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©2004 FASEB
The FASEB Journal express article 10.1096/fj.04-2637fje. Published online November 17, 2004.
Intracellular Aβ42 activates p53 promoter: a pathway to
neurodegeneration in Alzheimer’s disease
Yasumasa Ohyagi,* Hideaki Asahara,* De-Hua Chui,† Yuko Tsuruta,* Nobutaka Sakae,*
Katsue Miyoshi,* Takeshi Yamada,* Hitoshi Kikuchi,* Takayuki Taniwaki,*
Hiroyuki Murai,* Koji Ikezoe,* Hirokazu Furuya,* Takeshi Kawarabayashi,‡ Mikio Shoji,‡
Frederic Checler,§ Toru Iwaki,║ Takao Makifuchi,# Kazuya Takeda,** Jun-ichi Kira,* and
Takeshi Tabira**
*Department of Neurology, Neurological Institute, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan; †Division of Demyelinating Disease and Aging, National
Institute of Neuroscience, NCNP, Tokyo, Japan; ‡Department of Neurology, Neuroscience,
Biophysiological Science, Graduate School of Medicine and Dentistry, Okayama University,
Okayama, Japan; §Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France;
║Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences,
Kyushu University, Fukuoka, Japan; #Department of Clinical Research, National Saigata
Hospital, Niigata, Japan; and **National Institute for Longevity Sciences, Aichi, Japan
Corresponding author: Yasumasa Ohyagi, Department of Neurology, Neurological Institute,
Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka
812-8582, Japan. E-mail: ohyagi@neuro.med.kyushu-u.ac.jp
ABSTRACT
The amyloid β-protein (Aβ) ending at 42 plays a pivotal role in Alzheimer's disease (AD). We
have reported previously that intracellular Aβ42 is associated with neuronal apoptosis in vitro
and in vivo. Here, we show that intracellular Aβ42 directly activated the p53 promoter, resulting
in p53-dependent apoptosis, and that intracellular Aβ40 had a similar but lesser effect. Moreover,
oxidative DNA damage induced nuclear localization of Aβ42 with p53 mRNA elevation in
guinea-pig primary neurons. Also, p53 expression was elevated in brain of sporadic AD and
transgenic mice carrying mutant familial AD genes. Remarkably, accumulation of both Aβ42
and p53 was found in some degenerating-shape neurons in both transgenic mice and human AD
cases. Thus, the intracellular Aβ42/p53 pathway may be directly relevant to neuronal loss in AD.
Although neurotoxicity of extracellular Aβ is well known and synaptic/mitochondrial
dysfunction by intracellular Aβ42 has recently been suggested, intracellular Aβ42 may cause
p53-dependent neuronal apoptosis through activation of the p53 promoter; thus demonstrating an
alternative pathogenesis in AD.
Key words: apoptosis • amyloid β-protein • neurotoxicity
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lzheimer’s disease (AD) is the most common dementia in the elderly and is
pathologically characterized by remarkable neuronal loss, neurofibrillary tangles, and
senile plaques. Amyloid β-protein (Aβ) is the major insoluble protein in senile plaques,
and Aβ ending at 42 (Aβ42) is the major depositing species (1, 2). Extracellular Aβ42 is
neurotoxic through multiple pathways (3); it is likely that increased extracellular Aβ42 is a major
cause of neuronal death in AD. Strong evidence of this is the fact that Aβ42 production is
increased by early onset familial AD (FAD)-related mutations in presenilin (PS) 1, PS2, and
amyloid β-protein precursor (APP; 1, 4, 5, 6, 7). Therefore, inhibition or clearance of
extracellular Aβ42 deposition is a potential treatment for AD (8, 9). Also, because anti-Aβ
antibodies slow cognitive decline in AD (10), immunotherapeutic approaches may be a useful
treatment (11). However, the validity of the extracellular Aβ cascade theory is still in debate
because extracellular Aβ may have neuroprotective effects under physiological conditions (12).
An alternative Aβ42 pathogenesis should also be noted. Although Aβ42 produced in
endoplasmic reticulum (ER) is physiologically secreted to extracellular space (1, 9, 13), recent
pathological studies suggest that Aβ42 accumulates in neurons before plaque formation in AD
(14, 15) and Down syndrome (DS; 16, 17). Therefore, an intrinsic Aβ pathway as well as an
extrinsic Aβ pathway are widely considered important (18). We also have reported Aβ42
accumulation in neurons, but not in the extracellular space in aged mice carrying a mutant PS1
gene (19), and selective Aβ42 accumulation in neurons undergoing apoptosis in vitro (20). Thus,
some perturbation in the intracellular Aβ metabolism might promote neurodegeneration.
Recently, Bückig et al. (21) have shown that Aβ42 overproduced in the ER is exported to
cytosol, where Aβ42 forms aggresome-like structures, and is partly transferred to the nucleus, an
unusual compartment for Aβ42 to reside. Although the incidence of neuronal apoptosis is
increased in AD (22, 23), intracellular Aβ42, but not extracellular Aβ, induces neuronal
apoptosis in vitro (24). We have found intraneuronal Aβ42 to be linked to apoptosis in AD (25).
Although intraneuronal Aβ42 is reported to be associated with dysfunction of mitochondrias
(26), lysosomes (27, 28), and synapses (29), intranuclear Aβ42 might have a novel pathogenesis
related to apoptosis. P53 protein, which inhibits the cell cycle and also induces apoptosis (30),
may play an important role in AD pathology and be associated with cytosolic/nuclear Aβ42.
Because, p53-associated neuronal death in cytosolic Aβ42-transgenic (Tg) mice (31), elevated
p53 protein levels in AD (32) and DS (33) brains, p53-dependent apoptosis of primary human
neurons by Aβ42 injected in cytosol (34), and extracellular Aβ42 neurotoxicity independent on
p53 in vitro (35, 36) have been reported. Here, we show that intracellular Aβ42 has a novel
effect on the p53 promoter and that this might contribute to neurodegeneration in AD.
MATERIALS AND METHODS
Constructs of Aβ40 and Aβ42, transfection, and RT-PCR
Oligonucleotides encoding Aβ40, Aβ42, and reverse-sequence Aβ42 (rAβ42) peptides with
initiation (ATG) and stop (TAA) sequences were subcloned into the Hind III site of pTet-Splice
(GibcoBRL, Gaithersburg, MD). Transient transfection was performed with
lipofectAMINE-PLUS reagent (Gibco BRL). All control cells were transfected with pTet-splice
vector only. SKN-SH cells were cultured in serum-free Opti-MEMI (Gibco BRL). Saos2
(p53−/−) and U2OS (p53+/+) were cultured in DMEM (Gibco BRL) containing 5% fetal bovine
A
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serum (FBS) after transfection. For semiquantitative RT-PCR, total RNA was obtained from
cells using RNeasy® Mini kit (Qiagen) and treated with RNase-free DNase I (Takara Co.,
Osaka, Japan) at 25°C for 30 min. cDNA was prepared from 1–5 µg total RNA using a First
Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Uppsala, Sweden), followed by
amplification in a Perkin Elmer GeneAmp PCR system 9700. Each 50–200 ng of cDNA and 10
pmole primer DNAs mixed in the 20 µl HS-Taq reaction solution (Takara Co.) was subjected to
25–30 cycles of 1 min at 94°C, 1 min at 53, 60, or 65°C (annealing) and 1 min at 72°C.
Quantitative analyses were done at each appropriate cycle that showed linear amplification (data
not shown). Each PCR produced specific bands of 158 (Aβ40), 194 (Aβ42 and rAβ42), 216
(human p53), 232 (guinea-pig p53), 216 (mouse p53), 298 (human β actin), 282 (guinea-pig β
actin), 302 (mouse β actin), 77 (MDM2), 97 (p21), 90 (Bax), or 88 (PIG3) nucleotides.
Densitometry analysis for quantification was performed using NIH Image 1.62b7.
Immunocytochemical staining and immunoblotting of cultured cells
For immunocytochemical staining, cells on Cell Disks (Sumitomo Chemical Co., Osaka, Japan)
were fixed in methanol–acetone (1:1). Fixed cells were treated with 3% H2O2 and blocked with
non-immune goat serum, followed by incubation with BA-27 at 1:500 and BC-05 at 1:2,000 in
PBS. After incubation at 4°C overnight, cells were incubated with anti-mouse IgG antibody and
then with peroxidase-anti-peroxidase antibody (DAKO, Carpinteria, CA). The red color was
developed with aminoethylcarbazole (AEC). For double immunofluorescent staining, after
incubation with the primary antibodies of BC-05 (monoclonal) and anti-p53 antibody
(polyclonal, FL393, Santa Cruz Biotech, Santa Cruz, CA) at 1:200, FITC-conjugated anti-mouse
IgG (Cappel, Aurora, OH) and Texas Red-conjugated anti-rabbit IgG (Cappel) antibodies were
used at 1:100 as the secondary antibodies. Fluorescence was observed with a confocal laser
microscope (FLUOVIEW FV300, Olympus Optical Co., Tokyo, Japan). For immunoblotting
analyses, the cells were lysed in 2% SDS, and 20 µg of protein was electrophoresed in a 15%
SDS-polyacrylamide gel, followed by transfer onto a PVDF membrane (Millipore, Bedford,
MA). To markedly enhance the detection of Aβ, the transblotted membrane was incubated in
2.5% glutaraldehyde for 30 min at room temperature and washed with PBS containing 50 mM
monoethanolamine for 5 min. The membrane was incubated in 5% skim milk in TBST (25 mM
Tris [pH7.6], 150 mM NaCl, 0.1% Tween-20) for 30 min. The membrane was then incubated
overnight at 4°C with each primary antibody, i.e., anti-Aβ42 (BC-05), anti-Aβ40 (BA-27),
anti-Aβ17-24 (4G8, Signet Pathology Systems, Dedham, MA), anti-β-actin (AC-15, Sigma, St.
Louis, MO), anti-p53 (DO-1, Santa Cruz Biotech), anti-MDM2 (Santa Cruz Biotech), anti-p21
(C-19, Santa Cruz Biotech), anti-Bax (Santa Cruz Biotech), anti-PIG3 (Santa Cruz Biotech),
anti-human PS1 (N-terminal, Chemicon, Temecula, CA), anti-APP (C-terminal, Sigma), or
anti-NSE (H14, DAKO) antibodies in TBST at each recommended concentration. The
peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL) were used at 1:20,000, and
the membranes were developed using Super Signal West Dura Extended Duration Substrate
(Pierce). To obtain Aβ-enriched nuclear extract proteins (NEP), NEP were prepared by a
conventional method (37), followed by the removal of high molecular weight proteins and
~10-fold concentration of the proteins performed using Ultrafree tubes with cut-off molecular
weights of 10 and 3 kD (Millipore).
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Cell viability assay and TUNEL staining
Cell viability was measured with a Cell Counting Kit-F (Wako, Osaka, Japan). Calcein-AM,
which specifically produces fluorescence in living cells, was added to the cell cultures, and the
fluorescence intensity measured with a multi-well plate reader (CytoFluor II, PerSeptive
Biosystems GMI, Clearwater, MN). To test for the specific inhibition of p53-dependent
apoptosis, sense and antisense DNAs for p53 mRNA were added to the medium at a
concentration of 15 µM (38). Statistical analysis was performed using Kruskal-Wallis and
Scheffé tests. For terminal dUTP nick-end labeling (TUNEL) staining, a Cell Death Detection
Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used and developed with AEC.
Gel mobility shift assay
Synthetic Aβ1-40 and Aβ1-42 were both obtained from BACHEM (Philadelphia, PA), and a
BandShift Kit (Amersham Pharmacia Biotech) was used. Both sense and antisense 48-mer
oligonucleotides of the p53 promoter region were synthesized (pp53, see Fig. 3B). The sense
pp53 end-labeled with 32P was annealed with antisense oligonucleotides to form double-stranded
pp53 (ds-pp53). About 10,000 cpm of labeled ds-pp53 (~0.1–5 ng DNA) was incubated with
synthetic Aβ40 or Aβ42 in 20 µl of 10 mM Tris (pH7.5), 50 mM NaCl, 0.5 mM DTT, 10%
glycerol, 0.05% NP-40, and 1 µg poly(dI-dC) for 20 min at room temperature. The incubated
samples were then electrophoresed in a 5% polyacrylamide gel, followed by autoradiography. To
study the differential binding affinity of Aβ42 in pp53, each region-specific 40-mer sense and
antisense oligonucleotides was synthesized.
Magnetic bead collection of Aβ42 and chromatin-immunoprecipitation (ChIP) assay of p53
promoter DNA
To collect Aβ42 using the oligonucleotide and magnetic beads, biotinylated oligonucleotides of
HSE-B, HSE-MT, nonHSE-3′, and their antisense oligonucleotides were synthesized. The
double-stranded oligonucleotides were coupled with streptavidin-conjugated magnetic beads
(Dynabeads® M-280, DYNAL, Oslo, Norway). Approximate 50 µg of coupled beads was mixed
with synthetic Aβ40 or Aβ42 peptide under the same conditions as for the gel mobility shift
assay. To collect intranuclear Aβ42 from Aβ42-transfected cells, 100 µg of NEP (34) was
dialyzed to remove the salts and then was incubated with ~40 µg beads in a total volume of 100
µl. A ChIP assay was performed according to previous reports (39, 40) with minor
modifications. For each immunoprecipitation, 1 µg of anti-Aβ antibody was used. PCR for the
p53 promoter DNA containing the HSE region was performed for 35 cycles of 1 min at 94°C, 1
min at 53°C and 1 min at 72°C, and produced a specific band of 123 nucleotides.
Luciferase assay
A Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) was used. Wild-type
(WT) or mutant (MT) pp53 was subcloned into the Xho-I site of the pGL3-Enhancer vector
expressing Firefly luciferase (pGL3-E-pp53WT, MT). The pGL3-E-pp53WT/MT with
pTet-Aβ40/42, pTet-tTAk and Renilla luciferase-expressing pRL-CMV were co-transfected into
SKN-SH. Luciferase assay was performed according to the manufacturer’s instructions, and
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luciferase activities were measured with a luminometer (LUMATLB9501/16, PerkinElmer Inc.,
Wellesley, MA) 48 h after transfection.
Primary brain cell cultures from fetal guinea pig
Mixed primary brain cell cultures were prepared from fetal guinea pig as previously reported
(20). Briefly, embryonic day-30 brains were minced and treated with 0.03% trypsin/0.02%
EDTA at 37°C for 3 min, followed by sieving through 67 µm mesh. Cells were then plated on
Cell Disks (Sumitomo Chemical Co., Osaka, Japan) and maintained in Opti-MEMI containing
5% fetal bovine serum for 14 days prior to the experiments.
Immunocytochemical staining of Tg mice and human brain tissues
For immunocytochemical staining of human brain, tissues were fixed in 10% buffered formalin.
Frontal and temporal cortical tissue blocks were then transferred into 30% sucrose, followed by
quick-freezing with dry ice. Each section (10 µm thick) was prepared at the time of use. Tg2576
mice were purchased from Taconic (Germantown, NY). Tg mice sections were fixed in either
10% formalin (Tg2576) or 4% paraformaldehyde (mutant PS1-Tg), followed by paraffin
embedding. After blocking endogeneous peroxidase by 0.3% H2O2 in methanol for 30 min,
Mouse to Mouse Block (SCYTEK Laboratories, Logan, UT) was used to reduce the background
in immunostained mice brain tissues. Sections were autoclaved at 120°C for 20 min in 0.01M
citrate buffer (pH 6.0), and incubated with primary antibodies in TBS containing 5% skim milk
and 0.1% Tween-20 overnight at 4°C. The primary antibodies were diluted at 1:2,000 (BC-05),
1:1,000 (4G8), and 1:200 (anti-p53, FL393, Santa Cruz). Incubation with HRP-conjugated
ENVISION+ secondary antibodies (DAKO) at room temperature for 1 h and developing with
DAB were used to detect antigens. Counterstaining with hematoxylin was performed for 10–30
s. For immunofluorescent staining, as well as in primary cultures, FITC-conjugated anti-mouse
IgG (Cappel) and Texas Red-conjugated anti-rabbit IgG (Cappel) antibodies were used.
Lipofuscin autofluorescence was blocked by 0.1% Sudan Black B in 70% ethanol (25). For
counting numbers of Aβ42 plaques and neurons, hippocampal tissues from six AD and four
age-matched control brains were immunostained with BC-05 after autoclave treatment. Numbers
of Aβ42 plaques, cytosolic Aβ42 positive neurons, and both nuclear and cytosolic Aβ42 positive
neurons were counted in six separated fields (×200 magnification) at the CA1 sector.
Human brain tissues for immunoblotting
Human brain tissues (frontal cortices) were obtained from five AD cases and five age-matched
normal controls. The ages (years) and sex (M, male; F, female) of AD cases A1-A5 were 79F,
82F, 83M, 79M and 79F, respectively; while those of control cases C1-C5 were 72M, 78M,
90M, 74F, and 76F, respectively. Clinical features and pathological findings were used for the
diagnosis of AD. Tissues were homogenized in 2% SDS, and 30 µg of total protein was
immunoblotted.
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RESULTS
Elevation of p53 mRNA levels by intracellular Aβ42
To study the effects of cytosolic Aβ accumulation, we made constructs that express Aβ40, Aβ42,
or reverse-sequence Aβ42 (rAβ42) in cytosol under a tetracycline (TC)-sensitive promoter and
transiently expressed them in the SKN-SH human neuroblastoma cell line (Fig. 1A, upper). Each
Aβ mRNA was expressed in the respective culture and was clearly inhibited by TC (Fig. 1A,
lower). ELISAs showed no increases in extracellular Aβ (data not shown). Cytosolic
accumulation of Aβ40 or Aβ42 was clearly seen in the cells (Fig. 1B). The specificity of BA-27
and BC-05 for Aβ40 and Aβ42, respectively, was shown by immunoblotting (Fig. 1B, right).
Interestingly, Aβ42 immunoreactivity was also seen in the nucleus of the cells 48 h after
transfection (Fig. 1B, right, lower panel). These cells may have been degenerating, because lots
of Aβ42-transfected cells were undergoing apoptosis at 48 h (see Fig. 2B, C). The
immunoreactivities were absorbed by the respective Aβ peptides (data not shown). To check
whether some amounts of Aβ were present in the nucleus of each transfected cell, we performed
immunoblotting of NEP after enrichment of 3–10 kD proteins (see Materials and Methods). Both
4 kD soluble Aβ40 and Aβ42 were similarly detected in NEP of the cells 24 h after transfection
(Fig. 1C). However, oligomeric or fibrillar Aβ42 was not detected in NEP (data not shown). In
addition, we found approximately six- and twofold elevations of the p53 mRNA level in Aβ42-
and Aβ40-transfected cells, respectively, whereas rAβ42 did not have this effect (Fig. 1D). Aβ42
mRNA levels were elevated from 5 h after transfection, followed by a parallel elevation of p53
mRNA levels (Fig. 1E). Taken together, an increase in p53 mRNA expression seems to be
tightly linked to cytosolic Aβ expression, especially Aβ42. In other human neuroblastoma cell
lines (LAN-5, SH-SY5Y), smaller increases in p53 mRNA levels were observed (data not
shown).
P53-dependent apoptosis caused by intracellular Aβ42
MDM2, p21 (WAF-1), Bax, and PIG3 are well-known target genes for p53 (39). We found 2- to
4-fold increases in their mRNA levels, and 1.6-fold (MDM2, Bax, PIG3) or 3-fold (p21)
increases in their protein levels in Aβ42-transfected cells (Fig. 2A). Since p53 overexpression
causes apoptosis in SKN-SH (38), we measured the cell viability, which was significantly
(P<0.01) reduced to ~20 and 40% in Aβ42- and Aβ40-transfected cells, respectively, but not in
rAβ42-transfected cells (Fig. 2B). Cell death was inhibited by TC. Further, consistent with
Zhang et al. (34), the addition of actinomycin D (ActD) or a caspase inhibitor (CI), Z-VAD-fmk,
inhibited cell death (P<0.01), indicating that de novo protein synthesis and caspase activation
were required and that it was apoptosis. Moreover, antisense p53 (AS-p53), but not sense p53
(S-p53), DNA inhibited cell death, suggesting p53 dependence. TUNEL staining showed many
positive cells in Aβ42-transfectants, a lesser number of positive cells in Aβ40-transfectants and
no positive cells in control cultures (Fig. 2C). To further confirm the p53 dependence, we studied
Saos2 (p53−/−) and U2OS (p53+/+) osteosarcoma cells. In U2OS, p53 mRNA was increased
~2-fold by Aβ40, and ~3-fold by Aβ42 and p53 protein was increased ~1.2-fold by Aβ40 and
~1.5-fold by Aβ42; however, p53 mRNA and protein did not appear in Saos2 (Fig. 2D). The
relatively lower alteration in p53 protein levels compared with mRNA levels may be due to the
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effect of p53 protein degradation. Viability of Aβ42-transfected U2OS cells decreased
significantly to ~60% (P<0.01), but Aβ42-transfected Saos2 cells were unaffected (Fig. 2E).
Antisense p53 DNA inhibited cell death in U2OS. As Saos2 and U2OS readily died in the
serum-free medium that were used for SKN-SH, we cultured them in 5% FBS-containing
medium after transfection; the relatively lower effect on p53 mRNA expression and vulnerability
of U2OS compared with SKN-SH would be due to the differences in medium conditions.
Aβ42 bound to heat-shock elements in the p53 promoter
Aβ theoretically forms a β-hairpin shape followed by a helix-turn-helix (HTH) motif (41), an
essential motif in the DNA binding domain of heat-shock transcription factors (HSF) (42). Since
the p53 promoter contains heat-shock elements (HSE) (43), Aβ42 might directly bind the p53
promoter. Thus, we performed a gel mobility shift assay using a 48-mer p53 promoter
oligonucleotide (pp53, see Fig. 3B). Aβ42, but not Aβ40, bound pp53 dose-dependently (Fig.
3A). Also, the reverse sequence Aβ42 peptide (Aβ42-1) did not bind (data not shown). However,
when incubated overnight, Aβ40 did bind pp53 (data not shown). Such Aβ42 binding was not
found in two other promoters, namely Oct-1 and EBNA (Fig. 3A, left). These bands of the
Aβ42-pp53 complex disappeared when electrophoresed on SDS-PAGE, indicating that this
complex was not SDS-insoluble (data not shown). An excess of cold pp53, but not non-specific
calf thymus DNA, clearly diminished the binding of Aβ42 to labeled pp53, providing evidence
of the sequence specificity (Fig. 3B, upper). As shown in Fig. 3B lower, studies using
region-specific 40-mer oligonucleotides revealed that oligonucleotides containing the middle 10
nucleotides in HSE (see HSE-A, B, C), but not nonHSE-5′ or nonHSE-3′, bound Aβ42 showing
the same size bands (arrow on the lower right panel), and that HSE-B showed the most
remarkable binding. The data indicated that Aβ42 prominently bound the middle part of HSE.
Further evidence for the Aβ42 binding site came from the observation that the replacement of the
10-nucleotide sequence from -58 to -49 markedly diminished the Aβ42 binding affinity (see Fig.
4A). We next made biotinylated HSE-B to co-precipitate Aβ42 using streptavidin-conjugated
magnetic beads. The collecting efficiency of synthetic Aβ42 by HSE-B magnetic beads was
much improved when NEP, but not bovine serum albumin (BSA), was co-incubated (Fig. 3C,
upper). Aβ40 was not recovered because of its lower affinity (data not shown). Consistent with
the gel mobility shift assay, nonHSE-3′ collected no Aβ42, and mutant (MT) HSE collected
minimal Aβ42 (Fig. 3C, middle). Approximately 10 pg Aβ42 was recovered from 100 µg NEP
of Aβ42-transfected cells (Fig. 3C, lower). Thus, Aβ42 may bind the p53 promoter in
cooperation with other unknown nuclear proteins in vivo and might not form SDS-insoluble
fibrils in the nucleus. We also performed a chromatin-immunoprecipitation (ChIP) assay. P53
promoter DNA was detected by PCR in eluates recovered from the nuclei of transfected cells by
immunoprecipitation with each specific anti-Aβ antibody (Fig. 3D). Anti-Aβ40 antibody
co-precipitated the p53 promoter, though HSE-B did not co-precipitate Aβ40, which may be due
to the cross-linking step and PCR detection in the ChIP assay. Other two different heat-shock
promoters of HSP32 (heme oxygenase-1, HO-1) (44) and HSP70 (45) were not detected by PCR,
suggesting Aβ binding specificity for the p53 promoter (data not shown).
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Activation of p53 promoter by Aβ42 in cells
We then made two constructs in which wild-type (WT) or mutant (MT) pp53 were subcloned
upstream of Firefly luciferase (Fig. 4A). The binding affinity of MT pp53, in which 10
nucleotides of the putative Aβ42 binding site were replaced with the sequence-reversed DNA,
was 50–60% of that of WT pp53. The luciferase activity in WT pp53 + Aβ42-coexpressed cells
increased about sevenfold compared with controls (Fig. 4B, left), corresponding well to the
approximately sixfold elevation in p53 mRNA levels induced by Aβ42 transfection (see Fig.
1D), while Aβ40 showed a smaller effect. This effect was inhibited by TC. In MT
pp53-transfected cells, the baseline luciferase activity was dramatically reduced to ~10% of that
of WT pp53, and the luciferase activity of MT pp53 + Aβ42-coexpressed cells increased only
approximately threefold, which did not reach statistical significance (Fig. 4B, right). Thus, the
reduction in pp53 activation from sevenfold to threefold corresponded well to the ~50% decrease
in the binding affinity of MT pp53 in vitro (Fig. 4A).
Hydrogen peroxide-induced intranuclear Aβ42 accumulation and elevated p53 expression
in guinea-pig primary neurons
We previously reported that H2O2 treatment induces selective Aβ42 accumulation in guinea-pig
primary neurons (20). Such cultures provide a suitable system for Aβ study because guinea-pig
Aβ has the same sequence as human Aβ, and the authenticity of the intracellular Aβ species has
been confirmed by HPLC-mass spectrometry (46). H2O2 is known to cause p53-related apoptosis
in neuronal cells (47). To check the biological significance of intracellular Aβ42 on the
regulation of p53 mRNA expression in native neurons, we examined the alteration of Aβ42
localization after H2O2 treatment. As shown in Fig. 5A, although intracellular Aβ42 was hard to
see under normal conditions (0 h), cytosolic and partly nuclear accumulation of Aβ42 was
significantly observed at 6 h after treatment with 1 mM H2O2. Subsequently, marked localization
of Aβ42 in nucleus was observed at 12 h. In association with intranuclear Aβ42 accumulation
(green), p53 protein (red) accumulated in these cells. Neurons intensely positive for both
intranuclear Aβ42 and p53 looked to be degenerating because dendrites and axons disappeared
and the cells became round-shape (Fig. 5A, 12 and 24 h). In fact, in an earlier report we
demonstrated many TUNEL-positive cells in these H2O2-treated cultures (20). Also, Aβ42
became detectable in NEP by immunoblotting at 12 h (Fig. 5B), corresponding to the marked
immunoreactivity in the nucleus at 12 h (Fig. 5A). Protein levels of both p53 and Bax, a target of
p53, started to be elevated at 6 h after treatment; however, levels of β-actin, the internal standard,
were not changed (Fig. 5B). Similarly, p53 mRNA levels started to be elevated at 6 h after
treatment (Fig. 5C). Thus, Aβ42 may effect p53 mRNA expression at 6 h, when intranuclear
Aβ42 was detectable by immunostaining but not yet by immunoblotting. The data indicate that
oxidative DNA damage may induce Aβ42 accumulation in cytosol and then sequentially in the
nucleus activating p53 cascade.
Intracellular Aβ42 accumulation associated with p53 expression in Swedish mutant
APP-Tg and L286V mutant PS1-Tg mice brain
To study whether similar neurodegeneration occurs in brain, we next checked intracellular Aβ42
and p53 in FAD mutant gene-Tg mice brain. It is known that intracellular Aβ accumulates with
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aging from 4 months old (28), whereas extracellular Aβ is deposited from 8 to 12 months old
(48) in Swedish 670/671 mutant APP-Tg mice (Tg2576) brain. However, cognitive impairment
as memory loss in these mice was found to be present at ~6 months old (49). As shown in Fig.
6A–C, Aβ42 accumulation was found in some neurons in 3-, 6- and 15-month-old mice,
respectively. Neurons positive for intracellular Aβ42 showed abnormal shapes that looked to be
degenerating. In 15-month-old mice, in which marked extracellular Aβ42 deposition was
observed (data not shown), we found sporadic intense Aβ42 immunoreactivity in neurons, which
were similar in size and shape (Fig. 6C, D). Moreover, double staining revealed that p53
coincides with intense Aβ42 immunoreactivity (Fig. 6F–H) in the degenerating-shaped neurons
(Fig. 6E, arrows). A similar linkage between intracellular Aβ42 and p53 was found in
17-month-old L286V mutant PS1-Tg mice brain (Fig. 6I–L), which we previously reported to
have marked intracellular Aβ42 accumulation and apoptosis without plaque formation (19).
Subsequently, we checked p53 mRNA levels in Tg mice brain by semiquantitative RT-PCR.
Before checking these Tg mice, we also checked 3-month-old APP-knockout (APP-KO) mice
brain. As shown in Fig. 7A, p53 mRNA levels were similar in APP-KO, which lacked APP (Fig.
7A, upper), and control mice brain. Therefore, intracellular Aβ42 may not be obligatory for basic
p53 mRNA expression. In contrast, p53 mRNA expression was stepwise elevated in
17-month-old non-Tg, wild-type PS1-Tg and L286V mutant PS1-Tg mice brain (Fig. 7B), while
32-kD N-terminal fragments of human PS1 were found in wild-type PS1-Tg and L286V mutant
PS1-Tg but not in non-Tg mice brain (Fig. 7B, upper). Also, increased full-length APPs were
found in wild-type and mutant APP-Tg mice brain (Fig. 7C, upper), and a similar increase in p53
mRNA was found in Tg2576. Interestingly, these increases were observed in 6- and
10-month-old mice, but little was found in 3-month-old mice, indicating that aging may alter the
effect of APP mutation on p53 mRNA expression. In all RT-PCR studies in Fig. 7, β-actin
mRNAs were used as the internal standards.
Intracellular Aβ42 accumulation associated with elevated p53 expression in AD brain
We then checked the connection between cytosolic/nuclear Aβ42 and increased p53 expression
in AD brain. First, since accurate levels of p53 mRNA in human brain tissues could not be
measured because of mRNA degradation (data not shown), we checked p53 protein expression
levels. Consistent with a previous report (32), the p53 protein levels in AD frontal cortices were
apparently elevated compared with age-matched control brains (Fig. 8A). Since neuron-specific
enolase (NSE) bands were similarly seen in both AD and normal brains, p53 may be increased in
areas where many neurons still remain. Next, we studied immunostaining of intracellular Aβ. We
autoclaved the sections before staining to enhance intracellular immunoreactivity of both Aβ42
and p53 (compare Fig. 8B, C, left two panels); conventional 99% formic acid pretreatment
enhanced senile plaques markedly but little intracellular Aβ42 (data not shown). Intracellular
Aβ42 (BC-05), but not Aβ40 (BA-27, data not shown), was observed as cytosolic granular
immunoreactivities in lots of neurons (Fig. 8B). Other anti-Aβ42 antibodies, i.e., polyclonal
anti-Aβ35-42 (FCA3542), polyclonal anti-Aβ37-42 (Chemicon), and QCB42 (Biosource) also
showed similar staining patterns (data not shown). Interestingly, neuronal Aβ42
immunoreactivities were sometimes variable (Fig. 8B, right two panels). Slightly positive (white
arrows), cytosolic granular-positive (blue arrows), or markedly whole cell body-positive (red
arrows) neurons were observed. Remarkably, the markedly whole cell body-positive neurons
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looked to be degenerating. Consistent with a previous report (32), double immunostaining
showed that p53 was positive in both neurons and astrocytes (data not shown). As shown in the
right three panels of Fig. 8C, some apoptotic nuclei (arrows) were TUNEL-positive (green) and
p53-positive (red) and show overlapping (yellow). Pre-embedded immuno-electron microscopic
observation revealed no apparent amyloid fibrils in these immuno-labeled neurons (data not
shown), which may be consistent with previous reports (21, 29). Finally, double immunostaining
of p53 and Aβ42 (BC-05) showed some neurons strikingly positive for both antigens in
putatively degenerating neurons (Fig. 8D, black arrows), phenomena similar to FAD mutant Tg
mice brains (Fig. 6) and cultured neurons (Fig. 5A), while extracellular Aβ42 depositions were
p53-negative (Fig. 8D, white arrow). The data appear consistent with our previous findings that
TUNEL positivity was associated with intraneuronal Aβ42 accumulation in AD brain (25).
Relationships among the Aβ42 plaques, cytosolic Aβ42 positive neurons, and both nuclear
and cytosolic Aβ42 positive neurons in AD brain
To further study the relative populations of Aβ42 plaques, cytosolic Aβ42 positive neurons and
nuclear Aβ42 positive neurons, we counted their numbers in the CA1 sector of the hippocampus
in six AD brains. As shown in the left panel of Table 1, numerous cytosolic Aβ42 positive
neurons (C), but just a small number of both nuclear and cytosolic Aβ42 positive neurons (N),
were noted in AD brains. The number of nuclear Aβ42 positive neurons was relatively stationary
(13–24, average 20.7) compared with those of cytosolic Aβ42 positive neurons (91–273, average
192) and Aβ42 plaques (7–80, average 27.7). In contrast, apparently smaller numbers of
cytosolic Aβ42 positive neurons (9–73, average 32.3) and only a few nuclear Aβ42 positive
neurons and Aβ42 plaques were noted in age-matched control brain. Also, the intensity of
cytosolic Aβ42 immunoreactivity in control brain was much less prominent than in AD brain
(data not shown). Interestingly, there was a tendency for the number of Aβ42 plaques and
cytosolic Aβ42-positive neurons to be correlated negatively with each other.
DISCUSSION
Our present study indicates a novel effect of intracellular Aβ42 like HSF (42). Support for this
hypothesis comes from one of our major findings; that H2O2 treatment, an inducer for genomic
DNA damage and expression of some heat-shock proteins (50), induced nuclear localization of
Aβ42 and p53 mRNA expression in guinea-pig primary neurons. Because HSF binds HSE in a
trimeric form and the carboxyl termini are important for binding DNA (42), the differences in
effects of Aβ40 and Aβ42 on transcription may be due to the differences in their carboxyl
termini and aggregative natures. The possibility that intranuclear Aβ42 induces direct DNA
damage causing an indirect effect on the p53 promoter can be excluded by the following facts.
We found 4 kD soluble Aβ42 but not putatively toxic oligomeric or fibrillar Aβ42 in the nucleus,
Aβ42 bound and activated the p53 promoter in a sequence specific manner, and a novel cytosolic
protein that may transport Aβ42 to the nucleus (described following). However, Aβ42 may not
be an authentic HSF, but just a minor transcription co-factor. Because, Aβ42 may work with
other nuclear proteins, a marked Aβ-independent decrease in p53 promoter activity was found
when Aβ42 binding sequences were mutated, and p53 mRNA did not decrease in APP-KO mice.
In fact, it was previously reported that the vulnerability of neurons against H2O2 treatment was
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not altered in APP-KO mice (51). It is unclear whether Aβ has a nuclear localization signal like
HSF (52). Nevertheless, as Johnstone et al. (53) suggested the possibility that Aβ may be
actively transported to the nucleus. Although just preliminary, we have found that a novel
cytosolic protein, which contains a nuclear localization motif, specifically binds Aβ in vitro and
induces apoptosis when overexpressed with Aβ (unpublished data). Such a chaperon protein
would regulate the Aβ42 effect on p53 mRNA levels. Primary neurons but not neuroblastoma
cells have been shown to be vulnerable to injected Aβ42 (34), though our transfection may have
produced more than enough Aβ42 to cause apoptosis even in neuroblastoma cells. The different
effects of Aβ42 in various cells might be due to differences in such a regulating system.
Just how relevant cytosolic/nuclear Aβ42 is for AD is an important issue. Intraneuronal Aβ42
has been shown to have cytosolic granular immunoreactivities in AD or DS sections (14, 15, 17),
and recent immunoelectron microscopic studies have shown that Aβ42 accumulates in
multivesicular bodies (29, 54). However, no reports have shown an apparent intranuclear Aβ42
immunoreactivity. In this study, we found Aβ42 immunoreactivity in both cytosol and nuclei in
some degenerating neurons in Tg mice and AD brain. However, careful observation is essential
since it is difficult to find these neurons as they are soon cleared by microglias in brain. Also, we
postulated two other reasons for our successful detection of such whole positive neurons in Tg
mice and AD brain. First, we used fixed frozen human brain sections that preserve intracellular
proteins much better than paraffin-embedded sections, which are often used for
immunocytochemical studies (data not shown). Second, we autoclaved mice and human brain
sections before immunostaining (55). Recent reports have demonstrated that heating pre-fixed
frozen material greatly enhances immunoreactivity especially for nuclear proteins (56) and that
heating pretreatment also enhances detection of p53 (57). In fact, we found nuclear Aβ42
positive neurons to be 6.2–20.9% of total Aβ42 positive neurons after autoclave treatment (see
Table 1). Thus, our method seems an appropriate one to stain both intracellular Aβ42 and p53.
Cytosolic granular Aβ42-accumulating neurons in AD brain may be at an early degenerating or
proapoptotic stage, which corresponds to primary neurons at 6 h after H2O2 treatment in vitro
(Fig. 5A). Consistent with this, apparent accumulation of p53 was found only in markedly
Aβ42-accumulating and putatively degenerating neurons in Tg mice (Fig. 6), patterns, which
were quite similar in AD brain (Fig. 8B–D). Moreover, cytosolic Aβ42 accumulation and
extracellular Aβ42 deposits may be pathologically reciprocal phenomena (Table 1), while
nuclear Aβ42 accumulation and apoptosis may occur continuously during the progression of AD.
It is unclear whether increased intracellular Aβ42 is simply neurotoxic or might potentially be
protective against genotoxic damage. P53 induces cell-cycle arrest to repair DNA (30, 58, 59),
protecting cells from genomic DNA damage; regulation of the p53 promoter may control p53
function (60, 61), as well as the widely known post-translational regulation of p53 protein.
However, an overload of pathogenic stress as oxidative stress (62) or overproduction of Aβ42
due to FAD gene mutation may cause an inappropriate increase in cytosolic and nuclear Aβ42,
resulting in enhancement of neuronal apoptosis in AD. Thus, intracellular Aβ42 may not be
indispensable for p53 mRNA expression, but excessive Aβ42 accumulation in cytosol may
increase the risk for p53-dependent apoptosis. At this cytosolic stage, synaptic function might be
affected by Aβ42, as recently reported in triple-Tg mice (63).
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Vast numbers of reports attribute neurodegeneration in AD to extracellular Aβ neurotoxicity (3).
However, we suggest here a novel neurodegeneration pathway, one of multiple intracellular
Aβ42 pathogeneses. Mitochondrial damage may be important for neuronal loss in AD (64) and
DS (26) brains. Thus, it is of great interest that p53 may directly induce permeabilization of
mitochondrial membrane and cytochrome c release (65–67). In addition, p53 is also reported to
be associated with synaptic degeneration as well as mitochondrial dysfunction (68). Although a
recent report has suggested a direct linkage between Aβ42 and mitochondrial toxicity (69), p53
might be involved in toxicity for mitochondria and synapses in AD. Moreover, neuronal stress
increases p38 kinase (70), which mediates phosphorylation of p53 (71) and is activated in early
stages of AD (72), indicating that p53 phosphorylation may be activated as well as p53
expression in AD. As well, it has very recently been reported that the p53 homologue p73
accumulates in nuclei and is located in dystrophic neuritis and tangles in AD hippocampus (73),
which may imply a p53-like pathogenesis of p73 in AD neurons. Accordingly, appropriately
combined treatments against Aβ pathogeneses, that is, Aβ neurotoxicity, inside and outside, may
be promising for prevention of neuronal loss in AD, though the neurodegeneration process
remains quite complicated (74). Indeed, protective strategies against neuronal apoptosis related
to Aβ may be different between intracellular (75) and extracellular Aβ (76). Some p53
cascade-blocking drugs, which actually attenuate neuronal loss in a model of Parkinson’s disease
(77), could thus inhibit intracellular Aβ42-related neurodegeneration in AD brain. Alternatively,
reducing cytosolic Aβ42 may be beneficial for neuronal survival. Although the lysosomal system
may be associated with degradation of intracellular Aβ42 (15, 28), the ubiquitin-proteasome
pathway may also degrade cytosolic Aβ42 (21, 78). Because the ubiquitin-proteasome system
may be affected in AD brain (79) and contribute to neuronal degeneration (80), activation of
Aβ42 degradation by proteasome would attenuate intracellular Aβ42 pathogenesis.
Interaction between FAD-related proteins and the intracellular Aβ42/p53 pathway remains to be
elucidated. Wild-type, but not FAD mutant APP, prevents p53-dependent neuronal apoptosis by
controlling p53 activation (81), and so a decrease in the anti-apoptotic effect of the FAD mutant
APP might thus be mediated by an increase in intracellular Aβ42. In addition, FAD mutant PS1
and PS2 are known to increase Aβ42 generation (1); p53 was reported to inhibit PS1 expression
(82), and PS2 reported to trigger p53-dependent apoptosis down-regulating PS1 expression (83).
In AD model mice; FAD mutant PS1 (19), FAD mutant APP (28), PS1/APP double-Tg (84), and
PS1/APP/tau triple-Tg mice (63), all showed intraneuronal Aβ42 accumulation without or before
extracellular Aβ42 deposition, implying that neuronal loss may precede the accumulation of
extracellular Aβ42. In support, in FAD mutant APP mice (Tg2576), intracellular Aβ
accumulation at 4 months (28), cognitive impairment at ~6 months (49), and extracellular Aβ
deposition at 8–12 months (48) are reported. Moreover, we have recently found enhanced
generation of intracellular Aβ42 by FAD mutations in PS1 and PS2 in cultured cells (85). Thus,
APP, Aβ42, PS1, PS2, and p53 may regulate each others level in neurons, and the disruption of
their balance may increase the risk for p53-dependent apoptosis. Intracellular Aβ42, especially in
cytosol and nuclei, may be an important target in the pathogenesis and therapeutics for FAD as
well as sporadic AD.
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ACKNOWLEDGMENTS
We thank N. Suzuki (Takeda Pharmaceutical Co.) for BC-05 and BA-27, N. Araki (Kumamoto
University) for Saos-2 and U-20S, H. Zheng (Baylor College of Medicine) for APP-KO mice
brains. This work was supported by a Grant-in-Aid for Scientific Research, by Research for
Comprehensive Promotion of Study of Brain (Japan Ministry of Education, Science, Sports and
Culture), by the Health Science Research Grants (Japan Ministry of Health and Welfare), by
Japan Health Sciences Foundation, and by Kyushu University Interdisciplinary Programs in
Education and Projects in Research Development.
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Received July 8, 2004; accepted October 13, 2004.
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Table 1
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Fig. 1
Figure 1. RT-PCR analysis of Aβ and p53 mRNA levels, immunoblotting and immunocytochemical staining of Aβ.
A) (upper) Constructs for transfection of Aβ40 (pTet-Aβ40), Aβ42 (pTet-Aβ42), or reverse Aβ42 (pTet-rAβ42). ATG and
TAA indicate initiation (methionine) and stop codons, respectively. Bars indicate the primers for RT-PCR. (lower) RT-
PCR of exogenous Aβ40, Aβ42, rAβ42, and β-actin mRNA 24 h after transfection. B) (left panel) Immunocytochemical
staining of cells transfected with Aβ40 (Aβ40-Tf) or Aβ42 (Aβ42-Tf) using anti-Aβ40 (BA-27) and anti-Aβ42 (BC-05)
antibodies 24 h after transfection. (right upper panel) Immunoblotting of the respective antibodies for 20 pg each of
synthetic Aβ40 and Aβ42. (Right lower panel) Immunostaining of Aβ42-Tf with BC-05 48 h after transfection. Scale bars,
20 µm. C) Immunoblotting of NEP from transfected cells. After concentration of 3–10 kD proteins, 40 µg protein was
electrophoresed in each lane. D) Quantitative RT-PCR analysis of p53 mRNA with normalization by β-actin mRNA 24 h
after transfection (n=3). E) RT-PCR time course analysis of the expressions of Aβ42 and p53 mRNAs (n=3). Control cells
were transfected with the pTet-splice vector only. TC was added at 1.0 µg/ml, a concentration at which cells remain
healthy.
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Fig. 2
Figure 2. Analyses of immunoblotting, cell viability, TUNEL staining, and RT-PCR of neuroblastoma (SKN-SH)
and osteosarcoma (Saos2 [p53−/−], U2OS [p53+/+]) cells transfected with Aβ40 or Aβ42. A) RT-PCR (upper) and
immunoblotting (lower) of MDM2, p21, Bax, PIG3, and β actin in Aβ42-transfected SKN-SH cells 0 and 24 h after
transfection. B) Relative cell viability of SKN-SH cells 24 and 48 h after transfection (n=5). TC, ActD, CI (Z-VAD-fmk),
and sense/antisense DNA were added at 1.0 µg/ml and 5, 5, and 15 µM, respectively. C) TUNEL staining 48 h after
transfection. Scale bars, 50 µm. D) RT-PCR and immunoblotting of p53 in osteosarcoma cells 48 h after transfection.
E) Relative cell viability of osteosarcoma cells 24 and 48 h after transfection (n=5).
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Fig. 3
Figure 3. Gel mobility shift assays of the p53 promoter and Aβs and immunoblotting of Aβ collected by magnetic
beads. A) Gel mobility shift assay of synthetic Aβ40 or Aβ42 with labeled p53 promoter (pp53, right panel), and with
Oct-1/EBNA (left panel). B) (upper) Binding of labeled pp53 and Aβ42 was inhibited by an excess of cold (unlabeled)
pp53 but was not inhibited by cold calf thymus (CT) DNA. X15, X30, and X60 indicate the ratios of cold DNA to labeled
pp53. (lower) Binding of labeled region-specific oligonucleotides (indicated as bars) and Aβ42. The gel shift images on
the bars are isolated from the lower right panel (indicated by the arrow). C) (upper) Immunoblotting detection of Aβ42
(BC-05) collected by biotinylated HSE-B and streptavidin-conjugated magnetic beads. Note that addition of nuclear
extract proteins (NEP), but not bovine serum albumin (BSA), markedly increases the amount of Aβ42 recovered. (middle)
Immunoblotting detection of 4-kD Aβ42 (BC-05) collected by oligonucleotides (nonHSE-3′, HSE-MT, and HSE-B, see
Figures 3B and 4A). All samples contained 20 µg NEP during incubation. Note that HSE-B recovered Aβ42 much more
efficiently than the other two oligonucleotides. (Lower) Immunoblotting detection of endogenous Aβ42 (BC-05)
recovered by HSE-B from the NEP of transfected cells. Note that a small amount of Aβ42 is detectable in the NEP of
Aβ42-transfected cells. D) Chromatin immunoprecipitation (ChIP) assay of p53 promoter DNA using PCR. PCR detected
p53 promoter DNA in the eluates immunoprecipitated with each specific anti-Aβ antibody (4G8, both Aβ40 and 42; BA-
27, Aβ40; BC-05, Aβ42).
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Fig. 4
Figure 4. Luciferase assays of wild-type (WT) and mutant (MT) p53 Promoter activities in cells transfected with
Aβ40 or Aβ42. A) Constructs for luciferase assays assessing the promoter activity of WT and MT pp53. The 10
nucleotides in the middle HSE of the MT pp53 were replaced with DNA in the reverse sequence. Each binding affinity is
shown in the gel mobility shift assay presented under each construct. B) Relative luciferase activities induced by WT (left)
and MT (right) pp53 in transfected cells 48 h after transfection (n=5). TC was added at 0.5 or 1.0 µg/ml. The basal activity
level of MT pp53 decreased to ~10% of WT pp53.
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Fig. 5
Figure 5. Time-course double immunostaining, immunoblotting, and RT-PCR analyses of 1 mM H2O2-treated
primary cultured fetal guinea-pig brain cells. A) Double immunostaining of Aβ42 (BC-05, green) and p53 (FL393,
red). Aβ42 becomes positive (green) in cytosol and nuclei at 6 h, then localizes to nuclei at 12 h. P53 becomes positive
(red) in the whole cell body at 12 h. Scale bars, 20 µm. B) Immunoblotting of Aβ42 (BC-05) in NEP and intracellular
p53, Bax, and β-actin. C) RT-PCR of p53 and β-actin. Consistent with the result of immunostaining in (A), p53 mRNA
starts to increase at 6 h.
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Fig. 6
Figure 6. Immunocytochemical staining of Swedish 670/671 mutant APP-Tg mice (Tg2576, A–H) and L286V
mutant PS1-Tg mice (I–L). A) Aβ42 staining (BC-05) in 3-month-old mice. B) Aβ42 staining (BC-05) in 6-month-old.
C) Aβ42 staining (BC-05) in 15-month-old. D) Higher magnification of (C). Arrows indicate markedly positive cells that
may originally have been neurons. E) Features of 6-month-old brain neurons. Arrows indicate both Aβ42 and p53 positive
neurons that look to be degenerating. F) Aβ42 staining (BC-05, green) in 6-month-old. G) P53 staining (red) in 6-month-
old. H) Merging of Aβ42 and p53 immunoreactivity in the 6-month-old; overlapping of green and red shows yellow. I)
Features of the 17-month-old L286V mutant PS1-Tg mice brain neurons. Arrow indicates a neuron that is both Aβ42 and
p53 positive and shows a degenerating shape. J) Aβ42 staining (BC-05, green) in 17-month-old. K) P53 staining (red) in
17-month-old. L) Merging of Aβ42 and p53 immunoreactivity in the 17-month-old. Overlapping of green and red shows
yellow. Scale bars, 50 (A–C) and 20 µm (D–L).
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Fig. 7
Figure 7. RT-PCR analyses of p53 mRNA in APP-KO (A), PS1-Tg (B), and APP-Tg mice (C). A) RT-PCR (p53 and
β-actin mRNA) and immunoblotting (full-length APP) in 3-month-old control and APP-KO. B) RT-PCR (p53 and β-actin
mRNA) and immunoblotting (32 kD N-terminal of human PS1) in 17-month-old non-Tg, WT PS1-Tg, and L286V mutant
(MT) PS1-Tg. C) RT-PCR (p53 and β-actin mRNA) in 3-, 6-, and 10-month-old non-Tg, WT APP-Tg, and Tg2576 and
immunoblotting (full-length APP) in 3-month-old mice.
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Fig. 8
Figure 8. Immunoblotting analysis, immunocytochemical staining, and double immunostaining of AD brain tissue
(temporal cortices). A) P53 and NSE in the frontal cortices of 5 AD and 5 normal cases. The immunoblotting images of
p53 and NSE are from the same gel. B) BC-05 staining without or with autoclaving and higher magnification of BC-05 or
4G8 staining. Red arrows indicate markedly immunopositive neurons that appear to be degenerating; blue arrows indicate
cytosolic granular immunopositive neurons, and white arrows indicate immunonegative neurons that look healthy. Scale
bars, 20 (right two panels) and 50 µm (left two panels). C) P53 staining (FL393) without or with autoclaving, and double
immunostaining of p53 and TUNEL staining. Overlapping of TUNEL (green) and p53 (red) shows yellow. Scale bars, 20
(right three panels) and 50 µm (left two panels). D) Overlapping of p53 (FL393, red) and Aβ42 immunoreactivity (BC-
05, green) in some cells in AD brain. Black arrows indicate neurons positive for both antibodies, and white arrows
indicate extracellular Aβ42 deposition that is negative for p53. Scale bars, 20 µm.
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