Prostate Apoptosis Response-4 Enhances Secretion of Amyloid
Peptide 1-42 in Human Neuroblastoma IMR-32 Cells by a
Caspase-dependent Pathway*
Qing
Guo
,
Jun
Xie,
Xiaowei
Chang, and
Huimin
Du
From the Department of Neurobiology and Pharmacology, Northeastern
Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
Received for publication, December 6, 2000, and in revised form, February 21, 2001
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ABSTRACT |
Prostate apoptosis response-4 (Par-4) is a
leucine zipper protein that promotes neuronal cell death in
Alzheimer's disease (AD). Neuronal degeneration in AD may result from
extracellular accumulation of amyloid
peptide (A
) 1-42. To
examine the effect of Par-4 on A
secretion and to reconcile
amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that
overexpress Par-4 and/or its leucine zipper domain. Overexpression of
Par-4 did not significantly affect levels of the endogenously expressed
amyloid precursor protein but drastically increased the
A
1-42/A
total ratio in the
conditioned media about 6-8 h after trophic factor withdrawal. Time
course analysis of caspase activation reveals that Par-4 overexpression
exacerbated caspase activation, which is detectable within 2 h
after trophic factor withdrawal. Furthermore, inhibition of caspase
activity by the broad spectrum caspase inhibitor BD-fmk significantly
attenuated the Par-4-induced increase in A
1-42 production. In
addition, the effects of Par-4 on secretion of A
1-42 were
consistently blocked by co-expression of the leucine zipper domain,
indicating that the effect of Par-4 on A
secretion may require its
interaction with other protein(s). These results suggest that Par-4
increases secretion of A
1-42 largely through a
caspase-dependent pathway after apoptotic cascades are initiated.
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INTRODUCTION |
Mutations in familial Alzheimer's disease genes, such as
-amyloid precursor protein
(APP)1, presenilin-1, and
presenilin-2, have been shown to regulate the processing of APP and
result in increased production of the longer form of amyloid
peptide (A
), A
1-42 (1-5). It is widely accepted that neuronal
degeneration in AD is caused by extracellular accumulation of A
1-42. On the other hand, all three familial Alzheimer's disease genes
have been shown to regulate neuronal apoptosis, suggesting that
dysregulation of apoptotic pathways may play an important role in
neuronal degeneration in AD (6-10). Importantly, abnormal processing
of APP and increased production of A
may be induced by apoptotic
insults (11-14). Several studies show that APP in neuronal cells can
be processed by caspase-6 and -8 and that this processing can be
blocked by caspase inhibitors (12, 15).
We recently identified Par-4 as a novel cell death-promoting protein
associated with neuronal degeneration in AD (16, 17). The pro-apoptotic
actions of Par-4 require its interaction with other proteins via the
leucine zipper domain (Leu.zip; Refs. 16 and 17). Overexpression of
Leu.zip of Par-4 has been used as a dominant negative regulator of
Par-4 function because it competitively blocks the interaction of Par-4
with other proteins, thereby blocking the pro-apoptotic actions of
Par-4. Blockade of Par-4 function by Par-4 antisense treatment
or overexpression of Leu.zip significantly decreases neuronal apoptosis
induced by A
, trophic factor withdrawal, or overexpression of
Alzheimer's mutant presenilin-1 proteins in PC12 cells (16). In recent
studies in mutant presenilin-1 M146V knock-in (PS1mv KI) transgenic
mice (18-20), we found that the Alzheimer's presenilin-1 mutation
enhances Par-4 expression, resulting in increased vulnerability of
neurons to apoptosis and increased production of A
1-42 (18-20).
These data strongly suggest that induction of Par-4 is an important and
necessary event in the pathogenic mechanisms of Alzheimer's
presenilin-1 mutations and is very likely involved in the abnormal
processing of APP during the apoptotic process. To provide better
solutions for the treatment of Alzheimer's disease, it is very
important to reconcile amyloid/apoptosis hypotheses of AD and examine
how Par-4 might alter APP processing during apoptosis, leading to
increased production of the neurotoxic A
1-42.
The cell types responsible for overproduction of A
1-42 are not
completely known, although it has been suggested that A
1-42 is
produced by human central nervous system neurons from APP695 (1-5).
Human neuroblastoma IMR-32 cells have many features that resemble human
neurons in the central nervous system (21-23). Importantly, compared
with many other human neuronal cell lines, IMR-32 cells have been shown
to be able to synthesize a large amount of endogenous
APP751 and APP695 and secrete a significant amount A
1-42 together with A
1-40 (21-23). In addition, A
secreted by IMR-32 cells can be effectively detected using highly
sensitive sandwich ELISAs (21-23). These findings suggest that IMR-32
cells may provide a unique system to examine the possible role of Par-4 in APP processing. We report in this study that overexpression Par-4
increases secretion of A
1-42 from IMR-32 cells after trophic factor withdrawal by a mechanism involving interaction of Par-4 with
other proteins via the leucine zipper domain. In addition, we provide
evidence showing that the increase in secretion of A
1-42 induced
by Par-4 is a caspase-dependent event and occurs when
apoptotic cascades are initiated.
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MATERIALS AND METHODS |
Generation and Characterization of IMR-32 Cell Lines and Trophic
Factor Withdrawal--
Human neuroblastoma IMR-32 cells (American Type
Culture Collection) were maintained at 37 °C in an atmosphere of
95% air and 5% CO2 in Eagle's minimum essential medium
supplemented with nonessential amino acids and 10% heat-inactivated
fetal bovine serum. To express the full-length Par-4 in IMR-32 cells,
we subcloned a full-length Par-4 cDNA into the expression vector
pRc/CMV, yielding a recombinant construct pCMV-Par4, in which the
expression of the transgene was driven by enhancer-promoter sequences
from the immediate early gene of the human cytomegalovirus. This
construct encodes a 1.2-kilobase RNA species and a full-length 38-kDa
Par-4 protein. To address the role of the leucine zipper domain of
Par-4 in IMR-32 cells, we subcloned the cDNA of the leucine zipper
domain of Par-4 into the pREP4 expression vector, where the expression
of the transgene is under the control of the Rous sarcoma virus long
terminal repeat promoter. pREP-Leu.zip encodes an ~400-base pair RNA
species and the ~10-kDa leucine zipper domain of Par-4 protein.
IMR-32 cells were transfected with pCMV-Par4 or pREP-Leu.zip or
co-transfected with pCMV-Par4 and pREP-Leu.zip using LipofectAMINE
(Life Technologies, Inc.), and stable transfectants were selected for 4 weeks using G418 (for pCMV-Par4-transfected cells) and/or hygromycin
(for pREP-Leu.zip-transfected cells). Overexpression of Par-4 and/or Leu.zip in IMR-32 cells did not significantly affect the viability of
these cells under basal conditions. Selected clones of transfected cells were maintained in the culture medium containing G418 (400 µg/ml; for pCMV-Par4-transfected cells) or hygromycin (400 µg/ml; for pREP-Leu.zip-transfected cells) or both for co-transfected cells.
For control purposes, parallel cultures of IMR-32 cells were stably
transfected with the pRc/CMV and pREP4 vectors alone. After the cells
became confluent in the culture flasks, the culture medium was replaced
with fresh media, and cells were incubated for 48 h at
37 °C to condition the medium for A
measurement. Trophic factor
withdrawal was initiated by washing cultures four times with Locke's
buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2,
3.6 mM NaHCO3, 5 mM glucose, and 5 mM HEPES, pH 7.2) with subsequent incubation in 1 ml of
Locke's buffer.
Western Blot Analysis--
Levels of Par-4 in cultured IMR-32
cells were determined by Western blot analysis as described in our
previous studies (16). Western blot images were acquired and quantified
using Kodak Image Station 440 CF and Kodak Digital Science 1D 3.0.2. software. Generation and characterization of the affinity-purified
anti-Par-4 antibody have been described previously (16). The total
amount of cellular APP in various transfected cell lines was measured
by Western blot analysis using a monoclonal antibody (22C11; Roche
Molecular Biochemicals) that recognizes the N terminus of human
APP. Equal loading was verified by probing with the
anti-
-tubulin antibody (Sigma).
Quantification of Levels of A
1-40 and
A
1-42 by Sandwich ELISAs--
A
1-40 and A
1-42
levels in the conditioned media were measured using a
fluorescence-based sandwich ELISA described previously (19). The
C-terminal-specific sandwich ELISAs use a monoclonal antibody directed
against the N-terminal region of human A
and two other antibodies
specific for A
1-40 and A
1-42
(BIOSOURCE). Conditioned culture media were
collected and subjected to the sandwich ELISAs according to the
manufacturer's instructions. Standard curves were generated using
lyophilized synthetic human A
40 and human A
42 standards.
The standard A
40 and A
42 peptides were chemically modified by
the manufacturer to reduce aggregation of the peptides. Parallelism
between the standard A
peptides used in this study and the native
A
40 and A
42 peptides in terms of their immunoreactivity and
reliability in the ELISA has been well characterized by the
manufacturer. The specificity and sensitivity of the ELISAs have also
been characterized by the manufacturer. Fluorescence was quantified
with a fluorescence plate reader with excitation at 460 nm and emission
at 560 nm. Because absolute values of A
1-40 and A
1-42
secreted by IMR-32 cells under basal culture conditions showed some
variability among and even within cell lines, the ratio (percentage) of
A
1-42 to the total amount of A
(A
1-40 plus A
1-42),
which remains statistically consistent under basal culture conditions
among the various cell lines, was used to measure the changes in the relative amount of A
1-42 secreted from transfected IMR-32 cells.
Assessments of Apoptosis and Quantification of Caspase-3
Activity--
Apoptosis was quantified in cultures stained with the
fluorescent DNA-binding dye Hoechst 33342 as described previously (16). Hoescht 33342-stained cells were visualized and photographed under epifluorescence illumination (340 nm excitation and 510 nm barrier filter) using a ×40 oil immersion objective (200 cells per culture were counted, and counts were made in at least six separate cultures per treatment condition). Samples were analyzed without knowledge of
the treatment history of the cultures. Levels of caspase-3 activity
were quantified using a method that employed DEVD, a pseudosubstrate and inhibitor of caspase-3 (16). After treatment, cells
were incubated for 20 min in the presence of 0.02% digitonin plus 10 µg/ml biotinylated DEVD-CHO (Calbiochem, La Jolla,
CA), washed three times with phosphate-buffered saline (2 ml/wash), and fixed for 30 min in a cold solution of 4%
paraformaldehyde in phosphate-buffered saline. Cells were then
incubated for 5 min in phosphate-buffered saline containing 0.2%
Triton X-100, followed by a 30-min incubation in phosphate-buffered
saline containing 5 µg/ml Oregon Green-streptavidin (Molecular
Probes, Eugene, OR). Confocal images of cellular fluorescence
(corresponding to conjugates of activated caspase-3 with
DEVD-biotin) were acquired (488 nm excitation and 510 nm
emission). All images were acquired using the same laser intensity,
offset, and electronic gain to allow quantitative comparisons of
relative levels of fluorescence in cells. The average pixel
intensity/cell body was determined using Fluoview 2.0 software.
 |
RESULTS |
Generation and Characterization of IMR-32 Cell Lines Overexpressing
Full-length Par-4 and/or the Leucine Zipper Domain--
To establish
the role of Par-4 in A
secretion and its possible relationship to
apoptotic cell death and to address the ability of the leucine zipper
domain to mediate Par-4 actions in neural cells, we generated IMR-32
cell lines overexpressing full-length Par-4, Leu.zip alone, or a
combination of full-length Par-4 and Leu.zip. In previous studies, we
established that the leucine zipper domain fragment of Par-4 acts as a
dominant negative regulator of Par-4 activity when expressed in neural
cells, possibly by competitively abrogating the binding of Par-4 to
other proteins (16). On Western blots, the Par-4 polyclonal antibody
recognized full-length Par-4 (38 kDa) and Leu.zip (10 kDa) (Fig.
1). We chose for study two to three
clones of each of the cells overexpressing full-length Par-4, Leu.zip,
and Par-4+Leu.zip, based on their similar levels of expression of Par-4
and/or Leu.zip (Fig. 1). Western blot analyses showed that levels of
full-length Par-4 protein were ~8-fold higher in cell lines
transfected with Par-4 cDNA compared with untransfected,
vector-transfected, and Leu.zip-transfected cell lines. Bands of the
leucine zipper domain of Par-4 were clearly identified in cells
transfected or co-transfected with Leu.zip cDNA. Note that
overexpression of Par-4 and/or Leu.zip did not significantly affect the
levels of the endogenously expressed APP in the various cell lines used
in this study.

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Fig. 1.
Representative Western blots showing levels
of expression of Par-4 and the leucine zipper domain in transfected
IMR-32 cells used in the present study. Proteins from the
indicated cell lines were separated by SDS-polyacrylamide gel
electrophoresis (50 µg protein/lane), transferred to a nitrocellulose
sheet, and immunoreacted with a polyclonal anti-Par-4 antibody
(top), a monoclonal antibody that recognizes the N terminus
of human APP (middle), or a monoclonal
anti- -tubulin antibody (bottom).
Untransfected, untransfected parent cell line; Vector
alone, cell line transfected with empty vector; Par-4, C9,
Par-4, C6, and Par-4, C3, cell lines overexpressing
full-length Par-4; Par4+Leu.zip, C1, Par4+Leu.zip, C3, and
Par4+Leu.zip, C12, cell lines overexpressing both Par-4 and
Leu.zip; Leu.zip, C8 and Leu.zip, C10, cell lines
overexpressing Leu.zip. Note that transfected cell lines that
overexpress Par-4 express comparable levels of full-length Par-4 and
that transfected cell lines that overexpress Leu.zip express comparable
levels of Leu.zip. The total amount of cellular APP was at similar
levels among the various transfected cell lines.
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Effect of Overexpression of Par-4 on Secretion of A
1-42 from
IMR-32 Cells under Basal Culture Conditions and after Trophic Factor
Withdrawal--
Overexpression of Par-4 did not result in a
significant increase in spontaneous apoptosis in IMR-32 cells. To
examine whether overexpression of Par-4 and/or Leu.zip alters A
secretion from IMR-32 cells under basal conditions, levels of A
1-40 and A
1-42 in the conditioned media of various cell lines
were measured using a highly sensitive fluorescence-based sandwich
ELISA. As shown in Fig. 2, values of the
A
1-42/A
total ratio in the conditioned
culture media of wild type and vector-transfected control IMR-32 cells
were about 11-12%. Overexpression of Par-4, overexpression of
Leu.zip, or co-expression of Par-4 and Leu.zip did not result in
significant changes in the values of the
A
1-42/A
total ratio in the conditioned
culture media of various transfected IMR-32 cell lines, indicating that
Par-4 does not alter APP processing under basal culture conditions.
Because Par-4 increases the vulnerability of neuronal cells to
apoptosis induced by trophic factor withdrawal and A
(16), and
because APP has been reported to be processed in neuronal cells during
apoptosis by some cell death proteases (12, 15), we sought to examine
whether overexpression of Par-4 alters A
secretion from IMR-32 cells
after exposure of the cells to apoptotic insults, such as trophic
factor withdrawal. Time course analyses of secretion of A
peptides
in IMR-32 cells showed that values of the
A
1-42/A
total ratio in untransfected and
vector-transfected control cells and in cells overexpressing Leu.zip
remained statistically unchanged for up to 8 h after trophic factor withdrawal. This indicates that trophic factor withdrawal itself
is not sufficient to induce a significant increase in secretion of A
1-42 in IMR-32 cells (Fig.
3). However, overexpression of Par-4
drastically increased the A
1-42/A
total
ratio in the conditioned media about 6-8 h after trophic factor
withdrawal (Fig. 3). Consistent with a dominant negative mode of action
of Leu.zip involving protein-protein interactions, co-overexpression of
Leu.zip completely abolished the adverse effect of Par-4 on A
secretion, indicating that actions of Par-4 in A
secretion require
its interaction with other protein(s) via the leucine zipper domain
(Fig. 3).

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Fig. 2.
Under basal culture conditions,
overexpression of Par-4 does not alter the relative amount of
A 1-42 secreted by IMR-32 cells. The
percentage of A 1-42 as a fraction of total A (A 1-40 + A
1-42) secreted under basal culture conditions by the indicated clones
of transfected PC12 cells was quantitated by sandwich ELISAs as
detailed under "Materials and Methods." Untransfected,
untransfected parent cell line; Vector Alone, cell line
transfected with empty vector; Par-4, C9, cell line
overexpressing full-length Par-4; Leu.zip, C8, cell line
overexpressing the Leu.zip of Par-4; Par4+Leu.zip, C3, cell
line that overexpresses both full-length Par-4 and the Leu.zip of
Par-4. Values are the mean and S.E. of determinations made in six
separate cultures. ***, p < 0.001 compared with
values of the A 1-42/A total ratio in
untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell
groups. Similar data were obtained from cell lines Par-4, C6 and
Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12.
ANOVA was performed with Scheffe's post hoc tests.
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Fig. 3.
Par-4 significantly increases secretion of
A 1-42 from transfected IMR-32 cells after
trophic factor withdrawal: complete blockade by co-overexpression of
Leu.zip. Cultures of the indicated clones of transfected IMR-32
cells were deprived of trophic support for the indicated time periods,
and values of the A 1-42/A total ratio in
the conditioned culture media of transfected IMR-32 cells were measured
by sandwich ELISAs. Note that the values of the
A 1-42/A total ratio in untransfected and
vector-transfected control cells and in cells overexpressing Leu.zip
remained statistically unchanged after trophic factor withdrawal.
However, overexpression of Par-4 drastically increased the
A 1-42/A total ratio in the conditioned
media. This effect of Par-4 was not observed until ~6 h after trophic
factor withdrawal. Co-overexpression of Leu.zip completely abolished
the adverse effect of Par-4 on A secretion. Values are the mean and
S.E. of determinations made in six separate cultures. ***,
p < 0.001 compared with the corresponding values of
the A 1-42/A total ratio in untransfected,
vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data
were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip C10; and
Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with
Scheffe's post hoc tests.
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Par-4 Increases Secretion of A
1-42 after Initiation of
Apoptotic Cascades and by a Caspase-dependent
Pathway--
To test the hypothesis that Par-4 increases A
1-42
secretion by initiating early apoptotic cascades, we performed
additional experiments to examine whether Par-4 increases the
vulnerability of IMR-32 cells to apoptosis induced by trophic factor
withdrawal. Fig. 4 shows the impact of
overexpression of Par-4 on apoptosis of IMR-32 cells. Each of the cell
lines was deprived of trophic support for up to 48 h, and cells
with apoptotic nuclei were counted. Basal levels of apoptosis ranged
from 2-5% among the various cell lines. Forty-eight h after trophic
factor withdrawal, apoptosis was found in 33-42% of the control cell
lines and in cells overexpressing Leu.zip. Levels of trophic factor
withdrawal-induced apoptosis were significantly increased to about 74%
in cells overexpressing Par-4. Co-overexpression of Leu.zip completely
blocked trophic factor withdrawal-induced apoptosis and the
pro-apoptotic actions of Par-4. These results clearly demonstrate that
Par-4 does promote apoptosis in IMR-32 cells after trophic
factor withdrawal. Because cysteine proteases of the caspase family
have a prominent role in apoptosis, we examined the levels of caspase-3
activity and determined whether Par-4 played a role in caspase
activation in IMR-32 cells after trophic factor withdrawal. As shown in
Fig. 5, trophic factor withdrawal-induced
caspase activation was exacerbated in cells overexpressing Par-4, and
Leu.zip suppressed caspase activation induced by Par-4, indicating an
important role for Par-4 in IMR-32 cells in the early period of the
apoptotic cascades before caspase activation. To determine whether the
exacerbated caspase activation was responsible for the increase in A
1-42 secretion after trophic factor withdrawal, we examined whether the broad spectrum caspase inhibitor BD-fmk could block the
Par-4-induced increase in A
1-42 secreted by IMR-32 cells. As shown
in Fig. 6, the Par-4-induced increase in
secretion of A
1-42 after trophic factor withdrawal was
significantly attenuated by inhibition of caspase activation. Thus,
8 h after trophic factor withdrawal, the
A
1-42/A
total ratio in the conditioned
medium of cells overexpressing Par-4 was reduced from 57% without
BD-fmk treatment to 23% with BD-fmk treatment. These results strongly
suggest that Par-4 increases secretion of A
1-42 largely through a
caspase-dependent pathway.

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Fig. 4.
Par-4 increases the vulnerability of IMR-3
cells to apoptosis induced by trophic factor withdrawal: protection by
co-overexpression of Leu.zip. Cultures of the indicated clones of
transfected IMR-32 cells were deprived of trophic support for the
indicated time periods, and the percentage of cells with apoptotic
nuclei was then quantified. Values are the mean and S.E. of
determinations made in six separate cultures (measurements were made in
300-600 cells/culture). The cell lines used were the same as those
described in the Fig. 1 legend. ***, p < 0.01 compared with the corresponding values for untransfected,
vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data
were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip, C10;
and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with
Scheffe's post hoc tests.
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Fig. 5.
Early and exacerbated activation of caspase
activity in cells overexpressing Par-4 after trophic factor
withdrawal. Cultures of the indicated clones of transfected IMR-32
cells were deprived of trophic support for the indicated time periods,
and levels of cellular DEVD fluorescence, a measure of caspase-3
activity, were quantified. Note that trophic factor withdrawal-induced
caspase activation was significantly exacerbated in cells
overexpressing Par-4 as early as 2 h after trophic factor
withdrawal. Co-overexpression of Leu.zip completely blocked the effect
of Par-4 on caspase activation. Values are the mean and S.E. of
determinations made in six separate cultures. ***,
p < 0.001 compared with corresponding values in
untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell
groups. Similar data were obtained from cell lines Par-4, C6 and Par-4,
C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was
performed with Scheffe's post hoc tests.
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Fig. 6.
The broad spectrum caspase inhibitor BD-fmk
counteracts the adverse effect of Par-4 on APP processing after trophic
factor withdrawal. Cultures of the indicated clones of transfected
IMR-32 cells were pretreated with 100 µM of the broad
spectrum caspase inhibitor BD-fmk (Enzyme Systems Products, Livermore,
CA) for 2 h and then deprived of trophic support for 8 h.
Parallel cultures were prepared and deprived of trophic support for
8 h without BD-fmk pretreatment. Values of the
A 1-42/A total ratio in the conditioned
culture media of transfected IMR-32 cells were measured by sandwich
ELISAs. Note that overexpression of Par-4 drastically increased the
A 1-42/A total ratio. Inhibition of
caspase inhibition by BD-fmk significantly inhibited the adverse effect
of Par-4 on A 1-42 secretion. Values are the mean and S.E. of
determinations made in six separate cultures. ***,
p < 0.001 compared with corresponding values of the
A 1-42/A total ratio in untransfected,
vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. ###,
p < 0.001 compared with the value of the
A 1-42/A total ratio in Par-4 cells
without BD-fmk pretreatment. Similar data were obtained from cell lines
Par-4, C6 and Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and
Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post
hoc tests.
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DISCUSSION |
To examine the possible effect of Par-4 on APP processing, we
generated and characterized stable transfected IMR-32 cell lines that
overexpress Par-4 and/or Leu.zip. Notably, overexpression of Par-4
and/or Leu.zip did not significantly affect the levels of the
endogenously expressed APP in the various cell lines used in this
study. We found that Par-4 increases secretion of A
1-42 from
IMR-32 cells after trophic factor withdrawal. We further addressed some
key questions about the mechanisms by which Par-4 causes the observed
increase in A
1-42 production. We found that Par-4 induced an early
and exacerbated activation of caspase-3 activity that preceded
the increase in A
1-42 secreted by IMR-32 cells. Inhibition of
caspase activity by the broad spectrum caspase inhibitor BD-fmk
significantly attenuated the Par-4-induced increase in A
1-42
production. These results strongly suggest that Par-4 is a novel
mediator of aberrant APP processing and that Par-4 increases secretion
of A
1-42 largely through a caspase-dependent pathway
after apoptotic cascades are initiated.
Overexpression of Par-4 did not result in a significant increase in
spontaneous apoptosis in IMR-32 cells, indicating that Par-4
overexpression alone is not sufficient to induce apoptosis. These
findings are consistent with those of our previous study of cultured
prostate tumor cells and PC12 cells, in which overexpression of Par-4
in transiently or stably transfected cells did not induce apoptosis
(16, 17). In addition, overexpression of Par-4 or Leu.zip or
co-expression of Par-4 and Leu.zip did not result in significant
changes in the values of the
A
1-42/A
total ratio in the conditioned
medium of various transfected IMR-32 cell lines under basal culture
conditions. This result is in sharp contrast to the reported effect of
mutant presenilins, where secretion of A
1-42 from human as well as
rodent neural cells was significantly increased by overexpression of
mutant presenilins even under basal culture conditions (1-5). Most
importantly, however, overexpression of Par-4 drastically increased the
A
1-42/A
total ratio in the conditioned
media about 6-8 h after trophic factor withdrawal (Fig. 3), indicating
that Par-4 may have a significant impact on APP processing under
apoptotic conditions. It is noteworthy that the Par-4-induced increase
in A
1-42 secretion did not occur until 6-8 h after trophic factor
withdrawal (Fig. 3). This is the time when most IMR-32 cells
overexpressing Par-4 were still alive and morphologically well
preserved, but the apoptotic process should have been irreversibly
triggered in most of the cells after trophic factor withdrawal. Time
course analysis of caspase activation demonstrated that exacerbated
caspase activation induced by Par-4 in IMR-32 cells was apparent as
early as 2 h after trophic factor withdrawal (Fig. 5), indicating
that Par-4 increases secretion of A
1-42 after initiation of
apoptotic cascades. To determine whether the exacerbated caspase
activation was responsible for the increase in A
1-42 secretion
after trophic factor withdrawal, we examined whether the broad spectrum
caspase inhibitor BD-fmk could block the Par-4-induced increase in A
1-42 secreted by IMR-32 cells. Our results clearly demonstrated that
the Par-4-induced increase in secretion of A
1-42 is largely a
caspase-dependent event.
The effects of Par-4 on secretion of A
1-42 were consistently
blocked by co-expression of the dominant negative regulator of Par-4
activity, the leucine zipper domain. The results demonstrate that the
actions of Par-4 on APP processing, like its actions in apoptotic
pathway (16), require its interaction with other proteins via the
leucine zipper domain. The proteins that interact with Par-4 and
regulate (directly or indirectly) APP processing are unknown. However,
recent study showed that Par-4 interacts with the regulatory domains of
the
and
forms of protein kinase C and inhibits their enzyme
activity (24). The possible implications of these interactions in APP
processing need to be examined carefully because several studies have
indicated that protein kinase C-dependent pathways might
participate in regulation of APP processing (25-27).
All known genetic factors of familial AD, including mutations in APP
and presenilins, lead to increased production of amyloidogenic A
1-42 (1-5). Recent evidence suggests that the presenilin proteins may
be
-secretase, although their function as
-secretase may require
help from additional proteins, such as nicastrin (28-32). Interestingly, all three familial Alzheimer's disease genes have also
been shown to regulate neuronal apoptosis (1-5), suggesting that
dysregulation of apoptotic pathways may play a role in neuronal degeneration in AD. The data provided in this study reconcile the
amyloid/apoptosis hypotheses of AD and suggest that abnormal processing
of APP and increased production of A
1-42 may occur as a result of
initiation and execution of apoptotic cascades, such as aberrant
induction of Par-4 expression and activation of caspases after trophic
factor withdrawal.
 |
FOOTNOTES |
*
This work was supported by grants from the American
Federation for Aging Research and the Alzheimer's Association (to
Q. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Neurobiology
and Pharmacology, Northeastern Ohio Universities College of Medicine,
4209 State Route 44, P. O. Box 95, Rootstown, OH 44272-0095. Tel.:
330-325-6655; Fax: 330-325-5916; E-mail: qguo@neoucom.edu.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010996200
 |
ABBREVIATIONS |
The abbreviations used are:
Par-4, prostate
apoptosis response-4;
Leu.zip, leucine zipper domain;
A
, amyloid
peptide;
APP,
-amyloid precursor protein;
AD, Alzheimer's disease;
ELISA, enzyme linked-immunosorbent assay;
ANOVA, analysis of
variance.
 |
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