ABCA1 Agonist Reverses the ApoE4-Driven Cognitive and Brain Pathologies

Abstract

The allele ɛ4 of apolipoprotein E (apoE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD) and is therefore a promising therapeutic target. Human and animal model studies suggest that apoE4 is hypolipidated; accordingly, we have previously shown that the retinoid X receptor (RXR) agonist bexarotene upregulates ABCA1, the main apoE-lipidating protein, resulting in increased lipidation of apoE4, and the subsequent reversal of the pathological effects of apoE4, namely: accumulation of Aβ₄₂ and hyperphosphorylated tau, as well as reduction in the levels of synaptic markers and cognitive deficits. Since the RXR system has numerous other targets, it is important to devise the means of activating ABCA1 selectively. We presently utilized CS-6253, a peptide shown to directly activate ABCA1 in vitro, and examined the extent to which it can affect the degree of lipidation of apoE4 in vivo and counteract the associated brain and behavioral pathologies. This revealed that treatment of young apoE4-targeted replacement mice with CS-6253 increases the lipidation of apoE4. This was associated with a reversal of the apoE4-driven Aβ₄₂ accumulation and tau hyperphosphorylation in hippocampal neurons, as well as of the synaptic impairments and cognitive deficits. These findings suggest that the pathological effects of apoE4 in vivo are associated with decreased activation of ABCA1 and impaired lipidation of apoE4 and that the downstream brain-related pathology and cognitive deficits can be counteracted by treatment with the ABCA1 agonist CS-6253. These findings have important clinical ramifications and put forward ABCA1 as a promising target for apoE4-related treatment of AD.

Keywords

ABCA1 Alzheimer’s disease apoE CS-6253 lipidation lipids

INTRODUCTION

Apolipoprotein E, the most ubiquitous brain lipoprotein, exists as three major alleles, termed APOE ɛ2, APOE ɛ3, and APOE ɛ4. Genetic studies of sporadic Alzheimer’s disease (AD) and of families with late onset AD revealed that the allele APOE ɛ4 (apoE4) is a genetic risk factor for AD [1 –3]. More than half of the AD patients carry this allele, which renders it the most prevalent genetic risk factor for AD [2]. Although the mechanisms underlying the pathological effects of apoE4 are not fully understood, emerging evidence suggests that the pathological effects of apoE4 are mediated by lipid-related mechanisms [4 –6]. Studies performed in humans [7] and in the corresponding mouse models [8 –10] suggest that apoE4 is less lipidated than apoE3, the most common allele of apoE. Furthermore, apoE4 is markedly less potent than apoE3 in promoting the efflux of cholesterol and phospholipids from both astrocytes and neurons in culture [11 –13].

ApoE lipidation is regulated by the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1, respectively) [14, 15]. ABCA1 preferentially stimulates the efflux of cholesterol and phospholipids and their binding to lipid-free acceptors, such as lipid-free apoE, whereas ABCG1 is more selective for partially lipidated lipoprotein complexes [16, 17]. The expression of these proteins is controlled by the retinoid X receptor (RXR-LXR) transcription regulating system [18 –20]. We have previously shown that activation of the RXR system with the ligand bexarotene increases the expression of ABCA1 and ABCG1 in the hippocampus of targeted replacement (TR) mice that express either human apoE4 or apoE3, and that this is associated with reversal of the lipidation deficiency of apoE4 and of the cognitive impairments of the apoE4 mice [21]. Furthermore, bexarotene reversed the apoE4-driven accumulation of amyloid-β (Aβ)₄₂ and hyperphosphorylated tau in hippocampal neurons, as well as the apoE4-induced reduction in the levels of the presynaptic marker VGluT1 [21]. These findings provide a proof of principle that the pathological effects of apoE4 in vivo can be counteracted by the activation of ABCA1 and ABCG1 and the associated increase in the lipidation of apoE4. However, since the bexarotene-driven activation of the RXR system stimulates the expression of a variety of genes other than ABCA1 [22], there is a need for a more direct and specific on-target approach to activate this protein lipidation system.

Hafiane et al. have recently reported that ABCA1 activity in vitro can be stimulated and increased by treatment with a non-toxic peptide CS-6253, which is derived from the carboxyl terminal of apoE [23]. Accordingly, it was shown in several cell culture systems that CS-6253, like apoA-I, oligomerizes ABCA1 and enhances the rate of the ABCA1-mediated efflux of cholesterol and phospholipids, resulting in desorption of cholesterol and phospholipids from rafts and non-rafts regions of cell membranes. Furthermore, competition assays with apoE and apoA-I show high affinity interactions of CS-6253 with ABCA1, resulting in the formation and secretion of nascent HDL-like particles [23]. We presently investigated the extent to which CS-6253 can activate ABCA1 in vivo in apoE4-TR mice and reverse the impaired lipidation of apoE4 mice and the associated cognitive deficits and brain pathology. The results obtained revealed that intraperitoneal (i.p.) injections of CS-6253 for 6 weeks every 48 h to 2.5-month-old apoE3 and apoE4 mice result in the accumulation of this peptide in the brains of these mice and elevate the levels of ABCA1. Importantly, this was associated with increased lipidation of apoE4 and with a reversal of apoE4-driven cognitive impairments and the associated brain pathology.

MATERIALS AND METHODS

Mice

ApoE-TR mice, in which the endogenous mouse apoE was replaced by either human apoE3 or apoE4, were created by gene targeting as described previously [24]. The mice used were purchased from Taconic, who back-crossed them to wild-type C57BL/6J mice (2BL/ 610; Harlan Laboratories) for 7 generations and were homozygous for the apoE3 (3/3) or apoE4 (4/4) alleles. In order to minimize genetic drift, the mice were further back-crossed to wild-type C57BL/6J mice (2BL/610; Harlan Laboratories) in our lab for 3 more generations. These mice are referred to herein as apoE3 and apoE4 mice, respectively. The apoE genotype of the mice was confirmed by PCR analysis, as described previously [25, 26]. All experiments were performed on age-matched male animals (4 months of age) and were approved by the Tel Aviv University Animal Care Committee. Every effort was made to reduce animal stress and to minimize animal usage. CS-6253 was kindly provided by Artery Therapeutics, Inc. and was administered according to an in vivo protocol previously described [27]. Namely, CS-6253 was injected intraperitoneally (i.p.) to 2.5-month-old male mice for 6 weeks (20 mg/kg/48 h, which translates to 0.5 mg dissolved in 400μl of phosphate buffered saline (PBS) per mouse weighing 25 grams). Corresponding control mice were injected with PBS (P5493, Sigma) in a similar manner. After treatment, the mice were anesthetized with ketamine and xylazine, after which they were perfused transcardially with PBS. The brains were then removed and halved and each hemisphere was further processed for either biochemical or histological analysis, as outlined in the succeeding paragraphs.

Immunohistochemistry and immunofluorescence confocal microscopy

One brain hemisphere was fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and then placed in 30% sucrose for 48 h. Frozen coronal sections (30μm) were then cut on a sliding microtome, collected serially, placed in 200μl of cryoprotectant (containing glycerin, ethylene glycol, and 0.1 M sodium-phosphate buffer, pH 7.4), and stored at –20°C until use. The free-floating sections were immunostained with the following primary antibodies (Abs): rabbit anti-CS-6253 (1:1000, kindly provided by Artery Therapeutics, Inc.); mouse anti-GFAP (1:2000, Pharmingen); rabbit anti-Aβ₄₂ (1:500; Millipore); mouse anti-202/205 phosphorylated tau (AT8, 1:200; Innogenetics); guinea pig anti-vesicular glutamatergic transporter 1 (VGluT1, 1:2000; Millipore); rabbit anti-apoE receptor 2 (apoER2, 1:1000, kindly provided by Prof. Joachim Herz, UT Southwestern); and goat anti-Doublecortin (DCX, 1:200; Santa Cruz).

Immunohistochemistry was performed as described previously [28]. Briefly, sections were washed with 10 mM PBS, pH 7.4, and blocked for 1 h in 20% serum diluted in PBS with 0.1% Triton X-100 (PBST), after which the primary Ab, diluted in PBST containing 2% of the appropriate serum, was applied overnight at 4°C. The sections were then rinsed in PBST and incubated for 1 h at room temperature with the corresponding secondary Ab (Vector Laboratories) diluted 1:1000 in PBST containing 2% of the appropriate serum. After several additional rinses in PBST, the sections were incubated for 0.5 h in avidin-biotin-horseradish peroxidase complex (ABC Elite; Vector Laboratories) in PBST. Following rinses in PBST, sections were placed for up to 10 min in diaminobenzidine chromagen solution (Vector Laboratories). To minimize variability, sections from all animals of the same cohort were stained simultaneously. The reaction was monitored visually and stopped by rinses in PBS. The sections were mounted on a dry gelatin-coated slide and then dehydrated and sealed with coverslips. Aβ staining was performed similarly except that the sections were preincubated with 70% formic acid for 7 min to increase antigen retrieval prior to staining. The immunostained sections were viewed using a Zeiss Axioskop light microscope interfaced with a Kodak Megaplus CCD video camera. Photographs of stained slices were obtained at 10×magnification. Analysis and quantification of the staining (2 hippocampal sections per animal at bregma –1.7 to –2.06) were performed using the Image-Pro plus system for image analysis (version 5.1; Media Cybernetics). The images were analyzed by marking the area of interest (e.g., the hippocampal CA3 or the dentate gyrus subfields) and setting a threshold for all sections subjected to the same staining. The stained area above the threshold relative to the total area was then determined for each section. All of the groups were stained together and the results presented correspond to the mean±SEM of the percent area stained normalized relative to the control apoE3 mice.

Immunofluorescence staining was performed using fluorescent chromogens. Accordingly, sections were first blocked (incubation with 20% of either goat or donkey serum in PBST for 1 h at room temperature) and then reacted for 48 h at 4°C with the primary Abs (dissolved in 2% of the appropriate serum in PBST). Next, the bound primary Abs were visualized by incubating the sections for 1 h at room temperature with the appropriate fluorescently labeled secondary Abs (1:1000; Invitrogen), as previously described [28 –30]. The sections were then mounted on dry gelatin-coated slides. Sections stained for immunofluorescence were visualized using a confocal scanning laser microscope (LSM 510; Zeiss). Images (1024×1024 pixels, 12 bit) were acquired by averaging eight scans. Control experiments revealed no staining in sections lacking the first Ab. The intensities of immunofluorescence staining were calculated using the Image-Pro Plus system (version 5.1; Media Cybernetics), as described previously [28]. All images for each immunostaining were obtained under identical conditions, and their quantitative analyses were performed with no further handling. Moderate adjustments for contrast and brightness were performed similarly on all the presented images of the different mouse groups. The images were analyzed by setting a threshold for all sections stained with a specific labeling. The area of staining over the threshold relative to the total area of interest was determined and normalized to the control apoE3 mouse group.

Immunoblots

Preparation of brain protein extracts. The hippocampus was rapidly removed from one freshly excised hemisphere and stored frozen at –70°C until use. Frozen hippocampi were thawed, homogenized with a Teflon-glass homogenizer in cold Tris-buffered saline (TBS) containing protease inhibitor mixture (P8340; Sigma), and phosphatase inhibitor mixture (P5726; Sigma) and then aliquoted and frozen at –70°C until use.

For apoE SDS-electrophoresis, the hippocampal homogenates were boiled for 10 min with 0.5% SDS and immunoblotted as described previously [28, 31]. Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore). For ABCA1 SDS-electrophoresis staining, the homogenates were prepared utilizing the XT Tricine Buffer Kit (1610797, Bio-Rad) and were run on 3–8% Tris-Acetate gels (3450131, Bio-Rad). The gels were then transferred to a nitrocellulose membrane and stained with rabbit anti-ABCA1 Ab (1:1000; Novus). The immunoblot bands were all visualized using the ECL chemiluminescent substrate (Pierce), after which their intensity was visualized and quantified utilizing EZQuantGel software (EZQuant). GAPDH levels (mouse anti-GAPDH, 1:1000; Abcam) were used as gel-loading controls and the results are presented relative to the control apoE3 mice.

For blue native gels, the homogenates were run on 4–16% gels purchased from Novex in the NativePAGE Novex Bis-Tris Gel System according to the manufacturer’s instructions, and as previously described [9]. Gels were next transferred to PVDF membranes and stained with goat anti-apoE Ab (1:10,000; Millipore). The immunoblot bands were all visualized using the ECL chemiluminescent substrate (Pierce), after which their intensity was visualized and quantified utilizing the EZQuantGel software (EZQuant). The lipidated apoE bands were divided according to the emerging bands into high (>750 kDa), medium (200–600 kDa), and low (<66kDa) molecular weights (M.W.), and their intensities were analyzed separately for each sizerange.

Behavioral testing

The behavioral tests were performed following 5 weeks of treatment, and the mice were administered either CS-6253 or PBS throughout this testing period for an additional week. The Morris water maze was performed on a distinct cohort, in order to avoid the effects of the learning and stress on biochemical markers in mice’s brains [32].

Novel object recognition test

This test was performed as described previously [33]. In brief, the mice were first placed in an arena (60×60 cm with 50 cm walls) in the absence of objects, after which two identical objects were added. Twenty-four hours later, the mice were re-introduced to the arena in which one of the familiar objects was replaced by a novel one. The behavior of the mice was then monitored using the EthoVision XT 9 program for 5 min, and the time and number of visits that the mice paid to each of the objects were measured. The results are presented as the ratio in percent of the time spent near the novel object relative to the total time spent near both new and old objects.

Morris water maze

The Morris water maze test was performed as described previously [33]. Mice were placed in a 140 cm circular pool with the water rendered opaque with milk powder. A 10 cm circular platform submerged 1 cm below the surface of the water was placed at a fixed position. The mice were subjected to 4 trials per day for 5 days such that, for each trial, the mice were placed in one of equally spaced locations along the perimeter of the pool. The inter-trial interval was 30 min and the location of the platform was unchanged between days. The mice were introduced to the arena from four random locations, the order of which was unchanged between days. The performance of the mice was monitored by measuring the time they took to reach the platform. A probe test was performed after the last trial of the fifth day, in which the hidden platform was removed from the arena and the amount of time the mice spent in the quadrant in which the platform was previously located and in the other quadrants was measured. Measurements were performed using the computerized EthoVision XT 9 program.

Statistical analysis

The experimental design consisted of two genotypes (apoE3 and apoE4) and two treatments (control and CS-6253) and the results were analyzed using 2-way ANOVA testing with STATISTICA software (version 8.0; StatSoft). Only after 2-way ANOVA retrieved significant results, further post hoc Fisher analysis was performed to test for individual effects, and these findings are depicted in the figures. The experiments were performed on several different cohorts of mice; each of the four groups contained either 5–7 or 8–10 mice. The histological, biochemical, and behavioral results obtained with the different cohorts were similar, and the results presented are from a single cohort.

RESULTS

Reversal of apoE4-driven brain pathology

The extent to which CS-6253 can reverse key AD-related pathologies in the apoE4-TR mice was first examined by focusing on Aβ₄₂ and phosphorylated tau whose levels in CA3 hippocampal are elevated isoform-specifically in apoE4-TR mice [30]. These experiments were performed immunohistochemically in order to examine a specific hippocampal location, in which the apoE4-driven effects were most robust. This revealed, in accordance with previous findings [21, 30], that CA3 hippocampal neurons of control apoE4 mice have higher levels of Aβ₄₂ compared to the corresponding control apoE3 mice, and that following CS-6253 treatment, the levels of Aβ₄₂ in the apoE4 mice decreased and were rendered similar to those of the apoE3 mice, whose Aβ₄₂ levels were not affected by the CS-6253 treatment (Fig. 1A). Two-way ANOVA of these results revealed a significant effect of genotype×treatment (p = 0.04). Further post hoc analysis revealed that the levels of Aβ₄₂ were significantly higher in the control apoE4 mouse group compared to the control apoE3 mice (p = 0.02), and that the CS-6253 treatment significantly lowered the levels of Aβ₄₂ in the apoE4-treated mice (p = 0.005). The effect of CS-6253 on the levels of phosphorylated tau was examined utilizing AT8 mAb. This revealed that the levels of phosphorylated tau in hippocampal CA3 neurons were higher in the control apoE4 mice compared to the apoE3 mice, as previously shown [21, 30] and that treatment with CS-6253 markedly reduced the levels of phosphorylated tau in the apoE4 mice and induced a similar reduction in the corresponding apoE3 mice. Two-way ANOVA revealed a significant effect of treatment (p = 0.03). Further post hoc analysis revealed significantly higher levels of AT8 in the control apoE4 mice compared to the corresponding apoE3 mice (p = 0.01), and that CS-6253 significantly lowered the levels of AT8 in the treated apoE4 mice (p = 0.04). The corresponding effect of CS-6253 on the apoE3 mice was not significant.

The effects of CS-6253 treatment on the apoE4-driven synaptic and cellular pathologies were examined next (Fig. 2). We first focused on the vesicular glutamatergic transporter 1 (VGluT1). This showed, in accordance with previous findings [21, 30], that the levels of VGluT1 in CA3 neurons were lower in the control apoE4 mice compared to the corresponding apoE3 mice and that the CS-6253 treatment reversed this apoE4 phenotype and had no effect on the VGluT1 levels of the apoE3 mice (Fig. 2A). Two-way ANOVA of these results revealed a significant effect of genotype×treatment (p = 0.03). Further post hoc analysis revealed that the levels of VGluT1 were significantly lower in the control apoE4 mice compared to the corresponding apoE3 mice (p = 0.009), and that treatment with CS-6253 significantly elevated the VGluT1 levels in the apoE4 mice (p = 0.009). Similar results were obtained with immunoblot staining utilizing an anti-VGluT1 Ab (data not shown). The effect of CS-6253 on the levels of the apoE receptor apoER2 was examined next. As can be seen in Fig. 2B, the control apoE4 mice displayed lower levels of apoER2 compared to the corresponding apoE3 mice, as was previously published [29]. This phenotype was counteracted by treatment with CS-6253, which elevated the levels of apoER2 in both the apoE3 and apoE4 mice. Two-way ANOVA of these results revealed a significant effect of treatment (p = 0.008). Further post hoc analysis revealed that this effect was more pronounced in the CS-6253-treated apoE4 mice, where an increase of over 3-fold was observed (p = 0.005). Lastly, the effect of CS-6253 on the levels of the neurogenesis marker Doublecortin (DCX) was measured. As can be seen in Fig. 2C, and in accordance with previous findings [34], the levels of DCX were higher in the control apoE4 mice as compared to the corresponding apoE3 mice. Following the CS-6253 treatment, the DCX levels of the apoE4 mice decreased to match the level observed in the control apoE3 mice, whose DCX levels were not affected by this treatment. Two-way ANOVA of these results revealed a significant effect of genotype×treatment (p = 0.009). Further post hoc analysis revealed that the levels of DCX were significantly higher in the apoE4 control mice compared to the corresponding apoE3 mice (p = 0.001), and that treatment with CS-6253 significantly reduced the levels of DCX in the apoE4 mice (p = 0.0001).

Reversal of the apoE4-driven cognitive impairments

The extent to which the CS-6253 treatment affected the cognitive performance of the apoE4 mice was assessed utilizing the Morris water maze and object recognition test, in both of which the apoE4 mice’s performance has been shown to be impaired [33]. In the Morris water maze paradigm, the mice were subjected to 4 daily trials for 5 days in which their latency to reach a hidden platform was measured. This revealed, in accordance with previous observations in different spatial learning and memory paradigms [33 , 35–37], that the apoE4 control mice took longer to learn the location of the platform compared to the control apoE3 mice, and that the CS-6253 treatment significantly improved the performance of the treated apoE4 mice and rendered it similar to that of the apoE3 mice, whose performance was unaffected by this treatment (Fig. 3A, left panel). Repeated measurements ANOVA for days 2–4 revealed a significant effect for genotype×treatment (p = 0.02). Further post hoc analysis revealed that the control apoE4 mice took significantly longer to learn the position of the platform compared to the corresponding apoE3 mice (p = 0.03), and that treatment of the apoE4 mice with CS-6253 significantly improved their performance in the Morris water maze (p = 0.007). Following the last trial on day 5 of the Morris water maze, a probe test was performed, in which the platform was removed and the time the mice spent in the quadrant, in which the platform was previously located, was measured. This revealed that the control apoE4 mice spent less time in the quadrant of the platform (19.1 sec out of 60) compared to the control apoE3 mice (26.3 sec out of 60), and that this effect was reversed by the CS-6253 treatment in the apoE4 mice (28.6 sec out of 60), but had no effect on the apoE3 mice (25.7 sec out of 60, see Fig. 3A, right panel). Two-way ANOVA of these results revealed a significant effect of genotype×treatment (p = 0.02). Further post hoc analysis revealed that the apoE4 control mice spent significantly less time in the platform’s quadrant compared to the control apoE3 mice (p = 0.02), and that treatment with CS-6253 significantly improved the apoE4 mice’s performance (p = 0.003). The mice were next subjected to the novel object recognition test, in which they are first exposed to two identical objects followed by a session in which one of the objects is replaced by a new one, and their tendency to explore the novel object is assessed. As depicted in Fig. 3B, the control apoE4 mice spent a smaller fraction of time near the new object compared to the apoE3 mice, and this effect was abolished by the CS-6253 treatment, which increased the fraction of time that the apoE4 mice spent near the new object, but had no effect on the corresponding behavior of the apoE3 mice. Two-way ANOVA of these results revealed a significant effect of genotype×time (p = 0.02). Further post hoc analysis revealed that the control apoE4 mice spent significantly less time near the novel object compared to the control apoE3 mice (p = 0.0007), and that treatment with CS-6253 significantly improved the treated apoE4 mice’s behavior compared to that of the control apoE4 mice (p = 0.002).

Localization of CS-6253 in the brain

The extent to which CS-6253 accumulates in the brain and co-localizes with astrocytes, which are enriched with ABCA1 and are the major source of brain apoE [15 , 39] was assessed immunohistochemically by double staining of the hippocampus utilizing the anti-CS-6253 Ab and an Ab directed at the astrocytic marker GFAP. This revealed that the CS-6253 treatment resulted in the accumulation of the peptide in the treated mice that was co-localized with astrocytes (Fig. 4A). Quantitation of this results revealed that CS-6253 accumulates in the hippocampal neurons of the CA3 of both the treated apoE4 and apoE3 mice and that the accumulation of the peptide was more pronounced in the treated apoE4 mice, though not significantly. In contrast, the GFAP staining was not affected by CS-6253 and was similar in the apoE4 and apoE3 mice (Fig. 4B). Measurements of the co-localization of the two markers revealed that 30–40% of the total CS-6253 was co-stained with GFAP and that 20–25% of the GFAP-stained astrocytes co-localized with CS-6253(Fig. 4C).

The effect of CS-6253 on the levels of ABCA1 and of apoE and lipidated apoE

These experiments examined the hypothesis that the CS-6253-driven reversal of the brain pathology and the associated behavioral deficits of the apoE4 mice are related to up-regulation of the levels of ABCA1 and the consequent reversal of the hypolipidation of apoE4. Immunoblot measurements revealed, in accordance with previous findings, that the levels of ABCA1 are lower in the control apoE4 mice compared to the corresponding apoE3 mice [34] and that the CS-6253 treatment elevated ABCA1 levels in both treated mouse groups (Fig. 5A). Two-way ANOVA of these results revealed a significant effect of treatment (p = 0.009). Further post hoc analysis revealed that control apoE4 mice had significantly lower levels of ABCA1 compared to the control apoE3 mice (p = 0.003), and that treatment with CS-6253 significantly elevated the levels of ABCA1 in the treated apoE4 mice (p = 0.003).

The effect of CS-6253 on the levels and extent of lipidation of apoE are presented in Fig. 6. As can be seen, and in accordance with previous studies [13 , 40], the total apoE levels of the apoE4 mice were lower than that of the corresponding apoE3 mice. Importantly, the total levels of apoE were unchanged in both mouse groups following the CS-6253 treatment. Two-way ANOVA revealed p < 0.0001 for the effect of genotype and p < 0.01 for post hoc comparison between apoE4 and apoE3 mice in both the control and the CS-6253-treated groups. The effects of the apoE genotype and the CS-6253 treatment on the levels of lipidated apoE are presented in Fig. 6B. As can be seen, apoE electrophoresed as high M.W. (>700kDa) and medium M.W. (∼200–600kDa) lipoprotein complexes, whose levels in the control mice were, in accordance with previous reports [9 , 21], lower in control apoE4 mice than in the corresponding apoE3 mice. Importantly, the CS-6253 treatment markedly elevated the levels of the high and medium sized apoE4, but not of the corresponding apoE3. In addition to these apoE bands, another weak low M.W. band emerged (<66kDa), whose levels were higher in the control apoE4 than in the apoE3 mice and whose levels were specifically reduced in the apoE4 mice following treatment with CS-6253. Quantitation of these results is presented in Fig. 6C. Two-way ANOVA revealed a significant effect of group×treatment for the high M.W. apoE (p = 0.04) and a specific effect of treatment for the medium sized apoE (p = 0.001). Further post hoc analysis revealed that in the control mice, the levels of both the high and medium M.W. apoE were significantly lower in the apoE4 than in the apoE3 mice (p < 0.0001 and p < 0.001 for the high M.W. and medium M.W., respectively), and that they were significantly elevated in the apoE4 mice following the CS-6253 treatment (p = 0.04 and p = 0.01 for the high and medium M.W. apoE, respectively). Two-way ANOVA of the low M.W. band revealed p = 0.01 for the effect of genotype×treatment. Further post hoc analysis revealed that the levels of the low M.W. apoE were significantly greater in the control apoE4 mice compared to the corresponding control apoE3 mice (p = 0.002), and that treatment with CS-6253 significantly reduced these levels in the treated apoE4 mice (p = 0.0004).

DISCUSSION

This study investigated the extent to which the pathological effects of apoE4 in vivo can be ameliorated by treatment with an agonist of the key lipidation transporter protein ABCA1. This revealed that the levels of ABCA1 and the extent of lipidation of apoE are significantly lower in the brains of apoE4-targeted replacement mice than in those of the corresponding apoE3 mice and that both of these effects are reversed by i.p. injections of the ABCA1 agonist CS-6253 (20 mg/kg/48 h for 6 weeks). This effect was associated with a reversal of the accumulation of Aβ₄₂, tau phosphorylation, and neuronal pathologies of the control apoE4 mice and of the associated cognitive impairments. Taken together, these findings suggest that the pathological effects of apoE4 are driven by insufficient ABCA1 activity leading to hypolipidation of apoE4, and that these effects can be reversed by treatment with an ABCA1 agonist.

These findings are in agreement with a previous study in which activation of ABCA1 by bexarotene, an agonist of the RXR system that controls the expression of ABCA1, was shown to result in the reversal of the hypolipidation of apoE4 and of the associated brain and cognitive deficits [21]. CS-6253 was able to counteract the accumulation of both Aβ₄₂ andphosphorylated tau, as depicted by the AT8 marker, in accordance with the results from the bexarotene study [21]. The specific mechanism by which the amendment of the impaired lipidation of apoE4 results in this reduction in Aβ₄₂ is unclear, but may be due to improved binding efficiency of lipidated apoE4 to Aβ, which is compromised in control apoE4, causing diminished clearance of Aβ [41]. Alternatively, the increased lipidation of apoE4 by CS-6253 may affect Aβ production, which is increased in the presence of control apoE4 [22, 42], or may counteract the impaired Aβ-degrading efficiency of apoE4 [43]. Previous studies have shown that apoE3 binds unphosphorylated tau, while apoE4 does not [44]. This isoform-dependent direct interaction may explain the increased levels of phosphorylated tau in control apoE4 mice. Accordingly, the present results, showing that increased lipidation of apoE4 can decrease tau phosphorylation, are consistent with the interpretation that the impaired lipidation of apoE4 causes the deficient interaction with tau, and that the reversal of this impairment renders tau less prone to phosphorylation. High levels of DCX have been observed in both apoE4 mice, as well as in an apoE4 in vitro model [34 , 46], and may represent a compensatory mechanism to damage and stress caused by apoE4. The fact that CS-6253 treatment decreased the DCX levels in apoE4 mice may reflect a general state in which the apoE4-induced damage in diminished, and so the necessity of a compensatory mechanism is reduced.

It should, however, be noted that whereas the CS-6253 treatment counteracted all the examined brain and cognitive pathological effects of apoE4 (Figs. 1–3), the apoE4-driven reduction in the levels of the apoE receptor apoER2, which was abolished by CS-6253, was not affected by bexarotene [47]. The fact that CS-6253 was able to abolish the downregulation of apoER2, while bexarotene did not, might stem from the unspecific mode of action of bexarotene, which induces the expression of multiple genes, accounting for the side effects of the drug and the possible masking of such an effect [22 , 49].

The results described in the current experiments have been obtained from male TR mice. However, preliminary experiments with female TR mice which were subjected to CS-6253 treatment yielded similar results, namely that CS-6253 was able to counteract apoE4-driven brain pathologies in a similar manner as was observed in the corresponding male TR mice.

Previous in vitro cell culture experiments revealed that CS-6253 binds to the ABCA1 transporter, allowing for the oligomerization of ABCA1 and the ABCA1-mediated efflux of cholesterol and phospholipids in the form of apoE containing HDL-like particles [23]. In view of the findings that CS-6253 is present in the brain following 6 weeks of i.p. treatment and that it co-localizes with astrocytes, which contain both apoE and ABCA1 [15 , 50], we will first consider the possibility that the observed activation of brain ABCA1 and the associated increased lipidation of apoE4 are mediated by CS-6253. Accordingly, we suggest that, as was shown in vitro [23], CS-6253 binds to ABCA1 in the apoE4 brain resulting in elevated levels and the subsequent activation of ABCA1, which in turn increased the lipidation of apoE4 and thereby reversed the pathological effects of apoE4. Since ABCA1 activity can also be increased by enhancing the formation of dimers and higher oligomers, which are the activated form of this protein [51 –53], it is possible that the presently observed effects of CS-6252 on the lipidation of apoE4 are also related to increased dimerization of ABCA1. In addition to the direct apoE mimetic effect of CS-6253 on ABCA1, the reduced levels of ABCA1 in control apoE4 mice and the reversal of this effect by CS-6253 could be induced via a different indirect mechanism. Such a hypothetical pathway could be that the apoE4-driven reduced levels of apoER2 lead to inactivation of ABCA1, which in turn leads to hypolipidation of apoE4, and that CS-6253 and/or the CS-6253/apoE4 complex reverses this effect by interacting with apoER2 or any other non-ABCA1 target. Recent ex vivo findings revealed that CS-6253 HDL particles are remodeled in plasma [23], suggesting that apoE-HDL remodeling could also occur in the brain. In addition, preliminary results suggest that the levels of apoE-HDL are lower in the serum of the apoE4 mice than in the corresponding sera of the apoE3 mice and that this effect is reversed by i.p. injection of CS-6253 (data not shown). It can thus not be excluded that the peripheral effects of CS-6253 also contribute to the observed effects of CS-6253 in counteracting the apoE4-driven pathologies in the brain.

The finding that the CS-6253 treatment reverses all of the tested brain apoE4 phenotypes (Figs. 1–3) suggests that the decreased activation of ABCA1 and the hypolipidation of apoE4 are early upstream factors in the apoE4-driven cascade, leading to the observed brain pathology and cognitive deficits. However, the possibility that these effects are triggered by an even earlier and more primary effect of apoE4 cannot be excluded. Future studies of the extent to which additional apoE4-driven phenotypes can be rescued by the activation of ABCA1, and of the effects of apoE4 in ABCA1-deficient mice are expected to further our understanding of this issue. The downstream mechanisms by which the hypolipidated apoE4 triggers the observed brain pathology and cognitive deficits remain to be determined. However, in view of the wide range of apoE4 brain phenotypes including the upregulation of the AD hallmarks, Aβ₄₂, and hyperphosphorylated tau, and modulation of neuronal parameters, such as reduced levels of VGluT1 and the elevated levels of neurogenesis, as well as the reduced levels of the apoE receptor apoER2, and their different spatial localization in the hippocampus, it is likely that these downstream effects of hypolipidated apoE4 are mediated via different mechanisms. These are expected to emanate from the direct on-target effects of apoE4, as has been suggested in the case of Aβ [10].

In conclusion, this study presents an effective novel anti-apoE4 treatment in a mouse model of apoE4-related neurodegenerative pathology. We show that the pathological effects of apoE4 in vivo are associated with decreased activation of ABCA1 and impaired lipidation of apoE4 and that these effects and the resulting downstream brain-related pathology and cognitive deficits are counteracted by treatment with an ABCA1 agonist. These findings have important AD-related therapeutic implications and suggest ABCA1 as a promising therapeutic target.

Footnotes

ACKNOWLEDGMENTS

We thank Alex Smolar for his technical assistance. This research was supported in part by grants from the Legacy Heritage Bio-Medical Program of the Israel Science Foundation (grant No. 1575/14), from the Joseph K. and Inez Eichenbaum Foundation, from the Harold and Eleanore Foonberg Foundation, and from Teva Pharmaceutical Industries, Ltd as part of the Israeli National Network of Excellence in Neuroscience (NNE). DMM is the incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration.

Authors’ disclosures available online ().

References

Corder

, Saunders

, Strittmatter

, Schmechel

, Gaskell

, Small

, Roses

, Haines

, Pericak-Vance

(1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261, 921–923.

Roses

(1996) Apolipoprotein E alleles as risk factors in Alzheimer’s disease. Annu Rev Med 47, 387–400.

Saunders

, Strittmatter

, Schmechel

, George-Hyslop

, Pericak-Vance

, Joo

, Rosi

, Gusella

, Crapper-MacLachlan

, Alberts

(1993) Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 43, 1467–1472.

Puglielli

, Tanzi

, Kovacs

(2003) Alzheimer’s disease: The cholesterol connection. Nat Neurosci 6, 345–351.

Vance

, Hayashi

(2010) Formation and function of apolipoprotein E-containing lipoproteins in the nervous system. Biochim Biophys Acta 1801, 806–818.

Michel

, Liang

, Depner

, Klopp

, Ruether

, Kumar

, Schedel

, Vogelberg

, von Mutius

, von Berg

, Bufe

, Rietschel

, Heinzmann

, Laub

, Simma

, Frischer

, Genuneit

, Gut

, Schreiber

, Lathrop

, Illig

, Kabesch

(2010) Unifying candidate gene and GWAS Approaches in Asthma. PLoS One 5, e13894.

Hanson

, Bayer-Carter

, Green

, Montine

, Wilkinson

, Baker

, Watson

, Bonner

, Callaghan

, Leverenz

, Tsai

, Postupna

, Zhang

, Lampe

, Craft

(2013) Effect of apolipoprotein E genotype and diet on apolipoprotein E lipidation and amyloid peptides: Randomized clinical trial. JAMA Neurol 70, 972–980.

Tai

, Bilousova

, Jungbauer

, Roeske

, Youmans

, Yu

, Poon

, Cornwell

, Miller

, Vinters

, Van Eldik

, Fardo

, Estus

, Bu

, Gylys

, Ladu

(2013) Levels of soluble apolipoprotein E/amyloid-beta (Abeta) complex are reduced and oligomeric Abeta increased with APOE4 and Alzheimer disease in a transgenic mouse model and human samples. J Biol Chem 288, 5914–5926.

, Liu

, Chen

, Zhang

, Xu

, Bu

(2015) Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Abeta metabolism in apoE4-targeted replacement mice. Mol Neurodegener 10, 6.

10.

Tai

, Koster

, Luo

, Lee

, Wang

, Collins

, Ben Aissa

, Thatcher

, LaDu

(2014) Amyloid-beta pathology and APOE genotype modulate retinoid X receptor agonist activity in vivo. J Biol Chem 289, 30538–30555.

11.

Michikawa

, Fan

, Isobe

, Yanagisawa

(2000) Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture. J Neurochem 74, 1008–1016.

12.

Minagawa

, Gong

, Jung

, Watanabe

, Lund-Katz

, Phillips

, Saito

, Michikawa

(2009) Mechanism underlying apolipoprotein E (ApoE) isoform-dependent lipid efflux from neural cells in culture. J Neurosci Res 87, 2498–2508.

13.

Riddell

, Zhou

, Atchison

, Warwick

, Atkinson

, Jefferson

, Xu

, Aschmies

, Kirksey

, Hu

, Wagner

, Parratt

, Xu

, Li

, Zaleska

, Jacobsen

, Pangalos

, Reinhart

(2008) Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels. J Neurosci 28, 11445–11453.

14.

Hirsch-Reinshagen

, Zhou

, Burgess

, Bernier

, McIsaac

, Chan

, Tansley

, Cohn

, Hayden

, Wellington

(2004) Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain. J Biol Chem 279, 41197–41207.

15.

Wahrle

, Jiang

, Parsadanian

, Legleiter

, Han

, Fryer

, Kowalewski

, Holtzman

(2004) ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE. J Biol Chem 279, 40987–40993.

16.

Kim

, Weickert

, Garner

(2008) Role of ATP-binding cassette transporters in brain lipid transport and neurological disease. J Neurochem 104, 1145–1166.

17.

Karten

, Campenot

, Vance

(2006) Expression of ABCG1, but not ABCA1, correlates with cholesterol release by cerebellar astroglia. J Biol Chem 281, 4049–4057.

18.

Chawla

, Boisvert

, Lee

, Laffitte

, Barak

, Joseph

, Liao

, Nagy

, Edwards

, Curtiss

, Evans

, Tontonoz

(2001) A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 7, 161–171.

19.

Laffitte

, Repa

, Joseph

, Wilpitz

, Kast

, Mangelsdorf

, Tontonoz

(2001) LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci U S A 98, 507–512.

20.

Liang

, Lin

, Beyer

, Zhang

, Wu

, Bales

, DeMattos

, May

, Li

, Jiang

, Eacho

, Cao

, Paul

(2004) A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes. J Neurochem 88, 623–634.

21.

Boehm-Cagan

, Michaelson

(2014) Reversal of apoE4-driven brain pathology and behoral deficits by bexarotene. J Neurosci 34, 7293–7301 avi.

22.

Lefterov

, Schug

, Mounier

, Nam

, Fitz

, Koldamova

(2015) RNA-sequencing reveals transcriptional up-regulation of Trem2 in response to bexarotene treatment. Neurobiol Dis 82, 132–140.

23.

Hafiane

, Bielicki

, Johansson

, Genest

(2015) Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of prebeta-1 HDL in vitro. PLoS One 10, e0131997.

24.

Sullivan

, Mezdour

, Aratani

, Knouff

, Najib

, Reddick

, Quarfordt

, Maeda

(1997) Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J Biol Chem 272, 17972–17980.

25.

Levi

, Jongen-Relo

, Feldon

, Roses

, Michaelson

(2003) ApoE4 impairs hippocampal plasticity isoform-specifically and blocks the environmental stimulation of synaptogenesis and memory. Neurobiol Dis 13, 273–282.

26.

Belinson

, Michaelson

(2009) ApoE4-dependent Abeta-mediated neurodegeneration is associated with inflammatory activation in the hippocampus but not the septum. J Neural Transm 116, 1427–1434.

27.

Bielicki

, Zhang

, Cortez

, Zheng

, Narayanaswami

, Patel

, Johansson

, Azhar

(2010) A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice. J Lipid Res 51, 1496–1503.

28.

Belinson

, Lev

, Masliah

, Michaelson

(2008) Activation of the amyloid cascade in apolipoprotein E4 transgenic mice induces lysosomal activation and neurodegeneration resulting in marked cognitive deficits. J Neurosci 28, 4690–4701.

29.

Gilat-Frenkel

, Boehm-Cagan

, Liraz

, Xian

, Herz

, Michaelson

(2014) Involvement of the Apoer2 and Lrp1 receptors in mediating the pathological effects of ApoE4 in vivo. Curr Alzheimer Res 11, 549–557.

30.

Liraz

, Boehm-Cagan

, Michaelson

(2013) ApoE4 induces Abeta42, tau, and neuronal pathology in the hippocampus of young targeted replacement apoE4 mice. Mol Neurodegener 8, 16.

31.

Haas

, Liraz

, Michaelson

(2012) The effects of apolipoproteins E3 and E4 on the transforming growth factor-beta system in targeted replacement mice. Neurodegener Dis 10, 41–45.

32.

Salomon-Zimri

, Liraz

, Michaelson

(2015) Behavioral testing affects the phenotypic expression of APOE epsilon3 and APOE epsilon4 in targeted replacement mice and reduces the differences between them. Alzheimers Dement (Amst) 1, 127–135.

33.

Salomon-Zimri

, Boehm-Cagan

, Liraz

, Michaelson

(2014) Hippocampus-related cognitive impairments in young apoE4 targeted replacement mice. Neurodegener Dis 13, 86–92.

34.

Salomon-Zimri

, Liraz

, Michaelson

(2015) Behavioral testing affects the phenotypic expression of APOE ɛ3 and APOE ɛ4 in targeted replacement mice and reduces the differences between them. Alzheimers Dement (Amst) 1, 127–135.

35.

Rodriguez

, Burns

, Weeber

, Rebeck

(2013) Young APOE4 targeted replacement mice exhibit poor spatial learning and memory, with reduced dendritic spine density in the medial entorhinal cortex. Learn Mem 20, 256–266.

36.

Bour

, Grootendorst

, Vogel

, Kelche

, Dodart

, Bales

, Moreau

, Sullivan

, Mathis

(2008) Middle-aged human apoE4 targeted-replacement mice show retention deficits on a wide range of spatial memory tasks. Behav Brain Res 193, 174–182.

37.

Villasana

, Acevedo

, Poage

, Raber

(2006) Sex- and APOE isoform-dependent effects of radiation on cognitive function. Radiat Res 166, 883–891.

38.

DeMattos

, Brendza

, Heuser

, Kierson

, Cirrito

, Fryer

, Sullivan

, Fagan

, Han

, Holtzman

(2001) Purification and characterization of astrocyte-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice. Neurochem Int 39, 415–425.

39.

Pitas

, Boyles

, Lee

, Foss

, Mahley

(1987) Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 917, 148–161.

40.

Sullivan

, Han

, Liu

, Mace

, Ervin

, Wu

, Koger

, Paul

, Bales

(2011) Reduced levels of human apoE4 protein in an animal model of cognitive impairment. Neurobiol Aging 32, 791–801.

41.

Tokuda

, Calero

, Matsubara

, Vidal

, Kumar

, Permanne

, Zlokovic

, Smith

, Ladu

, Rostagno

, Frangione

, Ghiso

(2000) Lipidation of apolipoprotein E influences its isoform-specific interaction with Alzheimer’s amyloid beta peptides. Biochem J 348, Pt 2, 359–365.

42.

, Cooley

, Chung

, Dashti

, Tang

(2007) Apolipoprotein receptor 2 and X11 alpha/beta mediate apolipoprotein E-induced endocytosis of amyloid-beta precursor protein and beta-secretase, leading to amyloid-beta production. J Neurosci 27, 4052–4060.

43.

Jiang

, Lee

, Mandrekar

, Wilkinson

, Cramer

, Zelcer

, Mann

, Lamb

, Willson

, Collins

, Richardson

, Smith

, Comery

, Riddell

, Holtzman

, Tontonoz

, Landreth

(2008) ApoE promotes the proteolytic degradation of Abeta. Neuron 58, 681–693.

44.

Strittmatter

, Saunders

, Goedert

, Weisgraber

, Dong

, Jakes

, Huang

, Pericak-Vance

, Schmechel

, Roses

(1994) Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: Implications for Alzheimer disease. Proc Natl Acad Sci U S A 91, 11183–11186.

45.

Adeosun

, Hou

, Zheng

, Stockmeier

, Ou

, Paul

, Mosley

, Weisgraber

, Wang

(2014) Cognitive deficits and disruption of neurogenesis in a mouse model of apolipoprotein E4 domain interaction. J Biol Chem 289, 2946–2959.

46.

Levi

, Michaelson

(2007) Environmental enrichment stimulates neurogenesis in apolipoprotein E3 and neuronal apoptosis in apolipoprotein E4 transgenic mice. J Neurochem 100, 202–210.

47.

Michaelson

, Boehm-Cagan

, Luz

(2014) Focusing on ApoE: A viable strategy for Alzheimer therapy. Neurobiol Aging 35, S15–S16.

48.

Tachibana

, Shinohara

, Yamazaki

, Liu

, Rogers

, Bu

, Kanekiyo

(2015) Rescuing effects of RXR agonist bexarotene on aging-related synapse loss depend on neuronal LRP1. Exp Neurol 277, 1–9.

49.

Lalloyer

, Pedersen

, Gross

, Lestavel

, Yous

, Vallez

, Gustafsson

, Mandrup

, Fievet

, Staels

, Tailleux

(2009) Rexinoid bexarotene modulates triglyceride but not cholesterol metabolism via gene-specific permissivity of the RXR/LXR heterodimer in the liver. Arterioscler Thromb Vasc Biol 29, 1488–1495.

50.

Ito

, Nagayasu

, Miura

, Yokoyama

, Michikawa

(2014) Astrocytes endogenous apoE generates HDL-likelipoproteins using previously synthesized cholesterol through interaction with ABCA1. Brain Res 1570, 1–12.

51.

Denis

, Haidar

, Marcil

, Bouvier

, Krimbou

, Genest

(2004) Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles. J Biol Chem 279, 41529–41536.

52.

Trompier

, Alibert

, Davanture

, Hamon

, Pierres

, Chimini

(2006) Transition from dimers to higher oligomeric forms occurs during the ATPase cycle of the ABCA1 transporter. J Biol Chem 281, 20283–20290.

53.

Nagata

, Nakada

, Kasai

, Kusumi

, Ueda

(2013) ABCA1 dimer-monomer interconversion during HDL generation revealed by single-molecule imaging. Proc Natl Acad Sci U S A 110, 5034–5039.