Differential Cellular Accumulation/Retention of Apolipoprotein E
Mediated by Cell Surface Heparan Sulfate Proteoglycans
APOLIPOPROTEINS E3 AND E2 GREATER THAN E4*
Zhong-Sheng
Ji
,
Robert E.
Pitas
§, and
Robert W.
Mahley
§¶
From the
Gladstone Institute of Cardiovascular
Disease, Cardiovascular Research Institute, and the Departments of
§ Pathology and ¶ Medicine, University of California,
San Francisco, California 94141-9100
 |
ABSTRACT |
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
-very low density
lipoproteins (
-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
-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
-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 |
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,
-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
-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
-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
-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.
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MATERIALS AND METHODS |
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
-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
-VLDL ranged from
~15:1 to 20:1. Human apoE-enriched
-VLDL were prepared by
incubating apoE with
-VLDL at 37 °C for 1 h. For some
experiments, the apoE-enriched
-VLDL were reisolated by fast
performance liquid chromatography as follows. Either
125I-
-VLDL and unlabeled apoE or 125I-apoE
and unlabeled
-VLDL were mixed in a 1:1.5 ratio of
-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
-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
-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
-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
-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
-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
-VLDL--
Cultured cells were grown to ~100% confluence, washed
twice with fresh serum-free medium, and incubated at 37 °C with
apoE-enriched
-VLDL. Before the addition to the cells, the
-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
-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
-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-
-VLDL or 125I-apoE in the medium were
assayed as described previously (35).
Uptake of DiI-labeled
-VLDL by Cultured Cells--
Neuro-2a
cells were incubated for 2 h at 37 °C with DiI-labeled
-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
-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
-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
-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
-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
-counter, and
protein concentration was determined by Lowry's method (37).
 |
RESULTS |
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
-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
-VLDL by cultured neurons (Neuro-2a cells) was
examined in three ways: by measuring the cell association (binding and
internalization) of apoE-enriched 125I-
-VLDL, by
examining the metabolism of apoE-enriched DiI-labeled
-VLDL (DiI
serving as a fluorescent marker for the lipid moieties of the
lipoprotein particle), and by quantitating the ability of the
apoE-enriched
-VLDL to increase the content of cellular cholesterol.
Binding and Internalization of ApoE-enriched
-VLDL
Particles--
The cell association of 125I-
-VLDL or
125I-
-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
-VLDL alone was ~225 ng/mg of cell
protein. The cell association of
-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-
-VLDL, suggesting that a similar
amount of
-VLDL was internalized. In addition, DiI-labeled
-VLDL
were used to examine the uptake of the
-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
-VLDL (~1.8-2-fold) compared with the
amount of DiI-labeled
-VLDL internalized in the absence of apoE
(apoE4 stimulated
-VLDL uptake to a slightly greater extent than
apoE3 (p < 0.02)).

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Fig. 1.
Cell association by Neuro-2a cells of
125I- -VLDL or DiI- -VLDL alone or together with either
apoE3 or apoE4. A, Neuro-2a cells were incubated at 37 °C
for the times indicated with 125I- -VLDL alone ( ) or
together with apoE3 ( ) or apoE4 ( ). 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- -VLDL alone or enriched in apoE3 or apoE4 ( -VLDL:apoE, 1:1.5
protein). The DiI-labeled -VLDL cell association was significantly
increased by the -VLDL plus apoE4 compared with the -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.
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To establish further that apoE3 and apoE4 stimulated similar
-VLDL
particle uptake, the cells were incubated in medium alone, medium
containing
-VLDL, or medium containing
-VLDL and either apoE3 or
apoE4, and the cholesterol content of the cells was determined (Fig.
2). The
-VLDL alone increased the
cellular cholesterol content ~4.7-fold, compared with the control
cells maintained in the absence of lipoprotein. The
-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
-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-
-VLDL or
DiI-labeled
-VLDL and the ability of the cells to accumulate
-VLDL-derived cholesterol demonstrate that apoE3 and apoE4 stimulate
-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
-VLDL (20).

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Fig. 2.
Effect of -VLDL and -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),
-VLDL (40 µg of cholesterol/ml) alone, or -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 -VLDL plus
apoE4 than in those incubated with -VLDL plus apoE3
(p < 0.005). The results are the mean ± S.D. of
three separate experiments.
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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
-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).

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Fig. 3.
Comparison of apoE3 and apoE4
immunoreactivity in Neuro-2a cells. The cells were incubated with
-VLDL (40 µg of cholesterol/ml) enriched with either apoE3 ( )
or apoE4 ( ) (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).
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The accumulated intracellular apoE was primarily intact protein (Fig.
4). Cells were incubated with
apoE-enriched
-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).

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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 -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.
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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
-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).

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Fig. 5.
Cell association and degradation of
125I-apoE-enriched -VLDL by Neuro-2a cells and human
skin fibroblasts. A and B, Neuro-2a cells were
incubated with either 125I-apoE3-enriched -VLDL ( ) or
125I-apoE4-enriched -VLDL ( ) at 37 °C for 2, 4, 8, or 24 h. The concentrations of 125I-apoE and -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 -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 -VLDL ( ) or
125I-apoE4-enriched -VLDL ( ) 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 -counting. The
results are the mean ± S.D. of two independent experiments
performed in duplicate.
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The differential cellular accumulation of apoE3 and apoE4 from
apoE-enriched
-VLDL was also observed in hepatocytes. As shown in
Table I, HepG2 cells incubated with
125I-apoE3 plus
-VLDL displayed about 2.5-fold greater
cell association of apoE compared with cells incubated with
125I-apoE4 plus
-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
-VLDL.
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Table I
Cell association of 125I-apoE3- or 125I-apoE4-enriched
-VLDL by HepG2 cells
Means ± S.D. obtained from two independent experiments performed
in duplicate are shown.
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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
-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
-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
-VLDL particles (data not shown). One possible
reason for the differential accumulation might be that more apoE3 than
apoE4 associates with the
-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
-VLDL after isolation of apoE-enriched
-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
-VLDL cholesterol). Furthermore,
using the fast performance liquid chromatography-purified
125I-apoE-enriched
-VLDL, we demonstrated that the
differential apoE accumulation occurred with apoE on the
-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
-VLDL than in those incubated with
purified 125I-apoE4-enriched
-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
-VLDL
binding and uptake occurred at a protein ratio of 1.5:1 for apoE to
-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:
-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
-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
-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
-VLDL.

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Fig. 6.
Internalization of
125I-apoE-enriched -VLDL by fibroblasts and Neuro-2a
cells at 18 °C. A, human skin fibroblasts were incubated
with 125I-apoE3- or 125I-apoE4-enriched
-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 -VLDL at increasing
concentrations (ratio of -VLDL to apoE was 1:1.5, protein:protein).
The apoE and -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 -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
-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
-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
-VLDL, further suggesting the importance
of HSPG alone in mediating the enhanced metabolism of apoE-enriched
lipoproteins.

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Fig. 7.
Internalization of
125I-apoE-enriched -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 -VLDL or
125I-apoE4-enriched -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.

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Fig. 8.
Internalization of
125I-apoE-enriched -VLDL by wild-type and mutant CHO
cells. Wild-type, HSPG-deficient, and proteoglycan-deficient CHO
cells were incubated with -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
-VLDL with fibroblasts. Human
fibroblasts were incubated with 125I-apoE3- or
125I-apoE4-enriched
-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
-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
-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).
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Table II
Metabolism of 125I-apoE3- and 125I-apoE4-enriched
-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
-VLDL and 125I-apoE4 plus
-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.

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Fig. 9.
Internalization and degradation of
125I-apoE-enriched -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 -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 |
We have previously shown that in Neuro-2a cells incubated for
48 h with apoE3-enriched
-VLDL, neurite outgrowth was
stimulated and cellular microtubules were intact, whereas in cells
treated with apoE4-enriched
-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
-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
-VLDL to determine if apoE3 and apoE4 stimulate the
same level of uptake of
-VLDL particles. Second, we examined the
cellular uptake (retention or accumulation) of the apoE from
apoE-enriched
-VLDL more directly by immunocytochemistry and by
following the metabolism of iodinated apoE.
Incubation of Neuro-2a cells with either apoE3- or apoE4-enriched
-VLDL resulted in a similar cell association of
-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
-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
-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
-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
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
-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.
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).
 |
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