 |
INTRODUCTION |
Alzheimer's disease is a devastating neurological disorder
characterized by progressive memory loss
and cognitive deficits. To date, four genes have been reported to be
associated with Alzheimer's disease phenotypes including the amyloid
precursor protein gene on chromosome 21 (1-4), the presenilin-1 gene
on chromosome 14 (5), the presenilin-2 gene on chromosome 1 (6-7), and
the apolipoprotein-E (APOE) 1 gene on chromosome
19 (8-10). There are three common alleles of the APOE gene:
3 is the most common representing ~78% of all APOE
alleles,
4 representing 15% and
2 representing 7% (11). Although these alleles give six possible genotypes, the risk of Alzheimer's disease is increased, and the age of onset distribution is
earlier, with each dose of
4 (8). While the influence of the
APOE-
4 genotype was clearly demonstrated, the biological mechanism mediated by apoE4, the translated protein isoform derived from the
4 allele of APOE gene remains speculative.
Several hypotheses have been proposed regarding potential mechanisms
where apoE4 enhances the risk for Alzheimer's disease. One interesting
hypothesis is that apoE4 itself causes toxicity to hippocampal neurons,
but that apoE3 does not (12-13). A 22-kDa thrombin cleavage fragment
of apoE4 known as "truncated apoE4" and an apoE peptide consisting
of a tandem repeat of amino acid residues 141-149 of apoE showed
higher toxicity to neurons than apoE4 itself (14-15). Their toxicity
was apoE receptor-mediated (14) and involved calcium influx (16). Based
on these reports, we started to study the mechanism of apoE toxicity
using the synthetic apoE peptide.
We employed the Mercury Pathway Profiling System
(CLONTECH) to elucidate the mechanistic pathways
activated in the presence of apoE peptide. This system evaluates
several cis-acting enhancer elements such as
AP1, CRE, HSE, Myc, NF
B, and serum
response element and among them, transcription from the CRE element was found to be activated. In addition to the apoE peptide, a specific isoform of the apoE protein was found to stimulate the phosphorylation that activates CREB. We then examined the pathway upstream of CREB
activation and found that ERK and the apoE receptor are involved in
this same pathway. Downstream of CREB, the expression of
c-fos and Bcl-2 genes that are modulated by
activated CREB, were also increased. Our finding that apoE4 stimulates
phosphorylation of CREB through an ERK pathway to increase
transcription of specific genes while apoE3 does not, suggests a
mechanism of apoE isoform-specific function on neurons.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant apolipoprotein E3 and
apolipoprotein E4 were purchased from Calbiochem (San Diego,
CA); U0126 and cAMP-dependent protein kinase-specific
inhibitor from Promega (Madison, WI); nifedipine and dantrolene from
Sigma; (+)-MK801 hydrogen malate and KN62 from Research Biochemicals
International (Natick, MA); RAP and anti-apolipoprotein E antibody
(IB5-E1) from Progen Biotechnik (Heidelberg, Germany); anti-CREB
antibody, anti-phospho-CREB antibody, anti-ERK antibody, and
anti-phospho-ERK antibody from New England Biolabs (Beverly, MA);
anti-c-Fos antibody from Santa Cruz Biotechnology (Santa Cruz,
CA); mouse anti-actin monoclonal antibody (C4) from Chemicon
International (Temecula, CA) and anti-Bcl-2 antibody from MBL (Nagoya, Japan).
ApoE Peptide Synthesis--
A 30-amino acid apoE peptide,
which is a tandem repeat of apoE amino acid residues 141-155
(LRKLRKRLLRDADDL) was synthesized by the solid-phase method as
described previously (17) and greater than 95% purity was determined
by high performance liquid chromatography and mass spectrometry.
Cell Culture--
Primary cultures of rat hippocampal neurons
were prepared from Wistar rats at embryonic day 18 as described
previously (18). Cells were routinely propagated in Dulbecco's
modified Eagle's medium (Sigma) with 10% fetal calf serum (Life
Technologies, Inc.). All experiments were performed on cells cultured
for 5 to 8 days.
Primary astrocyte cultures were prepared from neonatal
apoE-deficient mice as described previously (19) with minor
modifications. In brief, apoE-deficient mice were purchased
from Taconic Farms (Germantown, NY) and backbred to C57BL/6 for at
least six generations, brains were removed from neonatal
apoE-deficient mice within 24 h of birth, meninges were
removed, and brain tissues were digested at 37 °C with Dispase II
(Rochem Molecular Biochemicals, Germany) in PBS. The mixture was
centrifuged (3000 × g for 10 min), plated in
175-cm2 culture flasks (two brains/flask), and cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. After 10 days, cells were incubated for 48 h
with cytosine arabinofuranoside (10 µg/ml; Wako, Osaka, Japan) to
prevent fibroblast overgrowth. Astrocytes were then separated from
microglia and oligodendroglia by agitation on a shaking platform
(Bioshaker BR-30L, Tautek, Tokyo, Japan) and identified by
immunoreactivity with an anti-GFAP antibody. Cultures used for
experiments were >98% astrocytes based on these techniques.
Stimulation of Primary Astrocytes and Quantification of
TNF
--
Inhibition of secretion of TNF
from primary astrocytes
by apoE was quantified as described previously (20). Briefly, primary astrocytes from apoE-deficient mice were plated on 96-well
tissue culture dishes at a density of 20,000 cells/well and incubated in serum-free media (Opti-MEM I, Life Technologies, Inc.) containing 1% N2 supplement (Life Technologies, Inc.). The following day, recombinant apoE3, apoE4, or control protein (denatured apoE3 by
boiling for 30 min) was added. 24 h later, 100 ng/ml
lipopolysaccharide (Sigma) was added. 60 h later, 50 µl of
medium was removed and TNF
secretion measured by Quantikine M Mouse
TNF
ELISA kit (R & D systems) as described in the manufacturer's protocol.
Promoter-reporter Assay--
To detect the pathway activated in
the presence of apoE peptide, we utilized the Mercury Pathway Profiling
Systems (CLONTECH). Briefly, rat hippocampal
neurons were cultured in 6-well dishes, transiently transfected with
promoter-reporter plasmids using LipofectAMINE 2000 (Life Technologies,
Inc.) as described in the manufacturer's protocol. These plasmids
contained the secreted alkaline phosphatase (SEAP) reporter gene
downstream of several copies of specific transcription factors binding
sequences such as AP1, CRE, HSE, Myc, NF
B, and serum response
element. Transfected neurons were allowed to recover for 24 h
before 2 µM apoE peptide was added to the media. Alkaline
phosphatase activities in the media, at indicated times after addition
of apoE peptide, were measured using Great EscApe SEAP
Chemiluminescence Detection Kit (CLONTECH)
following the manufacturer's protocol.
Western Blot Analysis--
Cells were cultured in 6-well dishes,
washed three times with PBS, and lysed with 200 µl of lysis buffer
(50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% SDS,
and 100 mM NaCl). 20-µg samples were boiled for 5 min,
electrophoresed on 12.5% SDS-polyacrylamide electrophoresis gels and
transferred onto Immobilon membrane (Millipore Corp., Bedford, MA). The
membrane was incubated in blocking buffer (1 × PBS, 5% nonfat
dried milk) for 1 h at room temperature and then probed with a
primary antibody in blocking buffer overnight at 4 °C. After four
washes in PBS containing 0.3% Tween 20, blots were probed with the
secondary antibody in blocking buffer for 1 h at room temperature,
and washed again in PBS containing 0.05% Tween 20. Detection of signal
was performed with an enhanced chemiluminescence detection kit
(Amersham International, Little Chalfont, United Kingdom).
Protein Quantification--
The amount of protein (CREB, ERK,
phosphorylated CREB, phosphorylated ERK, c-fos, Bcl-2, and actin) was
quantified by scanning the density of immunodetected bands on Immobilon
membrane using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Measurement of Intracellular Calcium Levels--
Fura-2 AM
(Molecular probes, Eugene, OR) and an Argus 50/CA system (Hamamatsu
Photonics, Japan) were used to quantify the cytoplasmic free calcium as
described in the manufacturer's protocol.
 |
RESULTS |
Recombinant ApoE4 and Recombinant ApoE3 Were Equally Active in
Suppressing the Secretion of TNF
from Astrocytes--
The
recombinant apoE4 and apoE3 proteins used for this study were not
degraded as determined by Western blot analysis of denatured and
reduced apoE proteins (Fig.
1A). To show that these
recombinant proteins also retained biological activity, we examined
whether they equally inhibited the secretion of TNF
from astrocytes
as described by Laskowitz et al. (20). Preincubation of
primary astrocytes cultures prepared from neonatal
apoE-deficient mice with recombinant apoE3 or with
recombinant apoE4, prior to stimulation with 100 ng/ml
lipopolysaccharide, each decreased the concentration of TNF
released
into the conditioned medium in a dose-dependent fashion as
compared with untreated cells (Fig. 1B). There was no
significant difference in TNF
levels between cells treated with the
same concentration of apoE3 or apoE4. Boiled and denatured apoE3 was
used as control protein and it did not suppress the secretion of
TNF
.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of recombinant apoE3 and
apoE4. 5 ng of recombinant apoE3 and apoE4 were electrophoresed on
denaturing and reducing polyacrylamide gels, Western blotted, and
probed with anti-apolipoprotein E antibody, demonstrating that both are
not degraded (A). Preincubation with recombinant apoE3 or
apoE4 inhibited the secretion of TNF from astrocytes prepared from
apoE-deficient mice in a dose-dependent fashion as compared
with heat-denatured apoE3. The difference between apoE3 and apoE4 was
not significant at any concentration (Student's t test).
All results are plotted as a mean ± S.E. of four data
(B).
|
|
ApoE Peptide Facilitates CRE-driven Transcription--
To detect
the signaling pathway activated in the presence of apoE peptide, we
utilized the Mercury Pathway Profiling Systems (CLONTECH). Several cis-acting enhancer elements,
such as AP1 (activator protein 1), CRE (cAMP responsive element), HSE
(heat shock element), Myc, NF
B (nuclear factor of
B), and serum
response element included in this system were assessed in rat primary
hippocampal neurons exposed to 1 µM apoE peptide. Among
these elements, transcription from the CRE element was found to be
activated (Fig. 2).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
ApoE-peptide treatment increases
CREB-responsive reporter activity (transcriptional activity of
CREB). A CRE-SEAP reporter plasmid containing three copies of CRE
elements linked to a TATA-like promoter from the HSV-TK gene and fused
to the SEAP gene (CLONTECH) was transfected into
rat primary hippocampal neurons on day 7 of in vitro culture
using the LipofectAMINE 2000 procedure (Life Technologies, Inc.). The
activity of SEAP measured as chemiluminescence (Great EscApe,
CLONTECH) in the medium increased in a
time-dependent fashion after treatment with 2 µM apoE peptide indicating that apoE peptide facilitates
activation of the transcriptional activity of CREB. Error
bars represent the S.E. (n = 6). *,
p < 0.05 compared with time 0 control using Student's
t test.
|
|
ApoE Peptide and ApoE4, but Not ApoE3, Facilitated Phosphorylation
of CREB at Ser-133--
Since apoE peptide stimulated transcription
from the CRE element, then the CREB transcription factor could be
activated by apoE treatments. Phosphorylation of CREB at Ser-133 leads
to its activation as a transcription factor (21, 22). To monitor the
activation of CREB directly, Western blots of cell lysates from rat
hippocampal neurons stimulated with 1 µM apoE peptide for
the indicated times were probed with anti-CREB and anti-phospho-CREB (p-CREB) antibodies. 15 min after addition of apoE peptide,
phospho-CREB levels substantially increased followed by a gradual
decrease over the next 12 h while the total amount of CREB did not
appear to change (Fig. 3A). In
addition to apoE peptide, treatment with 1 µM apoE4
protein also appeared to significantly increase phospho-CREB levels by
1 h after treatment, while 1 µM apoE3 protein did
not appear to change phospho-CREB levels significantly at any time (Fig. 3, B-D). Although the molar concentration of apoE4 and
apoE peptide added to the media was equivalent, the kinetics (time course) of CREB phosphorylation following treatment with apoE4 protein
appeared to be slower than that stimulated by apoE peptide treatment.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Treatment with apoE4 or apoE-peptide
increases phospho-CREB levels. Rat hippocampal neurons at day 7 in
culture were treated with 2 µM apoE peptide
(A), 2 µM apoE3 (B), or 2 µM apoE4 (C) for the indicated times. Cell
lysates (20 µg/lane) were electrophoresed on SDS-PAGE, Western
blotted, and probed with anti-phospho-CREB antibody or anti-CREB
antibody. The amount of phosphorylated CREB and total CREB was
quantified by scanning densitometry of immunoreactive bands. The ratios
of phosphorylated CREB to total CREB are plotted as a mean ± S.E.
of at least four separate experiments. *, p < 0.05 compared with unstimulated control using Student's t test
(A and D). **, p < 0.05 using
Student's t test. apoE4 > apoE3 (D).
|
|
ApoE Peptide and ApoE4, but Not ApoE3, Facilitated ERK Activity in
Rat Hippocampal Neurons--
Since activated CREB is phosphorylated at
Ser-133 and apoE treatment increases levels of phosphorylated CREB,
then apoE may stimulate a pathway resulting in kinase-mediated
phosphorylation of CREB. One candidate for CREB phosphorylation at
Ser-133 is through the ERK pathway that is activated by a double
phosphorylation of ERK at Thr-202 and Tyr-204. We examined ERK
activation by measuring phosphorylated ERK levels following treatment
with apoE peptide, apoE3, and apoE4 proteins. Treatment with apoE
peptide or with apoE4 protein resulted in significantly increased
levels of phosphorylated ERK while apoE3 treatment failed to increase
levels (Fig. 4). ApoE peptide increased
phospho-ERK levels by 15 min of treatment while apoE4 protein increased
them by 1 h of treatment. Like the phosphorylation of CREB,
phosphorylation of ERK following apoE4 protein treatment appeared to be
slower than that following apoE peptide treatment.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Treatment with apoE4 or apoE-peptide
increases phosphorylated ERK levels. Rat hippocampal neurons at
day 7 in culture were treated with 2 µM apoE-peptide
(A), 2 µM apoE3 (B), or 2 µM apoE4 (C) for the indicated times. Cell
lysates (20 µg/lane) were electrophoresed on SDS-PAGE, Western
blotted, and probed with anti-phospho-ERK antibody or anti-ERK
antibody. The amount of phosphorylated ERK and total ERK was quantified
by scanning densitometry of immunoreactive bands. The ratios of
phosphorylated ERK to total ERK are plotted as a mean ± S.E. of
four separate experiments. *, p < 0.05 compared with
unstimulated control using Student's t test (A, D); **,
apoE4 > apoE3, p < 0.05 using Student's
t test; ***, apoE4 < apoE3 (D).
|
|
ApoE Peptide Facilitated the Expression of c-fos and Bcl-2 in Rat
Hippocampal Neurons--
To confirm that the apoE peptide stimulated
phosphorylation that resulted in increased functional activity of CREB,
we examined whether apoE peptide
treatment led to transactivation of the
CRE-dependent genes, c-fos and Bcl-2.
As shown in Fig. 5, increased levels of both c-fos and Bcl-2
result from treatment with apoE peptide, whereas the level of a control
protein, actin, does not appear to increase.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Treatment with apoE-peptide increases c-fos
and Bcl-2 levels. Rat hippocampal neurons at day 7 of
culture were treated with 2 µM apoE-peptide for the
indicated times. Cell lysates were electrophoresed, Western blotted,
and probed with anti-c-fos antibody (A), anti-Bcl-2 antibody
(B), or anti-actin antibody as a control. (C) *,
p < 0.05 compared with unstimulated control using
Student's t test.
|
|
ApoE Receptor, NMDA Receptor, and L-type
Voltage-dependent Ca2+ Channel Are Involved in
the Pathway--
To further elucidate the pathway where CREB is
phosphorylated after apoE treatment, we examined the effects of RAP
which is a competitive blocker of apoE for its receptor; MK801 which is a selective antagonist of the NMDA receptor; and nifedipine which is a
selective antagonist of the L-type voltage-dependent
calcium channel (LVDCC). Pretreatment with RAP followed by apoE
appeared to attenuate the increase in phospho-CREB levels seen with
apoE treatment alone suggesting that the apoE receptor is involved in
the pathway (Fig. 6A).
Similarly, pretreatment with MK801 or with nifedipine appeared to
attenuate the increase in phospho-CREB levels, suggesting that the NMDA
receptor and/or the LVDCC are also involved in the pathway (Fig.
6B).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
Various agents inhibit apoE-peptide-induced
increases in phospho-CREB levels. Rat hippocampal neurons at day 7 of culture were treated with 2 µM apoE peptide for 30 min
in the presence or absence of 1 µM RAP(A), the
presence or absence of 100 µM MK801 or 20 µM nifedipine (B), the presence or absence of
10 µM U0126 or 10 µM
cAMP-dependent protein kinase inhibitor (C), and
the presence or absence of 10 µM SB203580, 10 µM KN62 or 10 µM U0126 (D). Cell
lysates (20 µg/lane) were electrophoresed on SDS-PAGE, Western
blotted, and probed with anti-phospho-CREB antibody or anti-CREB
antibody. The ratios of phosphorylated CREB to total CREB are plotted
as a mean ± S.E. of at least four separate experiments.
Significance of the difference between apoE-peptide alone
(bar labeled a) and treatment conditions were
calculated with Student's t test where double
asterisks represent p < 0.05 (A-D).
|
|
Activation of MEK and Protein Kinase A Is Involved in the
Pathway--
To find out which of the possible pathways involving apoE
receptors, LVDCC and/or NMDA receptors, contribute to the
phosphorylation of CREB, we examined the effect of inhibiting kinases
associated with these receptors by treatment with the U0126 inhibitor
of MAPK/ERK kinase (MEK) and with the c-AMP-dependent
protein kinase inhibitor of protein kinase A (PKA). Pretreatment with
one of these selective inhibitors, as well as pretreatment with both, mostly inhibited CREB phosphorylation suggesting that activation of MEK
and PKA are involved in this pathway (Fig. 6C).
ERK Cascade Is a Major Pathway Leading to the ApoE
Peptide-stimulated Phosphorylation of CREB--
To further investigate
the apoE stimulation pathway, we examined the effect of inhibiting
kinases associated with the receptor signaling by treatment with the
SB203580 inhibitor of p38 MAP kinase, KN62 inhibitor of
calmodulin-dependent protein kinases (CaMK II/IV), and the
U0126 inhibitor of MEK. Of these selective inhibitors, CREB
phosphorylation was mostly inhibited by pretreatment with U0126 while
pretreatment with KN62 or SB203580 did not appear to have any
significant effect on the phosphorylation status of CREB. This result
suggests that the ERK cascade is a major pathway leading to the
apoE-stimulated phosphorylation of CREB (Fig. 6D).
Elevation of Intracellular Ca2+ following ApoE Peptide
Treatment Involves the ApoE Receptor, NMDA Receptor, and L-type
Voltage-dependent Ca2+ Channel--
To
demonstrate if the elevation of intracellular Ca2+
parallels the phosphorylation of CREB, we measured the concentration of intracellular Ca2+ under unstimulated or apoE
peptide-stimulated conditions. Treatment with apoE peptide
significantly raised the concentration of intracellular calcium in rat
hippocampal neurons. Preincubation with RAP, MK801, or nifedipine
significantly attenuated this elevation, suggesting that the apoE
receptor, NMDA receptor, and LVDCC are involved in the pathway to
calcium elevation as was observed for the activation of CREB (Fig.
7A).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Intracellular calcium levels increase after
apoE-peptide treatment. As a measure of intracellular calcium
levels, Fura 2 fluorescence in 7-day-old rat hippocampal neuronal
cultures increases rapidly after treatment with apoE-peptide
(bar labeled a). This apoE-peptide stimulated
fluorescence is inhibited by pretreatment with RAP, MK801 at 10 or 100 µM, or nifedipine at 10 or 100 µM. Peak
values of intracellular calcium were observed about 40 s after
exposure to apoE-peptide in at least 10 neurons per experiment in each
of four separate experiments. Compared with the apoE-peptide alone,
significant inhibition of calcium fluorescence at the p < 0.01 are calculated with a Student's t test and marked
by a double asterisk (A). ApoE-peptide induces a
rapid elevation of intracellular calcium fluorescence in rat
hippocampal neurons (bar a) that is inhibited by culturing
in Ca2+-free growth medium or in the presence of growth
medium containing calcium and 10 µM dantrolene. Peak
concentrations of intracellular calcium in at least 10 neurons for each
of four experiments are plotted. Significance of the difference between
apoE-peptide alone and treatment conditions were calculated with
Student's t test where double asterisks
represent p < 0.01 (B).
|
|
A Major Part of the Intracellular Ca2+ Elevation May Be
from an Intracellular Source--
To determine whether calcium influx
from the extracellular space or from the endoplasmic reticulum (ER)
contributes to our apoE-mediated Ca2+ increases, we
measured the intracellular Ca2+ elevation in rat primary
hippocampal neurons cultured in medium lacking Ca2+ and
containing 1 mM EGTA (Fig. 7B). Under these
conditions, apoE-stimulated calcium elevation was significantly
attenuated suggesting that calcium influx from extracellular sources
significantly contributes to the elevation of intracellular
Ca2+. To confirm this finding, we also measured the
contribution of calcium release from the endoplasmic reticulum to the
increase in intracellular Ca2+. Rat primary hippocampal
neurons were preincubated with 10 µM dantrolene, an
inhibitor of ryanodine-sensitive Ca2+ channel found on the
ER membrane, followed by apoE peptide treatment. Interestingly,
preincubation with dantrolene also significantly attenuated the
elevation of intracellular Ca2+. Combining extracellular
Ca2+-free conditions and preincubation with dantrolene also
attenuated the calcium elevation, even though there was no significant
difference between the calcium levels measured under extracellular
Ca2+-free conditions without dantrolene preincubation and
the extracellular calcium-free condition with dantrolene preincubation.
Taken together, we hypothesize that the calcium influx from
extracellular sources triggers the release of Ca2+ from
intracellular endoplasmic reticulum sources through its ryanodine-sensitive Ca2+ channels. Calcium influx from
extracellular space seems to be essential for signaling the
apoE-stimulated elevation of intracellular Ca2+ levels, but
contributes only a small amount to the rise in intracellular calcium
levels compared with the calcium released from the ER.
 |
DISCUSSION |
Although evidence of the association between the
4 allele of
the APOE gene and Alzheimer's disease is overwhelming, the
mechanism by which the apoE4 protein isoform influences onset and
progression of the disease and its pathology is unknown. Of the many
suggested mechanisms, we have focused on reports that apoE3 and apoE4
protein isoforms have differential effects on neuronal plasticity and survival (13, 23, 24). Compared with the apoE3 protein isoform, apoE4
protein, proteolytic fragments of apoE4 protein, and peptides corresponding to the receptor-binding domain of apoE proteins appear to
actively injure, and certainly do not support maintenance of healthy
neurites and neuronal cells (13-16). On the larger scale, the failure
of apoE4 protein isoforms and their related fragments to support
neuronal plasticity and maintenance may infer a mechanism that
underlies the association between APOE gene alleles and disease.
Under many conditions that eventually result in neuronal death, the
cell struggles to induce protective mechanisms even though destructive
forces inevitably march forward. In this report, we have demonstrated
one such scenario where apoE4, but not apoE3, activates an ERK cascade
that results in activation of CREB and induction of many different
genes including the cell-protective gene, Bcl-2. In prior
reports, we showed that overexpression of only Bcl-2 protein could
inhibit neuronal death following a toxic insult (25). In this case,
treatment of neurons with recombinant apoE4 protein or with synthetic
apoE peptide resulted in CREB phosphorylation and induced Bcl-2
expression, events which were not observed following recombinant apoE3
protein treatments. Time course experiments showed that the increase in
phospho-CREB levels following apoE4 protein treatments appeared to be
slower than that by apoE peptide, although their molar concentrations
were the same. This time lag is consistent with previous results where protease inhibitors reduced the neurotoxicity of apoE4 (16) through a
presumed mechanism where the full-length apoE4 protein must be digested
over time to produce a toxic fragment of apoE4. Despite the increases
in phospho-CREB and Bcl-2, we also observed apoE4/apoE peptide-mediated
increases in calcium levels that were also observed by others in the
context of their association with neuronal death (16, 26).
The apoE4/apoE-peptide-mediated increase of intracellular
Ca2+ can be generated by calcium influx from the
extracellular space and release of calcium from intracellular stores.
Significant attenuation of increased intracellular calcium levels was
observed when we cultured rat primary hippocampal neurons in the medium lacking Ca2+ and containing EGTA as a Ca2+
chelator, suggesting that calcium influx from extracellular sources follows apoE4/apoE peptide treatments. When we measured the
intracellular Ca2+ elevation following preincubation with
10 µM dantrolene, an inhibitor of ryanodine-sensitive
Ca2+ channel on ER membranes, Ca2+ elevation
was also significantly attenuated, suggesting the participation of the
calcium influx from ER in the elevation of intracellular Ca2+. Interestingly, extracellular Ca2+-free
conditions did not seem to have any additional effect on attenuating
the Ca2+ elevation following preincubation with dantrolene,
suggesting that calcium influx from the extracellular space may serve
to trigger a larger amount of Ca2+ release from the ER
through ryanodine-sensitive Ca2+ channel in a
"Ca2+-induced Ca2+-release" fashion. Thus,
calcium influx from extracellular space may be less than calcium
release from intracellular sources such as the endoplasmic reticulum.
The apoE peptide-mediated increase in phospho-CREB levels and of
intracellular Ca2+ levels was inhibited by pretreatment
with RAP, MK801, or nifedipine, suggesting that apoE receptors, NMDA
receptor, and LVDCC are involved in rat hippocampal neuron responses to
apoE. Tolar et al. (16) reported that RAP and MK801
attenuated the elevation of intracellular Ca2+ caused by
truncated apoE. They also reported that nifedipine did not provide any
protection against the apoE-mediated rise in intracellular
Ca2+ in rat hippocampal neurons, but data was not shown.
Wang et al. (26) reported that RAP, MK801, and diltiazem,
another inhibitor of LVDCCs, failed to block the apoE peptide-induced
calcium influx in rat primary cultured neurons. Assuming that
neurotoxicity is caused by calcium influx following apoE treatment, our
data are consistent with the data provided by Tolar et al.
(16) in that RAP and MK801 attenuated calcium influx (16). This result
suggests that LRP or some other apoE receptors found in neurons may be linked to NMDA receptor. Our finding that preincubation with nifedipine almost completely inhibited the apoE peptide-induced calcium influx is
reasonable because calcium entry through NMDA receptors may depolarize
the plasma membrane and open LVDCCs that results in larger amount of
calcium influx. Compared with the calcium entry from LVDCCs, calcium
entry from NMDA receptors may be negligible (27). Nevertheless, this
calcium elevation can activate kinases such as ERK and calmodulin
kinase II that function to phosphorylate CREB (27).
Further inhibition experiments showed that pretreatment with a MEK
inhibitor and/or a PKA inhibitor mostly prevented the apoE-peptide stimulated phosphorylation of CREB, suggesting that activation of both
MEK and PKA kinases are involved in the pathway. Ca2+
elevation stimulates CREB phosphorylation by activation of a PKA-dependent, Rap1-MEK-ERK pathway (28). Pretreatment with KN62 or SB203580 did not appear to have any effect on the
phosphorylation status of CREB, suggesting that the ERK cascade is a
major pathway leading the phosphorylation of CREB following apoE treatment.
Upon binding to the CRE element of a gene's promoter, the
transcription factor known as CREB facilitates transcription of many
different genes, such as c-fos, Bcl-2, and
BDNF (27, 29-31). CREB that actively binds its DNA element,
CRE, is typically phosphorylated on serine at position 133. Activation
of CREB by its phosphorylation at serine residue 133 is modulated by a
variety of effectors.
-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA) /kainate receptors and NMDA receptors are
associated with synapses and LVDCCs are involved in neuronal functions
like long-term potentiation (32-34). Stimulation of these receptors
has also been associated with increased intracellular Ca2+
levels, potentiation of kinase activities, and CREB activation.
To clarify if calcium influx caused by apoE treatment causes
neurotoxicity and parallels phosphorylation of CREB, we measured the
elevation of intracellular Ca2+ caused by apoE peptide in
rat hippocampal neurons upon exposure to these receptor antagonists. As
others has reported (16), treatment of apoE peptide to rat primary
hippocampal neurons caused calcium influx and intracellular
Ca2+ levels to rise. Preincubation with RAP, MK801, or
nifedipine clearly attenuated the Ca2+ elevation (Fig.
7A), which parallels our data on CREB phosphorylation mediated by apoE peptide, suggesting the involvement of apoE receptor.
We have shown a possible mechanism whereby apoE4 facilitates the
expression of CRE-driven genes and apoE3 does not (Fig.
8). We hypothesize that apoE4, which
stimulates CREB phosphorylation, may be associated with synaptic
plasticity. This apparent isoform specificity of the effects might be
relevant to the isoform-specific association of apoE4 as a significant
risk factor in late-onset Alzheimer's disease.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 8.
A model of apoE-mediated CREB activation in
hippocampal neurons. When apoE4, but not apoE3, binds to apoE
receptor on the surface of hippocampal neuron, NMDA receptors open. Its
mechanism is unclear. Activation of NMDA receptor causes small amounts
of Ca2+ entry into postsynaptic spines. The resulting
depolarization triggers the opening of L-type
voltage-dependent calcium channels exist in cytoplasmic
membranes of the neurons. The activation of L-type
voltage-dependent calcium channels, which is open during
strong depolarization, permit the entry of larger amount of
Ca2+ along the dendrites and into the cell bodies, causing
the opening of ryanodine-sensitive Ca2+ channel on ER
membrane and the influx of larger amount of Ca2+ from ER in
Ca2+-induced Ca2+ release fashion. The
Ca2+ entry from extracellular space may be negligible,
comparing that from ER calcium elevation allows activation of
PKA-dependent Rap1-ERK pathway and phosphorylation of
CREB.
|
|