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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: qing-guo@ouhsc.edu

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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