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AATF Inhibits Aberrant Production of Αβ 1-42 by Interacting Directly with Par-4
Qing Guo, * and Jun Xie
Department of Physiology, The University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73104
Running Head: AATF, Par-4, and Amyloid beta Peptide
*Correspondence: Qing Guo, Ph.D., Department of Physiology, The University of
Oklahoma Health Sciences Center, Oklahoma City, OK 73104 U.S.A.
Phone: (405) 271-2226 ext. 243. FAX: (405) 271-3181. E-mail: [email protected]
32 text pages, 9 figures, 3 tables, 48,794 characters (with spaces).
Abbreviations: AATF, apoptosis antagonizing transcription factor; Par-4, prostate apoptosis
response-4; Leu.zip, leucine zipper domain; Αβ, amyloid beta peptide; APP, β-amyloid
precursor protein; AD, Alzheimer’s disease; ELISA, enzyme linked-immuno-sorbent assay; PS-1,
presenilin-1; SE, standard error;
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ABSTRACT Aggregation of the neurotoxic amyloid beta peptide 1-42 (Αβ 1-42) in the brain is
considered to be an early event in the pathogenesis of Alzheimer’s disease (AD). Par-4
(Prostate apoptosis response-4) is a leucine zipper protein that is pro-apoptotic and
associated with neuronal degeneration in AD. Overexpression of Par-4 significantly
increased production of Αβ 1-42 after initiation of apoptotic cascades, indicating factors
regulating apoptotic pathways may also affect processing of β-amyloid precursor protein
(APP). AATF (apoptosis antagonizing transcription factor) was recently identified as an
interaction partner of DAP-like kinase (Dlk), a member of the DAP (Death Associated
Protein) kinase family. AATF antagonizes apoptosis induced by Par-4, suggesting that
AATF might directly or indirectly participate in regulation of Par-4 activity. We now
report that AATF colocalizes with Par-4 in both cytoplasmic and nuclear compartments,
and it interacts directly and selectively with Par-4 via the leucine zipper domain in neural
cells. Par-4 induced an aberrant production and secretion of Αβ in neuroblastoma IMR-
32 cells after apoptotic cascades are initiated. Co-expression of AATF completely
blocked aberrant production and secretion of Αβ 1-42 induced by Par-4, and AATF/Par-4
complex formation was essential for the inhibitory effect of AATF on aberrant Αβ
secretion. These results indicate that AATF is an endogenous antagonist of Par-4 activity
and an effective inhibitor of aberrant Αβ production and secretion under apoptotic
conditions.
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INTRODUCTION It is widely accepted that neuronal degeneration in Alzheimer’s disease (AD) may
be caused by extracellular accumulation of aggregated, neurotoxic amyloid beta peptide
1-42 (Αβ 1-42, refs 1-13). Support for this amyloid hypothesis of AD came from several
lines of experimental evidence. In particular, mutations in familial Alzheimer’s disease
(FAD) genes, such as β-amyloid precursor protein (APP) and presenilin-1 and presenilin-
2, have been shown to regulate the processing of APP and result in increased production
and aggregation of Αβ 1-42 (14-20). Notably, increased production of Αβ 1-42 was also
observed in neural cells undergoing apoptosis, and mutations in all three FAD genes have
been shown to regulate neuronal apoptosis (21-27). Indeed, 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 (21-27). Par-4 (Prostate apoptosis response-4) is a
leucine zipper protein that was associated with neuronal degeneration in Alzheimer’s
disease (28-49). The leucine zipper domain of Par-4 mediates protein-protein interactions,
and is essential for pro-apoptotic actions of Par-4 in neuronal cells (46). Overexpression
of Par-4 in neuroblastoma IMR-32 cells significantly increased production of Αβ 1-42
through a caspase-dependent pathway (40). These results suggest that Par- is involved in
the abnormal processing of APP during the apoptotic process.
AATF (apoptosis antagonizing transcription factor) was recently identified as an
interaction partner of DAP-like kinase (Dlk), a member of the DAP (Death Associated
Protein) kinase family of pro-apoptotic serine/threonine kinases (50-58). AATF contains
a putative leucine zipper domain and several putative phosphorylation sites for protein
kinases CKII, PKA and PKC (50-58). Human AATF has an open reading frame of 560
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amino acids and contains several potential phosphorylation sites, a leucine zipper
structure, putative nuclear localization signals (NLS1 and NLS2), and three nuclear
receptor-binding motifs. Importantly, AATF antagonizes apoptosis induced by Par-4 (50-
58), suggesting that AATF might participate in inhibition of pro-apoptotic and/or
activation of anti-apoptotic pathways. We now report that AATF interacts directly with
Par-4 via the leucine zipper domain, and blocks Par-4 mediated aberrant production of
Αβ 1-42 in IMR-32 cells. Our data suggest that AATF may function as an endogenous
inhibitor of Par-4 activity in APP processing. Selective enhancement of AATF expression
in neuronal cells may therefore provide a potential therapeutic approach for aberrant
production of Αβ 1-42 in AD.
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MATERIALS AND METHODS Hippocampal Neuronal Cultures. Dissociated hippocampal cell cultures were
prepared from postnatal day 1 mouse pups using methods similar to those described
previously (47). Briefly, hippocampi were removed and incubated for 15 min in Ca2+-
and Mg2+-free Hank's Balanced Saline Solution (Gibco BRL) containing 0.2% papain.
Cells were dissociated by trituration and plated into polyethyleneimine-coated plastic or
glass-bottom culture dishes containing Minimum Essential Medium with Earle's salts
supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM
pyruvate, 20 mM KCl, 10 mM sodium bicarbonate and 1 mM Hepes (pH 7.2).
Following cell attachment (3-6 hr post-plating), the culture medium was replaced with
Neurobasal Medium with B27 supplements (Gibco BRL). Experiments were performed
in 7 day-old cultures.
Generation and Characterization of IMR-32 Cell Lines and Trophic Factor
Withdrawal. Methods used in transfection experiments and induction of apoptosis by
trophic factor withdrawal were described in our previous studies (40-41, 46-48). In brief,
human neuroblastoma IMR-32 cells (ATCC) were maintained at 37 ºC in an atmosphere
of 95% air and 5% CO2 in Eagle’s minimum essential medium supplemented with non-
essential amino acids, and 10% heat-inactivated fetal bovine serum. A full length Par-4
cDNA was subcloned into the expression vector pRc/CMV, yielding a recombinant
construct pCMV-Par-4 which encodes a 1.2 kb RNA species and a full-length 38kD Par-
4 protein. A cDNA fragment containing Par-4 lacking the leucine zipper domain (Par-
4∆Leu.zip) was similarly subcloned into pRc/CMV expression vector, yielding a
recombinant construct pCMV-Par4∆Leuzip that encodes an 862-bp RNA species and an
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approximately 30-kD protein. A cDNA fragment containing full-length wild type AATF
and cDNAs containing various deletion mutants of AATF (see ref. 50), including AATF
390 (deletion of the carboxy terminus NLS2), AATF 279 (deletion of the carboxy
terminus NLS2 and NSL1), AATF 179 (deletion of the carboxy terminus NLS2, NSL1
and the putative leucine zipper domain), were subcloned into the pREP4 expression
vector (Invitrogen), yielding recombinant constructs pREP4-AATF, pREP4-AATF390,
pREP4-AATF279, and pREP4-AATF179. Human IMR-32 cell lines stably expressing
Par-4 or Par4∆Leuzip were established by transfection using LipofectAMINE
(GIBCO/BRL) with pCMV-Par-4 or pCMV-Par4∆Leuzip. Transfected cells were
selected with G418 (400 µg/ml) for 4 weeks, and surviving clones were selected. IMR-
32 cells expressing AATF, AATF 390, AATF 279 and AATF 179 were similarly
established except that the transfected cells were selected with hygromycin (400 µg/ml).
Additional double transfected IMR-32 cell lines were generated where two proteins (Par-
4/AATF, Par4∆Leuzip/AATF, Par-4/AATF390, Par-4/AATF279 or Par-4/AATF179)
were co-expressed. For control purposes, parallel cultures of IMR-32 cells were stably
transfected with pRc/CMV and pREP4 vectors alone. After the cells became confluent in
the culture flasks, the culture medium were replaced with fresh media and incubated for
48 h at 37 ºC to condition the medium for Αβ measurement. Trophic factor withdrawal
was initiated by washing cultures four times with Locke’s buffer (154mN NaCl, 5.6 mM
KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5mM glucose, and 5 mM
HEPES, pH 7.2) with subsequent incubation in 1 ml of Locke’s buffer.
Generation of AATF antibody, Immunoprecipitation, and Western Blot
Analysis. Levels of expression of Par-4 and AATF in hippocampal neurons were
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determined by Western blot analysis as described (46-48). A suitable peptide sequence
(LDTDKRYSGKTTSRKAWKE, corresponding to rat AATF terminus amino acids 64-
82) from AATF was chosen based on its antigenicity and other considerations. The
BLAST program was used to compare the peptide sequence against known sequences in
the major databases to avoid similarity and cross reactivity with other related and
unrelated proteins. The peptide was conjugated to the carrier protein Keyhole Limpet
Hemocyanin (KLH) and used to immunize rabbits to generate AATF antisera (Sigma-
Genosys, the Woodlands, Texas) according to standard protocols. Crude antisera were
affinity purified further by adsorption of antibodies to the peptide that had been
immobilized to Sepharose. Purified antibodies were eluted with ImmunoPure Gentle
Ag/Ab elution buffer. The antibody was finally dialyzed, concentrated, and used at a
dilution of 1:100 in Western blots. The specificity of the AATF antisera was analyzed by
passing it down affinity columns containing the immobilized peptide and analyzing both
the unbound fractions and eluted fractions in Western blots. We have found that the
antibody recognizes rat, mouse and human AATF on Western blots. It revealed a strong
band at about 70 kD in whole cell protein extracts from both human IMR-32 cells and
mouse primary neurons. The antibody recognizes AATF390, AATF 279, and AATF 179
as well as full length AATF. The Par-4 antibody is a mouse monoclonal antibody raised
against full length rat Par-4 (Santa Cruz Biotechnology, Inc). This antibody reacts with
Par-4 of mouse, rat and human origin, and recognizes both full-length Par-4 as well as the
deletion mutant of Par-4 (Par-4∆Leu.zip) that lacks the leucine zipper domain. In addition,
the antibody recognizes both human and rodent Par-4 proteins at about 38 kD. For
immunoprecipitation, aliquots of cell lysates containing 200 µg of protein were incubated
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for 1 h at 4 °C with appropriate dilutions of mouse anti-Par-4 or rabbit anti-AATF
antibodies in immunoprecipitation buffer (150 mM NaCl, 2 mM EDTA, 1% Nonidet P-
40, 5 µg /ml leupeptin, 5 µg /ml aprotinin, 2 µg /ml pepstatin A, 0.25 mM
phenylmethylsulfonyl fluoride, 50 mM Tris, pH 7.6). Protein A-Sepharose resin
(Pharmacia, 30 µl/sample) was then added to the extracts for collection of the
immunocomplexes, which was then washed three times in immunoprecipitation buffer,
and solubilized by heating in Laemmli buffer containing 2-mercaptoethanol at 100 °C for
4 min. The solubilized proteins were separated by electrophoresis on a 4-12% gradient
SDS-polyacrylamide gel and then transferred to a nitrocellulose sheet. For Western blot
analysis, the nitrocellulose sheet was blocked with 5% milk followed by a 1-h incubation
in the presence of primary anti-AATF or anti-Par4 antibody. The membrane was further
processed using horseradish peroxidase conjugated secondary antibody and
immunoblotted proteins were detected by chemiluminescence using the ECL system
(Amersham). To examine if AATF or Par-4 interacts with c-Jun, a leucine zipper
containing transcription factor of about 39 kD, a mouse monoclonal anti-c-Jun antibody
(Oncogene Research Products, San Diego, CA) was used in the
immunoprecipitation/Western blot analysis. Equal loading was verified by probing the
blots with the anti-tubulin antibody (Sigma). Western blot images were acquired and
quantified using Kodak Image Station 2000R and Kodak Digital Science 1D 3.6.
software.
Immunocytochemistry by Confocal Laser Scanning Microscopy: The cultured
cells were fixed for 30 min in 4% paraformaldehyde/PBS, and membranes were
permeabilized by incubation in 0.2% Triton-X100 in PBS. Cells were incubated for 1 h in
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blocking serum (5% normal goat serum in PBS). Cells were then exposed to primary
antibodies (1:100 dilution of rabbit anti-AATF polyclonal antibody and 1:100 mouse
anti-Par-4 monoclonal antibody) overnight at 4 °C, followed by incubation for 1 h with a
mixture of Texas Red-labeled anti-rabbit and fluorescein-labeled anti-mouse secondary
antibodies (Vector Laboratories, Burlingame, California). Images of Par-4 and/or AATF
immunofluorescence were acquired using a confocal laser scanning microscope (dual
wavelength scan) with a 60X oil immersion objective. All images were acquired using
the same laser intensity and photodector gain, to allow quantitative comparisons of
relative levels of fluorescence in the cells. The average pixel intensity per cell and sites of
colocalization of immunoreactivities were determined using the Fluoview 2.0 software.
Preparation of Whole Cell, Cytosolic, Membrane, and Nuclear Extracts. After
washing 3 times with cold PBS, whole cell extracts were prepared in lysis buffer as
described in our previous studies (37, 46). Nuclear, cytosolic and membrane fractions
from IMR-32 human neuroblastoma cells were isolated as described previously (59), with
some modifications. Briefly, cells were washed with cold PBS and homogenized in a
tight-fitting Dounce homogenizer in cold lysis buffer containing 5 mM Tris-HCl, pH 7.4,
1 mM EGTA, 1 mM EDTA, 2 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 1 mM 4-
(2-aminoethyl) benzenesulfonyl fluoride, 1 µM leupeptin, 1 µM aprotinin, 1 µM
pepstatin, 1 µM bestatin, and 1 µM E64. 0.5 ml of the cell lysate was then layered onto a
solution of 1.1 M sucrose in lysis buffer (0.5 ml) and centrifuged at 1500 X g for 10 min
at 4 °C. The supernatant from this step contains cytosolic as well as membrane fractions,
while the pellet contains nuclear extract. The supernatant was further centrifuged at
15,000 X g for 30 min at 4 °C. The resulting supernatant and pellet were cytosolic and
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membrane fractions, respectively. The nuclear pellet was washed by resuspension in 1 ml
of 1.1 M sucrose in lysis buffer and recentrifugation at 1500 X g for 5 min at 4 °C. The
identity of these fractions has been confirmed previously by immunoblotting studies
using various specific organelle markers (59).
Quantification of Extracellular and Intracellular Levels of Αβ l-40 and Αβ l-42
by Sandwich-ELISAs: Αβ 1-40 and Αβ 1-42 levels in the conditioned culture media
were measured using a fluorescence-based sandwich ELISA described in detail in our
previous studies (40). The C-terminal specific sandwich-ELISAs use a monoclonal
antibody directed against the N-terminal region of human Αβ and two other antibodies
specific for Αβ 1-40 and Αβ 1-42 (see ref. 40). The ratio (%) of Αβ 1-42 to the total
amount of Αβ (Αβ 1-40 plus Αβ 1-42) was used to measure the changes in the relative
amount of Αβ 1-42 secreted from transfected IMR-32 cells. To measure levels of
intracellular Αβ, various lines of transfected IMR-32 cells were subject to trophic factor
withdrawal, then rinsed twice in PBS, and incubated on ice for 20 min either with PBS
alone, or with 10 mg/ml of trypsin in PBS. Under such conditions, trypsin has been
reported to destroy cell surface-associated Αβ, while keep intracellular Αβ intact. The
trypsin in the cultures was inactivated by the addition of 100mg/ml soybean trypsin
inhibitor. The treated IMR-32 cells were then washed with ice-cold PBS, scraped into
cell lysis buffer, and processed for Sandwich ELISA.
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RESULTS Par-4 and AATF colocalize in both cytoplasmic and nuclear compartments:
Since both Par-4 and AATF contain leucine zipper domain critically involved in protein-
protein interactions, it is important to examine if Par-4 and AATF colocalize and interact
in cytoplasmic and/or nuclear compartments in neuronal cells. Immunocytochemical
analysis of cultured hippocampal neurons using a polyclonal antibody against AATF or a
monoclonal antibody against Par-4 revealed that Par-4 and AATF are localized in both
cytoplasmic and nuclear compartments (Fig. 1 A1 and B1). These results were confirmed
by Western blotting analysis of AATF and Par-4 expressions in total cell homogenates,
nuclear extract and cytosol-membrane extract (Fig.1 A2 and B2). Double labeling
immunocytochemistry using confocal laser scanning microscope further showed that
AATF and Par-4 largely colocalize in both nuclear and cytoplasmic compartments (Fig.
2).
AATF and Par-4 directly interact in transfected IMR-32 cells and in primary
neurons: Since basal levels of AATF and Par-4 in untransfected wild-type IMR-32 cells
are low, co-immunoprecipitation studies were performed on homogenates of transfected
IMR-32 cell clones overexpressing Par-4 and AATF. When immunoprecipitation was
performed using the AATF antibody, a 38-kD Par-4 band was clearly detected on the
immunoblot (Fig. 3a). Reverse order of immunoprecipitation/Western blot analysis of the
same transfected cell lines showed similar AATF/Par-4 complex formation. As shown in
Fig. 3b, when immunoprecipitation was performed using the Par-4 antibody, a major
AATF band of about 70 kD could be clearly detected on the immunoblot. Further
immunoprecipitation/Western blot analysis confirmed similar AATF/Par-4 complex
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formation in primary hippocampal neurons under physiological concentrations of these
proteins (Fig. 4). These results demonstrate the interaction between the endogenous
AATF and Par-4, and indicate that AATF/Par-4 complex formation is physiologically
relevant. Because of the potential promiscuous nature of protein interactions mediated by
leucine zipper domains, it is critical to see if AATF or Par-4 would interact with any
leucine zipper containing proteins. To this end, we examined if AATF or Par-4 interacts
with c-Jun using a specific anti-c-Jun antibody. c-Jun is a leucine zipper domain
containing transcription factor of about 39 kD whose expression has been shown to be
drastically increased following neuronal injury. Immunoprecipitation/Western blotting
experiments in transfected IMR-32 cells co-expressing human AATF and Par-4 showed
that c-Jun was detected in proteins from total lysate but not from those
immunoprecipitated with anti-AATF or anti-Par-4 antibody, indicating that neither AATF
nor Par-4 interacts with c-Jun (Fig. 5). These negative control experiments demonstrate
the selectivity of AATF/Par-4 complex formation.
Mapping of AATF/Par-4 interaction domain to leucine zipper: Next, we sought
to determine if AATF interacts with Par-4 via the leucine zipper domain. For this purpose,
a series of deletion mutants of AATF and Par-4 were employed in immunoprecipitation
studies in transfected IMR-32 cells. Generation of the leucine zipper deletion mutant of
Par-4 that encodes a 265-amino-acid Par-4 protein lacking the leucine zipper domain
(designated Par-4∆Leu.zip) has been described in our previous studies (46-48). The
AATF deletion mutants were also reported in a previous study (50), and included AATF
390 (deletion of the carboxy terminus NLS2), AATF 279 (deletion of the carboxy
terminus NLS2 and NSL1), AATF 179 (deletion of the carboxy terminus NLS2, NSL 1
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and the putative leucine zipper domain). In Fig. 6a, the indicated double transfected IMR-
32 cell lines were lysed and precipitated with the Par-4 antibody, followed by Western
blotting with AATF antibody. Par-4 interacted with only wild-type AATF (a major band
of wild type AATF was detected at about 70 kD) and AATF deletion mutants containing
the leucine zipper domain (an AATF390 band at about 52 kD, and an AATF279 band at
about 37 kD were clearly detected on immunoblots), but not the AATF deletion mutant
lacking the leucine zipper domain ( a band of AATF179 at about 24 kD was missing on
immunoblot), indicating that the leucine zipper domain of AATF is necessary for
interaction with Par-4. In untransfected or vector transfected control cell lines,
AATF/Par-4 complex formation was weak or undetectable because endogenous levels of
AATF and Par-4 in IMR-32 cells are low under basal culture conditions. The requirement
for the leucine zipper domain in AATF/Par-4 complex formation was further confirmed
by reverse order of immunoprecipitation/Western blot analysis, as shown in Fig. 6b.
When immunoprecipitation was performed using the AATF antibody, the 38-kD Par-4
band was detected on immunoblots only in cells co-expressing Par-4 and AATF
containing the leucine zipper domain (Par-4/AATF wt, Par-4/AATF 390, and Par-
4/AATF 297). Deletion of the leucine zipper domain of AATF (as is the case in cells co-
transfected with Par-4/AATF179) abolished the interaction between AATF and Par-4,
indicating that the leucine zipper domain of AATF is required for interaction with Par-4.
Note that, in Fig. 6b, the 38kD Par-4 band was absent in cells co-transfected with Par-
4∆Leu.zip/AATFwt, demonstrating that deletion of the leucine zipper domain of Par-4
also abolished AATF/Par-4 complex formation. These results suggest that the leucine
zipper domains of both AATF and Par-4 are required for AATF/Par-4 complex formation.
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AATF abolishes aberrant Αβ production induced by Par-4: We previously
reported that levels of Par-4 expression were significantly increased in vulnerable brain
regions of human Alzheimer’s disease patients, and that Par-4 promotes neuronal cell
death, and significantly increases the aberrant production of Αβ 1-42 in IMR-32 cells
under apoptotic conditions. Several lines of previously reported evidence indicated that
AATF counteracted the pro-apoptotic actions of Par-4. Together with our observation
that AATF and Par-4 colocalize in both cytoplasmic and nuclear compartments, and
physically interact with each other via the leucine zipper domain, it is imperative to
determine if AATF counteracts the adverse effect of Par-4 in APP processing. For this
purpose, we examined the effect of AATF on both extracellular and intracellular levels of
Αβ 1-42 in transfected IMR-32 cells. Consistent with our previous observations (40),
overexpression of Par-4 drastically increased Αβ 1-42 secretion and therefore Αβ 1-42/Αβ
total ratio in the conditioned media about 8 hours following trophic factor withdrawal
(Table 1, and Fig. 7). To examine if Par-4 also affect intracellular production of Αβ,
various lines of transfected IMR-32 cells were incubated on ice for 20 min with 10 mg/ml
of trypsin in PBS. The trypsin in the cultures was then inactivated by the addition of
100mg/ml soybean trypsin inhibitor. As shown in table 2 and Fig. 8, eight hours
following trophic factor withdrawal, levels of intracellular Αβ 1-42 were significantly
increased by Par-4. Most importantly, co-overexpression of AATF completely abolished
the adverse effects of Par-4 on Αβ production (Table 1 and 2, Fig. 7 and 8). These results
demonstrate that Par-4 alters both intracellular production and extracellular secretion of
Αβ 1-42, and that these adverse effects of Par-4 can be effectively blocked by AATF.
Notably, overexpression of Par-4 and/or AATF did not significantly affect levels of the
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endogenously expressed APP or Αβ production in various cell lines used in this study
(Table 1 and 2, Fig. 7 and 8, and data not shown).
AATF/Par-4 complex formation via the leucine zipper domain is essential for
the role of AATF in reducing aberrant Abeta production: Next, we examined if
blocking AATF/Par-4 complex formation by removing the leucine zipper domain in
AATF would affect actions of AATF on Αβ production and secretion. As shown in table
3 and Fig. 9, eight hours following trophic factor withdrawal, Par-4 significantly
increased Αβ 1-42 secretion from IMR-32 cells, which was completely blocked by co-
expression of AATF. Furthermore, the inhibitory effect of AATF on Par-4 induced Αβ
secretion was observed only in cells expressing AATF containing the leucine zipper
domain (AATF wt, AATF 390, or AATF 279). Blocking AATF/Par-4 complex formation
by removing the leucine zipper domain of AATF (in cells expressing AATF179)
completely abolished the effect of inhibitory effect of AATF on Αβ secretion. These
results suggest that AATF is an endogenous antagonist of Par-4 activity in APP
processing, and it blocks Par-4 activity in IMR-32 cells by binding directly to Par-4 via
the leucine zipper domain.
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DISCUSSION
Extensive loss of neurons and synapses are often observed in AD brains, and an
increased production and aggregation of Αβ 1-42 in vulnerable regions of AD brain has
been shown to be an early, and possibly the primary event in the pathogenesis of the
disease (1-13). Aggregated Αβ 1-42 has been shown to be neurotoxic, and may cause
apoptotic neuronal cell death in many experimental settings (1-20). Mutations in APP and
presenilins, which are implicated in the pathogenesis of familial AD and neuronal
apoptosis, also significantly increase the production of Αβ 1-42 (14-27). Par-4, a leucine
zipper protein that promotes neuronal cell death, also plays important roles in aberrant
production of Αβ 1-42 (28-46). These results indicate that factors regulating apoptotic
pathways may also affect APP processing.
In an effort to reconcile the apoptotic and amyloid hypotheses of AD, and to
identify factors that may effectively block aberrant APP processing during apoptosis, we
have examined the effect of AATF on Par-4 mediated Αβ production using a series of
transfected IMR-32 cells that express wild-type or various deletion mutants of AATF
and/or Par-4. We now report that AATF, a novel transcription factor with anti-apoptotic
properties, is an effective inhibitor of aberrant production and secretion of Αβ in human
IMR-32 cells. AATF inhibits aberrant production of Αβ in cells undergoing apoptosis by
binding directly to Par-4 via the leucine zipper domain and blocking the activity of Par-4
in regulating APP processing.
AATF was initially found to be an interaction partner of Dlk (also known as zipper
interacting protein kinase), a pro-apoptotic member of the DAP kinase family (50-58).
Indeed, Dlk interacts with Par-4 via the leucine zipper domain and promotes Par-4
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activity in apoptotic pathways (50-58). Importantly, AATF was shown to antagonize
apoptosis induced by co-expression of Par-4 and Dlk (50-58), suggesting that AATF
might directly or indirectly participate in regulation of Par-4 activity. Previous studies in
our lab found that levels of Par-4 expression were significantly increased in vulnerable
regions of human AD brain (46). Subsequent in vitro studies documented that Par-4
promotes neuronal cells death and increase production of Αβ 1-42 (37, 40-41, 46-48).
Blocking Par-4 activity in neurons by pharmacological and/or genetic manipulations may
therefore have significant implications in treating AD. We have found in this study that:
(1) AATF colocalizes with Par-4 in both cytoplasmic and nuclear compartments in
primary neurons; (2) AATF interacts directly with Par-4 in transfected IMR-32 cells as
well as primary neurons, indicating that AATF/Par-4 complex formation is
physiologically relevant; (3) Neither AATF nor Par-4 interacts with leucine zipper
protein c-Jun, demonstrating the selectivity of AATF/Par-4 complex formation; (4)
Immunoprecipitation studies further mapped the interaction domain to leucine zipper; (5)
Co-expression of AATF completely blocks aberrant production and secretion of Αβ 1-42
induced by Par-4; (6) AATF/Par-4 complex formation is essential for the inhibitory effect
of AATF on aberrant Αβ secretion induced by Par-4. These results suggest that AATF
may function as an endogenous antagonist of Par-4 activity.
It is important to note that Par-4 induced a significant in increase in Αβ production
only at about 6-8 hours after trophic factor withdrawal, a time when most IMR-32 cells
expressing overexpressing Par-4 were still alive and morphologically well preserved but
apoptotic process should have been irreversibly triggered in most of the cells. It is also
noteworthy that this significant increase in production and secretion of Αβ 1-42 following
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trophic factor withdrawal was observed only in cells overexpressing Par-4, and trophic
factor withdrawal itself was not sufficient to significantly increase Αβ production in
untransfected control IMR-32 cells. Inhibition of Par-4 activity by co-expressing AATF
effectively blocked the aberrant Αβ production and secretion. These data demonstrated
that aberrant production of Αβ in IMR-32 cells following loss trophic support occurs only
after apoptotic cascades are initiated, and that Par-4 plays a significant and essential role
in this process. These results also indicate that, in the pathogenesis of AD, apoptosis and
aberrant APP processing may be intimately linked processes. Therefore, identification of
AATF as a novel interaction partner and endogenous blocker of Par-4 activity in APP
processing and apoptotic pathways may have significant implications for developing
novel therapeutic strategies for AD.
Acknowledgments. We thank W. Chen of Case Western Reserve University for his
excellent technical assistance. This work was supported by grants to Q.G. from the
National Institute of Neurological Disorders and Stroke of NIH (R01NS043296), The
Alzheimer’s Association, and American Federation for Aging Research (AFAR).
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Figure Legends.
Figure 1. AATF and Par-4 are localized in both cytoplasmic and nucleus
compartments in hippocampal neurons. Primary hippocampal neurons from new born
mice were cultured for 7 days and then examined for AATF (A1) and Par-4 (B1)
immunoreactivity by confocal laser scanning microscopy using specific polyclonal
AATF and Par-4 antibodies respectively. Note that AATF and Par-4 immunoreactivity
was observed in both nuclear (arrow) and cytosol (arrow head) compartments. Total cell
homogenates, nuclear extract and cytosol-membrane extract were also prepared and
AATF (A2) and Par-4 (B2) expressions in each preparation were analyzed by Western
blot analysis. Note that AATF and Par-4 were detected in both nuclear and cytosolic
fractions.
Figure 2. Immunocytochemistry analysis showing colocalization of AATF and Par-
4 in primary neurons. Representative confocal laser scanning micrographs showing
images of primary hippocampal neurons double-labeled with AATF and Par-4 antibodies.
(A) Green fluorescence, AATF immunoreactivity; (B) Red fluorescence, Par-4
immunoreactivity; (C) A merged image (anaglyph) of A and B. Note yellow fluorescent
areas indicating sites of colocalization of AATF and Par-4 immunoreactivity in both
cytoplasmic (arrow head) and nuclear compartments (arrow).
Figure 3. AATF/Par-4 complex formation in transfected IMR-32 cells. (a)
Transfected IMR-32 cells co-expressing human AATF and Par-4 were lysed and
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precipitated with the AATF antibody, followed by Western blotting with Par-4 antibody
(lanes 3-4). Input lanes (lanes 1-2) show 10% of the total protein used in
immunoprecipitation experiments. The preimmune serum from rabbits immunized with
AATF was used as a control (lanes 5-6). AATF/Par-4 complex was clearly observed in
two separate clones (C12 and C7) of transfected cells (lane 3-4). (b) Reverse order of
immunoprecipitation/Western blot analysis of the same transfected cells showed similar
AATF/Par-4 complex formation (lanes 3-4). Input lanes (lanes 1-2) show 10% of the total
protein used in immunoprecipitation experiments.
Figure 4. Interaction between endogenous AATF and Par-4 in primary
hippocampal neurons. (a) Cultures of primary hippocampal neurons were lysed and
proteins from total lysates were immunoprecipitated with rabbit anti-AATF antibody
(lane 2), followed by Western blotting with anti-Par4 antibody. The preimmune serum
from rabbits immunized with AATF was used as a control (lane 3). Input lane (lane 1)
show 10% of the total protein used in immunoprecipitation experiments. (b) Reverse
order of immunoprecipitation/Western blot analysis of the same hippocampal neurons
showed similar endogenous AATF/Par-4 complex formation.
Figure 5. Absence of interaction between c-Jun and AATF or Par-4. (a) Transfected
IMR-32 cells co-expressing human AATF and Par-4 (clones C12 and C7) were lysed and
proteins from total lysates were immunoprecipitated with rabbit anti-AATF antibody
(lanes 3-4), followed by Western blotting with anti-c-Jun antibody. Input lanes (lanes 1-2)
show 10% of the total protein used in immunoprecipitation experiments. Similar data
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were obtained in reverse order of immunoprecipitation/Western blot analysis of the same
transfected cells (data not shown). (b) Transfected IMR-32 cells co-expressing human
AATF and Par-4 (clones C12 and C7) were lysed and proteins from total lysates were
immunoprecipitated with anti-Par-4 antibody (lane 3-4), followed by Western blotting
with anti-c-Jun antibody. Input lanes (lanes 1-2) show 10% of the total protein used in
immunoprecipitation experiments. Similar data were obtained in reverse order of
immunoprecipitation/Western blot analysis of the same transfected cells (data not shown).
Note that, in both (a) and (b), c-Jun was detected in proteins from total lysate but not
from those immunoprecipitated with anti-AATF or anti-Par-4 antibody, indicating that
neither AATF nor Par-4 interacts with c-Jun.
Figure 6. Mapping of the binding region of AATF and Par-4 to the leucine zipper
domain by immunoprecipitation/Western blotting analysis in transfected IMR-32
cells. (a) Representative immunoprecipitation/Western blot analysis showing AATF
interacts with Par-4 via the leucine zipper domain of AATF. Double transfected IMR-32
cells used immunoprecipitation studies include: Par-4/AATFwt: cells co-transfected with
full-length Par-4 and wild-type AATF; Par-4/AATF390: cells co-transfected with full-
length Par-4 and the AATF deletion mutant AATF390; Par-4/AATF 279: cells co-
transfected with full-length Par-4 and the AATF deletion mutant AATF279; Par-
4/AATF179: cells co-transfected with full-length Par-4 and the AATF deletion mutant
AATF179; Par-4∆Leu.zip/AATFwt: cells co-transfected with Par-4∆Leu.zip and the full-
length wild-type AATF. The indicated double transfected IMR-32 cell lines were lysed
and precipitated with the Par-4 antibody, followed by Western blotting with AATF
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antibody. Note that Par-4 interacted with only wild-type AATF (AATFwt band at about
70 kD) and AATF deletion mutants containing the leucine zipper domain (AATF390
band at about 52 kD, and AATF279 band at about 37 kD), but not the AATF deletion
mutant lacking the leucine zipper domain, indicating that the leucine zipper domain of
AATF is necessary for interaction with Par-4. In untransfected or vector transfected
control cell lines, AATF immunoreactivity on Western blots was weak, most likely due
to the fact that endogenous levels of AATF and Par-4 in IMR-32 cells are relatively low
under normal basal conditions. (B) Reverse order of immunoprecipitation/Western blot
analysis showing that Par-4 interacts with AATF via its leucine zipper domain. The
indicated double transfected IMR-32 cell lines were lysed and precipitated with the
AATF antibody, followed by Western blotting with Par-4 antibody. Note that full length
wild-type AATF exhibited a functional interaction only with full length Par-4, but not
with the Par-4 deletion mutant Par-4∆Leu.zip lacking the leucine zipper domain,
indicating that the leucine zipper domain of Par-4 is involved in interaction with AATF.
Also note that, consistent with the results in (A), AATF390 and AATF 279, both of
which contain the leucine zipper domain of AATF, also interacted with the full-length
Par-4.
Figure 7. AATF blocks Par-4 induced secretion of Αβ 1-42 from transfected IMR-
32 cells 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 values
of Αβ 1-42/Αβ 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
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Αβ 1-42/Αβ total ratio in the conditioned media 6-8 hours following trophic factor
withdrawal. Co-overexpression of AATF completely abolished the adverse effect of Par-
4 on Αβ secretion. Values are the mean and SE. of determinations made in six separate
cultures. ***P<0.001 compared with corresponding values of Αβ 1-42/Αβ total ratio in
untransfected, vector transfected, and AATF cell groups. ### P<0.001 compared with
corresponding values of Αβ 1-42/Αβ total ratio in cells transfected with Par-4 alone. Similar
data were obtained from cell lines Par-4 C6 and C3, AATF C19, and Par4+AATF C23
and C32. ANOVA with Scheffe’s post-hoc tests.
Figure 8. Par-4 increases intracellular Αβ 1-42 production after trophic factor
withdrawal: inhibition by co-transfection of AATF. Cultures of the indicated clones of
transfected IMR-32 cells were deprived of trophic support (TFW) for 8 hours, and values
of Αβ 1-42/Αβ total ratio in cell lysates of transfected IMR-32 cells were measured by
sandwich ELISAs. Note that Αβ is produced intracellularly in IMR-32 cells and that
overexpression of Par-4 drastically increased intracellular Αβ 1-42/Αβ total ratio 8 hours
following trophic factor withdrawal. Co-overexpression of AATF significantly inhibited
the adverse effect of Par-4 on Αβ production. Values are the mean and SE. of
determinations made in six separate cultures. ***P<0.001 compared with the
corresponding value of Αβ 1-42/Αβ total ratio before TFW. ### P<0.001 compared with the
value of Αβ 1-42/Αβ total ratio in cells transfected with Par-4 alone after TFW. Similar data
were obtained from cell lines Par-4 C6 and C3, AATF C19, and Par4+AATF C23 and
C32. ANOVA with Scheffe’s post-hoc tests. Αβ found in IMR-32 cell lysates was
produced intracellularly since Αβ could be completely eliminated if the cells were
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incubated with 10 mg/ml of trypsin in PBS in the presence of 0.1% Triton X100 (data not
shown).
Figure 9. Interaction of AATF with Par-4 via the leucine zipper domain is required
for the inhibitory effect of AATF on Abeta secretion. Cultures of the indicated IMR-
32 cell lines were deprived of trophic support for 8 hours, and values of Αβ 1-42/Αβ total
ratio in the conditioned culture media of transfected IMR-32 cells were measured by
sandwich ELISAs. Note that the inhibitory effect of AATF on Par-4 induced Abeta
secretion was retained in cells expressing AATF390 and AATF297 (both of which
contain the leucine zipper domain), but was completely abolished in cells expressing
AATF179 (which lacks the leucine zipper domain). *** P<0.001 compared with
corresponding values of Αβ 1-42/Αβ total ratio in untransfected and vector transfected
control cell groups. ### P<0.001 compared with corresponding values of Αβ 1-42/Αβ total
ratio in cells transfected with Par-4 alone (Par-4) and those co-transfected with Par-4 and
AATF179 (Par-4/AATF179). Similar data were obtained from at least 3 separate clones
of each of the transfected cell lines. ANOVA with Scheffe’s post-hoc tests.
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TABLES
Table 1. Αβ ELISA showing secretion of Αβ 1-40 and Αβ 1-42 from transfected
IMR-32 cells before and after trophic factor withdrawal. Cultures of the indicated
clones of transfected IMR-32 cells were subjected to trophic factor withdrawal for 8
hours, and amount of Αβ 1-40 and Αβ 1-42 in the conditioned culture media were
measured by sandwich ELISAs. Note that overexpression of Par-4 drastically increased
secretion of Αβ 1-42 following trophic factor withdrawal. Co-overexpression of AATF
completely abolished the adverse effect of Par-4 on Αβ secretion. Values are the mean
and SE. of determinations made in six separate cultures. ***P<0.001 compared with
corresponding values of Αβ 1-42 in untransfected, vector transfected, AATF transfected,
and Par-4/AATF co-transfected cell groups. Similar data were obtained from cell lines
Par-4 C6 and C3, AATF C19, and Par4+AATF C23 and C32. ANOVA with Scheffe’s
post-hoc tests.
Before TFW 8 hours After TFW Cells
Αβ 1-40 (fmol/ml)
Αβ 1-42 (fmol/ml)
Αβ 1-40 (fmol/ml)
Αβ 1-42 (fmol/ml)
Untransfected 116.3 ± 7.2 16.0 ± 0.9 123.1 ± 7.9 18.9 ± 0.7
Vector alone 113.6 ± 6.2 14.8 ± 0.6 118.6 ± 8.1 17.4 ± 0.8
Par-4, C9 118.9 ± 8.1 17.1 ± 0.7 183.2 ±12.3 240.9 ± 15.9***
AATF, C3 106.5 ± 6.5 12.9 ± 0.4 109.8 ± 7.2 13.2 ± 0.6
Par4+AATF, C8 112.4 ± 8.7 15.8 ± 0.5 139.8 ± 8.5 29.4 ± 1.3
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Table 2. Αβ ELISA showing levels of intracellular Αβ 1-40 and Αβ 1-42 in
transfected IMR-32 cells before and after trophic factor withdrawal. Cultures of the
indicated clones of transfected IMR-32 cells were subjected to trophic factor withdrawal
for 8 hours, and levels of intracellular Αβ 1-40 and Αβ 1-42 in cell lysate were measured
by sandwich ELISAs. Note that overexpression of Par-4 drastically increased
intracellular production of Αβ 1-42 following trophic factor withdrawal. Co-
overexpression of AATF completely abolished the adverse effect of Par-4 on Αβ
production. Values are the mean and SE. of determinations made in six separate cultures.
***P<0.001 compared with corresponding values of Αβ 1-42 in untransfected, vector
transfected, AATF transfected, and Par-4/AATF co-transfected cell groups. Similar data
were obtained from cell lines Par-4 C6 and C3, AATF C19, and Par4+AATF C23 and
C32. ANOVA with Scheffe’s post-hoc tests.
Before TFW 8 hours After TFW Cells
Αβ 1-40 (fmol/mg)
Αβ 1-42 (fmol/mg)
Αβ 1-40 (fmol/mg)
Αβ 1-42 (fmol/mg)
Untransfected 31.8 ± 1.7 8.6 ± 0.5 33.1 ± 1.6 9.9 ± 0.7
Vector alone 28.6 ± 1.3 6.7 ± 0.5 36.3 ± 2.1 9.8 ± 0.6
Par-4, C9 36.3 ± 2.1 11.2 ± 0.8 46.5 ± 3.2 53.7 ± 3.9***
AATF, C3 29.7 ± 1.4 6.6 ± 0.4 30.9 ± 1.3 7.4 ± 0.5
Par4+AATF, C8 28.5 ± 1.6 6.8 ± 0.3 38.8 ± 2.0 15.5 ± 0.8
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Table 3. Αβ ELISA showing secretion of Αβ 1-40 and Αβ 1-42 from transfected
IMR-32 cells expressing different deletion mutants of AATF and/or Par-4. Cultures
of the indicated clones of transfected IMR-32 cells were subjected to trophic factor
withdrawal for 8 hours, and amount of Αβ 1-40 and Αβ 1-42 in the conditioned culture
media were measured by sandwich ELISAs. Values are the mean and SE. of
determinations made in six separate cultures. ***P<0.001 compared with corresponding
values of Αβ 1-42 in untransfected, vector transfected, AATF transfected, and Par-
4/AATF, Par-4/AATF390, Par-4/AATF279 co-transfected cell groups. Similar data were
obtained from at least 3 separate clones of each of the transfected cell lines. ANOVA
with Scheffe’s post-hoc tests.
Cells Αβ 1-40 (fmol/ml)
Αβ 1-42 (fmol/ml)
Untransfected 123.1 ± 7.9 18.9 ± 0.7
Vector alone 118.6 ± 8.1 17.4 ± 0.8
Par-4, C9 183.2 ±12.3 240.9 ± 15.9***
AATF, C3 109.8 ± 7.2 13.2 ± 0.6
Par4+AATF, C8 139.8 ± 8.5 29.4 ± 1.3
Par4+AATF390, C2 142.5 ± 8.2 33.9 ± 1.6
Par4+AATF279, C12 133.2 ± 7.7 25.0 ± 1.2
Par4+AATF179, C16 181.9 ± 11.1 210.1 ± 13.3***
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Fig. 1 (Below)
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Fig. 2 (Below)
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Fig. 3 (Below)
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Fig. 4 (Below):
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Fig. 5 (Below)
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Fig. 6 (Below)
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Fig. 7 (Below)
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Fig. 8 (Below)
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Fig. 9 (Below)
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Qing Guo and Jun XieAATF inhibits aberrant production of Abeta 1-42 by interacting directly with Par-4
published online November 18, 2003J. Biol. Chem.
10.1074/jbc.M309811200Access the most updated version of this article at doi:
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