(Received for publication, August 22, 1995)
From the
Previously, we demonstrated in cultured dorsal root ganglion
neurons that, in the presence of -migrating very low density
lipoproteins (
-VLDL), apolipoprotein (apo) E4, but not apoE3,
suppresses neurite outgrowth. In the current studies, murine
neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3
or apoE4 cDNA, and the effect on neurite outgrowth was examined. The
stably transfected cells secreted nanogram quantities of apoE
(44-89 ng/mg of cell protein in 48 h). In the absence of
lipoproteins, neurite outgrowth was similar in the apoE3- and
apoE4-secreting cells. The apoE4-secreting cells, when incubated with
-VLDL, VLDL, cerebrospinal fluid lipoproteins (d <
1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions,
showed a reduction in the number of neurites/cell, a decrease in
neurite branching, and an inhibition of neurite extension, whereas in
the apoE3-secreting cells in the presence of a lipid source, neurite
extension was increased. Uptake of
-VLDL occurred to a similar
extent in both the apoE3- and apoE4-secreting cells. With low density
lipoproteins or with dimyristoylphosphatidylcholine emulsions, either
alone or complexed with cholesterol, no differential effect on neurite
outgrowth was observed. A slight differential effect was observed with
apoE-containing high density lipoproteins. The differential effect of
apoE3 and apoE4 in the presence of
-VLDL was blocked by incubation
of the cells with heparinase and chlorate, with lactoferrin, or with
receptor-associated protein, all of which prevent the uptake of
lipoproteins by the low density lipoprotein receptor-related protein
(LRP). The data suggest that the secreted and/or cell surface-bound
apoE interact with the lipoproteins and facilitate their
internalization via the heparan sulfate proteoglycan-LRP pathway. The
mechanism by which apoE3 and apoE4 exert differential effects on
neurite outgrowth remains speculative. However, the data suggest that
apoE4, which has been shown to be associated with late onset familial
and sporadic Alzheimer's disease, may inhibit neuronal remodeling
and contribute to the progression of the disease.
Apolipoprotein (apo) ()E, a 34-kDa protein coded for
by a gene on chromosome 19, plays a prominent role in the transport and
metabolism of plasma cholesterol and triglyceride through its ability
to interact with the low density lipoprotein (LDL) receptor and the LDL
receptor-related protein (LRP) (1, 2) . Apolipoprotein
E also may play an important role in the redistribution of lipids
within the central nervous system(3) . More than three-fourths
of the apoE in the plasma is synthesized by the liver(4) ,
whereas apoE in cerebrospinal fluid (CSF) is produced primarily by
astrocytes within the brain(5, 6, 7) . Three
common isoforms of apoE, distinguished by differing mobility on
isoelectric focusing gels, differ in amino acids at positions 112 and
158(1) . The most common isoform, apoE3, has cysteine at
position 112 and arginine at 158, whereas apoE2 has cysteine at both
positions, and apoE4 has arginine at both. The isoforms are encoded by
three alleles at the same gene locus. Apolipoprotein E4 has recently
been shown to be associated with Alzheimer's disease
(AD)(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
Late onset familial AD is linked to the proximal long arm of
chromosome 19(10) , which contains the apoE locus(1) .
Saunders and co-workers at Duke University (14) were the first
to demonstrate that the apoE4 allele is associated with both late onset
familial and sporadic forms of AD, which together account for 90% of
cases(14) . The association of apoE4 with AD has been confirmed
by several groups (9, 12, 19, 20) .
Whereas the allele frequency for apoE4 is 0.143 in the general
population(21) , it is 0.36-0.57 in various populations
of patients with late onset
AD(9, 12, 14, 19, 20) . In
families with late onset AD, the risk for AD increases from 20 to 90%,
and the mean age of onset decreases from 84 to 68 years with an
increasing number of apoE4 alleles. Homozygosity for apoE4 is virtually
certain to result in AD by age 80(11) . The defining
neuropathological features of AD are the presence of extracellular
deposits of
-amyloid in the cerebral cortex and vascular amyloid
around blood vessels as well as intracellular deposits of
hyperphosphorylated
in the form of neurofibrillary tangles in
neurons of the cortex, hippocampus, and amygdala(22) .
Apolipoprotein E is associated with both the senile plaques and
neurofibrillary tangles present in the brains of AD
patients(10) .
The mechanism by which apoE4 is related to AD
is unknown and could involve a direct effect of apoE on neurons. We
therefore examined the effect of apoE3 and apoE4 on neurons in
vitro. When fetal rabbit dorsal root ganglion (DRG) neurons were
incubated with -migrating very low density lipoproteins
(
-VLDL), which are rich in apoE and cholesterol, neurite outgrowth
and branching were increased (23) . Addition of rabbit apoE
(structurally similar to human apoE3) together with the
-VLDL
reduced neurite branching but very significantly increased neurite
extension(23) . In an extension of these studies, we found that
human apoE3 and apoE4, when added together with
-VLDL, have
differential effects on the outgrowth of neurites from DRG neurons in
culture(24) . Compared with cells incubated with
-VLDL
alone, cells treated with human apoE3 plus
-VLDL showed decreased
branching and increased neurite extension, whereas cells treated with
apoE4 plus
-VLDL had decreased neurite outgrowth(24) .
Similar observations showing an isoform-specific effect of exogenous
apoE3 and apoE4 in the presence of a source of lipid have been
described using a murine neuroblastoma cell line (Neuro-2a) in
culture(25) .
In the present studies, we stably transfected
the Neuro-2a cells with human apoE3 or apoE4 cDNA and then examined the
effect of endogenously produced apoE on neurite outgrowth. When
incubated with -VLDL, VLDL, or CSF lipoproteins, these cells,
expressing small amounts of endogenous apoE3 and apoE4, displayed
differences in neurite outgrowth. The enhanced neurite outgrowth in the
apoE3-secreting cells and inhibition of neurite outgrowth in the
apoE4-secreting cells were abolished by reagents known to block binding
and internalization of apoE-enriched lipoproteins by the heparan
sulfate proteoglycan (HSPG)-LRP
pathway(26, 27, 28) .
Each pellet was washed 3 times with sterile water and dissolved in gel-loading buffer. Cellular apoE was extracted from the cells, following suramin removal of surface-bound apoE, using STEN buffer (50 mM Tris-Cl, pH 7.6, containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 20 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin). Samples were electrophoresed on 5-20% polyacrylamide gradient gels containing sodium dodecyl sulfate, as described previously(40) . The proteins were transferred to nitrocellulose paper by blotting and treated with an anti-human apoE polyclonal antiserum (1:1,000 dilution) raised in rabbit (generously provided by Dr. K. H. Weisgraber, Gladstone Institutes). The nitrocellulose immunoblot then was incubated with donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5,000 dilution) (Amersham Corp.). After washing to remove unbound antibody, the immunocomplex was detected using an ECL kit (Amersham Corp.), according to the manufacturer's instructions. Quantitation of the level of apoE bound, internalized, and secreted by the cells was accomplished by densitometric scanning (Ambis Scanner, San Diego, CA) and based on a standard curve of purified human plasma apoE3 and apoE4.
In studies on the effect of
the inhibitors of lipoprotein binding to the LRP, cells were incubated
for 1 h at 37 °C in N2 medium containing the indicated
concentrations of either lactoferrin, chlorate, or heparinase or with
the receptor-associated protein (RAP). Then the -VLDL were added,
and the incubation was continued for a total of 96 h. The reagents,
except for
-VLDL, were re-added every 24 h. The media and
-VLDL were replaced after 48 h.
The levels of apoE secreted into the medium, bound to the
cell surface, and accumulating intracellularly by the stably
transfected Neuro-2a cells expressing human apoE3 or apoE4 were
assessed by Western blot analysis and quantitated by densitometry (Table 1). The cells secreted 44-54 ng of apoE3 and
60-89 ng of apoE4/mg of cell protein in 48 h. The apoE3- and
apoE4-secreting cells had similar amounts of apoE bound to the cell
surface (releasable by suramin treatment), ranging from 4.9 to 8.0 ng
of apoE/mg of cell protein. The intracellular content of apoE in the
two apoE3-expressing cell lines was 140 and 259 ng of apoE/mg of cell
protein. Similar amounts of intracellular apoE (111-215 ng/mg)
were seen in the apoE4-expressing cell lines. The addition of
-VLDL to the cells did not have a significant effect on the amount
of apoE secreted, surface-bound, or present within the apoE3- or
apoE4-secreting cells (Table 1).
In initial experiments, two
Neuro-2a cell lines that secreted similar amounts of apoE3 (clone 1, 54
ng/mg of cell protein) and apoE4 (clone 4, 60 ng/mg of cell protein) (Table 1) were used to examine the growth of neurites. When these
cells were grown in N2 medium in the absence of -VLDL, there were
no apparent differences in neurite outgrowth between the apoE3- and
apoE4-secreting cells. However, incubation of the cells in N2 medium
containing
-VLDL resulted in a markedly different pattern in the
outgrowth of neurites from these cells. Apolipoprotein E3-secreting
cells incubated with
-VLDL developed long neurites (Fig. 1A), whereas in apoE4-secreting cells, neurite
outgrowth was suppressed (Fig. 1B).
Figure 1:
Photomicrographs of representative
Neuro-2a cells stably transfected with apoE3 (A) or apoE4 (B) cDNA and grown for 96 h in N2 medium containing -VLDL
(40 µg of cholesterol/ml).
Differences in
neurite outgrowth in the absence and presence of increasing
concentrations of -VLDL were quantitated by measuring the number
of neurites per cell, neurite branching, and neurite extension (Fig. 2, A-C, respectively). The values for the
non-apoE-transfected control cells incubated for 96 h in N2 medium in
the absence of
-VLDL are set at 100%. The expression of either
apoE3 or apoE4 by the transfected Neuro-2a cells did not influence
neurite number, branching, or extension when the cells were grown in N2
medium in the absence of added lipoprotein (Fig. 2,
A-C). However, as shown in Fig. 2A, the
addition of
-VLDL resulted in an increase in the number of neurons
in the control cells and in the cells secreting apoE3 (significantly
increased at 40 µg of
-VLDL cholesterol/ml compared with
apoE3-secreting cells in N2 medium). On the other hand, in the presence
of high concentrations of
-VLDL, the Neuro-2a cells secreting
apoE4 showed a significant reduction in the number of neurites/cell as
compared with the apoE4-secreting cells in the N2 medium.
Figure 2:
Effect of -VLDL on the number of
neurites/cell (A), neurite branching (B), and neurite
extension (C) from control Neuro-2a cells and from cells
stably transfected to express apoE3 or apoE4. Cells (clone 1 for
apoE3-expressing and clone 4 for apoE4-expressing) were incubated for
96 h in N2 medium alone or in medium containing increasing
concentrations of
-VLDL. The media were changed at 48 h. The cells
were stained with DiI and fixed, and the indicated parameters were
measured. Each data point was obtained by the measurement of
20-50 cells expressing neurites in four separate experiments. The
data are presented as the percentage of the value obtained with control
cells with N2 medium alone. The data are the mean ± the S.E. The
average values obtained with control cells incubated with N2 medium
alone were as follows: A, neurites/cell = 3; B, branch points/neurite = 2; C, average neurite
length = 155 µm. For calculation of the level of
significance for the effect of added
-VLDL, the results in the
presence of
-VLDL are compared with the data obtained with the
same cells in the absence of
-VLDL (i.e. grown in N2
medium alone). *, p < 0.025; **, p < 0.010;***, p < 0.005
As
described previously for DRG cells(23, 24) , the
addition of -VLDL alone resulted in increased branching of
neurites. As shown in Fig. 2B, addition of
-VLDL
to the non-apoE-transfected cells resulted in a significant increase in
neurite branching. In addition, at the highest concentration of
-VLDL cholesterol, the apoE3-secreting cells displayed enhanced
branching by comparison with the apoE3-secreting cells grown in N2
medium alone. In contrast, the apoE4-secreting cells tended to show
decreased branching when incubated with
-VLDL; however, this
decrease did not reach statistical significance.
Neurite extension
was increased in the Neuro-2a cells secreting apoE3 when they were
incubated with the highest concentrations of -VLDL. In contrast,
in the apoE4-secreting cells, neurite extension was very significantly
suppressed, even at the lowest concentration of
-VLDL used (Fig. 2C).
The results described in Fig. 2were based on a comparison of cells having neuritic
outgrowths and did not take into account those Neuro-2a cells without
neuritic extensions. Approximately 25-30% of the Neuro-2a cells
in N2 medium possessed neurite extensions (defined as a cell-surface
projection of at least one-half the cell diameter). However, as shown
in Fig. 3, it was apparent that in the presence of -VLDL,
the number of apoE3-secreting cells developing neurites increased
markedly to 60-70% of the total. On the other hand, the number of
apoE4-secreting cells developing neuritic extensions was significantly
reduced, compared with the control or apoE3-secreting cells. Thus, the
apoE3-secreting cells incubated with
-VLDL not only had longer
neuritic extensions but also showed an increase in the number of cells
with neurites. The apoE4-secreting cells grown in the presence of
-VLDL showed fewer neurites, and those that were produced were
much shorter.
Figure 3:
Effect of -VLDL on the percentage of
cells expressing neurites. The cells were incubated as described in Fig. 2. Four different fields in each dish were selected, and
the percentage of cells displaying neurites was measured. Data are the
means of three different experiments performed in duplicate (±
S.E.). The percentages of cells expressing neurites in the absence of
-VLDL were as follows: control cells (Control), 35
± 11; apoE3-expressing cells (ApoE3), 32 ± 9;
apoE4-expressing cells (ApoE4), 25 ± 13. *, p < 0.025 versus control;**, p < 0.005 versus control
To ensure that the differential effect of -VLDL
on neurite outgrowth in the apoE3- and apoE4-secreting cells was not
due to clonal variation or to differences in the secretion or
intracellular content of apoE in the various cell lines, additional
experiments were performed with the other stably transfected cell lines
secreting apoE3 or apoE4. Incubation of these cells with
-VLDL
also resulted in differential effects of apoE3 and apoE4 on neurite
outgrowth. As summarized in Table 2, in the presence of
-VLDL, all of the apoE4-secreting cells showed a significant
reduction in the number of neurites expressed, branching, and neurite
extension, whereas the apoE3-secreting cells displayed an increased
number of neurites, increased branching, and increased extension as
compared with cells grown in N2 medium lacking a source of lipoprotein.
To determine whether apoE4 blocks neurite extension in the presence
of -VLDL or whether it induces neurite retraction, the cells were
incubated for 48 h in N2 medium alone to stimulate neurite outgrowth.
The medium then was changed, and the cells were incubated for an
additional 48 or 96 h in media containing
-VLDL (40 µg of
cholesterol per ml). The addition of
-VLDL did not decrease the
extension of neurites of apoE4-expressing cells compared with cells
incubated in N2 medium alone (data not shown). Therefore, it appears
that apoE4 in the presence of
-VLDL inhibits neurite extension
directly and does not cause a retraction of neurites that have already
extended.
Other lipoproteins were used to determine if any lipid
vehicle carrying apoE would substitute for -VLDL. Incubation of
the apoE3- or apoE4-expressing cells with rabbit VLDL, a lipoprotein
rich in triglyceride, resulted in similar effects on neurite extension
as obtained with
-VLDL. As shown in Table 3, when the
Neuro-2a cells secreting apoE3 were incubated with VLDL, they showed an
increase in neurite extension, whereas the apoE4-secreting cells in the
presence of VLDL showed an inhibition of neurite extension. In other
experiments, human LDL and canine apoE HDL
, an
apoE-enriched plasma HDL induced by cholesterol feeding and resembling
apoE-containing lipoproteins in the CSF(3) , also were used.
The apoE3- and apoE4-secreting Neuro-2a cells did not respond to LDL
(40 µg of cholesterol/ml) (i.e. there was no difference in
neurite extension as compared with control cells grown in N2 medium
alone (data not shown)). On the other hand, incubation of apoE
HDL
(40 µg of cholesterol/ml) with the apoE4-secreting
or apoE3-secreting cells resulted in only a small reduction or increase
in neurite extension, respectively (control cells in N2 medium, 100%;
apoE4-secreting cells plus HDL
, 85-90% of the value
obtained with N2 medium; apoE3-secreting cells plus HDL
,
110% of the value obtained with N2 medium).
Liposomes and lipid
emulsions also were used in an attempt to define the type of lipid
vehicle required for the delivery of the apoE. The DMPC emulsion alone
or DMPC complexed with cholesterol were incubated with the apoE3- and
apoE4-secreting cells for 96 h at increasing phospholipid
concentrations of up to 45 µg of phospholipid and 5 µg of
cholesterol/ml of medium (higher concentrations were toxic to the
cells). In these studies, there was no effect on neurite outgrowth with
either of the apoE-transfected Neuro-2a cells (data not shown).
Previously, we have shown that apoE complexes with DMPC and mediates
high affinity binding to the LDL receptor(46) . On the other
hand, a lipid emulsion particle (emulsion A in Table 3), which
was a triglyceride- and phospholipid-containing spherical particle
(35.8 nm), caused a significant enhancement of neurite extension
in the apoE3-secreting cells and was associated with an inhibition of
outgrowth in the apoE4-secreting cells.Thus, specific combinations of
lipids and/or a unique particle size may be required to elicit the apoE
isoform-specific effects on neurite outgrowth. It is interesting to
note that the delivery of cholesterol to the cells does not appear to
be required for the differential effect.
Additional studies using
the lipoproteins from bovine CSF suggest that natural lipoproteins in
the central nervous system may mediate the isoform-specific effects of
apoE3 and apoE4. As shown in Fig. 4, addition of lipoproteins
isolated from CSF (d < 1.21 g/ml) to the cells caused an
inhibition of neurite outgrowth from the apoE4-expressing cells and an
increase in outgrowth from the apoE3-expressing cells. When CSF
lipoproteins were used at a concentration of 40 µg of lipoprotein
cholesterol/ml, the effect was similar to that obtained using
-VLDL at the same concentration.
Figure 4:
Effect of CSF lipoproteins on neurite
extension from Neuro-2a cells stably transfected to express apoE3 or
apoE4. Cells were incubated with -VLDL or bovine CSF lipoproteins (d < 1.21 g/ml) under the conditions described in the
legend to Fig. 2. Each data point represents the measurement of
20-40 neurons. The data are reported as the mean ± S.E.
The calculation of the level of significance of the differences
observed was performed as described in the legend to Fig. 2. *, p < 0.025;**, p < 0.01;***, p <
0.005
Cerebrospinal fluid lipoproteins (d < 1.21 g/ml) were analyzed for protein and cholesterol content and apolipoprotein composition. The ratio of cholesterol to protein was approximately 1:1, similar to data reported for canine CSF(3) . The bovine CSF lipoproteins (d < 1.21 g/ml) contained only apoE and apoA-I when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue staining. These results are similar to those reported previously for human and canine CSF lipoproteins(3, 47) .
The ability of the
neuroblastoma cells to bind, internalize, and degrade -VLDL was
examined to determine whether the differences in neurite outgrowth in
the apoE3- and apoE4-expressing cells were due to a different ability
of the secreted apoE3 and apoE4 to stimulate the delivery of apoE
and/or lipoprotein lipids to the cells. In these studies,
I-
-VLDL were used to quantitate the binding, uptake,
and degradation of the lipoproteins in the Neuro-2a cells (Table 4). The total amount of cell-associated (bound and
internalized)
I-
-VLDL was very similar in the apoE3-
and apoE4-secreting cells (both were slightly lower than that seen in
the non-apoE-transfected control cells). The degradation of
I-
-VLDL by the apoE3- and apoE4-secreting cells was
similar. There was a small (but statistically significant) decrease in
the degradation of
I-
-VLDL by the apoE4-secreting
cells when compared with the non-apoE-transfected control Neuro-2a
cells.
In a parallel experiment, the cells were incubated with
DiI-labeled -VLDL to visualize the internalization of the
lipoproteins in the apoE3- and apoE4-secreting cells by fluorescence
microscopy. Following internalization, DiI is trapped in the lysosomes,
and the fluorescent intensity of the cells, therefore, is proportional
to the total amount of lipoprotein internalized and
degraded(42) . In these studies, no difference in the uptake of
DiI-labeled
-VLDL was observed in the apoE3- and apoE4-secreting
cells. Extraction and quantitation of the DiI from cells incubated with
DiI-labeled
-VLDL (40 µg of cholesterol/ml) for 16 h at 37
°C confirmed the visual impression that the uptake of DiI-labeled
-VLDL was similar in the apoE3- and apoE4-secreting cells. The
control cells incorporated 8.9 ± 0.4 ng of DiI/mg of cell
protein, while the apoE3- and apoE4-expressing cells incorporated 10.2
± 1.0 and 10.8 ± 0.3 ng of DiI/mg of cell protein,
respectively.
To demonstrate that apoE binds to the lipid particles
when it is present at the concentrations secreted by the cells, we
incubated radiolabeled apoE3 or apoE4 with the -VLDL, VLDL, or
emulsion A for 1 h at 37 °C (100 ng of apoE with 40 µg of
-VLDL cholesterol or 100 ng of apoE with either 5 µg of VLDL
or emulsion A triglyceride) and fractionated them by FPLC.
Approximately 70% of the apoE was associated with the
-VLDL and
50% with the VLDL and emulsion A. There was no difference in the amount
of apoE3 or apoE4 associated with the lipid particles.
To determine
which receptor was involved in mediating the differential effects of
apoE3 and apoE4 on neurite outgrowth, we used inhibitors that block the
binding and internalization of apoE-enriched lipoproteins by the
HSPGLRP pathway, but not by the LDL receptor pathway, and
determined the effect on neurite outgrowth. Prior to the addition of
-VLDL, the cells were preincubated for 1 h with either heparinase
(20 units/ml) and chlorate (20 mM), with the RAP (5
µg/ml), or with lactoferrin (10 µg/ml). The binding of
apoE-enriched lipoproteins to the LRP requires their initial binding to
cell-surface HSPG. Heparinase and chlorate cleave and reduce the
sulfation of cell-surface HSPG, respectively(28, 48) .
Lactoferrin blocks binding of lipoproteins to both HSPG and LRP,
whereas RAP primarily blocks the binding of apoE-enriched lipoproteins
to the LRP. All of these reagents previously have been shown to inhibit
the uptake of apoE-enriched
-VLDL by the
LRP(26, 28, 49, 50) . As previously
shown in Fig. 2,
-VLDL alone stimulated the outgrowth of
neurites. The stimulation of neurite outgrowth by
-VLDL was
further enhanced in the apoE3-expressing cells and markedly inhibited
in the apoE4-secreting cells ( Fig. 2and Table 5). The
addition of chlorate and heparinase or RAP did not block the
stimulatory effect of
-VLDL on neurite outgrowth in the control
cells (Neuro-2a cells not expressing apoE), suggesting that the effect
of
-VLDL alone is mediated by the LDL receptor; however, these
reagents blocked the isoform-specific effects in the cells secreting
apoE (Table 5). Chlorate and heparinase treatment of the cells or
the addition of RAP prevented the stimulation of neurite extension in
the apoE3-expressing cells incubated with
-VLDL (that is,
significantly decreased the
-VLDL-induced neurite extension in the
Neuro-2a cells secreting apoE3). Moreover, chlorate and heparinase or
RAP blocked the inhibition of neurite extension seen in the
apoE4-expressing cells (that is, the apoE4-expressing cells in the
presence of
-VLDL did not demonstrate inhibition of neurite
extension but, in fact, showed increased extension) (Table 5). In
the presence of heparinase and chlorate or RAP, in the apoE-secreting
cells, neurite outgrowth was similar to that observed when
-VLDL
were added to the control cells in the absence of apoE (Table 5).
Therefore, in the presence of these reagents, the LDL receptor-mediated
effect of
-VLDL was not blocked. Lactoferrin also blocked the
effects of apoE3 and apoE4 on neurite outgrowth; however, it also
slightly suppressed the effect of
-VLDL on neurite extension in
the control cells. These data show that inhibition of the interaction
between
-VLDL and the HSPG
LRP pathway prevents the
differential effects of apoE3 and apoE4 on neurite outgrowth (Table 5).
We have previously shown that human apoE3 and apoE4 have a
differential effect on the outgrowth of neurites from DRG neurons and
Neuro-2a cells in culture when added exogenously to the cells together
with -VLDL(23, 24, 25) . Compared with
cells treated with
-VLDL alone, cells incubated with
-VLDL
and apoE3 had decreased neurite branching and increased neurite
extension, whereas cells incubated with apoE4 and
-VLDL showed a
decrease in both neurite branching and extension(24) . In the
current studies, we have demonstrated that in the presence of
-VLDL, VLDL, CSF lipoproteins, or specific lipid emulsions
(containing triglyceride and phospholipid), the endogenous synthesis
and secretion of nanogram quantities of apoE3 and apoE4 by transfected
murine neuroblastoma cells (Neuro-2a) had differential effects on
neurite outgrowth. Stably transfected cells secreting comparable
amounts of human apoE3 or apoE4 display similar patterns of neurite
outgrowth when incubated in the absence of lipoproteins. However, the
addition of as little as 5-10 µg of
-VLDL cholesterol/ml
of medium or 5 µg of VLDL triglyceride/ml of medium caused a
dramatic difference in the neurite extension and branching in the
apoE3- and apoE4-secreting cells. Whereas both branching and extension
were increased in the apoE3-producing cells, these processes were very
significantly inhibited in the apoE4-producing cells. This differential
effect was not due to clonal variation, because similar results were
obtained using several different stably transfected lines that secreted
different levels of apoE. The inhibitory effect on neurite outgrowth in
the apoE4-secreting cells was dependent upon the presence of a source
of lipid during the initiation of neurite formation, since
-VLDL
added to apoE4-secreting cells after the neurites had already formed
did not result in neurite retraction.
It has been shown that the
addition of a lipid source (-VLDL, VLDL, CSF lipoproteins,
apoE-containing HDL, or specific lipid emulsions) to the apoE3- and
apoE4-secreting cells is necessary for the isoform-specific effect on
neurite outgrowth to occur. This pathway may involve a
secretion-capture role for apoE, as has been postulated to occur with
other cell types secreting apoE(1, 6, 27) .
We have shown that the apoE secreted by the transfected Neuro-2a cells
interacts with the lipoproteins in the medium. In addition, the apoE
may interact with cell-surface HSPG and, by either or both of these
processes, may mediate lipoprotein or lipid emulsion uptake by the LDL
receptor or the LRP(1, 26, 27, 51) .
Our data demonstrate that it is the HSPG
LRP pathway that is
important for the differential effects of apoE3 and apoE4 on neurite
outgrowth. These data are consistent with the studies of Holtzman et al.(52) , in which the addition of RAP and anti-LRP
antibody blocked the stimulatory effect of apoE3 on NGF-induced neurite
extension in an immortalized central nervous system-derived neuronal
cell line in culture. The data also support a role for the LDL receptor
in mediating the stimulatory effect of
-VLDL alone on neurite
outgrowth. The VLDL receptor that also binds apoE-containing
lipoproteins and could be involved in mediating the uptake of the
-VLDL is not present in the Neuro-2a cells. (
)
It has
been shown previously that apoE-enriched lipoproteins bind initially to
cell-surface HSPG and are then transferred to LRP prior to
internalization or that the HSPGLRP complex itself is
internalized by the
cells(1, 6, 26, 27, 28) .
Lipid-free apoE is a poor ligand for any of the lipoprotein receptors,
including the LRP. Thus, a specific lipoprotein vehicle (e.g.
-VLDL, VLDL, CSF lipoproteins, specific lipid emulsions, or
apoE-containing HDL), capable of being enriched in apoE, may be
required to target apoE3 and apoE4 into a specific intracellular
pathway to allow for the differential effects of the apoE isoforms.
Once they are internalized, it is necessary to postulate that the apoE
isoforms are handled differently by the cells in such a way that apoE3
has a stimulatory role and apoE4 has an inhibitory role in regulating
neurite outgrowth.
There are several lines of evidence suggesting
that the differential effects of the apoE isoforms on neurite outgrowth
are secondary to an effect on the cytoskeleton, possibly by modulating
microtubular assembly. In Neuro-2a cells, the addition of exogenous
apoE3 plus -VLDL results in an abundance of well formed
microtubules in the presence of neurite extension, whereas cells
incubated with apoE4 and
-VLDL primarily contain monomeric
tubulin, and neurite outgrowth is impaired(25) . In vitro biochemical studies have shown that apoE3 interacts avidly with
the microtubular-associated proteins
and MAP 2C, whereas apoE4
does not(53, 54) . It has been postulated that apoE3
facilitates the ability of
and MAP 2C to interact with and
stabilize microtubules. These observations suggest one possible
mechanism whereby apoE3 may be protective in preventing late onset AD
and apoE4 may be detrimental(55, 56) . If this is, in
fact, the mechanism by which apoE3 and apoE4 affect neurite outgrowth,
the question remains as to whether it is a direct or indirect effect of
apoE on the cytoskeleton. If it is a direct effect, apoE must escape
intracellular endosomes and enter the cytoplasm by an unknown
mechanism. It has been suggested that apoE may occur in the cytoplasm (57) . Alternatively, apoE3 and apoE4 may have an indirect
effect on the cytoskeleton and signal changes in activity that support
or suppress extension. This could occur following internalization of
the apoE or through differential signaling events induced by apoE3 and
apoE4 following binding to the HSPG
LRP on the cell surface.
Interpretation of these data remains speculative at the present time.
The -VLDL and VLDL, which in the presence of apoE3 or apoE4
give the maximum differential effect on neurite outgrowth in both the
current studies and in those previously
reported(23, 24) , do not occur in the central nervous
system. The CSF, however, does contain HDL. It has been shown
previously that human and canine CSF contain separate populations of
HDL containing either apoE or apoA-I(3, 47) . The
apoE-containing HDL in the CSF are similar to the apoE HDL
used in our studies that were able to mediate a small inhibitory
effect of apoE4 on neurite outgrowth. Moreover, the addition of CSF
lipoproteins mimicked the effect of the
-VLDL. Thus, the CSF
lipoproteins could serve as vehicles for the delivery of the apoE3 or
apoE4 to neurons.