Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Free Radical Biology and Medicine 67 (2014) 195–210

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Original Contributions

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury Min Jea Shin a,1, Dae Won Kim b,1, Yeom Pyo Lee a, Eun Hee Ahn a, Hyo Sang Jo a, Duk-Soo Kim c, Oh-Shin Kwon d, Tae-Cheon Kang e, Yong-Jun Cho f, Jinseu Park a, Won Sik Eum a,n, Soo Young Choi a,n a

Department of Biomedical Sciences and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea Department of Biochemistry and Molecular Biology, Research Institute of Oral Sciences, College of Dentistry, Kangnung-Wonju National University, Gangneung 210-702, Korea c Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan-si 330-090, Korea d School of Life Sciences, College of Natural Sciences, Kyungpook National University, Taegu 702-702, Republic of Korea e Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea f Department of Neurosurgery, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 8 July 2013 Received in revised form 8 October 2013 Accepted 18 October 2013 Available online 16 November 2013

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT22 neuronal cells and protected cells against MG- and H2O2-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H2O2-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced TatGLO protein protects neuronal cells by inhibiting MG- and H2O2-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases. & 2013 Elsevier Inc. All rights reserved.

Keywords: Methylglyoxal (MG) Glyoxalase (GLO) Oxidative stress Protein therapy Ischemic damage

Introduction Methylglyoxal (MG) is a highly reactive carbonyl compound derived from oxidative and nonoxidative reactions, increases in which lead to oxidative stress and tissue damage [1]. MG induces

Abbreviations: AGE, glycation end products; CV, cresyl violet; DAPI, 4′,6-diamidino-2-phenylindole; DCF-DA, 2′,7′-dichlorofluorescein diacetate; F-JB, Fluoro-Jade B; GLO, glyoxalase; IPTG, isopropyl-β-D-thiogalactoside; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MG, methylglyoxal; PBS, phosphate-buffered saline; PTDs, protein transduction domains; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfated-polyacrylamide gel electrophoresis n Corresponding authors. Fax: þ 82 33 241 1463. E-mail addresses: [email protected] (W.S. Eum), [email protected] (S.Y. Choi). 1 These authors contributed equally to this work. 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.10.815

protein glycation, leading to the formation of AGE, which play important roles in the pathogenesis of aging as well as diseases including diabetes and neurodegeneration [2–4]. MG is efficiently detoxified by the glyoxalase system. In this system, glyoxalase 1 (GLO1) metabolizes MG to S-D-lactoylglutathione, and GLO2 converts S-D-lactoylglutathione to D-lactate [4]. Several studies have shown that the overexpression of GLO1 lessens the effects of various disorders such as diabetes, hyperglycemia, and Alzheimer’s disease, as well as aging [5–8]. Oxidative stress in neuronal cells is one of the major causes of neurodegenerative diseases and is due to the production of highly reactive oxygen species (ROS). Oxidative stress also plays an important role in the pathological processes of various human diseases including ischemic injury [9–15]. Therefore, antioxidant enzymes such as CuZn-SOD and catalase are well known for their protective effects against oxidative stress and ischemic injury

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[15,16]. However, the contribution of MG toxicity and the exact mechanisms of neuronal damage in ischemia remain unclear. Also, the neuroprotective effects of GLO proteins in ischemia are not yet well documented. Although antioxidant enzymes play a pharmacologic role in cells, the therapeutic application of proteins is very difficult owing to low delivery efficiency [17]. Protein transduction technology is a commonly used method for delivering exogenous proteins into living cells and tissues via protein transduction domains (PTDs). Among the various PTD peptides, Tat peptide is well known for its ability to deliver exogenous proteins into cells and tissues and has been used for a number of clinical applications [18–20]. In previous studies, we have shown that various PTD fusion proteins efficiently transduced into cells and tissues, where they protected against cell damage in vitro and in vivo [21–30]. In this study, we investigated the protective effects of Tat-GLO proteins in MG- and H2O2-induced HT-22 neuronal cell death and in vivo in an animal model of ischemia. Tat-GLO proteins directly transduced into HT-22 neuronal cells, where they protected against cell death. In addition, the neuroprotective effects of Tat-GLO proteins against ischemic damage were evident in vivo. Therefore, we suggest that Tat-GLO proteins could be a potential therapeutic agent for the treatment of transient forebrain ischemia.

Cell culture and transduction of Tat-GLO proteins Mouse hippocampal HT-22 cells were cultured in Dulbecco’s minimum essential medium (DMEM; Lonza BioWhittaker, MD, USA) containing 10% fetal bovine serum (FBS) and antibiotics (100 mg/ml streptomycin, 100 U/ml penicillin) at 37 1C under humidified conditions of 95% air and 5% CO2. For transduction of Tat-GLO protein, cells were grown on a sixwell plate for 12 h, after which they were exposed to various concentrations (0.5–3 μΜ) of Tat-GLO proteins over various durations (10–120 min). The cells were treated with trypsin-EDTA, washed with phosphate-buffered saline (PBS), and harvested for the preparation of cell extracts to perform Western blot analysis.

Western blot analysis The equal cell lysates were separated by 12% sodium dodecyl sulfated-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. After blocking with 5% nonfat dry milk in TBS buffer including 0.1% Tween 20 (TBST), the membrane was immunoblotted with the indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The protein bands were detected with enhanced chemiluminescent reagents [25,26].

Materials and methods

Fluorescence microscopic analysis

Materials

Transduced protein distribution fluorescence was performed as described previously [22,23]. Briefly, the cells were grown on coverslips and treated with 3 mM Tat-GLO protein for 2 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 5 min at room temperature. After the cells were permeabilized, blocked, and washed, they were exposed to the primary antibody (His-probe, 1:2000; Santa Cruz Biotechnology) for 1 h at room temperature. The secondary antibody (Alexa-Fluor 488, 1:15000; Invitrogen) was applied for 45 min at room temperature in the dark. Nuclei were stained for 5 min with 1 mg/ml 4′,6-diamidino-2phenylindole (DAPI; Roche, Basel, Switzerland). The distribution of fluorescence was analyzed by confocal microscopy using a Model FV-300 microscope (Olympus, Tokyo, Japan).

MG was purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies and actin were obtained from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Ni2 þ -nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen (Valencia, CA, USA). Isopropyl-β-D-thiogalactoside (IPTG) was obtained from Duchefa (Budapest, Hungary). Plasmid pET-15b and Escherichia coli strain BL21 (DE3) were obtained from Novagen (La Jolla, CA, USA). Human GLO1 and GLO2 cDNA were isolated using the polymerase chain reaction (PCR) technique. The GLO1 mutant (M157A) and GLO2 mutant (Y175F) cDNA were obtained from Bioneer (Daejeon, Korea). A D-lactate colorimetric assay kit was obtained from BioVison (Milpitas, CA, USA). AGE ELISA and MG ELISA kits were purchased from Cell BioLabs (San Diego, CA, USA). All other chemicals and reagents, unless otherwise stated, were of the highest analytical grade available.

Purification of Tat-GLO fusion proteins In a previous study, we demonstrated Tat-GLO protein construction and purification [30]. Briefly, Tat-GLO proteins were constructed using the following sense and antisense primers: wild-type GLO1 and mutant GLO1 (M157A) sense primers, 5′-CTCGAGATGGCAGAACCGCAGCCCCCGTCC-3′; wild-type GLO1 and mutant GLO1 (M157A) antisense primer, 5′-GGATCCCTACATTAAGGTTGCCATTTTGTT-3′; wild-type GLO2 and mutant GLO2 (Y175F) sense primers, 5′-CTCGAGATGA AGGTAGAGGTGCT;GCCTGCC-3′; wild-type GLO2 and mutant GLO2 (Y175F) antisense primer, 5′-GGATCCTCAGTCCCGGGGCATCTTGAACTG-3′. After TatGLO proteins were overexpressed by 0.5 mM IPTG for 4 h, they were purified using a Ni2 þ -nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography. The Bradford procedure was used to estimate protein concentration using bovine serum albumin as a standard [31].

Cell viability assay The biological activity of transduced Tat-GLO proteins was assessed by measuring the cell viability of HT-22 cells treated with MG and H2O2. A colorimetric 3-(4,5-dimethylthiazol-2-yl)2,5-dipheyltetrazolium bromide (MTT) assay was performed as described previously [21–23]. The cells were pretreated with TatGLO protein (0.5–3 mM) for 2 h before MG (1 mM) and H2O2 (0.65 mM) was added to the culture medium for 12 h. Cell viability was defined as the percentage of untreated control cells.

Measurement of D-lactate, AGE, and MG The cells (1  106) were pretreated with Tat-GLO proteins (3 mM) for 2 h, after which they were treated with MG (1 mM) and H2O2 (0.65 mM) for 6 h. Then, the D-lactate assay was performed by using a D-lactate colorimetric assay kit (BioVison) as per the manufacturer’s instructions. AGE and MG levels were analyzed using a AGE and MG enzyme-linked immunosorbent assay ELISA kit (Cell BioLabs) according to the manufacturer’s instruction.

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TUNEL assay Terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) staining was performed as described previously [25,26]. HT-22 cells were incubated in the absence or presence of Tat-GLO proteins (3 mM) for 2 h, and then treated with MG (1 mM) and H2O2 (0.65 mM) for 12 h. TUNEL staining was performed using a Cell Death Detection kit (Roche Applied Science) according to the manufacturer’s instructions. Images were taken using an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan). The percentage of TUNEL-positive cells was counted by phase-contrast microscopy at  200 magnifications for the cells. Determination of intracellular ROS Intracellular ROS levels were measured using the dye 2′,7′dichlorofluorescein diacetate (DCF-DA) as described previously [26]. The cells, pretreated with Tat-GLO protein (3 μM) for 2 h, were treated with MG (1 mM) and H2O2 (0.65 mM) for 6 h and were washed twice with PBS and incubated with 20 μΜ DCF-DA for 60 min. The fluorescence images were taken using a fluorescence microscope (Nikon Eclipse 80i, Tokyo, Japan). The cellular fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems Oy, Helsinki, Finland). Measurement of caspase-3 and mitogen-activated protein kinase (MAPK) activation HT-22 cells were incubated in the absence or presence of TatGLO proteins (3 mM) for 2 h, and then treated with MG (1 mM) and H2O2 (0.65 mM) for 3 h. Following that, the expression of caspase-3, cleaved caspase-3, cleaved caspase-9, and MAPKs in whole cell lysates was analyzed by Western blotting using the indicated specific antibodies [26]. Experimental animals Male gerbils (65–75 g) were obtained from the Experimental Animal Center at Hallym University. The animals were housed at a constant temperature (23 1C) and relative humidity (60%) with a fixed 12 h light/dark cycle and free access to food and water. All experimental procedures involving animals and their care conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea and were approved by the Hallym Medical Center Institutional Animal Care and Use Committee. To determine whether transduced Tat-GLO proteins protect against ischemic damage, gerbils were divided into seven groups (n ¼5 per group) as follows; sham-control group, vehicle-treated group, control GLO1-treated group, control GLO2-treated group, Tat-GLO1-treated group, Tat-GLO2-treated group, and combined Tat-GLO1 and Tat-GLO2-treated group. Tat-GLO proteins (each 2 mg/kg) were injected intraperitoneally 30 min before ischemic insult. Transient forebrain ischemia induction was carried out as described previously [21–23,25]. Seven days after induction of ischemia, gerbils were euthanized for immunohistochemistry staining. The same experiment was also performed 14 days after induction of ischemia. Immunohistochemistry Immunohistochemistry was performed as described previously [21–23,25]. Brains were removed and postfixed in the same fixative for 4 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight, after which the tissues were frozen

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and sectioned with a cryostat at a thickness of 50 μm. Consecutive sections were collected in six-well plates containing PBS. Cresyl violet (CV) and Fluoro-Jade B (F-JB) staining were performed according to the previously described [22]. Quantification of data and statistical analysis To verify the effects of Tat-GLO1, Tat-GLO2 and combined TatGLO1 and Tat-GLO2 proteins against ischemic damage, neuronal number, and the intensity of immunoreactivity were assessed using an image analyzing system. Five sections from each animal were selected for quantitative analysis. The staining intensity of the immunoreactive structures was evaluated as the relative optical density (ROD): The relative percentage of control level is shown in Fig. 10, the graph. Data are expressed as the mean 7 SEM of three experiments. The data were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. P o 0.05 was considered statistically significant.

Results Purification and transduction of Tat-GLO protein The construction, expression, and purification of cell-permeable Tat-GLO (GLO1, GLO2) proteins are described in a previous study [30]. We also constructed mutant Tat-GLO proteins in which methionine157 and tyrosine175 were replaced by alanine (M157A) and phenylalanine (Y175F) located in active sites of GLO1 and GLO2 proteins, respectively. As shown in Fig. 1A, the Tat-GLO expression vector contained consecutive cDNA sequences encoding human GLO and six histidine residues at the amino-terminus. The Tat-GLO proteins were expressed in E. coli and purified by affinity chromatography. The purified Tat-GLO proteins had estimated molecular masses of approximately 22 kDa (Tat-GLO1) and 31 kDa (Tat-GLO2) by SDS-PAGE, and were confirmed by Western blot analysis using an anti-rabbit polyhistidine antibody (Fig. 1B and C). To examine the transduction of Tat-GLO protein into HT-22 cells, Tat-GLO proteins were added to the cell culture medium for various periods of time (10–120 min, 3 mM) and at various concentrations (0.5–3 mM, 120 min). The levels of transduced proteins were then measured by Western blotting. As shown in Fig. 2A and B, Tat-GLO proteins transduced into HT-22 cells in time- and dose-dependent manners, whereas the control GLO protein did not transduce into the cells. Cells were saturated at more than 3 mM of transduced Tat-GLO protein, suggesting that the optimal dose of transduced Tat-GLO proteins is around 3 mM. We also examined the intracellular stability of transduced TatGLO protein in HT-22 cells. Cells treated with Tat-GLO proteins (3 mM) were harvested at various times and analyzed by Western blotting (Fig. 2C). Significant levels of transduced Tat-GLO protein persisted in the cells for 48 h in Tat-GLO1-treated cells and 24 h in those treated with Tat-GLO2. To further clarify the transduction of Tat-GLO protein into the cells, the transduced cells were stained with fluorescent marker DAPI and Alexa-Fluor 488. As shown in Fig. 2D, fluorescence signals were markedly increased in the cells treated with TatGLO protein, whereas control GLO protein did not show the fluorescence signals. These results indicate that Tat-GLO protein efficiently transduced into the cells and was stable for at least 48 and 24 h, respectively. We also examined the transduction of mutant Tat-GLO protein into HT-22 cells by the same method described for wild-type TatGLO protein. As shown in Fig. 3, mutant Tat-GLO (M157A and Y175F) proteins were transduced into HT-22 cells in time- and

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Fig. 1. Purification of Tat-GLO protein. Diagram of expressed control GLO and Tat-GLO proteins. The coding frame of human GLO is represented by an open box along with 6His and the Tat peptide. Amino acid sequences of the active site Met-157 GLO1 and Tyr-175 of human GLO1 and GLO2 (A). Purification of Tat-GLO proteins was analyzed by 12% SDS-PAGE (B) and subjected to Western blot analysis with an anti-rabbit polyhistidine antibody (C).

dose-dependent manners. In addition, the intracellular stability of transduced mutant Tat-GLO proteins showed patterns similar to those of wild-type Tat-GLO protein. These results indicate that mutant Tat-GLO protein also efficiently transduced into the cells and was stable in the cells.

Tat-GLO protein inhibits cell death under oxidative stress To determine whether the transduced Tat-GLO protein had a functional role in HT-22 cells against MG- and H2O2-induced oxidative stress, we performed a cell viability assay. As shown in Fig. 4A and B, after treatment with MG (1 mM) and H2O2 (0.65 mM) for 12 h, cell viability were decreased, 59 and 38% compared to control cells. However, cell viability markedly increased in a dose-dependent manner when the cells were pretreated with transduced Tat-GLO1 and Tat-GLO2 proteins. In addition, cell viability significantly increased up to 88 and 86% in the group treated with combined Tat-GLO1 and Tat-GLO2 proteins (each 3 mM). A 3 mM was used because similar results were observed at a higher dose than 3 mM of combined Tat-GLO1 and Tat-GLO2 protein (data not shown). However, control GLO protein and Tat peptide-treated cells did not show protective effects under the same conditions. These results indicate that transduced Tat-GLO protein plays a defensive role against MG- and H2O2-induced cell death. We also examined cell viability after cells were treated with Tat-GLO (3 mM) and mutant Tat-GLO (3 mM) proteins. As shown in Fig. 4C and D, mutant Tat-GLO1 protein showed slightly protective effects and mutant Tat-GLO2 protein did not show any protective effects. The protective effect did not change even though cells were treated with combined mutant Tat-GLO proteins (Tat-GLO1 þ Tat-GLO2). These results indicated that mutant Tat-GLO protein

has a slight defensive function against oxidative stress-induced cell death. Furthermore, we determined the biological activity of Tat-GLO proteins after transduction into the cells. In the MG- and H2O2treated cells, D-lactate levels were markedly increased by transduced wild-type Tat-GLO protein whereas it was slightly increased by mutant Tat-GLO protein compared to the control. Also, combined wild-type Tat-GLO proteins (Tat-GLO1 þ Tat-GLO2) significantly increased D-lactate levels compared to the combined mutant TatGLO proteins (Fig. 5A). Next, we examined that effect of transduced Tat-GLO protein on AGE and MG levels in the MG- and H2O2-treated cells. As shown in Fig. 5B and C, AGE and MG levels were markedly increased after treatment with MG and H2O2. However, transduced wild-type Tat-GLO protein significantly reduced AGE and MG levels whereas mutant Tat-GLO proteins reduced AGE and MG levels slightly less than wild-type Tat-GLO protein. Also, combined wild-type Tat-GLO (Tat-GLO1 þ Tat-GLO2) proteins more significantly reduced AGE and MG levels than mutant Tat-GLO proteins. These results indicate that transduced wild-type Tat-GLO protein demonstrated increased biological activity whereas by comparison mutant Tat-GLO protein demonstrated relatively lower activity. Reactive oxygen species (ROS) are well known to cause cellular macromolecule damage including DNA fragmentation which leads to cell death [15]. To examine ROS generation in MG- and H2O2treated HT-22 cells, the degree of fluorescence signals was measured by fluorescence microscopy. As shown in Fig. 6, strong fluorescence signals appeared in the MG- and H2O2-treated cells, whereas the fluorescence signals markedly decreased in those treated with transduced Tat-GLO proteins. In addition, in the combined Tat-GLO protein-treated cells, the fluorescence signals decreased further compared to those treated with only one

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Fig. 2. Transduction of Tat-GLO proteins into HT-22 cells. Tat-GLO and control GLO (3 mM) were added to the culture media for 10–120 min (A), Tat-GLO and control GLO (0.5–3 mM) were added to the culture media for 2 h (B), and cells pretreated with 3 mM Tat-GLO were incubated for 1–72 h and analyzed by Western blot (C). The band intensities were measured by a densitometer. Each bar represents the mean 7 SEM of three experiments. Cellular distribution of transduced Tat-GLO protein (D). The distribution of transduced Tat-GLO was observed by confocal microscopy. After transduction of Tat-GLO protein, the cells were examined by DAPI and Alexa staining. Scale bar ¼ 20 μm.

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Fig. 3. Transduction of mutant Tat-GLO proteins into HT-22 cells. Mutant Tat-GLO and control GLO (3 mM) were added to the culture media for 10–120 min (A), mutant TatGLO and control GLO (0.5–3 mM) were added to the culture media for 2 h (B), and cells pretreated with 3 mM mutant Tat-GLO were incubated for 1–72 h and analyzed by Western blot (C). The band intensities were measured by a densitometer.

protein. However, the fluorescence signals in cells treated with control GLO protein and Tat peptide were similar to those of MGand H2O2-treated cells. Next, the protective effect of transduced Tat-GLO protein against DNA fragmentation was determined via TUNEL staining. As shown in Fig. 7, MG- and H2O2-induced DNA fragmentation was markedly inhibited by transduced Tat-GLO protein. No control cells were TUNEL positive, and so did not display morphological changes associated with cellular apoptosis. MG and H2O2 markedly

increased the number of TUNEL-positive cells compared with untreated control cells. The cells treated with transduced TatGLO protein demonstrated significantly suppressed MG- and H2O2-mediated DNA damage, whereas control GLO protein and Tat peptide did not show the protective effects. Furthermore, DNA fragmentation was markedly inhibited in the combined Tat-GLO1 and Tat-GLO2 protein-treated cells. These results indicate that transduced Tat-GLO protein protects against MG- and H2O2-induced cell death by inhibiting ROS generation and DNA fragmentation.

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Fig. 4. Effect of transduced Tat-GLO protein on cell viability. MG (1 mM) and H2O2 (0.65 mM) for 12 h treated to HT-22 cells pretreated with Tat-GLO protein (0.5–3 mM) for 2 h (A and B). Under the same experimental conditions, mutant Tat-GLO proteins (3 mM) were treated to the cells (C and D). Cell viabilities were estimated by with a colorimetric assay using MTT. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

It is well known that caspase-3 plays central roles in mammalian cellular apoptosis [32]. Therefore, we examined whether transduced Tat-GLO protein could inhibit caspase-3 and caspase-9 activation in cells. Caspase-3 and caspase-9 activation in MG- and H2O2-treated cells was determined by Western blotting using suitable antibodies. MG and H2O2 increased the caspase-3 and caspase-9 activation compared with the control cells as well as control GLO protein and Tat peptide-treated cells. However, transduced Tat-GLO protein significantly inhibited caspase-3 and caspase-9 activation. Further, combined Tat-GLO proteins demonstrated markedly lower caspase-3 and caspase-9 activation levels compared to MG- and H2O2-treated cells (Fig. 8). We attempted to clarify the signal mechanisms responsible for the MG- and H2O2-induced apoptosis. To examine the effect of TatGLO on MG- and H2O2-induced MAPK activation, Tat-GLO protein was transduced into MG- and H2O2-treated cells after which MAPK activation was analyzed by Western blotting using specific antibodies against MAPK proteins. As shown in Fig. 9, Tat-GLO protein significantly inhibited activation of MG- and H2O2-induced phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK.

However, control GLO protein and Tat peptide failed to suppress the MAPK activation. These results indicate that transduced TatGLO protein inhibits MG- and H2O2-induced apoptosis and MAPK activation. Effects of transduced Tat-GLO protein against ischemic damage Protein therapy is particularly important in the treatment of neuronal diseases because of the difficulty in traversing the blood– brain (BBB) barrier. Therefore, we examined whether Tat-GLO protein was delivered to the gerbil brains using immunohistochemistry. Animals were ip injected with Tat-GLO protein (2 mg/kg). After 6 h, the animals were scarified and hippocampal CA1 region samples were collected. Then the delivery of Tat-GLO protein into brain was confirmed using a His antibody. As shown in Fig. 10A, TatGLO protein was found in animals injected with Tat-GLO protein. However, none was detected in sham control- and control GLOinjected animals. To examine the effects of transduced Tat-GLO protein on neuronal cell viability after transient forebrain ischemia in vivo,

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Fig. 5. Determination of biological activity on transduced Tat-GLO protein. The cells (1  106) were pretreated with Tat-GLO proteins (3 mM) for 2 h, and then treated with MG (1 mM) and H2O2 (0.65 mM) for 6 h. D-Lactate assay (A) was performed by using a D-lactate colorimetric assay kit. Cellular AGE (B) and MG (C) levels were analyzed by using an AGE and MG assay ELISA kit. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

neuronal cell viabilities were evaluated by CV and F-JB histochemistry at 7 days and 14 days after ischemic insults. As shown in Fig. 10B, in the vehicle-treated group, the percentage of positive

neurons was 13% compared to that of the sham-operated group. Control GLO protein-treated groups were similar to the vehicletreated group. However in the Tat-GLO1-treated groups, the

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Fig. 6. Protective effect of Tat-GLO protein against MG- and H2O2-induced ROS generation in HT-22 cells. MG (1 mM) and H2O2 (0.65 mM) for 6 h treated to HT-22 cells pretreated with Tat-GLO protein (3 mM) for 2 h. Intracellular ROS levels were measured by DCF-DA staining. Scale bar ¼ 50 μm. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

percentages of positive neurons were 57% (7 days) and 59% (14 days) and in the Tat-GLO2-treated groups, the percentages of positive neurons were 48% (7 days) and 51% (14 days) of the

sham-operated group. However, in the combined Tat-GLO-treated groups, neuronal cell viability increased significantly up to 83% (7 days) and 86% (14 days) compared to that in the vehicle-treated

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Fig. 7. Protective effect of Tat-GLO protein against MG- and H2O2-induced DNA fragmentation in HT-22 cells. The cells were treated with Tat-GLO (3 mM) for 2 h and then exposed to MG (1 mM) and H2O2 (0.65 mM) for 12 h, and then DNA fragmentation was detected by TUNEL staining. The percentage of TUNEL-positive cells is indicated on the Y axis. Scale bar ¼ 50 μm. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

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Fig. 8. Tat-GLO protein protects against MG- and H2O2-induced caspase-3 and -9 in HT-22 cells. The cells were treated with Tat-GLO (3 mM) for 2 h, and exposed to MG (1 mM) and H2O2 (0.65 mM) for 3 h and 30 min, respectively. Then the caspase-3 and -9 expression levels were measured by Western blotting and band intensity was measured by a densitometer. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

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Fig. 9. Tat-GLO protein inhibits activation of MAPK in MG- and H2O2-treated HT-22 cells. The cells were treated with Tat-GLO (3 mM) for 2 h, and exposed to MG (1 mM) and H2O2 (0.65 mM) for 3 h and 30 min, respectively. Then the activation of MAPK (JNK and p38) expression levels was measured by Western blotting and band intensity by a densitometer. nP o 0.05 and nnP o 0.01 compared with MG- and H2O2-treated cells. #P o 0.01, compared to control.

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Fig. 10. Effects of transduced Tat-GLO protein on neuronal cell viability of ischemic insults. Transduction of Tat-GLO protein into gerbil brain. Gerbils were treated with a single injection Tat-GLO (2 mg/kg) proteins and killed after 6 h. Then the transduced Tat-GLO protein was analyzed by immunohistochemistry using anti-histidine antibody (A). Scale bar ¼ 280 μm. Hippocampus stained with CV and F-JB in sham-, vehicle-, control GLO-, Tat-GLO1, Tat-GLO2, and combined Tat-GLO-treated ischemic animals 7 and 14 days after I/R (B). Scale bar ¼ 400 and 50 μm. The relative numeric analysis of CV positive neurons values is significantly different from that of the vehicle group, nn P o 0.01. #P o 0.01, compared to control sham group.

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groups. Under the same experimental conditions, we performed F-JB staining. In the vehicle-treated group, F-JB-positive neurons were abundant in the CA1 region compared to that in the shamoperated group. However, combined Tat-GLO-treated groups were markedly lower in the CA1 region compared to the vehicle-treated group. These results indicate that Tat-GLO protein efficiently transduced across the gerbil blood–brain barrier and significantly inhibits neuronal cell death against ischemic insults.

Discussion ROS such as superoxide anion and hydrogen peroxide are constantly generated under the normal conditions of aerobic metabolism. Increased production of these ROS mediates many of the pathophysiological events that cause several diseases [33–36]. MG, an α-oxoaldehyde generated from the oxidation of carbohydrates and glycolysis, is a reactive precursor of protein glycation and leads to AGEs. MG accumulation induces oxidative stress and cell damage [8,37–40]. In addition, AGE is an intracellular hallmark of aging as well as various diseases such as Alzheimer’s disease, Parkinson’s disease, and diabetes [2–4,41]. MG is detoxified by the glyoxalase system that is composed of GLO1 and GLO2 [4]. GLO enzymes play an important role in cells by suppressing AGE formation [5–8]. As such, these enzymes are considered to be potential therapeutic agents for such ailments. However the application of these enzymes is limited owing to the difficulty of delivering them into cells [17]. Thus, we prepared cell-permeable Tat-GLO protein using the protein transduction domain (PTD), Tat peptide. Several studies have demonstrated that Tat-mediated protein transduction can be used as a tool for therapeutic protein application [20,42,43]. In a previous study, we demonstrated that Tat-GLO protein efficiently transduced into RINm5F cells and protected cells against SNP-induced cell death and inhibited STZ-mediated toxicity in an animal model by suppressing blood glucose levels as well as regulating serum biochemical parameters [30]. In this study, we investigated the protective effects of Tat-GLO protein against MG- and H2O2induced cell death and ischemic insults in an animal model. After purified Tat-GLO protein was confirmed by Western blot analysis using an anti-rabbit polyhistidine antibody, we examined the transduction ability and biological functions of Tat-GLO proteins in cells. Purified Tat-GLO proteins were efficiently transduced into HT-22 cells in time- and dose-dependent manners and remained stable for 48 and 24 h in transduced cells, respectively. In addition, transduced Tat-GLO protein significantly increased cell viability against MG- and H2O2-induced cell death. Although mutant TatGLO proteins were transduced into the cells, mutant Tat-GLO proteins showed only slight protective effects. Mutants such as M157A and Y175F in which methionine157 and tyrosine175 were replaced by alanine (M157A) and phenylalanine (Y175F) located in active sites of GLO1 and GLO2 proteins influence the functional properties of the protein and have minimal enzymatic activity compared to wild-type GLO proteins [44,45]. Although these reports suggest that amino acid Met157 and Tyr175 residues in active sites have important roles in the function of the proteins, in this study, the changes of amino acids did not have any effect on transduction. The biological functions of the transduced Tat-GLO protein in the cells were assessed by D-lactate, AGE, and MG assay. Mutant Tat-GLO protein, which has lower enzymatic activity, could not detoxify the cytotoxic effects of MG- and H2O2-induced AGE and MG levels. However, transduced wild-type Tat-GLO protein effectively reduced the AGE and MG levels under the same conditions. The lower activity and lower influence of protein function of

mutant Tat-GLO proteins compared to wild-type Tat-GLO proteins are consistent with results observed in Ridderstrom et al. [44,45]. Reactive oxygen species are well known to be potentially damaging to cellular macromolecules and altered signaling leading to cell death [15]. Therefore, we examined whether transduced Tat-GLO proteins can inhibit MG- and H2O2-induced ROS generation and DNA fragmentation, as well as activation of MAPKs. Our results show that transduced Tat-GLO protein significantly inhibits ROS generation and DNA damage caused by MG- and H2O2induced oxidative stress. Furthermore, we observed that transduced Tat-GLO protein inhibited the activation levels of caspase-3 as well as MAPKs such as p38 and JNK. MG causes cellular stress that is sufficient to induce apoptosis through the activation of p38 MAPK in Schwann cells [46]. Also, ROS-induced DNA damage can be mitigated by antioxidants, suggesting that antioxidants are potential therapeutic agents of ROS associated with neurodegenerative disorders [47]. Recent studies have demonstrated that MG can increase cellular ROS levels and the activation of the p38 MAPK pathway in intestinal cells. Our experiments demonstrated results similar to the effects shown by MG [46,48]. These results indicate that transduced Tat-GLO protein efficiently protects against cell death caused by MG. To examine the ability of transduced Tat-GLO to protect against ischemic neuronal damage after transient ischemia, we used a TatGLO protein gerbil ischemia model. Tat-GLO proteins were intraperitoneally administered 30 min prior to the induction of ischemia. At 7 days and 14 days following ischemia, the protective effects of the Tat-GLO proteins were confirmed by CV and F-JB immunohistochemistry. We showed that cotreatment with TatGLO1 and Tat-GLO2 proteins had a greater neuroprotective effect than treatment with individual Tat-GLO proteins. The number of damaged neurons was markedly decreased in the hippocampal CA1 regions of animals treated with transduced Tat-GLO protein. These results indicate that Tat-GLO protein is associated with delayed neuronal death in the hippocampal CA1 region after ischemia, and attenuates neuronal damage after ischemic insults. In a previous study, we demonstrated that various antioxidant fusion proteins can be efficiently delivered into neurons in the ischemic hippocampus, and that fusion protein treatment in animals with ischemic damage reduces that damage [21–23,25]. Oxidative stress is an important underlying factor in delayed neuronal death induced by ischemic insult [15,49]. MG can cause neurotoxicity by carbonyl stress [41]. The authors suggest that antioxidant or carbonyl scavengers counteract the detrimental effects caused by carbonyl compounds, which might be exploited as a therapeutic tool to reduce the risk of pathophysiological changes associated with carbonyl stress in neuronal diseases including ischemia [41]. In addition, overexpression of GLO1 prevents renal ischemia–reperfusion (I/R) injury by reducing MG levels, suggesting that GLO may be an effective therapeutic tool in renal I/R injury [50]. Inhibition of ROS and inflammatory reaction also may reduce I/R injury [51]. These protective effects are in agreement with our protective effect results of Tat-GLO protein. Therefore, we suggest that transduced Tat-GLO protein may be a useful therapeutic agent for a variety of neuronal diseases including ischemia. In summary, we have demonstrated that Tat-GLO protein protects against MG- and H2O2-induced induced cell death and ischemic insults. Moreover, combined Tat-GLO proteins bestow a greater protective effect than treatment with Tat-GLO protein alone in vitro and in vivo. Therefore, our results suggest that TatGLO protein may provide a new strategy to protect against ischemic neuronal cell death damage. Also, it may be a potential therapeutic agent for the treatment of various MG- and H2O2related diseases.

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Acknowledgments This work was supported by a Priority Research Centers Program grant (2009-0093812) and in part by a Research grant (2012R1A2A1A03006155) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning in the Republic of Korea, and also it was supported by a BioGreen21 Program (PJ009051) of Rural Development Administration.

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