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Intracellular Aß42 activates p53 promoter: a pathway to neurodegeneration in Alzheimer’s disease

Yasumasa Ohyagi^*,1, 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,
^|| Department of Neuropathology, 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 Clinical Research, National Saigata Hospital, Niigata, Japan; and
National Institute for Longevity Sciences, Aichi, Japan

¹Correspondence: 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{at}neuro.med.kyushu-u.ac.jp

SPECIFIC AIMS

Alzheimer’s disease (AD) is the major cause of dementia; amyloid ß-protein (Aß), especially Aß42, plays an important role in AD pathology. Our aims are to find a molecular mechanism of p53-dependent apoptosis induced by intracellular Aß42 and to determine the significance of intracellular Aß42 and p53 expression in AD pathology.

PRINCIPAL FINDINGS

1. Cytosolic Aß expression and induction of p53-dependent apoptosis
We made constructs that express Aß40, Aß42, or reverse-sequence Aß42 (rAß42) in cytosol under a tetracycline (TC) -sensitive promoter. Each Aß mRNA was transiently expressed in the SKN-SH human neuroblastoma cell line. We found 6- and 2-fold elevations of the p53 mRNA level in Aß42- and Aß40-transfected cells, respectively; rAß42 did not have this effect. Aß42 mRNA levels were elevated from 5 h after transfection, followed by a parallel elevation of p53 mRNA levels. In Aß42-transfected cells, 2- to 4-fold increases in MDM2, p21 (WAF-1), Bax, and PIG3 mRNAs, targets of p53, were found. Cell viability was reduced to 20% in Aß42-transfected cells (P<0.01). Addition of actinomycin D (ActD) or a caspase inhibitor (CI), Z-VAD-fmk, inhibited cell death (P<0.01); antisense p53 DNA, inhibited cell death; and TUNEL staining showed many positive cells in Aß42 transfectants. p53 mRNA and protein were increased 3-fold and 1.5-fold by Aß42 respectively in U2OS (p53+/+) but not in Saos2 (p53–/–) cells; viability of Aß42-transfected U2OS cells decreased to 60% (P<0.01), suggesting p53 dependent apoptosis.

2. Direct binding of Aß42 to p53 promoter and activation of promoter activity
Aß is reported to form a ß-hairpin shape followed by a helix-turn-helix (HTH) motif, an essential motif of heat shock transcription factors (HSF) and the p53 promoter contains heat shock elements (HSE). A gel mobility shift assay using a 48-mer p53 promoter oligonucleotide (pp53, see Fig. 1 B) revealed that Aß42, but not Aß40, bound pp53 dose dependently (Fig. 1A ). The rAß42 peptide did not bind pp53; but Aß40 bound pp53 only when incubated overnight (data not shown). Such Aß42 binding was not found in Oct-1 or EBNA promoter (Fig. 1A , left). An excess of cold pp53, but not nonspecific calf thymus DNA, diminished the binding of Aß42 to labeled pp53, showing sequence specificity (Fig. 1B , upper). Further study using the region-specific 40-mer oligonucleotides revealed that oligonucleotides containing the middle 10 nucleotides in HSE (HSE-A, B, C), but not non-HSE-5' or non-HSE-3', bound Aß42; HSE-B showed the most remarkable binding (Fig. 1B , lower). We next made biotinylated HSE-B to coprecipitate Aß42 using streptavidin-conjugated magnetic beads. The collecting efficiency of synthetic Aß42 by HSE-B magnetic beads was much improved when nuclear extract proteins (NEP), but not BSA, was coincubated (Fig. 1C , upper). Aß40 was not recovered because of its lower affinity (data not shown). Non-HSE-3' collected no Aß42 and mutant (MT) HSE collected minimal Aß42 (Fig. 1C , middle). Approximately 10 pg Aß42 was recovered from 100 µg NEP of Aß42-transfected cells (Fig. 1C , lower); thus, Aß42 may bind the p53 promoter in cooperation with other unknown nuclear proteins in vivo and may not form SDS-insoluble fibrils in the nucleus. A further chromatin immunoprecipitation (ChIP) assay showed that the p53 promoter DNA was detected by PCR in eluates recovered from the nuclei of transfected cells by immunoprecipitation with each specific anti-Aß antibody (Fig. 1D ). Anti-Aß40 antibody coprecipitated the p53 promoter; perhaps due to the cross-linking step and PCR detection in the ChIP assay. We made two constructs in which wild-type (WT) or a mutant (MT) pp53 were subcloned upstream of Firefly luciferase. The binding affinity of MT pp53 was 50–60% that of WT pp53. Luciferase activity in WT pp53 + Aß42-coexpressed cells increased 7-fold; that of MT pp53 + Aß42-coexpressed cells increased only 3-fold, which coresponded to lowered binding affinity in vitro. Thus, Aß42 may directly bind and activate p53 promoter in the nucleus.

View larger version (33K): [in this window] [in a new window]   Figure 1. Gel mobility shift assays of the p53 promoter and Aßs and immunoblotting of Aß collected by magnetic beads. A) Gel mobility shift assay of synthetic Aß40 or Aß42 with labeled p53 promoter (pp53, right panel) and Oct-1/EBNA (left panel). B) Upper: binding of labeled pp53 and Aß42 was inhibited by an excess of cold (unlabeled) pp53 but not by cold calf thymus (CT) DNA. x15, x30, and x60 = ratios of cold DNAs to labeled pp53. Lower: binding of labeled region-specific oligonucleotides (bars) and Aß42. Gel shift images on the bars are isolated from the lower right panel (arrow). C) Upper: immunoblotting detection of Aß42 (BC-05) collected by biotinylated HSE-B and streptavidin-conjugated magnetic beads. Note that addition of NEP, but not BSA, markedly increased the amount of recovered Aß42. Middle: immunoblotting detection of Aß42 (BC-05) collected by oligonucleotides (non-HSE-3', HSE-MT, and HSE-B). All samples contained 20 µg NEP during incubation. Note that HSE-B recovered Aß42 much more efficiently than the other two oligonucleotides. Lower: immunoblotting detection of endogenous Aß42 (BC-05) recovered by HSE-B from the NEP of transfected cells. A small amount of Aß42 is detectable in the NEP of Aß42-transfected cells. D) Chromatin immunoprecipitation (ChIP) assay of p53 promoter DNA using PCR. PCR detected p53 promoter DNA in the eluates immunoprecipitated with 4G8, Aß40, and 42; BA-27, Aß40; BC-05, Aß42.

3. Oxidative stress-induced cytosolic/nuclear localization of Aß42 in primary brain cells
To check the biological significance of intracellular Aß42 on regulating p53 mRNA expression in native neurons, we examined alteration of Aß42 localization after H₂O₂ treatment. As shown in Fig. 2 A, significant cytosolic and partly nuclear accumulation of Aß42 was observed 6 h after treatment with 1 mM H₂O₂. In association with marked Aß42 localization (green) in nucleus at 12 h, p53 protein (red) accumulated in these cells. Neurons intensely positive for intranuclear Aß42 and p53 appeared to be degenerating: dendrites and axons disappeared and the cells became round-shaped (Fig. 2A , 12 and 24 h). We earlier found many TUNEL-positive cells in these H₂O₂-treated cultures. Aß42 became detectable in NEP by immunoblotting at 12 h (Fig. 2B ), corresponding to marked immunoreactivity in the nucleus at 12 h (Fig. 2A ). Protein levels of p53 and Bax, a target of p53, began to be elevated 6 h after treatment, but levels of ß-actin were not changed (Fig. 2B ). Similarly, p53 mRNA levels began to be elevated 6 h after treatment (Fig. 2C ). Thus, Aß42 may effect on p53 mRNA expression at 6 h, when intranuclear Aß42 was detectable by immunostaining. The data indicate that oxidative stress may induce Aß42 accumulation in cytosol, then sequentially in nucleus activating p53 cascade.

View larger version (39K): [in this window] [in a new window]   Figure 2. Time course analysis of 1 mM H2O2-treated guinea pig primary brain cells. A) Double immunostaining of Aß42 (BC-05, green) and p53 (FL393, red). Aß42 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 Aß42 (BC-05) in NEP and intracellular p53, Bax, and ß-actin. C) RT-PCR of p53 and ß-actin. Consistent with results of immunostaining in panel A, p53 mRNA starts to increase at 6 h.

4. Intracellular Aß42 accumulation and p53 expression in degenerating neurons in AD-model mice brain
Intracellular Aß accumulates with aging from 4 months whereas extracellular Aß begins to deposit over 10 months in Swedish 670/671 mutant APP-Tg mice (Tg2576) brains. However, memory loss in these mice was found to be present at 6 months of age. In our study, Aß42 accumulation was found in some degenerating-shaped neurons in 3-, 6-, and 15-month-old mice. Double staining revealed that p53 coincides with intense Aß42 immunoreactivity in the degenerating-shaped neurons. A similar linkage between intracellular Aß42 and p53 was found in 17-month-old L286V mutant PS1-Tg mice brains. We checked p53 mRNA levels in 3-month-old APP knockout (APP-KO) mice and these Tg mice brains by semiquantitative RT-PCR. Similar p53 mRNA levels were found in APP-KO and control mice brains, indicating that Aß42 may not be obligatory for basic p53 mRNA expression. P53 mRNA expression was stepwise elevated in 17-month-old non-Tg, wild type PS1-Tg and L286V-mutant PS1-Tg mice brains. A similar increase in p53 mRNA was found in Tg2576 in 6- and 10-month-old mice; little was found in 3-month-old mice, indicating that aging may enhance the effect of APP mutation on p53 mRNA expression.

5. Intracellular Aß42 accumulation and p53 expression in degenerating neurons in AD brain
Immunoblotting indicated that the p53 protein levels in AD frontal cortices were elevated vs. age-matched control brains. Immunocytochemically, intracellular Aß, but not Aß40, was observed in many neurons; and neuronal Aß42 immunoreactivities were sometimes variable. Slightly positive, cytosolic granular-positive, or markedly whole cell body-positive neurons were observed. Remarkably, the markedly whole cell body-positive neurons appeared to be degenerating. Some apoptotic nuclei were TUNEL- and p53-positive. Double immunostaining of p53 and Aß42 showed some neurons strikingly positive for both antigens in putatively degenerating neurons, similar to Tg mice brains and cultured neurons.

CONCLUSIONS AND SIGNIFICANCE

Our present study suggests a novel biological effect of intracellular Aß42 as a transcription factor for the p53 promoter. An overload of pathogenic stress as oxidative stress or overproduction of Aß42 may cause an inappropriate increase in cytosolic and nuclear Aß42, resulting in enhancement of p53-dependent neuronal apoptosis in AD (Fig. 3 ). Since the ubiquitin-proteasome system is reported to be affected in AD brain, activation of Aß42 degradation by proteasome may attenuate intracellular Aß42 pathogenesis.

View larger version (21K): [in this window] [in a new window]   Figure 3. Scheme of intracellular Aß42 accumulation and p53-dependent apoptosis. Age-related oxidative stress or overproduction of Aß42 in endoplasmic reticulum (ER) induces Aß42 accumulation in cytosol. A part of cytosolic Aß42 may localize to nucleus and activate p53 promoter, enhancing p53-dependent apoptosis. Since p53 protein is degraded by the proteasomal pathway, activation of proteasomal function may attenuate Aß42 accumulation and p53-dependent apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2637fje;

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