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Amyloid-β (Aβ) accumulation and fibril formation are key pathologic characteristics of Alzheimer’s disease (AD). We have previously found that sulfatide depletion occurs at the earliest stages of AD. To further identify the role of sulfatides in the pathogenesis of Alzheimer’s disease as well as the interactions between apolipoprotein E (apoE), sulfatides, and Aβ peptides, we examined alterations in the clearance of apoE-mediated Aβ peptides after sulfatide supplementation to cell culture systems. We demonstrated that sulfatides markedly facilitate apoE-mediated clearance of Aβ peptides endogenously generated from H4-APPwt cells through an endocytotic pathway. Moreover, we found that the uptake of Aβ42 mediated by sulfatides was selective in comparison to that of Aβ40. We excluded the possibility that the supplementation of sulfatides and/or apoE altered the production of Aβ peptides from H4-APPwt cells through determination of the clearance of Aβ peptides from conditioned H4-APPwt cell media by neuroblastoma cells which do not appreciably generate Aβ peptides. Finally, we demonstrated that the sulfate galactose moiety of sulfatides is essential for the sulfatide-facilitated clearance of Aβ peptides. Collectively, the current study provides insight into a molecular mechanism leading to Aβ clearance/deposition, highlights the significance of sulfatide deficiency at the earliest clinically recognizable stage of Alzheimer’s disease, and identifies a potential new direction for therapeutic intervention for the disease.
Although the accumulation of amyloid-β peptide (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD), the underlying biochemical mechanism(s) of this devastating disease still remains undefined. Prior work has demonstrated that the ε4 allelic variant of apolipoprotein E (apoE) is a major genetic risk factor for “sporadic” AD, which accounts for > 99% of AD cases (Strittmatter and Roses 1996; Cedazo-Minguez and Cowburn 2001). However, a relationship between the genetic risk conveyed by the apoE4 allele and the accumulation of Aβ peptides in the development of AD remains to be elucidated.
Both in vitro and more recent in vivo data strongly suggest that the ability of apoE to modify Aβ deposition may explain the role of apoE4 as an AD risk factor (Bales et al. 2002). For example, in transgenic animal models that develop AD-like amyloidosis and neuritic plaques, the absence of apoE results in little or no fibrillar amyloid formation and neuritic dystrophy (Bales et al. 1997; Holtzman et al. 2000b; Holtzman et al. 2000a). Furthermore, the expression of different human apoE isoforms in mice alters the deposition of amyloid within neuritic plaques in decreasing order: apoE4 > E3 > E2 (Holtzman et al. 2000b; Fagan et al. 2002). Together, these data strongly suggest that apoE-associated lipoproteins produced in the brain are somehow required for Aβ deposition in these animal models. Two non-mutually exclusive possibilities could explain the influence of apoE on Aβ deposition: (1) interactions of apoE with Aβ peptides facilitate conversion of soluble Aβ to fibrillar Aβ and (2) interactions of apoE with Aβ peptides influence Aβ clearance. However, it is unclear whether apoE is 1) able to directly modify the deposition of Aβ peptides to form neuritic plaques, 2) differentially involved in the clearance of Aβ peptides in an isoform-dependent manner, or 3) contributing to Aβ accumulation through an unknown mechanism (Wisniewski and Frangione 1992; Strittmatter et al. 1993; LaDu et al. 1994; Bales et al. 1997; Holtzman et al. 2000b).
The mechanism(s) by which Aβ peptides are normally cleaned from the brain remains undefined, but may involve transport (likely via lipoproteins) through the interstitial space between neuronal cells in the brain to the cerebrospinal fluid and plasma (Kang et al. 2000; Bales et al. 2002; Hartman et al. 2005; Bateman et al. 2006). In addition, the clearance of Aβ may involve local uptake and processing by cells (glia and/or neuronal) via receptor-mediated endocytosis or phagocytosis by brain macrophages (e.g., microglia). It has recently been demonstrated that increases in the uptake of Aβ peptides can be facilitated by the expression of low density lipoprotein (LDL) receptor-related protein, thereby supporting an endocytotic process (Zerbinatti et al. 2006). Furthermore, the LDL receptor-related protein expressing cells could be protected from Aβ peptide-induced apoptosis through this endocytotic pathway (Hayashi et al. 2007) while endocytosis is involved in the amyloidogenic process in neurons (Schneider et al. 2008). Indeed, reactive astrocytes, microglia, and neuronal processes have been found to be in direct contact with Aβ-containing plaques in the AD brain (see (McGeer and McGeer 2003) for recent review).
We have recently demonstrated a novel role of apoE in the central nervous system (CNS) through its involvement in the metabolism, trafficking, and homeostasis of sulfatides, a class of myelin-specific sphingolipid (Han et al. 2003a). We have demonstrated that (1) sulfatides are specifically associated with apoE-containing high-density lipoprotein-like particles; (2) apoE modulates sulfatide levels in the CNS; and (3) the modulation of sulfatide content in the CNS is apoE-isoform dependent (Han et al. 2003a). The important cellular role of sulfatides in the nervous system has been identified by the establishment of sulfatide-null mice in which multiple myelin developmental abnormalities, including myelin sheath degeneration and deterioration of nodal/paranodal structures, are manifest (Marcus et al. 2006).
Accumulation of sulfatides, due to a deficiency in sulfatidase or its co-enzymes (e.g., saponin B) in the lysosome compartment of neurons, is responsible for the development of metachromatic leukodystrophy (von Figura et al. 2001; Molander-Melin et al. 2004). The accumulation of sulfatides in neuronal lysosomes only can occur after the myelin-specific sulfatides (Vos et al. 1994) are transported and absorbed by the neurons through an endocytotic pathway. A profound depletion of sulfatides is specifically associated with AD pathology, present even at the earliest clinically-recognizable stage of the disease (Han et al. 2002; Cheng et al. 2003; Han et al. 2003b). The underlying cause(s) of this sulfatide depletion remains undefined. Recently, we have demonstrated through shotgun lipidomic analyses that the brain sulfatide contents of transgenic mice expressing mutant human isoforms of amyloid precursor protein (APP), APPsw and APPV717F, are specifically depleted in an apoE-dependent fashion (Cheng et al. 2008). Collectively, these lines of evidence support a working model of apoE-mediated sulfatide trafficking and metabolism (Han 2005; Han 2007) and suggest that apoE is likely involved in the sulfatide loss in AD patients, providing insights into the mechanism by which apoE4 is a major risk factor for AD.
These lines of evidence also indicate that the trafficking and metabolism of sulfatides mediated by apoE-associated lipoproteins occur in parallel with Aβ clearance/deposition since endogenously-produced apoE-associated lipoproteins which contain sulfatides (Han et al. 2003a) have been found to bind Aβ peptides (Hartman et al. 2005; Bateman et al. 2006). However, it is unknown whether sulfatide plays a direct role in the clearance of Aβ peptides, although it is anticipated that the transport of myelin-specific sulfatide between neuronal cells via apoE-medicated trafficking must play important role(s) in neuronal function. We therefore hypothesized that sulfatide can facilitate the process of apoE-mediated Aβ peptide clearance. In the current study, by employing membrane vesicles and cell culture models, we demonstrated that sulfatides enhanced the binding of Aβ peptides to apoE-associated vesicles and facilitated the clearance of Aβ peptides through an endocytotic pathway in an apoE isoform-dependent manner. In summary, the study identified an essential role of sulfatides in neuronal function and the results may provide insights into the metabolism of Aβ peptides which may aid in the development of therapeutic interventions for AD.
Bovine brain sulfatides (C42H81NO11S) were purchased from Matreya, Inc. (Pleasant Gap, PA, USA). 1-Hexadecanoyl-2-octadec-9′-enoyl-sn-glycero-3-phosphocholine (PO PtdCho) and bovine liver phosphatidylinositol (PtdIns) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Enzyme-Linked Immuno Sorbent Assay (ELISA) kits for Aβ40 and Aβ42, rabbit polyclonal anti Aβ40 biotin conjugate, streptavidin FITC conjugate, and LysoTracker Red DND-99 were purchased from Invitrogen Corporation (Carlsbad, CA, USA). BACE polyclonal antibody, β-actin polyclonal antibody, and β-secretase fluorometric assay kit were purchased from BioVision Corporate (Mountain View, CA, USA). All other chemical reagents were of the best grade available and were obtained from either Fisher Scientific (Pittsburgh, PA, USA) or Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) or as indicated.
Equal amounts of protein (determined by BCA protein assay) from the cellular lysates were separated by SDS-PAGE. The separated protein bands were transferred by electro-elution to immobilon-P membranes. Nonspecific binding sites were blocked by powdered milk (5%, w/v) prior to incubation with primary BACE polyclonal antibody (1:100 dilution). Beta-actin polyclonal antibody was applied as a gel loading control at a 1:1000 dilution. Horseradish peroxidase linked secondary antibody was used in combination with an ECL detection system to visualize immunoreactive bands.
BACE activity present in cells treated with or without sulfatides was measured by using a β-secretase fluorometric assay kit according to the manufacturer’s instructions. Briefly, equal amounts of H4-APPWT cells (5 × 106 cells/assay) were treated with or without sulfatides (0, 30, and 60 μM) for 24 hrs. The cells were then collected from the culture dish after treatment with 0.25% trypsin-EDTA for 2 min followed by centrifugation at 200 × g for 5 min at 4 °C. Each cell pellet was then homogenized on ice in 0.1 ml/assay of ice-cold extraction buffer. The homogenates were incubated on ice for 10 min and centrifuged at 10,000 × g for 5 min. The supernatants were transferred to a new tube and kept on ice. Fifty μl of cell lysate was added to each well of a 96-well plate. For a positive control assay, 2 μl of reconstituted active β-secretase was added to 50 μl of extraction buffer. For a negative control assay, 2 μl of the β-secretase inhibitor was added to 50 μl of extraction buffer. Fifty μl of 2X reaction buffer and 2 μl of β-secretase substrate were added to the samples. The plate was covered, tapped gently to mix, and incubated in the dark at 37 °C for 2 hrs. The samples were analyzed using a fluorescence plate reader with excitation/emission wavelength at 350/500 nm. The background reading from substrate without secretase was subtracted from all treated and untreated samples. Beta-secretase activity was expressed as the relative fluorescence units per μg of protein sample.
Neuroblastoma (NB) cells were obtained from the American Type Culture Collection (CRL-2768, ATCC, Manassas, VA, USA) and were grown in GIBCO’s modified minimum essential medium (MEM) supplemented with 4% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids in a 5% CO2/95% air atmosphere at 37 °C (GIBCO’s MEM, Invitrogen Corporation, Carlsbad, CA, USA). H4-APPwt cells were kindly provided by Dr. Golde’s research group from Mayo Clinic Jacksonville (Jacksonville, FL, USA) and were grown in GIBCO’s MEM supplemented with 4% fetal bovine serum, 2 mM glutamine, and 100 μg/ml zeocin in a 5% CO2/95% air atmosphere at 37 °C. While NB cells yielded undetectable Aβ42 and a very low level of Aβ40, H4-APPwt cells produced very high levels of both Aβ40 and Aβ42 peptides as previously described (Murphy et al. 2003).
For sulfatide supplementation experiments, cells were incubated with the modified GIBCO’s MEM containing various amounts of sulfatides and 50 μM methyl-β-cyclodextrin. Sulfatides were freshly added to the media from a concentrated stock solution (40 mM in dimethyl sulfoxide (DMSO), the final DMSO concentration was less than 0.2% (v/v)). Methyl-β-cyclodextrin (50 μM) was included in the media to enhance sulfatide solubility. Control experiments were conducted with cells incubated with media containing 50 μM of methyl-β-cyclodextrin and an appropriate concentration of DMSO.
PO PtdCho or its mixture with bovine brain sulfatides or bovine liver PtdIns (8 μmol in total) in chloroform, were dried under a nitrogen stream, and subsequently subjected to high vacuum (less than 6.67 Pa (50 mTorr)) for at least 2 h. The dried lipid film was suspended in 2 ml of cell culture medium by vigorous vortexing for 2 min. Small unilamellar vesicles were prepared by sonication for 4 min at 40% duty cycle utilizing a Vibra Cell Model VC 600 sonicator (Sonics Material, Danbury, CT, USA) under a nitrogen atmosphere. Large unilamellar vesicles were prepared by an extruder as previously described (Zeng et al. 1999). The vesicles were diluted to the indicated concentrations in the culture medium. ApoE isoforms were then added to the vesicle suspension while stirring.
Endosome- and lysosome-enriched fractions were isolated using a flotation-gradient fractionation method as described previously (Zeng et al. 2008) with minor modifications. Briefly, H4-APPwt cells were treated with or without sulfatides (60 μM) for 24 hours. The cells from fifteen 100-mm dishes (approximately 1.5 × 108 cells) were then collected from the culture dish after treatment with a solution of 0.25% trypsin-EDTA for 2 min followed by centrifugation at 200 × g for 5 min at 4 °C. The cell pellets were then washed with 50 ml of homogenization buffer (HB, i.e., 250 mM sucrose, 20 mM HEPES, 0.5 mM EGTA, pH 7.0), resuspended gently in HB, and homogenized with a ground glass cell homogenizer (15 strokes). The homogenate was centrifuged at 800 × g for 10 min at 4 °C to isolate the post-nuclear supernatant. To separate mitochondria from the endosome/lysosome-enriched fraction, the post-nuclear supernatant was centrifuged at 50,000 × g for 5 min at 4 °C. The supernatant was subsequently centrifuged at 198,000 × g for 15 min at 4 °C to isolate the microsomal fraction which was then subsequently resuspended and diluted 1:1 (v/v) with a 62% sucrose solution to produce a solution of 40.6% sucrose. The diluted microsomal fraction containing lysosomes/endosomes (1 ml) was transferred to the bottom of a transparent 14 × 89 mm centrifuge tube (Beckman) and was overlaid sequentially with 1.5 ml of a 35% sucrose solution, 1.5 ml of a 30% sucrose solution and then 2 ml of a 25% sucrose solution. The remainder of the tube was then filled with HB and centrifuged at 125,000 × g utilizing a SW41-Ti rotor (Beckman) at 4 °C for 2 h to yield the different subcellular fractions. Each fraction was collected separately using a syringe with a 22Gx4″ needle and stored at −20 °C.
The concentrations of Aβ40 and Aβ42 peptides in the culture media or purified endosomal or lysosomal fractions were determined by using ELISA kits according to the manufacturer’s instructions. Samples and standards were prepared at the appropriate dilution. Fifty μl/well of the diluted samples, standards, or controls were mixed with 50 μl/well of detection antibody solution in the ELISA plate prior to incubation overnight at 4 °C. The plate was washed 4 times with wash buffer, and incubated with 100 μl/well of anti-rabbit Ig’s-HRP for 30 min at room temperature. The plate was washed 4 times with wash buffer and incubated with 100 μl/well of stabilized chromogen for 10 to 30 minutes at room temperature in the dark. Stop solution (100 μl/well) was added and the side of the plate was tapped gently to mix. The absorbance at 450 nm was determined and compared to a chromogen blank composed of 100 μl each of the stabilized chromogen and the stop solution.
Cells were probed with a rabbit polyclonal anti-Aβ40 biotin conjugate and/or with LysoTracker Red DND-99 (for the detection of lysosomes) according to the manufacturer’s instructions. Specifically, H4-APPwt cells (approximately 2 × 105 cells/well) were plated on a chromic acid-treated glass cover slip (18 × 18-mm2) in individual wells of a 6-well culture plate. Following an additional 24-h incubation period with or without sulfatides (60 μM) in the culture media, cells were washed twice with phosphate buffered saline (PBS), fixed for 20 min with 2 % paraformaldehyde in PBS, and rinsed three times (5 min each) with PBS. The cells were permeabilized for 20 min with Blocker (PBS, 0.1 M NH4Cl, 0.2% gelatin, and 0.05% Triton X-100), rinsed three times (5 min each) in wash buffer (PBS, 0.02% azide, and 0.2% gelatin), and incubated with a rabbit polyclonal anti-Aβ40 biotin conjugate (at 1:100 dilution) in wash buffer for 1 h. The antibody-treated cells were rinsed in wash buffer four times (for a total of 20 min) and incubated with a streptavidin-FITC conjugate in wash buffer for 1 h in the dark. After rinsing the cells in wash buffer four times (for a total of 20 min), all samples were mounted and viewed using a Bio-Rad laser confocal fluorescence microscope. The specificity of the primary antibodies was confirmed by the absence of fluorescence for antigen-negative control cells. Negative control experiments were conducted in cell culture media supplemented with all components (including DMSO and methyl-β-cyclodextrin) except sulfatides.
Protein concentration was determined with a bicinchroninic acid protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Electrospray ionization mass spectrometric analysis of lipids was performed as described previously (Cheng et al. 2007). Quantitative data are presented as the means ± SD. Differences between mean values were determined by an unpaired Student’s t test. P < 0.01 was considered significant.
Previous work has shown that H4-APPwt cells can generate large amounts of Aβ40 and Aβ42 and secret into the culture media (Murphy et al. 2003). In initial experiments, we determined the altered amounts of Aβ40 and Aβ42 peptides in H4-APPwt cell culture media in response to a range of supplemented sulfatide concentrations in the presence of 50 μM methyl-β-cyclodextrin. Intriguingly, the addition of sulfatides to the cell culture media markedly lowered the amounts of both Aβ40 and Aβ42 peptides in the media in a dose-dependent manner (Figure 1A and 1B). Specifically, after H4-APPwt cells were incubated for 24 h after supplementation of sulfatides to the culture media, the levels of Aβ42 present in the cell culture media decreased from 160 ± 8 to 63 ± 9 pg/ml and the levels of Aβ40 decreased from 1412 ± 58 to 993 ± 88 pg/ml. Notably, the relative decrease in the amount of Aβ42 was more substantial than that of Aβ40 at all the concentrations of sulfatide tested (Figure 1C).
To better understand the potential mechanism(s) underlying the decreased levels of Aβ peptides after media sulfatide supplementation, we performed three additional experiments. First, we added different amounts of sulfatides as a component of vesicles comprised of a fixed amount of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PO PtdCho). A similar decreasing pattern in the levels of Aβ peptides present in the culture media occurred in a sulfatide-dose dependent manner (Figure 2), although the magnitude of the decline in Aβ peptides in the culture media was relatively smaller than that observed after direct supplementation of sulfatides with methyl-β-cyclodextrin as carrier (compared Figure 2 to Figure 1). For example, after incubation of the cells in media containing sulfatides (30 μM) in vesicles for 24 h, the level of Aβ42 present in the cell culture media decreased from 154 ± 5 to 101 ± 8 pg/ml and the level of Aβ40 decreased from 1424 ± 58 to 1212 ± 54 pg/ml. These results suggest that the decreases in Aβ levels in the culture media are largely caused by the supplemented sulfatides.
Next, we tested whether alterations in the anionic charge density of the sulfatide-containing vesicles would have an impact on the levels of the altered Aβ peptides in the culture media. Specifically, we included increasing amounts of phosphatidylinositol (PtdIns) in the PO PtdCho vesicles and determined the altered levels of Aβ peptides induced with anionic charge density under these conditions. The anionic inositol phosphate moiety is analogous to galactose sulfate with the exception of the positions of the anion and the six-membered ring in PtdIns and sulfatides. We found no significant decreases in the levels of both Aβ40 and Aβ42 in the culture media occurred after supplementation with PtdIns-containing vesicles in comparison to those of sulfatide-containing vesicles (Figure 2). These results indicate that the anionic charge density on the surface of the vesicles is not a determinant factor in mediating the sulfatide-induced decreases in the levels of Aβ peptides in the media.
Finally, we thought the sulfatide-dependent decreases in the levels of Aβ peptides in the media could possibly be due to the alterations in membrane physical properties induced by sulfatide supplementation, which caused changes in cellular secretase activities responsible for release of Aβ peptides from H4-APPwt cells. To exclude this possibility, we performed two additional experiments. First, we determined whether the levels and activities of β-secretase were altered after treatment with sulfatides. Western blot analysis and β-secretase activity assay did not show any significant difference between the homogenates of cells treated with or without sulfatide (data not shown). Next, we performed an identical experiment with NB cells which hardly produce Aβ peptides. In the experiment, the media of NB cells was replaced with the media with which H4-APPwt cells had been culturing for 24 h at 37 °C prior to exchanging and prior to addition of vesicles containing 0 or 10 mol% of bovine brain sulfatides to the media. Therefore, the exchanged media for NB cells contained high levels of Aβ40 and Aβ42 peptides as well as vesicles. A modestly less degree of decreases in the levels of Aβ peptides in NB cell culture media to that in H4-APPwt cell media was determined after supplementation of sulfatide-containing PO PtdCho vesicles relative to PO PtdCho vesicles (Figure 3). This study indicates that the decreased levels of Aβ peptides in cell culture media did not result from the reduction of Aβ production, but mainly from the increased uptake (see below).
Very recently, we have demonstrated that sulfatides present in the cell culture media undergo rapid uptake by cells through an endocytotic process (Zeng et al. 2008). Therefore, it is logical to conclude that the decrease in the levels of Aβ peptides in the cell culture media is likely due to sulfatide-assisted uptake through an endocytotic pathway. To determine if this pathway occurred in the sulfatide-facilitated Aβ clearance, we first examined whether the uptaken Aβ40 peptide co-localized with the lysosomal compartment. We employed dual staining confocal immunofluorescence microscopy for both lysosomes and Aβ40 in H4-APPwt cells after supplementation with sulfatides in the culture medium. These experiments demonstrated that (1) the lysosomes became enlarged (compare panels A and D in Figure 4) in cells treated with sulfatides as reported previously in a different cell type (i.e., NB cells and primary neurons) (Zeng et al. 2008); (2) the intracellular levels of Aβ40 increased substantially (compare panels B and E in Figure 4); and (3) Aβ40 was largely co-localized with the lysosomal compartment in H4-APPwt cells (panels C and F in Figure 4).
Next, we isolated the endosome- and lysosome-enriched fractions from H4-APPwt cells after treatment with sulfatides (60 μM) for 24 h and determined the levels of Aβ40 in these fractions by ELISA analysis. It should be pointed out that the concentration of sulfatide used in the experiment was selected for our convenience to isolate these organelles and in the absence of cell apoptosis (Zeng et al. 2008). Our experiments demonstrated profound increases in Aβ40 content present in both the endosomal and lysosomal compartments after supplementation with sulfatides in the cell culture media (Figure 5). More specifically, after incubation of H4-APPwt cells in the media supplemented with sulfatides (60 μM) for 24 h, the levels of Aβ40 in the endosome fraction dramatically increased from 748 ± 90 to 1765 ± 160 pg/mg proteins and the levels of Aβ40 in the lysosome fraction increased from 1440 ± 90 to 1960 ± 160 pg/mg proteins (Figure 5A). Since the volume of the lysosomal fraction was considerably larger than that of endosomal fraction, the majority of Aβ40 was present in this organelle (Figure 5B). Flow cytometric analyses further confirmed the cellular accumulation of Aβ40 after treatment with sulfatides (data not shown).
It is intriguing to point out that the levels of Aβ40 peptide present in H4-APPwt endosomal and lysosomal fractions were substantially higher than those secreted into the culture media. In stark contrast, the amount of Aβ42 in the endosomal/lysosomal fractions was essentially undetectable despite additional attempts utilizing up to three-fold more cells to isolate these fractions. This result is especially surprising since the magnitude of the sulfatide-induced decrease in the relative content of Aβ42 in the media was greater than that of Aβ40. Moreover, we did not detect the presence of Aβ42 in other isolated membrane fractions (e.g., mitochondria). These results indicate that Aβ42 was selectively degraded after uptake under the experimental conditions examined.
The role of apoE in Aβ peptide clearance has been well established (Kang et al. 2000; Bales et al. 2002; Hartman et al. 2005; Bateman et al. 2006). Since sulfatides are present in the apoE-associated lipoproteins in the CNS, we next investigated the role of sulfatides in the apoE-mediated cellular clearance of Aβ peptides. Specifically, we determined the decreases in the levels of Aβ peptides in the culture media (which were secreted by H4-APPwt cells) following addition of PO PtdCho vesicles containing 0 or 10 mol% sulfatides as well as the indicated amounts of apoE3. ELISA analyses demonstrated that both Aβ42 and Aβ40 levels in the culture media modestly decreased after addition of only PO PtdCho and apoE3 to the media in an apoE3-dose dependent manner (Figures 6A and 6B). However, the effects of apoE3 in the absence of sulfatides on the clearance of Aβ peptides were relatively modest in comparison to those induced by sulfatides (Figures 6A and 6B). Similar experiments were also conducted with NB cells cultured in the media from H4-APPwt cells as described above to confirm whether the decreased levels of Aβ peptides were due to the influence of the added reagents on Aβ production. We showed that similar results were obtained with NB cells to those with H4-APPwt cells (Figure 6) suggesting that uptake of Aβ peptides through endocytosis facilitated by sulfatides and/or mediated by apoE is the major pathway of Aβ clearance under the experimental conditions examined.
Next, we examined the effects of sulfatides/apoE isoform complexes on the clearance of Aβ peptides in the culture media supplemented with sulfatide-containing PO PtdCho vesicles. The rank order of the ability of apoE isoforms to decrease levels of both Aβ40 and Aβ42 in H4-APPwt cell culture media in the presence of PO PtdCho vesicles containing 10 mol% (30 μM) sulfatides was, in increasing effectiveness: apoE2 < apoE3 < apoE4 (Figure 7). These results led us to postulate that the differential effectiveness of apoE isoforms to facilitate the removal Aβ peptides from the H4-APPwt media may reflect differences in the abilities of the apoE isoforms to bind Aβ peptides and to interact with LDL receptor superfamily members. To address these possible mechanisms contributing to the apoE isoform dependent order of Aβ clearance, we examined the specific interactions between apoE isoforms, sulfatides, and Aβ peptides by employing a vesicular model system containing sulfatides as guest in a host phospholipid matrix. After mixing of recombinant human apoE2, E3 or E4 (5 μg), Aβ40 or Aβ42 (0.5 μg), and PO PtdCho small unilamellar vesicles containing sulfatides (0 or 10 mol%) (25 μM of final vesicle concentration) and subsequent incubation at 37 °C for 2h, the samples were ultracentrifuged at 120,000 × g for 30 min to separate the components into soluble and vesicle-associated (pellet) fractions. The nearly complete recovery (> 95%) of PO PtdCho vesicles was confirmed by electrospray ionization mass spectrometric analysis of lipids in both the pellet and supernatant fractions. Only residual amounts of apoE were present in the sample supernatants as assessed by Western blot analysis. Of particular interest was the impact of sulfatides on the interaction between apoE isoforms and Aβ peptides in the membrane microenvironment of the PtdCho vesicles. Specifically, sulfatide significantly enhanced Aβ binding to apoE-associated PtdCho vesicles which ranged from a 35% increase in binding of Aβ40 to apoE3/PtdCho vesicles to an over 2-fold enhancement of Aβ42 association with apoE4/PtdCho vesicles (Figure 8). These results indicate that the isoform-specific apoE differences in the clearance of Aβ peptides were dependent upon 1) the differential binding of Aβ peptides to the apoE isoform-associated lipoproteins in the absence of sulfatides and 2) increased affinity of the Aβ peptides/apoE complexes to interact with lipoprotein receptors in the presence of sulfatides. Collectively, these results indicate that one role of sulfatides is to promote the clearance of Aβ peptides through enhancing Aβ binding to membrane surfaces.
Although lipids and/or apoE-associated lipids in general have been previously examined with respect to the interaction with Aβ peptides (Yanagisawa et al. 1995; Tokuda et al. 2000; Matsuzaki 2007), the effects of sulfatides in particular on the interactions between apoE isoforms and Aβ peptides have not been previously investigated. Since both Aβ peptides and sulfatides are associated and contained, respectively, with apoE-associated lipoproteins, our goal in this study was to test the hypothesis that sulfatides could facilitate the apoE-mediated process of Aβ peptide clearance. By employing cell culture and vesicular membrane models, we have demonstrated that sulfatides enhance the binding of Aβ peptides to vesicles containing apoE and facilitate the clearance of Aβ peptides through an endocytotic pathway in an apoE isoform-dependent manner.
To the best of our knowledge, interactions between sulfatides and Aβ peptides have not been explored previously. However, prior studies have demonstrated that sulfate ions and the sulfate moieties of proteoglycans are critical for facilitating Aβ peptide fibril formation in vitro and in vivo (Fraser et al. 1992; Castillo et al. 1999). Other studies have found that heparan sulfate and other proteoglycans accumulate in neurons and in the amyloid deposits of AD and Down syndrome patients (Snow et al. 1990; van Horssen et al. 2002). Since the galactosyl sulfate moiety in sulfatides is analogous to that in heparan sulfate, we believe that the galactosyl sulfate group might play a critical role in sulfatide-facilitated Aβ binding to apoE-associated lipoproteins. Consistent with this notion is the demonstration by the present study of the minimal effects of other negatively charged lipids (e.g., PtdIns) present on the model membrane surface on Aβ peptide binding/clearance. It is conceivable that sulfatides bound to the apoE-associated lipoproteins form a matrix on the particle surface that likely mimics some of the properties of proteoglycans to facilitate the association of Aβ peptides. We believe that sulfatide-facilitated Aβ peptide binding is likely due to specific electrostatic interactions of the galactosyl sulfate moiety in sulfatides with apoE which is enriched in basic amino acids and Aβ peptides in which both acidic and basic amino acids are abundant.
In this study, we demonstrated that the sulfatide-facilitated decreases in cell culture media Aβ42 levels were larger than those for Aβ40, strongly suggesting a selective clearance of Aβ42 relative to Aβ40. Intriguingly, the concentration of Aβ42 in the isolated endosomal/lysosomal fractions was below detectable levels while we found a higher concentration of Aβ40 in these fractions than in the cell culture media, thereby suggesting a selective degradation of Aβ42 peptide in these organelles in the examined neuronal cells. Our previous studies showed that apoE4-associated lipoproteins in human cerebrospinal fluid contain more sulfatides than apoE3-associated lipoproteins under normal physiological conditions (Han et al. 2003a; Han et al. 2003b). Our current results support a strong interaction of apoE4 with lipoprotein receptors. These results highlight the importance of the interactions between Aβ42, sulfatides, and apoE in Aβ clearance and sulfatide metabolism while direct binding assays showed that Aβ42 has comparatively less affinity for apoE4 than apoE3. Thus, when abnormal Aβ42 production occurs in the CNS, sulfatide/apoE-induced Aβ42 aggregation may initiate and propagate Aβ fibrillogenesis.
Following this line of reasoning, we would anticipate that sulfatide-facilitated Aβ42 accumulation would occur in the endosomal/lysosomal compartment if the degradation machinery for Aβ peptides becomes inefficient due to protein mutation and/or misfolding in these organelles. Presumably, the aggregated Aβ42 peptides would serve as a nucleation seed for Aβ fibrillogenesis eventually leading to neuronal cell death. This mechanism is consistent with previous recognition of the role of endosomal/lysosomal dysfunction in AD pathogenesis (Kalanj-Bognar et al. 2002; Nixon 2005). Therefore, maintenance of normal lysosomal function during aging and reversal of abnormal lysosomal function in diseased states potentially represent a new direction in the therapeutic intervention for AD.
Prior work has established that over-production of Aβ peptides is associated with early onset AD (Selkoe 2006). The clearance pathway for Aβ peptides would be predicted to become overwhelmed under this pathological condition, potentially leading to the concomitant depletion of sulfatides through at least two possible mechanisms. First, sulfatides are consumed through accelerated trafficking and metabolism as demonstrated by previous studies in which Aβ accumulation induced increases in the expression levels of apoE and its receptors (e.g., LRP) (e.g., (Qiu et al. 2001; Arelin et al. 2002)), thereby increasing the processing of apoE-mediated sulfatide trafficking/metabolism. Second, sulfatides are depleted through the process of Aβ clearance as demonstrated in this work. Our very recent study using animal models of AD has unambiguously verified this hypothesis (Cheng et al. 2008) through demonstrating that the sulfatide levels in various brain tissues are reduced in both APPsw and APPV717F transgenic mice in an age-dependent manner relative to their respective wild type littermates. In contrast, we have found that sulfatide depletion does not occur in APP transgenic animals with an apoE null background relative to the apoE null littermates.
Collectively, this study reports the novel finding that sulfatides, a class of myelin-specific sphingolipids which are present in apoE-associated lipoproteins in the CNS, facilitate apoE-mediated clearance of Aβ peptides. In addition, the current results have also revealed specific interactions between sulfatides, apoE isoforms, and Aβ peptides and have demonstrated the importance of these interactions in Aβ clearance/fibrillogenesis. Accordingly, our results provide novel insights into the molecular mechanism leading to Aβ aggregation/deposition in AD and identify a potential new direction for therapeutic intervention for AD.
This work was supported by National Institute on Aging Grants R01 AG23168 and R01 AG31675. The authors are grateful to Drs. Christopher M. Jenkins and Ari Cedars for their comments on the preparation of the manuscript and to Dr. Rebecca Miller for her technique help during the study.