Systemic treatment with liver X receptor agonists raises apolipoprotein E, cholesterol, and amyloid-β peptides in the cerebral spinal fluid of rats
- Sokreine Suon†1,
- Jie Zhao†1,
- Stephanie A Villarreal2,
- Nikesh Anumula1,
- Mali Liu1,
- Linda M Carangia3,
- John J Renger1 and
- Celina V Zerbinatti1Email author
© Suon et al; licensee BioMed Central Ltd. 2010
Received: 8 July 2010
Accepted: 29 October 2010
Published: 29 October 2010
Apolipoprotein E (apoE) is a major cholesterol transport protein found in association with brain amyloid from Alzheimer's disease (AD) patients and the ε4 allele of apoE is a genetic risk factor for AD. Previous studies have shown that apoE forms a stable complex with amyloid β (Aβ) peptides in vitro and that the state of apoE lipidation influences the fate of brain Aβ, i.e., lipid poor apoE promotes Aβ aggregation/deposition while fully lipidated apoE favors Aβ degradation/clearance. In the brain, apoE levels and apoE lipidation are regulated by the liver X receptors (LXRs).
We investigated the hypothesis that increased apoE levels and lipidation induced by LXR agonists facilitates Aβ efflux from the brain to the cerebral spinal fluid (CSF). We also examined if the brain expression of major apoE receptors potentially involved in apoE-mediated Aβ clearance was altered by LXR agonists. ApoE, cholesterol, Aβ40, and Aβ42 levels were all significantly elevated in the CSF of rats after only 3 days of treatment with LXR agonists. A significant reduction in soluble brain Aβ40 levels was also detected after 6 days of LXR agonist treatment.
Our novel findings suggest that central Aβ lowering caused by LXR agonists appears to involve an apoE/cholesterol-mediated transport of Aβ to the CSF and that differences between the apoE isoforms in mediating this clearance pathway may explain why individuals carrying one or two copies of APOE ε4 have increased risk for AD.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by the progressive loss of memory and cognitive function . The presence of amyloid-β (Aβ) peptide deposits in the hippocampal and cortical regions of the brain is a major hallmark of AD pathology. Aβ peptides, mainly Aβ40 and Aβ42, are released from the transmembrane amyloid precursor protein (APP) following sequential cleavage by β- and γ-secretases and have been shown to cause toxicity to both neurons and glia cells in vitro and in vivo [1, 2]. The most significant genetic association reported for late-onset AD is with apolipoprotein E (apoE), the main lipid transporter protein in the central nervous system (CNS) [3, 4]. Three human apoE isoforms arise from polymorphisms within the APOE gene, named E2, E3 and E4. While only 15% of the normal population carries apoE4, up to 70% of AD patients have one or two copies of apoE4.
The mechanism by which apoE4 increases the risk for AD is not yet clear. It has been established that both healthy individuals and AD patients carrying apoE4 have increased brain amyloid burden [5, 6]. Likewise, APP transgenic (APP-Tg) mice expressing human apoE4 in replacement of mouse apoE (apoE4 knock-in, KI) have increased brain amyloid load compared to apoE3 KI mice . Furthermore, it has been previously shown that binding of apoE to Aβ in vitro is isoform- and lipid-dependent [8, 9]. When associated with lipids, apoE3 binds Aβ more efficiently than apoE4. However, apoE's ability to bind Aβ is reduced and the isoform-dependent Aβ binding differences are lost when apoE is lipid-free. Therefore, due to its ability to bind Aβ, particularly the more amyloidogenic Aβ42, apoE appears to play an important role in both the aggregation and the clearance of Aβ within and out of the brain parenchyma.
In addition to increased brain amyloid load, apoE4 KI mice have reduced levels of CNS apoE and it has been postulated that the overall lower levels of apoE protein may explain the increased amyloid load and risk for AD in apoE4 carriers [7, 10, 11]. Astrocyte-secreted apoE4 also has decreased association with lipids when compared to apoE3, a difference that may underlie the reduced ability of apoE4 to bind Aβ and to promote its clearance [11, 12]. The majority of lipids associated with astrocyte-secreted apoE are contributed by the ATP-binding cassette protein A1 (ABCA1), and both apoE and ABCA1 are liver X receptors (LXR)-target genes [13, 14].
LXRα and LXRβ are nuclear hormone receptors involved in lipid metabolism and inflammatory signaling throughout the body . It has been previously shown that synthetic LXR agonists regulate the expression of apoE and ABCA1 in astrocytes suggesting that LXRs play an important role in the regulation of lipid metabolism in the CNS . Increased levels of brain ABCA1 and apoE observed with short-term (6-10 days) and prolonged (4 months) systemic treatment with synthetic LXR agonists was also associated with reduced brain Aβ burden in mice [17–23]. The mechanism by which LXR activation reduces brain amyloid appears to be via increased Aβ clearance since APP processing was not altered by LXR agonist compounds [22, 23]. Potential clearance pathways may involve the endocytosis of Aβ/apoE-lipid complexes via apoE receptors such as the low-density lipoprotein receptor (LDLR) and the low-density lipoprotein receptor-related protein 1 (LRP1), which are highly expressed in neurons and glia and have been shown to regulate the overall levels of CNS apoE [24–27]. Other Aβ clearance pathways are through elimination at the blood-brain barrier (BBB) and the CSF [28, 29]. These pathways are not well understood and may also involve Aβ/apoE-lipid particles binding to apoE receptors.
In the present study, we investigated the modulation of apoE and Aβ levels by LXR agonists in the CSF. Because this Aβ clearance pathway is likely to be saturated in APP overexpressing mice and CSF samples free of blood contamination can be difficult to obtain from mice, we used rats for these studies. We show, for the first time, that systemic administration of LXR agonists causes a robust and fast increase in apoE, cholesterol, and endogenous Aθ in the CSF of rats. Consistently, LXR agonist treatment markedly increases the levels of LXR target genes apoE and ABCA1 in cells of the choroid plexus and ependyma, suggesting that central engagement of LXRs may improve the clearance of parenchymal amyloid to the ventricular system, contributing to the overall reduction of brain amyloidosis.
Soluble apoE levels were also significantly elevated in the brain following treatment with LXR agonists. DEA-soluble extracts obtained from the right forebrain (cortex and hippocampus) were analyzed for apoE content by Western blotting (Figure 1B). Up-regulation of soluble apoE was clearly observed after 6 days of dosing, but the differences were only statistically significant after 10 days of treatment. This LXR agonist-mediated effect was specific to apoE as there were no significant changes in the soluble levels of brain apoJ, which is not directly regulated by LXRs. Consistent with the findings for the CSF, T1317 showed a more potent effect in increasing soluble apoE brain levels than GW3965. The delayed increase in brain apoE when compared to CSF apoE levels was also observed in the plasma, where elevation in apoE levels were more evident following 10 days of treatment with LXR agonists (Figure 1C). In summary, up-regulation of the LXR target gene apoE was observed in the CSF, the brain parenchyma and plasma within a few days of systemic administration with LXR agonists.
When CSF samples were separated in native gels, immunoreactivity associate with apoE in T1317 treated rats was markedly increased in bands at higher molecular weight than the bands present in the CSF of vehicle control rats (Figure 2C). While vehicle CSF showed faint apoE-immunoreactive bands at 240 and around 400 kD (←), CSF from T1317-treated animals had strong apoE-immunoreactivity in bands detected at approximately 400 and 650 kD (◀). These results suggested that larger apoE-lipid particles were likely present in the rat CSF following LXR agonist treatment.
Genetic and biochemical evidence suggest that apoE plays a crucial role in the pathology of AD [3, 32–34]. In the CNS, apoE is the major component of HDL-like particles that deliver lipids to neurons via uptake by apoE receptors such as LRP1 and LDLR. ApoE and the cholesterol efflux pump ABCA1 are LXR-target genes and LXRs are highly expressed in glia cells, which are the primary site of apoE and lipid synthesis in the brain. Both short- and long-term administration of synthetic LXR agonists have been shown to upregulate apoE and ABCA1 levels and to reduce brain amyloid load [17–23]. We have shown here for the first time that systemic administration of LXR agonists also leads to a marked and rapid up-regulation of apoE, cholesterol, and Aβ peptides in the CSF of rats, suggesting that this pathway can contribute significantly to the elimination of Aβ from the brain.
In addition to providing cholesterol needed for synaptic plasticity and membrane maintenance, apoE is involved in the elimination of excess cholesterol from the brain and this pathway is upregulated in the neurodegenerative state . In agreement with this important apoE role, administration of LXR agonist T1317 increased brain cholesterol excretion by almost 3-fold and slowed neurodegeneration in Niemann-Pick type C (NPC1) mutant mice . Deletion of LXRs in mice results in several brain abnormalities, most notably the lateral ventricles are closed, lined with lipid-laden cells, and deprived of CSF content by 12 months of age . Deletion of LXRs also significantly increases brain amyloid burden in APP-Tg mice . Taken together, these studies implicate LXRs in the clearance of excess cholesterol and Aβ from the brain, likely involving transport to the CSF.
Amyloid-binding apoE and apoAI are the most abundant apolipoproteins in the CSF . However, while CSF apoAI is derived from the plasma, CSF apoE is believed to originate entirely within the CNS . CSF is produced by the choroid plexus, the ependymal cells lining the ventricles, and the brain parenchyma . Besides offering support and cushion to elements of the CNS, the CSF also provides an effective pathway for elimination of metabolites from the brain via drainage to the vascular system . In young and adult rats, CSF is secreted at a rate of approximately 1.3 μl/min and is replaced in its total volume about 10 times per day . In humans, the total CSF volume is approximately 130 ml and is completely replaced about four times per day . CSF secretion rate decreases with aging, but the overall CSF volume increases in older individuals due to a reduced rate of turnover [42, 43]. These age-related changes may translate into reduced Aβ elimination via changes in CSF fluid dynamics. We found that apoE and ABCA1 mRNAs were highly upregulated by LXR agonists in the ventricular region of the mouse brain. In support of our findings, it has been previously shown that treatment with the endogenous LXR ligand 24S-hydroxycholesterol led to a marked increase in ABCA1 levels and cholesterol release from the apical side of the rat choroid plexus epithelial cell line TR-CSFB3 . Furthermore, LXR-mediated increase in cholesterol release was greater when apoE3 instead of apoE4 was used as an acceptor . Taken together with these previous published findings, our results suggest that LXRs play an important role in controlling the excretion of lipid-associated apoE into the CSF and that this mechanism may underlie changes in the ability of the CNS to control amyloid clearance via lipid particle transport out of the brain parenchyma.
The steady-state levels of brain ABCA1 were also rapidly increased with LXR agonist treatment in rats, consistently with the overall changes in apoE and cholesterol seen in the CSF. The changes induced by GW3965 treatment for ABCA1 in the mouse brain were of similar magnitude to that observed in GW3965-treated rats, and about 1.5-fold greater than the levels observed in vehicle treated controls. Correspondingly with LXR agonist treatment, a 6-fold genetic overexpression of ABCA1 in the brain led to a striking decrease of amyloid burden in APP-Tg mice . Overexpression of ABCA1 was further associated with increased lipidation of astrocyte-secreted apoE, but overall reduced levels of brain apoE, suggesting that highly lipidated apoE particles have an improved clearance rate from the brain parenchyma. In contrast, ABCA1 deletion from APP-Tg mouse lines decreased apoE secretion by astrocytes and brain apoE levels with concomitant increase in amyloid deposition [46–48], further implicating apoE and lipid transport as an important route for amyloid clearance in the CNS.
Upon T1317 treatment, the CCF-STTG1 human astrocytoma cell line released cholesterol to exogenously added HEK-apoE3 conditioned media but not to HEK-control vector conditioned media suggesting that apoE is a putative acceptor for ABCA1-mediated cholesterol efflux stimulated by LXR agonists. In addition, the knock-down of ABCA1 prevented cholesterol release to HEK-apoE in the presence of T1317. These results suggest that ABCA1 up-regulation by LXR agonists leading to increased lipidation of brain apoE could modulate apoE's ability to interact with Aβ as well as its catabolism and transport within the CNS.
It has been previously proposed that the primary mechanism by which LXR agonists decrease Aβ burden is via increased degradation of apoE-lipid by microglia [22, 23]. This mechanism may involve apoE receptors that are highly expressed in microglia, particularly LDLR. In agreement with the ability of apoE-lipid particles to bind Aβ and promote its clearance, overexpression of LDLR reduced brain apoE levels and Aβ deposition in APP Tg mice . However, LDLR deletion did not result in increased amyloid deposition in the same APP Tg model . Unchanged levels of amyloid could have resulted from increased transport of apoE-bound Aβ out of the brain in the absence of LDLR. Nonetheless, due to the high levels of Aβ production in APP-Tg mice, this transport mechanism was likely saturated, which prevented an overall decrease in the brain Aβ burden from occurring.
Even though LDLR is not directly regulated by LXRs, recent findings implicated the LXR-responsive ubiquitin-ligase Idol in the degradation of LDLR and VLDLR in peripheral tissues other than the liver [52, 53]. Although it did not reach statistical significance, a down-regulation of LDLR and VLDLR levels was observed in the brain of rats treated with LXR agonists for 10 days, in agreement with the Idol mechanism previously described for other tissues. Taken together with these recent findings, our results suggest that LXR agonists not only increased apoE synthesis and lipidation, but also led to reduced levels of apoE receptors in the brain, which could result in further improvement of apoE-bound amyloid clearance from the CNS. This regulatory mechanism is consistent with the pivotal role of LXRs in promoting reverse cholesterol transport from tissues to the liver for excretion.
Our novel findings suggest that LXRs play an important role in the elimination of excess brain cholesterol via secretion of apoE-lipid particles into the CSF. This transport mechanism is also likely to be involved in the elimination of amyloid, particularly of Aβ42. It has been recently shown that low CSF Aβ42 levels are more closely linked to the presence of the ε4 allele of APOE than to clinical status of AD patients, which suggests that Aβ42 levels in the human CSF are highly influenced by apoE genotype [54, 55]. The APOE4 ε4 genotype is associated with increased brain amyloid load in AD patients and normal individuals, as well as in APP Tg mice [55, 56]. Recent studies have also shown that apoE4 KI mice have decreased steady-state levels of apoE protein in the CNS when compared to apoE3 KI mice [7, 11]. Therefore, reduced levels of Aβ42 in the CSF and increased brain amyloid load in apoE4 carriers could be explained in part by the reduced capacity of apoE4 to bind and transport amyloid out of the brain.
In summary, our original findings provide new insights into the function of LXRs in brain cholesterol homeostasis and suggest that the CSF is likely a major pathway for elimination of CNS-originated apoE-lipid particles and Aβ. Our results also provide further evidence to the hypothesis that the increased risk for AD associated with apoE4 is related to its reduced ability to associate with lipids and therefore to bind and remove toxic Aβ42 from the CNS, supporting the rationale for the development of safe LXR agonists for the treatment and prevention of AD.
Sprague Dawley male rats (~1 month of age) were dosed subcutaneously with vehicle (propylene glycol) or with synthetic LXR agonists at 30 mg/Kg (mpk) daily for 3, 6 or 10 days. T0901317 was purchased from Cayman Chemical and GW3965 was synthesized as previously described . CSF, plasma, and brain were collected from non-fasted animals approximately 18 hours after last dose. C57Bl6/SJL male mice (6 months of age) were dosed subcutaneously with vehicle (propylene glycol) or GW3965 at 50 mpk for 10 days. Brains were harvested from non-fasted animals approximately 18 hours after last dose following transcardial perfusion with ice-cold phosphate buffer saline (PBS) containing 3 U/ml of heparin. All animals were handled according to the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals guidelines and the study protocol was approved by the Institutional Animal Care and Use Committee (IACUC).
Rat forebrain samples including the cortex and hippocampus from the right hemisphere were homogenized in 4X volume of RIPA buffer (Sigma-Aldrich) containing complete protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF), followed by incubation with RIPA buffer for 30 additional minutes at 4°C. One ml volume of each sample was spun at 14,000 rpm for 15 minutes at 4°C. Protein concentration was measured in the supernatant using a BCA assay kit (BioRad). Equal amounts of sample protein in Laemmli buffer were separated on 4-15% Tris-HCl polyacrylamide SDS gels (BioRad) and transferred to polyvinylidene fluoride (PVDF) membranes. Blots were placed in blocking solution with 10% non-fat milk in phosphate buffer saline with 0.05% Tween-20 (PBS-T) for 1 h, followed by incubation with various primary antibodies diluted in 5% nonfat milk in PBS-T for 3 h at room temperature (RT) or overnight at 4°C. Primary antibodies were from: Calbiochem 178479 for apoE, Signet 9080 for apoJ, Abcam ab18180 for ABCA1, R&D Systems AF2255 for LDLR, R&D Systems MAB2258 for VLDLR, and Sigma-Aldrich A3853 for actin. Blots were then washed with PBS-T and incubated with HRP-conjugated secondary antibodies at 1:2000 dilution (Amersham Biosciences) for 1 h at RT. Immunoreactive bands were visualized using ECL Plus (Amersham Biosciences) on the Typhoon 9410 (Amersham Biosciences). Data was analyzed using ImageQuant (Molecular Dynamics). Undiluted rat CSF samples (1-2 μl) and 10 μl of 500-fold diluted plasma samples were separated on 4-15% Tris-HCl SDS-PAGE gels and transferred to PVDF membranes. Western blots were carried out as described above. Fresh rat CSF samples (5 μl) were also separated on 4-16% NativePAGE Novex Bis-Tris gel (Invitrogen) and transferred to PVDF membranes. Blots were placed in blocking solution with 5% non-fat milk in phosphate buffer saline with 0.1% Tween-20 (0.1% PBS-T) for 1 h, followed by incubation with primary antibodies to apoE (Calbiochem 178479) and to albumin (Sigma-Aldrich) with 5% nonfat milk in 0.1% PBS-T for 1 h at RT. Blots were washed with 0.1% PBS-T and incubated with HRP-conjugated secondary antibodies at 1:2000 dilution for 1 h at RT. Immunoreactive bands were visualized using SuperSignal West Femto Substrate (34095, Pierce) on the VersaDoc Imaging System (BioRad). Data was analyzed using QuantityOne Analysis Software (BioRad).
Rat forebrain samples including the cortex and hippocampus from the left hemisphere were homogenized in 4X volume of 50 mM NaCl buffer containing 0.2% diethylamine (DEA) and complete protease inhibitor cocktail (Roche). After homogenization, 1 ml of each sample was boiled at 100°C for 15 min and spun at 14,000 rpm for 30 minutes at 4°C. The supernatant was neutralized to pH 7.4 with 10% volume of 0.5 M Tris-HCL pH 6.8. Black polystyrene plates (3925, Corning) were coated overnight with 2 μg/ml of rodent N-terminal polyclonal antibody (Sig-39153, Covance) in 50 mM carbonate-bicarbonate buffer pH 9.4 (C3041, Sigma). Plates were washed in PBS-T and then blocked with 0.1% Tween 20-Superblock in TBS (37545, Pierce) at RT for 3 h. For CSF Aβ42 detection, 50 μl of undiluted samples were added per well in duplicate and for CSF Aβ40, 50 μl of 5-fold diluted samples were added per well in triplicate. For DEA-soluble brain extracts, 50 μl of undiluted sample were added per well in quadruplicate, followed by 50 μl of Aβ40 G2-10 or Aβ42 12F4 alkaline phosphatase-conjugated antibody. After overnight incubation at 4°C, plates were washed and developed using alkaline phosphatase CDP-star substrate (Applied Biosystems). Luminescence counts were measured using the LJL Analyst (Molecular Devices).
Cholesterol measurement was performed using the Amplex Red cholesterol kit according to manufacturing instructions (A12216, Invitrogen). Briefly, the cholesterol standard was prepared using a 5.17 mM stock solution of cholesterol in 1X reaction buffer. 50 μl of 20-fold diluted CSF or 50 μl of 10-fold diluted DEA brain extracts were added per well in triplicate followed by 50 μl of the complete reaction mixture containing 150 mM Amplex Red, 1 U/ml HRP, 1 U/ml cholesterol oxidase, and 0.1 U/ml cholesterol esterase. The reaction mixture was incubated at 37°C for 30 minutes and fluorescence intensity was measured using a fluorescence microplate reader (Molecular Devices).
In Situ Hybridization
Mouse ABCA1 cDNA was obtained from Open Biosystems (Clone ID 5388972) and mouse apoE cDNA was obtained from Invitrogen (5136415). The plasmids were linearized with EcoRV and PvuII respectively and used to generate antisense 35S-UTP labeled cRNA probes (1015 bp and 785 bp) for in situ hybridization . Briefly, sagittal 20 μm slide-mounted brain sections were fixed in 4% paraformaldehyde, acetylated with acetic anhydride in triethanolamine and dehydrated. Slides were then hybridized overnight in a sealed humidification chamber with the antisense riboprobe for ABCA1 or apoE mRNA, stringently washed with decreasing concentrations of SSC and treated with RNase A to remove non-specific label. After dehydration, slides were opposed to BioMax MR x-ray film (Kodak) for 18 hours (ABCA1) or 4 hours (apoE), dipped in NTB-2 nuclear emulsion (Kodak) and stored in desiccated lightproof boxes for 3 weeks (ABCA1) or 1 week (apoE) at 4°C. Slides were then developed, counterstained with hematoxylin, and cover-slipped. Film autoradiographs were used to assess the regional distribution of apoE and ABCA1 mRNA expression in the mouse brain. Densitometric images were acquired using the MCID image analysis system (Imaging Research Inc) and Adobe Photoshop was used to extract images from background. A digital camera (Nikon DSM1200C) and a slide scanning system (Aperio ScanScope XT) were used in conjunction with bright-field microscopy to take images of emulsion-coated slides for evaluation of the cellular localization of silver grains.
Cholesterol efflux assay
Human embryonic kidney (HEK) 293 cells were transiently transfected with apoE3 cDNA or empty vector control using the Lipofectamine reagent (Invitrogen). After overnight incubation, cells were rinsed three times with pre-warmed serum-free media and incubated for 48 h in serum-free media. Conditioned media was concentrated using 10,000 MW cut-out Centricons (Amicon) and protein was measured using the BioRad protein assay reagent. ApoE concentration in the media was estimated by Western blotting against a standard curve of recombinant human apoE3. CCF-STTG1 human astrocytoma cells were plated in 96-well plates and incubated with media containing 12.5 μM of cholesterol for 24 h. Cell were then rinsed three times with pre-warmed serum-free media and treated with serum-free media containing approximately 50 μg apoE/ml or the equivalent total protein amount of concentrated media from empty vector-transfected HEK cells. Following addition of 5 μM T1317 or vehicle (0.05% DMSO), cells were incubated at 37°C for 48 h. Media was then collected and analyzed for total cholesterol (AmplexRed cholesterol kit, Invitrogen) and protein content (BioRad). For ABCA1 knock-down experiments, CCF-STTG1 cells were transfected by eletrophoration (Amaxa) with siRNA to ABCA1 or non-targeting siRNA control (Dharmacon) prior to cholesterol loading and addition of apoE plus 5 μM T1317 as described above. Cell monolayers were lysed with ice-cold PBS containing 1% Triton X-100 and the protease inhibitors Complete (Roche) and PMSF. Equal amounts of total lysate protein were separated on SDS-PAGE and blotted with antibodies to ABCA1 (Abcam) and actin (Sigma).
The authors are thankful to the Merck West Point Laboratory Animal Resources staff for support with dosing and sample collection and to Jill Williams from Merck Visual Communications/Creative Services for helping with figures.
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