Proteomic identification of hippocampal proteins vulnerable to oxidative stress in excitotoxin-induced acute neuronal injury

Neurobiology of Disease 43 (2011) 706–714

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y n b d i

Proteomic identification of hippocampal proteins vulnerable to oxidative stress in excitotoxin-induced acute neuronal injury Ayako Furukawa a, Yoshiyuki Kawamoto b, Yoichi Chiba a, Shiro Takei a, Sanae Hasegawa-Ishii a, Noriko Kawamura a, Keisuke Yoshikawa a, Masanori Hosokawa a, Shinji Oikawa c, Masashi Kato b, Atsuyoshi Shimada a,⁎ a b c

Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi, Japan Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie, Japan

a r t i c l e

i n f o

Article history: Received 27 January 2011 Revised 2 May 2011 Accepted 28 May 2011 Available online 6 June 2011 Keywords: Kainic acid Excitotoxicity Oxidative stress Proteomics Carbonylated proteins

a b s t r a c t Excitotoxicity is involved in seizure-induced acute neuronal death, hypoxic–ischemic encephalopathy, and chronic neurodegenerative conditions such as Alzheimer's disease. Although oxidative stress has been implicated in excitotoxicity, the target proteins of oxidative damage during the course of excitotoxic cell death are still unclear. In the present study, we performed 2D-oxyblot analysis and mass spectrometric amino acid sequencing to identify proteins that were vulnerable to oxidative damage in the rat hippocampus during kainic acid (KA)-induced status epilepticus. We first investigated the time course in which oxidative protein damage occurred using immunohistochemistry. Carbonylated proteins, a manifestation of protein oxidation, were detected in hippocampal neurons as early as 3 h after KA administration. Immunoreactivity for 8-hydroxy-2′-deoxyguanosine (8-OHdG) was also elevated at the same time point. The increase in oxidative damage to proteins and DNA occurred concomitantly with the early morphological changes in KA-treated rat hippocampus, i.e., changes in chromatin distribution and swelling of rough endoplasmic reticulum and mitochondria, which preceded the appearance of morphological features of neuronal death such as pyknotic nuclei and hypereosinophilic cytoplasm. Proteomic analysis revealed that several hippocampal proteins were consistently carbonylated at this time point, including heat shock 70 kDa protein 4, valosin-containing protein, mitochondrial inner membrane protein (mitofilin), α-internexin, and tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein (14-3-3 protein). We propose that oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death in KA-treated rat hippocampus, and these proteins may be targets of therapeutic intervention for seizureinduced neuronal death. © 2011 Elsevier Inc. All rights reserved.

Introduction

Abbreviations: CA, cornu ammonis; CaMK II, Calcium/calmodulin-dependent kinase II; CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; DAB, 3′3′diaminobenzidine tetrahydrochloride; 2-DE, 2-dimensional gel electrophoresis; 2DDIGE, 2-dimensional fluorescence difference gel electrophoresis; DNP, 2,4-dinitrophenylhydrazone; DNPH, 2,4-dinitrophenylhydrazine; DTT, dithiothreitol; ER, endoplasmic reticulum; H&E, hematoxylin and eosin; HRP, horseradish peroxidase; IEF, isoelectric focusing; KA, kainic acid; LC–MS/MS, liquid chromatography–tandem mass spectrometry; mtDNA, mitochondrial DNA; NGS, normal goat serum; NMDA, N-methyl-D-aspartate; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; PB, phosphate buffer; PBS, phosphate-buffered saline; PSD, postsynaptic density; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; RT, room temperature; SDS-PAGE, SDSpolyacrylamide gel electrophoresis; VCP, valosin-containing protein. ⁎ Corresponding author at: Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan. Fax: + 81 568 88 0829. E-mail address: [email protected] (A. Shimada). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2011.05.024

Excitotoxicity is involved in acute and chronic neurodegenerative conditions such as hypoxia–ischemia (Hattori and Wasterlain, 1990), status epilepticus (Vincent and Mulle, 2009), and Alzheimer's disease (Hynd et al., 2004). Increasing in vitro (Bondy and Lee, 1993; Fukui et al., 2009) and in vivo (Ueda et al., 2002) evidence indicates that oxidative stress contributes to neurotoxicity induced by the activation of ionotropic glutamate receptors. Pretreatment with antioxidants such as curcumin or resveratrol showed a protective effect against excitotoxin-induced neuronal cell death (Shin et al., 2007; Wang et al., 2004) and cerebral ischemia-induced brain lesions (Ritz et al., 2008), suggesting that oxidative stress is involved in excitotoxin-induced cell death. One of the most well documented excitotoxins is kainic acid (KA), an analog of glutamic acid. A single intraperitoneal injection of KA has been known to produce convulsions through activation of the

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excitatory amino acid receptors. Therefore, KA has been used to make a model for the study of temporal lobe epilepsy (Ben-Ari, 1985; Vincent and Mulle, 2009). Activation of KA receptor has been shown to induce increases in the intracellular Ca 2+ (Frandsen and Schousboe, 1993) and production of reactive oxygen species (ROS) (Ueda et al., 2002), leading to neuronal cell death (Wang et al., 2005). We previously studied KA-induced seizures using 3-week old rats, in which we found a dual phase regulatory mechanism of eicosanoid production during seizure activities (Yoshikawa et al., 2006). We recently found that a single KA injection to 3-week old rats caused long-lasting progressive neuronal death in the hippocampus (unpublished data), which has not been documented in adult rats following KA injection (Lenz et al., 1997). These may represent some vulnerability of hippocampal neurons to excitotoxicity in rats of this particular age. Several studies have reported that oxidative damage to DNA occurred following excitotoxic insults. Formation of 8-hydroxyl-2′deoxyguanosine (8-OHdG), a hallmark of oxidative DNA damage, was detected 8 h after KA administration by using high performance liquid chromatography with electrochemical detection (Lan et al., 2000). Oxidative DNA lesions induced Poly (ADP-ribose) polymerase-1 activation and resulted in severe reduction in neuroprotective glutamate uptake capacity in astrocytes (Tang et al., 2010). These reports suggest that oxidative damage to DNA may be an early event in the cascade from activation of glutamate receptors to DNA fragmentation leading to cell death. Despite well-known involvement of oxidative DNA damage in excitotoxin-induced neuronal death, the role of oxidative damage to proteins during excitotoxicity remains unknown. Protein carbonylation, one form of the protein oxidation induced by ROS, has attracted a great deal of attention (Nystrom, 2005). Carbonyl derivatives are formed by a direct metal-catalyzed oxidative attack on the amino-acid side chains of proline, arginine, lysine, and threonine (Maisonneuve et al., 2009). In contrast to methionine sulfoxide and cysteine disulfide bond formation, carbonylation is an irreversible oxidative process (Dalle-Donne et al., 2003). These features make protein carbonyls a useful marker for oxidative damage to proteins. Previous studies have reported that specific proteins were carbonylated in animal models of Alzheimer's disease (Sultana et al., 2009), ischemia (Oikawa et al., 2009), and brain aging (Nabeshi et al., 2006). An increase in the oxidative protein damage has been reported also in an animal model of epileptic seizure (Gluck et al., 2000). Total protein carbonyls were elevated in the rat hippocampus 4 h after KA administration. In addition, an increase in the oxidative protein damage has been reported in the plasma of patients with epilepsy (Ercegovac et al., 2010). However, it is still unclear which proteins are especially vulnerable to excitotoxin-induced oxidative damage. The aim of the present study was to identify the proteins specifically damaged in an animal model of status epilepticus. We performed proteomic analysis of hippocampal proteins prepared from KA-treated rats. By 2-dimensional (2D)-oxyblot and liquid chromatography– tandem mass spectrometry (LC–MS/MS), we identified several specific proteins that were carbonylated in the KA-treated rat hippocampus. Oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death. We propose that these proteins may be the targets of therapeutic or preventive intervention for seizure-induced hippocampal neuronal death.

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the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, USA). KA was purchased from Sigma (St. Louis, MO, USA), dissolved in saline, and administered by intraperitoneal injection at a dose of 10 mg/kg. Animals were observed for 2 h following KA administration, and only those that displayed continuous convulsive seizure activities were used for the following histological and proteomic analyses. Control rats were injected with the same volume of saline. For histology, brains were fixed 1 h, 3 h, 24 h, 72 h, and 7 days after KA injection (n=4 per experimental group), and 1 h, 3 h, 24 h, 72 h, and 7 days after saline injection (n=3 per experimental group). For electron microscopy, brains were fixed 3 h after KA injection (n=3) and 3 h after saline injection (n=3). For the detection of carbonyl-modified proteins and protein identification, hippocampi were dissected out 3 h after KA injection (n=4) and 3 h after saline injection (n=4). For 2-dimensional difference gel electrophoresis, hippocampi were dissected out 3 h after KA injection (n=5) and 3 h after saline injection (n=5). Histology and immunohistochemistry Saline- or KA-treated rats were deeply anesthetized with diethyl ether. After decapitation, brains were quickly removed and bisected along the midline. For each brain, the hippocampus from one of the hemispheres was dissected out and stored immediately at −80 °C for protein sample preparation. Brains were frozen within 15 min after decapitation. The other halves of the brains were immersion-fixed with Carnoy's fixative (ethanol:chloroform:acetic acid = 6:3:1) at 4 °C overnight. Then, brains were cut coronally at several levels, embedded in paraffin wax, and 6-μm-thick sections were cut using a sliding microtome. Hematoxylin and eosin (H&E) and cresyl violet staining was performed according to standard protocols. For immunological detection of carbonylated proteins, the carbonyl group was derivatized by 2,4-dinitrophenylhydrazine (DNPH). Deparaffinized sections were preincubated with 0.01% DNPH in 2 N HCl for 30 min at room temperature (RT) and were sequentially washed with 2 N HCl, 80% ethanol, 100% ethanol, 50% ethanol/50% ethyl acetate, 80% ethanol, and distilled water. After these pretreatments, sections were incubated in a 0.3% solution of H2O2 in methanol for 30 min at RT to block endogenous peroxidase activity. Sections were then incubated with 10% normal goat serum (NGS) in phosphate-buffered saline (PBS) for 20 min at RT to block non-specific binding sites, followed by the incubation with rabbit anti-2,4-dinitrophenylhydrazone (DNP) antibody (1:100 dilution in 10% NGS; Shima Laboratories Co., Ltd., Tokyo, Japan) at 4 °C overnight. Sections were washed three times in PBS and incubated with a polymer solution conjugated with anti-rabbit IgG secondary antibodies and horseradish peroxidase (HRP) (Envision™ +rabbit/HRP, Dako, Glostrup, Denmark) at RT for 60 min. Reactions were visualized by incubating sections with 0.2 mg/ml 3′3′-diaminobenzidine tetrahydrochloride (DAB) (Wako, Osaka, Japan) in 0.05 M Tris–HCl, pH 7.4, containing 0.02% H2O2 for 15 min. To detect 8-OHdG in nuclear DNA, deparaffinized sections were treated with 5 mg/ml ribonuclease (Sigma), washed in 0.3% Triton X-100 in PBS, and immersed in H2O for 10 min (Kajitani et al., 2006). The sections were treated with 2 N HCl at RT for 30 min to denature the nuclear DNA, and then the sections were treated with Tris–HCl, pH 7.5, for 10 min. After these pretreatments, sections were subjected to immunohistochemistry with mouse anti-8-OHdG antibody (1:200 dilution in 10% NGS; N45.1, JaICA, Shizuoka, Japan) and Envision™ + mouse/HRP (Dako) as described above.

Experimental procedures Electron microscopy Animals and KA administration Three-week-old male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan). All animals were handled in accordance with the Guide for Animal Experiments at our institute and the Guide for

For transmission electron microscopic observation, saline- or KAtreated rat were perfused with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), then postfixed with 2.5% glutaraldehyde in PB for 4 h and 1% OsO4 in PB for 1 h at 4 °C. Specimens were

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dehydrated using a graded series of alcohols and QY-1 (Nisshin EM Co., Ltd., Tokyo, Japan), and embedded in Quetol 812 (Nisshin EM). Ultrathin sections were cut with an LKB ultramicrotome, and sections were counterstained with aqueous TI-blue (Nisshin EM) and Sato's lead citrate (Inaga et al., 2007). Sections were observed with a JEOL 1200EX transmission electron microscope (Nisshin EM). Protein sample preparation Each hippocampus was homogenized in 150 μl of lysis buffer [7 M urea, 2 M thiourea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), 30 mM Tris, supplemented with protease inhibitor cocktail tablets (Complete; Roche Applied Science, Mannheim, Germany)]. The tissues were gently ground using Sample Grinding Kit (GE Healthcare, Piscataway, NJ, USA), and incubated for 30 min on ice, followed by centrifugation at 30,000 ×g at 4 °C for 30 min to remove debris. The supernatant was extracted. The protein concentration was determined by Bradford assay, using bovine serum albumin as the standard. Detection of carbonyl-modified proteins by 2D-oxyblot analysis Quantitative analysis of protein carbonyl level was performed as described previously (Levine et al., 1990; Nakamura and Goto, 1996). One hundred micrograms of proteins precipitated with 10% trichloroacetic acid were suspended and incubated in 10 mM DNPH in 2 N HCl for 30 min at RT. DNPH-derivatized proteins were washed three times with 1 ml of ethanol. The final precipitates were dissolved in 30 μl of lysis buffer. Immobiline DryStrips (18 cm, pH 4–7; GE Healthcare) were rehydrated with rehydration buffer (6 M urea, 2 M thiourea, 2% Triton X-100, 1% Pharmalyte, 25 mM acetic acid, 0.0025% Orange G) at RT overnight. The first-dimension isoelectric focusing (IEF) was run using CoolPhoreStar IPG-IEF (Anatech, Tokyo, Japan) at 500 V for 1 kVh, at 700 V for 700 Vh, 1 kV for 1 kVh, 1.5 kV for 1.5 kVh, 2 kV for 2 kVh, 2.5 kV for 2.5 kVh, 3 kV for 3 kVh, and at 3.5 kV for 35 kVh. After reduction and alkylation of disulfide bonds with 5 mg/ml of dithiothreitol (DTT) and 45 mg/ml of iodoacetamide, respectively, the second-dimension 10% SDS-polyacrylamide gel electrophoresis (SDSPAGE) was run on a CoolPhoreStar SDS-PAGE Dual-200 (Anatech). After 2-dimensional gel electrophoresis (2-DE), proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Billerica, MA, USA) and carbonylated proteins were detected by a primary rabbit anti-DNP antibody (1:250 dilution in Tris-buffered saline (TBS) containing 0.1% polyoxyethylene sorbitan monolaurate (Tween20) (TBS-T); Oxyblot Protein Oxidation Detection Kit, Millipore), a secondary HRP-conjugated goat anti-rabbit IgG antibody (1:500 dilution in TTBS; Oxyblot Protein Oxidation Detection Kit, Millipore), and ECL western blotting detection reagents (GE Healthcare). Autoradiograms were obtained by exposing an X-ray film to the membrane and were scanned and analyzed with the Image-Pro™ Plus computerassisted image analyzer (Media Cybernetics, Silver Spring, MD, USA). The signal intensity of each spot and adjacent background were represented by gray level expressed as integers ranging from 0 (representing black) to 255 (representing white). We measured area (A), the mean signal intensity of each spot (S) and the signal intensity of the background (B). The amount of protein carbonylation in each spot was calculated with the formula ∣S − B∣ × A. For each spot, we determined the ratio of the amount of protein carbonylation relative to the mean carbonylation level of corresponding control spots (relative carbonylation level). Protein identification Mixed 600-μg protein samples from control or KA-administrated rat hippocampus were subjected to 2-DE as described above. Spots of

interest were manually excised from 2D gels stained with Coomassie Brilliant Blue R-350 (CBB, GE Healthcare). Gel pieces were washed three times with 50% acetonitrile in 25 mM ammonium bicarbonate at RT, and then dehydrated with 100% acetonitrile and dried in a centrifugal vacuum-evaporator (CE 1, Hitachi Koki, Tokyo, Japan). The gel pieces were digested with 10 μg/ml trypsin (Promega, Madison, WI, USA) solution at 37 °C overnight. The resultant peptide mixtures were extracted twice with 5% trifluoroacetic acid/50% acetonitrile. The extracted peptides were concentrated in a CE 1 and subjected to LC–MS/MS analysis. Peptides were separated on a 75-μm inner diameter× 15-cm C18 PepMap column (LC Packings). The flow rate was set at 300 nl/min. Peptides were eluted using a 5–60% linear gradient of solvent B in 30 min (solvent A was 0.1% formic acid in 5% acetonitrile, and solvent B was 0.1% formic acid in 95% acetonitrile). Mass analysis of peptides was performed using a LTQ XL linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA). The scan routine was a data-dependent experiment consisting of a full MS scan (200–2000 m/z) followed by a MS/MS scan for the seven most abundant ions. Dynamic exclusion allowed fragmentation of co-eluting peptides. The data were analyzed with the Xcalibur 2.0.5 and BioworksBrowser 3.3 software (Thermo Scientific), and the proteins were identified against rat protein database obtained from NCBI Reference Protein Sequence Database (October 2007) modified with carbamidomethylation on cysteine. All peptide identification with stringent BioWorksBrowser filtering criteria (peptide probability N1 × 10 − 6 and Xcorr vs. Charge State ≥1.5, 2.0 and 2.5 for singly, doubly, and triply charged peptides, respectively) was manually examined, and all peptides had to be identified by consecutive b- or y-ions so that false identifications were eliminated. Protein labeling and separation by 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE) Twenty-five micrograms of extracted proteins were labeled with 200 pmol of either Cy3 or Cy5 for comparison on the same 2D gel. The labeling reaction was performed for 30 min and quenched with 10 mM lysine for 10 min on ice in the dark. An internal standard for every gel electrophoresis was generated by combining equal amounts of all samples and was labeled with Cy2. Equal amounts (25 μg) of quenched Cy3-labeled and Cy5-labeled samples, together with the 25 μg of Cy2labeled internal standard, were mixed and added to an equal volume of 2 × sample buffer [7 M urea, 2 M thiourea, 4% w/v CHAPS, 130 mM DTT, 2% IPG buffer (pH 3–11; GE Healthcare), and a protease inhibitor cocktail]. After incubation on ice for 10 min in the dark, the samples were added to rehydration buffer [7 M urea, 2 M thiourea, 4% w/v CHAPS, 13 mM DTT, 1% IPG buffer (pH 3–11)] and a trace of bromophenol blue to make 450 μl of total sample volume. Ingel rehydration of the IPG strips (Immobiline DryStrips, 24 cm, pH 3–11 NL; GE Healthcare) with the samples was performed at RT for 12 h. The first-dimension IEF was run using an Ettan IPGphor II (GE Healthcare) for a total of 51,500 Vh (Gradient mode). IEF strips were prepared for the second dimension gels as described above. Standard SDS-PAGE was performed on 12.5% gels (Ettan™ DALT six Large Format Vertical System, GE Healthcare). The 2D gels were scanned on a Typhoon 9400 imager (GE Healthcare). Intragel matching was performed using DeCyder software version 6.5 (GE Healthcare). Statistics Quantified densitometric data representing protein carbonylation were analyzed by two-way analysis of variance (ANOVA, treatment × protein spot) to examine the main effect of treatment (KA vs. saline) in each spot. Analyses were performed using the multivariate ANOVA procedure of STATISTICA (Stat Soft, Inc., Tulsa, OK). Post hoc test were performed using Fischer's procedure.

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Results Immunohistohemical detection of oxidative damage to proteins and DNA after KA administration We examined the time course of neuronal morphology following KA administration by H&E staining. A single systemic injection of KA induced cell death in pyramidal cells of the cornu ammonis (CA) 1 and CA3 sectors of the hippocampus. At 1 h after KA, there was no morphological change in hippocampal neurons (Fig. 1B). We noticed the earliest morphological change in neurons 3 h after KA administration; i.e., hematoxylin-positive granules scattered in the nuclei (Fig. 1C, arrowheads), which suggested chromatin clumps. Abnormal distribution of chromatins in the neuronal nuclei was also detected by cresyl violet stain 3 h after KA (data not shown). At 24 h after KA, vacuolation of cell bodies (Fig. 1D, asterisks) and some dying neurons with pyknotic nuclei and hypereosinophilic cytoplasms (Fig. 1D, arrows) were observed. At 72 h and 7 days after KA, most of the pyramidal neurons showed characteristics of dying neurons (Figs. 1E, F). To investigate when free radicals damage proteins and which cells are especially vulnerable to KA-induced oxidative stress in the hippocampus, we detected carbonylated proteins by immunostaining. At 1 h after KA, no carbonylated proteins were observed in the hippocampus (Fig. 1H). At 3 h after KA, DNP immunoreactivity was detected in the hippocampus (Fig. 1I). There were many DNPimmunopositive nuclei in the CA1 pyramidal neurons (Fig. 1I, arrowheads). At 24 h, 72 h, and 7 days after KA, surviving neurons were still DNP-positive (Figs. 1J, K, L, arrowheads), but carbonylated proteins disappeared in neurons with vacuoles (Fig. 1J, asterisks) or pyknotic nuclei (Figs. 1J, K, L, arrows). We also examined the formation of 8-OHdG, an indicator of oxidative damage to DNA, in the KA-treated rat hippocampus. Like carbonylated proteins, generation of 8-OHdG in DNA was first observed 3 h after KA administration

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(Fig. 1O). At 24 h, 72 h, and 7 days after KA, glial cell nuclei were still 8-OHdG positive (Figs. 1P, Q, R, bold arrows), but few or no 8-OHdGpositive neurons were observed. These immunohistochemical results revealed that free radicals damage neuronal proteins and DNA as early as 3 h after KA administration, much earlier than the emergence of morphological features of neuronal cell death. Ultrastructural changes of the hippocampal neurons after KA administration To further examine the morphological changes of the CA1 pyramidal neurons 3 h after KA, we used electron microscopy to show that multiple condensed chromatins were scattered in the nuclei 3 h after KA (Fig. 2B, long arrows). In addition, KA administration caused swelling of the rough endoplasmic reticulum (ER) in the CA1 pyramidal neurons 3 h after KA (Fig. 2D, short arrows). Compared to control mitochondria (Fig. 2C, arrowheads), some mitochondria in CA1 pyramidal neurons exhibited swelling, relatively low electron density, and diffuse cristae (Fig. 2D, asterisks) 3 h after KA, although other mitochondria retained intact structures (Fig. 2D, arrowheads). Identification of carbonyl-modified proteins by 2D-oxyblot To identify oxidatively damaged hippocampal proteins induced by KA administration, we performed proteomic comparison of carbonylated protein levels in the hippocampus between control and KAtreated rats. The total amount of carbonylated proteins in the hippocampus from KA-treated rats was 1.77-fold as much as that from saline-treated control rats (Student's t test, P b 0.05). We focused on 16 protein spots in which the amount of carbonylated proteins in KA-treated rats was consistently more than 1.77-fold that found in control rats. Among these 16 protein spots, we successfully identified

Fig. 1. Time course of morphological changes and oxidative damage to protein and DNA in the hippocampus following kainic acid treatment. Three-week-old male Wister rats received intraperitoneal injection of 10 mg/kg of kainic acid (KA) and were subjected to histological analysis at 1 h (B, H, N), 3 h (C, I, O), 24 h (D, J, P), 72 h (E, K, Q) and 7 days (F, L, R) after KA administration. Control rats were treated with saline (A, G, M). At 1 h after KA injection, neurons in the hippocampal cornu ammonis (CA) 1 sector exhibited no morphological changes based on hematoxylin and eosin (H&E) staining (B), and no immunoreactivity for carbonylated proteins (H) or 8-OHdG (N). At 3 h after KA injection, hematoxylin-positive granules increased in numbers in neuronal nuclei (C, arrowheads). There was strong immunoreactivity for carbonylated proteins (I, arrowheads) and 8-OHdG (O) in neuronal nuclei at this time point. Vacuolation of cell bodies (asterisks in D and J) and neuronal acute cell death (arrows in D and J) were detected 24 h after KA. At this time point, some neuronal cell bodies were immunoreactive for carbonylated proteins (arrowheads in J), whereas neurons with pyknotic nuclei were devoid of immunoreactivity (arrows in J). At 72 h and 7 days after KA, most neurons showed features characteristic of cell death (E and F). At this stage, immunoreactivity for carbonylated proteins (K and L) and 8-OHdG (Q and R) was inconspicuous in CA1 pyramidal neurons. Arrowheads in K and L indicate rare neuronal nuclei immunopositive for carbonylated proteins at 72 h and 7 days, respectively after KA. At 24 h, 72 h, and 7 days after KA, some glial cell nuclei were immunopositive for 8-OHdG (P, Q, and R, bold arrows). Scale bars, 5 μm.

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inner membrane protein (mitofilin, spot #3; Figs. 3C, G), α-internexin (spot #4; Figs. 3D, H), chaperonin containing TCP1, subunit 5 (spot #5; Figs. 3D, H), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3 protein, spot #6, 7, 8, 9; Figs. 3E, I), and βsynuclein (spot #10; Figs. 3F, J). Densitometric quantification followed by statistical analyses revealed that relative carbonylation levels of spots #1, #2, #3, and #4 were significantly increased and that of spot #9 tended to be increased in KA-treated rat hippocampus (Fig. 4). To rule out the possibility that the increase in the relative carbonylation level was due to an increase in protein expression level, we performed 2D-DIGE and quantified protein expression levels. We detected approximately 2000 protein spots per gel (Fig. 5). There was no protein spot in which the mean expression level of KA-treated rats exhibited a 1.77-fold or more increase with statistical significance, compared to that of control rats. Thus, the results from 2D-oxyblot together with those from 2D-DIGE indicated that the increase in the carbonyl content in the KA-treated rats was attributable to increases in the oxidative protein damage, but not to increases in the expression level of each proteins, but. Discussion

Fig. 2. Ultrastructural changes of pyramidal neurons 3 h after kainic acid treatment. Compared to hippocampal CA1 neurons in saline-treated control rats (A, intact nucleus; C, intact rough endoplasmic reticulum, short arrows. Intact mitochondria, arrowheads), nuclear chromatins were condensed (B, long arrows), cisterns of rough endoplasmic reticulum (D, short arrows) and mitochondria (D, asterisks) were swollen in KA-treated CA1 pyramidal neurons. Some mitochondria showed intact structure (D, arrowhead) in KA-treated hippocampus. Scale bars, 2 μm (A and B) and 0.5 μm (C and D).

10 spots by the analysis of amino acid sequence (Table 1). These proteins included heat shock 70 kDa protein 4 (spot #1; Figs. 3C, G), valosin-containing protein (VCP, spot #2; Figs. 3C, G), mitochondrial

Excitotoxicity is a process by which excitatory amino acids induce neuronal cell death. In the present study, a single systemic injection of KA induced neuronal cell death in the pyramidal layer of CA1 and CA3 sectors of the hippocampus (Figs. 1D, E, F). Before KA-induced neuronal cell death occurred, an abnormal distribution of hematoxylin-positive granules in the neuronal nuclei was detected 3 h after KA (Fig. 1C). Concomitant with these histological findings, we found chromatin condensation and swelling of ER and mitochondria in the hippocampal neurons on electron micrographs (Fig. 2). These ultrastructurally detected alterations have been reported in cultured neurons exposed to N-methyl-D-aspartate (NMDA) or a KA receptor agonist (Regan et al., 1995) and may be an early morphological manifestation of excitotoxic neuronal death. Some studies have reported that glutamate receptor agonists induced oxidative stress through mitochondrial dysfunction (Bondy and Lee, 1993; Li et al., 2010; Waldbaum and Patel, 2010). Oxidative stress has also been linked to the neuropathology of several neurodegenerative disorders (Butterfield et al., 2006; Halliwell, 2006), stroke (Alexandrova and Bochev, 2005; Ozkul et al., 2007), trauma (Ansari et al., 2008; Awasthi et al., 1997), and seizures (Patel, 2004; Waldbaum and Patel, 2010). In the present study, we detected the earliest oxidative damage to proteins and DNA in the KA-treated rat hippocampus concurrently 3 h after KA injection. Some studies

Table 1 Identification of proteins based on LC–MS/MS. Spota

Protein nameb

Accession no.c

Scored

% Coveragee

Nominal mass

Significance

1 2 3 4 5 6 7 8 9 10

Heat stroke protein 4 Valosin containing protein Inner membrane protein, mitochondrial Internexin, alpha Chaperonin containing TCP1, subunit 5 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase Synuclein, beta

24025637 17865351 77917546 9506811 51890219 13928824 62990183 6981712 9507245 77404215

1.15E−10 1.08E−10 2.65E−12 1.78E−14 3.21E−14 1.41E−11 7.72E−12 5.11E−14 1.40E−11 6.88E−14

18.90 27.54 22.50 25.30 36.78 36.50 31.00 25.70 32.00 38.06

93,997.1 89,292.9 67,135.3 56,081.7 59,498.9 29,103.3 27,753.7 27,760.8 28,284.9 14,213.0

p = 0.0068 p = 5.965E−09 p = 0.0003 p = 0.0004 n.s.f n.s. n.s. n.s. p = 0.0551 n.s.

a b c d e f

activation activation activation activation

Spot number as indicated in Fig. 3. Proteins identified by LC–MS/MS. Accession numbers from NCBI database. The probability-based score. Coverage of the matched peptides in relation to the full-length sequence. n.s. not significant.

protein protein protein protein

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Fig. 3. Two-dimensional profiles of carbonyl-modified proteins in the hippocampus from control and kainic acid-treated rats. Hippocampi from control (A) and KA-treated rats (B) were extracted 3 h after treatments, and subjected to 2D-oxyblot analysis. Protein spots were analyzed using a LC–MS/MS, if the spots met the criteria that the amount of carbonylated proteins prepared from KA-treated rats were consistently 1.77-fold more than those from control rats. As a result, 10 protein spots were successfully identified (arrows in C–J). Squares in A and B indicate the location of identified protein spots. Magnified views of individual squares are shown in C–J in lower insets. Spot numbers in C–J correspond to those in Table 1.

reported that 8-OHdG was formed prior to excitotoxic cell death (Jarrett et al., 2008; Lan et al., 2000). Total protein carbonyl content was increased in the hippocampus 4 h after KA administration in rat (Gluck et al., 2000). These findings together with our results indicate that oxidative damage to proteins and DNA is an upstream event of neuronal cell death induced by excitotoxicity. Carbonyl modifications of proteins may change protein structures and impair their functions (Dalle-Donne et al., 2006). We previously reported that Arg469 in HSP70, which plays an important role in maintaining the substrate-bound conformation of this protein (Chang et al., 2001), was carbonylated in ischemiareperfusion injured brain (Oikawa et al., 2009). HSP70 acts as a cell survival protein by inhibiting the death-associated permeabilization of lysosomes (Nylandsted et al., 2004). Thus, we proposed that dysfunction of HSP70 by carbonylation at Arg469 may contribute to ROS-induced neuronal cell death via release of cathepsins B and L due to lysosomal rupture (Oikawa et al., 2009). In addition to the loss of protein functions, it has recently been reported that competition between carbonylation and acetylation occurred (Sharma et al., 2006). Competition of these two modifications on lysine and arginine residues in histone proteins can alter the chromatin structure and function (Sharma et al., 2006). Thus, identification of the proteins that are specifically carbonylated under specific pathological conditions would be valuable to clarify the mechanism of disease and may contribute to the discovery of new targets for therapeutic intervention. We therefore compared specific carbonyl-modified proteins in the hippocampus between saline- or KA-treated rats by 2-DE with immunochemical detection of protein carbonyls. We successfully identified several specific

carbonylated proteins in the hippocampus at 3 h after KA administration (Fig. 3, Table 1). Among these proteins, heat shock 70 kDa protein 4, VCP, mitofilin, α-internexin showed significant increase in protein carbonylation level in KA-treated hippocampus (Fig. 4). In addition, the amount of protein carbonylation of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (spot #9) tended to increase in KA-treated rat hippocampus (Fig. 4). Several of the identified proteins act as molecular chaperones, including heat shock 70 kDa protein 4, VCP, and tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein. VCP is a ubiquitin-dependent ATPase that plays central roles in the ubiquitin–proteasome system-mediated protein degradation pathways in neurodegenerative disorders (Halawani et al., 2010). It was reported that VCP (K524A), an ATPase activity-negative VCP mutant, induced the accumulation of ubiquitinated proteins together with the elevation of ER stress markers and cell death (Hirabayashi et al., 2001; Kobayashi et al., 2002). In addition, RNA interference of VCP in HeLa cells promoted extensive cellular vacuolization due to swelling of the ER (Wojcik et al., 2006). Since lysine residue is one of the carbonylation sites, carbonylated VCP may cause ER stress due to impaired ATPase activity. In accordance with this idea, our electron microscopic observation showed abnormal ER expansion 3 h after KA administration (Fig. 2), suggesting increased ER stress. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins (also known as 14-3-3 protein) control the function of a wide array of cellular proteins and promote cell survival (Fu et al., 2000; Henshall et al., 2002; Meller et al., 2003). The K53E mutation in 14-3-3 disrupts the interaction between this chaperone protein and various

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Fig. 4. Relative carbonylation levels of each protein spot. Among identified protein spots, heat shock protein 4 (A), valosin-containing protein (B), mitochondrial inner membrane protein (C), and alpha internexin (D) showed significant increase in the amount of protein carbonylation in KA-treated hippocampus (P b 0.01). (E) Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein tended to increase in the amount of protein carbonylation in KA-treated rat hippocampus (P = 0.055).

binding targets (Fu et al., 2000). Thus, carbonylation of lysine residues at critical sites may affect the function of 14-3-3 protein. In addition, 14-3-3 proteins are necessary to activate tyrosine 3-monooxygenase and tryptophan 5-monooxygenase by calcium/calmodulin-dependent kinase II (CaMK II) (Ichimura et al., 1987; Yamauchi et al., 1981). Tryptophan 5-monooxygenase and tyrosine 3-monooxygenase are the rate-limiting enzymes in the biosynthesis of serotonin and noradrenaline in the brain, respectively. Thus, 14-3-3 proteins are also important to maintain normal neurotransmitter biosynthesis. Heat shock 70 kDa protein 4 (also known as ischemia responsive protein 94 kDa (IRP94)) is a member of the heat shock protein 110 family (Yagita et al., 1999). IRP94 was constitutively expressed in the normal rat hippocampus (Kaneko et al., 1997; Yagita et al., 1999) and the level of expression was apparently enhanced after transient forebrain ischemia (Lee et al., 2002; Yagita et al., 1999). These studies suggest that IRP94 may

play an essential role in the ischemic brain. In the present study, IRP94 carbonylation was increased without an increase in the amount of IRP94, suggesting that oxidative stress may interfere with the function of IRP94. In addition to these molecular chaperones, two proteins associated with the maintenance of the cellular or mitochondrial structure were consistently carbonylated following KA treatment. α-Internexin is primarily an axonal intermediate filament protein found in most neurons in the central nervous system of rat (Kaplan et al., 1990). α-Internexin is also localized in the postsynaptic density (PSD) of rat brains (Suzuki et al., 1997), and is a substrate for CaMK II in the PSD (Yoshimura et al., 2000). Mitofilin is a critical organizer of the mitochondrial cristae morphology and indispensable for normal mitochondrial function (John et al., 2005). We observed swelling of mitochondria 3 h after KA administration (Fig. 2D), suggesting mitofilin oxidation may affect mitochondrial structure. It has recently been reported that mitofilin promotes and is required for mitochondrial localization of PARP-1 (Rossi et al., 2009). The depletion of either PARP-1 or mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage (Rossi et al., 2009). Thus, mitofilin is required to sustain normal mitochondrial function. Carbonylation of these two proteins may affect the axonal function, synaptic plasticity and mitochondrial morphology and function in the hippocampus of KA-treated rats. In the present study, we identified several proteins that were specifically carbonylated in KA-treated rat hippocampus. Carbonylation of these proteins may contribute to hippocampal neuronal death. Several studies reported that pretreatment with antioxidant (Shin et al., 2007; Wang et al., 2004) or induction of antioxidative enzymes (Rojo et al., 2008) can protect against KA-induced neuronal cell death. Our results suggested that specific proteins identified in the present study could be therapeutic targets in seizure disorders. Upregulation or exogeneous supplementation of these proteins might protect neurons from oxidative stress induced by excitotoxicity. Conclusions In summary, our results suggest that oxidative damage to certain proteins such as molecular chaperones and proteins maintaining cellular or mitochondrial structure may be involved in KA-induced neuronal cell death in the hippocampus. This is the first report to identify the proteins that are especially vulnerable to oxidative stress under KA-induced excitotoxic conditions. Since oxidative damage to several specific proteins occurred prior to neuronal cell death, these proteins may be promising targets of therapeutic intervention for seizure-induced neuronal death.

Fig. 5. Comparison of protein expression levels in the hippocampus between control and kainic acid-treated rats by two-dimensional difference gel electrophoresis (2D-DIGE). We performed 2D-DIGE to evaluate changes in protein expression in KA-treated rat hippocampi. We detected approximately 2000 spots per gel. There was no protein spot prepared from KA-treated rat hippocampi that showed a 1.77-fold or more increase in the expression level (B) compared to control hippocampi (A).

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Acknowledgments This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (contract grant numbers: 20790780 to AF and 21590458 to AS) and in part by COE Project (Health Science Hills) for Private Universities from Ministry of Education, Culture, Sports, Science and Technology and Chubu University (No. S0801055). The authors declare that they have no conflicts of interest.

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