Apoptosis signal-regulating kinase 1 (Ask1) targeted small interfering RNA on ischemic neuronal cell death

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Research Report

Apoptosis signal-regulating kinase 1 (Ask1) targeted small interfering RNA on ischemic neuronal cell death Hyun-Woo Kim, Kyoung-Joo Cho, Su Kyoung Lee, Gyung W. Kim⁎ Department of Neurology, Yonsei University College of Medicine, Seoul, Republic of Korea

A R T I C LE I N FO

AB S T R A C T

Article history:

Apoptosis signal-regulating kinase 1 (Ask1) is one of mitogen-activated protein kinase

Accepted 7 July 2011

kinase kinase (MAPKKK) for cell differentiation and apoptosis. The aim of the present study

Available online 18 July 2011

is to evaluate whether RNA interference against Ask1 (Ask1-siRNA) down-regulates the expression of Ask1 and prevents apoptotic neuronal cell death after ischemia/reperfusion (I/

Keywords:

R) in mice. Mice were subjected to intraluminal suture occlusion of the middle cerebral

Stroke

artery for 1 h, followed by reperfusion. The Ask1-siRNA or a control-siRNA was introduced

Cerebral infarction

using osmotic pump intracerebroventricularly at 3 days before I/R. The expression and

Apoptosis signal-regulating kinase 1

mRNA of Ask1 were evaluated by Western blot and RT-PCR after I/R with time.

RNA interference

Immunohistochemistry and TUNEL assay were also investigated to evaluate the effect of

Mice

Ask1 on cerebral infarction by Ask1-siRNA treatment. The expression of Ask1 was increased significantly at 8 h after I/R. The level of mRNA and protein of Ask1 down-regulated after treatment of Ask1-siRNA and subsequently cerebral infarction volume was reduced. Our results suggest the increased Ask1 expression induce apoptotic cell death after I/R. And we also demonstrated that Ask1-siRNA attenuates upregulation of Ask1, which was followed by the reduction of infarction in ischemic brain after I/R. Ask1-siRNA could represent a molecular target for prevention of ischemic stroke. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Numerous studies have reported that reactive oxygen species (ROS) can cause cell injury, directly or indirectly, through the signal transduction system. ROS plays an important role in the pathophysiology of cerebral ischemia through apoptosis (Chan, 2001; Fujimura et al., 1999, 2000; Murakami et al., 1997; Saito et al., 2004). The mitogen-activated protein kinase (MAPK) cascade is activated in response to diverse external stimulation, ROS, ultraviolet light (UV), and growth factors. MAPK has been shown to be involved in cell differentiation, growth, and apoptosis (Errede and Levin, 1993; Widmann et al., 1999).

Apoptosis signal-regulating kinase 1 (Ask1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, is widely distributed in various cells and is thought to be essential for cell differentiation and apoptosis (Chang et al., 1998; Hayakawa et al., 2006; Nishitoh et al., 1998; Saitoh et al., 1998; Takeda et al., 2003; Tobiume et al., 1997). In addition, Ask1 has been reported to activate under the state of stress and induce apoptosis through the MAPK cascade, SEK1–JNK signal pathway (c-Jun NH2-terminal kinase), and MAPKK3/MAPKK6p38 signal pathway (Ichijo et al., 1997). Several studies have indicated that the JNK signaling (MAPK) is involved in neuronal cell death after cerebral ischemia (Gu et al., 2001; Ozawa et al.,

⁎ Corresponding author at: Department of Neurology, College of Medicine, Yonsei University, 134, Sinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea. Fax: +82 2 393 0705. E-mail address: [email protected] (G.W. Kim). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.07.018

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1999). However, the role of Ask1 (MAPKKK), which is an upstream signal transduction participant in the MAPK cascade, is not well understood in brain. In this study, we investigated the association between the temporal expression profile of Ask1 and apoptotic cell death in a murine model of transient focal cerebral ischemia. In addition, the effectiveness of Ask1 down-regulation in preventing ischemic cell death was evaluated.

2.

Results

2.1. I/R

Change in middle cerebral artery (MCA) blood flow during

The amount of blood flow in the middle cerebral artery was measured 10 min prior to ischemia, 10 min after ischemia, and 10 min after reperfusion. There were no statistically significant differences in regional cerebral blood flow before occlusion versus after reperfusion (%: 10 min prior to ischemia, 100 ± 0, 10 min after ischemia, 14.4 ± 2.8, 10 min after reperfusion, 97.3 ± 1.8, mean ± SEM, n = 5).

2.2. Expression pattern of Ask1 and phosphorylated Ask1 (pAsk1) Western blot analysis was used to assess changes in expression patterns of Ask1 and pAsk1 after I/R with time. Ask1 and pAsk1 immunoreactivity was detected at molecular weight 155 kDa (Fig. 1A). Ask1 expression was slightly increased and maintained for 4 h, but became significantly elevated at 8 h. Levels of pAsk1 also rapidly increased at 8 h after I/R, compared to normal controls (Ask1 O.D.: Nor., 127.66 ± 16.01; 1 h, 272.38± 28.47; 2 h, 197.42 ± 19.81; 4 h, 286.99± 35.41; 8 h, 596.63 ± 106.88; 24 h 572.53 ± 91.86; pAsk1 O.D.: Nor., 113.36± 34.38; 1 h, 157.34 ± 39.55; 2 h, 219.06 ± 41.07; 4 h, 181.25± 43.54; 8 h, 465.75 ± 130.68; 24 h 323.37 ± 323.37; mean ± SEM, n = 5, Nor., Normal control; *P < 0.05; Fig. 1).

2.3. Suppression of Ask1 and pAsk1 protein and mRNA by treatment with Ask1-siRNA To assess outcome at 24 h after I/R, ask1 inhibition by Ask1siRNA treatment was tested by Western blot analysis and RT-PCR in both the Ask1-siRNA and Cont.-siRNA treatment groups (Fig. 2). RT-PCR analysis showed that Ask1 gene expression in the Ask1-siRNA treatment group was significantly suppressed 24 h after I/R (O.D.: Cont.-siRNA, 100± 0; Ask1-siRNA, 11.1 ± 6.8; mean ± SEM, n = 5, *P < 0.05; Fig. 2A). Western blot analysis demonstrated that the expression of Ask1 protein was reduced in the Ask1-siRNA treatment group compared with the Con.-siRNA treatment group (O.D.: Cont.-siRNA, 100 ± 0; Ask1-siRNA, 26.8 ± 6.0; mean ± SEM, n = 5, *P< 0.05; Fig. 2B). Furthermore, pAsk1 was barely expressed (O.D.: Cont.-siRNA, 100 ± 0; Ask1-siRNA, 24.1 ± 8.7; mean± SEM, n = 5, *P < 0.05; Fig. 2C). These results suggest that our synthetic Ask1-siRNA suppresses Ask1 expression, and subsequently pAsk1, after I/R.

2.4. Reduction of cell death and infarct size by Ask1-siRNA treatment To examine whether suppression of Ask1 expression protects the brain from ischemic injury, apoptotic cell death and lesion size were compared between the two groups, using immunohistochemistry and TTC staining. To determine the relationship between Ask1 expression and DNA fragmentation in neuronal cells, double/triple fluorescence labeling was performed for Ask1 (green/red), TUNEL (green), nuclei (blue) and NeuN (red) staining in mouse brain (Fig. 3A and B). In the Ask1-siRNA group, there was a significant decrease in Ask1 immunopositive cells. No remarkable TUNEL-positive cells were detected at 24 h after I/R (Ask1; Cont.-siRNA, striatum, 680.0 ± 191.1, cortex, 600.4 ± 157.5; Ask1-siRNA, striatum, 22.1 ± 1.9, cortex, 7.4 ± 5.7; mean ± SEM, n = 5, *P< 0.05; Fig. 3Ab) (TUNEL; Cont.-siRNA, striatum, 789.9 ± 179.8, cortex, 981.8 ± 206.7; Ask1siRNA, striatum, 97.8 ± 39.2, cortex, 39.4 ± 18.3; mean ± SEM, n = 5, *P < 0.05; Fig. 3Ad). TTC staining showed results consistent with

Fig. 1 – Western blot analyses for Ask1 and pAsk1. Expression of Ask1 and pAsk1 protein was quantified at times after Ischemia/Reperfusion. Nor, normal control; mean ± SEM; *P < 0.05.

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Fig. 2 – Effects of Ask1 targeted siRNA. Semi-quantitative RT-PCR for Ask1 mRNA and Western blot assay for Ask1/pAsk1 protein were performed 24 h after Ischemia/Reperfusion in Ask1-siRNA treated or Cont.-siRNA treated mice. The gene and protein expressions of Ask1/pAsk1 were quantified as mean ± SEM; *P < 0.05.

TUNEL staining. Lesion volume was smaller in the Ask1-siRNA group than the Cont.-siRNA group (Cont.-siRNA, 98.1 ± 15.6 mm3; Ask1-siRNA, 29.2 ± 7.4 mm3; mean ± SEM, n = 6, *P< 0.05; Fig. 3C).

3.

Discussion

These experiments demonstrated that expression of Ask1 protein increased slightly during the first 4 h after I/R and increased significantly at 8 and 24 h (Fig. 1). Apoptotic cell death was induced 24 h after I/R (Fig. 3). The Ask1 protein and mRNA were suppressed by Ask1-siRNA (Fig. 2). Moreover, Ask1-siRNA treatment resulted in reduced phosphorylation of Ask1, apoptotic cell death, and decreased size of cerebral infarction (Figs. 2 and 3). I/R resulted in cerebral elevation of Ask1 protein with time. Accumulation of Ask1 promoted apoptotic cell death signal, which was blocked by suppression of Ask1 by RNA interference. This may indicate a therapeutic strategy for minimizing ischemic neuronal cell death. In this study, we observed that the expression of Ask1 protein was involved in the apoptotic cell death process caused by transient cerebral ischemia (Fig. 3). These results concur with previous findings concerning the role of Ask1 in apoptotic cell death. Apoptotic cell death was suppressed in Ask1(−/−) embryonic fibroblasts (Tobiume et al., 2001). Overexpressed Ask1 accelerated apoptosis induced by H2O2 or TNF-α (Gotoh and Cooper, 1998; Ichijo et al., 1997; Nishitoh et al., 1998). Overexpression of Ask1 increased apoptosis in vitro (Hatai et al., 2000). Moreover, after spinal cord injury, Ask1 expression peaked within 24 h (Nakahara et al., 1999). Although previous studies reported that activated Ask1 was increased in response to cerebral ischemia (Gotoh and Cooper, 1998; Ichijo et al., 1997; Zhang

et al., 2003; Zhang and Zhang, 2002), our data demonstrate that the amount of protein expression also increases with time after I/ R (Fig. 1). Furthermore, expression of phosphorylated Ask1 was showed. It is known that siRNA degrades mRNA in a sequencespecific manner. Therefore, siRNA could control the expression of specific protein. Recent studies reported that cell death was controlled by knock-down of Ask1 using RNA-silencing techniques (Pan et al., 2010; Sekimukai et al., 2009). In our study, RNA-silencing blocked the expression of Ask1 mRNA and protein expression. Consequently, phosphorylated Ask1 levels were decreased and there was a significant reduction in cerebral ischemic cell death (Figs. 2 and 3). Immunohistochemical results provided insights into the role and function of Ask1 in neuron. In the Cont.-siRNA group, Ask1- and TUNEL-positive signals were detected in the same cells. In contrast, in Ask1-siRNA treated mice, few TUNELpositive cells were detected in Ask1-negative cells. There were very few Ask1-positive cells present, in response to Ask1 down-regulation by Ask1-siRNA treatment (Fig. 3A and B). These data suggest that our Ask1-siRNA technique efficiently knocked downed the Ask1. It is interesting to note that some TUNEL-positive cells were also found in Ask1-negative cell of the Ask1-siRNA treatment group. It is believed that inhibition of Ask1 did not completely suppress another cell death signal transduction cascade, since Ask1-independent cell death signal transduction cascades exist. Taken together, our results show that increased amounts of Ask1 protein induces apoptotic cell death, playing an important role in ischemic neuronal cell death. This evidence shows that after cerebral ischemia, Ask1 is directly associated with one of cell death signal. In addition, under the hypothesis that the Ask1-mediated cell death signal pathway is active after

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Fig. 3 – (A and B) Immunohistochemistry of Ask1 and TUNEL in neuron. Ask1 (green/red), TUNEL (green), hoechst (nuclei, blue), and NeuN (red) were stained with fluorescence labeling and counted. Scale bars = 20 μm; mean± SEM; n = 5; *P < 0.05. (C) The comparison of infarct volume between Cont.-siRNA and Ask1-siRNA treated mice. Scanned representative images of an entire TTC stained in ischemic brain sections. The infarcted areas remain unstained (white). The graph represents quantified infarct volume in two groups. Mean ± SEM, n = 6, *P < 0.05.

ischemic injury, such injury should increase Ask1 protein levels to sufficient levels for initiating cell death signal transduction. We suppose that Ask1 can lead ischemic neuronal cell death only when the two factors, quantity and quality of Ask1, are satisfied. If only activation of Ask1 could induce cell death, ready-made Ask1 protein, under normal conditions and before Ask1-siRNA treatment, should induce apoptotic cell death. Nonetheless, our data show that after ischemic injury, increased amounts and the phosphorylated form of Ask1 were inhibited, blocking cell death. This indicates that sufficient elevation in the amount of Ask1 protein is required for induction of cell death. Therefore, although this study remains to elucidate for Ask1, this study introduces the possibility that blocking Ask1 may be an effective therapeutic strategy for stroke.

4.

Experimental procedure

4.1.

Transient cerebral ischemia model

All animal experiments were performed in compliance with the guidelines of the Animal Experiment Committee, Yonsei University, College of Medicine, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Male C57BL/6J mice weighing approximately 25– 30 g (3 months, Central Laboratory Animal Inc., Seoul, Korea) were used to model transient cerebral ischemia. Transient MCAO occlusion (MCAO) was induced using nylon sutures (Kim et al., 2002). Mice were anesthetized with 2% isoflurane and the air mixture of nitrogen and oxygen (70%/30%) using a

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facial mask. Body temperature was maintained as 37 ± 0.5 °C using a heating pad. The external carotid artery (ECA) was exposed with an incision of the median neck. Then, surgical nylon suture 11.0 mm in length (Ethicon, Edinburgh, UK), rounded at the distal end with heat treatment, was inserted into the internal carotid artery (ICA) through the ECA. This resulted in a blockage of blood flow in the MCA. After 1 h, MCA blood flow was restored by the removal of the nylon (Kim et al., 2001). To confirm obstruction and restoration of blood flow in the MCA, blood flow was measured by a Laser Doppler Flowmeter (n = 5; Transonic Systems, Inc., Ithaca, NY).

4.2. Preparation of the Ask1 targeting siRNA (Ask1-siRNA) and application to mice To suppress mRNA controlling the expression of Ask1, Ask1siRNA was prepared (Ambion, Austin, USA; sense, GCUCGUAAUUUAUACACUGtt; antisense, CAGUGUAUAAAUUACGAGCtt; conc, 5 μM). Using the SiPORT NeoFX (Ambion) and an osmotic minipump (Alzet, Cupertino, CA), 5 μM Ask1siRNA was administered to the cerebral ventricle for 3 days prior to the MCAO (1 μL/h/3 days, mediolateral = 1.0 mm; anteroposterior = 0.2 mm; dorsoventral = 3.1 mm). Ask1siRNA was infused to the experiment group (n = 9). Scramble siRNA was used as a control (Cont.-siRNA; n = 9). Additionally, RT-PCR was performed to measure the expression level of Ask1 mRNA after siRNA treatment.

4.3. Expression measurement using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) Total RNA was extracted from the ischemic penumbra using guanidine isothiocyanate, phenol, chloroform. Then, cDNA was synthesized and used as a PCR template. The primer of Ask1 was determined by applying the cDNA sequency provided by the nucleotide data base of the National Center for Biotechnology Information (NCBI; primer, 5′-TGC TCA CAG CGA TGC CAA AG-3′; reverse, 5′-GAA GCT ACT GCA GGA GGG TA-3′). For semi-quantitative PCR, 30 cycles (94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min) were performed. Then 1% agarose gel electrophoresis was performed and stained with ethidium bromide. Ask1 was examined at the 949-bp band, while GAPDH was examined at the 285-bp band. The density of Ask1 was analyzed with an imaging analysis program (TINA) and compared with GAPDH. The relative expression levels of Ask1 were calculated.

4.4.

Detection of Ask1 and pAsk1 by Western blot analysis

Experimental animals were decapitated at various times after the start of reperfusion and samples from the ischemic penumbra were taken. The tissues were homogenized in cold homogenizing buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 12 mM b-glycerophosphate, 3 mM dithiolthreitol (DTT), 2 mM sodium orthovanadate, 1 mM EGTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, and 5 mg/mL each of leupeptin, pepstatin A, and aprotinin (Sigma-Aldrich, St Louis, MO). The homogenized tissues were centrifuged at 15,000 ×g for 20 min, the supernatant collected, and protein content analyzed.

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To samples containing the same amount of protein, 2× sample buffer (125 mM Tris–HCl, 2% SDS, 10% glycerin, 1 mM DTT, 0.002% bromophenol blue, pH 6.9) was added and boiled for 5 min. Proteins were separated by 6% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF; Millipore, MA). Molecular weights of proteins were determined by measuring the migration distance of proteins relative to markers (Fermentas Inc.; Burlington, Ontario, Canada). The membranes containing transferred proteins were washed twice with TBS (50 mM Tris–HCl, 140 mM NaCl, pH 7.3) containing 0.1% Tween 20. Nonspecific reactions were blocked by soaking the membranes in TBS containing 5% skim milk at room temperature for 1 h. A polyclonal antibody to Ask1 (1:1000, Cell Signaling Technologies, Beverly, MA) or pAsk1 (1:500, Cell Signaling Technologies) was used as a primary antibody and reacted at room temperature for 1 h. Subsequently, membranes were washed three times with TBS containing 0.1% Tween 20 for 10 min. The membranes were reacted with a secondary antibody conjugated to horseradish peroxidase (1:1000 in TBS plus 5% skim milk). Membranes were then stained with a chemiluminescence detection system ECL plus kit (Amersham International, Buckinghamshire, UK).

4.5.

Immunohistochemical staining

Mice in the experimental and control groups were anesthetized with urethane. After MCAO reperfusion, mice were perfused with 0.9% saline containing 10 U/mL and 3.7% formaldehyde. The brains were extracted, fixed for 16 h, and stored in 30% sucrose until used. The brain tissues were frozen on dry ice. Coronal sections 20 μm in thickness were prepared using a cryostat. Sections were blocked with PBS containing 5% BSA at room temperature for 1 h and reacted with the primary antibody polyclonal rabbit anti-Ask1 antibody (1:100, Cell Signaling technologies). Sections were washed with PBS, and reacted with the secondary antibody FITC- or Cy3.18-conjugate (1:200, Jackson Immuno Research Laboratories, West Grove, PA) at room temperature for 1 h to provide detection and visualization information.

4.6. Double/triple immuno fluorescence staining of Ask1, TUNEL, Hoechst, and NeuN Immunohistochemical staining for Ask1 was performed using the above method. After washing, tissues were reacted with 50 μL terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate biotin nick-end labeling (TUNEL) reaction solution (terminal deoxynucleotidyl transferase and fluorescein-dUTP; Roche Diagnostics GmbH, Penzberg, Germany) in a 37 °C dark room for 1 h. The nucleus was counterstained with Hoechst (Molecular Probes, Eugene, OR). Tissues were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined under a microscope (BX51; Olympus, Tokyo, Japan). And the neuronal cell-staining was performed by using NeuN-monoclonal antibody and MOM kit (Vector Laboratories, Burlingame, CA).

4.7.

Measurement of cerebral infarct size

Mice in the Cont.-siRNA infusion group and the Ask1-siRNA infusion group were compared. Twenty-four hours after

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cerebral ischemia, mice were sacrificed and brains were extracted. The extracted brains were prepared as coronal sections by sectioning from the anterior brain area at 1-mm slices. Each section was stained by immersion in 2% 2,3,5triphenyltetrazolium chloride (TTC; Sigma-Aldrich) and incubated for 15 min (Kim et al., 2002). Stained sections were scanned at 1200 dpi with a flatbed scanner.

4.8.

Statistical analysis

All data are presented as mean± SEM. Statistical analyses were performed using a t-test or one-way analysis of variance (ANOVA), followed with Mann–Whitney U-test (StatView; SAS Institute Inc., Cary, NC). Statistical significance was defined as P < 0.05.

Acknowledgments This study was supported by a grant from the Korea Healthcare technology R&D Project, Ministry of health & Welfare, Republic of Korea (A110023), and the National Research Foundation of Korea (NRF-20100012644) and the College of Medicine, Yonsei University (6-2010-0030).

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