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Journal of Alzheimer’s Disease 28 (2011) 1–14 DOI 10.3233/JAD-2011-110506 IOS Press
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Formononetin Protects Neurons Against Hypoxia-Induced Cytotoxicity Through Upregulation of ADAM10 and sAPP␣
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Miao Suna,1 , Ting Zhoua , Liang Zhoua,1 , Qiang Chenb,∗ , Yan Yua , Huan Yanga , Kaiyin Zhonga , Ximeng Zhanga , Feng Xuc , Shaoqing Caic , Albert Yua , Hui Zhangd , Ruizhong Xiaod , Dongsheng Xiaod and Dehua Chuia,d,∗
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a Neuroscience
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Handling Associate Editor: Chengxin Gong
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Research Institute and Department of Neurobiology, Key Laboratory for Neuroscience, Ministry of Education and Ministry of Public Health, Health Science Center, Peking University, Beijing, China b Department of Neurology, Ningbo Beilun Hospitals, Ningbo, China c Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, China d Department of Neurology, Peking University Third Hospital, Beijing, China
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Keywords: ADAM10, amyloid- protein precursor (APP), formononetin, soluble-APP␣
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Supplementary data available online: http://www.j-alz.com/issues/28/vol28-4.html#supplementarydata03
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Abstract. Formononetin, an active constituent of the Chinese herb Astragali Radix, has been reported to have beneficial effects for Alzheimer’s disease (AD). Yet the mechanism of this effect remains to be elucidated. The present study shows that formononetin increases soluble-APP␣ (sAPP␣) secretion and thus protects human-APP Swedish mutation cell (N2a-APP cell) from hypoxia-induced apoptosis. Using hypoxic N2a-APP cell as an in vitro model of AD-like pathology, we confirmed that regular treatment with formononetin could have neuroprotective effects, followed respectively by reduced caspase 3 activity and increased cell viability. Strikingly, our data revealed that the caspase 3-blocking effect of formononetin was largely mediated by stimulation of ␣-secretase cleavage of APP, and increasing the secretion of its soluble form, sAPP␣. Moreover, the protective effect of formononetin was totally inhibited by TAPI-2, an ␣-secretase complex inhibitor, suggesting the role of the sAPP␣ pathway in the neuroprotective response to formononetin. We also found that the stimulative effect of formononetin on ␣-secretase activity was mainly conducted by upregulating ADAM10 expression at the transcriptional level. Altogether, our study provides novel insights into how formononetin mediates stimulation of the ADAM10-sAPP␣ pathway and exerts a neuronal protective effect.
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INTRODUCTION
1 These
authors contributed equally to this paper. ∗ Correspondence to: Dr. Dehua Chui, Neuroscience Research Institute, Peking University Health Science Center, 38 Xueyuan Road, Hai Dian District, 100191, Beijing, China. E-mail:
[email protected] or Dr. Qiang Chen, Department of Neurology, Ningbo Beilun Hospitals, Ningbo 315806, China.
Formononetin [7-hydroxy-3 (4-methoxypheny) chromone or 4 -methoxy daidzein] is a soy isoflavonoid found abundantly in traditional Chinese medicine Astragalus mongholicus (Bunge) and Trifolium pretense L. (red clover). It belongs to the phytoestrogens due to its similar chemical structure to gonadal steroid estrogen [1]. Studies have found
ISSN 1387-2877/11/$27.50 © 2011 – IOS Press and the authors. All rights reserved
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MATERIALS AND METHODS
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Formononetin extraction
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Fig. 1. Structure of formononetin. 13 C-MNR.
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The purity of formononetin by HPLC-UV analysis was above 98% (Fig. 1). Reagents
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), and N, N-dimethyl sulfoxide (DMSO), were purchased from Sigma (St. Louis, MO, USA). The ␣-secretase inhibitor TAPI-2 was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA, USA). The LDH kit was purchased from Promega (Madison, WI, USA), and the sAPP␣ activity kit was purchased from R&D Systems (Minneapolis, MN, USA).
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that formononetin can promote endothelial repair and wound healing [2], prevent lipopolysaccharideinduced injury in dopaminergic neurons [3], and protect PC12 cells as well as cultured cortical and hippocampal neurons from oxidative stress and toxicity induced by L-glutamate or amyloid- [4–7], suggesting a neuroprotective effect of formononetin. The soluble fragment of the amyloid- protein precursor (sAPP␣) is the product of APP cleavage by ␣-secretase in the non-amyloidogenic pathway. It has been suggested that decreased sAPP␣ correlates with memory loss during aging in a rat model [8], and generation of sAPP␣ has been found to be down-regulated in aging human fibroblasts [9]. Moreover, sAPP␣ formation tends to be down-regulated in response to ischemia [10, 11]. In neuronal precursors and nonneuronal cells, sAPP␣ acts as a proliferative factor [12–14], involved in both cell differentiation induction [15] and neurite outgrowth [16]. Besides these proliferative effects, sAPP␣ also plays an important role in neuronal plasticity and memory [17, 18]. Furthermore, sAPP␣ has been found to have a neuroprotective effect against cell apoptosis [20] and to improve the outcome of severe brain injury [19]. Therefore upregulating sAPP␣ could be a potential target for AD treatment, and to find out whether and how formononetin could regulate APP processing and sAPP␣ formation would be of great interest. Here, we used a mouse neuroblastoma cell line stably transfected with human-APP Swedish mutation (N2a-APP) to evaluate the effects of formononetin on hypoxia-induced reduction of sAPP␣ secretion and subsequent neuron injury. Results of the study provide a novel neuroprotective mechanism of formononetin mediated by increasing sAPP secretion and ␣secretase and A-Disintegrin-And-Metalloproteinase (ADAM) 10 expression.
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Astragali Radix was collected from Hunyuan County, Shanxi Province of China, and authenticated by Professor Shao-Qing CAI as the roots of Astragalus membranaceus (Fisch.) Bge. var. mongholicus 11/4/2011(Bge.) Hsiao. Formononetin was isolated from Astragali Radix by chromatographic methods, including column chromatography on D-101 macroporous adsorption resin, silica gel, and was identified on the basis of spectral data, including UV, 1 H-NMR, and
Cell culture and hypoxia treatments N2a cells stably transfected with humanAPP695sw (N2a-APP) were grown in DMEM supplemented with 10% FBS, at 37◦ C in an atmosphere of 5% CO2 . For formononetin and hypoxia treatment, cells were pre-treated with formononetin for 4 h and then transferred into an anaerobic chamber (model 1029, Forma Scientific, Marietta, OH, USA, [21]) containing 85% N2 /10% H2 /5% CO2 , for various durations. Cell viability assay MTT assay was carried out as previously described [22]. Cells were plated in 96-well plates, and the cell viability was determined by the conventional MTT reduction assay. The cells were exposed to hypoxic condition at different time points with/without various concentrations of formononetin for the indicated time. After incubation, cells were treated with the MTT solution (0.5 mg/ml) for 4 h at 37◦ C in the dark. 200 l DMSO was added to the culture medium and mixed with a pipette until the blue formazan dissolved completely. The optical density of formazan was measured at 570 nm using a micro plated reader
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As the key index for apoptosis detection, Annexin V-PI (Sigma) staining was performed to measure phosphatidylserine externalization in N2a-APP cells. Briefly, cells were processed with different treatments, then washed twice with PBS and resuspended in 200 l of binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM MgCl2 , 5 mM KCl, 2.5 mMCaCl2 ). 10 l FITC-conjugated AnnexinV was then added to give a final concentration of 0.5 g/ml. The staining sample was incubated at room temperature for 20 min, without light. Subsequently, 5 l of propidium iodine was added to the samples (final concentration of 1 g/ml) and 10,000 cells were immediately analyzed using a FACSCalibur flow cytometer (Becton Dickinson). Results were calculated as a percentage of apoptotic cells. Lactate dehydrogenase release assay
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Immunoprecipitation, electrophoresis, and Western blot analysis Western blotting and immunoprecipitaion assay were performed as previously described with minor modifications [23]. After the indicated treatments, conditioned medium was collected, mixed with the complete protease inhibitor cocktail (Roche), and centrifuged at 3,000 g for 10 min to remove cell debris. Cells were harvested and lysed on ice in Western blot lysis buffer containing 50 mM Tris-HCl, pH 6.8, 8 M urea, 5% -mercaptoethanol, 2% SDS, and protease inhibitors. The lysates were collected, centrifuged at 12,000 g for 5 min, and quantified for total proteins by the BCA protein assay kit. To detect sAPP␣, equivalent volumes of conditioned medium were directly analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), based on the protein concentration of cells in each plate. sAPP␣ were immunoprecipitated from conditioned medium using a monoclonal Aspecific antibody 6E10 as described previously [24], and culture supernatants were normalized for total cellular protein to correct for variations in cell number. For analysis of flAPP, and CTF␣, cell lysates were separated on 10% T 5% C Bicine/Tris, 8 M urea, SDS-PAGE [25]. For western blot analysis, protein was transferred to 0.45 m polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA), blocked for 1 h with 5% (m/v) nonfat milk in Tris-buffered saline (pH 7.5), supplemented with 0.1% Tween 20. Antibodies and their dilutions used in this study include A8717 (1 : 10000) for APP derivatives and anti--actin mouse monoclonal antibody (1 : 5,000) as an internal reference control. For ADAMs analysis, cells were harvested on ice, homogenized, and suspended in RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and complete protease inhibitor cocktail). Extracts (60 g of protein) were subjected to electrophoresis, and separated
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Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme present in most eukaryotic cells, released into the culture medium upon cell death due to the damage of plasma membrane. The increase in LDH activity in the culture supernatant is proportional to the number of lysed cells. After cells were exposed to hypoxia in the absence or presence of formononetin for 18 h, the supernatant and cell lysates were collected, and the amount of LDH release was determined using an assay kit according to the manufacturer’s protocol. Briefly, the supernatant and cell lysates were transferred to 96-well plates and incubated with 1 mg/ml NADH in pyruvate substrate solution at 37◦ C for 15 min. After additional incubation at 37◦ C for 15 min with 2, 4dinitrophenylhydrazine, the reaction was stopped by addition of 0.4 M NaOH. The changes in absorbance were determined at 440 nm using a spectrophotometer microplate reader. LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the supernatant + LDH in the cell lysate), according to the equation: %LDH released = (LDH activity in the medium/total LDH activity) × 100%.
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USA). Cells treated with formononetin in the presence or absence of hypoxia treatment were allowed to swell by adding 100 l of lysis buffer. The protein concentration was determined, and equal amount of protein and primary antibody were added to each well for 1 h in a 37◦ C incubator. Then the wells were washed 5 times and secondary antibody was added, the plate was then placed in a 22◦ C incubator for another 20 min. Stop buffer was added and the final value was determined at 450 nm using a spectrophotometer microplate reader.
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(Sunrise®, Tecan, Sweden). Three control wells of medium and DMSO alone was used to provide the blanks for absorbance readings.
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Measurement of caspase-3 activity Caspase activity was assayed using caspase-3 activity assay kit (MBL) according to the manufacturer’s instructions. Briefly, cells were collected and washed with PBS, then resuspended in a cell lysis buffer. After incubation on ice for 10 min, the lysates were centrifuged for 20 min at 12,000 × g, and the supernatants were collected and protein concentrations were determined. Cell lysates (100 g) were mixed with reaction buffer containing the DEVD-pNA substrate (200 M) for caspase-3 activity. The absorbance was measured in the wells at 460 nm using an ELISA reader. RT-PCR measurement
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To detect mRNA levels, total RNA was extracted with TRIZOL (Invitrogen), and converted to cDNA by reverse kit (Takara) according to the manufacturer’s instructions. ADAM10 sense primer, 5 CC ATgCTCATggAAgACAgTT-3 ; ADAM10 antisense primer, 5 -CCTTCTTCSCCSTSSSTSTgTCCA-3 ; actin sense primer, 5 -TGTACGCCTCTGGCCG TACC-3 ; and -actin antisense primer, 5 -CCACG TCACACTTCATGATGG-3 , PCR reactions were performed at 94◦ C for 30 s, 60◦ C for 1 min, and 68◦ C for 2 min during 40 cycles, followed by a final extension of 7 min at 68◦ C. The resulting PCR products were analyzed on a 1% agarose gel stained with ethidium bromide.
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ference MAX reagent (Invitrogen) according to the manufacturer’s instructions. The target sequences of the siRNAs specific for mouse ADAM10, or scrambled siRNA (Genechem, Shanghai, China) were as follows: ADAM10, 5 -GCAAAGATGATTGCTGCTTCG-3 ; Scramble 5 -GGTATATGCGCCATACACTACCC-3 . At 24 h post-siRNA treatment, cells were collected for different measurements.
All data in the text and figures are expressed as mean ± S.D. of at least three independent experiments. A one-way analysis of variance (ANOVA) followed by Dunnett’s or Tukey-Kramer’s post hoc tests was performed to compare groups. Mean values were considered significantly different at *p < 0.05, **p < 0.01, or ***p < 0.001. RESULTS
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proteins were transferred onto PVDF membranes, subsequently immunostained with the following primary antibodies against ADAM9 (1 : 500, Sigma), ADAM10 (1 : 2000, Abcam), ADAM17 (1 : 500, Sigma), and GAPDH (1 : 5000, Calbiochem). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1 : 2,000) for 1 h at ambient temperature, the immunoblots were developed using the ECL system. Quantitative densitometric analyses were performed on digitized images of immunoblots with Quantity One software (Bio-Rad, Hercules, CA, USA). Representative blots from at least three independent experiments were shown.
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The siRNA method was performed according to previous work [48]. N2a-APP cells were reverse transfected with 20 nM small interfering RNA (siRNA) duplexes by the use of Lipofectamine RNA inter-
Formononetin protects cells against hypoxia-induced cytotoxicity Using a cell culture model with hypoxic treatment, we confirmed the impairments of hypoxia treatment to cell viability by MTT and flow cytometry assay as previously reported [26]. To examine the neuroprotective effects of formononetin against hypoxia-induced cell viability loss, N2a-APP cells were treated with hypoxia in the presence and absence of formononetin at different doses (0.1, 1, 10, 20, 50, and 100 M) for 18 h, and results showed formononetin markedly attenuated hypoxia-induced cytotoxicity in a concentration-dependent manner (Fig. 2A). As the 10 M formononetin was the minimum effective concentration in our system, it was used in the following experiments. To determine whether this effect was time-dependent, N2a-APP cells were exposed to hypoxia condition at different time points in the presence or absence of 10 M formononetin and results showed formononetin significantly attenuated hypoxia-induced cytotoxicity after 18 h of hypoxia treatment with cell viability 25% higher in formononetin group than control group (Fig. 2B). Similar results were obtained by flow cytometry experiments: hypoxia treatment significantly reduced cell viability, while pretreatment with formononetin notably increased living cell number and reduced cell death rate (Fig. 2C). Using EGb, a generally recognized antioxidant which could protect cells from oxidative damage [27] as positive control, we found formononetin also
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Fig. 2. Protective effect of formononetin (FRM). A) N2a-APP cells treated with different concentrations of FRM were collected for MTT assay 18 h later. B) N2a-APP cells were treated at different hypoxia time points (0, 4, 8, 12, 18, 24 h) with or without 10 M FRM pre-treatment and then measured by MTT assay. C) N2a-APP cells were pre-treated with FRM, EGb, or the same amount of DMSO for 4 h and then processed with hypoxia treatment for another 18 h. At the end, cells were collected for flow cytometry assay. D) N2a-APP cells were pre-treated with FRM, EGb, or the same amount of DMSO for 4 h and then processed with hypoxia treatment for another 18 h. Cells lysates were collected for LDH assay, and (E) for caspase-3 activity assay. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
had a protective effect similar with that of EGb (Fig. 2C). Accordingly, we also evaluated LDH activity in culture medium as an indicator of cell damage and showed that 10 M formononetin pretreatment reduced hypoxia-mediated LDH release by nearly 40%, which further confirmed its protective role (Fig. 2D). As caspase-3 activity is an indicator of apoptosis [28], we measured its level under hypoxia in the presence or absence of different chemicals. In agreement with a previous report [29], we also observed a
significant increase of caspase-3 activity after hypoxia treatment; meanwhile in formononetin pretreatment groups, we found that the casapse-3 activity showed a significant decrease compared with the levels of those without formononetin pretreatment (Fig. 2E). Formononetin could stimulate α-secretase activity Previous reports have shown that hypoxia treatment would induce a calcium influx, activate down-stream
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Formononetin increases α-secretase activity by upregulating ADAM10 protein level, but not ADAM 9 or ADAM17 As we have found formononetin treatment increases ␣-secretase activity, we further investigated its effect
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level was significantly reduced while the total amount of APP did not change (Fig. 3B). In addition to the changes in sAPP␣, we showed that formononetin could also upregulate CTF-␣ protein levels compared to the hypoxia treatment group (Fig. 3B, C). We next measured ␣-secretase activity and noticed that after treatment with formononetin, ␣-secretase activity showed a threefold increase compared to the hypoxia group (Fig. 4A). Combined with the elevated CTF-␣ protein level, our results above indicated that formononetin could shift APP processing toward the non-amyloid pathway by increasing ␣-secretase activity.
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caspase-3 activity, and down-regulate sAPP␣ levels [30, 34]. Moreover, sAPP␣ is also known to be a potent mediator that attenuates excessive calcium entry and excitotoxicity [32, 33]; therefore, formononetin might protect cells from hypoxia-induced toxicity by upregulating sAPP␣. Indeed, we found formononetin could upregulate sAPP␣ in a dose-dependent manner under normoxia condition (data not shown). So we continued to examine the effect of formononetin on sAPP␣ levels under hypoxia treatment and found, as previously reported, that sAPP␣ was significantly decreased under hypoxic conditions [34], while formononetin pretreatment significantly increased sAPP␣ secretion (Fig. 3A). The results above suggest that formononetin may exert its protective effect by increasing sAPP␣ secretion under hypoxic conditions. Furthermore, we found this increase in sAPP␣ was conducted by shifting APP processing to the nonamyloid cleavage pathway. As shown in Fig. 3B, in agreement with previous reports [31, 45, 58], we also found that after hypoxia treatment the CTF-␣ protein
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Fig. 3. APP processing was measured after formononetin (FRM) administration. N2a-APP cells were pre-treated with FRM, EGb, or same amount of DMSO for 4 h and then processed with hypoxia treatment for another 18 h. At the end, cells lysates were collected for western blot or immuno-precipitation to detect (A) sAPP␣ secretion level; (B) full length APP protein level; and (C) CTF-␣ protein level. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 4. Formononetin (FRM) could increase ␣-secretase by upregulating ADAM10 mRNA level. N2a-APP cells were pre-treated with or without FRM for 4 h and then hypoxia treatment for an additional 18 h. After that, cells lysates were collected for different measurements as follows: (A) ␣-secretase activity; (B) pre-mature form of ADAM10 protein level; (C) mature form of ADAM10 protein level. D) The ratio of mature form of ADAM10 versus pre-mature form of ADAM10; (E) ADAM 9 protein level, and (F) ADAM17 protein level. Total mRNA was also isolated for detecting (G) ADAM10 mRNA level. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
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Since we found that formononetin could upregulate ADAM10 under hypoxic condition, followed by increasing sAPP␣ secretion levels, we next wondered whether formononetin’s effect on ADAM10 expression was hypoxia dependent. As shown in Fig. 5, we found under normoxia condition, when N2a-APP cells were treated with formononetin, the total protein level of ADAM10 and its mRNA level were both increased (Fig. 5C, D, lane 1 compared with lane 2). Furthermore, in addition to increasing ADMA10, formononetin administration could also upregulate ␣secretase activity (Fig. 5B, lane 1 and lane 2), which indicated that formononetin could increase sAPP␣ by elevating ␣-secretase activity and ADAM10 transcriptional levels independent of hypoxia. At last, we wanted to confirm whether the cell protective effect under hypoxic conditions was conducted by the ADAM10-sAPP␣ pathway activated by formononetin treatment. For this purpose, we used the ␣-secretase specific inhibitor, TAPI-2, which could
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on the components of ␣-secretase complex to clarify the underlying mechanism. As shown in Fig. 4B and C, we found that the levels of both pre-mature and mature ADAM10 were decreased under hypoxia treatment and that formononetin treatment could significantly attenuate this decrease, as well as increase the mature/immature ratio of it compared with the hypoxia group (Fig. 4D). Meanwhile, other potential ␣-secretase candidates, ADAM9 and ADAM17, did not exhibit a statistically significant change under different experimental treatments (Fig. 4E, F). Since our results showed that not only the mature form of ADAM10 was increased by formononetin treatment but also the pre-mature form, a question was raised regarding whether or not formononetin treatment could increase ADAM10 at the transcriptional level. As shown in Fig. 4G, employing conventional PCR, we found that the mRNA level of ADAM10 was significantly higher in the formononetin treatment group compared to the hypoxia group, suggesting that under hypoxic condition, formononetin treatment could recover ADAM10 at the transcriptional level.
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Fig. 5. sAPP␣ secretion and ␣-secretase activity were totally abolished by TAPI-2. A) N2a-APP cells were treated with formononetin (FRM) in the presence or absence of TAPI-2 for 24 h and then cells lysates were collected for different measurements as follows: (A) sAPP␣ protein level; (B) ␣-secretase activity; and (C) ADAM10 protein level. Total mRNA was also isolated for detecting (D) ADAM10 mRNA level. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
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ment (Fig. 6C). Meanwhile both MTT and flow cytometry assays showed a decrease of cell viability in formononetin with the TAPI-2 group compared with the formononetin alone group under hypoxic conditions (Fig. 6D–I). Furthermore, ADAM10 siRNA treatment also appeared to give a similar result. As shown in Fig. 7D–J, flow cytometry assay showed a nearly 40% decrease of living cell number in the hypoxia group compared with control, and formononetin treatment could attenuate hypoxia-induced cell impairment, while ADAM10 siRNA could almost
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significantly inhibit ␣-secretase activity and further decrease sAPP␣ production (Fig. 5A, B) without influencing ADAM10 expression (Fig. 5C, D), and ADAM10 siRNA which could inhibit sAPP␣ secretion while diminish ADAM10 expression (Fig. 7A, B, C). Treating the cells with TAPI-2 and formononetin for a total of 6 h followed by an additional 18 h of hypoxia treatment, we found while the sAPP␣ secretion and ␣-secretase activity were almost totally abolished (Fig. 6A, B), the mRNA level of ADAM10 was hardly influenced by TAPI-2 under hypoxia treat-
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Fig. 6. The neuroprotective effect of formononetin (FRM) was inhibited by TAPI-2. N2a-APP cells were administrated with hypoxia treatment in the presence or absence of pre-treatment of FRM and TAPI-2. 18 h later, cells lysates or total mRNA was collected for different measurements as follows: (A) sAPP␣ protein level; (B) ␣-secretase activity; (C) ADAM10 mRNA level. After 18 h hypoxia treatment, cells were also collected for detecting apoptosis rate or cell viability. D–H) Flow cytometry assay for neuro-apoptosis rate measurement in the presence or absence of FRM or TAPI-2. I) Viability of N2a-APP cell after 18 h hypoxia treatment with FRM and TAPI-2 was measured by MTT assay. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 7. The neuroprotective effect of formononetin (FRM) was inhibited by ADAM10 siRNA. N2a-APP cells were transfected by ADAM10 siRNA for 24 h, and then (A) ADAM10 protein level; (B) ␣-secretase activity; and (C) ADAM10 mRNA level was detected. Cells were exposed to hypoxia in the presence or absence of FRM pre-treatment for 18 h, and then cells were collected for different measurements as follows: D–H) flow cytometry assay for neuro-apoptosis rate measurement in the presence or absence of FRM and ADAM10 siRNA. I) MTT assay for cell viability measurement after 18 h hypoxia treatment in the presence or absence of FRM and ADAM10 siRNA. J) Caspase-3 activity after 18 h hypoxia treatment with FRM and ADAM10 siRNA was measured. All data were represented as a mean ± S.D. from triplicate independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
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abolish formononetin -induced protective effects and reduction in caspase-3 activity. MTT assay also showed a similar result (Fig. 7I). All results above indicated that the protective effect of formononetin against hypoxia was mainly conducted by the ADAM10sAPP␣ pathway.
DISCUSSION In the APP non-amyloid cleavage pathway, APP would be cleaved by ␣-secretase and thus produce the neuroprotective fragment sAPP␣ [35, 43]. Induced sAPP␣ secretion might have additional advan-
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nificantly affected by ischemic insult [55], while another study showed that BACE1 levels were unchanged in human neuroblastoma SH-SY5Y cells subjected to chronic hypoxic treatments [34]. Recently, researchers have found both BACE-1 [31, 53] and APH-1 [45, 46] are upregulated by elevated HIF1␣ after hypoxia treatment, and the amount of C99 as well as the ratio of C99/C83 would also be increased. In agreement with the latter two studies, we also found a significant increase in BACE activity and CTF- level (supplementary Figure 1; available online: http://www.j-alz.com/issues/28/vol284.html#supplementarydata03) as well as a decrease of CTF-␣. Enzymes acting as ␣-secretase complex in the APP non-amyloid pathway have been identified as members of ADAM family. It has also been found that under hypoxic condition ␣-secretase activity and sAPP␣ would decrease significantly [45, 56, 57]. Three of these membrane-anchored zinc-dependent metalloproteinases, ADAM10 as well as ADAM17 and ADAM9, are the candidate enzymes that show ␣-secretase activity [47]. Among them, although ADAM10 has been extensively researched, the results are still controversial. Some studies found ADAM10 and TACE level significantly decreased after hypoxia treatment [34, 58], yet other studies, using primary cell of AD transgenic mice overexpressing mutant APP or cerebral microvascular smooth muscle cells as a model, found that ADAM10 was markedly increased in the early stage of ischemic insult or hypoxia treatment and afterwards decreased [55, 57]. Furthermore, another study found in the SH-SY5Y cell line that hypoxia treatment would change APH-1 and BACE-1 levels but not ADAM10 or TACE [49]. While it is hard to reconcile these results, differing experimental procedures, such as cells/tissues examined, culture conditions, the percentage of O2 utilized, or duration of hypoxic treatment, could all contribute to discrepant results. In agreement with some previous works, we found a significant decrease in ADAM10 protein after 18 h of hypoxic treatment coincidently with a decrease in sAPP␣ secretion and CTF-␣ level. SIRT1 is one of the seven mammalian proteins of the sirtuin family of NAD (+)-dependent deacetylases, and its hyperactivity might be able to prevent AD pathology both in vitro and in vivo. It has been recently found that SIRT1 could directly activate transcription of ADAM10, elevating both mature and immature forms of ADAM10, but not ADAM9 or ADAM17, and further increasing the activity of ␣secretase [50]. Additionally, there was a study showing
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tages, for various studies have strongly established that secreted sAPP␣ possesses potent neurotrophic and neuroprotective activities against excitotoxic and oxidative assaults [32, 36], JNK-mediated apoptosis [37], and the pro-apoptotic action of mutant presenlin1 by activating the transcription factor NF-B [38]. Moreover, sAPP␣ stimulates neurite outgrowth [39], regulates synaptogenesis [40], has trophic effects on cerebral neurons in culture [41], stabilizes neuronal calcium homeostasis, and protects hippocampal and cortical neurons against the toxic effects of glutamate and A peptides [42]. Also, it has been shown that intracerebroventricular administration of secreted forms of sAPP␣ to amnestic mice had potent memory-enhancing effects and blocked learning deficits induced by scopolamine [18]. Therefore, increased production of sAPP␣ protein by upregulating ADAMs would potentially be beneficial for AD [44, 50]. Cerebral hypoxia results from an insufficient oxygen supply to the brain and causes neuronal damage in vulnerable brain areas [60]. Accumulating evidence indicates that cerebral hypoxia/stroke significantly increases AD risk [61–63]. Multiple studies have confirmed that hypoxia treatment could cause cell damage [review see 68], one of the mechanisms being the activation of caspase-3. Caspase-3 is the most abundant of the known caspases in neural cells, and appears to play a crucial role both during normal development and in situations of hypoxic injury [64]. It has been found that under hypoxic conditions, caspase-3 activity is stimulated by increased calcium influx, which is the common event after exposure to hypoxia [65, 66]. As sAPP␣ is also known to be a potent mediator attenuating excessive calcium entry and excitotoxicity [32, 67], we found that formononetin’s inhibition effects on caspase-3 activity was mainly conducted by increasing sAPP␣ secretion level. Administration of ADAM10 siRNA, which would further inhibit sAPP␣ secretion, could largely abolished formononetin’s inhibition effect on caspase-3 activity and its neuroprotective effect against hypoxia. Although hypoxia/ischemia is known to be a risk factor for AD, the detailed mechanism is still unclear, and there are conflicting results concerning the relationship between hypoxia and APP processing pathways. Some studies [54, 59] found that both activity and expression of BACE1 were significantly increased in rats under transient cerebral ischemia. Another study using AD transgenic mice overexpressing mutant APP, however, showed that neither expression nor activity of BACE was sig-
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ACKNOWLEDGMENTS
We thank Dr. Sangram S. Sisodia (University of Chicago, Chicago) for providing Mouse N2a neuroblastoma cells stably expressing APPsw. This work was supported by the National Natural Science Foundation of China (NSFC; Grants No. 30670414, No.30830120 No.30973145 and No.81171015), Doctoral Fund of Ministry of Education (20090001 110058).and the National High Technology Research and Development Program of China (973 Program No. 2012CB911004). Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1037). REFERENCES [1]
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that, as one of the chemicals belonging to isoflavone, formononetin could increase both the activity and expression of SIRT1 [51]. Combined with the aforementioned results, the fact that formononetin could increase SIRT1 expression and that SIRT1 had no effects on ADAM9 or ADAM17 gives us a clue that formononetin probably regulates ADAM10 by modulating SIRT1 level. Here we report that after formononetin administration, the ␣-secretase activity of N2a-APP cells was significantly higher than the hypoxia treatment group. Our results also show both that mature and immature forms of ADAM10 were elevated while ADAM9 and ADAM17 did not change. Interestingly, we also found that the ratio of ADAM10 mature to immature form was increased by formononetin treatment, which indicates that formononetin may not only regulate ADAM10 on a transcriptional level, but also influence ADAM10 protein trafficking and subcellular location. In summary, we demonstrated for the first time that formononetin prevented hypoxia-induced neurotoxicity by increasing sAPP␣ expression in N2a-APP cells, and we further found that the effect of formononetin on sAPP␣ level was mainly mediated by upregulating ADAM10 at the transcriptional level and further increasing ␣-secretase activity. These findings provide novel insights on formononetin’s neuroprotective mechanisms and the signaling pathways involved, and could be valuable to future therapeutic research.
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