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INTRODUCTION |
Apoptosis, or programmed cell death, plays an important role not
only in neuronal development and differentiation of the central nervous
system but also in the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer's disease. However, the molecular events
or cascades underlying neuronal death regulated by the genetic program
still remain unclear. Elucidation of the molecular mechanisms
underlying neuronal death could contribute to understanding of the
pathophysiology of neurodegenerative disorders such as Alzheimer's disease.
Previously, we isolated a novel gene named DP5 that is induced during
neuronal apoptosis using rat sympathetic neurons in culture deprived of
NGF (1).1 This gene has the
following unique features: 1) the encoded protein has a BH3 domain,
which is essential for interaction with Bcl-2 and Bcl-xl, and a
transmembrane region at its C-terminal; 2) its expression shows marked
induction with peak levels at 15 h after NGF withdrawal,
concurrent with the time at which neurons are committed to die in the
sympathetic culture model; and 3) overexpression of full-length DP5 in
cultured neurons was sufficient to induce apoptosis. In the developing
murine nervous system, DP5 mRNA was localized in several tissues
such as the trigeminal and dosal root ganglia and the anterior horn of
the spinal cord, which are known to contain a number of apoptotic cells
in the mouse embryo. These observations suggested that DP5 could be
associated with the phenomena of neuronal death in vivo
(2).
Recently, Inohara et al. (3) cloned the human gene Harakiri
(Hrk), which physically interacts with Bcl-2 and Bcl-xl. The polypeptide encoded by Hrk has a BH3 domain and transmembrane region
and is highly homologous with DP5 (72% identity), suggesting that DP5
and Hrk are homologues from different species. Nbk/Bik (4, 5) and Bid
(6) were identified as proteins that contain only BH3, and both
interact with members of the Bcl-2 family and have death-promoting
activities. However, these proteins do not show any significant amino
acid homology beyond the conserved BH3 domain.
Amyloid
protein (A
) damages and kills cultured neurons by a
mechanism involving oxidative stress and disruption of cellular calcium
homeostasis (7-12). Morphologically, this type of neuronal death shows
hallmarks of apoptosis including cellular shrinkage, blebbing of the
plasma membrane, nuclear condensation, and nucleosomal fragmentation
(13-15). To gain insight into the neuronal responses to A
at the
molecular level, analyses of the effects of A
treatment on neuronal
gene expression in vitro have been carried out (16). Some
gene expression patterns induced by A
treatment were markedly
similar to those of sympathetic neurons deprived of NGF, suggesting
that a genetic cascade is necessary for neuronal death following
exposure to A
similarly to NGF-deprived neuronal death.
In the present study, we determined whether DP5 gene expression was
closely associated with the process of neuronal apoptosis induced by A
in addition to sympathetic neuronal death as reported previously
(1). Furthermore, the changes in binding between DP5 and the death
repressor protein Bcl-xl were also examined in the culture model. We
report here that DP5 mRNA expression was induced and the encoded
protein interacted specifically with Bcl-xl during neuronal death
following exposure to A
. Our in vitro results suggested
that DP5 may have an important role in neuronal apoptosis induced by
treatment with A
and in the neuronal loss associated with
Alzheimer's disease.
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EXPERIMENTAL PROCEDURES |
Primary Culture and Treatment with A
--
Primary cultures
of neuronal cells were prepared from the cortex of fetal rats at 18 days of gestation. The dissected tissues were treated with papain
(Sigma), 0.02% DL-cysteine-HCl, 0.02% bovine serum
albumin, 0.5% glucose, and 0.1% DNase to dissociate the cells.
Aliquots of 1×107 cells were plated in 10-cm dishes coated
with poly-L-lysine and maintained at 37 °C in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
On the next day, the medium was changed to Dulbecco's modified
Eagle's medium containing B27 supplement (Life Technologies, Inc.) in
place of fetal calf serum, and culture was maintained for 5 days before
A
stimulation. A
25-35, A
1-40, and
A
40-1 (Bachem) stock solutions were prepared as
1-mM stocks in sterile distilled water. Cultured rat primary neurons were exposed to A
by the culture medium replacing with Dulbecco's modified Eagle's medium/B27 containing 20 or 40 µm
A
. The numbers of living cells were counted based on morphological criteria and trypan blue staining at various time points after treatment with A
.
RT-PCR Analysis--
Aliquots of 3 µg of total RNA purified
from preplated cortical neuron cultures were reverse-transcribed using
300 units of Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) in 60-µl reaction mixtures in the presence of 2.5 µM oligo(dT) primer and 20 µM dNTP mixture
for 60 min at 37 °C. For PCR amplification, specific oligonucleotide
primer pairs (0.5 µM each) were incubated with 1 µl of
cDNA, 1 unit of Taq polymerase, 1 × Taq
buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2), 10 µM dNTP mixture, and 10 µCi of [
-32P]dCTP in a 20-µl reaction
mixtures. The sequences of primers used in this study were as follows:
DP5 sense primer, 5'-AGACCCAGCCCGGACCGAGCAA-3', and DP5 antisense
primer, 5'-ATAGCACTGAGGTGGCTATC-3'; neurofilament-M sense primer,
5'-TGGCTTAGATGTGAGCCCTG-3', and neurofilament-M antisense primer,
5'-GACTATGGCATGTGAAGTGACC-3'; Bcl-2 sense primer, 5'-CTGGTGGACAACATCGCTCTG-3', and Bcl-2 antisense primer,
5'-GGTCTGCTGACCTCACTTGTG-3'; Bcl-xl sense primer,
5'-AGGCTGGCGATGAGTTTGAA-3', and Bcl-xl antisense primer,
5'-CGGCTCTCGGCTGCTGCATT-3'; Bax sense primer,
5'-TGGTTGCCCTTTTCTACTTTG-3', and Bax antisense primer,
5'-GAAGTAGGAAAGGAGGCCATC-3'. Typical PCR parameters were 1 min at
94 °C, 1 min at 60 °C, and 30 s at 72 °C for 22-24
cycles followed by 72 °C for 5 min. Aliquots of 10 µl of each
reaction mixture were electrophoresed through 5% polyacrylamide gels,
and the dried gels were subjected to autoradiography. Control
experiments were performed to determine the range of PCR cycles over
which amplification efficiency remained constant. The identity of each
PCR product was confirmed by subcloning the amplified cDNAs into
the pGEM-T vector (Promega) and sequencing.
Interaction of DP5 and Bcl-2 Family--
Aliquots of
1×107 primary neurons were plated on 10-cm dishes. Two
dishes were used per immunoprecipitation. Cells were harvested and
rinsed twice with PBS and then lysed in 1 ml of buffer containing 10 mM Tris-HCl (pH 7.8), 0.2% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 10 µg/ml aprotinin.
Lysates were centrifuged at 13,000 rpm for 5 min to remove large
cellular debris. For each immunoprecipitation experiment, 3 µg of
antibody was used. Samples were incubated for 1 h at 4 °C on a
rocker with the antibodies. Recombinant protein G agarose (Life
Technologies, Inc.) was added to each sample followed by another 1-h
incubation at 4 °C on a rocker. The beads were then washed five
times in lysis buffer to reduce nonspecific binding. After the last
wash, all buffer was removed, and reducing sample buffer was added to
each reaction mixture. Samples were boiled and loaded onto a 5-20%
gradient SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred onto Immobilon P membranes (Millipore). Blots were
preblocked in PBS containing 5% nonfat milk, and washes were performed
using PBS containing 0.1% Tween 20 (PBS-T). Primary antibodies were
used at 0.2-0.5% (v/v), and detection was performed with 0.1%
alkaline phosphatase-conjugated goat anti-rabbit IgG (Boehringer
Mannheim) or goat anti-mouse IgG (Sigma) in PBS-T with 5% nonfat milk
by the alkaline phosphatase method. Anti-Bcl-2 and Bcl-xl monoclonal
antibodies were purchased from MBL (Japan). Clone 9E10 monoclonal
antibody was used for detection of Myc epitope sequence. Anti-DP5
polyclonal antibodies were raised against the recombinant glutathione
S-transferase-DP5 fusion protein (1) and peptide
corresponding to amino acid residues 25-58 of DP5.
Plasmid Construction--
The mammalian expression plasmids
SFFV-human Bcl-2 and mouse Bcl-xl were provided by Prof. Nunez
(University of Michigan Medical School). The Myc epitope sequence was
attached to the rat DP5 cDNA to generate Myc-rat DP5 by PCR and
cloned into pCDNA3 or pIND (Invitrogen).
Human embryonic kidney 293T cells were used for transient transfection.
10-cm culture dishes containing 5×106 cells were
transfected with 5 µg of plasmid DNA by lipofection (LipofectAMINE,
Life Technologies, Inc.). The levels of expression of each protein were
determined in total lysates by Western blotting. For inducible
expression, transfection of plasmids and induction of expression were
performed according to the supplier's recommendations. Briefly,
Myc-DP5 cloned into pIND was cotransfected with pVgRXR into 293T cells.
On the next day, cells were treated with 3 µM muristerone
A (Invitrogen) to induce intracellular expression from pIND.
X-Gal Staining--
For X-gal staining of cells expressing
-galactosidase, cells were fixed in 1% glutaraldehyde for 3 min and
stained with X-gal solution (100 mM sodium phosphate buffer
(pH 7.2), 10 mM KCl, 1 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM
K4Fe(CN)6, 0.1% Triton X-100, and 0.1% X-gal)
at 37 °C.
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RESULTS |
Structure of DP5 Polypeptide and Its Interaction with Members of
the Bcl-2 Family--
DP5 has a BH3 domain, which was shown to be
essential for the interaction with Bcl-2 and Bcl-xl proteins (Fig.
1). Recently, Hrk, which was considered
to be a human DP5 homologue, was shown to interact with Bcl-2 and
Bcl-xl by in vitro transfection analysis (3). To confirm
that rat DP5 interacts with members of the Bcl-2 family, 293T cells
were transiently co-transfected with expression plasmids producing
Myc-tagged DP5 and Bcl-xl. Immunoprecipitates were prepared using
anti-Myc monoclonal antibody and subjected to immunoblotting with
anti-Bcl-xl antibody. Western blotting with anti-Bcl-xl antibody
revealed that 30-kDa Bcl-xl was co-immunoprecipitated with Myc-DP5
(Fig. 2A). As the reverse
experiment, we performed immunoprecipitation using anti-Bcl-xl
antibody, followed by blotting with anti-Myc antibody. The 10-kDa
Myc-DP5 was co-immunoprecipitated with Bcl-xl (Fig. 2B). To
examine the interaction of Bcl-2 and DP5, we performed
immunoprecipitation experiments similar to those used to assess the
DP5-Bcl-xl interaction. Our results confirmed that DP5 specifically
interacted with Bcl-2 (data not shown). However, we detected only a
faint band of Bcl-2, which interacted with Myc-DP5 in contrast with the
results of immunoprecipitation with anti-Bcl-xl antibody. Accordingly,
we examined only the interaction of DP5 with Bcl-xl in the next set of
experiments.

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Fig. 1.
Structure of DP5 polypeptide.
A, amino acid sequence of DP5 polypeptide. DP5 consists of
92 amino acids. The conserved BH3 domain and putative transmembrane
region are indicated by a box and single
underline, respectively. B, schematic structures of DP5
and Bcl-2 family. C, comparison of the BH3 domains between
DP5 and Bcl-2 family members. The amino acids surrounded by
dotted lines are conserved in all members.
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Fig. 2.
Interaction of DP5 and Bcl-xl in 293T
cells. 293T cells were transiently transfected with the indicated
plasmids. The DP5 expression plasmid was tagged with the human Myc
amino acid sequence at the N terminus of DP5. Transfection was
performed with equal amounts of plasmid DNA using empty plasmid as a
control. A, lysates were immunoprecipitated (IP)
with anti-Myc antibody. Immunoprecipitates were immunoblotted with
anti-Bcl-xl antibody (upper panel) and anti-Myc antibody
(lower panel). B, immunoprecipitates with
anti-Bcl-xl antibody were immunoblotted with anti-Bcl-xl antibody
(upper panel) and anti-Myc antibody (lower
panel).
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Neuronal Death by A
Toxicity--
To determine the neuronal
toxicity of A
, cortical neurons were exposed to A
25-35 or A
1-40 at a concentration of
40 µM. Viability was quantified by trypan blue staining
and morphological criteria at various time points. Neurons began to degenerate asynchronously, exhibiting shrinkage and irregularly shaped
cell bodies with dystrophic neurites from about 12 h after A
exposure (Fig. 3). At 24 h, cultures
treated with both A
25-35 and A
1-40
showed 40% cell survival compared with controls. Neuronal death was
prevented by treatment with cycloheximide, a protein synthesis
inhibitor, at the same time as addition of A
. These results
suggested that A
-induced cell death is dependent on macromolecular
synthesis and is controlled by a genetic program. Our observations were
consistent with those of previous studies (13, 16, 17).

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Fig. 3.
A neurotoxicity in
rat cortical neuronal culture. Representative phase-contrast
micrographs of cortical neurons. A, control culture.
B, culture exposed to 40 µM A
25-35 for 24 h. A -treated neurons underwent
degeneration; degenerating neurons showed disruption of neurites,
shrinkage and irregularly shaped cell bodies. C, time course
of A neurotoxicity. Culture neurons were exposed to 40 µM A 25-35 and 40 µM A
1-40 with or without cycloheximide (1 µg/ml). Changes
in survival of neurons at the specified time points were determined.
Results are presented as the mean percentages of surviving cells ± SD observed in five independent experiments.
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Expression of DP5 mRNA during Neuronal Death--
To determine
the temporal changes in levels of DP5 mRNA during neuronal death
induced by A
, we performed reverse transcription of mRNAs
isolated from cortical neuronal cultures before and at various time
points after A
treatment and analyzed the proportions of DP5
cDNAs obtained using RT-PCR (Fig. 4).
We also analyzed the expression patterns of members of the Bcl-2 family
including Bcl-2, Bcl-xl, and Bax. DP5 expression level was relatively
low before A
stimulation. This signal increased at 6 h after
addition of 40 µM A
25-35, and the level
was maintained at least until 12 h, showing a subsequent reduction
after this time point that appeared to be consistent with neuronal loss
(Fig. 4A). Stimulation with A
25-35 and A
1-40 showed almost equivalent induction of DP5 mRNA
(Fig. 4B). Moreover, when cortical neurons were exposed to A
25-35 or A
1-40 at a concentration of
20 µM, the pattern of expression of DP5 mRNA was the
same as that at 40 µM (data not shown). On the other
hand, the levels of Bcl-2 family mRNA expression did not change or
were slightly diminished during the course of cell death, and DP5
mRNA expression was not induced by treatment with 40 µM A
40-1 (Fig. 4C). These
results suggested that within the Bcl-2 family, DP5 mRNA was
selectively induced during A
-induced neuronal death.

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Fig. 4.
RT-PCR analysis of DP5 mRNA expression
after treatment with A . Cortical neurons
were exposed to 40 µM A 25-35
(A), A 1-40 (B), and A
40-1 (C) for the indicated intervals. Total
RNA was isolated at each time point and then reverse-transcribed
followed by PCR analysis for DP5 and Bcl-2 family members as described
under "Experimental Procedures." Neurofilament-M (NF)
was used as an internal marker.
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Previous studies showed that disruption of cellular calcium homeostasis
occurs in neuronal apoptosis induced by A
(7, 11, 17-19) or
deprivation of NGF (20, 21). Indeed, nifedipine, a blocker of
L-type voltage-dependent calcium channels (22), and dantrolene, an inhibitor of calcium release from ER stores (23),
prevent neuronal death induced by A
. We next examined the changes
in DP5 mRNA expression following treatment of cortical neurons with
nifedipine or dantrolene. To test the abilities of these two agents to
promote survival, cortical neurons treated with 40 µM A
25-35 were cultured for 24 h with or without these
agents, and the numbers of surviving neurons were counted. Without
additives, approximately 60% of neurons died following exposure to A
, whereas neuronal death was prevented in the presence of nifedipine
or dantrolene (Fig. 5A). After
cortical neurons were treated with 40 µM A
for 6 h in the presence or absence of these agents, total RNA was extracted
and analyzed for changes in DP5 gene expression by RT-PCR. Levels of
products amplified by DP5-specific primers were markedly decreased in
all cases treated with these two agents (Fig. 5B).
Quantification of the DP5 signals showed that the expression levels of
DP5 in cortical neurons treated with calcium blockers were decreased to
approximately the same levels as in nontreated controls (Fig.
5C).

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Fig. 5.
DP5 mRNA expression on treatment of
cortical neurons with agents that prevent neuronal death induced by
A 25-35. A, after
cortical neurons were treated with 40 µM A
25-35 for 12 h in the presence or absence of 2 or
5 µM nifedipine or 2 or 5 µM dantrolene,
the numbers of dying cells were counted. The data (means ± S.D.)
shown are percentages of dead cells. Data were collected from at least
five independent experiments. B, RT-PCR analysis of changes
in DP5 expression 6 h after exposure to 40 µM A
25-35 in the presence of cell death blockers.
Neurofilament-M was used as an internal control. C,
quantification of changes of DP5 expression suppressed by cell death
blockers. Changes in the levels of DP5 were quantified by Scanning
Imager (Molecular Dynamics) analysis of polyacrylamide gels such as
that shown in B. Cont, control.
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Interaction of DP5 and Bcl-2 Families during Neuronal Death Induced
by A
--
To determine whether DP5 interacts with members of the
Bcl-2 family during neuronal apoptosis following exposure to A
, we performed immunoprecipitation followed by immunoblotting analysis of
DP5 and Bcl-xl after treatment with A
(Fig.
6). The expression of DP5 protein was
increased in cortical neurons 6 h after exposure to A
25-35, consistent with the DP5 mRNA expression
pattern (Fig. 6A). DP5 did not bind with Bcl-xl before A
stimulation. At 6 h after treatment with A
, a 30-kDa band of
Bcl-xl was detected in immunoprecipitates by anti-DP5 antibody (Fig.
6C). The expression levels of Bcl-xl proteins were
equivalent in A
-stimulated and nonstimulated cultured neurons (Fig.
6B). These results suggested that DP5 specifically
interacted with Bcl-xl during neuronal death induced by A
.

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Fig. 6.
Interaction of DP5 and Bcl-xl during neuronal
death induced by A 25-35.
Primary cultures were maintained in the presence or absence of 40 µM A for 6 h before harvesting. Following
immunoprecipitation with anti-recombinant DP5 antibody, samples were
subjected to immunoblotting analysis using anti-Bcl-xl antibody.
Although control lysates did not show a 30-kDa Bcl-xl band, lysates
from neurons treated with A for 6 h showed a single Bcl-xl
band (C). Western blotting showed that DP5 protein was
induced in cortical neurons treated with A (A), and the
amounts of Bcl-xl protein in both cell lysates were equivalent
(B). The asterisk indicates a nonspecific
band.
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Mechanisms of Cell Death Induced by the Expression of
DP5--
Stimulation with A
resulted in an increase in level of
DP5 mRNA within 6 h. A specific interaction between DP5 and
Bcl-xl also occurred in cell death induced by A
. Our previous study indicated that overexpression of DP5 in cultured neurons was sufficient to induce cell death. Taken together, these observations suggested that
increased expression of DP5 and its interaction with Bcl-xl play a
significant role in the process of neuronal death. To examine the
events involved in DP5-induced cell death after interaction with
Bcl-xl, we analyzed the characteristics of DP5
expression-dependent cell death using ecdysone-inducible
expression systems (24). 293T cells were transiently transfected with a
pIND-derived plasmid expressing Myc-DP5 and pVgRXR plasmid. After
culture for 24 h, cells were incubated in the presence of 3 µM muristerone A, an ecdysone analogue, for various
periods. On addition of muristerone A, 293T cells rapidly began to
undergo apoptosis within 6 h and showed <40% viability at
24 h (Fig. 7). The cell death was
inhibited by the expression of Bcl-xl. Western blotting analysis
revealed induction of DP5 by 1 h, and the level of expression was
maintained up to 24 h after treatment with muristerone A.

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Fig. 7.
DP5 induces apoptosis in 293T cells.
A and B, phase-contrast photographs of 293T cells
after induction of DP5 in response to muristerone treatment (X-gal
staining). X-gal-stained DP5-expressing 293T cells showed shrinkage and
blebbing (B), and these cells increased in number with time
after induction, whereas the Bcl-xl expression plasmid pSFFV/Bcl-xl
that was co-transfected with the DP5-inducible plasmid showed intact
morphology (A). C, time course of changes in
number of living cells. The percentages of blue intact cells are shown.
The data are the means ± SD from 10 fields counted in three
independent transfection experiments. D, Protein
immunoblotting analysis of DP5 expression after induction by
muristerone. Lysates of cells at various time points after
muristerone-induction were subjected to Western blotting analysis using
anti-DP5 antibody.
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Cell death was significantly attenuated in the
Ca2+-depleted state by exposure to 1 mM EGTA
(Fig. 8B), which provided
preparations with low cytosolic and low sequestered Ca2+
(22). Dantrolene also prevented cell death. In contrast, depolarization by high potassium (35 mM) or nifedipine did not prevent
cell death induced by expression of DP5, suggesting that the cell death
involves calcium release from ER and the resultant disruption of
calcium homeostasis causes cell death.

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Fig. 8.
The effects of various agents on DP5-induced
cell death. A, the expression of DP5 protein 6 h
after addition of muristerone with dantrolene or nifedipine. The
amounts of DP5 protein in each cell lysate were equivalent.
B, cell death prevented by various agents. Living cells were
counted 24 h after addition of muristerone in the presence of 35 mM KCl, 1 mM EGTA, 2 or 5 µM
dantrolene, or 2 or 5 µM nifedipine (means ± S.D.).
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DISCUSSION |
Previous studies showed that A
induces apoptosis in neurons in
primary culture (13, 16, 17). The mechanisms of A
neurotoxicity
involve membrane lipid peroxidation and impairment of intracellular
calcium homeostasis (7-12). However, the detailed mechanisms are still
unclear. We found that the messenger RNA of DP5, which has been cloned
as a gene induced in programmed cell death of sympathetic neurons
deprived of NGF (1), was selectively elevated after A
stimulation.
Recently, gene expression patterns during neuronal death induced by
treatment with A
were reported to be markedly similar to those
observed in the models of sympathetic neurons deprived of NGF,
i.e. induction of c-jun begins in the early stage
followed by c-fos, fosB at the time of commitment
to cell death in both culture models (16, 25). The temporal patterns of
expression of several genes including DP5 during neuronal death after A
treatment indicated that A
stimulus activates a cellular
genetic program for cell death similarly to NGF deprivation.
Calcium influx contributes to A
-induced neuronal degeneration
because removal of extracellular calcium (26) and calcium channel
blockers (18, 19) protect neurons against A
toxicity. So we
examined whether the DP5 gene induction after exposure to A
was
changed following treatment with these agents. Cell death was prevented
by treatment with nifedipine and dantrolene, which are blockers of
L-type voltage-dependent calcium channels and of calcium release from the ER, respectively. In these cases, the
expression of DP5 mRNA was significantly suppressed, suggesting that induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A
. We considered that A
stimulus caused the influx of extracellular calcium, calcium release from the ER, and accumulation of reactive oxygen species, followed by activation of the apoptosis cascade involving induction of
cell death-promoting genes such as DP5.
The protein encoded by DP5 mRNA contains a BH3 domain that is
critical for interaction with members of the Bcl-2 family and regulation of apoptosis. Harakiri (Hrk), which was reported
to bind to members of the Bcl-2 family (3), is considered to be a human
homologue of DP5 because it shows 72% overall amino acid sequence
identity and the sequence of the BH3 region is completely conserved.
Hrk physically interacts with Bcl-2 and Bcl-xl at the BH3 region. We
confirmed that DP5 also possessed the ability to interact with Bcl-2
family members. Nbk/Bik, Bid, and Hrk are known as proteins that
contain only BH3 and were identified recently as interacting partners
with Bcl-2 family members (3-6). Overexpression of these proteins
including DP5 is sufficient for induction of apoptosis in several types
of cells. The mechanisms by which these molecules induce apoptosis are
not well known. There are two possible mechanisms as follows: proteins
that contain only BH3 may be death effector molecules, i.e.
Bcl-2 or the other Bcl-2 family members may be dominant negative
regulators; alternatively, these molecules may promote cell death by
inhibiting the death-suppressing activities of the Bcl-2 family. In the
present study, the level of expression of DP5 in neurons was low under
normal conditions. Death signals such as A
stimulus caused the
accumulation of DP5 mRNA, but levels of expression of Bcl-2 family
members were not changed or were diminished. Furthermore, the protein
encoded by DP5 mRNA interacted with Bcl-xl during cell death. These
observations lead us to hypothesize the scenario as follows: A
stimulus causes the selective accumulation of DP5 mRNA by
increasing intracellular calcium concentration involved in activation
of the cell death cascade followed by interaction of Bcl-2 family
members and DP5 protein via the BH3 region. Binding to the BH3 domain
could impair the survival-promoting activities of the Bcl-2 family (27,
28). However, we cannot exclude the possibility that DP5 could be an effector molecule because overexpression of Bcl-xl inhibited the killing activity of DP5.
To examine the molecular events involved in DP5
expression-dependent cell death, we established an
ecdysone-inducible expression system for DP5. 293T cells expressing DP5
rapidly underwent apoptosis in this system, and this cell death was
blocked by perturbation of intracellular calcium concentration.
Dantrolene, which is an inhibitor of calcium release from the ER and
which is known to protect neurons against A
toxicity (29), and EGTA
prevented cell death induced by DP5. These agents provide the situation of low intracellular concentrations of calcium. In contrast,
nifedipine, a blocker of L-type
voltage-dependent calcium channels, had no effect. These
results indicated that DP5-induced cell death involves calcium release
from ER, but not calcium influx through plasma membrane channels.
Previous studies have shown that ER calcium regulation contributes to
apoptosis of neuronal (29) and nonneuronal cells (30, 31). Moreover,
Bcl-2 protects lymphoma cells against apoptosis induced by
thapsigargin, an inhibitor of ER calcium-ATPase, and suppresses release
of calcium from the ER (32, 33). Collectively, overexpression of DP5
could affect intra- and extra-ER calcium homeostasis, and the resultant
increase in cytosolic calcium concentration could lead to
apoptosis. The localization of DP5 and its human homologue, Hrk,
to the membranes of intracellular organelles (3) supports the above
hypothesis. However, it is unclear whether DP5 can itself generate
channels on the membranes similarly to Bax (34) or activate the IP3
pathway and how DP5 accelerates the calcium release from ER after
increase of cytosolic calcium concentration in neuronal death induced
by A
.
In conclusion, the present study strongly suggested that DP5 plays a
significant role in neuronal apoptosis followed by exposure to A
.
We are currently engaged in generation of DP5 knockout mice to examine
the resistance to A
toxicity using neurons derived from the mouse
brain. These experiments will help to elucidate the molecular
mechanisms underlying neurodegeneration induced by A
and may allow
for the development of therapeutic strategies for Alzheimer's disease.