(Received for publication, April 3, 1995)
From the
Evidence is presented for the differential effects of two isoforms of apolipoprotein (apo) E, apoE3 and apoE4, on neurite outgrowth and on the cytoskeleton of neuronal cells (Neuro-2a) in culture. In the presence of a lipid source, apoE3 enhances and apoE4 inhibits neurite outgrowth. Immunocytochemical studies demonstrate that there is a higher concentration of apoE3 than apoE4 in both the cell bodies and neurites. Cells treated with apoE4 showed fewer microtubules and a greatly reduced ratio of polymerized to monomeric tubulin than did cells treated with apoE3. The effect of apoE4 on depolymerization of microtubules was shown by biochemical, immunocytochemical, and ultrastructural studies. The depolymerization of microtubules and the inhibition of neurite outgrowth associated with apoE4 suggest a mechanism whereby apoE4, which has been linked to the pathogenesis of Alzheimer's disease, may prevent normal neuronal remodeling from occurring later in life, when this neurodegenerative disorder develops.
Apolipoprotein (apo) ()E is a 34-kDa protein
component of lipoproteins that mediates their binding to the low
density lipoprotein (LDL) receptor and to the LDL receptor-related
protein (LRP)(1, 2, 3, 4) .
Apolipoprotein E is a major apolipoprotein in the nervous system, where
it is thought to redistribute lipoprotein cholesterol among the neurons
and their supporting cells and to maintain cholesterol
homeostasis(5, 6, 7) . Apart from this
function, apoE in the peripheral nervous system functions in the
redistribution of lipids during
regeneration(8, 9, 10) .
There are three
common isoforms of apoE (apoE2, apoE3, and apoE4) that are the products
of three alleles (2,
3, and
4) at a single gene locus on
chromosome 19(11) . Apolipoprotein E3, the most common isoform,
has cysteine and arginine at positions 112 and 158, respectively,
whereas apoE2 has cysteine at both of these positions and apoE4 has
arginine at both(1, 12) .
Accumulating evidence
demonstrates that the apoE4 allele (4) is specifically associated
with sporadic and familial late-onset Alzheimer's disease and is
a major risk factor for the disease (13, 14, 15, 16) . In accord with
these findings, apoE immunoreactivity is associated with both the
amyloid plaques and the intracellular neurofibrillary tangles seen in
postmortem examinations of brains from Alzheimer's disease
patients(17, 18) . The mechanism by which apoE4 might
contribute to Alzheimer's disease is unknown. However, our recent
data demonstrating that apoE4 stunts the outgrowth of neurites from
dorsal root ganglion (DRG) neurons suggest that apoE may have a direct
effect on neuronal development or remodeling(19, 20) .
In an extension of these previous studies, we have now examined the
effects of the apoE isoforms on neurite outgrowth and on the
cytoskeleton of Neuro-2a cells, a murine neuroblastoma cell line.
Apolipoprotein E4 inhibits neurite outgrowth from these cells, and this
isoform-specific effect is associated with depolymerization of
microtubules.
Immunocytochemistry to detect tubulin was performed on
Neuro-2a cells and fibroblasts grown for 48 h in medium alone, with
-VLDL alone, or with
-VLDL and either apoE3 or apoE4.
Following incubation with test reagents, the medium was aspirated, and
the cells were washed twice with PBS. The cells were fixed for 1 h at
room temperature in 10 mM HEPES, pH 7.2, containing 100 mM KCl, 3 mM MgCl
, 300 mM sucrose, 1
mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 0.5%
Triton X-100, 2% paraformaldehyde, and 0.1% glutaraldehyde, followed by
several quick washes with PBS. The cells were quenched with 0.05 M ammonium chloride in PBS for 5 min at room temperature and blocked
for 1 h at room temperature with 3% bovine serum albumin (BSA) in PBS.
Immunocytochemistry was performed for 45 min at room temperature, using
a monoclonal antibody to
-tubulin (Boehringer Mannheim) at a
concentration of 1 µg/ml in PBS containing 1% BSA. Following
incubation, the cells were washed five times with PBS containing 0.1%
BSA and then incubated for 30 min in the dark with goat anti-mouse IgG
(Zymed Laboratories Inc., South San Francisco, CA) conjugated to
fluorescein isothiocyanate (10 µl/ml) in PBS containing 1% BSA.
Cells were coverslipped, and optical sections of 0.5 µm thickness
were made using a Bio-Rad MRC-600 confocal laser scanning microscope;
the sections were overlaid to obtain a composite image.
For localization of actin, the medium was aspirated, and the cells were washed twice with PBS. Cells were fixed with 3% paraformaldehyde in PBS, washed twice with PBS, and permeabilized for 5 min at room temperature with 0.25% Triton X-100 in PBS containing 1% BSA. The permeabilized cells were washed twice with PBS and incubated for 30 min at room temperature in PBS containing 5 units/ml of rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR). The cells were washed twice with PBS and coverslipped, and optical sections were made as described above.
In immunocytochemistry experiments, 15-20% of the cells did not respond to the treatments and were similar to control neurons. These cells may represent cells injured during plating.
In this study, we found
a similar differential effect of apoE3 and apoE4 on the outgrowth of
neurites from Neuro-2a cells. In the presence of -VLDL, apoE3 and
apoE4 had dramatic isoform-specific effects on neurite outgrowth from
Neuro-2a cells, as assessed by phase contrast microscopy (Fig. 1). Incubation of the cells with
-VLDL (Fig. 1B) stimulated neurite outgrowth slightly as
compared with cells grown in N2 medium alone (Fig. 1A);
however, a more dramatic effect was seen with the addition of apoE.
Cells incubated with apoE3 and
-VLDL (Fig. 1C) had
more neurite extension than cells incubated with
-VLDL, whereas
cells incubated with apoE4 and
-VLDL had less neurite extension (Fig. 1D). These observations were confirmed when
neurite outgrowth was quantitated using an image analysis system (Fig. 2). Incubation of the cells with
-VLDL enhanced
neurite extension, as compared with cells maintained in N2 medium alone (Fig. 2). Addition of human apoE3 with
-VLDL further
increased the extension (p < 0.005), whereas human apoE4
along with
-VLDL significantly reduced neurite extension as
compared with the extension observed from cells incubated with
-VLDL (p < 0.001).
Figure 1:
Effect of apoE on neurite outgrowth
from Neuro-2a cells. Neuro-2a cells were grown for 2 days in N2 medium
alone (A), in N2 medium with -VLDL (40 µg of
cholesterol/ml) (B), or in N2 medium with
-VLDL (40
µg of cholesterol/ml) and 30 µg/ml of either human apoE3 (C) or human apoE4 (D). Cells were photographed using
a phase contrast microscope. Scale bar = 25
µm.
Figure 2: Quantitation of the effect of apoE on neurite extension from Neuro-2a cells. Neuro-2a cells were incubated with test reagents as described in the legend to Fig. 1. Neurite extension then was measured for 50-60 neurons from each group as described under ``Experimental Procedures.'' Data were calculated as the percent difference between each treatment group and the matched control (N2 medium alone) for each experiment. The percent differences for the various experiments then were averaged. The value for the N2 medium alone was set at 100% (dashed line). Data are presented as the mean ± S.E.
Three different studies were
performed to rule out a general toxic effect of apoE4 on neurons. We
assayed lactate dehydrogenase activity, a commonly used indicator of
cell death, measured thymidine incorporation into DNA as an indication
of cell replication, and examined the ability of the cells to develop
neurites following incubation with apoE4 and -VLDL. No significant
differences in lactate dehydrogenase activity (apoE3 +
-VLDL,
212 ± 26.2 units/ml; apoE4 +
-VLDL, 205 ± 22
units/ml) or [
H]thymidine incorporation into DNA
(apoE3 +
-VLDL, 17.37 ± 1.66 counts/min
10
; apoE4 +
-VLDL, 18.13 ± 1.42 counts/min
10
) were seen with cells incubated with
-VLDL
and either apoE3 or apoE4. The inhibitory effect of apoE4 was
reversible, since removal of apoE4 and
-VLDL from the cells in
culture and addition of medium alone or medium containing
-VLDL
resulted in normal outgrowth (data not shown). These findings taken
together demonstrate that the inhibitory effect of apoE4 on neurite
outgrowth is not due to a cytotoxic effect of apoE4 on the cells.
To determine if lipoproteins were required for the differential effects of apoE3 and apoE4, the cells were incubated with free apoE for 48 h and the effects on neurite outgrowth examined. In the absence of lipoprotein, neither apoE3 nor apoE4 had an effect on neurite extension (data not shown). This result suggests that receptor-mediated endocytosis of apoE is necessary for the differential effects on neurite outgrowth, as previous studies have shown that apoE is a ligand for the LDL receptor and the LRP only when it is present on lipoproteins(1, 2, 3, 29) .
We first examined the
ability of -VLDL together with either apoE3 or apoE4 to deliver
lipids to the cells. Incubation of the cells with
-VLDL and apoE3
or apoE4 for 48 h did in fact lead to similar levels of lipid
accumulation (apoE3 +
-VLDL, 115 ± 3.9 µg
cholesterol/mg of cell protein; apoE4 +
-VLDL, 121 ±
5.8 µg cholesterol/mg of cell protein), suggesting that the
stimulation of
-VLDL uptake was similar with the two apoE
isoforms. In comparison, Neuro-2a cells accumulated 20 ± 0.9 and
86 ± 2.6 µg of cholesterol/mg of cell protein when incubated
with N2 medium and
-VLDL alone, respectively. Examination of the
uptake of fluorescently labeled
-VLDL in the presence of either
apoE3 or apoE4 supported the conclusion that apoE3 and apoE4 stimulated
-VLDL uptake similarly.
To determine whether apoE3 and apoE4
were processed differently by neurons and by fibroblasts, we incubated
Neuro-2a cells or murine fibroblasts with apoE3 or apoE4 in the
presence of -VLDL and examined the accumulation of apoE in the
cells. Immunocytochemical detection of apoE in Neuro-2a cells incubated
with
-VLDL and either apoE3 or apoE4 revealed that both isoforms
were present within neurons (Fig. 3). There was, however, a
substantial difference in the intensity of reactivity of apoE3 (Fig. 3A) and apoE4 (Fig. 3B).
Apolipoprotein E3 was present both in the cell body and in the neurites
at a substantially higher concentration than was apoE4 (Fig. 3, A and B). Both apoE3 and apoE4 were observed in
nearly all serial optical sections made throughout the cell, suggesting
that apoE was intracellular (Fig. 3, A and B).
Figure 3:
Immunocytochemical localization of apoE in
Neuro-2a cells. Neuro-2a cells were grown for 2 days in medium
containing -VLDL (40 µg of cholesterol/ml) together with 30
µg/ml of either human apoE3 (A) or human apoE4 (B). Immunocytochemistry was performed as described under
``Experimental Procedures.'' Serial optical sections (
1
µm in thickness) were made from the top (section 1) to the
bottom (section 6) of the cells using a confocal laser
scanning microscope. Scale bar = 15
µm.
The possibility that apoE was localized intracellularly was examined
using two additional approaches. First, Neuro-2a cells were incubated
with -VLDL and either apoE3 or apoE4 and treated with suramin (a
polyanion known to remove lipoproteins nonspecifically bound to the
cell surface and specifically bound to their receptors), followed by
immunocytochemistry for apoE. Treatment with suramin did not
significantly reduce the apoE immunoreactivity associated with the
cells. Second, immunocytochemical studies were performed in cells that
were not permeabilized to permit access of antibody to the cytoplasm.
No immunoreactivity of apoE was observed in nonpermeabilized cells
incubated with
-VLDL together with either apoE3 or apoE4. These
results demonstrated that the apoE was intracellular. The observed
intracellular accumulation of apoE was unexpected, since lipoproteins
and their apoproteins, when internalized by nonneuronal cell types, are
rapidly degraded. To determine if the accumulation of apoE was specific
to neurons, we performed similar immunocytochemistry experiments in
murine fibroblasts. In these studies no apoE immunoreactivity was
observed in the fibroblasts incubated with
-VLDL and either apoE3
or apoE4, suggesting either that apoE enters neurons and fibroblasts
through different pathways or that in neurons apoE, especially apoE3,
can escape lysosomal degradation. These results demonstrate that apoE3
is retained in Neuro-2a cells to a greater extent than apoE4 and that
the metabolism of apoE in neuronal and nonneuronal cells is different.
The differences observed in the accumulation of apoE3 and apoE4 were
confirmed by incubating the neurons with I-labeled apoE
and
-VLDL at 37 °C for 48 h, and the amount of cell-associated
apoE (bound and internalized) was quantitated. A differential
accumulation of
I-apoE was observed, with twice as much
I-apoE3 as
I-apoE4 being associated with
the cells at the end of the incubation period (Fig. 4).
Figure 4:
Cell
association of I-apoE with Neuro-2a cells. Neuro-2a cells
were grown for 2 days in medium containing
-VLDL (40 µg of
cholesterol/ml) together with 30 µg/ml of either
I-apoE3 or
I-apoE4. Following incubation,
the radioactivity associated with the cells (representing bound and
internalized apoE) was determined as described under
``Experimental Procedures.'' The experiment was repeated five
times, each time with a fresh preparation of
I-apoE and
-VLDL. The data are presented as the mean ±
S.E.
Figure 5:
Immunocytochemical localization of tubulin
in Neuro-2a cells. Neuro-2a cells were incubated with test reagents as
described in the legend to Fig. 1. Immunocytochemistry was
performed using a monoclonal antibody to -tubulin and visualized
using a fluorescein-labeled secondary antibody. Optical sections (0.5
µm in thickness) were made using a confocal microscope, and
sections were overlaid to obtain a reconstructed image. Reconstructed
images of cells incubated in medium alone (A), in medium
containing
-VLDL (40 µg cholesterol/ml) (B), or in
medium containing
-VLDL together with 30 µg/ml of either human
apoE3 (C) or human apoE4 are shown (D).
Representative individual optical sections near the top (E and G) and center (F and H) of cells incubated
with
-VLDL and either apoE3 (E and F) or apoE4 (G and H) also are shown. Scale bar =
7 µm.
The photomicrographs in Fig. 5(A-D) are composites of all optical
sections. However, we also analyzed individual optical sections
visualizing various levels through the cells (Fig. 5, E-H) to ensure that the presence of microtubules in
the apoE4-treated cells was not obscured by the diffuse tubulin
staining. In cells treated with -VLDL and apoE4 (Fig. 5, G and H), none of the optical sections contained
well-formed microtubules, whereas in cells incubated with
-VLDL
and apoE3 (Fig. 5, E and F), numerous
microtubules were seen in nearly all of the sections. When similar
experiments were performed with murine fibroblasts, no significant
difference in microtubular morphology was observed among cells treated
with medium alone (Fig. 6A), medium containing
-VLDL (Fig. 6B), or medium containing
-VLDL
together with either apoE3 (Fig. 6C) or apoE4 (Fig. 6D).
Figure 6:
Immunocytochemical localization of tubulin
in fibroblasts. BALB/c fibroblasts were incubated for 2 days in medium
alone (A), in medium containing -VLDL (40 µg
cholesterol/ml) (B), or in medium containing
-VLDL
together with 30 µg/ml of either human apoE3 (C) or human
apoE4 (D). Immunocytochemistry for
-tubulin was performed
as described in the legend to Fig. 5. Scale bar = 7 µm.
Additional experiments were performed to determine if apoE4 disrupts other cytoskeletal structures, such as actin filaments. As shown in Fig. 7, no significant difference in actin morphology was evident among Neuro-2a cells from the four treatment conditions when actin was detected using rhodamine-phalloidin.
Figure 7:
Localization of actin in Neuro-2a cells.
Neuro-2a cells were incubated with test reagents as described in the
legend to Fig. 1. Actin was detected using rhodamine-phalloidin.
Optical sections (1 µm in thickness) were made using a confocal
microscope, and sections were overlaid to obtain a reconstructed image.
Reconstructed images of cells incubated in medium alone (A),
in medium containing -VLDL (40 µg cholesterol/ml) (B), or in medium containing
-VLDL together with 30
µg/ml of either human apoE3 (C) or human apoE4 are shown (D). Scale bar = 8
µm.
To confirm the fluorescence microscopy data
and to examine the effect of apoE on the microtubular architecture in
the neurites of Neuro-2a cells, we performed electron microscopic
studies. Long parallel arrays, identified by morphologic appearance and
size (22 nm in width) as microtubules, were present in cells grown
in medium alone (Fig. 8A),
-VLDL alone (Fig. 8B), and
-VLDL and apoE3 (Fig. 8C). In contrast, in cells treated with apoE4 and
-VLDL, only a few fragments of microtubules were observed both in
the cell body and neurites (Fig. 8D). All of these data
taken together demonstrate that the differences in neurite outgrowth
induced by apoE3 and apoE4 are associated with differences in
microtubular formation and suggest that apoE4 depolymerizes
microtubules in neuronal cells.
Figure 8:
Electron microscopy of microtubules in
Neuro-2a cells. Neuro-2a cells were incubated for 2 days in medium
alone (A), in medium containing -VLDL (40 µg
cholesterol/ml) (B), or in medium containing
-VLDL
together with 30 µg/ml of either human apoE3 (C) or human
apoE4 (D). Following incubation, electron microscopy was
performed, as described under ``Experimental Procedures,'' to
detect microtubules (arrows) in the neurites. Scale bar = 0.5 µm.
Figure 9:
Binding of I-apoE to
partially purified microtubules in vitro. Partially purified
microtubules were prepared from Neuro-2a cells grown in N2 medium, as
described under ``Experimental Procedures.'' Aliquots of
microtubular preparations were incubated with
I-apoE3 or
I-apoE4 (2 µg/ml), followed by centrifugation to
separate the free
I-apoE from that bound to microtubules.
The radioactivity associated with the supernatant and pellet was
determined, and the percentage of total radioactivity in the pellet was
calculated. The data are the mean ± S.E. for three independent
experiments.
The effect of apoE on
the ratio of polymerized to monomeric tubulin was examined
biochemically. Total tubulin, monomeric tubulin, and polymerized
tubulin were extracted from Neuro-2a cells incubated under the various
conditions and then were quantitated by Western blotting and
densitometry. The amounts of total tubulin in extracts from the cells
grown under the four treatment conditions were similar (Fig. 10A). Incubation of cells with N2 medium
containing -VLDL did not significantly affect the quantity of
monomeric or polymeric forms of tubulin as compared with cells grown in
N2 medium alone (Fig. 10A). However, a dramatic
isoform-specific difference in the polymerization state of the
microtubules was observed when the cells were grown in medium
containing
-VLDL and apoE. In contrast to cells treated with apoE3
and
-VLDL, incubation of the cells with apoE4 and
-VLDL
resulted in an increase in the amount of monomeric tubulin and a
decrease in the amount of polymeric tubulin (microtubules).
Figure 10:
Immunoblotting of total, polymeric, and
monomeric forms of tubulin from Neuro-2a cells. Neuro-2a cells were
incubated for 2 days in medium alone (control), in medium containing
-VLDL (40 µg cholesterol/ml), or in medium containing
-VLDL together with 30 µg/ml of either human apoE3 or human
apoE4. Following incubation, cell extract was prepared as described
under ``Experimental Procedures.'' A, an aliquot of
the cell extract containing 50 µg of total protein from each
treatment condition was immunoblotted using a monoclonal antibody to
-tubulin. Monomeric and polymeric forms of tubulin were separated
from the cell extract by centrifugation, as described under
``Experimental Procedures,'' and immunoblotted for
-tubulin as described above. B, densitometric scanning of
immunoblots obtained from three independent experiments performed as
described in A. The value for the N2 medium alone was set at
100% (dashed line), and data were calculated as the percent
difference between each treatment group and the matched control (N2
medium alone) for each experiment. The percent differences for the
different experiments then were averaged. Data are presented as the
mean ± S.E.
Quantitation of the immunoblots from three independent experiments
by densitometry revealed that the incubation of Neuro-2a cells with
-VLDL resulted in a slight increase in the amount of monomeric and
polymeric tubulin (Fig. 10B) as compared with cells
grown in N2 medium alone. However, cells incubated with
-VLDL plus
apoE3 had a significant decrease in monomeric tubulin and a significant
increase in polymeric tubulin as compared with cells grown in
-VLDL alone (p < 0.001) (Fig. 10B). On
the other hand, the opposite effect was observed in cells treated with
apoE4 and
-VLDL, as monomeric tubulin increased significantly and
polymeric tubulin decreased (p < 0.001). These biochemical
results confirm and extend the ultrastructural studies and suggest that
apoE alters the state of tubulin polymerization in an isoform-specific
manner.
The present study demonstrates that the isoform-specific effect of apoE in association with a lipid source on neurite outgrowth that was previously seen in rabbit DRG neurons also is observed in Neuro-2a cells, a murine neuroblastoma cell line derived from the central nervous system(19, 20) . Furthermore, this study demonstrates that the isoform-specific effect of apoE on neurite outgrowth correlates with a differential accumulation of apoE3 versus apoE4 within the neurons and with a differential effect of the apoE3 and apoE4 on cellular microtubules.
Previous studies of nonneuronal cells have shown that apoE-containing lipoproteins are taken up and degraded by receptor-mediated endocytosis. Apolipoprotein E3- and apoE4-containing lipoproteins have a similar binding affinity and cause a similar degree of lipoprotein internalization(1, 22, 30) . The LDL receptor and the LRP have been implicated in this process(1, 2, 3, 29) . Neuronal cells possess both of these major receptors(19, 39, 40) , and apoE3-induced neurite extension in DRG neurons has been suggested to be mediated, at least in part, by the LRP(19) .
In the present study, using
immunocytochemistry of apoE, we have observed a differential
accumulation of apoE3 and apoE4 in Neuro-2a cells. Apolipoprotein E3,
incubated with the cells together with -VLDL, accumulated widely
throughout the cell bodies and the neurites in a diffuse pattern,
suggesting that the internalized apoE3 is not restricted to a specific
organelle. Confocal microscopy revealed that the apoE3 is
intracellular, an observation confirmed by the fact that the pattern of
staining did not change when the cells were treated with suramin to
remove surface-bound apoE and by the fact that the immunoreactivity was
observed only after membrane permeabilization to allow the antibodies
to enter the cells. In comparison to apoE3, less apoE4 accumulates
within the Neuro-2a cells, even though equal amounts of
lipoprotein-derived lipid are delivered to the cells. The data suggest
a differential processing of apoE3 and apoE4, resulting in a greater
accumulation of apoE3. How apoE escapes lysosomal degradation remains
to be determined. The ability of apoE to escape lysosomal degradation
is supported by the recent observations of Han et
al.(31) , which suggest that apoE is present in the
cytoplasm of human neurons.
The differential effect of the apoE
isoforms on neurite extension and their differential intracellular
accumulation suggested that apoE might alter the neuronal cytoskeleton,
specifically the microtubular system. Microtubules have been shown to
have several important functions in neurons, including the development
and maintenance of neuronal polarity, neurite extension, and
retraction, the transport of macromolecules, and the release of
neurotransmitters(32, 33, 34, 35, 36, 37, 41, 42) .
In fact, microtubular stability is linked to several neurodegenerative
disorders, including Alzheimer's disease(43) . We have
shown by three different criteria that apoE3 and apoE4, when incubated
with the cells together with -VLDL, have differential effects on
microtubular structure. The apoE3 clearly supports microtubule
formation in the Neuro-2a cells, whereas apoE4 is associated with
microtubular depolymerization. By immunocytochemistry, the cells
incubated with apoE3 along with
-VLDL displayed an extensive
microtubular system as visualized using an anti-tubulin antibody. In
the apoE4-treated cells, the microtubules were poorly formed and
revealed a diffuse immunoreactivity to tubulin, suggesting the
depolymerization of the microtubules. These observations were confirmed
by electron microscopy of the Neuro-2a cells. Furthermore, quantitation
of monomeric and polymeric tubulin extracted from the Neuro-2a cells
revealed that incubation of the cells with apoE3 along with
-VLDL
resulted in a reduction in monomeric tubulin and an increase in
polymeric tubulin, whereas the opposite results were observed with
apoE4.
The mechanism whereby apoE may alter the microtubular system is unclear. However, apoE3 binds to crude microtubule preparations from Neuro-2a cells to a greater extent (2-fold) than does apoE4. These results are consistent with those of Huang et al.(44) and Strittmatter et al.(45) , who demonstrated that apoE3 bound much more avidly to tau and MAP2c, two microtubule-associated proteins, than did apoE4. Tau, MAP2c, or other microtubule-associated proteins may be mediating the binding of apoE to the crude microtubule preparations observed in our studies. In fact, the accumulation and retention of apoE3 in the Neuro-2a cells may reflect an interaction of apoE3 with the microtubules. Based on their biochemical studies, Roses (46) and Strittmatter et al.(45) postulated that the interaction of apoE3 with tau might support and stabilize microtubule formation and prevent hyperphosphorylation of tau. Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the characteristic lesions of Alzheimer's disease, suggesting a role for the microtubular system in the pathogenesis of the disease.
Even though apoE4 does
not support neurite outgrowth, it is important to note that apoE4 does
not have a general toxic effect on the Neuro-2a cells. Removal of the
apoE4 from the cells allows neurite extension to occur. Furthermore,
the effect of apoE4 on microtubules does not reflect a general
disruption of the cytoskeleton. The apoE4 did not affect actin
stability, and actin filaments appeared identical in cells incubated
with -VLDL and either apoE3 or apoE4. Furthermore, apoE4 did not
effect cell replication, as determined by thymidine incorporation.
In conclusion, these studies suggest that apoE4 might play a role in the pathogenesis of Alzheimer's disease by destabilizing microtubules. In the aging brain, it is known that tubulin concentrations are low, favoring microtubular disassembly(47, 48) . In combination with a low tubulin level, the expression of apoE4, which appears to stimulate microtubular depolymerization, may prevent normal neuronal remodeling from occurring later in life, when the disease process occurs. Ongoing studies aimed at elucidating the mechanism responsible for the apoE4-mediated inhibition of neurite extension and the possible involvement of the microtubular system in this inhibition may shed light on the pathogenesis of Alzheimer's disease and other neurodegenerative disorders.