Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia

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COMBINED METABOLIC AND TRANSCRIPTIONAL PROFILING IDENTIFIES PENTOSE PHOSPHATE PATHWAY ACTIVATION BY HSP27 PHOSPHORYLATION DURING CEREBRAL ISCHEMIA

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TAICHIRO IMAHORI, a KOHKICHI HOSODA, a* TOMOAKI NAKAI, a YUSUKE YAMAMOTO, a YASUHIRO IRINO, b MASAKAZU SHINOHARA, c,d NAOKO SATO, a TAKASHI SASAYAMA, a KAZUHIRO TANAKA, a HIROAKI NAGASHIMA, a MASAAKI KOHTA a AND EIJI KOHMURA a

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a Department of Neurosurgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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b Division of Evidenced-based Laboratory Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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c The Integrated Center for Mass Spectrometry, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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d Division of Medical Education, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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Abstract—The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD+ ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly

reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: cerebral ischemia, glucose 6-phosphate dehydrogenase, heat shock protein 27, middle cerebral artery occlusion, omics, pentose phosphate pathway. 22

*Corresponding author. Fax: +81-78-382-5979. E-mail address: [email protected] (K. Hosoda). Abbreviations: ATM, ataxia telangiectasia mutated; F6P, fructose 6phosphate; G6PD, glucose 6-phosphate dehydrogenase; HSP27, heat shock protein 27; MAPK, mitogen-activated protein kinase; MCAO, middle cerebral artery occlusion; PPP, pentose phosphate pathway; R5P, ribulose 5-phosphate; ROS, reactive oxygen species. http://dx.doi.org/10.1016/j.neuroscience.2017.02.036 0306-4522/Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Stroke is estimated to be the second leading cause of death and the third most common cause of permanent disability worldwide (Donnan et al., 2008). Ischemic stroke accounts for more than 90% of all strokes. However, the metabolic pathophysiology underlying ischemic stroke remains poorly understood. The development of high-throughput ‘‘omic” methods, such as transcriptomics, which permits the screening of large numbers of genes for involvement in biological processes, has provided powerful tools for addressing complex issues related to human health (Barr et al., 2010). Metabolome analyses using omics methods have recently been reported. In this context, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have garnered the most attention because of their ability to simultaneously profile a large number of metabolites (Lewis et al., 2008). These technologies provide comprehensive information on thousands of lowmolecular-mass compounds (less than 2 kDa), including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates. Metabolomics renders the metabolic profile of a system and the end points of biological events and reflects the state of a cell or a group of cells at a given time point (Gerszten and Wang, 2008). Gas-chromatography/mass-spectrometry (GC–MS) is one of the wide-spread techniques that enables researchers to determine analyte masses with high precision and accuracy, such that peptides and metabolites can be unambiguously identified even in complex fluids (Lewis and Gerszten, 2010).

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The profiling of low-molecular-weight biochemicals that serve as substrates and products in metabolic pathways is particularly relevant to cardiovascular diseases (Lewis et al., 2008). To date, only a few studies have reported metabolic profiling of strokes. In the current study, we used a combination of an unbiased and global metabolic approach and a transcriptional approach to assess the effects of cerebral ischemia induced by middle cerebral artery occlusion (MCAO) on the metabolism of the cerebral cortex in a rat model. We explored ischemia-specific metabolic pathways that might serve as potential therapeutic targets for stroke treatment and found that heat shock protein 27 (HSP27) was hyperphosphorylated at serine 85 (S85) after MCAO. We also observed a corresponding elevation in glucose 6-phosphate dehydrogenase (G6PD) activity in the pentose phosphate pathway (PPP) and an increase in the NADPH/NADP+ ratio after MCAO. Furthermore, administration of ataxia telangiectasia mutated (ATM) kinase inhibitor significantly reduced HSP27 phosphorylation after MCAO. These findings suggest that the PPP is activated via HSP27 phosphorylation by ATM kinase as part of the endogenous defense system against oxidative stress during the early stages of ischemia, even without reperfusion.

EXPERIMENTAL PROCEDURES

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Animals and MCAO

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All procedures involving animals were performed under protocols approved by the Animal Care and Use Review Committee of Kobe University Graduate School of Medicine. Male Wistar rats weighing 220–260 g (Clea Japan, Inc.; Osaka, Japan) were used for this study. The rats were housed in a controlled environment (alternating 12-h light/dark cycle, 22 ± 2 °C, 55 ± 5% relative humidity) and were fasted overnight before surgery and given free access to water. These rats were randomly allocated to a sham-operated group and ischemia groups. The rats were anesthetized with 5% halothane and maintained under 1% halothane in 70% nitrous oxide and 30% oxygen via a face mask to allow spontaneous breathing. Rectal temperatures were maintained at 37.0 ± 0.5 °C with a feedback-regulated heating pad throughout the procedure. The right femoral artery was cannulated to monitor the arterial blood pressure using a pressure transducer (AP-601G; Nihon Koden, Tokyo, Japan) and to obtain blood samples before and after ischemia to evaluate blood gas, electrolytes and blood glucose using a blood gas analyzer (iSTATÒ). To monitor changes in the regional cerebral blood flow (rCBF) of the right hemisphere, a thin laser Doppler flowmetry probe (TBF-LN1; Unique Medical Inc., Tokyo, Japan) was placed between the right temporal muscle and the right temporal bone (Harada et al., 2005). Focal cerebral ischemia was induced with the suture occlusion technique, with some modifications (Longa et al., 1989; Chiba et al., 2008). Briefly, the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA)

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were exposed through a ventral cervical midline incision. The pterygopalatine artery and ECA were ligated with a 7-0 silk suture. The CCA and ICA were closed with a microvascular clip. The ECA was cut and a 4-0 monofilament nylon suture coated with silicone-rubber (Doccol Corporation, Sharon, MA, USA) was introduced into the ECA lumen and was gently advanced to the ICA until the laser Doppler signal showed a steep decrease. After the desired period of occlusion (30 min, 60 min, or 120 min), the rats were sacrificed under deep anesthesia with 30 mg of pentobarbital sodium administered intraperitoneally. The oxygen saturation, PaO2, PaCO2, glucose, hemoglobin, and hematocrit levels did not differ significantly between the groups before and after the MCAO (data not shown). We used the following criterion to achieve consistent ischemic injury using laser Doppler flowmetry: a more than 70% decrease of rCBF was necessary for the successful induction of ischemia. Shamoperated rats underwent the same procedure but without occlusion. For the GC–MS analysis, real-time polymerase chain reaction (RT-PCR), immunoblotting, and measurements of enzyme activity and NADPH levels, brains were extracted quickly after transcardial perfusion with 150 mL of cold saline. After the olfactory bulbs were discarded, the brains were laid in an ‘‘ad hoc” frame and cut into 2-mm-thick coronal sections. The 3 slices located between 4 and 10 mm from the front were used. Samples of 30 mg from ischemic regions of the cortex were then collected from these slices. The ischemic regions were predetermined using 2,3,5-triphenyltetrazolium chloride (TTC) staining images in a pilot study to assure that the samples were taken from the same cortical regions. The samples were immediately frozen and stored at 80 °C until use. For microarray hybridization, the removed brains were immediately immersed in ice-cold RNAlater (Thermo Fisher Scientific, MA, USA) for 1 min. Then, 200 mg of the cortex was collected. The samples were immersed in RNAlater overnight at 4 °C. The next day, the samples were transferred to a new tube and stored at 80 °C until use.

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Gas-chromatography/mass-spectrometry analysis

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Extraction of low-molecular-weight metabolites from 30 mg of the rat cortex was performed according to a previously described method, with some modifications (Nishiumi et al., 2012; Nakamizo et al., 2013). A 30-mg sample of the frozen cerebral cortex was sonicated and mixed with 1 mL of a solvent mixture (MeOH:H2O: CHCl3 = 2.5:1:1) containing 10 lL of 2.84 mmol/L (0.5 mg/mL) 2-isopropylmalic acid (Sigma–Aldrich, Tokyo, Japan) dissolved in distilled water as an internal standard. The solution was shaken at 1,200 rpm for 30 min at 37 °C, and then centrifuged at 15,000g for 3 min at 4 °C. A 1 mL aliquot of the supernatant was transferred to a new tube, and 0.5 mL of CHCl3 was added to the tube. After being mixed, the solution was centrifuged at 15,000g for 3 min at 4 °C. Then, 0.5 mL of distilled water was added and mixed in. The solution was subsequently centrifuged at 15,000g for 3 min at

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4 °C, and 0.5 mL of the resultant supernatant liquid was transferred to a clean tube and lyophilized using a freeze dryer. For oximation, 50 lL of 0.24 mol/L (20 mg/mL) methoxyamine hydrochloride (Sigma–Aldrich, Tokyo, Japan) dissolved in pyridine was mixed with a lyophilized sample, and this was followed by sonication for 20 min, and shaking at 1,200 rpm for 90 min at 30 °C. Next, 25 lL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) (GL Science, Tokyo, Japan) was added for derivatization, and the mixture was incubated at 1,200 rpm for 30 min at 37 °C. The mixture was then centrifuged at 15,000g for 5 min at 4 °C, and the resultant supernatant was subjected to GC–MS measurement. GC–MS analysis of the collected cortex samples was performed using a GCMS-QP2010 Ultra (Shimadzu Co., Kyoto, Japan) according to a previous report (Nishiumi et al., 2012; Tanaka et al., 2015). A fused silica capillary column (CP-SIL 8 CB low bleed/MS; 30 m  0.25 mm inner diameter, film thickness: 0.25 mm; Agilent Co., Palo Alto, CA, USA) was used for this analysis. The column temperature was maintained at 80 °C for 2 min and then increased at 15 °C/min to 330 °C, where it was held there for 6 min. The transfer line and ion-source temperatures were 250 °C and 200 °C, respectively. Twenty scans per second were recorded over a mass range of 85–500 m/ z using the Advanced Scanning Speed Protocol (ASSP, Shimadzu Co.). Data processing was performed as described in a previous report (Nishiumi et al., 2012). The MS data were exported in netCDF format. Peak detection and alignment were conducted using MetAlign software (Wageningen UR, The Netherlands). The resultant data were exported in CSV format and then analyzed with in-house analytical software (AIoutput) (Tsugawa et al., 2011). This software enables peak identification and semi-quantification using an in-house metabolite library. To perform the semiquantitative assessment, the peak height of each quantified ion was calculated and normalized using the peak height of 2-isopropylmalic acid as an internal standard.

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Microarray hybridization

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The ischemia rat group (120-min MCAO) and the shamoperated rat control group (n = 5 rats/group) were subjected to microarray analysis with the GeneChipÒ Rat Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). Total RNA (including small RNAs) from the rat cerebral cortex was extracted using the Ambion mirVanaTM miRNA Isolation Kit (Thermo Fisher Scientific, MA, USA). Complementary DNA (cDNA) was synthesized with the AmbionÒ WT Expression Kit (Life Technologies) according to the manufacturer’s instructions. Biotinylated cDNA was prepared according to the standard Affymetrix protocol (GeneChipÒ WT Terminal Labeling and Hybridization User Manual for use with the AmbionÒ WT Expression Kit, 2009, Affymetrix). After the fragmentation of the biotinylated cDNA, 5.5 mg of single-strand cDNA was hybridized onto the GeneChip Rat Gene 2.0 ST Array in a hybridization oven for 17 h at 45 °C. Then, the GeneChips were washed and stained in an Affymetrix Fluidics Station 450. The GeneChips were scanned

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using a GeneChipÒ Scanner 3000 7G. The probe intensities were exported as Affymetrix cel files. The cel files were imported into a personal computer, where data pre-processing was performed using free opensource software (Bioconductor 3.0 and R3.1.1; R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org). Each Affymetrix dataset was background adjusted and normalized and the log2 probe-set intensities were calculated using the Robust Multichip Averaging (RMA) algorithm in the R affy package (Gautier et al., 2004).

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Gene set enrichment analysis (GSEA)

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We utilized gene set enrichment analysis (Subramanian et al., 2005) for the microarray data to identify groups of related genes that were differentially expressed between the cerebral ischemia (rats with 120-min MCAO) and control (sham-operated rats) groups (n = 5/group) using GSEA (version 5.0, http://software.broadinstitute.org/ gsea/index.jsp), which was provided by the Broad Institute of the Massachusetts Institute of Technology (Cambridge, MA, USA). In the current analysis, gene sets represented by <15 genes or >500 genes were excluded. The t-statistic mean of the genes was computed for each Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway in the KEGG database (http:// www.genome.jp/kegg/). The ranking metric measures the correlation between a gene and a phenotype (ischemia and control). A positive value indicates a correlation with the first phenotype (ischemia), and a negative value indicates a correlation with the second phenotype (control). The enrichment score is a measure of the degree to which a gene set is over-represented at the top or bottom of the ranked list of genes in the expression dataset (ratio of ischemia/control expression values). The statistical significance of the nominal P values of the enrichment scores was assessed by permuting (1000 times) class labels (i.e., ischemia versus control) and calculating the enrichment scores for the permuted datasets that yielded a null distribution.

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Real-time polymerase chain reaction (RT-PCR)

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Quantitative real-time PCR analysis was performed on the rats in the ischemia group (30-min MCAO, 60-min MCAO, 120-min MCAO; n = 5/group) and the shamoperated control group (n = 6). Total RNA was extracted from the rat cerebral cortex using a mirVanaTM miRNA Isolation Kit (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 20 ng of total RNA using a HighCapacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR was conducted with 3 mL of diluted cDNA using TaqMan geneexpression assays (Applied Biosystems) following the manufacturers instructions. b-Actin RNA was used as an endogenous control. Quantitative mRNA expression data were acquired and analyzed via the ΔΔCt method using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). TaqMan gene-expression

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assays with FAM-MGB dye were conducted for the following genes in this study: G6PD (Rn01529640_g1), HSP27 (Rn00583001_g1), and b-actin (Rn00667869_m1).

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Immunoblotting analysis

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Immunoblotting analysis was performed on the rats in the ischemia group (60-min MCAO and 120-min MCAO; n = 4/group) and the sham-operated control rat group (n = 4). The rat cerebral cortex was homogenized using lysis buffer AM1 containing 10 mM dithiothreitol (DTT) and a phosphatase inhibitor and protease inhibitor cocktail (Active Motif). Equal amounts of protein extracts were separated via electrophoresis on 4 to 12% NuPAGE BisTris Gels (Invitrogen, Carlsbad, CA, USA), and this was followed by transfer to a nitrocellulose membrane (GE Healthcare, Milwaukee, WI, USA) using an XCell II Blot Module (Invitrogen). The membrane was blocked for 1 h in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% skim milk and then probed with various primary antibodies diluted in Can Get Signal (TOYOBO, Osaka, Japan) at 4 °C overnight. The following antibodies were used: rabbit polyclonal antibodies against G6PD (Cell Signaling Technologies, Danvers, MA, USA; #8866, 1:1000), HSP27 (Cell Signaling Technologies; #2442; 1:1000), and phosphorylated HSP27 (S85) (Abcam, Cambridge, MA, USA; ab5594; 1:4000) and a mouse monoclonal antibody against b-actin (Thermo Fisher Scientific; AM4302; 1:4000). After 3 washes of 10 min each in TBST, secondary antibodies conjugated to horseradish peroxidase (HRP) were added, and this was followed by incubation for 1 h in Can Get Signal at room temperature. The membrane was then washed 3 times for 10 min each in TBST and the immunoblots were developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

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G6PD activity

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G6PD activity was assessed in the rat cerebral cortex (60min MCAO, 120-min MCAO and control; n = 4/group) using a Glucose 6 Phosphate Dehydrogenase Assay Kit (Abcam; ab102529) essentially according to the manufacturer’s instructions. The protein concentration was determined for each sample and enzyme activity was calculated using reduced nicotinamide adenine dinucleotide (NADH) standard curve and expressed as nmol/min/mg protein.

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NADPH/NADP

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Reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ levels in the cerebral cortices of rats (60-min MCAO, 120-min MCAO and control; n = 4/group) were measured using an NADP/NADPH Assay kit (Abcam; ab65349) essentially according to the manufacturer’s instructions. The protein concentration was determined for each sample, and the values are presented as NADPH/NADP+ ng/mg protein.

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Intracerebroventricular injection of protein kinase inhibitors

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Rats were anesthetized and placed in a stereotaxic apparatus. After a small hole was drilled in the right parietal bone (coordinates: 1.0 mm posterior to bregma and 1.8 mm lateral [right] from the midline), a Hamilton syringe (Hamilton, Reno, NV, USA) was lowered 4.0 mm below the brain surface. ATM kinase inhibitor (KU-55933; 5, 25, 50, 100 mM in 10 mL; Abcam; ab120637), protein kinase D (PKD) inhibitor (CID755673; 5, 25, 50, 100 mM in 10 mL; MERCK; 476495, Germany) or vehicle (Dimethyl sulfoxide: DMSO; Sigma; D8418, St. Louis, MO, USA) was injected 60 min before MCAO. After the desired period of MCAO, the rats were sacrificed as described above.

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Statistical analysis

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All statistical analyses were performed with R. The statistical significance between two groups was determined using the Mann–Whitney U test. The statistical significance among more than two groups was determined using the Steel–Dwass test. The statistical significance between each experimental mean and the control mean was determined with the Dunnett’s test. Probability (P) values less than 0.05 were considered statistically significant. In the multivariate analysis, Pareto scaling was applied to the data processing. To determine the multivariate structure, we performed a principal component analysis (PCA). A volcano plot was also used to identify metabolites that were differentially expressed in the ischemia group compared with the control group. Differences in metabolite levels were detected using the limma package in R with the false discovery rate (FDR) (Ritchie et al., 2015). Pathway analysis of the metabolites was performed using MetaboAnalyst 3.0 (http://www.metaboanalyst.ca) (Xia et al., 2015). The pathway impact was calculated as the sum of the importance measures of the matched metabolites normalized by the sum of the importance measures of all metabolites in each pathway (Xia and Wishart, 2010).

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RESULTS

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Changes in the cerebral cortex metabolic profile after MCAO-induced ischemia

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Using the current GC–MS-based metabolomic analysis system, which mainly targets water-soluble metabolites, 92 metabolites were detected in the cerebral cortex of rats subjected to 120-min MCAO or the sham-operation (n = 10/group). In the multivariate analysis, the differences in the levels of the 92 metabolites that showed changes between 120- min MCAO and control groups were assessed using PCA. The PCA score plot showed clear separation between the 2 groups in the first component, which explained 67% of the observed variance (Fig. 1A). Heatmap representation of the hierarchical clustering also showed clear differences in metabolites between the 2 groups (Fig. 1C). The changes in the metabolite time-course were also

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GABA Alanine Ethanolamine Glycerol Citrate Uracil Glycine Hypoxanthine 3 Hydroxybutyrate Serine Leucine Fumarate Malate Threonine Phenylalanine Lysine Isoleucine Valine Alanine Sucrose Hypotaurine Succinate Ribose Coniferyl alcohol Ribitol Arabitol Galactosamine Oxalate 2 Aminoisobutyrate Asparagine Methionine Tryptophan Acetoacetic acid Citramalic acid Cytosine Xylitol Glycerol 2 phosphate Methionine sulfone Cysteine sulfonic acid N Acetyl D glucosamine 2 Hydroxybutyrate 2,3 Bisphospho glycerate Deoxyribose 5 phosphate Nonanoic acid Glycolic acid Threitol N Methylethanolamine Isobutylamine Ketovaline Tyrosine 1,5 Anhydro D glucitol Erythritol Sorbopyranose Fructose Allose Glutamate Sorbitol Pyruvate Arabinose N Acetyl L glutamate Tagatose Dimethylbenzimidazole Ribulose 5 phosphate Fructose 6 phosphate Lauric acid Adenine Creatinine Aconitate 1 Aminocyclopropane 1 carboxylate Allothreonine Homoserine 1 Hexadecanol 3 Hydroxy 3 methylglutarate Lactitol Mannitol Galactose Mannose Glutaric acid Phosphoenolpyruvic acid threo Hydroxyaspartic.acid Ascorbic acid trans 4 Hydroxy L proline Glucose Xylose Arabinose 5 phosphate Nicotinamide Pyroglutamic acid Glutamate Aspartate O Phosphoethanolamine Lactate Urea

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N Acetyl L aspartate Taurine Glutamate Pyroglutamic acid Aspartate Urea GABA Alanine Ethanolamine Glycerol Glycine Hypoxanthine 3 Hydroxybutyrate Uracil Citrate Leucine Tyrosine Lysine Glutamine Serine Fumarate Malate Lactate O Phosphoethanolamine Phenylalanine Methionine Ornithine Cysteine sulfonic acid Phenylglycine Tryptophan 2 Hydroxybutyrate Ribitol 1,5 Anhydro D glucitol Alanine Isoleucine Valine Erythritol Hypotaurine Threonine Succinate Thiouracil Adenine Aconitate Oxalate Asparagine Proline trans 4 Hydroxy L proline Ribose Acetoacetic acid Homoserine lactone Pantothenate 2,3 Bisphospho glycerate Caprylic acid Nonanoic acid Creatinine Galactitol Cytosine Fructose 6 phosphate threo Hydroxyaspartic acid Cysteine Ribulose 5 phosphate N Acetyl L glutamate Sucrose 1,3 Propanediamine Threitol Deoxyribose 5 phosphate Lactitol Rhamnose N2 Acetyl L lysine Homoserine Allothreonine Glucose Xylulose Cystathionine N Methylethanolamine Propylamine Arabitol Lyxose N Acetyl L glutamine Glucuronate Ascorbic acid Xylitol Dopamine Tagatose Xylose Pyruvate Arabinose 5 phosphate Fructose Sorbopyranose Galactosamine Sorbitol

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Fig. 1. Overview of the metabolite profile data from the cerebral cortices of rats with middle cerebral artery occlusion (MCAO) and sham-operated rats (control) obtained through GC–MS analysis. (A) Score plot of the principal component analysis (PCA) of the control (cyan circle) and 120-min MCAO groups (magenta circle) (n = 10 rats/group). Ellipses represent 95% confidence intervals. (B) PCA score plot of the control (cyan circle, n = 14), 30min MCAO (green circle, n = 6) and 120-min MCAO (magenta circle, n = 4) groups. Both score plots demonstrate clear separation among groups. (C) Heatmap representation of a 2D hierarchical clustering of metabolites identified as differentially expressed between the control and 120-min MCAO groups (n = 10/group). Each column represents a metabolite, and the leftmost column represents the treatment groups (cyan, control; magenta, 120-min MCAO). Each row represents each subject. Metabolite features whose levels vary significantly between the groups are projected on the heatmap and used for sample clustering. The row Z-score or scaled expression value of each feature is plotted with a red-green color scale. Red tiles indicate high abundance, and green tiles indicate low abundance. (D) Heatmap representation of a 2D hierarchical clustering of metabolites identified as differentially expressed among the control (n = 14), 30-min MCAO (n = 6) and 120-min MCAO (n = 4) groups. Each column represents a metabolite, and the leftmost column represents the treatment groups (cyan, control; green, 30-min MCAO; magenta, 120-min MCAO). Each row represents one rat. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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variance (Fig. 1B). The heatmap clearly separated the samples into three groups (Fig. 1D). The time course changes in the major pathways involved in the catabolism of carbohydrates revealed significant differences in key metabolites (Fig. 2).

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15

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120

0

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5

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TCA cycle

6

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Acetyl-CoA Relative value

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Citrate

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PC

0.02

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PDH

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Pyruvate

Relative value

Alanine

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30

Malate-aspartate shuttle 30

ribulose-5-P

Fructose-1,6-P2

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30

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C

LDH

Lactate

Time (min)

Time (min)

G6PD

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Relative value 120

Time (min)

C

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Pentose phosphate pathway Ribulose 5P

HK

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C

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6 4

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8

Alanine

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2.0

Pyruvate

0.002

Relative value

0.004

Fructose 6P

0.3

407

0.2

406

Relative value

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investigated between the control (n = 14), 30-min MCAO (n = 6) and 120-min MCAO (n = 4) groups through GC– MS analysis. The PCA score plot showed clear separation between the three groups of time points for the first component, which explained 67% of the

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Fig. 2. Major pathways for the catabolism of carbohydrate and temporal changes in key metabolites after middle cerebral artery occlusion (MCAO) in rat cerebral cortices obtained from GC–MS analysis. Box-and-whisker plots represent the semi-quantitative levels of the metabolites. Thick horizontal lines divide the boxes at the median values. The bottom and top of the box are the first and third quartiles. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range beyond the box. Each circle represents the semi-quantitative level of one sample (control [n = 14], blue circle; 30-min MCAO [n = 6], green circle; 120-min MCAO [n = 4], magenta circle). The Steel-Dwass test was performed for multiple comparisons testing (*, P < 0.05; **, P < 0.01; ***, P < 0.001). CS, citrate synthase; fructose-6-P, fructose 6-phosphate; fructose-1,6-P2, fructose 1,6-bisphosphate; glucose-6-P, glucose 6-phosphate; G6PD, glucose 6-phosphate dehydrogenase; GAD, glutamic acid decarboxylase; GABA-T, 4-aminobutyrate transaminase; glyceraldehyde-3P, glyceraldehyde 3-phosphate; GS, glutamine synthetase; HK, hexokinase; LDH, lactate dehydrogenase; PAG, phosphate-activated glutaminase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PFK1, phosphofructokinase-1. Please cite this article in press as: Imahori T et al. Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.036

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Gamma-aminobutyric acid (GABA), alanine, and citrate were significantly elevated by 30-min and 120-min MCAO. Conversely, glutamate, aspartate, and pyruvate were significantly decreased after 30-min and 120-min MCAO. Fructose 6-phosphate (F6P) and ribulose 5phosphate (R5P) were significantly decreased after 120min MCAO (Figs. 2 and 6A). Fumarate and malate were significantly elevated after 30-min MCAO and significantly decreased, to the control level, after 120min MCAO. Aconitate, lactate, glutamine, and succinate did not significantly change during 120-min MCAO. The differences in metabolites that contributed to clear separation between the ischemia and control groups in the score plot were identified using volcano plots, which simultaneously measured differentially accumulated metabolites based on t-statistics and fold changes. Among the 34 metabolites with adjusted P values <0.001 (Fig. 3A), the volcano plot showed 32 differentially accumulated metabolites with a simultaneous an adjusted P value < 0.001 and an absolute value of fold change >1.5 (Fig. 3B, green circle). Many of these metabolites were associated with the major pathways of carbohydrate catabolism (Fig. 2). F6P and R5P in the PPP deserve special attention because they simultaneously showed an adjusted P value < 0.001 and a fold change <2 log2––1.5 (i.e., a

7

more than 33.3% decrease from the control level) (Fig. 3) and because the product of the PPP is the electron donor NADPH, which counters the damaging effects of reactive oxygen species (ROS) (Murphy, 2009). We also noted significant increases in ethanolamine, glycerol, glycine, hypoxanthine, ketone bodies (3hydoroxybutyrate), branched-chain amino acids (isoleucine, leucine, and valine), lysine, phenylalanine, tryptophan and uracil after MCAO (Fig. 4). In addition, we performed pathway analysis of the metabolites, using MetaboAnalyst 3.0. Twenty-one significantly changed pathways were associated with cerebral ischemia with impact >0 (Table 1). In the pathway analysis, the PPP is the 6th among the top ranked pathways, which is in accordance with the results of the volcano plots.

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Changes in the transcriptional profile of the cerebral cortex after MCAO-induced ischemia

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The dataset contained 29,489 native features. After collapsing the features into gene symbols, 11,633 genes remained. The applied gene set size filters (min = 15, max = 500) filtered out 26/186 gene sets based on the KEGG pathway maps. The remaining 160 gene sets were used in the analysis. In total, 23 significantly

457

Fig. 3. (A) Metabolites showing differential accumulation between the ischemia and control groups with adjusted P value (adj. P Val) <0.001 in the GC–MS analysis. The numbers (No.) represent the ascending order of the adjusted P value and correspond to the numbers in B. abs_logFC, absolute value of the logarithm (to the base 2) of the fold change. (B) Volcano plot of metabolite profile data from the control versus 120-min middle cerebral artery occlusion (MCAO) groups (n = 10/group). The dashed horizontal line shows where the adjusted P value = 0.001. The dashed vertical line shows where the absolute value of the logarithm (to the base 2) of the fold change (FC) = 1.5. Please cite this article in press as: Imahori T et al. Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.036

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Fig. 4. Box-and-whisker plots of 12 metabolites that were significantly increased during 120-min middle cerebral artery occlusion (MCAO). Thick horizontal lines divide boxes at the median value. The bottom and top of the box are the first and third quartiles. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box limits. Each circle represents the semi-quantitative level of each sample (control [n = 14], blue circle; 30-min MCAO [n = 6], green circle; 120-min MCAO [n = 4], magenta circle). The Steel-Dwass test was performed for multiple comparisons testing (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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upregulated pathways were associated with cerebral ischemia with FDR q values <0.25 (Table 2). Based on the KEGG pathway maps in the KEGG database (http://www.genome.jp/kegg/), these 23 significant pathways primarily mapped to 6 functional

classes: metabolism (No. 5 and 23 in Table 2), genetic information processing (No. 11, 14, and 18 in Table 2), environmental information processing (No. 2 and 9 in Table 2), cellular processes (No. 12 and 17 in Table 2), organismal systems (No. 1, 4, 7, 10, 19, and 21 in

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T. Imahori et al. / Neuroscience xxx (2017) xxx–xxx Table 1. Results of pathway analysis in the cerebral cortex of rats with 120-min MCAO compared with sham-operated rats (10/group) No.

Metabolite

Total

Hits

Raw p

FDR

Impact

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Synthesis and degradation of ketone bodies Glyoxylate and dicarboxylate metabolism beta-Alanine metabolism Alanine, aspartate and glutamate metabolism Valine, leucine and isoleucine biosynthesis Pentose phosphate pathway Arginine and proline metabolism Citrate cycle (TCA cycle) Porphyrin and chlorophyll metabolism Tryptophan metabolism Butanoate metabolism Glycerolipid metabolism Cysteine and methionine metabolism Glycolysis or Gluconeogenesis Drug metabolism – other enzymes Glycerophospholipid metabolism Starch and sucrose metabolism Galactose metabolism Purine metabolism Pyruvate metabolism Valine, leucine and isoleucine degradation

5 16 19 24 11 19 44 20 27 41 20 18 28 26 30 30 23 26 68 22 38

1 3 3 4 2 2 7 2 2 1 2 1 1 2 1 1 1 1 5 1 3

0.016286 0.00089972 2.09E 07 2.11E 07 0.00063343 8.45E 05 1.62E 07 0.00095593 0.0034697 1.72E 08 2.49E 07 0.015364 2.73E 06 1.56E 11 0.0074591 0.0083765 0.11949 0.11949 0.037841 0.073562 0.00060737

0.022801 0.0018524 9.22E 07 9.22E 07 0.0013856 0.00024641 9.22E 07 0.0018588 0.0063916 3.01E 07 9.69E 07 0.022801 9.56E 06 5.46E 10 0.013053 0.013961 0.1394 0.1394 0.049053 0.091953 0.0013856

0.6 0.44445 0.44444 0.35865 0.33333 0.17654 0.17339 0.16767 0.15824 0.15684 0.13044 0.10471 0.09464 0.08958 0.05291 0.04444 0.03778 0.03644 0.03268 0.01503 0.0119

Ranking based on the impact of pathways after 120-min middle cerebral artery occlusion (MCAO) in the rat cerebral cortex. Total is the total number of compounds in the pathway; Hits is the actually matched number from the current data; Raw p is the original p value calculated from the enrichment analysis; FDR p is the p value adjusted using False Discovery Rate; the Impact is the pathway impact value calculated from pathway topology analysis.

Table 2. Overrepresented pathways in the cerebral cortex of rats with 120-min MCAO compared with sham-operated rats (5/group) identified by GSEA No.

NAME

SIZE

ES

NES

NOM p-val

FDR

FWER

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Toll-like receptor signaling pathway MAPK signaling pathway Leishmaniasis B cell receptor signaling pathway Oxidative phosphorylation Colorectal cancer Cytosolic DNA-sensing pathway Pathogenic Escherichia coli infection VEGF signaling pathway Chemokine signaling pathway Aminoacyl-tRNA biosynthesis p53 signaling pathway Huntington’s disease Protein export Epithelial cell signaling in Helicobacter pylori infection Parkinson’s disease Cell cycle Ubiquitin mediated proteolysis NOD-like receptor signaling pathway Alzheimer’s disease T cell receptor signaling pathway Amyotrophic lateral sclerosis (ALS) Amino sugar and nucleotide sugar metabolism

69 211 49 54 76 46 30 32 57 122 27 49 120 18 50 75 93 89 36 113 81 41 30

0.619 0.500 0.546 0.533 0.496 0.541 0.583 0.558 0.489 0.434 0.555 0.487 0.407 0.597 0.469 0.436 0.413 0.406 0.490 0.392 0.414 0.471 0.493

2.256 2.122 1.845 1.832 1.816 1.799 1.793 1.739 1.699 1.698 1.669 1.631 1.620 1.612 1.606 1.590 1.547 1.543 1.542 1.536 1.512 1.511 1.495

0.000 0.000 0.004 0.005 0.002 0.006 0.014 0.014 0.015 0.003 0.029 0.030 0.008 0.031 0.031 0.023 0.029 0.026 0.054 0.023 0.027 0.048 0.057

0.004 0.023 0.226 0.185 0.165 0.154 0.138 0.175 0.199 0.180 0.193 0.223 0.217 0.211 0.203 0.209 0.248 0.240 0.228 0.223 0.243 0.233 0.243

0.004 0.043 0.475 0.503 0.537 0.574 0.591 0.74 0.822 0.823 0.872 0.918 0.933 0.94 0.941 0.953 0.976 0.977 0.977 0.979 0.989 0.989 0.992

Ranking based on the normalized enrichment score (NES) of pathways after 120-min middle cerebral artery occlusion (MCAO) in the rat cerebral cortex. ES, enrichment score; NOM p-val, nominal P value; FDR, false discovery rate; FWER, familywise error rate. Pathways with an FDR < 0.25 are shown. GSEA, gene set enrichment analysis.

473 474 475 476 477 478 479

Table 2) and human diseases (3, 6, 8, 13, 15, 16, 20, and 22 in Table 2). In the organismal systems category, all 6 pathways (Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, cytosolic DNA- sensing pathway, T cell receptor signaling pathway, B cell receptor signaling pathway and chemokine signaling pathway) belonged to the immune system. In human

disease category, the 4 pathways (Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease) belonged to neurodegenerative diseases. Among the 23 differentially expressed pathways, only the Toll-like receptor signaling pathway and mitogenactivated protein kinase (MAPK) signaling pathway

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GSEA results for MAPK signaling pathway GENE SYMBOL GENE TITLE FOS v-fos FBJ murine osteosarcoma viral oncogene homolog NR4A1 nuclear receptor subfamily 4, group A, member 1 GADD45G growth arrest and DNA-damage-inducible, gamma DUSP6 dual specificity phosphatase 6 DUSP1 dual specificity phosphatase 1 JUN jun oncogene HSPB1 heat shock 27kDa protein 1 GADD45B growth arrest and DNA-damage-inducible, beta KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog RASA1 RAS p21 protein activator (GTPase activating protein) 1 FGF9 fibroblast growth factor 9 (glia-activating factor) TNFRSF1A tumor necrosis factor receptor superfamily, member 1A DUSP5 dual specificity phosphatase 5 DUSP4 dual specificity phosphatase 4 HSPA2 heat shock 70kDa protein 2 RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) MAP3K2 mitogen-activated protein kinase kinase kinase 2 JUND jun D proto-oncogene MAPKAPK2 mitogen-activated protein kinase-activated protein kinase 2 MAP2K4 mitogen-activated protein kinase kinase 4 MAPK14 mitogen-activated protein kinase 14 NFKB2 nuclear factor of kappa light polypeptide gene enhancer MAPK8 mitogen-activated protein kinase 8 CACNG3 calcium channel, voltage-dependent, gamma subunit 3 DUSP2 dual specificity phosphatase 2 CRKL v-crk sarcoma virus CT10 oncogene homolog (avian)-like FLNC filamin C, gamma (actin binding protein 280) PLA2G2A phospholipase A2, group IIA (platelets, synovial fluid) PRKCG protein kinase C, gamma TNF tumor necrosis factor (TNF superfamily, member 2) PPP3R1 protein phosphatase 3 (formerly 2B), regulatory subunit B NFKB1 nuclear factor of kappa light polypeptide gene enhancer ARRB2 arrestin, beta 2 ATF4 activating transcription factor 4 CACNG5 calcium channel, voltage-dependent, gamma subunit 5 FGF14 fibroblast growth factor 14 PLA2G4E phospholipase A2, group IVE IL1A interleukin 1, alpha MAPK1 mitogen-activated protein kinase 1 HSPA8 heat shock 70kDa protein 8 PAK1 p21/Cdc42/Rac1-activated kinase 1 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) CACNA2D1 calcium channel, voltage-dependent, alpha 2/delta subunit 1 RAP1B RAP1B, member of RAS oncogene family FGFR1 fibroblast growth factor receptor 1

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501

RANK RMS 1 0.653 3 0.562 6 0.502 14 0.346 19 0.252 25 0.229 26 0.228 37 0.196 125 0.095 175 0.083 176 0.082 221 0.076 228 0.075 250 0.071 276 0.070 309 0.067 360 0.063 370 0.062 377 0.062 426 0.060 471 0.058 579 0.053 588 0.053 619 0.052 635 0.051 753 0.048 763 0.047 776 0.047 930 0.044 939 0.043 945 0.043 961 0.043 1014 0.042 1051 0.041 1097 0.040 1151 0.039 1162 0.039 1164 0.039 1223 0.038 1241 0.038 1260 0.037 1277 0.037 1307 0.037 1319 0.036 1334 0.036

RES 0.081 0.151 0.214 0.256 0.287 0.316 0.344 0.368 0.372 0.378 0.388 0.394 0.402 0.410 0.416 0.422 0.425 0.432 0.439 0.443 0.446 0.443 0.449 0.453 0.458 0.454 0.459 0.464 0.456 0.461 0.466 0.470 0.470 0.472 0.473 0.473 0.477 0.482 0.482 0.485 0.488 0.491 0.493 0.497 0.500

et al., 1999), which is the first and rate-determining enzyme in the PPP. G6PD reduces NADP to NADPH, and NADPH is then utilized by glutathione reductase to reduce the oxidized form of glutathione (glutathione disulfide: GSSG) to the reduced form of glutathione (GSH) (Murphy, 2009), hence leading to oxidoresistance. We focused the subsequent investigation on the relationship between HSP27 and G6PD during cerebral ischemia, given the results obtained in our metabolic and transcriptional analyses and the importance of the PPP in combating ischemic and oxidative stress.

502

Temporal expression patterns of HSP27 and G6PD during MCAOinduced cerebral ischemia

518

The HSP27 gene, which was identified as differentially regulated in MCAO rats through microarray analysis, was validated via quantitative RT-PCR. The G6PD gene was also investigated. The HSP27 mRNA level did not change after 30-min MCAO, but was significantly elevated by 2.4fold after 60-min MCAO and by 4.4fold after 120-min MCAO, compared with the controls (Fig. 6B). The same trend was observed in the microarray data. Conversely, the G6PD mRNA level did not change during 120-min Fig. 5. Gene set enrichment analysis (GSEA) of microarray data from the rat cerebral cortex. The MCAO (Fig. 6B). heatmap on the left shows the top 50 features in the mitogen-activated protein kinase (MAPK) The immunoblotting analysis signaling pathway for the 120-min middle cerebral artery occlusion (MCAO) versus control groups showed no change in the G6PD and the correlation between the ranked genes and the phenotypes in the microarray data protein level and a slight increase in (n = 5/group). In the heatmap, expression values are represented as colors, and the range of colors (red, pink, light blue, and dark blue) indicates the range of expression values (high, HSP27 protein expression during moderate, low, and lowest, respectively). The table on the right shows the gene symbol, gene MCAO (Fig. 6C), although this title, RANK, rank metric score (RMS), and running enrichment score (RES) of each gene. RANK change was not significant according is the position of the gene in the ranked list of genes. RMS is the score used to position the gene to densitometric quantification in the ranked list. RES is the enrichment score for this set at this point in the ranked list of genes. (Fig. 6D). On the other hand, the phosphorylation of HSP27 at serine showed FDR q values < 0.05. Close examination of the 85 (S85) was significantly elevated significantly modulated genes in the MAPK signaling after 60-min MCAO and was maintained at a high level pathway revealed upregulation (52%) of the mRNA after 120-min MCAO compared with the controls encoding HSP27 [HSPB1], which was seventh among (Fig. 6C and D). the top-ranked genes in the MAPK signaling pathway (including 211 genes) and 27th in the total ranked list of Temporal changes in G6PD activity and the NADPH/ 11,633 genes (Fig. 5). The mRNA encoding HSP70 was + NADP ratio during MCAO-induced cerebral also significantly upregulated, but was 15th among the ischemia top ranked genes in the MAPK signaling pathway, and 277th in the total ranked list. The GC–MS analysis demonstrated that F6P and R5P HSP27 is of particular interest because it is a were significantly decreased after 120-min MCAO chaperone protein, and its primary functions are to (Fig. 6A). Continued recycling of the PPP ultimately provide cellular protection and support cell survival leads to the conversion of glucose 6-phosphate to six under stressful conditions (Arrigo, 2013). HSP27 has CO2 molecules. Therefore, the decrease in F6P and been reported to increase the activity of G6PD (Pre´ville R5P levels observed after 120-min MCAO suggested

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Fig. 6. Upregulation of the pentose phosphate pathway after middle cerebral artery occlusion (MCAO) in the rat cerebral cortex. (A) Fructose 6phosphate (Fructose-6P) and ribulose 5-phosphate (Ribulose-5P) levels were significantly decreased after 120-min MCAO in the GC–MS analysis. The Steel-Dwass test was performed for multiple comparisons testing (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) The glucose 6-phosphate dehydrogenase (G6PD) mRNA level did not change, but the level of heat shock protein 27 (HSP27) was significantly elevated after 60-min MCAO and 120-min MCAO in real-time polymerase chain reaction (RT-PCR) analysis. (C) Immunoblotting analysis of rat cerebral cortices after MCAO using the indicated antibodies. Images of G6PD, HSP27, HSP27 phosphorylated at S85 (pHSP27), and b-actin were obtained from the same gel. (D) The relative expression levels of the proteins were determined through densitometric evaluation of the immunoblots, normalized to b-actin. The pHSP27 protein level was significantly elevated after 60-min MCAO and 120-min MCAO compared with the control (sham-operated). (E) G6PD activity was not changed after 60-min MCAO, but was significantly elevated by 50% after 120-min MCAO, compared with the control. (F) The NADPH/NADP+ ratio did not change after 60-min MCAO, but was increased by almost 100% after 120-min MCAO, compared with the control. (G) NADPt (total of NADPH and NADP+) was significantly decreased after 60-min and 120-min MCAOs compared with the control. Dunnett’s test for many-to-one comparisons was performed for multiple comparisons testing with the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The columns represent the average of each group, and the bars represent the standard errors in B, D, E, F, and G.

558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577

activation of the PPP during cerebral ischemia. Additionally, the immunoblotting analysis showed a marked increase in phosphorylated HSP27 after MCAO, which has been reported to increase G6PD activity (Cosentino et al., 2011). To determine whether the increase in pHSP27 was associated with PPP activation, G6PD activity and the NADPH/NADP+ ratio in the rat cerebral cortex were measured after MCAO. G6PD activity did not change during 60-min MCAO. However, its activity was significantly increased by 50% during 120-min MCAO compared with the controls (Fig. 6E), which corresponded to the decrease of F6P and R5P. Consistently with the findings regarding G6PD activity, the NADPH/NADP+ ratio did not change during 60-min MCAO, but was increased by almost 100% during 120min MCAO, compared with the controls (Fig. 6F). Conversely, total NADPH and NADP+ (NADPt) levels were significantly decreased after 60-min and 120-min MCAO compared with the controls (Fig. 6G).

Inhibition of phosphorylation of HSP27 by ATM kinase inhibitor during MCAO-induced cerebral ischemia

578

G6PD and HSP27 have been shown to interact following ATM-dependent phosphorylation of HSP27 (Cosentino et al., 2011). PKD also has been shown to be associated with HSP27 phosphorylation (Stetler et al., 2012). To address which kinase phosphorylates HSP27 during the early stage of cerebral ischemia, we injected these inhibitors into the cerebral ventricle. Intracerebroventricular injection of ATM kinase inhibitor (KU-55933; 0, 5, 25, 50, 100 mM) 60-min before MCAO reduced phosphorylation of HSP27 after 60-min MCAO in a dose-dependent manner without any significant changes of HSP27 and G6PD (Fig. 7A left). However, intracerebroventricular injection of PKD inhibitor (CID755673; 0, 5, 25, 50, 100 mM) did not affect the phosphorylation of HSP27 (Fig. 7A right). To determine the effect of the inhibition of HSP27 phosphorylation on the enhancement of PPP during

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the PPP using a volcano plot because these metabolites were significantly decreased in the MCAO group compared with the control group, and the PPP is biologically important in oxidoresistance. In addition, the pathway analysis also demonstrated that the PPP was significantly changed after MCAO. The decreases of R6P and R5P under conditions where MCAO disrupts the supply of glucose and oxygen suggests activation of the PPP. The PPP generates NADPH, which is a cofactor for glutathione reductase. Glutathione Fig. 7. (A) ATM kinase is the major kinase responsible for phosphorylation of HSP27 during the early stage of cerebral ischemia by middle cerebral artery occlusion (MCAO). A panel of reductase regenerates reduced kinase inhibitors was applied to the rat brain 60 min before induction of MCAO, including the glutathione (GSH), which acts together ATM kinase inhibitor KU-55933 (0, 5, 25, 50, 100 mM) and the PKD inhibitor CID755673 (0, 5, with glutathione peroxidase in eliminating 25, 50, 100 mM) by intracerebroventricular injection. KU-55933 reduced HSP27 phosphoryROS (Murphy, 2009). Therefore, it is lation at Ser85 after 60-min MCAO. CID755673 did not affect HSP27 phosphorylation. (B) The intracerebroventricular injection of 50 or 100 mM of KU-55933 blocked the increase of highly likely that promotion of the PPP G6PD activity after 120-min MCAO. Dunnett’s test for many-to-one comparisons was forms part of the defense system against performed for multiple comparisons testing with the control (*, P < 0.05). The columns oxidative stress in the brain. represent the average of each group, and the bars represent the standard errors. C, control Accumulation of hypoxanthine, GABA, (only vehicle injection followed by sham-operation); M2, only vehicle injection followed by and 3-hydroxybutyrate has been reported 120-min MCAO; 50, 50 mM of KU-55933 injection followed by 120-min MCAO; 100, 100 mM of KU-55933 followed by 120-min MCAO. to occur during ischemia (Ha˚berg et al., 2001; Guzma´n and Bla´zquez, 2004; Abramov et al., 2007), and was also cerebral ischemia, G6PD activity in the rat cerebral cortex observed in the present study. However, these findings was also measured after MCAO under are beyond the scope of the current work. Future studies intracerebroventricular injection of ATM kinase inhibitor will focus on these metabolites. (KU-55933; 0, 50, 100 mM). The increase of G6PD activity during 120-min MCAO was almost completely blocked by intracerebroventricular injection of 50 or Transcriptional profiling suggests the upregulation 100 mM of KU-55933 (Fig. 7B). of various pathways, including the immune system and HSP27, during the early stages of cerebral DISCUSSION ischemia

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Through a combination of metabolic and transcriptional profiling, we performed a comprehensive assessment of the effects of cerebral ischemia on cerebral cortex metabolism in a rat MCAO model.

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Metabolic profiling suggests PPP activation during the early stages of cerebral ischemia In the GC–MS-based metabolomics analysis performed in the current study, the PCA score plot of the variation in rat cerebral cortex metabolites showed distinct clustering or clear separation of the control and 120-min MCAO groups. Furthermore, the metabolic variations in the 3 groups were clearly separated over the time course (control versus 30-min MCAO versus 120-min MCAO). Both score plots showed 67% of the total variance in the first component, indicating that the data for these metabolites in the rat cerebral cortex through the different time points of ischemia are interpretable. The heatmap representation of hierarchical clustering also clearly separated the samples into these groups. These findings indicate time-dependent changes in metabolic states in response to ischemic injury, even in ultra-early stages. Among the 34 metabolites exhibiting significant differences during MCAO, we highlight R6P and R5P in

The microarray analysis conducted in the current study showed that MCAO induced differential expression of a few dozen genes. The bioinformatics analysis indicated that a considerable portion of these genes are involved in the immune system, in line with the results of previous microarray studies on cerebral ischemicreperfusion injury (Feng et al., 2007; Chen et al., 2011b; Ramos-Cejudo et al., 2012; Cox-Limpens et al., 2014). Recent studies have indicated a complex role for the immune system in the pathophysiological changes that occur after an acute stroke (Chamorro et al., 2012). Sensors of the innate immune system, such as Toll- like receptors (TLRs), are activated by cerebral ischemia and lead to exacerbation of the inflammatory response. Inhibition of Toll-like receptors has been proposed to contribute to ischemic preconditioning (Feng et al., 2007); thus, suppression of TLR2 signaling may be a valuable approach to minimizing postischemic inflammation (Abe et al., 2010). Activation of the adaptive immune system is mediated by T and B cells in response to stroke and can lead to deleterious antigen-specific autoreactive responses but can also exert cytoprotective effects (Chamorro et al., 2012). The adaptive immune system functions in the delayed phase of ischemia (Iadecola and Anrather, 2011) and is therefore beyond the scope of the current study.

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In line with the results of previous studies on cerebral ischemic-reperfusion injury (Feng et al., 2007; Chen et al., 2011b; Cox-Limpens et al., 2014), the current study demonstrated that cerebral ischemia caused by 120-min MCAO without reperfusion induced upregulation of MAPK signaling pathway expression. MAPK signaling pathways have been implicated in the transduction of a variety of external signals leading to various cellular processes, and activated MAPKs primarily function as mediators of cellular stress by phosphorylating intracellular enzymes, transcription factors, and cytosolic proteins involved in cell survival, production of inflammatory mediators, and apoptosis (Cargnello and Roux, 2011; Kyriakis and Avruch, 2012). Increasing evidence has implicated the MAPK signaling pathways play vital roles in the inflammatory and apoptotic processes of cerebral ischemia and reperfusion injury (Jiang et al., 2014). During ischemic injury, the antioxidant response induces heat shock proteins and chaperones to inhibit proapoptotic signaling pathways (Chen et al., 2011b). HSP27 and HSP70 were significantly upregulated after 120-min MCAO in our analysis of MAPK signaling pathways, in line with the results of previous studies (Raghavendra Rao et al., 2002; Lu et al., 2003; Dhodda et al., 2004; Chen et al., 2011b; Ramos-Cejudo et al., 2012; Chelluboina et al., 2014). Most of the previous studies on this topic have consisted of investigations into ischemia/reperfusion injury and have relied on ischemia experiments lasting for 1–2 h, followed by reperfusion of 2 h to 7 days, with or without preconditioning. The current microarray study clearly demonstrated that even 120-min MCAO without reperfusion induced upregulation of HSP27 and HSP70 gene expression, which was confirmed via RT-PCR. HSP27 has been reported to form a complex with the first enzyme (G6PD) in the oxidative branch of the PPP. This interaction activates G6PD and increases its activity thereby supporting an elevated PPP flux (Cosentino et al., 2011). G6PD is a rate-limiting enzyme in the PPP that can regulate the production of NADPH through the PPP. Thus, G6PD is crucial for maintaining the NADPH concentration, which provides the redox power for antioxidant systems (Pollak et al., 2007). Given the results of the metabolic approach that suggested PPP activation, we focused our investigation on the relationship between HSP27 and G6PD during MCAO. In ischemic cerebral tissue, PPP activity appears to increase through orchestrated allosteric/post- translational and transcriptional regulation, although these pathways do not necessarily act at the same time (Stincone et al., 2014). Ultimately we used omics analysis as a discovery tool to build new biological hypotheses, which must be independently verified through other methods, such as RT-PCR, immunoblotting, and enzyme activity measurements. Cerebral ischemia may induce G6PD activation via HSP27 phosphorylation by ATM kinase We confirmed the upregulation of HSP27 observed in the microarray analysis via RT-PCR, which showed an elevation of the HSP27 mRNA level during 60-min

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MCAO and a further increase during 120-min MCAO. The HSP27 protein level was slightly increased, but this change did not reach statistical significance. These results are in line with those from previous ischemia/ reperfusion studies with or without preconditioning (Currie et al., 2000; Dhodda et al., 2004; Chelluboina et al., 2014). However, the pHSP27 level was significantly increased after 60-min and 120-min MCAO. The elevation of HSP27 phosphorylation during the early stages of cerebral ischemia has not been well studied previously. In tumor tissues, pHSP27 has been reported to serve as an indicator of ischemic changes (Zahari et al., 2015). Phosphorylation of HSP27 is critical for its oligomerization and interaction with specific protein targets, such as cytochrome c, to improve survival (Arrigo 2013; Arrigo and Gibert, 2014). Indeed, G6PD is known to exhibit increased activity when it interacts with highly phosphorylated small oligomers of HSP27 following ATM-dependent phosphorylation of HSP27 (Cosentino et al., 2011). Phosphorylation of HSP27 at serine 15 and serine 82 by protein kinase D (PKD) has also been shown to be necessary for HSP27-induced neuroprotection against ischemic neuronal injury in mouse ischemia/reperfusion models (Stetler et al., 2012). Phosphorylated HSP27 binds to apoptosis signal-regulating kinase 1 (ASK1) and prevents ASK1 signaling via mitogen-activated protein kinase kinase (MKK) 4/7 to c-Jun NH(2)-terminal kinase (JNK) and c-Jun, which would otherwise result in apoptosis (Stetler et al., 2012). S82 in humans corresponds to S85 in rats, which is the specific site that we identified as phosphorylated during the early stages of cerebral ischemia, even without reperfusion. It is possible that this phosphorylation increases the affinity of HSP27 for G6PD, which in turn increases G6PD activity (Cosentino et al., 2011). The current results clearly demonstrated that ATM kinase is the major kinase responsible for phosphorylation of HSP27 during the early stage of cerebral ischemia by MCAO. G6PD mRNA and protein levels did not change during 120-min MCAO, in line with results from previous studies (Li et al., 2014). Because metabolic pathways appear to be primarily regulated by post-translational mechanisms (Stincone et al., 2014), the available information concerning mRNA and protein levels is limited, thus hindering accurate assessment of changes in PPP activity and their potential causal importance for ischemic biology. Therefore, it is necessary to determine these values in concert with enzyme activity and concentrations of metabolites and cofactors (e.g., NADPH). The current study demonstrated that G6PD activity was significantly elevated by 50% after 120-min MCAO. In agreement with the changes in G6PD activity, the NADPH/NADP+ ratio was increased during cerebral ischemia. In contrast, total NADPH and NADP+ (NADPt) levels were significantly decreased after ischemia without reperfusion. The decrease in NADPt after ischemia suggested a decrease in NAD kinase activity or depletion of NAD or NADP pools (Pollak et al., 2007). Nevertheless, ischemia did not reduce the ability of the brain tissue to regenerate NADPH, which is required for defense against oxidative stress, as shown by the NADPH/NADP+ ratio in the current study. Further-

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more, the intracerebroventricular injection of ATM kinase inhibitor blocked the increase of G6PD activity during 120min MCAO. Together, these results suggest that ischemia induces phosphorylation of HSP27 by ATM kinase, even without reperfusion. In turn, this phosphorylation activates G6PD, stimulating the PPP to produce more NADPH, and, thus, decrease ROS (Fig. 8). ROS are generated during cerebral ischemia, and oxidative stress plays an important role in brain damage after stroke. Reperfusion injury aggravates ischemic brain damage primarily by markedly increasing ROS generation (Chen et al., 2011a; Zhao et al., 2012; Li et al., 2014). This ROS production is primarily in the form of superoxide, which is generated via the incomplete one electron reduction of oxygen at mitochondrial complexes I and III and is highly reactive. Superoxide production leads to the formation of H2O2 through superoxide dismutasecatalyzed dismutation (Murphy, 2009). H2O2 is converted to H2O by reduced glutathione (GSH) and glutathione peroxidase; then, oxidized glutathione (GSSG) is converted back to the reduced form (GSH) by glutathione reductase, which receives reducing equivalents from the NADPH pool. Accordingly, the antioxidant system depends on the production of NADPH to function properly (Stanton, 2012). Although NADPH is primarily produced by four enzymes in mammalian cells (G6PD, 6phosphogluconate dehydrogenase, malic enzyme, and isocitrate dehydrogenase), G6PD is the main supplier of NADPH (Stanton, 2012). Thus, to confer protection against ROS, it is reasonable that PPP activation be initiated before a burst of ROS upon reperfusion. Furthermore, a recent study has reported that administration of exogenous NADPH decreases ROS levels and significantly protects neurons against ischemia/reperfusioninduced injury (Li et al., 2016). Consequently, the activa-

+

Ischemia

Glycolysis

P HSP27

+

ATMK

HSP27

Glucose

+

Oxidative PPP

G6PD G6P

6-P-gluconolactone

6-P-gluconate +

NADP NADP+ NADPH

NADPH F6P

tion of G6PD via phosphorylation of HSP27 by ATM kinase may be part of an endogenous antioxidant defense mechanism in the earliest stages of ischemia. There are some limitations to the current study. First, the study setting was based on comparison of MCAO (30-, 60- and 120-min) and control groups. No follow-up experiments were performed in order to gain mechanistic insight into the observed differences. We now plan to perform the follow-up experiments including reperfusion. Second, the potential mechanism of increased NADPH/NAD+ ratio is that G6PD is activated via HSP27 phsophorylation, leading to decreased ROS production, which could be considered neuroprotective mechanism. However, decreased ROS production itself was not demonstrated in the present study. The mechanism behind the observed changes remains to be truly worked out.

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CONCLUSIONS

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The current data demonstrate the usefulness of omics approaches for the screening of potential targets of ischemia-related genes and metabolic pathways. The combination of metabolic and transcriptional approaches helped to focus the study on the PPP and contributed to the detection of G6PD activation via HSP27 phosphorylation by ATM kinase during the earliest stage of cerebral ischemia without reperfusion, resulting in an increase in the NADPH/NAD+ ratio. This process may represent an additional endogenous neuroprotection system against cerebral ischemia. These findings indicate the neuroprotective properties of HSP27 and its phosphorylation at serine 85 by ATM kinase and may have important therapeutic implications for the treatment of stroke.

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DISCLOSURE/CONFLICT OF INTEREST

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The authors declare no conflict of interest.

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Acknowledgments—We express our gratitude to Yukiko Takeuchi (Division of Evidenced-Based Laboratory Medicine, Kobe University Graduate School of Medicine) for helping with the GC-MS analysis. Hosoda K. is supported in part by a Grant-inAid for Scientific Research (C) KAKENHI Number 15K10302 from the Japan Society for the Promotion of Science and a medical research grant from the SENSHIN Medical Research Foundation. Sasayama T. and Kohmura E. are also supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) (25462258 and 25293309, respectively).

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R5P Non-oxidative PPP

F1,6P2

Fig. 8. Hypothetical model of how heat shock protein 27 (HSP27) may activate the pentose phosphate pathway (PPP) during ischemia. Ischemia induces HSP27 phosphorylation at serine 85 by ATM kinase. Phosphorylated HSP27 interacts with and activates glucose 6- phosphate dehydrogenase (G6PD), thereby stimulating the PPP to produce more NADPH. ATMK, ataxia telangiectasia mutated kinase; 6-P-gluconolactone, 6-phosphogluconolactone; 6-P-gluconate, 6phosphogluconate; F6P, fructose 6-phosphate; G6P, glucose 6phosphate; F1,6P2, fructose 1,6-bisphosphate; PKD, protein kinase D; R5P, ribulose 5-phosphate.

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(Received 5 November 2016, Accepted 17 February 2017) (Available online xxxx)

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