Abeta 42 (126%) were found from cells treated with TO-901317 and retinoic acid (Fig. 6). In all of the experiments, the increase in Abeta 42 was greater than the increase in Abeta 40. No marked differences were observed by Western blot analyses in the levels of cellular APP or secreted APP after treatment with LXR and RXR agonists; furthermore, no toxicity was observed by monitoring of released LDH activity.


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  		Fig. 6.   Induction of ABCA1 and secreted Abeta levels. Neuro2A cells were treated in triplicate with retinoic acid and either 22(R)-hydroxycholesterol (22) or TO-901317 (TO) for 1 day. Conditioned medium was analyzed for Abeta 40 and Abeta 42 and compared with levels found in sister cultures treated only with vehicle (defined as 100%). The cells treated with retinoic acid and 22(R)-hydroxycholesterol showed increased Abeta 40 (29%, p < 0.02) and Abeta 42 (65%, p < 0.005). The cells treated with retinoic acid and TO-901317 showed increased Abeta 40 (25%, p < 0.1, not significant) and Abeta 42 (126%, p < 0.001). Error bars represent S.E.

To determine whether induction of ABCA1 was partially responsible for the increased levels of secreted Abeta , we treated cells with RNAi to inhibit ABCA1 induction. Western blot analysis showed that RNAi inhibited ABCA1 induction by 30-41%. Consistent with the results above, the cells treated only with buffer showed significantly increased secretion of Abeta 40 and Abeta 42 after 22(R)-hydroxycholesterol/retinoic acid treatment. The cells treated with ABCA1 RNAi showed levels of secreted Abeta 40 and Abeta 42 that were not significantly increased after 22(R)-hydroxycholesterol/retinoic acid treatment (Fig. 7). As a control, we also treated cells with an RNAi against the human Hsp70 gene; these cells showed increased levels of secreted Abeta 40 and Abeta 42 similar to those seen in cells without RNAi. These data suggest that induction of ABCA1 increases levels of secreted Abeta .


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  		Fig. 7.   RNA inhibition of ABCA1 and secreted Abeta levels. Triplicate cultures of Neuro2A cells were treated with vehicle (ethanol) in the absence of RNAi (-), in the presence of RNAi directed against ABCA1 (ABC), or in the presence of RNAi directed against human Hsp70 (HSP). Conditioned medium was analyzed for Abeta 40 and Abeta 42 and defined as 100%. Sister cultures of each condition were treated with retinoic acid and 22(R)-hydroxycholesterol and secreted Abeta 40 and Abeta 42 levels compared with the vehicle-treated cultures. Control cells (no RNAi) showed significant increases in Abeta 40 (29%, p < 0.05) and Abeta 42 (62%, p < 0.01) after treatment with retinoic acid and 22(R)-hydroxycholesterol. These increases were diminished and not significant in cells treated with ABCA1 RNAi: Abeta 40, 4%, p < 0.9, not significant; Abeta 42, 37%, p < 0.2, not significant. Hsp70 RNAi did not diminish the soluble Abeta increases induced by retinoic acid and 22(R)-hydroxycholesterol: Abeta 40, 30%, p < 0.2, not significant; Abeta 42, 88%, p < 0.02. Error bars represent S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Translating knowledge gained about lipid metabolism in the periphery to the central nervous system is vital for understanding processes important to neuronal function, degeneration and repair. The cholesterol efflux molecule ABCA1 is expressed widely in the periphery (10, 31), but ABCA1 message and protein have also been detected in the brain (10, 31-33). Our in situ (Figs. 1 and 2) and cell culture data (Figs. 3 and 4) show that ABCA1 expression is high in both neurons and glia. We hypothesized that cholesterol efflux might be very important after neuronal damages, when excess membranes need to be cleared from the brain. Indeed, excitotoxic lesions of the hippocampus resulted in up-regulation of ABCA1 mRNA, beginning at 3 days and increasing through 11 days (Fig. 2). The time course of increase is similar to apoE and apoJ mRNA, as well as with the development of gliosis (34), suggesting coordinate regulation of these cholesterol transport proteins during repair. Both neurons and glia expressed ABCA1 in response to injury, in contrast to the glial-expressed apoE, suggesting a more ubiquitous role in repair mechanisms.

Oxysterols and agonists of LXR induce ABCA1 expression in many tissue types (6). We found in neuronal cells and glia that ABCA1 was increased by retinoic acid and LXR agonists (Figs. 3 and 4). Induction of the central nervous system ABCA1 message by LXR agonists was also recently demonstrated in vitro and in vivo (35). 24(S)-Hydroxycholesterol is the most prominent oxysterol in the brain, produced by the brain-specific enzyme cholesterol 24-hydroxylase (CYP46) (36). 24(S)-hydroxycholesterol induces ABCA1 expression through interactions with either LXRalpha or LXRbeta isoforms (37, 38); LXRbeta is the isoform more strongly expressed in the brain (35). A knock-out of LXRalpha and LXRbeta genes in mice demonstrated accumulation of lipid-laden cells in the brain, particularly associated with the ventricles and blood vessels (39), further emphasizing the importance of LXR in central nervous system lipid efflux.

Oxysterols, like cholesterol, are components of cell membranes and soluble lipoproteins (40). There are several classes of lipoproteins in the cerebrospinal fluid, defined by their content of apoE and apoAI, and all are high density (41-43). In the central nervous system, apoE is made by glia (44) and secreted as part of lipoproteins that are smaller and denser than cerebrospinal fluid lipoproteins (45). It was suggested that these glial lipoproteins accumulated lipids via cellular cholesterol efflux to become the larger lipoproteins found in the cerebrospinal fluid (45). Lipid-poor apolipoproteins, including apoAI and apoE, can interact with ABCA1 to promote cholesterol efflux to generate high density lipoproteins (46, 47). Indeed, cholesterol efflux has been demonstrated from fibroblasts to isolated cerebrospinal fluid lipoproteins (48) and from neurons and astrocytes to purified apoE (49). Thus, ABCA1 transfer of lipids to many of the classes of apoE- and apoAI-containing lipoproteins can occur in the central nervous system, allowing elimination and redistribution of membrane components during degeneration.

Epidemiological studies link high levels of plasma cholesterol with an increased risk of Alzheimer's disease (19-23). In vitro and in vivo mouse studies link high levels of cellular cholesterol with high levels of secreted Abeta (14-18). We hypothesized that some of the effects of high cholesterol on Abeta may be due to production of oxysterols from cholesterol, causing changes in gene transcription via interactions with LXR. We focused on changes in ABCA1 because of its significant role in cholesterol efflux and its strong expression in the brain. In neuroblastoma cells, oxysterol induction of ABCA1 was accompanied by increased secreted Abeta species (Fig. 6); a similar effect was seen in C6 glial cells (data not shown). Importantly, the Abeta produced in these experiments was from endogenous sources of APP and not from cells overexpressing APP.

The amino-terminal 12-14 amino acids of Abeta are hydrophobic, comprising part of the transmembrane domain of APP. Indeed, Abeta cleaved from APP is found in plasma membranes (50). We reasoned that soluble Abeta levels are not only dependent on Abeta production and Abeta degradation but also on the transfer of Abeta between intracellular and extracellular compartments. Our data that the induction of ABCA1 increased the levels of secreted Abeta (Figs. 6 and 7) suggests that ABCA1 is a mechanism of Abeta secretion from the cell. We hypothesize that in vivo, Abeta is secreted from the cells associated with cholesterol and phospholipids. Our finding that a more hydrophobic isoform of Abeta , Abeta 42, was increased to a greater extent than Abeta 40 is consistent with this hypothesis. Endogenous plasma Abeta has been found associated with high density lipoproteins (51), further supporting this hypothesis.

Increased extracellular Abeta has been observed after brain trauma (52, 53). Based on our findings here, we hypothesize that this increase may be due to increased ABCA1 after brain damage (Fig. 2) and increased ABCA1-related efflux of Abeta . The connection between acute brain injuries and chronic brain injuries such as Alzheimer's disease is unknown, although epidemiological studies have suggested a link (54). In both brain trauma and AD, genetic associations have been observed between APOE and high Abeta load (13, 55), suggesting that there may be biological links between trauma and AD related to cholesterol metabolism.

The anatomical pattern of ABCA1 in mice expression closely matches the regional expression of the genes required for Abeta generation: APP, presenilin-1, and BACE (25, 56). Although each of these genes is expressed at high levels in cortical and limbic areas that develop senile plaques in human AD, high expression is also seen in the cerebellum, which does not develop significant amyloid deposition, indicating that other region-specific factors are necessary for plaque formation. Because LXR agonists induce secreted Abeta species, particularly Abeta 42, in vitro, we hypothesize that an increase in LXR agonists in specific brain regions may increase Abeta deposition. Levels of 24(S)-hydroxycholesterol are increased in the cerebrospinal fluid of AD patients (57, 58) but not in healthy aged individuals (59), suggesting that this compound may increase risk of AD. Furthermore, 24(S)-hydroxycholesterol is reduced in individuals taking simvastatin (60), who are at decreased risk of AD (21-23). Thus, approaches that reduce brain oxysterol production or act as LXR antagonists may reduce the risk of AD.

In this study, we characterized the expression and regulation of the cholesterol efflux molecule ABCA1 in neurons and glia. We found that induction of ABCA1 increases secretion of Abeta from cells in culture, suggesting that molecules that regulate ABCA1 could regulate levels of Abeta in the brain. Thus, ABCA1 could constitute a new target in developing therapeutics for prevention of Alzheimer's disease.

    ACKNOWLEDGEMENTS

We thank Armando Mendez and Anne Cataldo for helpful discussions. Abeta antibodies for enzyme-linked immunosorbent assay were the generous gift of Takeda Chemical Industries.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01 AG14473 (to G. W. R.), K08 AG00793 (to M. C. I.), and HL68988 and HL10398 (to M. L. F.) and a grant from the Harvard Center for Neurodegeneration and Repair.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Alzheimer Research Unit, 114 16th St., Charlestown, MA 02129. Tel.: 617-724-8329; Fax: 617-724-1480; E-mail: rebeck@helix.mgh.harvard.edu.

Published, JBC Papers in Press, October 15, 2002, DOI 10.1074/jbc.M209085200

    ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; LXR, liver X receptor(s); RXR, retinoid X receptor(s); AD, Alzheimer's disease; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; APP, amyloid precursor protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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