Journal of Alzheimer’s Disease 43 (2015) 243–257 DOI 10.3233/JAD-140612 IOS Press

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ATBF1 is a Novel Amyloid-␤ Protein Precursor (A␤PP) Binding Protein that Affects A␤PP Expression Kyung-Ok Uhma,1 , Mi-Jeong Kima,1 , Makoto Kawaguchib , Hiroyasu Akatsuc,d , Yutaka Miurae , Sachiyo Misumif , Hideki Hidaf , Eun-Kyoung Choig , Yong-Sun Kimh , Makoto Michikawai and Cha-Gyun Junga,f,∗ a Department of Alzheimer’s Disease Research, Research Institute, National Center for Geriatrics and Gerontology

(NCGG), Morioka, Obu, Aichi, Japan b Department of Pathology, Niigata Rosai Hospital, Niigata, Japan c Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan d Department of Community-based Medical Education, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan e Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan f Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan g Laboratory of Cellular Aging and Neurodegeneration, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea h Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Gangwon-do, Korea i Department of Biochemistry, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan

Accepted 29 May 2014

Abstract. The cytoplasmic C-terminal domain of amyloid-␤ protein precursor (A␤PP) binds to several proteins that regulate the trafficking and processing of A␤PP and affects amyloid-␤ (A␤) production. We previously reported that levels of ATmotif binding factor 1 (ATBF1) are increased in the brains of 17-month-old Tg2576 mice compared with wild-type controls, and that A␤42 increases ATBF1 expression, inducing death in primary rat cortical neurons. Here, we show that ATBF1 levels are increased in the cytoplasm of hippocampal neurons in Alzheimer’s disease (AD) brains compared with non-AD brains. Furthermore, cotransfection of human embryonic kidney (HEK293T) and human neuroblastoma (SH-SY5Y) cells with ATBF1 and A␤PP695 increased steady-state levels of A␤PP via the binding of ATBF1 to the A␤PP cytoplasmic domain (amino acids 666–690), resulting in increased A␤ production and cellular and soluble A␤PP (sA␤PP) levels without affecting the activity or levels of A␤PP processing enzymes (␣-, ␤-, or ␥-secretase). Conversely, knockdown of endogenous ATBF1 reduced levels of cellular A␤PP, sA␤PP, and A␤ in HEK293 cells overexpressing human A␤PP695. Our findings provide insight into the dynamics of A␤PP processing and A␤ production, and suggest that ATBF1 is a novel A␤PP binding protein that may be a suitable therapeutic target for AD. Keywords: A␤ production, A␤PP binding protein, A␤PP stabilization, Alzheimer’s disease, ATBF1 1 These

authors contributed equally to this work. to: Cha-Gyun Jung, Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Science, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8136; Fax: +81 52 842 3069; E-mail: [email protected]. ∗ Correspondence

ISSN 1387-2877/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved

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INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in the elderly. Although the molecular mechanisms underlying the selective neurodegeneration in AD are still poorly understood, the deposition of amyloid-␤ peptide (A␤) in senile plaques is a characteristic hallmark of the disease and is considered to play an important role in the pathogenesis of the condition [1–3]. A␤ is derived from the amyloid-␤ protein precursor (A␤PP), a type 1 transmembrane protein, by two sequential proteolytic cleavages. A␤PP is first cleaved by ␤-site A␤PP cleaving enzyme-1 (␤-secretase, BACE-1) to generate a membrane-bound C-terminal fragment of 99 amino acids (A␤PP-C99) [4–7]. This A␤PP-C99 is further cleaved within the transmembrane domain by ␥-secretase, which consists of a multicomponent complex composed of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, to generate A␤ [8, 9]. Alternatively, A␤PP can be cleaved in the middle of the A␤ sequence within the protein by ␣-site A␤PP -cleaving enzyme (␣-secretase, including ADAM10 and AMAM17) to generate nonamyloidogenic soluble A␤PP␣ (sA␤PP␣), thereby precluding A␤ generation [10–12]. Although the detailed characterization of the A␤PP proteolytic processing pathways has revealed important targets for drug discovery, the physiological functions of A␤PP and its cleavage products remain unclear. The cytoplasmic C-terminal domain of A␤PP plays critical roles in the trafficking and processing of the protein by binding to several cell surface proteins such as Fe65, X11, and X11L. For example, overexpression of Fe65, X11, or X11L in an A␤PP transgenic mouse model causes a reduction in A␤ generation and deposition [13–15]. Furthermore, X11-deficient mice exhibit amyloidogenic processing of endogeneous A␤PP, leading to an increase in A␤ generation [16]. In addition to cell surface proteins, transmembrane proteins, such as several low-density lipoprotein receptor family members, also affect A␤PP trafficking and processing [17]. Most A␤PP binding proteins interact with the cytoplasmic domain of the protein, which contains a phosphorylation site and functional motifs that play an important role in the regulation of its metabolism, trafficking and function. Thus, studies on A␤PP function and processing, including the identification and characterization of new A␤PP binding proteins, are important for our understanding of the physiological functions and processing of A␤PP, and

can provide potential targets for therapeutic intervention in AD. AT-motif binding factor 1 (ATBF1) is a 404-kDa transcription factor that contains four homeodomains and 23 zinc-finger motifs [18]. In addition to playing a critical role in transcriptional regulation, ATBF1 also participates in various protein-protein interactions [19]. We previously reported that ATBF1 is highly expressed in postmitotic neurons and that it induces cell cycle arrest associated with neuronal differentiation in the developing rat brain [20]. Furthermore, more recent results from our laboratory revealed that ATBF1 expression is increased in the brains of 17-month-old Tg2576 mice overexpressing human A␤PP with the Swedish mutation compared with age-matched wild-type mice. Moreover, our in vitro studies showed that A␤42 and the DNA-damaging drugs etoposide and homocysteine increase ATBF1 levels in primary rat cortical neurons, whereas the knockdown of ATBF1 in these neurons protects against neuronal death induced by A␤42 , etoposide, and homocysteine, indicating that ATBF1 mediates neuronal death triggered by these factors [21]. However, it is unknown whether ATBF1 levels are also altered in the brains of AD patients. In the present study, we show that ATBF1 levels are increased in hippocampal neurons of AD patients compared with hippocampal neurons in non-AD subjects. Furthermore, we demonstrate for the first time that ATBF1 is an A␤PP binding protein, and that overexpression of ATBF1 increases the steady-state levels of A␤PP, resulting in a significant increase in A␤ production.

MATERIALS AND METHODS Immunohistochemical study of AD and non-AD brains Hippocampus postmortem tissue samples of AD and non-AD were obtained under Committees on Human Research approval of National Center for Geriatrics and Gerontology and Choju Medical Institute of Fukushimura Hospital. The information of the brain specimens is shown in Table 1. Paraffin-embedded hippocampal tissue sections from 20 AD and 25 non-AD brains were deparaffinized in xylene and rehydrated in a graded ethanol series. Sections were incubated with anti-ATBF1 (D1-120, MBL, Nagoya, Japan) antibody for 1 h in a humid chamber at room temperature and subsequently incubated with a horseradish peroxidase-conjugated rabbit IgG antibody, and sig-

K.-O. Uhm et al. / ATBF1 is a Novel A␤PP binding protein Table 1 Information on the cases used for immunohistochemistry Diagnosis

Age (years)

Break stage

PMI (h)

Gender

AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD Non-AD

91 83 99 83 82 78 91 94 83 98 92 81 85 84 79 86 87 87 79 71 88 79 96 80 87 84 80 93 91 79 90 80

5 6 5 5 None None 5 5 None None None None 6 4 None None 2 2 1 1 None None None 1 2 None None 2 None None None None

18 2 8 3 3 3 8 43 1 2.5 9 2 2 3 3 2 3 10 3 44 None 3 10 3 10 3 6 18 2 3 12 2

Female Female Female Female Female Female Female Female Female Female Male Male Male Male Female Male Male Male Male Male Male Female Male Male Male Male Male Male Male Female Female Female

nals were visualized with 3,3 -diaminobenzidine (DAKO Cytomation, Denmark). Sections were then counterstained with hematoxylin and mounted in Super Mount medium. Images were obtained using an AX70 fluorescence microscope (Olympus). Plasmid constructs An ATBF1 expression construct, containing an 11kb full-length human cDNA [18], was inserted into the pCI vector (Promega, Madison, WI, USA) with a hemagglutinin (HA)-tag sequence at the 5 terminus of the insert (HA-ATBF1) [20]. Constructs of Flag-tagged full-length human A␤PP695 and of the three different Flag-tagged C-terminal A␤PP deletion mutants (Flag- A␤PP-CTF1,
652-665; Flag- A␤PP-CTF2,
666-680; Flag- A␤PP-CTF3,
681-690 [referring to A␤PP695 numbering]) were kindly provided by Dr. Toshiharu Suzuki (Hokkaido University, Japan, [22, 23]). The pcDNA A␤PP-C99 construct (C-terminal 99 amino acid residues of A␤PP) was kindly provided by

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Dr. Hiroto Komano (Iwate Medical University, Japan, [24]). Cell culture and transfection HEK293T (human embryonic kidney, 293T), SH-SY5Y (human neuroblastoma) and HEK293A␤PP695 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. HEK293-A␤PP695 cells stably expressing human A␤PP695 were kindly provided by Dr. Toshiharu Suzuki (Hokkaido University, Japan) [25, 26]. Transfections of HEK293T and SH-SY5Y were performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Quantification of Aβ40 and Aβ42 levels To quantify A␤ levels in transfected cells, medium was replaced with fresh medium 16 h after transfection, and the conditioned medium was collected 32 h later and centrifuged for 15 min at 10,000×g to remove cell debris. Levels of secreted A␤40 and A␤42 in the medium were measured with sandwich enzyme-linked immunosorbent assay (ELISA) kits (Wako Pure Chemical Industries, Osaka, Japan). The A␤ levels were normalized to the amount of total protein in the transfected cells. Western blot analysis The cells were washed with PBS and homogenized in lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The homogenates were agitated on a rocking shaker at 4◦ C for 30 min and centrifuged at 13,000 × g at 4◦ C for 30 min to remove cell debris. The resulting supernatant was collected and the protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were subjected to 7.5% or 5–20% gradient SDS polyacrylamide gel electrophoresis, and the separated products were transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were then blocked with 5% skim milk in lysis buffer for 1 h at room temperature. These membranes were then incubated with primary antibodies, namely, anti-Flag M2 (Sigma, Saint Louis, MO, USA), anti-HA (MBL), anti-ATBF1 (D1120; MBL), anti-N-terminal A␤PP (22C11; Millipore,

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Billerica, MA, USA), anti-sA␤PP␣ (6E10; Covance, Emeyryville, CA, USA), anti-sA␤PP␤ (IBL, Gunma, Japan), anti-C-terminal A␤PP (Sigma), anti-presenilin (PS1, Millipore), anti-BACE1 (Cell Signaling Technology, Cambridge, UK), anti-ADAM10 (Millipore), anti-GFP (Santa Cruz, CA, USA), or anti-actin (Sigma) antibody. The membranes were washed and then incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. Immunoreactive bands were visualized with the ECL™ or ECL Plus™ western blotting detection reagent (GE Life Sciences, Piscataway, NJ, USA) and analyzed with the LAS3000 Mini Bio-imaging Analyzer System (Fujifilm, Tokyo, Japan). Signal intensity was determined using MultiGauge software (Fujifilm). Immunoprecipitation HEK293T cells were grown in 10-cm2 dishes. After reaching 50–70% confluency, the cells were transiently cotransfected with various combinations of the FlagA␤PP, HA-ATBF1, A␤PP-C99 or Flag-A␤PP-CTF1, Flag-A␤PP-CTF2, Flag-A␤PP-CTF3 and pCI-HA plasmids. After transfection, the cells were washed twice with PBS, lysed in 1 ml lysis buffer (25 mM TrisHCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail, and centrifuged at 13,000×g at 4◦ C for 20 min. The resulting supernatant was immunoprecipitated overnight with anti-Flag (M2) or anti-HA antibody in the presence of protein G beads (Pierce) at 4◦ C. The immune complexes were washed four times with lysis buffer. The samples were subjected to 5–20% gradient SDS-PAGE and transferred to a PVDF membrane and used for western blot analysis using specific antibodies. Measurement of secretase activity HEK293T and SH-SY5Y cells were transiently cotransfected with Flag-A␤PP and either HA-ATBF1 or pCI HA (control vector). Forty-eight hours after transfection, cells were harvested and ␤-secretase activity was measured using a ␤-secretase activity kit (Biovision, Milpitas, CA, USA) following the manufacturer’s instructions. The activity of other secretases was measured as described previously, with some modifications [27]. Briefly, transfected cells were lysed in extraction buffer on ice for 10 min and centrifuged at 10,000 × g at 4◦ C for 5 min. The protein concentration in the supernatant was quantified, and ␤-secretase activity was measured using 100 ␮g (50 ␮l) of protein.

␤-secretase activity was measured using the fluorescence resonance energy transfer (FRET) method. The sample was mixed with EDANS/DABCYL and then incubated for 2 h at 37◦ C. The fluorescence was measured with an emission wavelength of 500 nm and an excitation wavelength of 350 nm using a Spectra Max Gemini EM spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Using the same lysate, ␣-secretase activity was measured using the fluorogenic ␣-secretase substrate II with EDANS/DABCYL (480 nm emission and 330 nm excitation; Millipore), and ␥-secretase activity was measured using a fluorogenic ␥-secretase substrate with NMA/DNP (435 nm emission and 330 nm excitation; Millipore) after 2 h of incubation. Determination of AβPP half-life HEK293T cells were transiently cotransfected with Flag-A␤PP and either HA-ATBF1 or pCI HA (control vector). After 24 h, cells were incubated in the presence or absence of cycloheximide (40 ␮g/ml) for 0, 4, 8, 12, or 24 h and then lysed. The cell lysates were analyzed by western blotting with anti-Flag, anti-HA, and antiactin antibodies. ATBF1 knockdown ATBF1 knockdown experiments were performed using predesigned Stealth™ siRNA against ATBF1 (ATBF1 siRNA) and Stealth siRNA negative control (Invitrogen) as previously described [21]. The ATBF1 siRNA sequences were as follows: ATBF1-siRNA sense (5 -UAC ACU GGU CAG ACC ACU GUC CUU G-3 ) and antisense (5 -CAA GGA CAG UGG UCU GAC CAG UGU-3 ). Briefly, The HEK293A␤PP cells were transiently transfected with 50 nM ATBF1 siRNA or control siRNA using Lipofectamine RNAiMAX (Invitrogen) in accordance with the manufacturer’s instructions. The knockdown effects were examined after 48 h of incubation. The cultures were then processed for western blot analysis. Statistical analysis Statistical analysis was performed using a statistical package, GraphPad prism software (GraphPad Software, San Diego, CA, USA). All values are presented as the mean ± SEM of at least three independent experiments.

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Non-AD

247

AD

Fig. 1. ATBF1 is highly expressed in the cytoplasm of hippocampal neurons in the AD brain. Coronal section of hippocampal tissue from non-AD (A, C) and AD brains (B, D). Sections were immunostained with anti-ATBF1 antibody (D1-120), and all sections were counterstained with hematoxylin. C and D are higher magnifications of the boxed area in A and B, respectively.

RESULTS ATBF1 is highly expressed in the cytoplasm of neurons in the AD brain Previously, we reported that ATBF1 expression is increased in the brains of 17-month-old Tg2576 mice overexpressing human A␤PP with the Swedish mutation compared with age-matched wild-type mice [21]. However, the distribution of ATBF1 in human brains, as well as whether levels of ATBF1 are altered in AD brains, had not been explored. Hence, we performed an immunohistochemical study of ATBF1 expression in the hippocampus, one of the most affected brain regions in AD. We found ATBF1 immunoreactivity was more intense in the cytoplasm of hippocampal pyramidal neurons in AD brains in comparison with non-AD brains (Fig. 1). Interestingly, ATBF1 immunoreactivity was also observed in amyloid plaques in AD brains. Overexpression of ATBF1 increases Aβ generation The presence of ATBF1 in amyloid plaques and the increased levels of this protein in the cytoplasm of hippocampal neurons in AD brains suggested a potential role of the protein in A␤PP processing and A␤ generation. To examine whether ATBF1 plays a role in A␤ production, we investigated whether ATBF1

Fig. 2. Overexpression of ATBF1 increases A␤ production. A and B, HEK293T cells (A) or SH-SY5Y cells (B) were transiently cotransfected with pcDNA3-Flag-tagged A␤PP695 (Flag-A␤PP) and either pCI-HA-tagged ATBF1 (HA-ATBF1) or pCI-HA (control vector). Medium was replaced with fresh medium 16 h after transfection, and conditioned medium was collected another 32 h later. Levels of secreted A␤40 and A␤42 in the medium were measured by sandwich ELISA. The A␤ levels were normalized to the amount of total protein in the transfected cells. All the values are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, versus control, as determined by Student’s t-test.

could modulate the amount of A␤ peptide in a cell culture model. For this purpose, we overexpressed human ATBF1 in HEK293T and SH-SY5Y (human neuroblastoma) cells and examined the effect of overexpression on the production of secreted A␤ peptide. HEK293T and SH-SY5Y cells were transiently cotransfected with either HA-tagged ATBF1 (HAATBF1) or empty vector control (pcDNA-HA) and Flag-tagged human A␤PP695 (Flag-A␤PP). Fortyeight hours after transfection, the conditioned media were analyzed with sandwich ELISA to specifically assess levels of human A␤40 and A␤42 . ELISA results revealed that levels of secreted A␤40 and A␤42 in HAATBF1-transfected HEKT293 cells were significantly increased by 77% and 67%, respectively, compared with the empty vector transfection control (Fig. 2A). Similar to HEK293T cells, overexpression of ATBF1 in SH-SY5Y cells also significantly increased A␤40 and A␤42 levels in media by 101% and 43%, respectively, compared with the empty vector-transfected control cells (Fig. 2B).

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Next, we measured the amounts of cellular A␤PP and secreted sA␤PP in media by western blot using Flag antibody to examine the effect of ATBF1 overexpression on A␤PP processing. When ATBF1 was overexpressed in HEK-293T cells, the levels of cellular A␤PP and sA␤PP were greatly increased compared with empty vector-transfected cells. Quantification of western blots from three independent experiments showed that the levels of cellular A␤PP and sA␤PP were increased 3.9 and 3.7-fold, respectively, compared with empty vector-transfected cells (Fig. 3A). Similar results were obtained with SH-SY5Y cells; the levels of cellular A␤PP and sA␤PP were increased 4 and 3.5-fold, respectively, compared with controls (Fig. 3B). We also measured the levels of sA␤PP␣ and sA␤PP␤ in the media of HEK-293T and SH-SY5Y cells transfected with HA-ATBF1 and Flag-A␤PP by western blot analysis using specific antibodies against sA␤PP␣ and sA␤PP␤, and we assessed the levels of C-terminal fragment (CTF) ␣ and CTF␤ using antiC-terminal A␤PP antibody (Supplementary Fig. 1). Consistent with the results for sA␤PP, the levels of sA␤PP␣ and sA␤PP␤ were increased by ATBF1 overexpression. The levels of CTF␣ and CTF␤ were also increased by ATBF1 overexpression. These results indicate that ATBF1 regulates levels of cellular A␤PP and sA␤PP and affects A␤ production, suggesting an important role of ATBF1 in A␤PP processing. To exclude the possibility that the transfection efficiency of Flag-A␤PP is higher when it is cotransfected with HA-ATBF1 than with empty vector, HEK293T cells were cotransfected with a GFP construct and with either HA-ATBF1 or empty vector. Forty-eight hours after transfection, GFP levels were evaluated by western blotting with an anti-GFP antibody. Cotransfection with either HA-ATBF1 or empty vector resulted in similar levels of GFP (Fig. 3C). This indicates that the increase in levels of cellular A␤PP and sA␤PP induced by ATBF1 overexpression is not an artifact of greater transfection efficiency. It has been reported that altered levels of A␤PP processing enzymes affect A␤ production. ATBF1 is a transcription factor that regulates the expression of several genes, including alpha-fetoprotein, p21, and neuroD1 [19, 20, 28]. Thus, it is possible that overexpression of ATBF1 may affect the expression levels of A␤PP processing enzymes, resulting in increased A␤ production. Therefore, we next examined whether ATBF1 affects the expression of A␤PP processing enzymes. We measured protein levels of ADAM10 (␣secretase), BACE1 (␤-secretase), and PS1 (␥-secretase component) after cotransfection of HEK293T and SH-

SY5Y cells with Flag-A␤PP and either HA-ATBF1 or empty vector. The levels of these proteins were not altered by ATBF1 overexpression (Fig. 3A, B). We also directly measured the activity of these secretases using a fluorescent substrate and the FRET method. The activities of the various secretases in HEK-293T or SH-SY5Y cells transfected with HA-ATBF1 were not changed compared with empty vector-transfected cells (Supplementary Fig. 2). Taken together, these results indicate that the increase in A␤ production induced by ATBF1 overexpression was not caused by changes in the activity or levels of these secretases. ATBF1 interacts with cytoplasmic domain of AβPP To gain insight into the mechanisms by which ATBF1 regulates A␤PP processing and A␤ production, we analyzed the subcellular localization of ATBF1 and A␤PP by immunofluorescence staining after cotransfection of HEK293T and SH-SY5Y cells with FlagA␤PP and either HA-ATBF1 or empty vector. Our previous studies showed that exogenous ATBF1 was localized in the nucleus in mouse neuroblastoma cells (N2A cells) and primary rat cortical neurons [20, 21]. Consistent with these previous results, we found that exogenous ATBF1 was localized in the nucleus in HEK-293T and SH-SY5Y cells when the HA-ATBF1 vector was transfected alone (Fig. 4A). However, we found that exogenous ATBF1 was localized in the cytoplasm of cells when the HA-ATBF1 vector was cotransfected with the Flag- A␤PP vector, and that ATBF1 and A␤PP co-localized in these cells (Fig. 4A, B). These observations indicate that A␤PP overexpression alters the localization of ATBF1, suggesting that these two proteins may interact. Therefore, we next examined whether ATBF1 could interact with A␤PP using an immunoprecipitation assay. As shown in Fig. 4C, ATBF1 has the ability to coimmunoprecipitate with A␤PP. Reverse immunoprecipitation experiments were also confirmed that A␤PP was coimmunoprecipitated with ATBF1 (Fig. 4D). Next, we also examined whether endogenous ATBF1 interacts with endogenous A␤PP using SH-SY5Y cells using an immunoprecipitation assay. As shown in Fig. 4E, endogenous ATBF1 has the ability to coimmunoprecipitate with endogenous A␤PP. Thus these results indicated that ATBF1 can interact with A␤PP in cultured cells and that the interaction between ATBF1 and A␤PP may affect the distribution of ATBF1 within the cells. Several studies suggest that the cytoplasmic domain of A␤PP is responsible for binding to several

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Fig. 3. Overexpression of ATBF1 increases the steady-state levels of cellular A␤PP and soluble A␤PP (sA␤PP) in the medium. HEK293T cells (A) or SH-SY5Y cells (B) were transiently cotransfected with pcDNA3-Flag-APP695 (Flag-A␤PP) and either pCI-HA-tagged ATBF1 (HA-ATBF1) or pCI-HA (control vector). Medium was replaced with fresh medium 16 h after transfection and cells were lysed another 32 h later. The cell lysates were analyzed by western blotting with anti-HA (to detect HA-tagged ATBF1), anti-Flag (M2; to detect Flag-tagged A␤PP), anti-PS1 (CT; C-terminal), anti-BACE1, anti-ADAM10, and anti-actin antibodies. Forty-eight hours after transfection, the culture medium was collected and proteins were analyzed by western blotting using an anti-Flag (M2) antibody to detect Flag-tagged A␤PP. C) HEK293T cells were transiently cotransfected with pcDNA3-GFP and either pCI-HA-tagged ATBF1 or pCI-HA (control vector). Medium was replaced with fresh medium 16 h after transfection, and cells were lysed another 32 h later. The cell lysates were analyzed by western blotting with anti-HA (to detect HA-tagged ATBF1), anti-GFP and anti-actin antibodies. A representative immunoblot is shown (left panel), and bands were quantified by densitometry, normalized to actin, and expressed as a value relative to that of control (right panel). All the values are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, versus control, as determined by Student’s t-test.

cell surface adaptor proteins. To test the ability of ATBF1 to bind to the C-terminal domain of A␤PP, we next performed a coimmunoprecipitation assay using HEK293T cells transiently cotransfected with A␤PP-C99 (C-terminal fragment of A␤PP) and either HA-ATBF1 or empty vector. As show in

Fig. 5A, ATBF1 could interact with C99. In a parallel experiment, we measured A␤40 and A␤42 levels in the media of these cotransfected cells by ELISA after cotransfection. We found that overexpression of ATBF1 increased A␤40 and A␤42 levels in the media of A␤PP-C99-transfected cells compared with

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A

C

B

D

E

Fig. 4. Overexpression of A␤PP alters ATBF1 localization by binding to the protein. A) HEK293 cells (upper picture) or SH-SY5Y cells (lower picture) were transiently transfected with pCI-HA-tagged ATBF1 (HA-ATBF1) alone. Twenty-four hours after transfection, cells were fixed and then incubated with the anti-HA polyclonal antibody to label the exogenous HA-ATBF1. Cells were subsequently labeled with the secondary antibody, Alexa-594-conjugated goat anti-rabbit IgG, for 1 h at room temperature. Nuclei were stained with DAPI. B) HEK293 cells (upper picture) or SH-SY5Y cells (lower picture) were transiently cotransfected with pcDNA3-Flag-A␤PP695 (Flag-A␤PP) and pCIHA-tagged ATBF1 (HA-ATBF1). Twenty-four hours after transfection, cells were fixed and then labeled with the anti-Flag monoclonal and anti-HA polyclonal antibodies for 1 h at room temperature. Immunoreactivity was visualized with an Alexa-488-conjugated goat anti-mouse IgG and Alexa-594-conjugated goat anti-rabbit IgG antibodies. The right panel shows the merged image of ATBF1 and A␤PP labeling. Nuclei were stained with DAPI. C, D) ATBF1 binds to A␤PP. C) HEK293T cells were transiently cotransfected with pcDNA3-Flag-tagged A␤PP695 (Flag-A␤PP) and either pCI-HA-tagged ATBF1 (HA-ATBF1) or pCI HA (control vector). D) HEK293T cells were transiently cotransfected with pCI-HA-tagged ATBF1 (HA-ATBF1) and either pcDNA3-Flag-tagged A␤PP695 (Flag-A␤PP) or pcDNA3 (control vector). Medium was replaced with fresh medium 16 h after transfection, and cells were lysed 32 h later. Cell lysates were immunoprecipitated with the anti-HA antibody in the presence of protein G beads (C) or with the anti-Flag antibody in the presence of protein G beads (D). The immunoprecipitated complex was then subjected to western blotting with the anti-Flag antibody (C) or with anti-HA antibody (D). The lower panel shows a western blot of lysates using anti-Flag, anti-HA and anti-actin antibodies. E) Endogenous ATBF1 also binds to endogenous A␤PP. Lysates from SH-SY5Y cells were immunoprecipitated with the anti-A␤PP or with anti-IgG (as control) antibody in the presence of protein G beads. The immunoprecipitated complex was then subjected to western blotting with the anti-ATBF1 antibody. The lower panel shows a western blot of lysates using anti-ATBF1, anti-A␤PP and anti-actin antibodies. Typical bands representative of three independent experiments with similar results are shown. IP, immunoprecipitation; WB, western blotting.

empty vector-transfected cells (Fig. 5B). These results indicate that ATBF1 binds to the C-terminal of A␤PP, which likely accounts for the ability of ATBF1 to increase A␤ levels. ATBF1 decreases AβPP degradation We hypothesized that the ability of ATBF1 to increase cellular A␤PP levels may result from the capacity of cytoplasmic ATBF1 to slow A␤PP turnover. Thus, A␤PP degradation was examined in ATBF1-overexpressing cells after treatment with

cycloheximide, an inhibitor of de novo protein synthesis. Consistent with our hypothesis, the A␤PP degradation was significantly decreased in cells overexpressing ATBF1 compared with the empty vector-transfected cells (Fig. 6). This data suggests that ATBF1 in the cytoplasm critically impacts A␤PP stability. Reducing endogenous ATBF1 levels decreases levels of Aβ and cellular AβPP To further clarify the role of ATBF1 in A␤PP processing and A␤ production, we analyzed the levels of

K.-O. Uhm et al. / ATBF1 is a Novel A␤PP binding protein

Fig. 5. ATBF1 interacts with A␤PP-C99. A) HEK293T cells were transiently cotransfected with pCI-HA-tagged ATBF1 (HAATBF1) and either pcDNA3-A␤PP-C99 or pcDNA3 (control vector). Medium was replaced with fresh medium 16 h after transfection, and cells were lysed another 32 h later. Cell lysates were immunoprecipitated with the anti-HA antibody in the presence of protein G beads. The immunoprecipitated complex was then subjected to western blotting with the anti-6E10 antibody against A␤PP. The lower panel shows a western blot using anti-6E10, anti-HA and anti-actin antibodies. Typical bands representative of three independent experiments with similar results are shown. B) Effect of ATBF1 on A␤40 and A␤42 production in the presence of A␤PP-C99. HEK293T cells were transiently cotransfected with pCI-HA-tagged ATBF1 (HA-ATBF1) and either pcDNA3-A␤PP-C99 or pcDNA3 (control vector). Medium was replaced with fresh medium 16 h after transfection, and conditioned medium was collected another 32 h later. The secreted A␤40 and A␤42 in the medium were measured by sandwich ELISA. The A␤ levels were normalized to the amount of total protein in the transfected cells. All the values are presented as mean ± SEM of three independent experiments (n = 4). ∗ p < 0.05, ∗∗ p < 0.01, versus control, as determined by Student’s t-test. IP, immunoprecipitation; WB, western blotting.

cellular A␤PP, sA␤PP, and A␤ in ATBF1 knockdown cells. For this experiment, we used HEK- A␤PP cells, which stably express human A␤PP695. In these cells, endogeneous ATBF1 is localized in the cytoplasm (Fig. 7A). ATBF1 or control siRNA oligonucleotide [21] was transfected into HEK-A␤PP cells, and the levels of cellular A␤PP, sA␤PP, and A␤ were examined by western blotting or sandwich ELISA. As shown in Fig. 7B, the transfection of ATBF1 siRNA decreased

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ATBF1 protein levels by approximately 85% in HEKA␤PP cells, compared with control siRNA-transfected cells. This result indicates that endogenous ATBF1 can be efficiently knocked down in these cells by transfection of ATBF1 siRNA. When ATBF1 expression was reduced by ATBF1 siRNA, the protein levels of cellular A␤PP and sA␤PP were greatly reduced, but protein levels of BACE1, PS1, and ADAM10 remained unchanged, compared with control siRNA-transfected cells (Fig. 7B). In a parallel experiment, we measured the levels of secreted A␤40 and A␤42 in the medium, and found that ATBF1 knockdown significantly reduced A␤40 and A␤42 levels to 22% and 20%, respectively, control siRNA-transfected cells (Fig. 7C). Taken together, these data suggest that cytoplasmic ATBF1 enhances the stability of A␤PP, resulting in increased A␤ production. The C-terminal domain of AβPP is required for the induction of the ATBF1-mediated Aβ production The A␤PP cytoplasmic domain has two functional motifs; the 667 VTPEER672 and 681 GYENPTY687 motifs [29–31]. The former is phosphorylated by several kinases, which induces structural change in the cytoplasmic region. The latter is responsible for binding to several cell surface adaptor proteins, including X11L. Therefore, we analyzed whether these motifs are required for the effects of ATBF1 on the steady-state levels of A␤PP and A␤ production using three different cytoplasmic deletion constructs (A␤PP-CTF1,
652–665; A␤PP-CTF2,
666–680; A␤PP-CTF3,
681–690 [referring to A␤PP695 numbering]). When A␤PP-CTF1 was transiently cotransfected with HA-ATBF1, levels of cellular A␤PP and secreted A␤40 and A␤42 in the medium were increased. These increases were compa-

Fig. 6. ATBF1 decreases A␤PP degradation. HEK293T cells were transiently cotransfected with pcDNA3-Flag-tagged A␤PP695 (Flag-A␤PP) and either pCI-HA-tagged ATBF1 (HA-ATBF1) or pCI HA (control vector). After 24 h, cells were incubated with or without cycloheximide (40 ␮g/ml) for 0, 4, 8, 12, or 24 h and then lysed. The cell lysates were analyzed by western blotting with anti-Flag, anti-HA and anti-actin antibodies. Typical bands representative of three independent experiments with similar results are shown.

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A

B

C

Fig. 7. ATBF1 knockdown decreases the levels of cellular A␤PP, soluble A␤PP (sA␤PP), and secreted A␤40 and A␤42 in HEK293 cells stably expressing A␤PP695 (HEK293-A␤PP cells). A) endogenous ATBF1 is localized in the cytoplasm of HEK293-A␤PP cells. HEK293-A␤PP cells were stained with anti-ATBF1 (D1-120) antibody. Cell nuclei were also stained with DAPI. B) Knockdown of ATBF1 in HEK293-A␤PP cells. HEK293-A␤PP cells were transiently cotransfected with ATBF1 siRNA or control siRNA for 48 h. The levels of ATBF1, cellular A␤PP, soluble A␤PP (sA␤PP), BACE1, PS1, ADAM10, and actin were determined by western blotting using anti-ATBF1 (D1-120), anti-A␤PP (22C11; for cellular and soluble A␤PP), anti-PS1, anti-ADAM10 and anti-actin antibodies, respectively. C) HEK293-A␤PP cells were transiently cotransfected with ATBF1 siRNA or control siRNA for 48 h, and levels of secreted A␤40 and A␤42 in the medium were measured by sandwich ELISA. The A␤ levels were normalized to the amount of total protein in the transfected cells. All the values are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, versus control, as determined by Student’s t-test.

rable to those following cotransfection of full-length A␤PP with HA-ATBF1. However, when A␤PP-CTF2 or A␤PP-CTF3 was transiently cotransfected with HAATBF1, levels of cellular A␤PP (Fig. 8C) and secreted A␤40 and A␤42 (Fig. 8B) in the medium were not changed. Next, we examined whether ATBF1 could interact with amino acids 666–690 of the A␤PP Cterminal. As shown in Fig. 9, ATBF1 interacts with full-length A␤PP and A␤PP-CTF1, but not with the

CTF2 or CTF3 deletion mutants. These results indicate that ATBF1 interacts with amino acids 666–690 of A␤PP, and that this interaction affects the steady-state levels of A␤PP as well as A␤ production. DISCUSSION ATBF1, a 404-kDa transcription factor that contains four homeodomains and 23 zinc finger motifs,

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Fig. 8. The C-terminal domain of A␤PP is involved in ATBF1-mediated stabilization of the protein and enhancement of A␤ production. A) Schematic representation of A␤PP mutant constructs. B) Flag-tagged full-length A␤PP (A␤PP695, control) and the three different Flag-tagged cytoplasmic deletion mutants of A␤PP (A␤PP-CTF1,
652–665; A␤PP-CTF2,
666–680; A␤PP-CTF3,
681–690 [referring to A␤PP695 numbering]) were cotransfected with either HA-tagged ATBF1 (HA-ATBF1) or empty vector control (HA) into HEK293T cells. Medium was replaced with fresh medium 16 h after transfection, and the conditioned medium was collected another 32 h later, and the amounts of A␤40 (left panel) and A␤42 (right panel) in the medium were measured by sandwich ELISA. The A␤ levels were normalized to the amount of total protein in the transfected cells. Furthermore, cells were lysed, and the lysates were analyzed by western blotting with anti-Flag antibody to detect Flag-tagged full-length A␤PP and the three different Flag-tagged cytoplasmic deletion mutants of A␤PP (C). A representative immunoblot is shown (left panel), and bands were quantified by densitometry, normalized to actin, and expressed as a value relative to that of the control (right panel). All the values are presented as mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, versus control, as determined by Student’s t-test.

is involved in transcriptional regulation and differentiation in many types of cells, including neurons and pubertal mammary gland cells [20, 32]. We previously reported that ATBF1 expression is increased in the brains of 17-month-old Tg2576 mice overexpressing human A␤PP with the Swedish mutation compared with age-matched wild-type mice [21]. However, it remained unknown whether ATBF1 levels were also elevated in AD brains. Therefore, in the present study,

we initially performed immunohistochemical staining for ATBF1 in hippocampal tissue of AD and non-AD brains. We found that ATBF1 is localized in the cytoplasm of neurons in both AD and non-AD brains, and that ATBF1 levels are increased in the AD brain compared with the non-AD brain. We previously reported that ATBF1 is localized in the nucleus in post-mitotic neurons, where it induces cell cycle arrest and promotes neuronal differentiation in the developing rat

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K.-O. Uhm et al. / ATBF1 is a Novel A␤PP binding protein

Fig. 9. ATBF1 binds to amino acid 666–690 of C-terminal A␤PP. A) Flag-tagged full-length A␤PP (A␤PP695, control) and the three different Flag-tagged cytoplasmic deletion mutants of A␤PP (CTF1, CTF2 and CTF3) were cotransfected with either HA-tagged ATBF1 (HA-ATBF1) or empty vector control (HA) into HEK293T cells. Medium was replaced with fresh medium 16 h after transfection, and cells were lysed 32 h later. Cell lysates were immunoprecipitated with the anti-HA antibody in the presence of protein G beads. The immunoprecipitated complex was then subjected to western blotting with the anti-Flag antibody. The lower panel shows a western blot of lysates using anti-Flag, anti-HA and anti-actin antibodies. Typical bands representative of three independent experiments with similar results are shown. IP, immunoprecipitation; WB, western blotting.

brain. However, ATBF1 is localized in the cytoplasm of proliferating cells, indicating that the nuclear localization of ATBF1 is required for cell cycle arrest [20, 33]. Therefore, it is possible that nuclear localization of ATBF1 is not required in post-mitotic neurons in AD or non-AD brains. The increase in ATBF1 levels in hippocampal neurons in AD brains could be triggered by the accumulation of extracellular A␤. Indeed, A␤ increases the levels of ATBF1 mRNA and protein in cultured rat cortical neurons [21]. It has been reported that the levels of proteins involved in cell cycle progression, such as Cdc2, Cdk4, Cyclin B1 and Cyclin D, are increased in vulnerable neurons in AD brains [34–37]. Although the cytoplasmic function of ATBF1 is unknown, A␤ may induce neurons to re-enter the cell cycle [38, 39], and increased cytoplasmic ATBF1 levels may enhance this process. It has been reported that ATBF1 degradation occurs through the ubiquitin-proteasome pathway [40]. Because the ubiquitin-proteasome pathway is impaired in AD [41, 42], ATBF1 may remain undegraded in the AD brain. Interestingly, ATBF1 immunoreactivity was observed in amyloid plaques in AD brains, suggesting that ATBF1 released from dying neurons may interact with

A␤ or amyloid fibrils in amyloid plaques. Alternatively, ATBF1 may interact with a different protein, which also accumulates in amyloid plaques. Future studies should address the localization of ATBF1 in amyloid plaques. Next, we examined whether the increased cytoplasmic ATBF1 levels could affect A␤PP processing and A␤ production. A␤ deposition appears to be a critical event in AD. However, although familial A␤PP mutations can enhance A␤ production, almost all sporadic cases of AD occur with wild-type A␤PP. Therefore, a detailed understanding of the mechanisms by which A␤ is generated from A␤PP is crucial for clarifying the pathogenesis of the disease. In the present study, we found that cotransfection of ATBF1 and A␤PP695 in HEK293T and SH-SY5Y cells increased cellular A␤PP levels through the binding of ATBF1 to the A␤PP cytoplasmic domain (A␤PP-C99), resulting in an increase in A␤ production. The quantity of pathogenic A␤ peptide generated in the brain is dependent on the levels and activities of the ␣-, ␤-, and ␥-secretases. However, our present findings show that ATBF1 has no effect on the activity or protein levels of the enzymes that increase A␤ production (BACE1, PS1) or of the enzyme that reduces A␤ generation (ADAM10). Interestingly, the localization of the exogenous ATBF1 shifted from the nucleus to the cytoplasm upon A␤PP overexpression in cultured cells. This change in localization was likely mediated by the binding of ATBF1 to A␤PP. Previously, we have indicated that exogenous ATBF1 was localized in the nucleus of many types of cells, including mouse neuroblastoma cells and primary neurons. Supporting these data, we found similar result using HEK293T and SHSY-5Y cells when exogenous ATBF1 was expressed alone. However, when ATBF1 was coexpressed with A␤PP, exogenous ATBF1 was redistributed from the nucleus to the cytoplasm of cells and colocalized with A␤PP in these cells. Moreover, our immunoprecipitation results revealed that ATBF1 interacts with A␤PP. Taken together, our findings indicate that A␤PP overexpression results in a change in ATBF1 localization through a binding interaction between these two proteins. Almost all members of A␤PP binding proteins, including cell surface adaptor proteins such as Fe65 and X11, bind to the A␤PP cytoplasmic domain and affect A␤PP internalization and processing to A␤. For example, Fe65 increases, whereas X11 decreases, A␤ secretion by regulating the cell surface levels of A␤PP [43–46]. Therefore, we performed a coimmunoprecipitation experiment to determine whether ATBF1 could

K.-O. Uhm et al. / ATBF1 is a Novel A␤PP binding protein

interact with the C-terminal fragment of A␤PP (A␤PPC99). We found that ATBF1 interacts with A␤PP-C99. In contrast, reducing ATBF1 levels using siRNA in HEK293-A␤PP cells, where the protein is expressed in the cytoplasm, decreases cellular A␤PP levels and A␤ production. We also examined whether ATBF1 could stabilize A␤PP, and we demonstrated in a time course experiment with cycloheximide that ATBF1 overexpression attenuates A␤PP degradation in a timedependent manner. These data demonstrate that cytoplasmic ATBF1 stabilizes A␤PP. Therefore, the higher cytoplasmic levels of ATBF1 seen in AD brains are also expected to increase the amount of A␤PP in vivo. Finally, we sought to identify the A␤PP binding site in ATBF1. The A␤PP cytoplasmic domain has two functional motifs; the 667 VTPEER672 and 681 GYENPTY687 motifs. The former is phosphorylated by several kinases, which induces structural changes in the cytoplasmic region. The latter is responsible for binding to several cell surface adaptor proteins, including X11L. In the present study, we found that deletion of the 666 AVTPEER672 and 681 GYENPTYKEF690 sequences does not increase steady-state levels of A␤PP or A␤. In addition, our immunoprecipitation results showed that deletion of amino acids 666–672 or 681–690 in A␤PP abolished interaction of ATBF1 with A␤PP, while deletion of 651–666 did not. These results indicate that amino acids 666–690 in the A␤PP cytoplasmic domain are required for ATBF1 to increase the steady-state levels of A␤PP and A␤, and that this region might be responsible for binding to ATBF1. The A␤PP cytoplasmic domain contains an NPXY motif that is important for the internalization of a variety of transmembrane proteins via clathrin-coated pits [29]. Deletion of NPXY in A␤PP decreases internalization and reduces accumulation of the protein in the lysosomal fraction [47, 48]. Furthermore, chimeric proteins containing the cytosolic domain of A␤PP are rapidly internalized [49]. Recently, it was demonstrated that binding of X11a to this region increases the half-life of A␤PP in cultured cells [48]. This interaction could have important biological consequences, because endocytosis of A␤PP via the clathrin pathway and targeting to endosomes is important for the processing of A␤PP and the generation of A␤. We have not yet demonstrated whether increased levels of ATBF1 in the cytoplasm affect endocytosis of A␤PP. Therefore, further studies are necessary to more fully clarify the role of ATBF1 in A␤PP processing. In summary, we found increased levels of ATBF1 in the cytoplasm of hippocampal pyramidal neurons in

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AD brains compared with non-AD brains. Moreover, our in vitro experiments revealed that ATBF1 binds to the C-terminal of A␤PP, thereby increasing the steadystate levels of A␤PP and resulting in increased A␤ production. Our findings provide insight into the pathogenesis of AD, and we anticipate that they will lay the foundation for novel therapeutic strategies based on reducing cytoplasmic ATBF1 levels. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (CGJ), by The Japan-Korea Basic Scientific Cooperation Program from the Japan Society for the Promotion of Science (JSPS), Japan (CGJ), by the framework of international cooperation program managed by the National Research Foundation of Korea (NRF2011-616-H00002, EKC), by the National Research Foundation of Korea Grant funded by the Korean Government(NRF-2011-619-E0001, YSK) and by a grant from the Ministry of Health, Labour and Welfare of Japan (Research on Dementia, Health and Labor Sciences Research Grants H23-005) (MM). DISCLOSURE STATEMENT Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2373). SUPPLEMENTARY MATERIAL The supplementary material is available in the electronic version of this article: http://dx.doi.org/10.3233/ JAD-140612. REFERENCES [1]

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