Differential Cellular Accumulation/Retention of Apolipoprotein E Mediated by Cell Surface Heparan Sulfate Proteoglycans
APOLIPOPROTEINS E3 AND E2 GREATER THAN E4*

Zhong-Sheng JiDagger , Robert E. PitasDagger §, and Robert W. MahleyDagger §parallel

From the Dagger  Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and the Departments of § Pathology and  Medicine, University of California, San Francisco, California 94141-9100

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isoform-specific effects of apolipoprotein E (apoE) on neurite outgrowth and the cytoskeleton are associated with higher intracellular levels of apoE3 than apoE4 in cultured neurons. The current studies, designed to determine the mechanism for the differential intracellular accumulation or retention of apoE, demonstrate that apoE3- and apoE4-containing beta -very low density lipoproteins (beta -VLDL) possess similar cell binding and internalization and delivery of cholesterol to the cells. However, as assessed by immunocytochemistry, analysis of extracted cellular proteins, or quantitation of 125I-apoE-enriched beta -VLDL, there was a 2-3-fold greater accumulation of apoE3 than apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes (HepG2) after 1-2 h, and this differential was maintained for up to 48 h. ApoE2 also accumulated in Neuro-2a cells to a greater extent than apoE4. The differential effect was mediated by the apoE-enriched beta -VLDL and not by free apoE. Neither the low density lipoprotein receptor nor the low density lipoprotein receptor-related protein was responsible for the differential accumulation of apoE3 and apoE4, since cells deficient in either or both of these receptors also displayed the differential accumulation. The effect appears to be mediated primarily by cell surface heparan sulfate proteoglycans (HSPG). The retention of both apoE3 and apoE4 was markedly reduced, and the differential accumulation of apoE3 and apoE4 was eliminated both in mutant Chinese hamster ovary cells that did not express HSPG and in HSPG-expressing cells treated with heparinase. The data suggest that cell surface HSPG directly mediate the uptake of apoE-containing lipoproteins, that the differential accumulation/retention of apoE by cells is mediated via HSPG, and that there is a differential intracellular handling of the specific apoE isoforms.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Apolipoprotein E (apoE)1 is a component of both plasma and cerebrospinal fluid lipoproteins (1-3). Lipoproteins containing apoE are ligands for members of the low density lipoprotein (LDL) receptor gene family, which includes the LDL receptor, the very low density lipoprotein (VLDL) receptor, the apoE receptor 2, and the LDL receptor-related protein (LRP) (1, 4-8). Plasma lipoproteins containing apoE participate in lipid transport and in the maintenance of cholesterol homeostasis (1). A similar role has been proposed for cerebrospinal fluid lipoproteins in the central nervous system (2, 9).

Apolipoprotein E occurs as three isoforms (apoE2, apoE3, and apoE4) that are products of different alleles at the same gene locus (10). These isoforms differ by single amino acid substitutions at positions 112 and 158 of the 299-amino acid protein (1). Whereas apoE3 has cysteine in position 112 and arginine at position 158, apoE2 has cysteine at both positions, and apoE4 has arginine. These single amino acid changes significantly affect the structure and function of the proteins (1, 11, 12). Apolipoprotein E2 is associated with the development of type III hyperlipoproteinemia (1). Subjects with apoE4 have higher plasma cholesterol levels and are more prone to developing atherosclerosis than subjects with apoE3, the most common form of apoE. The apoE4 allele is also associated with a greater risk of developing Alzheimer's disease, whereas the apoE3 and apoE2 alleles may be protective (13-17).

In initial attempts to understand how apoE4 contributes to the development of Alzheimer's disease, we examined the effect of apoE with lipoproteins on the outgrowth of neurites from neurons in culture. We found that both rabbit dorsal root ganglion neurons (18, 19) and murine neuroblastoma (Neuro-2a) cells (20) incubated with lipoproteins alone had enhanced neurite outgrowth and that neurite outgrowth was further enhanced by incubation with apoE3-enriched lipoproteins and inhibited by incubation with apoE4-enriched lipoproteins. Similar results were obtained with Neuro-2a cells stably transfected to secrete either apoE3 or apoE4 (21). When the cells were incubated with cerebrospinal fluid lipoproteins or with a plasma lipoprotein, beta -VLDL, the cells expressing apoE3 had long neurite extensions, whereas in the cells expressing apoE4 neurite extension was suppressed (21).

Various studies have demonstrated a role for the heparan sulfate proteoglycan (HSPG)·LRP pathway in the differential effects of the apoE isoforms on neurite outgrowth. In this pathway, cell surface HSPG first sequester apoE-enriched lipoproteins before their internalization via the HSPG·LRP complex (22-25). Treatment of the cells with chlorate, heparinase, lactoferrin, or the receptor-associated protein, all of which block the HSPG·LRP pathway, eliminates the differential effects of the apoE isoforms on neurite outgrowth (21). Furthermore, the LRP appears to be essential for the apoE3-mediated stimulation of nerve growth factor-induced neurite outgrowth in a central nervous system-derived neuronal cell line (26).

In nonneuronal cells, apoE3 and apoE4 show an equivalent ability to enhance binding and stimulate uptake of beta -VLDL by the LRP pathway (27), whereas apoE2 is ~50-90% as active (23, 27). In Neuro-2a cells, we found that incubation with apoE3 or apoE4-enriched beta -VLDL also led to a similar increase in cellular cholesterol content, suggesting that apoE3 and apoE4 stimulated the uptake of lipoprotein particles to a similar extent (20). By contrast, when the cellular content of apoE was examined by immunochemistry and confocal microscopy, a differential cellular accumulation of apoE3 and apoE4 was observed (20). After 48 h of incubation with apoE3- or apoE4-enriched beta -VLDL, Neuro-2a cells accumulated and retained more apoE3 than apoE4 (20). The accumulation of apoE3 by the cells was associated with enhanced neurite outgrowth and microtubule stability, whereas the lesser accumulation of apoE4 was associated with an inhibition of neurite outgrowth and disruption of the cellular microtubule network (12, 20, 28).

The current studies were performed to identify the mechanism by which the cells internalize and retain apoE and to determine if the differential accumulation of apoE in cells is specific for neurons. Our results demonstrate that there is a differential accumulation or retention of intact apoE3 and apoE4 by neurons, fibroblasts, and hepatocytes with a greater accumulation of apoE3 than apoE4. Using cells deficient in the LDL receptor, the LRP, or the expression of cell surface HSPG, we demonstrate that the differential accumulation is mediated, at least in part, by cell surface HSPG. These studies describe a new mechanism for the internalization and accumulation of apoE within cells and suggest that the HSPG pathway may process (bind, internalize, and/or sequester) the apoE isoforms differently.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Heparinase I and specific phospholipase C were purchased from Sigma. Suramin was obtained from Research Biochemicals International (Natick, MA). Purified human plasma apoE and sheep anti-human apoE antibody were provided by Dr. Karl Weisgraber (Gladstone Institute of Cardiovascular Disease, San Francisco, CA). Donkey anti-sheep IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Preparation of Lipoproteins-- Rabbit beta -VLDL (d < 1.006 g/ml) were isolated from the plasma of New Zealand White rabbits fed a high fat, high cholesterol diet for 4 days (29). The ratio of cholesterol to protein in this beta -VLDL ranged from ~15:1 to 20:1. Human apoE-enriched beta -VLDL were prepared by incubating apoE with beta -VLDL at 37 °C for 1 h. For some experiments, the apoE-enriched beta -VLDL were reisolated by fast performance liquid chromatography as follows. Either 125I-beta -VLDL and unlabeled apoE or 125I-apoE and unlabeled beta -VLDL were mixed in a 1:1.5 ratio of beta -VLDL protein to apoE and incubated at 37 °C for 1 h. The mixture (250 µl) was then fractionated by chromatography on a Superose 6 column (Pharmacia Fine Chemicals, Uppsala, Sweden; 10/50 HR). The flow rate was 0.5 ml/min, and 0.5-ml fractions were collected. The elution profile was monitored by quantitation of 125I and cholesterol.

Labeling of Lipoproteins and ApoE-- The beta -VLDL were iodinated by the method of Bilheimer et al. (30). Apolipoproteins E3 and E4 were iodinated by the Bolton-Hunter procedure (31). Free iodine was removed by P10 column chromatography. The beta -VLDL were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), as described previously (32).

Detection of Intact ApoE in Cell Extracts-- Murine neuroblastoma (Neuro-2a) cells were grown to ~100% confluence in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) containing 10% fetal bovine serum (FBS), washed with N2 medium, and incubated in N2 medium with beta -VLDL (40 µg of cholesterol/ml) alone or together with 30 µg/ml of iodinated apoE3 or iodinated apoE4. At the times indicated, the surface-bound apoE was removed by incubation with 10 mM suramin for 30 min at 4 °C. The cells were then washed three times with phosphate-buffered saline (PBS) at 4 °C and gently scraped with a rubber policeman. The cells were dissolved in SDS-sample buffer, and the cell proteins were separated by 3-20% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes; apoE was detected by autoradiography.

Cell Culture-- Neuro-2a cells were maintained in DMEM/F-12 (1:1) containing 10% FBS; this medium was replaced with serum-free medium ~16 h before use. Human skin fibroblasts were grown in DMEM containing 10% FBS. The LDL receptor-negative fibroblasts were grown in minimal essential medium supplemented with 10% FBS. Human hepatoma (HepG2) cells were maintained in minimal essential medium containing 10% FBS, 1% human nonessential amino acids, and 1% sodium pyruvate as described (25). Mutant Chinese hamster ovary (CHO) cells pgsA-745 (xylose transferase-deficient), which do not produce any glycosaminoglycans, and pgsD-677 (N-acetylglucosamine transferase-deficient and glucuronic acid transferase-deficient), which do not produce heparan sulfate (33), were kindly provided by Dr. J. D. Esko (University of Alabama, Birmingham). The CHO cells were maintained in F-12 medium containing 7.5% FBS. Mouse fibroblasts that are LRP-negative (LRP-/-), LRP-heterozygous (LRP+/-), and LDL receptor- and LRP-negative (LDLR-/-, LRP-/-), provided by Dr. J. Herz (University of Texas Southwestern Medical School, Dallas, TX) (34), were maintained in DMEM containing 10% FBS. The cholesterol content of the beta -VLDL or cultured cells was assayed as described previously (35).

Immunocytochemistry-- Neuro-2a cells or fibroblasts grown in tissue culture dishes were washed with serum-free medium and incubated at 37 °C with apoE3 (30 µg/ml) or apoE4 (30 µg/ml) plus beta -VLDL (40 µg of cholesterol/ml) for the time indicated. After incubation, the cells were placed immediately on ice and washed with phosphate buffer. Cells were then fixed with 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for immunofluorescence cytochemistry. Immunofluorescence from apoE was detected as described previously (20). The intensity of apoE immunofluorescence was quantitated by confocal microscopy.

Cell Association, Internalization, and Degradation of ApoE plus beta -VLDL-- Cultured cells were grown to ~100% confluence, washed twice with fresh serum-free medium, and incubated at 37 °C with apoE-enriched beta -VLDL. Before the addition to the cells, the beta -VLDL and apoE were incubated together (5 and 7.5 µg of protein, respectively, unless otherwise indicated) for 1 h at 37 °C. Some cells were incubated with 50 µM chloroquine, an inhibitor of lysosomal protease (35), at 37 °C for 2 h before the addition of the apoE-enriched beta -VLDL. At the times indicated, the cells were placed on ice, and the medium was assayed for protein degradation products (35). For the cell association studies, Neuro-2a cells were washed five times on ice with 0.1 M PBS containing 0.2% bovine serum albumin and once with 0.1 M PBS. Cell-associated ligand represents both bound and internalized material. The fibroblasts were washed three times with DMEM-Hepes on ice and incubated with 10 mM suramin at 4 °C for 30 min to remove surface-bound ligand (36). The radioactivity remaining within the cells represents that which was "internalized." After washing, the cells were dissolved in 0.1 N NaOH for measurement of radioactivity and protein concentration (37).

Internalization of 125I-apoE-enriched beta -VLDL by fibroblasts and by Neuro-2a cells was also studied at 18 °C. The cells were placed in an 18 °C incubator for 20 min before the addition of the lipoproteins and then incubated for an additional 3 h at 18 °C. After incubation, the cells were placed on ice, washed three times with DMEM-Hepes, and incubated with 10 mM suramin at 4 °C for 30 min to remove cell surface-bound 125I-apoE. Degradation products of 125I-beta -VLDL or 125I-apoE in the medium were assayed as described previously (35).

Uptake of DiI-labeled beta -VLDL by Cultured Cells-- Neuro-2a cells were incubated for 2 h at 37 °C with DiI-labeled beta -VLDL alone or together with either apoE3 or apoE4. The cells were then washed and solubilized with 0.1 N NaOH, and the cell-associated DiI, which is proportional to the total amount of lipoprotein metabolized (bound, internalized, and degraded), was assayed (38).

Heparinase and Specific Phospholipase C Treatment of Cells-- The cells were pretreated at 37 °C with heparinase I (10 units/ml) for 2 h or with specific phospholipase C (5 units/ml) for 30 min. The cells were then incubated in the presence of the enzymes with beta -VLDL together with either apoE3 or apoE4. A second addition of phospholipase C (5 units/ml) was made 1 h after the addition of the lipoproteins. The beta -VLDL (5 µg protein/ml) and apoE (7.5 µg/ml) were mixed and incubated together for 1 h at 37 °C before the addition to the cells.

Pulse-Chase of 125I-apoE plus beta -VLDL by Wild-type and HSPG-deficient CHO Cells-- Cultured cells were grown to ~100% confluence, placed on ice, and washed twice with cold DMEM-Hepes. The cells were then incubated with 125I-apoE plus beta -VLDL at 4 °C for 1 h to allow for cell surface binding (zero time-bound ligand). Cells were rinsed three times with cold F-12 medium to remove unbound ligands. Prewarmed F-12 medium was added, and the cells were incubated at 37 °C for the times indicated. At each time point, the cells were again placed on ice, and the culture medium was collected. To 0.5 ml of medium was added 0.4 ml of 0.2% bovine serum albumin (Sigma) and 0.4 ml of 50% trichloroacetic acid. The medium was then incubated at 4 °C for 30 min and centrifuged at 3,000 rpm for 10 min. The supernatant was collected for 125I-apoE degradation assay (35), and the pellet was counted as trichloroacetic acid-precipitable intact 125I-apoE. The cells were washed once with cold DMEM-Hepes, incubated with 10 mM suramin on ice in a cold room for 30 min, and then dissolved in 0.1 N NaOH. Cellular radioactivity (internalized apoE) was measured with a gamma -counter, and protein concentration was determined by Lowry's method (37).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies with cultured fibroblasts have shown that apoE3 and apoE4 mediate the binding and uptake of lipoproteins by LDL receptors to a similar extent (1) and that the binding and uptake of beta -VLDL enriched in apoE3 or apoE4 were greatly enhanced via the HSPG·LRP pathway (22, 25). In the present study, the metabolism of apoE-enriched beta -VLDL by cultured neurons (Neuro-2a cells) was examined in three ways: by measuring the cell association (binding and internalization) of apoE-enriched 125I-beta -VLDL, by examining the metabolism of apoE-enriched DiI-labeled beta -VLDL (DiI serving as a fluorescent marker for the lipid moieties of the lipoprotein particle), and by quantitating the ability of the apoE-enriched beta -VLDL to increase the content of cellular cholesterol.

Binding and Internalization of ApoE-enriched beta -VLDL Particles-- The cell association of 125I-beta -VLDL or 125I-beta -VLDL enriched with either human apoE3 or apoE4 by Neuro-2a cells was examined at 37 °C (Fig. 1A). In these studies, the maximal cell association of beta -VLDL alone was ~225 ng/mg of cell protein. The cell association of beta -VLDL was enhanced ~1.7-fold by apoE3 or apoE4. There was therefore no major isoform-specific difference in the ability of apoE3 or apoE4 to promote the binding and internalization of 125I-beta -VLDL, suggesting that a similar amount of beta -VLDL was internalized. In addition, DiI-labeled beta -VLDL were used to examine the uptake of the beta -VLDL particles by Neuro-2a cells (Fig. 1B). DiI internalized with lipoproteins is retained by cells and can be used to quantitate the total amount of lipoprotein metabolized (bound, internalized, and degraded) (38, 39). In these studies, at 2 h both apoE3 and apoE4 stimulated the uptake of DiI-labeled beta -VLDL (~1.8-2-fold) compared with the amount of DiI-labeled beta -VLDL internalized in the absence of apoE (apoE4 stimulated beta -VLDL uptake to a slightly greater extent than apoE3 (p < 0.02)).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Cell association by Neuro-2a cells of 125I-beta -VLDL or DiI-beta -VLDL alone or together with either apoE3 or apoE4. A, Neuro-2a cells were incubated at 37 °C for the times indicated with 125I-beta -VLDL alone (diamond ) or together with apoE3 (square ) or apoE4 (triangle ). The cell association data are from two separate studies performed in duplicate. B, Neuro-2a cells were incubated at 37 °C for 2 h with DiI-beta -VLDL alone or enriched in apoE3 or apoE4 (beta -VLDL:apoE, 1:1.5 protein). The DiI-labeled beta -VLDL cell association was significantly increased by the beta -VLDL plus apoE4 compared with the beta -VLDL plus apoE3 (p < 0.02). The cellular accumulation of DiI was assayed as described under "Materials and Methods." The results are the mean ± S.D. of two separate studies performed in triplicate.

To establish further that apoE3 and apoE4 stimulated similar beta -VLDL particle uptake, the cells were incubated in medium alone, medium containing beta -VLDL, or medium containing beta -VLDL and either apoE3 or apoE4, and the cholesterol content of the cells was determined (Fig. 2). The beta -VLDL alone increased the cellular cholesterol content ~4.7-fold, compared with the control cells maintained in the absence of lipoprotein. The beta -VLDL enriched with either apoE3 or apoE4 increased the cellular cholesterol content ~(1.5- and ~1.7-fold, respectively; the cholesterol content with apoE4 was significantly greater (p < 0.005)) compared with the cells incubated with beta -VLDL alone. Free apoE3 or apoE4 added without lipid had essentially no effect on the cellular cholesterol level (data not shown). Taken together, the results examining the effect of apoE3 and apoE4 on the uptake of 125I-beta -VLDL or DiI-labeled beta -VLDL and the ability of the cells to accumulate beta -VLDL-derived cholesterol demonstrate that apoE3 and apoE4 stimulate beta -VLDL internalization to a similar extent in Neuro-2a cells, with apoE4 being somewhat more active. These results are consistent with those previously reported in fibroblasts (25, 27). Differences in lipoprotein particle uptake could not therefore account for the difference in the accumulation of apoE3 versus apoE4 (apoE3 greater than apoE4) in Neuro-2a cells incubated with apoE-enriched beta -VLDL (20).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of beta -VLDL and beta -VLDL enriched with apoE on the cholesterol content of Neuro-2a cells. The cells were incubated for 48 h at 37 °C with medium alone (control), beta -VLDL (40 µg of cholesterol/ml) alone, or beta -VLDL (40 µg of cholesterol/ml) enriched with either apoE3 or apoE4 (30 µg/ml). Total cellular lipids were extracted and assayed for cholesterol using a commercially available kit (Boehringer Mannheim). The cholesterol content was significantly greater in cells incubated with beta -VLDL plus apoE4 than in those incubated with beta -VLDL plus apoE3 (p < 0.005). The results are the mean ± S.D. of three separate experiments.

Intracellular Accumulation of ApoE Isoforms-- The time course for differential accumulation of apoE3 and apoE4 was analyzed in the Neuro-2a cells (Fig. 3). The cells were incubated with apoE-enriched beta -VLDL for 2-48 h, permeabilized, and processed for immunocytochemistry with a polyclonal antibody that detects purified human apoE3 and E4 equally well on Western blots (data not shown). Immunoreactive apoE was detected and quantitated by confocal microscopy to measure the relative fluorescence intensity. At the earliest time point (2 h), the cells contained approximately 1.8-fold more apoE3 than apoE4. This difference in the level of immunoreactive apoE was maintained for up to 48 h (~1.6-fold more apoE3 than apoE4) (Fig. 3).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of apoE3 and apoE4 immunoreactivity in Neuro-2a cells. The cells were incubated with beta -VLDL (40 µg of cholesterol/ml) enriched with either apoE3 (square ) or apoE4 (triangle ) (30 µg/ml). After incubation, the cells were placed on ice, washed, and fixed in 3% paraformaldehyde in 0.1 M PBS, pH 7.4. Immunocytochemistry was performed as described under "Materials and Methods." Sheep anti-human apoE was used at a 1:1000 dilution, and fluorescein-labeled donkey anti-sheep IgG was used at a 1:100 dilution. The intensity of apoE immunofluorescence was measured by confocal microscopy in an average of 60 cells from at least six different fields for each time point. The results at each time point are the mean ± S.E. (in most cases the S.E. bars are obscured by the symbol for the mean).

The accumulated intracellular apoE was primarily intact protein (Fig. 4). Cells were incubated with apoE-enriched beta -VLDL for the times indicated; the cellular proteins were extracted, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose, and apoE was detected by autoradiography. Autoradiography demonstrated a greater cellular accumulation of apoE3 than apoE4 and no obvious accumulation of degradation products. Western blot analysis yielded similar results, revealing the differential intracellular accumulation of intact apoE (not shown).


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 4.   Differential accumulation of apoE3 and apoE4 in Neuro-2a cells. Cells grown in DMEM/F-12 medium to ~100% confluence were incubated with beta -VLDL (40 µg cholesterol/ml) enriched with iodinated apoE3 (A) or apoE4 (B) (30 µg/ml). At the times indicated, the medium was removed, and the cells were incubated with 10 mM suramin for 30 min at 4 °C. The cells were then washed three times at 4 °C with PBS, scraped from the plates, and dissolved in SDS-sample buffer. Cell proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, and apoE was detected by autoradiography.

To determine if the difference in accumulation or retention of apoE3 and apoE4 by cells was due to a difference in cell association (binding and internalization) or to a difference in degradation of internalized apoE3 or apoE4, studies were performed using beta -VLDL enriched with 125I-apoE3 or 125I-apoE4. In these studies, the differential cellular association or internalization of the iodinated apoE3 and apoE4 in both Neuro-2a cells (Fig. 5A) and human skin fibroblasts (Fig. 5C) was also apparent beginning at the earliest time point (2 h) and continuing to the end of the experiment (24 h). The difference in apoE3 and apoE4 content of the cells was maximal after 4-8 h of incubation. In the Neuro-2a cells, the amount of apoE3 associated with the cells was twice the amount of apoE4 associated with the cells (Fig. 5A), whereas in fibroblasts apoE3 was 3-fold more abundant than apoE4 in the cells (Fig. 5C). Likewise, 125I-apoE2 also accumulated intracellularly to a greater extent than apoE4 (~1.5-fold greater than apoE4 at 2 h) (data not shown). In contrast to the differential cell association or internalization of 125I-apoE3 and 125I-apoE4 in the Neuro-2a cells and fibroblasts, respectively, there was no significant difference in the degradation of the iodinated apoE3 or apoE4 by the cells (Fig. 5, B and D).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Cell association and degradation of 125I-apoE-enriched beta -VLDL by Neuro-2a cells and human skin fibroblasts. A and B, Neuro-2a cells were incubated with either 125I-apoE3-enriched beta -VLDL (square ) or 125I-apoE4-enriched beta -VLDL (triangle ) at 37 °C for 2, 4, 8, or 24 h. The concentrations of 125I-apoE and beta -VLDL in the medium were 7.5 and 5 µg of protein/ml, respectively. After incubation, the medium was collected to assay for apoE degradation. The cells were washed five times as described under "Materials and Methods" with cold 0.1 M PBS and then dissolved in 0.1 N NaOH. The 125I-apoE cell association (A) and the 125I-apoE degradation (B) were measured by gamma -counting. The results are the mean ± S.D. of two separate experiments performed in duplicate. C and D, fibroblasts were incubated with either 125I-apoE3-enriched beta -VLDL (square ) or 125I-apoE4-enriched beta -VLDL (triangle ) at 37 °C for 2, 4, 6, or 24 h. After incubation, the medium was collected for the 125I-apoE degradation assay, and the cells were washed, incubated with 10 mM suramin at 4 °C for 30 min, washed again, and dissolved in 0.1 N NaOH as described under "Materials and Methods." The internalization (C) and degradation (D) products were measured by gamma -counting. The results are the mean ± S.D. of two independent experiments performed in duplicate.

The differential cellular accumulation of apoE3 and apoE4 from apoE-enriched beta -VLDL was also observed in hepatocytes. As shown in Table I, HepG2 cells incubated with 125I-apoE3 plus beta -VLDL displayed about 2.5-fold greater cell association of apoE compared with cells incubated with 125I-apoE4 plus beta -VLDL. Data from the immunological and autoradiographic studies, as well as the binding and degradation experiments, showed differential accumulation of apoE3 and apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes incubated with apoE3- or apoE4-enriched beta -VLDL.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Cell association of 125I-apoE3- or 125I-apoE4-enriched beta -VLDL by HepG2 cells
Means ± S.D. obtained from two independent experiments performed in duplicate are shown.

Furthermore, the differential accumulation of apoE required the presence of lipid or a lipoprotein in the medium. Neuro-2a cells were incubated with lipid-free 125I-apoE3 or 125I-apoE4 (7.5 µg/ml) without added beta -VLDL at 37 °C for 2 h. Cell association of the lipid-free apoE3 and apoE4 was very low and did not demonstrate a differential accumulation (apoE3 and apoE4, 83.7 and 72 ng/mg of cell protein, respectively).

In the experiments described thus far, the apoE3 and apoE4 were incubated with the beta -VLDL at 37 °C for 1 h, and then the mixture was added to the cells. Separation of the mixture by fast performance liquid chromatography demonstrated that ~50% of the apoE was associated with beta -VLDL particles (data not shown). One possible reason for the differential accumulation might be that more apoE3 than apoE4 associates with the beta -VLDL and that more apoE3 is therefore delivered to the cells. This possibility was ruled out by examining the amount of 125I-apoE3 or 125I-apoE4 associated with beta -VLDL after isolation of apoE-enriched beta -VLDL by fast performance liquid chromatography. In fact, slightly more apoE4 than apoE3 was associated with the lipoprotein particles (7.0 versus 6.1 µg/mg of beta -VLDL cholesterol). Furthermore, using the fast performance liquid chromatography-purified 125I-apoE-enriched beta -VLDL, we demonstrated that the differential apoE accumulation occurred with apoE on the beta -VLDL particles and not with lipid-free or lipid-poor apoE. The cell association was greater in Neuro-2a cells incubated with purified 125I-apoE3-enriched beta -VLDL than in those incubated with purified 125I-apoE4-enriched beta -VLDL (58 versus 39 ng/mg of cell protein at 2 h; 101 versus 65 ng/mg of cell protein at 4 h).

As shown previously (23-25), the optimal enhancement of beta -VLDL binding and uptake occurred at a protein ratio of 1.5:1 for apoE to beta -VLDL (the ratio used in the present study). In cell association studies performed at 37 °C in Neuro-2a cells, we found that lower ratios of apoE:beta -VLDL markedly decreased the 125I-apoE3 and 125I-apoE4 internalization and abolished the apoE differential accumulation. When the ratio was changed to 0.1:1, internalization of apoE was reduced by ~3-4-fold, and at this level of internalization, the differential accumulation between apoE3 and apoE4 essentially disappeared. Thus, it appears that apoE enrichment of the beta -VLDL is important in directing the uptake to a specific pathway.

Mechanisms Responsible for Differential Accumulation of ApoE Isoforms-- To explore in more detail how differential processing of apoE3 versus apoE4 could explain the differential accumulation, we examined the internalization of iodinated apoE-enriched beta -VLDL by fibroblasts and Neuro-2a cells at 18 °C, a temperature at which lipoprotein internalization occurs but degradation does not (Fig. 6) (40). Analysis of the culture medium for degradation products of the 125I-apoE confirmed that degradation did not occur under the conditions used (data not shown). In these studies, apoE3 accumulated to a greater extent than apoE4 in both fibroblasts (Fig. 6A) and neurons (Fig. 6B), demonstrating that the differential accumulation was due to differential handling of at least a portion of the internalized apoE and not to differences in lysosomal degradation. This conclusion was supported by studies in fibroblasts, in which degradation was blocked by chloroquine (data not shown). Even in the absence of lysosomal degradation, the differential accumulation of apoE3 and apoE4 was apparent when the cells were incubated with apoE3- or apoE4-enriched beta -VLDL.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Internalization of 125I-apoE-enriched beta -VLDL by fibroblasts and Neuro-2a cells at 18 °C. A, human skin fibroblasts were incubated with 125I-apoE3- or 125I-apoE4-enriched beta -VLDL for 3 h at 18 °C, and internalized apoE was determined as described under "Materials and Methods." As indicated, the 125I-apoE was added along with the beta -VLDL at increasing concentrations (ratio of beta -VLDL to apoE was 1:1.5, protein:protein). The apoE and beta -VLDL were preincubated at 37 °C and then cooled to 18 °C before the addition to the cells. The results are the mean ± S.D. of two independent experiments performed in duplicate. B, Neuro-2a cells were incubated with 125I-apoE-enriched beta -VLDL (7.5 µg and 5 µg of protein, respectively) for 3 h at 18 °C, and internalization was assayed as described. The results are the mean ± S.D. of two independent experiments performed in duplicate.

To identify the mechanism of the differential cellular accumulation of apoE3 and apoE4, we made use of fibroblasts that lacked expression of the LDL receptor, the LRP, or specific cell surface proteoglycans. The differential cellular accumulation of apoE3 and apoE4 with added beta -VLDL occurred in both LDL receptor-expressing and LDL receptor-negative fibroblasts, demonstrating that the LDL receptor was not involved in the differential accumulation (Fig. 7A). On the other hand, the differential accumulation was blocked totally by prior treatment of the normal or FH fibroblasts with heparinase, and the total cell association was significantly decreased for both isoforms, suggesting that the differential effect might be mediated either by the HSPG·LRP complex or by HSPG alone (Fig. 7A). As shown in Fig. 7B, embryonic mouse fibroblasts either heterozygous for LRP expression (LRP+/-) or lacking LRP expression (LRP-/-) displayed differential accumulation of apoE3 and apoE4. Therefore, LRP expression is not required for the differential accumulation of apoE3 versus apoE4. Furthermore, the differential accumulation of 125I-apoE3 and 125I-apoE4 plus beta -VLDL occurred in mouse fibroblasts lacking the LDL receptor and the LRP (LDLR-/-, LRP-/-) (Fig. 7C). However, heparinase treatment of these cells blocked the effect, again suggesting a role for cell surface HSPG (Fig. 7, B and C). As indicated, heparinase markedly decreased total internalization of both apoE3- and apoE4-enriched beta -VLDL, further suggesting the importance of HSPG alone in mediating the enhanced metabolism of apoE-enriched lipoproteins.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Internalization of 125I-apoE-enriched beta -VLDL by LDL receptor-negative and LRP-negative fibroblasts. Human fibroblasts (A) expressing or lacking expression of the LDL receptor (FH fibroblasts), murine embryonic fibroblasts (B) heterozygous for or lacking expression of the LRP, or murine embryonic fibroblasts (C) that were wild-type or lacking both the LDL receptor and the LRP, were incubated at 37 °C for 2 h with or without heparinase (A and B, 10 units of heparinase/ml; C, 50 units heparinase/ml). The cells were then incubated with 125I-apoE3-enriched beta -VLDL or 125I-apoE4-enriched beta -VLDL (7.5 µg/ml and 5 µg of protein/ml, respectively) for an additional 2 h. After incubation, the cells were placed on ice, washed with DMEM-Hepes, and incubated with 10 mM suramin. A and B, results are the mean ± S.D. of two independent experiments performed in duplicate; C, results from one study performed in quadruplicate.

The role of HSPG in the apoE3 and apoE4 differential accumulation was examined further in control CHO cells, in mutant CHO cells specifically lacking HSPG expression, and in CHO cells lacking expression of all proteoglycans (Fig. 8). The differential cellular accumulation or retention of 125I-apoE3 versus 125I-apoE4 was apparent in the wild-type CHO cells; however, the differential accumulation or retention was completely abolished in both the HSPG-deficient and the proteoglycan-deficient CHO cells, conclusively demonstrating the importance of cell surface HSPG in this process. Likewise, the levels of apoE3 and apoE4 internalized by the CHO mutant cells were very significantly reduced.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 8.   Internalization of 125I-apoE-enriched beta -VLDL by wild-type and mutant CHO cells. Wild-type, HSPG-deficient, and proteoglycan-deficient CHO cells were incubated with beta -VLDL enriched with either 125I-apoE3 or 125I-apoE4 (7.5 and 5 µg of protein/ml, respectively) at 37 °C for 2 h. Next, the cells were incubated with 10 mM suramin at 4 °C for 30 min to remove cell surface-bound apoE. The results are the mean ± S.D. of two independent experiments performed in duplicate.

Proteoglycans associate with cell membranes either by glycerophosphatidylinositol (GPI) anchors or by transmembrane spanning of their core proteins (41, 42). These classes of proteoglycans undergo different rates of cellular processing (42). The GPI-anchored proteoglycans exhibit fast endosome to lysosome transport and undergo lysosomal degradation with an intracellular half-life of ~30 min, whereas the core protein-anchored proteoglycans exhibit slow endosome to lysosome transport (half-life of ~4 h) and undergo delayed processing. The retention of apoE by the cells would be consistent with use of the slow pathway for endosome to lysosome transport and would suggest that the differential accumulation of apoE3 and apoE4 in the cells is not due to internalization of apoE with GPI-anchored proteoglycans. To test this hypothesis, we examined the effect of specific phospholipase C, which removes GPI-anchored HSPG, on the cell association of iodinated apoE-enriched beta -VLDL with fibroblasts. Human fibroblasts were incubated with 125I-apoE3- or 125I-apoE4-enriched beta -VLDL at 37 °C for 2 h to determine the cell association of apoE with or without treatment of the cells with phospholipase C. Under the conditions used, the phospholipase removed ~15% of 35S from cells labeled for 24 h with 35SO4. Specific phospholipase C treatment of the cells did not affect the differential accumulation of apoE3 and apoE4 in the cells or the total binding and internalization of either the apoE3- or apoE4-enriched beta -VLDL (125I-apoE3, 679 ± 16 (no treatment) and 713 ± 38 (phospholipase C treatment) ng per mg of cell protein; 125I-apoE4, 385 ± 16 (no treatment) and 473 ± 43 (phospholipase C treatment) ng per mg of cell protein). These data indicate that GPI-anchored HSPG were not involved in the differential accumulation of apoE.

Consideration was given to the possibility that the apoE4 isoform differential resulted from shunting of apoE3 specifically into an intracellular compartment and/or retroendocytosis or retarded internalization of apoE4. To evaluate these possibilities, we conducted a modified pulse-chase study in which CHO cells were incubated with 125I-apoE-enriched beta -VLDL for 1 h at 4 °C, washed to remove unbound lipoproteins, and then warmed to 37 °C for various times to follow internalization, degradation, and retention (see "Materials and Methods"). At the specific times, the medium was removed for analysis of both degradation products (degraded apoE) and trichloroacetic acid-precipitable proteins (released intact apoE), and the cells were washed with suramin (suramin-releasable apoE) and then counted (internalized apoE). As summarized in Table II, the amounts of apoE3 and apoE4 bound at 4 °C (zero time) were similar; however, the amount of apoE3 in the cells (internalized = accumulated or retained) after 30, 60, and 120 min at 37 °C was approximately 2-fold greater than the amount of apoE4. At each time point, we found a small amount of the 125I-apoE that was suramin-releasable (i.e. apoE present on the cell surface). Between 30 and 120 min, the amount of 125I-apoE3 and apoE4 degraded increased and was approximately equal for both isoforms. Thus, similar fractions of internalized apoE3 and apoE4 were degraded. Of interest was the greater amount of apoE4 that appeared in the medium during the incubation period, especially at 30 and 60 min. This trichloroacetic acid-precipitable, intact apoE could represent apoE that is retroendocytosed or is on or near the cell surface and rapidly released upon warming. Thus, with time apoE4 is released to a greater extent or internalized to a lesser extent than apoE3, or, alternatively, more apoE3 is sequestered into a compartment and unavailable to be released. Therefore, more apoE3 accumulates and is retained by the cells. Typically, 80-90% of the total apoE bound to the cells at 4 °C at zero time was recovered in the various fractions of the medium and cells after the warm-up periods (Table II).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Metabolism of 125I-apoE3- and 125I-apoE4-enriched beta -VLDL by wild-type CHO cells
Similar amounts of 125I-apoE3 and 125I-apoE4 (399 ng/mg and 378 ng/mg of cell protein, respectively) were bound to the cells at 4 °C (i.e. zero time). Recovery of 125I-apoE (total) in the fractions analyzed after warming to 37 °C is also reported in the table. Data represent results from one experiment performed in quadruplicate. The experiment was repeated three times with similar results.

Data from this pulse-chase study are graphically illustrated in Fig. 9A. Three separate experiments were performed with this design and yielded comparable results. In wild-type CHO cells, apoE3 accumulated and was retained to a greater extent than apoE4, similar amounts of apoE3 and apoE4 were degraded at all time points, and more apoE4 reappeared in the medium at 30 and 60 min. By contrast, HSPG-deficient CHO cells bound much less 125I-apoE3 plus beta -VLDL and 125I-apoE4 plus beta -VLDL (77 and 75 ng/mg of cell protein) than wild-type CHO cells (399 and 378 ng/mg of cell protein); the HSPG-deficient cells internalized and degraded similar amounts of apoE3 and apoE4 at all time points. We also found similar amounts of suramin-releasable and trichloroacetic acid-precipitable 125I-apoE3 and 125I-apoE4 (Fig. 9B). Thus, HSPG-deficient cells not only have markedly reduced uptake of apoE but also do not show any isoform-specific differential accumulation, degradation, or retention.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 9.   Internalization and degradation of 125I-apoE-enriched beta -VLDL by wild-type and HSPG-deficient CHO cells. The wild-type (A) and HSPG-deficient (B) CHO cells were incubated with 2 µg/ml of beta -VLDL protein plus 3 µg/ml of 125I-apoE3 or 125I-apoE4 at 4 °C for 1 h. The cells were then washed and warmed to 37 °C for the indicated times. At each time point, the incubation medium was collected, and the degraded and nondegraded (trichloroacetic acid-precipitable) 125I-apoE was assayed. The cells were placed on ice, rinsed with cold DMEM-Hepes, and incubated with 10 mM suramin for 30 min at 4 °C. The medium was collected and counted for suramin-releasable 125I-apoE from the cell surface. The cells were then dissolved in 0.1 N NaOH, and the 125I-apoE radioactivity associated with the cells was measured (internalization). The results are from one experiment performed in quadruplicate.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that in Neuro-2a cells incubated for 48 h with apoE3-enriched beta -VLDL, neurite outgrowth was stimulated and cellular microtubules were intact, whereas in cells treated with apoE4-enriched beta -VLDL neurite outgrowth was suppressed and microtubules were disrupted (20). This difference in biological effect was associated with the cellular accumulation or retention of more apoE3 than apoE4 (20). Cellular accumulation of apoE internalized with lipoproteins had not been reported previously, and in fact apoE3- and apoE4-enriched lipoproteins have been shown to stimulate the same level of cholesterol esterification and cell association of beta -VLDL with fibroblasts (1, 27, 43), suggesting that apoE3 and apoE4 mediate similar levels of cellular uptake.

The current studies were undertaken to determine the mechanism for the differential cellular accumulation of apoE3 and apoE4 in neurons and to determine if differential apoE accumulation occurs in other cell types. Two types of studies were performed. First, we examined the metabolism of apoE-enriched beta -VLDL to determine if apoE3 and apoE4 stimulate the same level of uptake of beta -VLDL particles. Second, we examined the cellular uptake (retention or accumulation) of the apoE from apoE-enriched beta -VLDL more directly by immunocytochemistry and by following the metabolism of iodinated apoE.

Incubation of Neuro-2a cells with either apoE3- or apoE4-enriched beta -VLDL resulted in a similar cell association of beta -VLDL and a similar increase of cellular cholesterol. These studies demonstrated that in neurons, as in fibroblasts (27), apoE3 and apoE4 stimulate the uptake of similar numbers of lipoprotein particles. On the other hand, when the cellular accumulation specifically of apoE3 and apoE4 was examined in Neuro-2a cells by either immunofluorescence or analysis of extracted cellular proteins, a differential accumulation of apoE3 and apoE4 was observed. These observations were confirmed in Neuro-2a cells and extended to fibroblasts and hepatocytes by examining the cellular association or internalization of 125I-apoE3- or 125I-apoE4-enriched beta -VLDL. In all three cell types, intracellular apoE3 accumulated to a greater extent than apoE4 (~2-fold). Likewise, apoE2 also accumulated to a greater extent than apoE4 in Neuro-2a cells (~1.5-fold). The differential accumulation of apoE3 and apoE4 occurred in both LDL receptor-negative human fibroblasts and in LRP-negative murine embryonic fibroblasts, demonstrating that these receptors are not significantly involved. However, the differential accumulation or retention was abolished by treating the cells with heparinase. The role of the HSPG in this process was confirmed by the use of mutant CHO cells deficient in HSPG synthesis. In these cells, the accumulation of both apoE3 and apoE4 was reduced, and the differential accumulation of apoE3 and apoE4 was abolished. Treatment of the cells with specific phospholipase C, which releases phospholipid-anchored HSPG, had no effect on the differential accumulation of apoE3 and apoE4 from apoE-enriched beta -VLDL. Enhanced degradation of apoE4 was not the reason for the difference in cellular accumulation of apoE3 and apoE4 by the cells, since the differential accumulation occurred at 18 °C, a temperature at which endosome-lysosome fusion does not occur, as well as in the presence of chloroquine, which inhibits lysosomal degradation.

The pulse-chase studies (Table II, Fig. 9) suggest a possible mechanism for the differential accumulation or retention of apoE. After similar amounts of 125I-apoE3- and 125I-apoE4-enriched beta -VLDL were bound to the CHO cells at 4 °C, warming the cells to 37 °C resulted in internalization of more apoE3 than of apoE4. On the other hand, more apoE4 was found in the medium at the early time points (30 and 60 min), suggesting that the differential apoE accumulation and retention resulted from a preferential release of apoE4 from the cells. In these same studies, the HSPG-deficient CHO cells bound, internalized, and degraded much less apoE, and there was no differential between apoE3 and apoE4.

Cell surface HSPG bind a number of biologically important molecules (for a review, see Ref. 41). In addition, HSPG can function as a receptor directly involved in binding and internalization of specific ligands. This has been demonstrated for certain viruses (44), thrombospondin (41, 42, 45), lipoprotein and hepatic lipases (46-49), thrombin (50), and fibroblast growth factor (FGF) (51). Furthermore, HSPG facilitate the interaction of ligands with other receptors or serve as a bridge functioning like a co-receptor. For example, HSPG can facilitate the interaction of FGF with the FGF receptor (51). Likewise, we have demonstrated a co-receptor function for HSPG and the LRP in the binding and internalization of apoE- and hepatic lipase-containing lipoproteins (22, 25, 52, 53). Recently the syndecan-1 type of HSPG has been shown to mediate the uptake of lipoproteins directly, independent of other lipoprotein receptors (54, 55). As demonstrated in the present study, apoE-containing lipoproteins can be bound and apoE can be internalized in an HSPG-dependent process without participation of the LDL receptor or the LRP. Whether or not the internalization is mediated by HSPG alone or requires an unknown co-receptor remains to be determined; however, heparinase treatment alone abolishes the differential accumulation of apoE. Previously, we established that heparinase treatment of cultured cells does not interfere with LDL receptor-mediated LDL binding or LRP-mediated binding of alpha 2-macroglobulin (56).

The ability of HSPG alone or in complex with a co-receptor to function in the internalization of ligands suggests ways in which the intracellular processing of these molecules may differ. In fact, the intracellular fate of FGF is determined by which pathway is being used (51). When FGF is internalized by HSPG alone, it is degraded; however, when FGF is internalized via the HSPG·FGF receptor pathway, a portion of the FGF enters the cytoplasm and ultimately the nucleus (51). Clearly, apoE-enriched lipoproteins can be internalized by three cellular mechanisms: the LDL receptor, the HSPG·LRP pathway, and an HSPG-dependent/LRP-independent pathway (for reviews, see Refs. 1, 12, 22, and 53). Thus, the intracellular fate of apoE may depend on the proportion of the protein entering the cell via each of these pathways. Specifically, the HSPG-dependent/LRP-independent pathway accounts for the differential handling of apoE3 versus apoE4 that is responsible for the greater accumulation of apoE3 than apoE4. One can speculate that apoE3-enriched lipoprotein uptake via the HSPG pathway directs apoE3 to a separate (intracellularly sequestered) pool, allowing it to accumulate in the cells. On the other hand, apoE4-enriched lipoproteins taken up via the HSPG pathway may fail to escape the typical endosomal/lysosomal cascade, and thus apoE4 does not accumulate. Alternatively, apoE4 complexed to HSPG may be recycled and released at the cell surface (retroendocytosis). These possible mechanisms can be tested experimentally.

There is support for the hypothesis that apoE3 accumulation results from its intracellular sequestration. Lovestone et al. (57) recently demonstrated that COS cells transfected to express high levels of tau, a microtubule-associated cytosolic protein, retained apoE3 diffusely throughout the cells when incubated with apoE3-containing cerebrospinal fluid lipoproteins. However, when the cells were incubated with cerebrospinal fluid lipoproteins containing apoE4, the apoE4 remained primarily in small, discrete vesicular structures. Furthermore, there was apparent immunochemical co-localization of apoE3 with tau, presumably in the cytoplasm. In vitro studies have shown that apoE3, but not apoE4, interacts biochemically with the microtubule-associated proteins tau and MAP2C (58, 59). Furthermore, cytosolic apoE has been reported in neurons in the human brain (60, 61). However, there is no known mechanism whereby apoE could escape the endosomal pathway. Nevertheless, it is possible that apoE3 does enter the cytoplasm. Although the mechanisms remain obscure, it is possible that apoE3 internalized via the HSPG alone pathway is protected from degradation and escapes into the cytosol, where it may complex with various cytosolic proteins. Such a mechanism may account for the isoform-specific effect of apoE3 in promoting neurite outgrowth and microtubule stability and the lack of such an effect of apoE4. These mechanisms have been proposed by Strittmatter et al. and Roses (58, 59) and by our group (12, 20, 28, 62, 63). Alternatively, apoE4 may enter a pathway that preferentially favors retroendocytosis and release from the cells, thereby resulting in decreased accumulation and retention and decreased ability to modulate neurite outgrowth or remodeling.

In summary, we have shown that incubation of neurons, fibroblasts, and hepatocytes with beta -VLDL together with either apoE3 or apoE4 results in the retention of intact apoE by the cells and in a greater cellular accumulation of apoE3 than apoE4. Cell surface HSPG appear to play a primary role in both the retention of the apoE and the differential accumulation of apoE3 versus apoE4. The LRP and the LDL receptor are not primarily involved. The intracellular fate of the apoE remains to be determined; however, the retention of apoE by the cells is most likely due to association with the slow endosome to lysosome transport of HSPG. It remains to be determined whether or not apoE in this pathway can escape lysosomal degradation and enter the cytoplasmic compartment, where it might interact with microtubule-associated proteins or other cellular components that could account for the differential effects of apoE3 and apoE4 on neurite outgrowth and the cytoskeleton.

    ACKNOWLEDGEMENTS

We thank R. Dennis Miranda for excellent technical assistance; Kate Cook and Susannah Patarroyo for manuscript preparation; Gary Howard, Stephen Ordway, and Sylvia Richmond for editorial support; and Amy Corder, John C. W. Carroll, and Stephen Gonzales for graphics and photography. We also thank Dr. Joachim Herz (University of Texas Southwestern Medical School, Dallas, TX) for generosity in providing LRP and LRP-LDL receptor-negative fibroblasts.

    FOOTNOTES

* This work was supported in part by NIA, National Institutes of Health, Grant AG13619 and by National Institutes of Health Program Project Grant HL41633. Additional funding was obtained from a Cambridge Neuroscience/Gladstone collaborative research agreement.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632.

1 The abbreviations used are: apoE, apolipoprotein E; CHO, Chinese hamster ovary; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; GPI, glycerophosphatidylinositol; HSPG, heparan sulfate proteoglycan(s); LDL, low density lipoprotein(s); LRP; LDL receptor-related protein; PBS, phosphate-buffered saline; VLDL, very low density lipoprotein(s).

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Mahley, R. W. (1988) Science 240, 622-630[Medline] [Order article via Infotrieve]
  2. Pitas, R. E., Boyles, J. K., Lee, S. H., Hui, D., and Weisgraber, K. H. (1987) J. Biol. Chem. 262, 14352-14360[Abstract/Free Full Text]
  3. Borghini, I., Barja, F., Pometta, D., and James, R. W. (1995) Biochim. Biophys. Acta 1255, 192-200[Medline] [Order article via Infotrieve]
  4. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601-637[CrossRef][Medline] [Order article via Infotrieve]
  5. Kim, D.-H., Iijima, H., Goto, K., Sakai, J., Ishii, H., Kim, H.-J., Suzuki, H., Kondo, H., Saeki, S., and Yamamoto, T. (1996) J. Biol. Chem. 271, 8373-8380[Abstract/Free Full Text]
  6. Pitas, R. E., Innerarity, T. L., Arnold, K. S., and Mahley, R. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2311-2315[Abstract]
  7. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamoto, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9252-9256[Abstract]
  8. Kounnas, M. Z., Stefansson, S., Loukinova, E., Argraves, K. M., Strickland, D. K., and Argraves, W. S. (1994) Ann. N. Y. Acad. Sci 737, 114-123[Medline] [Order article via Infotrieve]
  9. Pitas, R. E. (1997) Nutr. Metab. Cardiovasc. Dis. 7, 202-209
  10. Zannis, V. I., and Breslow, J. L. (1981) Biochemistry 20, 1033-1041[Medline] [Order article via Infotrieve]
  11. Weisgraber, K. H. (1994) Adv. Protein Chem. 45, 249-302[Medline] [Order article via Infotrieve]
  12. Weisgraber, K. H., and Mahley, R. W. (1996) FASEB J. 10, 1485-1494[Abstract/Free Full Text]
  13. Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., and Pericak-Vance, M. A. (1993) Science 261, 921-923[Medline] [Order article via Infotrieve]
  14. Mayeux, R., Stern, Y., Ottman, R., Tatemichi, T. K., Tang, M.-X., Maestre, G., Ngai, C., Tycko, B., and Ginsberg, H. (1993) Ann. Neurol. 34, 752-754[Medline] [Order article via Infotrieve]
  15. Poirier, J., Davignon, J., Bouthillier, D., Kogan, S., Bertrand, P., and Gauthier, S. (1993) Lancet 342, 697-699[Medline] [Order article via Infotrieve]
  16. Saunders, A. M., Strittmatter, W. J., Schmechel, D., St. George-Hyslop, P. H., Pericak-Vance, M. A., Joo, S. H., Rosi, B. L., Gusella, J. F., Crapper-MacLachlan, D. R., Alberts, M. J., Hulette, C., Crain, B., Goldgaber, D., and Roses, A. D. (1993) Neurology 43, 1467-1472[Abstract]
  17. Corder, E. H., Saunders, A. M., Risch, N. J., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Jr., Rimmler, J. B., Locke, P. A., Conneally, P. M., Schmader, K. E., Small, G. W., Roses, A. D., Haines, J. L., and Pericak-Vance, M. A. (1994) Nat. Genet. 7, 180-184[Medline] [Order article via Infotrieve]
  18. Handelmann, G. E., Boyles, J. K., Weisgraber, K. H., Mahley, R. W., and Pitas, R. E. (1992) J. Lipid Res. 33, 1677-1688[Abstract]
  19. Nathan, B. P., Bellosta, S., Sanan, D. A., Weisgraber, K. H., Mahley, R. W., and Pitas, R. E. (1994) Science 264, 850-852[Medline] [Order article via Infotrieve]
  20. Nathan, B. P., Chang, K.-C., Bellosta, S., Brisch, E., Ge, N., Mahley, R. W., and Pitas, R. E. (1995) J. Biol. Chem. 270, 19791-19799[Abstract/Free Full Text]
  21. Bellosta, S., Nathan, B. P., Orth, M., Dong, L.-M., Mahley, R. W., and Pitas, R. E. (1995) J. Biol. Chem. 270, 27063-27071[Abstract/Free Full Text]
  22. Mahley, R. W., Ji, Z.-S., Brecht, W. J., Miranda, R. D., and He, D. (1994) Ann. N. Y. Acad. Sci. 737, 39-52[Medline] [Order article via Infotrieve]
  23. Ji, Z.-S., Fazio, S., and Mahley, R. W. (1994) J. Biol. Chem. 269, 13421-13428[Abstract/Free Full Text]
  24. Ji, Z.-S., and Mahley, R. W. (1994) Arterioscler. Thromb. 14, 2025-2032[Abstract]
  25. Ji, Z.-S., Fazio, S., Lee, Y.-L., and Mahley, R. W. (1994) J. Biol. Chem. 269, 2764-2772[Abstract/Free Full Text]
  26. Holtzman, D. M., Pitas, R. E., Kilbridge, J., Nathan, B., Mahley, R. W., Bu, G., and Schwartz, A. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9480-9484[Abstract]
  27. Kowal, R. C., Herz, J., Weisgraber, K. H., Mahley, R. W., Brown, M. S., and Goldstein, J. L. (1990) J. Biol. Chem. 265, 10771-10779[Abstract/Free Full Text]
  28. Pitas, R. E. (1996) Semin. Cell Dev. Biol. 7, 725-731[CrossRef]
  29. Kowal, R. C., Herz, J., Goldstein, J. L., Esser, V., and Brown, M. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5810-5814[Abstract]
  30. Bilheimer, D. W., Eisenberg, S., and Levy, R. I. (1972) Biochim. Biophys. Acta 260, 212-221[Medline] [Order article via Infotrieve]
  31. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529-539[Medline] [Order article via Infotrieve]
  32. Pitas, R. E., Innerarity, T. L., Weinstein, J. N., and Mahley, R. W. (1981) Arteriosclerosis 1, 177-185[Abstract]
  33. Esko, J. D. (1991) Curr. Opin. Cell Biol. 3, 805-816[Medline] [Order article via Infotrieve]
  34. Willnow, T. E., and Herz, J. (1994) J. Cell Sci. 107, 719-726[Abstract/Free Full Text]
  35. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260[Medline] [Order article via Infotrieve]
  36. Tabas, I., Lim, S., Xu, X.-X., and Maxfield, F. R. (1990) J. Cell Biol. 111, 929-940[Abstract]
  37. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  38. Pitas, R. E., Innerarity, T. L., and Mahley, R. W. (1983) Arteriosclerosis 3, 2-12[Abstract]
  39. Pitas, R. E., and Mahley, R. W. (1992) in Lipoprotein Analysis: A Practical Approach (Converse, C. A., and Skinner, E. R., eds), pp. 215-242, Oxford University Press, Oxford
  40. Dunn, W. A., Hubbard, A. L., and Aronson, N. N., Jr. (1980) J. Biol. Chem. 255, 5971-5978[Abstract/Free Full Text]
  41. Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef]
  42. Yanagishita, M. (1992) J. Biol. Chem. 267, 9505-9511[Abstract/Free Full Text]
  43. Weisgraber, K. H., Innerarity, T. L., and Mahley, R. W. (1982) J. Biol. Chem. 257, 2518-2521[Free Full Text]
  44. Shieh, M.-T., WuDunn, D., Montgomery, R. I., Esko, J. D., and Spear, P. G. (1992) J. Cell Biol. 116, 1273-1281[Abstract]
  45. Corless, C. L., Mendoza, A., Collins, T., and Lawler, J. (1992) Dev. Dyn. 193, 346-358[Medline] [Order article via Infotrieve]
  46. Williams, K. J., Fless, G. M., Petrie, K. A., Snyder, M. L., Brocia, R. W., and Swenson, T. L. (1992) J. Biol. Chem. 267, 13284-13292[Abstract/Free Full Text]
  47. Rumsey, S. C., Obunike, J. C., Arad, Y., Deckelbaum, R. J., and Goldberg, I. J. (1992) J. Clin. Invest. 90, 1504-1512[Medline] [Order article via Infotrieve]
  48. Kounnas, M. Z., Chappell, D. A., Wong, H., Argraves, W. S., and Strickland, D. K. (1995) J. Biol. Chem. 270, 9307-9312[Abstract/Free Full Text]
  49. Eisenberg, S., Sehayek, E., Olivecrona, T., and Vlodavsky, I. (1992) J. Clin. Invest. 90, 2013-2021[Medline] [Order article via Infotrieve]
  50. Ganguly, P. (1977) Br. J. Haematol. 37, 47-51[Medline] [Order article via Infotrieve]
  51. Reiland, J., and Rapraeger, A. C. (1993) J. Cell Sci. 105, 1085-1093[Abstract/Free Full Text]
  52. Ji, Z.-S., Lauer, S. J., Fazio, S., Bensadoun, A., Taylor, J. M., and Mahley, R. W. (1994) J. Biol. Chem. 269, 13429-13436[Abstract/Free Full Text]
  53. Mahley, R. W. (1996) Isr. J. Med. Sci. 32, 414-429[Medline] [Order article via Infotrieve]
  54. Williams, K. J., and Fuki, I. V. (1997) Curr. Opin. Lipidol. 8, 253-262[Medline] [Order article via Infotrieve]
  55. Fuki, I. V., Kuhn, K. M., Lomazov, I. R., Rothman, V. L., Tuszynski, G. P., Iozzo, R. V., Swenson, T. L., Fisher, E. A., and Williams, K. J. (1997) J. Clin. Invest. 100, 1611-1622[Abstract/Free Full Text]
  56. Ji, Z.-S., Brecht, W. J., Miranda, R. D., Hussain, M. M., Innerarity, T. L., and Mahley, R. W. (1993) J. Biol. Chem. 268, 10160-10167[Abstract/Free Full Text]
  57. Lovestone, S., Anderton, B. H., Hartley, K., Jensen, T. G., and Jorgensen, A. L. (1996) Neuroreport 7, 1005-1008[Medline] [Order article via Infotrieve]
  58. Strittmatter, W. J., Weisgraber, K. H., Goedert, M., Saunders, A. M., Huang, D., Corder, E. H., Dong, L.-M., Jakes, R., Alberts, M. J., Gilbert, J. R., Han, S.-H., Hulette, C., Einstein, G., Schmechel, D. E., Pericak-Vance, M. A., and Roses, A. D. (1994) Exp. Neurol. 125, 163-171[CrossRef][Medline] [Order article via Infotrieve]
  59. Roses, A. D. (1994) J. Neuropathol. Exp. Neurol. 53, 429-437[Medline] [Order article via Infotrieve]
  60. Han, S.-H., Hulette, C., Saunders, A. M., Einstein, G., Pericak-Vance, M., Strittmatter, W. J., Roses, A. D., and Schmechel, D. E. (1994) Exp. Neurol. 128, 13-26[CrossRef][Medline] [Order article via Infotrieve]
  61. Han, S.-H., Einstein, G., Weisgraber, K. H., Strittmatter, W. J., Saunders, A. M., Pericak-Vance, M., Roses, A. D., and Schmechel, D. E. (1994) J. Neuropathol. Exp. Neurol. 53, 535-544[Medline] [Order article via Infotrieve]
  62. Mahley, R. W., Nathan, B. P., Bellosta, S., and Pitas, R. E. (1996) in Apolipoprotein E and Alzheimer's Disease (Roses, A. D., Weisgraber, K. H., and Christen, Y., eds), pp. 49-58, Springer-Verlag, Berlin
  63. Mahley, R. W. (1997) in The Molecular and Genetic Basis of Neurological Disease (Rosenberg, R. N., Prusiner, S. B., DiMauro, S., and Barchi, R. L., eds) 2nd Ed., pp. 1037-1049, Butterworth-Heinemann, Boston


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.