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The Journal of Lipid Research, Vol. 40, 447-455, March 1999
Copyright © 1999 by Lipid Research, Inc.

Original Article

Competition of Aß amyloid peptide and apolipoprotein E for receptor-mediated endocytosis

Karl Winklera, Hubert Scharnagla, Ursula Tisljara, Heinz Hoschützkyb, Isolde Friedricha, Michael M. Hoffmanna, Manfred Hüttingerc, Heinrich Wielanda, and Winfried Märza
a Department of Clinical Chemistry, Albert Ludwigs-University, Freiburg, Germany
b NanoTools Antikörpertechnik, Teningen, Germany
c Department of Medical Chemistry, University of Vienna, Austria

Correspondence to: Karl Winkler


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

The genetic polymorphism of apolipoprotein E (apoE) is associated with the age of onset and relative risk of Alzheimer's disease (AD). In contrast to apoE3, the wild type allele, apoE4 confers an increased risk of late-onset AD. We demonstrate that the ß-amyloid peptide isoforms Aß (1–28), Aß (1–40), and Aß (1–43) compete for the cellular metabolism of apoE3 and apoE4 containing ß-very low density lipoproteins. An antibody raised against Aß (1–28) cross-reacted with recombinant apoE. Epitope mapping revealed positive amino acid clusters as common epitopes of Aß (13 through 17; HHQKL) and apoE (residues 144 through 148; LRKRL), both regions known to be heparin binding domains. Aß in which amino acids 13 through 17 (HHQKL) were replaced by glycine (GGQGL) failed to compete with the cellular uptake of apoE enriched ßVLDL.

These observations indicate that Aß and apoE are taken up into cells by a common pathway involving heparan sulfate proteoglycans.—Winkler, K., H. Scharnagl, U. Tisljar, H. Hoschützky, I. Friedrich, M. M. Hoffman, M. Hüttinger, H. Wieland, and W. März. Competition of Aß amyloid peptide and apolipoprotein E for receptor-mediated endocytosis. J. Lipid Res. 1999. 40: 447–455.

Supplementary key words: Aß amyloid, Alzheimer's disease, apolipoprotein E, heparin binding domain


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Accumulation of the ß-amyloid (Aß) peptide in the brain is a defining feature of all forms of Alzheimer's diseases (AD), irrespective of the genetic background (1). Aß is derived from the proteolytic processing of the ß-amyloid precursor protein (APP). APP is thought to be cleaved by at least three different proteolytic activities (2). Cleavage by alpha-secretase occurs between residues 688 and 689 of mature APP. As these residues correspond to positions 16 and 17 of the Aß peptide (3) (4), alpha-secretase prevents the formation of Aß (5) (6). Minute amounts of Aß are, however, generated constitutively through the action of two other putative endoproteolytic activities (7) (8): the amino terminal cleavage is mediated by beta-secretase, carboxy terminal cleavage by gamma-secretase (2). Mutations in the genes for APP, presenilin 1, and presenilin 2 have been shown to increase the formation of Aß and may thus cause early onset forms of AD (1). Apolipoprotein E (apoE) is another constituent of amyloid plaques in the brain of patients with AD. ApoE is genetically polymorphic. There are three frequent alleles at the apoE gene locus: E2, E3, and E4 (9). This polymorphism strongly affects the risk of developing AD. In individuals with late-onset AD, the E4 allele is 2- to 3-fold more frequent than in the general population (10) (11). Recently, we demonstrated that the E4 allele not only correlates with the development of amyloid deposits, but also with the formation of neurofibrillary tangles, another major histopathological hallmark of AD (12), strongly implicating apoE in the pathogenesis of AD.

The mechanistic link between the apoE polymorphism and AD has not been unraveled so far. There are several hypotheses that may account for the association between apoE4 and AD. ApoE and Aß form dodecyl sulfate-resistant complexes in vitro, apoE4 complexing more rapidly than apoE3 (13) (14). When Aß and apoE are co-incubated, unique monofibrillar structures evolve, apoE4 yielding a denser matrix than apoE3 (15). According to Castaño and co-workers (16), apoE enhances both the rate and the amount at which fibrils are generated from soluble amyloid in vitro, apoE4 being more effective compared to apoE3. In contrast to apoE3, apoE4 reduces the branching and outgrowth of neurites in different types of neuronal cells (17) (18) (19). It has been suggested that apoE4, but not apoE3 promotes depolymerization of microtubuli (20) (21). ApoE may thus intimately participate in maintaining the integrity of the cytoskeleton.

In the central nervous system, apoE mRNA is found in astrocytes and glial cells, but not in neurons (22) (23) (24) (25) (26). As neurons contain immunoreactive apoE (27), a metabolic pathway must exist by which apoE is taken up. This pathway most likely involves endocytosis by the LDL receptor-related protein (LRP) (18) (19). Current opinion predicates that this process is initiated by binding of apoE- containing particles to heparan sulfate proteoglycans (HSPG) on the cell surface from where they are subsequently transferred to LRP (28) (29). Aß has also been found to bind to heparan sulfate proteoglycans (30) (31) (32) (33) (34) (35) (36). Therefore, we examined the hypothesis that Aß and apoE-containing lipoproteins are taken up into cells by a common pathway. Our results indicate that Aß peptide and apoE compete with each other for receptor-mediated endocytosis by virtue of their heparin binding domains. These observations link the cellular metabolism of apoE and Aß and may provide another clue to the mechanism underlying the association between the apoE polymorphism and AD.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
Polyclonal anti-Aß(1–28) IgY raised against Aß(1–28) peptide in chicken was from Nanotools, Denzlingen, Germany, and affinity- purified using Aß(1–28) peptide covalently coupled to divinyl sulfone-activated agarose (37). Peroxidase-conjugated anti-chicken IgY from rabbit was from Jackson ImmunoResearch Laboratories, West Grove, PA. Alkaline phosphatase-conjugated anti-chicken IgG from goat was from Dianova, Hamburg, Germany. ßVLDL were prepared from plasma of 1% cholesterol-fed rabbits (38). Recombinant human apoE (E2, E3, and E4) expressed in baculovirus (39) (40) was from PanVera, Madison, WI. Aß(1–28) peptide, Aß(1–40) peptide, and Aß(1–43) peptide were from Bachem, Bubendorf, Switzerland. 125I-labeled Aß was from Immundiagnostik (Bensheim, Germany). Aß peptides were stored freeze-dried at -20°C. In each experiment they were freshly dissolved at a concentration of 1 mg/ml at pH 8 in PBS and then used immediately.

Binding, uptake, and degradation of lipoproteins
Human skin fibroblasts were from skin biopsies of normolipidemic individuals. Binding, uptake, and degradation of lipoproteins were measured according to Goldstein, Basu, and Brown (41) with slight modifications (42). ßVLDL and recombinant apoE were iodinated using the iodine monochloride method (43). Loading of ßVLDL with recombinant human apoE was accomplished by incubating ßVLDL at a protein concentration of 7.5 mg/ml with 2.4 mg/ml apoE in phosphate buffer at 37°C for 1 h (17). The apoE-loaded ßVLDL were then used for cell culture experiments at a final concentration of 7.5 µg/ml and 2.4 µg/ml of ßVLDL protein and apoE, respectively.

Fast protein liquid chromatography
Gel filtration of lipoproteins was performed with a column containing 120 ml of Superdex 200 HiLoad (Pharmacia Biotech, Uppsala, Sweden). The elution buffer contained 50 mmol/l Tris, 200 mmol/l NaCl, 0.2 g/l Na-azide, and 0.1% Brij35, pH 7.5. The flow rate was 60 ml per h and the size of the collected fractions was 2 ml.

Competitive enzyme immunoassay
Aß(1– 43) peptide (10 µg/well) was covalently bound to microtiter plates (Covalink NHR, Nunc, Roskilde, Denmark) according to the manufacturer's recommendations. Polyclonal anti-Aß antibodies were incubated overnight at room temperature with the respective competitors: recombinant apoE or Aß(1– 43) peptide in 10 mmol/l phosphate, pH 7.4, 137 mmol/l NaCl, 2.7 mmol/l KCl (PBS), containing 10 g/l bovine serum albumin, 0.5 g/l Tween 20, and 0.05 g/l EDTA. After incubation, the antigen–antibody mixtures were loaded to the coated microtiter plates. Antibodies not neutralized by the competitors and binding to the solid phase were measured using appropriate peroxidase-conjugated secondary antibodies and o-phenylenediamine as substrate.

Epitope mapping of an apoE peptide library
Peptides were synthesized by Fmoc chemistry on an activated cellulose membrane using an ABIMED Auto-Spot Robot ASP 222 (Abimed, Langenfeld, Germany) according to the manufacturer's instructions and standard spot synthesis protocols (44). The cellulose-bound peptide libraries were incubated and probed essentially like immunoblots. Blocking of membranes and antibody dilutions were performed in PBS containing, in addition, 10% (v/v) fetal calf serum and 2 g/l Tween 20. Primary antibodies were used at 200 µg/l; visualization was performed with goat anti-chicken alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as substrate.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

We examined whether Aß was able to compete with the uptake of apoE-containing lipoproteins in cultured human skin fibroblasts. Recombinant apoE isoforms expressed in the baculovirus system (39) (40) were loaded to 125I-labeled ßVLDL from cholesterol-fed rabbits. As expected, supplementation of apoE3, and apoE4, but not apoE2, enhanced cellular binding, uptake, and degradation approximately 2.5- and 3.0-fold, respectively, compared to VLDL alone ( Figure 1 A–C). When we added Aß(1–43) at increasing concentrations, the increases in binding, uptake, and degradation mediated by apoE3 and apoE4 were almost completely abrogated, suggesting that Aß and apoE competed for cellular uptake. This effect might have been non-specific as Aß is cytotoxic. To exclude this possibility, competition experiments were done with the less toxic and aggregatable Aß(1–28) and Aß(1– 40) (45) (46) (47). These Aß peptides had virtually equal effects on binding, uptake, and degradation of apoE3 compared to Aß(1– 43) ( Figure 2 A–C). In addition, lactate dehydrogenase activities were measured in the culture medium after incubation with Aß(1– 43) peptide. There was no change of lactate dehydrogenase activity when we exposed fibroblasts for 4 and 24 h, respectively, to those Aß(1– 43) concentrations used in cell culture experiments. These data suggest that the competition of Aß with the uptake of apoE-loaded ßVLDL was not due to non-specific effects of Aß. As Aß (1–28) was active as a competitor, the competing domain obviously resided within the aminoterminal part of Aß. To evaluate the possibility that the decreased uptake of apoE-containing particles was due to a displacement of apoE by Aß from the surface of the ßVLDL, we examined whether Aß was able to liberate apoE from ßVLDL under our experimental conditions. Cell culture medium was supplemented with 125I-labeled apoE-loaded ßVLDL and incubated with human skin fibroblasts as in the binding studies. Aß(1–43) peptide was then added to the medium ( Figure 3, panel A), but was omitted from the respective control (Figure 3, panel B). After incubation for 1 h, the culture medium was subjected to gel-filtration and the radioactivity was measured in the eluted fractions. There was no difference in the amounts of 125I-labeled apoE associated with the ßVLDL fraction, regardless of whether or not Aß was present in the incubation medium.



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  		Figure 1. Effect of Aß on binding, uptake, and degradation of 125I-labeled ßVLDL in cultured human skin fibroblasts. ßVLDL were prepared by ultracentrifugation from the plasma of cholesterol-fed rabbits, labeled with 125I, and complexed with recombinant apoE as described (17) (38) (39). Human skin fibroblasts were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum. Cells received 7.5 µg/ml 125I-labeled ßVLDL protein (open circles), ßVLDL complexed with 2.4 µg/ml recombinant apoE2 (closed circles), apoE3 (closed triangles), or apoE4 (closed squares), respectively. Aß was used as unlabeled competitor at the concentrations indicated. Binding (panel A), uptake (panel B), and degradation (panel C) were determined as described (41) (42). Each data point represents the average value from triplicates, error bars represent standard deviations.



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  		Figure 2. Effect of various Aß peptides on binding, uptake, and degradation of 125I-labeled ßVLDL in cultured human skin fibroblasts. ßVLDL were prepared by ultracentrifugation from the plasma of cholesterol-fed rabbits, labeled with 125I, and complexed with recombinant apoE as described (17) (38) (39). Human skin fibroblasts were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum. Cells received 7.5 µg/ml 125I-labeled ßVLDL protein (open circles). ßVLDL complexed with 2.4 µg/ml recombinant apoE3 was used with Aß of various lengths (Aß(1–28) solid circle; Aß(1–40) solid triangle and Aß(1–43) solid square) as unlabeled competitors at the concentrations indicated. Binding (panel A), uptake (panel B), and degradation (panel C) were determined as described (41) (42). Each data point represents the average value from triplicates; error bars represent standard deviations.



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  		Figure 3. Effect of Aß on the apoE contents of 125I-labeled apoE-enriched ßVLDL and interaction of 125I-labeled Aß with apoE enriched ßVLDL. ßVLDL and 125I-labeled apoE3 were incubated at room temperature for 1 h. ApoE3 loaded ßVLDL (final concentrations: 2.4 µg/ml 125I-labeled apoE and 7.5 µg/ml ßVLDL protein) were then added to cell culture medium and incubated at 4°C with human fibroblasts. Sixteen µg/ml (4 µM) Aß(1–43) peptide was added within 1 min after the addition of 125I-labeled apoE/ßVLDL to the cell culture, whereas in the control experiment Aß was omitted. After 60 min of incubation, the cell culture medium was removed from the cells and subjected to gel filtration. The amount of radiolabeled apoE was quantified in each of the eluted fractions. Panel A: incubation with Aß(1–43) peptide. Panel B: control experiment without Aß(1–43) peptide. Panel C: ßVLDL and unlabeled apoE3 were incubated at room temperature, then added to cell culture medium and incubated at 4°C with human fibroblasts as described above. Sixteen µg/ml (4 µM) 125I-labeled Aß(1–43) peptide was added within 1 min after the addition of apoE/ßVLDL to the cell culture. After 60 min of incubation, the cell culture medium was removed from the cells and subjected to gel filtration. The amount of 125I-labeled Aß was quantified in each of the eluted fractions.

We also wished to exclude the possibility that Aß bound to ßVLDL during the cell culture experiments, thereby masking the cell surface binding domains of apoE. Cell-culture medium was supplemented with apoE/ßVLDL, incubated with human skin fibroblasts, and 125I-labeled Aß(1–43) peptide was added to the medium. After 60 min of incubation the culture medium was withdrawn and subjected to gel filtration (Figure 3, panel C). No ßVLDL-associated radioactivity could be detected. Therefore, we concluded that Aß(1– 43) neither displaced radiolabeled apoE from ßVLDL nor did it bind to ßVLDL to a relevant extent under the conditions used here.

To investigate whether structural similarities of Aß and apoE exist which could explain a competition between Aß and apoE for cell surface binding sites, we raised a polyclonal antiserum in chicken, directed against Aß(1–28), the hydrophilic domain of Aß. Using a solid-phase competitive enzyme immunoassay, we analyzed the binding of this antiserum to recombinant apoE. Aß(1– 43) was immobilized to microtiter plates. Recombinant apoE isoforms and Aß peptide(1– 43) were pre-incubated at increasing concentrations with the antiserum and the mixtures were then loaded to the Aß-coated wells. In this assay, the polyclonal antiserum prepared against Aß(1–28) cross-reacted with apoE. The affinities of the antiserum for the Aß(1–43) peptide and apoE isoforms were all in the same order of magnitude (KD around 0.2 µmol/l). The three major apoE isoforms (E2, E3, E4) did not differ regarding their reactivities ( Figure 4). We wished to identify the epitope(s) of apoE producing cross-reactivity with the Aß antibodies. We generated a library of peptides containing hexameric sequential parts of the amino acid sequence of apoE, starting at spot number 12 with position 1 through 6 of mature apoE (44). This peptide library was probed with polyclonal anti-Aß(1–28) from chicken which was previously affinity purified using immobilized Aß(1–28). Immunostaining of the peptide library revealed that the antibody recognized multiple regions of the apoE molecule, all of which contain positively charged amino acids (48). As shown in Figure 5, the most intense staining was seen with peptides covering positions 144 through 148 of mature apoE (48). No staining was seen in the control incubations using either crude IgY from pre-immune chicken or affinity-purified anti-myoglobin IgY. We also screened a peptide library of dodecamers derived from Aß. Only one domain of antibody binding was detected, comprising amino acids 9 through 19 ( Figure 6). Amino acids 12 through 17 and 144 through 147 of Aß and of apoE, respectively, constitute heparin binding domains (30) (32) (33) (34) (35) (36) (49) (50). The uptake of lipoproteins mediated by apoE involves initial binding of the ligand to cell surface HSPG (28) (29). We reasoned that the homology of the heparin binding motifs of apoE and Aß revealed by the immunochemical studies was responsible for the competition of Aß for the uptake of apoE-enriched ßVLDL. To test this hypothesis, we generated an Aß variant (1– 43), designated Aß*, in which residues 13 through 16 (HHQK) were replaced by GGQG. This variant peptide was earlier shown to be completely defective in binding to heparin (30). In contrast to wild-type Aß, the Aß* peptide was also not able to compete with apoE-supplemented ßVLDL for binding, uptake, and degradation in cultured cells ( Figure 7), indicating that amino acids 13 through 16 of Aß are crucial for the displacement of apoE from cell surface binding sites.



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  		Figure 4. Apolipoprotein E binding of a polyclonal antibody raised against Aß(1–28). Panel A: ApoE2 (circles), apoE3 (triangles), apoE4 (squares), and Aß(1–43) amyloid (dashes) were preincubated overnight at the concentrations indicated with polyclonal anti-Aß(1–28) antiserum (40 µg/ml). The mixture was applied to microtiter plates coated with Aß(1–43). Antibodies bound to the solid phase were detected with peroxidase-conjugated anti-IgY (1.6 µg/ml). B0 (100%) is the absorbance with no competitor added and B is the absorbance in percent of B0 at the indicated concentrations of the respective competitors.



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  		Figure 5. Epitope mapping using a library of peptides derived from the apolipoprotein E amino acid sequence. Individual peptides were synthesized according to the following scheme: X-X-A1-A2-A3-A4-A5-A6-X-X-bA-bA-cellulose, where X = equimolar mixture of the natural amino acids with the exception of C and W, bA = ß-alanine, A1 to A6 = hexapeptides consisting of six subsequent amino acids. Spot numbers 1 through 11 represent a part of the leader sequence of immature apoE. Spot number 12 consists of amino acids 1 to 6 of the mature apoE protein, spot number 13 of amino acids 2 to 7, and so on. Binding of affinity-purified anti-Aß(1–28) antibody was visualized using goat anti-chicken antibodies conjugated to alkaline phosphatase and BCIP/MTT.



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  		Figure 6. Epitope mapping using a library of peptides derived from the Aß peptide amino acid sequence. Individual peptides were synthesized according to the following scheme: X-X-A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-X-X-bA-bA-cellulose, where X = equimolar mixture of the natural amino acids with the exception of C and W, bA = ß-alanine, A1 to A12 = dodecapeptides consisting of twelve subsequent amino acids. Spot numbers 1 through 9 represent amino acids of the amyloid precursor protein (APP) preceding the Aß peptide. Spot number 10 consists of amino acids 1 to 12 of the mature Aß peptide, spot number 11 of amino acids 2 to 13, and so on. Binding of affinity purified anti-Aß(1–28) antibody was visualized using goat anti-chicken antibodies conjugated to alkaline phosphatase and BCIP/MTT.



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  		Figure 7. Effects of Aß and Aß* on binding, uptake, and degradation of 125I-labeled ßVLDL in cultured human fibroblasts. ßVLDL were prepared by ultracentrifugation from the plasma of cholesterol-fed rabbits, labeled with 125I, and complexed with recombinant apoE as described (17) (38) (39). Human skin fibroblasts were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum. Cells received 7.5 µg/ml 125I-labeled ßVLDL protein (circles), ßVLDL complexed with 2.4 µg/ml recombinant apoE3 (triangles). Aß (positions 13 through 16 HHQK, closed symbols) and Aß* (positions 13 through 16 GGQG, open symbols) were used as unlabeled competitors at the concentrations indicated. Binding (panel A), uptake (panel B), and degradation (panel C) were determined as described (41) with slight modifications (42). Each data point represents the average value from triplicates; error bars represent standard deviations.


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

The cellular uptake of apoE-enriched lipoproteins is thought to occur in two steps. In the first step, apoE mediates binding of these particles to cell surface proteoglycans. In the second step, lipoproteins are transferred to lipoprotein receptors such as the LDL receptor-related protein (LRP) to undergo endocytosis (28) (29). So far, very little is known about the cellular catabolism of Aß. Both Aß and apoE possess heparin binding domains. We, therefore, examined whether Aß was able to affect the endocytosis of apoE-containing lipoproteins. Our results demonstrate that Aß(1–28), Aß(1– 40), and Aß(1– 43) decreased the uptake of apoE-loaded ßVLDL into cells. As Aß peptides are highly lipophilic, we wished to rule out that decreased binding and uptake of apoE-enriched ßVLDL was due to displacement of apoE by ßA4 directly interacting with the ßVLDL. When we co-incubated apoE-enriched ßVLDL and Aß, we were not able to demonstrate that Aß detached radiolabeled apoE from the ßVLDL. In addition, there was no evidence that Aß bound to apoE-containing ßVLDL under the conditions used here, thus excluding the possibility that Aß modulated the ability of apoE to interact with the surface of cultured cells. We conclude from this data that Aß decreased apoE-mediated binding to the cell surface by competition of Aß with apoE-loaded ßVLDL rather than by displacement of apoE from ßVLDL or by direct binding of Aß to apoE-containing ßVLDL. This assumption is further supported by the fact that Aß(1–28), which lacks the hydrophobic carboxy-terminus most likely interacting with apoE, was effective in diminishing cellular uptake of apoE-containing ßVLDL as well.

Competition of Aß and apoE for cellular binding would require that the two polypeptides share structural similarities. As there is no homology of the amino acid sequences of apoE and Aß, we decided to use an immunological approach to investigate whether domains of apoE might be similar to Aß by three-dimensional structure or charge distribution. A polyclonal antibody against the Aß(1–28) peptide, the hydrophilic region of Aß(1– 43), was raised in chicken. The anti-Aß(1–28) antiserum was immunologically reactive with both, Aß(1– 43) amyloid and recombinant apoE, recognizing both antigens with approximately equal affinities.

Epitope mapping suggested amino acids 9–19 of the amyloid-peptide and amino acids 144 through 148 of apoE as the common epitope. Both epitopes consist of clusters of positive amino acids. Amino acids 144 through 148 of apoE are part of the receptor binding domain of the molecule which extends between residues 136 and 150 (51) and they represent one out of at least two heparin binding sites of the apoE molecule (48) (49) (50). In the Aß peptide, residues 12 through 16 mediate heparin binding (32) (33) (34) (35) (36); replacing the positive amino acids between positions 13 and 16 of Aß (HHQK) by glycine (GGQG) completely abolishes the binding of Aß to heparin (30). Consistently, we found that the peptide containing G at positions 13, 14, and 16 lost the ability to compete with apoE-enriched ßVLDL for cellular binding, uptake, and degradation. Taken together, these data indicate that apoE and Aß share homology of their heparin binding sites and they suggest that the heparin binding site in the aminoterminal region of Aß is able to interact with cellular sites responsible for the endocytosis of apoE. Thus far, our findings are completely consistent with a report by Ida, Masters, and Beyreuther (51), who obtained evidence that the aminoterminus of Aß was involved in receptor-mediated uptake of this peptide.

What is the molecular nature of the cellular site binding Aß and apoE? ApoE is a ligand of the LDL receptor (52), the LRP (53) (54), and the VLDL receptor (55). The VLDL receptor is not expressed in fibroblasts. The uptake experiments were conducted in the presence of fetal calf serum as a source of cholesterol and LDL receptor activity was thus down-regulated. Finally, the increase in cellular uptake which is produced by supplementing ßVLDL with apoE is attributable to the HSPG/LRP pathway rather than to the LDL receptor (28) (29) (56). Together, these considerations imply that in this study Aß inhibited endocytosis of apoE by interacting with the HSPG/LRP complex.

Our observations may have implications for the mechanism underlying the association of the apoE polymorphism and the risk for AD. In the central nervous system there appears to be a surplus of about 100-fold of apoE over Aß (100–300 nM versus 3 nM, respectively (57) (58)). ApoE might therefore modify the clearance of Aß by pathways involving heparan sulfate proteoglycans even in situations where the generation of Aß is increased. In this respect, it is interesting that the three common apoE isoforms differ by their binding to heparan sulfate proteoglycans. Whereas apoE4 binds more avidly (about 125% of apoE3), binding of apoE2 is approximately one-third lower compared to apoE3 (40, cf. Figure 1, panel A; Scharnagl, J. Acar, G. Feussner, H. Wieland, and W. März, unpublished results). One might therefore speculate that the three apoE isoforms inhibit the decay of Aß in the order apoE4 > apoE3 > apoE2. This would provide an explanation for both the positive correlation between Aß deposition and apoE4 (10) (11) (12) and the apparent protective effect of apoE2 (59), but this hypothesis still needs to be proven.

Further, it may be of interest that amino acids 13 to 16 of Aß, the epitope shared with apoE, are adjacent to the site at which APP is cleaved by alpha-secretase (amino acids 16/17). One might, therefore, speculate that intracellular apoE is able to modify the processing of APP by `trapping' alpha-secretase. The possible implications of our findings are currently under further investigation.


  ACKNOWLEDGMENTS

Parts of this study were supported by a grant from Boehringer Ingelheim to Manfred Hüttinger and Winfried März, by a grant from the Commission of the European Community (BIOMED 2, project MICAD, grant number PL 950162), by the Forschungskommission der Albert Ludwigs-Universität, Freiburg, and by a research award from the Dr. Walter Freundlich and Luise Freundlich Foundation, Frankfurt, to Karl Winkler.

Manuscript received May 8, 1998; and in revised form September 28, 1998; and in revised form October 28, 1998.

Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DAB, diamino benzidine; EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatography; HSPG, heparan sulfate proteoglycans; LRP, LDL receptor-related protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; VLDL, LDL, very low and low density lipoproteins, respectively; AD, Alzheimer's disease


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

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K. Yamauchi, M. Tozuka, E. Hidaka, I. Ueno, K. Matsuda, and T. Katsuyama
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