Proteomic identification of carbonylated proteins in the monkey hippocampus after ischemia–reperfusion

Free Radical Biology & Medicine 46 (2009) 1472–1477

Contents lists available at ScienceDirect

Free Radical Biology & Medicine 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 / f r e e r a d b i o m e d

Original Contribution

Proteomic identification of carbonylated proteins in the monkey hippocampus after ischemia–reperfusion Shinji Oikawa a,⁎, Tomoko Yamada a, Toshikazu Minohata b, Hatasu Kobayashi a,c, Ayako Furukawa d, Saeko Tada-Oikawa a,c, Yusuke Hiraku a, Mariko Murata a, Mitsuru Kikuchi e, Tetsumori Yamashima f a

Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507, Japan Analytical and Measuring Instruments Division, Analytical Applications Department, Shimadzu Corporation, Kyoto 604-8511, Japan JSPS Research Fellow, Japan d Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Aichi 480-0392, Japan e Department of Psychiatry and Neurobiology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8641, Japan f Department of Restorative Neurosurgery, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8641, Japan b c

a r t i c l e

i n f o

Article history: Received 26 November 2008 Revised 12 February 2009 Accepted 18 February 2009 Available online 9 March 2009 Keywords: Carbonylation CA1 Ischemia–reperfusion Proteomics Hsp70 Reactive oxygen species Free radicals

a b s t r a c t Reactive oxygen species (ROS) are known to participate in neurodegeneration after ischemia–reperfusion. With the aid of ROS, the calpain-induced lysosomal rupture provokes ischemic neuronal death in the cornu Ammonis (CA) 1 of the hippocampus; however, the target proteins of ROS still remain unknown. Here a proteomic analysis was done to identify and characterize ROS-induced carbonyl modification of proteins in the CA1 of the macaque monkey after transient whole-brain ischemia followed by reperfusion. We found that carbonyl modification of heat shock 70-kDa protein 1 (Hsp70-1), a major stress-inducible member of the Hsp70 family, was extensively increased before the neuronal death in the CA1 sector, and the carbonylation site was identified to be Arg469 of Hsp70-1. The CA1 neuronal death conceivably occurs by calpain-mediated cleavage of carbonylated Hsp70 that becomes prone to proteolysis with the resultant lysosomal rupture. In addition, the carbonyl levels of dihydropyrimidinase-like 2 isoform 2, glial fibrillary acidic protein, and βactin were remarkably increased in the postischemic CA1. Therefore, ischemia–reperfusion-induced oxidative damage to these proteins in the CA1 may lead to loss of the neuroprotective function, which contributes to neuronal death. © 2009 Elsevier Inc. All rights reserved.

Overproduction of reactive oxygen species (ROS)1 during ischemia and reperfusion has been implicated as one of the leading causes of cell death in neurological diseases [1,2]. Hippocampal neurons, particularly in the cornu Ammonis (CA) 1 sector, are known to be vulnerable to oxidative damage induced by ROS [3–6]. ROS such as hydroxyl radicals (·OH) and singlet oxygen (1O2) participate in damage to cellular molecules including proteins, DNA, and lipids, all of which lead to cell death [7–9]. Oxidative damage to proteins causes several events such as loss of specific protein function, abnormal protein clearance, depletion of the cellular redox balance, and interference with the cell cycle and, ultimately, neuronal death [10]. Protein carbonylation, the

Abbreviations: ROS, reactive oxygen species; CA, cornu Ammonis; 2D oxyblot, twodimensional gel electrophoresis with immunochemical detection of protein carbonyls; PMF, peptide mass fingerprinting; 2D DIGE, two-dimensional difference gel electrophoresis; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DNPH, 2,4-dinitrophenylhydrazine; PVDF, polyvinylidene fluoride; DNP, 2,4-dinitrophenyl hydrazone; CHCA, α-cyanohydroxycinnamic acid; Hsp70-1, heat shock 70-kDa protein 1; DRP2, dihydropyrimidinase-like 2; GFAP, glial fibrillary acidic protein; Hsc70, heat shock cognate protein. ⁎ Corresponding author. Fax: +81 59 231 5011. E-mail address: [email protected] (S. Oikawa). 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.02.029

major and most common oxidative modification of proteins [11], was increased by ischemia–reperfusion injury in the hippocampus of gerbils [12]. Accordingly, protein carbonyls are likely to play an important role in ROS-mediated neuronal death in the postischemic CA1 sector. Accumulating evidence suggests a role for calpain and cathepsin in the development of neuronal necrosis from Caenorhabditis elegans to primates [13]. Activated calpain compromises the integrity of the lysosomal membrane and causes leakage of degrading hydrolytic enzyme cathepsins into the cytoplasm. Lysosomes are one of the major targets for oxidant injury [14]. Identification of specific targets of protein oxidation is crucial for establishing a relationship between oxidative modification and neuron death. In this study, to identify target proteins of ROS oxidation, we investigated oxidative damage of proteins in the CA1 of Japanese monkeys after transient whole-brain ischemia followed by reperfusion (ischemia–reperfusion). Twodimensional gel electrophoresis (2DE) with immunochemical detection of protein carbonyls (2D oxyblot) and peptide mass fingerprinting (PMF) were done to identify and characterize carbonyl-modified proteins in hippocampal CA1 sectors at 3, 5, and 7 days after ischemia– reperfusion.

S. Oikawa et al. / Free Radical Biology & Medicine 46 (2009) 1472–1477

Materials and methods Animals All experimental procedures were performed in strict adherence with the guidelines of the Animal Care and Ethics Committee of Kanazawa University and the NIH Guide for the Care and Use of Laboratory Animals. The monkeys (Macaca fuscata), with a body weight of 5–10 kg, were bred in air-conditioned cages and were allowed free daily access to food and water. Under general anesthesia, 12 adult (5–11 years of age) monkeys underwent transient, complete, whole-brain ischemia (n = 9) or a sham surgery (n = 3), according to the procedure previously described [15]. Briefly, after removal of the sternum, the innominate and left subclavian arteries were exposed in the mediastinum and were clipped for 20 min, then reperfusion was done. The effectiveness of clipping was demonstrated by an almost complete absence of cerebral blood flow, which was monitored by laser Doppler (Vasamedics, St. Paul, MN, USA). Hippocampal CA1 tissues were resected from both sham-operated controls and postischemic days 3, 5, and 7 monkeys under general anesthesia. Sample preparation The hippocampal CA1 tissues were collected [15,16] and immediately put into liquid nitrogen and stored at −80 °C until use. Frozen tissue (20–30 mg) was homogenized in lysis buffer (30 mM Tris–HCl, 7 M urea, 2 M thiourea, 4% w/v CHAPS, and a protease inhibitor cocktail, pH 8.5). After incubation for 60 min on ice, homogenates were centrifuged at 30,000 g for 30 min at 4°C and the supernatant was collected. Protein concentration of the supernatant was determined by the Bradford assay (Pierce), using bovine serum albumin as a standard [17]. The remaining hippocampal tissues were fixed in 4% paraformaldehyde for 2 weeks, embedded in paraffin, and stained with hematoxylin–eosin to confirm CA1 neuronal death. Two-dimensional difference gel electrophoresis (2D DIGE) Each soluble protein sample was minimally labeled with CyDye DIGE fluors according to the manufacturer's protocol (GE Healthcare). Internal pools were generated by combining equal amounts of all samples and were labeled with Cy2. Equal protein amounts of Cy2, Cy3, and Cy5-labeled samples were mixed and added to an equal volume of 2× sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 130 mM dithiothreitol (DTT), 2% IPG buffer (pH 4–7; 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 4–7)) and a trace of bromophenol blue to make 340 μl of total sample volume. Ingel rehydration of the IPG strips (Immobiline DryStrips, 18 cm, pH 4– 7; GE Healthcare) with the samples was performed at room temperature for 12 h. The first dimension of isoelectric focusing was run using an Ettan IPGphor II (GE Healthcare) at 500 V for 500 V h, at 1 kV for 1 kV h, and at 8 kV for 50 kV h. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was run on an Ettan DALT Six large electrophoresis 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). Detection of carbonyl modified proteins (2D oxyblot analysis) Derivatization with 2,4-dinitrophenylhydrazine (DNPH) was done according to the procedure of Nakamura and Goto [18]. The mixtures were prepared by combining equal amounts (35 μg) of three different samples from each group (control, day 3, day 5, and day 7), precipitated by

1473

trichloroacetic acid, and then incubated in10 mM DNPH in 2 N HCl at room temperature for 1 h. The pellets were washed three times with ethanol and dissolved in 1× sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 65 mM DTT, 1% IPG buffer (pH 4–7), and a protease inhibitor cocktail). Two-dimensional electrophoresis and subsequent immunoblotting for protein carbonyls were performed as described previously [19]. Briefly, aliquots of CA1 extracts containing 100 μg of DNPHtreated total protein were applied to Immobiline DryStrips (18 cm, pH 4–7) for the first-dimension isoelectric focusing. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 10% SDS–PAGE was run on an Ettan DALT Six large electrophoresis system. After the second-dimension, the proteins from the gels were transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon-P transfer membrane; Millipore) using a TE77 semidry transfer unit (GE Healthcare) at 50 V for 30 min. The 2,4-dinitrophenyl hydrazone (DNP) adduct of the carbonyls of the proteins was detected on the PVDF membrane using a primary rabbit antibody specific for DNP–protein adducts (1:250; Chemicon), and a secondary horseradish peroxidaseconjugated goat anti-rabbit IgG antibody (1:500; Chemicon) was applied. Then, the spot intensities of carbonylated proteins were quantified using ImageMaster 2D Platinum software (GE Healthcare). We calculated specific oxidation as relative carbonyl level (obtained from the 2D oxyblot) per relative protein expression (obtained from the 2D DIGE). Mass spectrometry For MS fingerprinting, the Coomassie Brilliant Blue-stained proteins of the 2D gel obtained with non-DNPH-treated samples of postischemic day 3 monkeys were excised and digested with trypsin, as described by Kondo [20]. The differential protein spots on the gel were cut out, decolorized twice, dehydrated twice, added to 12.5 µg/ml trypsin (Promega) solution, and digested overnight at 37°C. After its peptide segment was extracted, the sample was air-dried, mixed with 1 μl of saturated α-cyanohydroxycinnamic acid (CHCA) in 0.1% trifluoroacetic acid and 50% acetonitrile, and dripped on a stainless-steel target and was then ready for MS identification. The peptide mass maps produced by matrix-assisted laser doppler ionization time-of-flight (MALDITOF)/MS (Voyager B-RP; PerSeptive Biosystems) were searched against the published databases by means of the MS-Fit module in Protein Prospector (available at http://prospector.ucsf.edu/). The MS-Fit search was carried out with the following parameters: database, NCBInr; allow one missed cleavage; enzyme, trypsin; mass tolerance, ± 100 ppm; a minimum of four hits required for a match; fixed modification, cysteine carbamidomethylation; and variable modification, methionine oxidation. In addition, for protein samples that needed more precise confirmation of carbonylation sites, MALDI-TOF/TOF analysis was done. In-gel digestion of these protein samples was performed according to a standard protocol [21] with a few modifications. Briefly, the gel pieces were dehydrated and dried. The dried gel pieces were reswollen in 5 μl of 100 mM ammonium bicarbonate containing 10 μg/ ml trypsin for 3 h at 37 °C. After digestion, tryptic peptides were extracted twice in 50 μl of 66% acetonitrile and 0.1% trifluoroacetic acid in a sonicator. The extracted peptides were dried, redissolved in 0.1% trifluoroacetic acid, and injected onto a MonoCap 0.1 × 250-mm monolithic C18 column (Kyoto Monotech) with Prominence Nano (Shimadzu). Column eluent was spotted every 15 s onto a μFocus MALDI plate (Shimadzu GLC) with CHCA as the matrix, using AccuSpot (Shimadzu). The digests were analyzed on a MALDI-TOF/TOF instrument (AXIMA Performance; Shimadzu) with Kompact version 2.8. Protein identification was carried out using the MS/MS ion search of Mascot (http://www.matrixscience.com/; Matrix Science Ltd.). The following parameters were set for searching peptides containing carbonylated arginine using Mascot: database, NCBInr; allow one missed cleavage; enzyme, none; peptide mass tolerance,±0.3 Da;

1474

S. Oikawa et al. / Free Radical Biology & Medicine 46 (2009) 1472–1477

Fig. 1. Light microphotographs of the control and days 3, 5, and 7 postischemic CA1 neurons. Eosinophilic cell death of CA1 neurons starts on day 3 and is completed on days 5 and 7. Hematoxylin–eosin staining. Bar, 15 μm.

fragment mass tolerance, ±1.5 Da; fixed modification, cysteine carbamidomethylation; and variable modification, methionine oxidation and glutamic semialdehyde from arginine. A Mascot threshold of 63 was used to achieve a false identification rate of 5%. Results and discussion

developed eosinophilic coagulation necrosis that was characterized by reddish residual cytoplasm and negligible nuclear staining (Fig. 1) [14– 16]. On day 7, instead of the total CA1 neuronal loss, abundant cell debris and reactive glial proliferation were seen. In this neurodegenerative process, apoptotic bodies were not observed, and the cell death pattern was considered necrosis as previously reported [14–16].

Delayed neuronal death on days 5–7

Detection and identification of carbonyl protein in the CA1

Pyramidal cells of the control (nonischemic) CA1 sector showed round and vesicular nuclei. However, on day 3 after transient ischemia most of the pyramidal cells in the CA1 showed shrinkage of the cell body and mild pyknotic change in nuclei. On day 5 almost all pyramidal cells

2D oxyblot, which has recently been applied to detect carbonylated proteins in mainly mammalian brain tissue [18,22], was performed to obtain new information about actual number, pI, and mass of proteins prone to oxidation during the development of neuronal cell death

Fig. 2. 2D oxyblot and 2D DIGE analyses of the control and day 3 postischemic CA1. (A) Proteins (100 μg) were treated with DNPH and separated by 2DE, followed by transfer onto a PVDF membrane. Membranes were incubated with primary antibody specific to the DNP moiety of the proteins, followed by incubation with horseradish peroxidase-conjugated antibody directed against the primary antibody. The proteins were visualized by ECL. The resulting images were processed using ImageMaster 2D Platinum software (GE Healthcare) to match spots and provide spot volumes. The spots were identified by PMF. Molecular weight markers are on the left. (B) Proteins (25 μg) were labeled with Cy5 or Cy3 dye, mixed, and subjected to 2D DIGE analysis. Cy5 and Cy3 images are illustrated using red and green pseudocolors, respectively. Yellow spots represent proteins that were unchanged.

S. Oikawa et al. / Free Radical Biology & Medicine 46 (2009) 1472–1477

1475

Table 1 Identification of carbonylated proteins based on PMF Spota C2 C3 C4 C6 C7 C17 a b c d e f

Protein nameb Heat shock-70 kDa protein 1 (Hsp70-1) Hsp70-1 Dihydropyrimidinase-like 2 isoform 2 Glial fibrillary acidic protein β-Actin β-Actin

Accession No.c 147744565 147744565 109085951 55846684 4501885 4501885

MOWSE scored 6

1.14 × 10 6.95 × 107 9.80 × 106 9.01 × 106 1.11 × 106 4.73 × 106

Matched peptides/totale

% Coveragef

Theoretical molecular mass (Da)/pI

11/140 15/86 12/150 19/143 12/153 12/40

22.6 29.0 28.2 37.0 39.7 32.3

70,053/5.5 70,053/5.5 73,583/5.9 47,412/5.2 41,737/5.3 41,737/5.3

Spot number as indicated in Fig. 2. Proteins identified by PMF. Accession numbers from NCBInr database. The probability-based MOWSE score. Number of matched peptides versus total number of peptides. Coverage of the matched peptides in relation to the full-length sequence.

induced by ischemia–reperfusion. Fig. 2A shows carbonylated proteins in representative 2D oxyblots from the control and day 3 postischemic CA1. The corresponding spots in the 2D DIGE image are shown in Fig. 2B. Carbonyl levels of spots C2, C3, C4, C6, C7, and C17 were increased at 3 or 5 days in the CA1 region. Spots C2 and C3 were identified as heat shock 70-kDa protein 1 (Hsp70-1). Spots C4 and C6 were identified as dihydropyrimidinase-like 2 (DRP2) isoform 2 and glial fibrillary acidic protein (GFAP), respectively. Spots C7 and C17 were identified as βactin. These six spots are annotated and listed in Table 1. To normalize carbonylated protein levels, we calculated the specific oxidation of each spot as the ratio of carbonyl level to the protein expression level obtained from 2D DIGE. The time course of specific oxidation is shown in Fig. 3. Interestingly, the specific oxidation of spot C2 (Hsp70-1) in the CA1 was extensively increased on day 3 (Fig. 3). Microglial and astroglial proliferation was induced in the CA1 on postischemic days 4 and 9, respectively [23]. Thus, these results suggest that oxidative modifications of Hsp70-1 might be induced in neurons. Carbonyl levels of Hsp70-1 In addition to the specific oxidation of spot C2 (Hsp70-1), that of spot C3 (Hsp70-1) increased on postischemic day 3 (Fig. 3), before the neuronal cell death that was completed on days 5–7 (Fig. 1). The pI difference between spots C2 and C3 was approximately 0.05, which suggested posttranslational modification such as phosphorylation [24]. Heat shock proteins are known to be induced by stressful stimuli and to maintain cellular integrity and viability. After a variety of insults to the central nervous system, Hsp70 is synthesized abundantly and is localized in the cytosol, nucleus, and endoplasmic reticulum [25]. The downstream events of the ROS-induced cell death pathway in postischemic neurons have been unknown. In 1996 Yamashima et al. proposed, as a mechanism for postischemic neuronal death in primates, the “calpain–cathepsin hypothesis” that calpain-mediated lysosomal rupture, with the resultant release of cathepsins B and L, might gradually provoke degradation of certain cellular constituents [13–16]. Hsp70 acts as a cell survival protein, functioning by inhibiting the death-associated permeabilization of lysosomes [26]. In addition, several studies in nonneuronal cell lines have shown that the protective effect of Hsp70 derives also from its antiapoptotic mechanisms [27,28]. At any rate, Hsp70 plays an important role in the protection of cells against various stresses, including cerebral ischemia–reperfusion. Carbonylation of proteins is an irreversible oxidative damage, often leading to the loss of protein function [29,30]. Therefore, we speculate that carbonyl modification of Hsp70-1 may lead to the loss of its neuroprotective function.

the MS/MS data yielded 43% sequence coverage. Fig. 4 shows the spectrum of the monocarbonylated peptide 459-FELSGIPPAPR⁎G-470 (R⁎, carbonylated arginine). We confirmed the peptide sequence of the observed fragment ions and demonstrated the presence of a new carbonylation site at Arg469. This identified peptide was cleaved Cterminal to the glycine neighbor of the carbonylated arginine. It is widely accepted that trypsin treatment cleaves exclusively C-terminal to arginine and lysine. However, the specificity of trypsin cleavage for the peptide including the carbonylated arginine still remains unclear. Because previous reports have suggested that trypsin does not cleave oxidized arginine and lysine [31,32], it is probable that carbonylation of arginine may influence trypsin cleavage. Carbonyl derivatives are formed by oxidation of the side chains of lysine, proline, arginine, and threonine residues [33,34]. The presence of the y2 fragment ion at m/z 189.19 showed a carbonylation site at Arg469, whereas the y3 fragment ion at m/z 286.30, b7 fragment ion at m/z 744.85, and b8 fragment ion at m/z 841.97 indicated that Pro465, Pro466, and Pro468 were not carbonylated. Chang et al. reported that mutating Arg469 in the rat 70-kDa heat shock cognate protein (Hsc70), a constitutive member of the heat shock-induced Hsp70 protein family, provoked a reduced affinity for peptide substrates and an impaired refolding activity [35]. This mutant Hsc70 (R469C) is more accessible to proteolytic cleavage. These results suggested that the Arg469 is likely to be involved in the stabilization of the closed conformation of the C-terminal domain. Therefore, it is probable that carbonylation on Arg469 in Hsp70 may play an important role in ROS-induced neuronal cell death. Although calpain-mediated proteolysis may occur upon a wide variety of substrates [36], direct in vivo evidence of a key substrate at

Identification of a carbonylation site on Hsp70-1 by mass spectrometry analysis To identify carbonylated residues in Hsp70-1, we performed MALDI-TOF/TOF analysis with the Mascot search. A Mascot search of

Fig. 3. Specific oxidation levels of identified proteins 3, 5, and 7 days after the ischemia– reperfusion insult. Spot numbers are shown in Fig. 2. Specific oxidation was calculated as relative carbonyl level per relative protein expression.

1476

S. Oikawa et al. / Free Radical Biology & Medicine 46 (2009) 1472–1477

Fig. 4. Mass spectra of Hsp70-1. (A) MALDI-TOF/MS spectrum of the tryptic digest of spot C2 (Fig. 2). (B) Structures of arginine and carbonylated arginine. (C) MS/MS spectrum of 1197.66-Da peptide (FELSGIPPAPR⁎G) acquired with the MALDI-TOF/TOF/MS. R⁎, carbonylated arginine.

the lysosomal membrane has been lacking. It is probable that carbonylation of the Arg469 provokes a resultant susceptibility of Hsp70-1 to activated calpain. The depletion of Hsp70, localized to the lysosomal membrane of carcinoma cells, has been shown to result in lysosomal destabilization, release of its constituents into the cytosol, and caspase-independent cell death [26]. The depletion of Hsp70-2, which is highly homologous with Hsp70-1, also triggers lysosomal membrane permeabilization and cathepsin-dependent cancer cell death [25]. Thus, we speculate that Hsp70-1 carbonylation may contribute to the occurrence of lysosomal rupture in the postischemic CA1 neurons. Carbonyl levels of other proteins in the postischemic CA1 The specific oxidation of spot C4 (DRP2 isoform 2) was also increased on day 3 (Fig. 3). DRP2, a member of the dihydropyrimidinase-related protein family, is involved in axonal outgrowth and pathfinding through the transmission and modulation of extracellular signals [37]. Decreased expression of DRP2 has been observed in Alzheimer disease, Down syndrome, schizophrenia, and affective disorders [38,39]. Castegna et al. reported a significant increase in protein carbonyl levels of DRP2 in the Alzheimer brain [10]. In the adult brain, DRP2 plays a role in repair and regeneration of stress-damaged neurons [40]. These reports and the present results altogether suggest a potential involvement of oxidative modification of DRP2 in neurodegeneration of the CA1 sector after ischemia–reperfusion.

The specific oxidation of spots C7 (β-actin) and C17 (β-actin) was increased on day 5 after the ischemic insult (Fig. 3). β-Actin, present in neurons [41] and glial cells such as microglia [42], is a core subunit of microfilaments responsible for cell structure. Because microglial proliferation was observed, instead of neuronal loss, in the CA1 sector of the monkey hippocampus on day 4 after ischemia–reperfusion [23,43], carbonylation of β-actin on day 5 was considered to occur primarily in microglia. In addition, the carbonylation of spot C6 (GFAP, a specific marker protein in astrocytes) was maximal on day 5 (Fig. 3), although its protein expression level did not increase (data not shown). Glial cells, including microglia and astrocytes, play a key role in the survival of neuronal cells [44]. As carbonylation of β-actin and GFAP results in dysfunction of glia, this may be also associated with development of CA1 neuronal cell death. Conclusions Oxidative protein damage due to ischemia–reperfusion has been implicated as one of the leading causes of neuronal cell death. Carbonylation of proteins due to oxidative modification has been shown to affect their function and/or metabolic stability [45]. In this study, the proteome analysis revealed that the carbonyl levels of Hsp70-1, DRP2 isoform 2, GFAP, and β-actin were remarkably increased in the postischemic CA1. Thus, it is likely that carbonylation of these proteins, especially Hsp70-1, in CA1 is related to the development of neuronal cell death. We speculate that these carbonyl-modified proteins can be used as biomarkers for assessing

S. Oikawa et al. / Free Radical Biology & Medicine 46 (2009) 1472–1477

neurodegeneration of not only cerebral ischemia but also various brain disorders. Acknowledgment This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. References [1] White, B. C.; Sullivan, J. M.; DeGracia, D. J.; O'Neil, B. J.; Neumar, R. W.; Grossman, L. I.; Rafols, J. A.; Krause, G. S. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J. Neurol. Sci. 179:1–33; 2000. [2] Neumar, R. W. Molecular mechanisms of ischemic neuronal injury. Ann. Emerg. Med. 36:483–506; 2000. [3] Di Loreto, S.; Zimmitti, V.; Sebastiani, P.; Cervelli, C.; Falone, S.; Amicarelli, F. Methylglyoxal causes strong weakening of detoxifying capacity and apoptotic cell death in rat hippocampal neurons. Int. J. Biochem. Cell Biol. 40:245–257; 2008. [4] Serrano, F.; Klann, E. Reactive oxygen species and synaptic plasticity in the aging hippocampus. Ageing Res. Rev. 3:431–443; 2004. [5] Wang, X.; Pal, R.; Chen, X. W.; Limpeanchob, N.; Kumar, K. N.; Michaelis, E. K. High intrinsic oxidative stress may underlie selective vulnerability of the hippocampal CA1 region. Brain Res. Mol. Brain Res. 140:120–126; 2005. [6] Vornov, J. J.; Park, J.; Thomas, A. G. Regional vulnerability to endogenous and exogenous oxidative stress in organotypic hippocampal culture. Exp. Neurol. 149: 109–122; 1998. [7] Chan, P. H. Oxygen radicals in focal cerebral ischemia. Brain Pathol 4:59–65; 1994. [8] Coyle, J. T.; Puttfarcken, P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695; 1993. [9] Nakabeppu, Y.; Tsuchimoto, D.; Furuichi, M.; Sakumi, K. The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death. Free Radic. Res. 38:423–429; 2004. [10] Castegna, A.; Aksenov, M.; Thongboonkerd, V.; Klein, J. B.; Pierce, W. M.; Booze, R.; Markesbery, W. R.; Butterfield, D. A. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II. Dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J. Neurochem. 82:1524–1532; 2002. [11] Stadtman, E. R.; Berlett, B. S. Reactive oxygen-mediated protein oxidation in aging and disease. Chem. Res. Toxicol. 10:485–494; 1997. [12] Kwon, Y. S.; Ann, H. S.; Nabeshima, T.; Shin, E. J.; Kim, W. K.; Jhoo, J. H.; Jhoo, W. K.; Wie, M. B.; Kim, Y. S.; Jang, K. J.; Kim, H. C. Selegiline potentiates the effects of EGb 761 in response to ischemic brain injury. Neurochem. Int. 45:157–170; 2004. [13] Yamashima, T.; Tonchev, A. B.; Borlongan, C. V. Differential response to ischemia in adjacent hippocampal sectors: neuronal death in CA1 versus neurogenesis in dentate gyrus. Biotechnol. J. 2:596–607; 2007. [14] Yamashima, T. Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates. Prog. Neurobiol. 62:273–295; 2000. [15] Yamashima, T.; Kohda, Y.; Tsuchiya, K.; Ueno, T.; Yamashita, J.; Yoshioka, T.; Kominami, E. Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'. Eur. J. Neurosci. 10:1723–1733; 1998. [16] Yamashima, T.; Saido, T. C.; Takita, M.; Miyazawa, A.; Yamano, J.; Miyakawa, A.; Nishijyo, H.; Yamashita, J.; Kawashima, S.; Ono, T.; Yoshioka, T. Transient brain ischaemia provokes Ca2+, PIP2 and calpain responses prior to delayed neuronal death in monkeys. Eur. J. Neurosci. 8:1932–1944; 1996. [17] Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976. [18] Nakamura, A.; Goto, S. Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis. J. Biochem 119:768–774; 1996. [19] Nabeshi, H.; Oikawa, S.; Inoue, S.; Nishino, K.; Kawanishi, S. Proteomic analysis for protein carbonyl as an indicator of oxidative damage in senescence-accelerated mice. Free Radic. Res. 40:1173–1181; 2006. [20] Kondo, T.; Hirohashi, S. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics. Nat. Protoc 1: 2940–2956; 2006. [21] Toda, T.; Kimura, N. Standardization of protocol for Immobiline 2-D PAGE and construction of 2-D PAGE protein database on world wide web home page. Jpn. J. Electrophor. 41:13–19; 1997.

1477

[22] Butterfield, D. A.; Perluigi, M.; Sultana, R. Oxidative stress in Alzheimer's disease brain: new insights from redox proteomics. Eur. J. Pharmacol. 545: 39–50; 2006. [23] Tonchev, A. B.; Yamashima, T.; Zhao, L.; Okano, H. Differential proliferative response in the postischemic hippocampus, temporal cortex, and olfactory bulb of young adult macaque monkeys. Glia 42:209–224; 2003. [24] Yamagata, A.; Kristensen, D. B.; Takeda, Y.; Miyamoto, Y.; Okada, K.; Inamatsu, M.; Yoshizato, K. Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2:1267–1276; 2002. [25] Daugaard, M.; Kirkegaard-Sørensen, T.; Ostenfeld, M. S.; Aaboe, M.; H.yer-Hansen, M.; Ørntoft, T. F.; Rohde, M.; Jäättelä, M. Lens epithelium-derived growth factor is an Hsp70-2 regulated guardian of lysosomal stability in human cancer. Cancer Res. 67:2559–2567; 2007. [26] Nylandsted, J.; Gyrd-Hansen, M.; Danielewicz, A.; Fehrenbacher, N.; Lademann, U.; Hoyer-Hansen, M.; Weber, E.; Multhoff, G.; Rohde, M.; Jaattela, M. Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization. J. Exp. Med. 200:425–435; 2004. [27] Rokutan, K. Role of heat shock proteins in gastric mucosal protection. J. Gastroenterol. Hepatol. 15 (Suppl.):D12–19; 2000. [28] Yenari, M. A.; Giffard, R. G.; Sapolsky, R. M.; Steinberg, G. K. The neuroprotective potential of heat shock protein 70 (HSP70). Mol. Med. Today 5:525–531; 1999. [29] Dalle-Donne, I.; Aldini, G.; Carini, M.; Colombo, R.; Rossi, R.; Milzani, A. Protein carbonylation, cellular dysfunction, and disease progression. J. Cell. Mol. Med. 10: 389–406; 2006. [30] Nystrom, T. Role of oxidative carbonylation in protein quality control and senescence. EMBO J. 24:1311–1317; 2005. [31] Mirzaei, H.; Regnier, F. Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry. Anal. Chem. 77:2386–2392; 2005. [32] Wilmarth, P. A.; Tanner, S.; Dasari, S.; Nagalla, S. R.; Riviere, M. A.; Bafna, V.; Pevzner, P. A.; David, L. L. Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility? J. Proteome Res. 5: 2554–2566; 2006. [33] Bizzozero, O. A.; Reyes, S.; Ziegler, J.; Smerjac, S. Lipid peroxidation scavengers prevent the carbonylation of cytoskeletal brain proteins induced by glutathione depletion. Neurochem. Res. 32:2114–2122; 2007. [34] Aksenov, M. Y.; Aksenova, M. V.; Butterfield, D. A.; Geddes, J. W.; Markesbery, W. R. Protein oxidation in the brain in Alzheimer's disease. Neuroscience 103:373–383; 2001. [35] Chang, T. C.; Hsiao, C. D.; Wu, S. J.; Wang, C. The effect of mutating arginine-469 on the substrate binding and refolding activities of 70-kDa heat shock cognate protein. Arch. Biochem. Biophys. 386:30–36; 2001. [36] Rami, A. Ischemic neuronal death in the rat hippocampus: the calpain– calpastatin–caspase hypothesis. Neurobiol. Dis. 13:75–88; 2003. [37] Poon, H. F.; Vaishnav, R. A.; Getchell, T. V.; Getchell, M. L.; Butterfield, D. A. Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice. Neurobiol. Aging 27: 1010–1019; 2006. [38] Lubec, G.; Nonaka, M.; Krapfenbauer, K.; Gratzer, M.; Cairns, N.; Fountoulakis, M. Expression of the dihydropyrimidinase related protein 2 (DRP-2) in Down syndrome and Alzheimer's disease brain is downregulated at the mRNA and dysregulated at the protein level. J. Neural Transm. Suppl. 57:161–177; 1999. [39] Johnston-Wilson, N. L.; Sims, C. D.; Hofmann, J. P.; Anderson, L.; Shore, A. D.; Torrey, E. F.; Yolken, R. H. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder: the Stanley Neuropathology Consortium. Mol. Psychiatry 5:142–149; 2000. [40] Choi, J.; Forster, M. J.; McDonald, S. R.; Weintraub, S. T.; Carroll, C. A.; Gracy, R. W. Proteomic identification of specific oxidized proteins in ApoE-knockout mice: relevance to Alzheimer's disease. Free Radic. Biol. Med. 36:1155–1162; 2004. [41] Allison, D. W.; Gelfand, V. I.; Spector, I.; Craig, A. M. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J. Neurosci. 18:2423–2436; 1998. [42] Christensen, R. N.; Ha, B. K.; Sun, F.; Bresnahan, J. C.; Beattie, M. S. Kainate induces rapid redistribution of the actin cytoskeleton in ameboid microglia. J. Neurosci. Res. 84:170–181; 2006. [43] Tsukada, T.; Watanabe, M.; Yamashima, T. Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus. J. Neurochem. 79:1196–1206; 2001. [44] Shaham, S. Glia–neuron interactions in nervous system function and development. Curr. Top. Dev. Biol. 69:39–66; 2005. [45] Levine, R. L. Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic. Biol. Med. 32:790–796; 2002.