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J. Biol. Chem., Vol. 279, Issue 39, 41197-41207, September 24, 2004
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From the
Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver V5Z 4H4, Canada, the ¶Clinical Research Institute of Montreal, Montreal H2W 1R7, Canada, and the **Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver V5Z 4H4, Canada
Received for publication, July 14, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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ABCA1 is abundant in liver and macrophages, where its expression is induced by agonists of the liver X receptor/retinoid X receptor (LXR/RXR) pathways (10-16). ABCA1 is also expressed in both glia and neurons in the central nervous system (CNS), where it is particularly abundant in Purkinje cells and in large pyramidal cortical neurons (10, 17, 18). Similar to non-CNS cells, ABCA1 is induced by LXR/RXR stimulation in primary neurons, astrocytes, and microglia (18-20).
The importance of lipid homeostasis in the CNS is underscored by the fact that the brain is the most cholesterol-rich organ in the body, and contains 
25% of total body cholesterol in only 2% of the total body weight (21). Nearly all brain cholesterol is synthesized in situ, as quantitative analyses show that essentially no cholesterol crosses the blood-brain barrier (21). In contrast, the brain cannot degrade cholesterol, and excess cholesterol must be delivered to the peripheral circulation for eventual excretion via the liver. Approximately 6-7 mg of cholesterol leaves the human brain each day after conversion to 24S-hydroxycholesterol, which easily traverses the blood-brain barrier (22-24).
Glial cells play crucial roles in regulating lipid homeostasis in the CNS. Astrocytes and microglia are the cells within the CNS that synthesize and secrete apolipoproteins that transport lipids within the brain and cerebrospinal fluid (CSF) (25-27). Apolipoprotein E (apoE) is the major apolipoprotein in the brain, and is also a component of several lipoproteins in the peripheral circulation including HDL, very low density lipoproteins (VLDL), and chylomicrons (28-30). In humans, apoE exists as one of three allelic variants that contain either a cysteine or arginine residue at amino acids 112 and 158 (31). Liver transplantation experiments in humans have shown that the allelic variant of apoE in plasma is converted to that of the donor, whereas apoE in the CSF retains the allelic identity of the recipient, illustrating that CNS and non-CNS apoE pools are synthesized independently (32). Additionally, brain perfusion experiments in guinea pigs have shown that radiolabeled apoE administered into the peripheral circulation demonstrates very low blood-to-brain transport (33), providing additional support for independent regulation of CNS and non-CNS apoE pools. ApoJ is a second major CNS lipoprotein that, unlike apoE, easily traverses the blood-brain barrier and associates with HDL in the periphery (26, 33, 34). ApoE and apoJ are synthesized and secreted from astrocytes and microglia, and expression of both apoE and apoJ are induced in response to CNS injury or disease (24, 26). ApoE is thought to coordinate the mobilization and redistribution of cholesterol in the repair and maintenance of neuronal membranes and myelin (35-37), whereas apoJ may function as a cytoprotective extracellular chaperone (38). In contrast to apoE and apoJ, apoA-I is primarily taken up into the brain from outside the CNS, and is believed to be involved in the maturation of lipoprotein particles in the CSF (26, 39).
In 1993, apoE was discovered to be an important genetic risk factor for Alzheimer's disease (AD), a finding that has now been replicated in over 100 studies. Inheritance of apoE4 increases the risk of AD in a dosage-specific manner and may act by decreasing the age of onset of AD (40, 41). In contrast, inheritance of apoE2 appears to protect from AD and is associated with a later age of onset (42). Many hypotheses have been proposed to explain how apoE participates in the pathogenesis of AD, including roles for apoE in the formation, deposition, or clearance of the A
peptides that constitute amyloid plaques (43-51). For example, apoE binds A in vivo and is required for amyloid deposition (50-53). Furthermore, apoE affects deposition of A in an isoform-specific manner, such that mice expressing human apoE4 develop at least 2.5-fold more A deposits than mice expressing human apoE3 per unit area (45, 49, 53, 54). Interestingly, replacement of the murine apoE with any of the human apoE isoforms completely suppresses A deposition compared with wild-type mice expressing endogenous murine apoE (49, 55), suggesting that distinctions between human and murine apoE may play important roles in A deposition and/or clearance. Compared with apoE, the role of apoJ in AD is less well understood, although it is known that apoJ also binds A and has been suggested to play a major role in transporting A across the blood-brain barrier (33, 34).
Both ABCA1 and apoE have been shown to be induced in response to LXR/RXR stimulation (15, 16, 20, 56, 57) and to be repressed by ZN202 (58) in peripheral macrophages. Previous work has demonstrated that apoE is a robust acceptor of lipids from ABCA1 (59, 60), raising the question of whether ABCA1-mediated lipid efflux may play important roles in the brain where apoE is a predominant apolipoprotein. Furthermore, antisense inhibition of ABCA1 has been observed to inhibit the secretion of apoE from monocyte-derived macrophages (61), raising a second question of whether ABCA1 may also regulate apoE synthesis or secretion in other apoE-producing cells such as astrocytes or microglia.
To address these two questions, we sought to determine the relationship between ABCA1, cholesterol efflux and apoE metabolism specifically in glia. In this study, we report that ABCA1-deficient primary astrocytes and microglia are impaired in their ability to efflux lipids to exogenous apoE, which results in lipid accumulation under normal culture conditions. Furthermore, lack of ABCA1 expression decreases apoE secretion from glial cells in vitro. In vivo, this impairment of apoE secretion by astrocytes and microglia results in a decrease in apoE levels by 65% in whole brain lysates of ABCA1-deficient mice, with hippocampus and striatum being the most severely affected regions. Our observations show that ABCA1 plays key roles in cholesterol transport and apoE metabolism in the CNS.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Animal Tissues—Murine brains were harvested immediately after sacrifice, dissected into individual regions where indicated, and stored at -80 °C until use. Tissues were homogenized in a buffer containing 10% glycerol, 1% Triton X-100 and protease inhibitor (Roche Applied Science) in phosphate-buffered saline (PBS). Protein concentration was determined by Lowry assay.
Culture of Primary Astrocytes and Microglia—Primary mixed glial cultures were prepared from postnatal day 1-2 mice. Brains from individual animals were placed into ice-cold Hanks Buffered Salt Solution (Canadian Life Technologies) containing 6 mg/ml glucose and 10 mM HEPES. Meninges were removed, frontal cortices were dissected, and cells were dissociated by repeated passage through a series of wide to fine bored pipettes. Dissociated cells were plated in Dulbecco's modified Eagle's medium (DMEM) (Canadian Life Technologies) with 10% fetal bovine serum, 10% horse serum, 2 mM L-glutamine (Canadian Life Technologies), and 100 units/ml of penicillin-streptomycin (Canadian Life Technologies) at one T75 flask per mouse. Cells were cultured in the presence of 5% CO2 for 14 days with 3 medium changes. After reaching confluence, microglia were isolated by gently tapping the culture flasks and collecting the medium with detached cells. Subsequently, attached microglia were separated from astrocytes by mild trypsinization as previously described (62), and added to the previously isolated cells. Microglia and enriched astrocytes were reseeded as needed.
Immunofluorescence—Cells seeded on poly-D-lysine-coated coverslips were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed twice more, and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing three times in PBS, cells were blocked with 4% normal goat serum in PBS for 30 min at room temperature, and stained with the astrocyte marker GFAP-Cy3 (1:250, Sigma) or the microglial marker F4/80 (1:200, Serotec) for 1 h. F4/80-stained cells were visualized after incubation with Alexa-488 conjugated secondary antibody (Transduction Laboratories) for 30 min. Finally, cell nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI), and coverslips were mounted with Vectashield (Vector Laboratories). The percentage of stained cells was determined over at least 200 cells. Cells were viewed under a Zeiss Axioplan 2 microscope and images were captured using a CCD camera equipped with Metamorph (Universal Imaging Corporation) imaging software.
Western Blotting—Equal amounts of protein were electrophoresed through 7.5 or 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene fluoride membrane (Millipore), and immunodetected using a monoclonal anti-ABCA1 antibody raised against the second nucleotide binding domain (NBD2 of ABCA1) (10), a murine-specific apoE antibody (Santa Cruz Biotechnology, 1:500), a polyclonal antibody against murine apoJ (1:250, generously provided by Dr. David Holtzman, Washington University School of Medicine) or an anti-GAPDH antibody (Chemicon, 1:10,000) as a loading control. Blots were developed using enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's recommendations. Bands were quantitated by densitometry using NIH Image J. Where applicable, apoE, apoJ, and ABCA1 levels were normalized to GAPDH levels to control for protein loading.
Purification of Recombinant apoE Proteins—ApoE isoforms were purified from Escherichia coli harboring recombinant glutathione S-transferase (GST)-apoE2, -apoE3, or apoE4 fusion proteins as described (63). Briefly, logarithmic phase cells were induced with 0.5 mM isopropyl-1-thio--D-galactopyranoside for 1 h, harvested by centrifugation, lysed by sonication in 1.0% Triton X-100, 10% glycerol in PBS, 200 µg/ml lysozyme, and cleared by centrifugation. GST-apoE fusion proteins in the cleared lysates were purified over glutathione-Sepharose 4B beads and eluted upon cleavage of the GST moiety by Precision Protease (Amersham Biosciences). Where indicated, purified apoE proteins were delipidated in 6 M urea followed by dialysis against several changes of PBS.
Cholesterol Efflux—Microglia and enriched astrocytes were seeded at 50,000 cells/well in 96-well plates and labeled with 1 µCi/ml of 3H-cholesterol (PerkinElmer Life Sciences) for 18 to 24 h. Labeled cells were then washed and equilibrated in DMEM/F12 for 1 h, after which 10 µg/ml of lipid-free apoA-I (Calbiochem), or recombinant apoE2, apoE3, apoE4 were added for 8 h. Media was collected and centrifuged at 2000 rpm for 5 min. Cells were lysed by addition of 50 µl of 0.1 M NaOH and 0.2% SDS followed by incubation at room temperature for 20 min. 50 µl of media and cell lysate was added to scintillation vials and quantified. Percent cholesterol efflux was calculated as the total counts in the medium divided by the sum of the counts in the medium plus the cell lysate, as previously described (64).
Lipid Staining—Cells were seeded in 24-well plates on poly-D-lysine-coated coverslips at 100,000 cells/well for microglia and 200,000 cells/well for astrocytes. After at least 18 h in DMEM containing 10% fetal bovine serum and 10% horse serum, cells were air dried, fixed in neutral-buffered formalin, and stained with Oil-red-O. Nuclei were counterstained with hematoxylin. Cells were photographed on a Zeiss Axioplan 2 microscope using a CCD camera equipped with Metamorph (Universal Imaging Corporation) imaging software.
Measurement of Apolipoprotein Secretion—Microglia and astrocytes were seeded in 96-well plates at 50,000 cells/well and 100,000 cells/well, respectively. After 18 h, cells were washed once with PBS and cultured in serum-free DMEM/F12 (Canadian Life Technologies) for 6 h (microglia) or 8 h (astrocytes). Conditioned medium was collected, centrifuged at 2,000 rpm for 4 min and the supernatant was stored at -80 °C until use. Cells were scraped, lysed in a buffer containing 10% glycerol, 1% Triton X-100, and protease inhibitor (Roche Applied Science) in PBS and centrifuged at 9,000 rpm for 10 min. The supernatant was stored at -80 °C until use. Media and cell lysate samples were analyzed for apoE, apoJ, ABCA1, and GAPDH expression by Western blot as described above. Intracellular GAPDH levels were used to normalize measurements of cellular or secreted apoE.
Statistical Analysis—One-way ANOVA with a Newman-Keuls post-test or two-tailed unpaired Student's t-tests were used for statistical analyses. In the Student's t test analyses, Welch's correction for unequal variances was applied when variances were significantly different between groups. Where indicated, apoE and ABCA1 levels were correlated using linear regression analyses. All statistical analyses were performed using Graphpad Prism (version 3.0; Graphpad Software for Science Inc., San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The effect of different apolipoprotein acceptors on cholesterol efflux from wild-type and ABCA1-/- astrocytes and microglia are illustrated in Fig. 1 and summarized in Table I. As expected, ABCA1 is required for astrocytes to efflux cholesterol to exogenous apoA-I. Under our experimental conditions, wild-type astrocytes displayed a 2.5-fold increase in cholesterol efflux upon apoA-I addition, whereas ABCA1-/- astrocytes did not show higher efflux to apoA-I compared with baseline (p > 0.05) (Fig. 1B and Table I). In wild-type astrocytes, addition of apoE2, apoE3, and apoE4 resulted in a 2.3-2.6-fold increase in cholesterol efflux, which is similar in magnitude to that elicited by apoA-I. In contrast, the cholesterol efflux elicited by each apoE isoform was 30% less in ABCA1-/- cells compared with wild-type astrocytes when corrected for differences in baseline efflux (Table I). These observations suggest that ABCA1 in astrocytes facilitates lipid efflux to exogenous apoE. However, ABCA1 is not absolutely required to efflux cholesterol to apoE, as residual efflux activity between 1.5-1.8-fold remained in the absence of ABCA1. These observations suggest that genes in addition to ABCA1 also contribute to cholesterol efflux from astrocytes. Comparison among apoE isoforms suggested that apoE2 was a less effective cholesterol acceptor than apoE3 or apoE4 for both wild-type and ABCA1-/- astrocytes, whereas no significant differences were observed in efflux to apoE3 and apoE4 (Fig. 1B). Compared with wild-type astrocytes, ABCA1-/- astrocytes were significantly impaired in their ability to efflux cholesterol to apoA-I and each apoE isoform (p < 0.001, Table I).
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Although surprisingly low efflux to exogenous apoA-I (1.2-fold) was observed in primary wild-type microglia, these cells did display significant cholesterol efflux to apoE2 (1.6-fold) and apoE3 (1.8-fold). Wild-type microglia showed poor efflux in the presence of apoE4 (1.5-fold), which did not reach statistical significance over baseline (Fig. 1C, Table I). Deficiency of ABCA1 in microglia blocked efflux to apoA-I and impaired cholesterol efflux by 15% to apoE2 and apoE3 (p < 0.05) compared with wild-type cells. Each apoE isoform was still able to elicit residual efflux activity between 1.3-1.6-fold in ABCA1-/- microglia (Fig. 1C, Table I) suggesting that microglia also contain ABCA1-independent pathways to efflux lipid to apoE. For both wild-type and ABCA1-/- microglia, apoE3 tended to be the best acceptor, although no statistically significant difference was observed among any apoE isoform tested.
Taken together, these results suggest that ABCA1 facilitates cholesterol efflux to apoE, and is required for cholesterol efflux to apoA-I in astrocytes and microglia. Additionally, both astrocytes and microglia appear to contain ABCA1-independent pathways to efflux cholesterol to apoE. In astrocytes, apoE is as good a cholesterol acceptor as apoA-I, whereas apoE appears to be the preferred acceptor for microglia.
Lack of ABCA1 Results in Lipid Accumulation in Astrocytes and Microglia—To determine if elimination of ABCA1-dependent efflux pathways results in cellular lipid accumulation in glia, we subjected wild-type and ABCA1-/- primary astrocytes and microglia to Oil-Red-O staining. Under normal culture conditions, lipids did not accumulate in efflux-competent wild-type astrocytes (Fig. 2A), and only small lipid droplets were observed in wild-type microglia (Fig. 2C). In contrast, ABCA1-/- astrocytes and microglia both accumulated numerous cytoplasmic lipid droplets, which were particularly abundant in microglia (Fig. 2, B and D). These observations suggest that ABCA1 is required to prevent excessive accumulation of lipids in glial cells even in the absence of cholesterol loading.
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65% compared with wild-type DBA1/J littermate controls (p < 0.01) as well as age- and sex-matched wild-type C57Bl/6 mice (p < 0.01) (Fig. 6, A and B). There were no significant differences in total brain apoE levels between wild-type DBA1/J and C57Bl/6 mice (Fig. 6, A and B). Identical results were obtained for mice evaluated at 2.5 months as well as at 6 months of age (data not shown). Compared with wild-type mice, ABCA1-/- mice also had 75% less apoE in plasma (data not shown), similar to the results reported by Warhle et al. (84).
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To determine if ABCA1 affected apoE levels in a region-specific manner, individual brain regions from age- and sex-matched ABCA1-/- mice and wild-type littermate controls were then examined (Fig. 7). Lack of ABCA1 affected apoE levels most severely in the hippocampus and striatum where apoE levels were reduced by 76 and 79%, respectively (Fig. 7, A and B). ApoE levels were reduced by 61% in the thalamus and 41% in cortex (Fig. 7, A and B). In contrast, ABCA1 had the least impact in the cerebellum, where apoE levels were reduced by only 35% in ABCA1-/- compared with wild-type mice (Fig. 7, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, we provide evidence that lack of glial ABCA1 activity results in a 65% decrease in apoE levels in vivo, which is likely due to reduced apoE secretion from both astrocytes and microglia and a diminished pool of intracellular apoE in microglia. Additionally, we show that ABCA1 facilitates cholesterol efflux to exogenous apoE in primary astrocytes and microglia, as efflux is reduced in the absence of ABCA1 in both cell types. However, both astrocytes and microglia may also contain ABCA1-independent pathways to efflux cholesterol to apoE, as some residual efflux remains in ABCA1-/- cells. Our findings are in agreement with previous studies showing that macrophage ABCA1 facilitates cholesterol efflux to exogenous apoE (60, 72).
Under the experimental conditions used in our efflux studies, we did not observe marked apoE isoform specificity in a pattern that was consistent between astrocyte and microglial cultures. In astrocyte cultures, apoE2 was a slightly less potent acceptor than apoE3 or apoE4, but this trend was not observed in microglial cultures where no significant differences were observed in efflux among any of the apoE isoforms. Our findings are in contrast to a previous study suggesting that apoE2 elicits more cholesterol efflux than apoE3 or apoE4 (73). The reasons why our findings differ from previous work are not clear, but may be due to subtle differences in culture preparation or efflux protocol.
In this study, we show that ABCA1 plays a critical role in the secretion of apoE from both astrocytes and microglia. These observations confirm and extend a previous study in which ABCA1 was found to facilitate apoE secretion from human monocyte-derived macrophages (61). We also demonstrate that ABCA1 is positively correlated with steady-state levels of intracellular apoE as well as with apoE secreted into the medium, both of which are measures of dynamic processes. Steady-state intracellular apoE levels reflect a balance between apoE synthesis and degradation of both newly synthesized and recycled apoE, whereas secreted apoE levels reflect a balance between apoE released from the cell coupled with reuptake of apoE by apoE receptors that are expressed in both astrocytes and microglia. Although the mechanisms by which ABCA1 affect apoE metabolism in glial cells are not yet understood, it is clear that lack of ABCA1 impairs the proportion of total apoE that is secreted into the medium in both astrocytes and microglia.
There are several potential mechanisms by which ABCA1 may affect apoE metabolism, which may or may not be linked functionally to lipid efflux. ABCA1 is known to cycle between the plasma membrane and late endosomal compartments (74) and, in addition to the plasma membrane, it has been suggested that ABCA1 may efflux cholesterol from late endosomes and lysosomes (65). Moreover, apoA-I has been recently reported to be internalized and resecreted during ABCA1-mediated cholesterol efflux (75). An increasing body of evidence indicates that internalized apoE does not undergo complete degradation and that it is resecreted (75-77). For example, nascent lipoproteins recovered from livers of apoE-/- mice transplanted with wild type bone marrow, a model in which circulating apoE is derived exclusively from macrophages, showed that up to 60% of internalized apoE may be reutilized under physiological conditions (78). Recently, HDL3 was shown to stimulate the recycling of internalized apoE and to act as an extracellular acceptor for recycled apoE in hepatoma cells (76). In this study, apoE recycling was accompanied by cholesterol efflux and involved internalization of HDL3-derived apoA-I and its targeting to endosomes containing cholesterol and apoE (76). Taken together, these observations suggest the possibility that apoE secretion and recycling may be coupled to cholesterol efflux and that ABCA1 may play a role in this process.
It is also possible to speculate that ABCA1 could influence apoE secretion by affecting trafficking in secretory or recycling pathways. In this respect, plasma membrane-associated ABCA1 may have a distinct function compared with intracytoplasmic ABCA1. For example, plasma membrane-associated ABCA1 may function to transport cholesterol and phospholipids to various extracellular apolipoprotein acceptors, whereas intracytoplasmic ABCA1 may primarily participate in intracellular lipid trafficking. Additionally, it is known that apoE requires prenylation in order to be efficiently secreted (79), suggesting the possibility that any impact of ABCA1 on prenylation pathways might also affect apoE secretion. Although much remains to be learned, our results provide support for a role of ABCA1 in apoE secretion. Furthermore, Wahrle et al. have reported that nascent particles released into conditioned medium from ABCA1-/- astrocytes contain less cholesterol than wild-type particles, suggesting that ABCA1 affects both secretion and lipidation of apoE in the CNS.
In vivo, total apoE levels are reduced by 65% in whole brains of ABCA1-/- compared with wild-type animals, indicating a critical role for ABCA1 in maintaining normal apoE homeostasis in the CNS under physiological conditions. Loss of ABCA1 affects apoE levels most severely in the hippocampus and striatum, whereas cortex and thalamus are moderately affected by ABCA1 deficiency. ApoE levels in the cerebellum are minimally reduced in ABCA1-/- mice compared with wild-type controls. We hypothesize that defective secretion of apoE from astrocytes and microglia is a key factor that underlies the reduced apoE levels in vivo. In the absence of ABCA1, both astrocytes and microglia secrete less apoE, and the intracellular pool of microglial apoE available for secretion is also lower. Interestingly, elimination of ABCA1 had no effect on apoJ levels, suggesting that ABCA1 selectively regulates apoE metabolism rather than having a general effect on other brain-derived apolipoproteins. To our knowledge, whether deficiency of human ABCA1 results in changes in CNS or CSF levels of apoE has not been determined.
The in vivo reduction of brain apoE levels, coupled with the reduced ability of ABCA1-/- glia to maintain normal levels of cholesterol efflux and apoE secretion in vitro, suggests that ABCA1 deficiency may impair normal brain function, particularly in response to injury or disease. ApoE expression is known to be induced in glial cells in response to neuronal injury, where it is believed to participate in lipid transport processes involved in repairing neuronal membranes (36, 80). Because ABCA1 deficiency results in region-specific reductions in apoE levels in brain, it is possible to speculate that ABCA1 may play a role in recovery from a variety of neurological insults by facilitating lipid mobilization via apoE.
Our observation that ABCA1-/- mice have a significant reduction in apoE levels in brain creates a paradox for predictions about the role of ABCA1 in AD. In this study, we provide the first direct evidence that glial ABCA1 affects apoE metabolism in vivo and in vitro. Reduced levels of apoE in ABCA1-/- brains may predict a greater susceptibility to neuronal injury due to decreased lipid-mediated repair pathways, yet lower levels of apoE may also delay amyloid deposition. In addition to glial ABCA1, neuronal ABCA1 may also participate in AD by affecting A generation. At present, it is controversial whether ABCA1 increases or decreases A generation, as two in vitro studies reported that increased ABCA1 expression reduced A production (18, 81), whereas a third study reported the opposite finding (19). Genetic variations in ABCA1 have been reported to alter the susceptibility to AD (82, 83), although additional studies are necessary to fully validate these results. Clearly, understanding the contributions of ABCA1 to AD will require in vivo studies in which the contribution of glial and neuronal ABCA1 to the pathogenesis of AD can be evaluated in an appropriate physiological context.
In summary, we demonstrate that glial ABCA1 is a key participant in apoE metabolism. We show that selective disruption of ABCA1 reduces cholesterol efflux to exogenous apoE and impairs apoE secretion from both astrocytes and microglia. As a result, ABCA1-/- glia accumulate lipids under normal culture conditions. In vivo, ABCA1 is required to maintain normal levels of apoE in brain. Further, the effect of ABCA1 is specific to apoE and is independent of HDL and apoA-I levels. Taken together, our observations provide a clear and direct link between ABCA1 and apoE metabolism in brain.
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FOOTNOTES |
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Supported by the British Columbia Research Institute for Children's and Women's Health (BCRICWH).
|| Supported by an operating grant from the Canadian Institutes of Health Research (CIHR) and by a CIHR/Pfizer Investigator Salary Award.
Holds a Canada Research Chair in Human Genetics and is supported by grants from the Heart and Stroke Foundation of BC and Yukon (HSFBCY) and CIHR.
Supported by a CIHR New Investigator Salary Award and by operating grants from CIHR and from the Alzheimer's Society of Canada/CIHR/AstraZeneca. To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of British Columbia, 980 W. 28th Ave., Vancouver, BC V5Z 4H4, Canada. Tel.: 604-875-2000 (ext. 6825); Fax: 604-875-3819; E-mail: cheryl{at}cmmt.ubc.ca.
1 The abbreviations used are: HDL, high density lipoprotein; AD, Alzheimer's disease; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CNS, central nervous system; apoE, apolipoprotein E; WT, wild type.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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