reperfusion through the facilitation of energy utilization

Neurochemistry International 133 (2020) 104631

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Phosphoglycerate mutase 1 reduces neuronal damage in the hippocampus following ischemia/reperfusion through the facilitation of energy utilization

T

Woosuk Kima,b,1, Hyun Jung Kwonc,1, Hyo Young Jungb, Dae Young Yood, Dae Won Kimc,∗∗, In Koo Hwangb,∗ a

Department of Biomedical Sciences, Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon, 24252, South Korea Department of Anatomy and Cell Biology, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, South Korea c Department of Biochemistry and Molecular Biology, Research Institute of Oral Sciences, College of Dentistry, Gangneung-Wonju National University, Gangneung, 25457, South Korea d Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan, 31151, South Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Phosphoglycerate mutase 1 Oxidative stress Ischemia Gerbil Energy production

In a previous study, we observed the effect of phosphoglycerate mutase 1 (PGAM1) on proliferating cells and neuroblasts in the subgranular zone of mouse dentate gyrus. In the present study, we examined the roles of PGAM1 in the HT22 hippocampal cell line and in gerbil hippocampus after H2O2-induced oxidative stress and after ischemia/reperfusion, respectively. Control-PGAM1 and Tat-PGAM1 proteins were synthesized using Tat-1 expression vector since Tat-1 fusion proteins can easily cross the blood-brain barrier and cell membranes. We found that transduction of Tat-PGAM1 protein into HT22 cells was dose- and time-dependent. Delivery of the protein to the cytoplasm was confirmed by western blotting and immunocytochemistry. Treatment of HT22 cells with Tat-PGAM1 protein showed a concentration-dependent reduction in cell damage and decreased formation of reactive oxygen species after H2O2 exposure. Tat-PGAM1 administration significantly ameliorated the ischemia-induced hyperactivity in gerbils at 1 day after ischemia/reperfusion. Additionally, a pronounced decrease in neuronal damage and reactive gliosis were observed in the hippocampal CA1 region of the Tat-PGAM1treated group at 4 days after ischemia/reperfusion compared to that in the vehicle (Tat peptide) or controlPGAM1-treated groups. Administration of Tat-PGAM1 mitigated the changes in ATP content, succinate dehydrogenase activity, pH, and 4-hydroxynonenal levels in the hippocampus at 4 and 7 days after ischemia/reperfusion compared to that in the vehicle-treated group. In addition, administration of Tat-PGAM1 significantly ameliorated the ischemia-induced increases of lactate levels in the hippocampus at 15 min and 6 h after ischemia/reperfusion than in the vehicle or control-PGAM1-treated groups. These results suggest that TatPGAM1 can be used as a therapeutic agent to prevent neuronal damage from oxidative stress or ischemia.

1. Introduction Transient forebrain ischemia, caused by interruption of blood vessels to the brain, rapidly decreases the neuronal ATP concentration and subsequently impairs ion gradients across the neuronal membrane (Hayashi and Abe, 2004). The transmembrane distribution of Na+, K+, Cl−, and Ca2+ ions is tightly regulated in neurons, and the loss of maintenance is considered to be a critical event in the pathogenesis of brain ischemia (Hansen, 1985). During ischemia, extracellular K+ levels are biphasically increased while the extracellular Na+, Cl−, and

Ca2+ concentrations are substantially decreased (Hansen and Zeuthen, 1981; Harris et al., 1981; Silver and Erecinska, 1990). During reperfusion, the formation of reactive oxygen species (ROS) facilitates the neuronal entry of Na+ and Ca2+ (Kristián and Siesjö, 1996; Siesjö, 1992; Young, 1986). Finally, the impairment of ion gradients and overload of Ca2+ causes neuronal damage after ischemia/reperfusion. Hence, the depletion of ATP is one of the critical events in the process of cell damage after ischemia/reperfusion and amelioration of ATP decline and facilitation of ATP recovery can be a strategy to protect the neurons from ischemic damage.

∗

Corresponding author. Department of Anatomy, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea. Corresponding author. Department of Biochemistry and Molecular Biology, College of Dentistry, Gangneung-Wonju National University, Gangneung, 25457, South Korea. E-mail addresses: [email protected] (D.W. Kim), [email protected] (I.K. Hwang). 1 Woosuk Kim and Hyun Jung Kwon are contributed equally to this article. ∗∗

https://doi.org/10.1016/j.neuint.2019.104631 Received 12 July 2019; Received in revised form 15 November 2019; Accepted 8 December 2019 Available online 10 December 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved.

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1 h. For time optimization, HT22 cells were incubated with 3 μM protein for various periods (15–60 min). Cells were harvested and the delivery of the proteins was confirmed by Western blot analysis using anti-polyhistidine antibody as described previously (Jung et al., 2019).

The adult brain utilizes 10–12% of glucose as a source of energy (Powers et al., 2007; Vaishnavi et al., 2010). Glucose is metabolized by oxidative phosphorylation to generate large amounts of ATP, which is utilized in the maintenance of ion gradients across the membrane and in synaptic transmission (Raichle and Mintun, 2006). During glycolysis, glucose is metabolized into phosphoenolpyruvate by two subsequent enzymatic processes through the action of phosphoglycerate mutase (PGAM, EC 5.4.2.11) and α-enolase (EC 4.2.1.11) (Durany et al., 1997; Pancholi, 2001). Two PGAM genes have been identified in the human genome; PGAM1 is expressed in the liver, kidney, and brain (Betrán et al., 2002; Gao et al., 2013) and PGAM2 is expressed abundantly in the muscles (Zhang et al., 2001). PGAM1 expression is significantly increased in the cancer tissue and in human astrocytoma (Liu et al., 2018) and PGAM1 knockdown significantly reduces the mass and size of glioblastoma (Sanzey et al., 2015). Development of PGAM1 inhibitors is being considered as one of the strategies for cancer treatment (Huang et al., 2019; Li et al., 2017). Our previous proteomic study showed that hippocampal neurogenesis is enhanced by pyridoxine treatment via the upregulation of PGAM1 expression in the hippocampus (Jung et al., 2017). In addition, treatment with PGAM1 has positive effects on hippocampal functions such as novel object recognition and hippocampal neurogenesis (Jung et al., 2019). PGAM1 levels are lower in neuronal cultures derived from a senescence-accelerated mouse (SAMP8, an animal model of aging) than in controls (Díez-Vives et al., 2009). It has been shown that Alzheimer's disease oxidatively modifies the PGAM1 protein (Bigl et al., 1999; Butterfield et al., 2007; Sultana et al., 2006) and its levels are significantly decreased in a brain affected by Alzheimer's disease (Iwangoff et al., 1980; Meier-Ruge et al., 1984; Sultana et al., 2006). Although several studies have shown that PGAM1 level and activity are increased in the brain after hypoxia and ischemia (Datta et al., 2011; González-Cinca et al., 2003; Takahashi et al., 1998), the levels of glycolytic enzymes are decreased in the gerbil brain after ischemia/reperfusion (Djuricic et al., 1983; Tomimoto et al., 1993). It has been proposed that the high levels of PGAM1 offers a potential target to ameliorate the neuronal damage seen in ischemia. However, till date no studies have been conducted to elucidate the effects of PGAM1 against oxidative stress or ischemic damage. Therefore, in the present study, we examined the roles of PGAM1 in H2O2-induced oxidative damage in HT22 hippocampal cell lines and in transient forebrain ischemia in gerbils.

2.1.3. Visualization of the transduced Tat-PGAM1 protein in HT22 cells To visualize the delivery of Tat-PGAM1 and control-PGAM1 into HT22 cells, immunocytochemical staining was performed using antipolyhistidine antibody as described previously (Yoo et al., 2019). Briefly, HT22 cells were grown on coverslips and incubated with 3 μM Tat-PGAM1 or control-PGAM1 protein for 1 h. Thereafter, cells were subsequently incubated with rabbit anti-polyhistidine antibody (SantaCruz Biotechnology) followed by Alexa Fluor® 488-conjugated antirabbit IgG (1:1000; Jackson ImmunoResearch, PA, USA). Nuclei were counterstained with 1 μg/mL DAPI (Roche Applied Science, Mannheim, Germany) and the cells were observed under a confocal fluorescence microscope (LSM 510 META NLO; Zeiss GmbH, Jena, Germany). 2.2. Effect of Tat-PGAM1 on oxidative stress in HT22 cells 2.2.1. Cell death and DNA damage To assess the effects of Tat-PGAM1 and control-PGAM1 on oxidative stress-induced cells death and DNA damage, WST-1 and terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) assay were conducted as previously described (Yoo et al., 2019). Briefly, HT22 cells were incubated with different concentrations (0.25–1 μM) of Tat peptide, control-PGAM1 protein or TatPGAM1 protein for 1 h and thereafter the cells were exposed to 1 mM H2O2 for 5 h. Cell viability was evaluated by measuring the optical density at 450 nm using an ELISA microplate reader (Labsystems Multiskan MCC/340, Helsinki, Finland). For the TUNEL assay, cells were incubated with 1 μM Tat peptide, control-PGAM1 protein or TatPGAM1 protein for 1 h and then treated with 1 mM H2O2 for 3 h. Images were taken using a fluorescence microscope (Nikon Eclipse 80i, Tokyo, Japan) and fluorescence was measured using a Fluoroskan ELISA plate reader (Labsystems Oy) at 485 nm excitation and 538 nm emission. 2.2.2. ROS levels Intracellular ROS was assessed by the conversion of 2′,7′-dichlorofluorescein diacetate (DCF-DA) to DCF in cells as described previously (Yoo et al., 2019). Briefly, HT22 cells were incubated with 1 μM Tat peptide, control-PGAM1 protein or Tat-PGAM1 protein for 1 h and then treated with 1 mM H2O2 for 10 min. Afterwards, cells were incubated with 20 μM DCF-DA for 30 min, and the fluorescence levels were measured using a Fluoroskan ELISA plate reader (Labsystems Oy, Helsinki, Finland) at 485 nm excitation and 538 nm emission.

2. Materials and methods 2.1. Delivery of Tat-PGAM1 into HT22 cells 2.1.1. Purification of control-PGAM1 and Tat-PGAM1 protein Tat-PGAM1 was generated by cloning human PGAM1 cDNA in a TA vector and then sub-cloning in a Tat-1 expression vector containing polyhistidine-tag as described previously (Jung et al., 2019). ControlPGAM1 expression vector was constructed without Tat-1. Escherichia coli BL21 cells were transformed with either Tat-PGAM1 or controlPGAM1 plasmids and cultivated in broth media. Tat-PGAM1 and control-PGAM1 proteins were purified using Ni2+-nitrilotriacetic acid Sepharose affinity column (Qiagen, Inc.) and PD-10 column chromatography (GE Healthcare, Chicago, IL, USA). Purified Tat-PGAM1 and control-PGAM1 proteins were confirmed on Western blot using anti-polyhistidine antibody (1:2000, His-probe, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously (Jung et al., 2019). Bands were visualized using chemiluminescent reagents according to the manufacturer's recommendation (Amersham, Franklin Lakes, NJ, USA).

2.3. Effect of Tat-PGAM1 on ischemic damage in gerbils 2.3.1. Experimental animals Mongolian gerbils (male, 3-month old) were obtained from Japan SLC Inc. (Shizuoka, Japan) and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-160613-18). All animals were carefully handled to minimize the physiological stress caused by the procedures employed in the study. 2.3.2. Induction of transient forebrain ischemia and treatment with TatPGAM1 Ischemic surgery in gerbils was conducted as described previously by Yoo et al. (2019). Briefly, animals were anesthetized with 2.5% isoflurane (Baxtor, Deerfield, IL, USA) and a midline incision was made in the neck region. Both common carotid arteries were occluded with aneurysm clips for 5 min and reperfusion was confirmed using a stereoscope. During anesthesia, the body temperature of the animals was

2.1.2. Efficient delivery of Tat-PGAM1 protein into HT22 cells For dose optimization, HT22 cells were treated with different concentrations (0.5–5 μM) of Tat-PGAM1 or control-PGAM1 protein for 2

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mid-point of the hippocampal CA1 region using OPTIMAS software (version 6.5; CyberMetrics® Corporation, Phoenix, AZ, USA). The mean number of NeuN-immunoreactive neurons was obtained from five sections at 90 μm intervals and the data are presented as percent values compared to the control group (which was set as 100%).

maintained at 37 ± 0.5 °C (normothermic condition) using a thermometric blanket. The central artery in the retina was observed to confirm complete interruption of blood flow using an ophthalmoscope (HEINE K180®, Heine Optotechnik, Herrsching, Germany) and three animals were ruled out because of incomplete interruption and abnormal body temperature. Tat peptide (2 mg/kg), control-PGAM1 protein, or Tat-PGAM1 protein (10 mg/kg) was intraperitoneally administered to gerbils immediately after the onset of reperfusion. Based on our previous observation, the dose that showed a significant increase in neurogenesis was chosen (Jung et al., 2019).

2.4.2. Immunodensity of GFAP and Iba-1 immunoreactive structures For analysis of GFAP and Iba-1 immunoreactivity, five sections, 90 μm apart from each other, were obtained between 1.4 and 2.0 mm caudal to the bregma with reference to a gerbil atlas (Loskota et al., 1974). GFAP and Iba-1 immunoreactive structures in the hippocampal CA1 region were quantified under × 100 magnification as immunodensity (256 grayscale) with pixel number using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) as described previously (Jung et al., 2019; Yoo et al., 2019). Data are presented as percent values compared to the control group (set as 100%).

2.3.3. Spontaneous motor activity Transient forebrain ischemia causes neuronal death in the hippocampal CA1 region 4 days after ischemia. However, the ischemic gerbils exhibited a significant increase in activity (days 1 and 2 after ischemia) (Babcock et al., 1993). To observe the effects of Tat-PGAM1 and control-PGAM1 on ischemia-induced hyperactivity, spontaneous motor activity was measured in the gerbils for 60 min one day before and after the ischemic surgery, as described by Yoo et al. (2019). Distance traveled was measured during live observations and reanalyzed with video sequences by two independent observers to ensure objectivity.

2.4.3. Statistical analysis Data are presented as mean with the standard error of means (mean ± S.E.M). Differences of means were compared and statistically analyzed by a one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA).

2.3.4. Neuronal damage and reactive gliosis Neuroprotective effects of Tat-PGAM1 and control-PGAM1 were assessed by immunohistochemical staining for neuronal nuclei (NeuN). In addition, glial fibrillary acidic protein (GFAP), and ionized calciumbinding adapter molecule 1 (Iba-1) immunohistochemical staining was performed to observe the reactive gliosis of astrocytes and microglia as described previously (Yoo et al., 2019). Briefly, animals (n = 5 per group) used in the spontaneous motor activity test were sacrificed under anesthesia [1 g/kg body weight urethane (Sigma-Aldrich, St. Louis, MO, USA)] at 4 days after ischemia and subsequent transcardiac perfusion was performed. Brain serial coronal sections (30 μm thick) were obtained between 1.4 and 2.0 mm caudal to the bregma with reference to a gerbil atlas (Loskota et al., 1974). Five sections, 90 μm apart from each other, were obtained from each animal and incubated with mouse anti-NeuN antibody (1:1000; EMD Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; EMD Millipore), and rabbit anti-Iba-1 antibody (1:500; Wako, Osaka, Japan) for 48 h at 4 °C. Thereafter, the sections were sequentially treated with biotinylated goat anti-mouse IgG or anti-rabbit IgG and a streptavidin-peroxidase complex (1:200; Vector, Burlingame, CA, USA) for 2 h at 25 °C and visualized using 3,3′-diaminobenzidine tetrachloride (Sigma).

3. Results 3.1. Construction of Tat-PGAM1 fusion protein The successful construction of Tat-PGAM1 and control-PGAM1 proteins was confirmed by western blotting using anti-polyhistidine antibody. Coomassie brilliant blue staining demonstrated clear bands, which were identified as control-PGAM1 and Tat-PGAM1 approximately at 28 kDa and 30 kDa, respectively (Fig. 1A). 3.2. Confirmation of Tat-PGAM1 delivery into HT22 cells Delivery of Tat-PGAM1 protein was confirmed by Western blot analysis using anti-polyhistidine antibody. Polyhistidine immunoreactivity was not observed in control-PGAM1-treated group. In contrast, polyhistidine expression was increased in a concentrationdependent manner in Tat-PGAM1 treated cells (Fig. 1B). In addition, treatment with 3 μM control-PGAM1 did not show any polyhistidine expression at various time points of harvest in HT22 cells. In contrast, 3 μM Tat-PGAM1 treatment increased the expression of polyhistidine with time, from 30 min to 60 min after the treatment (Fig. 1C). Intracellular delivery of control-PGAM1 and Tat-PGAM1 was also confirmed by immunocytochemistry for polyhistidine at 1 h after the protein treatment. In the control (Tat-peptide) and control-PGAM1treated groups, no polyhistidine immunoreactivity was detected in the HT22 cells, while it was observed in Tat-PGAM1-treated cells. In the Tat-PGAM1-treated group, polyhistidine immunoreactivity was observed in the cytoplasm and in neurites (Fig. 1D).

2.3.5. SDH activity, pH, ATP, lactate, and HNE levels To elucidate the effect of Tat-PGAM1 and control-PGAM1 on ischemic damage in gerbils, succinate dehydrogenase (SDH) activity, pH, ATP, lactate concentration, and 4-hydroxynonenal (HNE) levels were measured in the hippocampal homogenates at 15 min, 6 h, 2, 4, and 7 days after ischemia. Briefly, animals (n = 6 per group) were sacrificed with an overdose of urethane and the hippocampi were dissected. The tissue samples were homogenized in the assay buffer and centrifuged at 10000 g. SDH activity colorimetric assay kit (BioVision, Milpitas, CA, USA), fluorometric intracellular pH assay kit (Merck, Darmstadt, Germany), ATP determination kit (Molecular Probes, USA), lactate colorimetric assay kit (Abcam, Cambridge, UK), and Bioxytech HAE586 spectrophotometric assay kit (OxisResearch, Portland, OR, USA) were used for measurement of SDH activity, pH, ATP concentration, and HNE levels by a spectrophotometric method (Schimadzu UV 1601 spectrophotometer), respectively, according to the manufacturer's protocol.

3.3. Role of Tat-PGAM1 in oxidative stress in HT22 cells Cell viability was assessed by WST-1 assay after subsequent treatment with vehicle, control-PGAM1, or Tat-PGAM1 for 1 h and 1 mM H2O2 for 5 h. In the vehicle-treated and H2O2-exposed groups, cell viability was significantly decreased to 62.6% of the control group. In the control-PGAM1-treated and H2O2-exposed groups, cell viability did not show significant changes at different concentrations of the protein and was similar to that seen in the vehicle-treated and H2O2-exposed groups. In the Tat-PGAM1-treated and H2O2 exposed groups, cell viability was increased in a concentration-dependent manner, and it was significantly higher in 0.75 and 1 μM Tat-PGAM1-treated groups than

2.4. Data quantification and analysis 2.4.1. Quantification of NeuN-immunoreactive neurons The number of NeuN-immunoreactive neurons was counted in the 3

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Fig. 1. Construction and delivery of control-PGAM1 and Tat-PGAM1 protein into HT22 cells. (A) Construction of control-PGAM1 and Tat-PGAM1 expression vectors and confirmation of protein expression in E. coli cells by Coomassie brilliant blue staining and Western blot analysis (using anti-polyhistidine antibody). (B) Concentration (0.5–5 μM) dependent uptake of control-PGAM1 and Tat-PGAM1 proteins by HT22 cells at 1 h after the treatment. (C) Time-dependent (0–60 min) uptake of control-PGAM1 and Tat-PGAM1 proteins (both 3 μM) by HT22 cells. (D) Confirmation of the cellular location of transduced control-PGAM1 and TatPGAM1 proteins at 1 h after treatment based on immunocytochemical staining for polyhistidine. Scale bar = 20 μm. Bar graph represents the mean ± S.E.M.

cells were observed and the fluorescence was significantly lower than that in the vehicle-treated and H2O2-exposed groups. However, it was significantly higher than that in the control group (Fig. 2C).

in the vehicle-treated and H2O2-exposed groups (Fig. 2A). DNA fragmentation was assessed by TUNEL staining after treatment with vehicle, control-PGAM1, or Tat-PGAM1 for 1 h and 1 mM H2O2 for 3 h. In the control group, very few TUNEL positive cells were observed in the HT22 cells. In the vehicle-treated as well as the control-PGAM1treated groups, abundant TUNEL positive cells were observed and the fluorescence intensity was significantly increased when compared to that in the control group. In the Tat-PGAM1-treated group, a few TUNEL positive cells were detected and the fluorescence intensity was significantly decreased when compared to that in the vehicle-treated or control-PGAM1-treated group (Fig. 2B). ROS formation was evaluated by DCF-DA fluorescence after subsequent treatment with vehicle, control-PGAM1, or Tat-PGAM1 for 1 h, 1 mM H2O2 for 10 min, and 20 μM DCF-DA for 30 min. In the control group, DCF fluorescence was rarely observed in the HT22 cells, while in the vehicle-treated and H2O2 exposed group, DCF stained cells were abundant and the fluorescence was significantly increased compared to that in the control group. In the control-PGAM1-treated and H2O2-exposed groups, the number of DCF stained cells and the fluorescence were similar to that in the vehicle-treated and H2O2-exposed groups. In the Tat-PGAM1-treated and H2O2-exposed groups, few DCF stained

3.4. Role of Tat-PGAM1 in ischemic damage in gerbils 3.4.1. Behavioral and morphological changes The neuroprotective effects of Tat-PGAM1 and control-PGAM1 were determined in ischemia-induced hyperactivity and neuronal damage at 1 and 4 days after ischemia/reperfusion, respectively. In the control group, the animals did not show any significant change in terms of the distance traveled in 60 min one day before and after ischemia and abundant NeuN positive neurons were observed in the hippocampal region. In the vehicle-treated group, distance traveled was significantly increased (2.88-folds) 1 day after ischemia as compared to that in the control group. In addition, very few NeuN positive neurons (4.68% of the control group) were observed in the hippocampal CA1 region, while they were abundant in other regions. In the control-PGAM1-treated group, the distance traveled was similar to that in the vehicle-treated group at 1 day after ischemia and a few NeuN immunoreactive cells (12.96% of the control group) were observed in the CA1 region. In the 4

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Fig. 2. Effect of control-PGAM1 and Tat-PGAM1 on H2O2-induced oxidative stress in HT22 cells. (A) HT22 cell viability after subsequent treatment with various doses of control-PGAM1 or Tat-PGAM1 for 1 h and 1 mM H2O2 for 5 h. (B) ROS production was measured by DCF fluorescence in HT22 cells after subsequent treatment with 3 μM control-PGAM1 or Tat-PGAM1 for 1 h, 1 mM H2O2 for 10 min, and 20 μM DCF-DA for 30 min. (C) TUNEL kit was used to assess DNA fragmentation in HT22 cells after treatment with 3 μM control-PGAM1 or Tat-PGAM1 for 1 h and 1 mM H2O2 for 3 h. Scale bar = 50 μm (B and C). Fluorescence intensities were measured and the data were analyzed by one-way ANOVA followed by a Bonferroni's post-hoc test (ap < 0.05, significantly different from the control group; bp < 0.05, significantly different from the H2O2 alone group). Data are expressed as mean ± S.E.M.

3.4.3. SDH activity, pH, ATP, lactate, and HNE levels In the control group, SDH activity, pH, ATP, and lactate levels did not show any significant changes after sham operation. In contrast, vehicle and control-PGAM1 treatment showed a significant increase in SDH activity and a decrease in ATP content at 4 and 7 days after ischemia as compared to that in the time-matched control group. TatPGAM1 treatment significantly ameliorated the changes in SDH activity and ATP content at 4 and 7 days after ischemia when compared to that in the vehicle-treated group and 7 days after ischemia as compared to that in the control-PGAM1-treated group. The pH levels steadily decreased with time after ischemia in ischemic groups, while the pH level in vehicle and control-PGAM1treated groups was significantly lower in the hippocampus 6 h, 2, 4, and 7 days after ischemia when compared to that in the time-matched control group. In Tat-PGAM1-treated group, the pH level was significantly higher in the hippocampal homogenates 6 h, 4, and 7 days after ischemia as compared to that in the vehicle-treated group and 4 and 7 days after ischemia as compared to that in the control-PGAM1treated group. Lactate levels dramatically increased at 15 min after ischemia in the vehicle-, control-PGAM1-, and Tat-PGAM1-treated groups when compared to that in the control group. Thereafter, lactate levels decreased with time after ischemia in vehicle-, control-PGAM1-, and Tat-PGAM1treated groups and did not show any significant differences between the groups at 2, 4, and 7 days after ischemia. However, the levels were

Tat-PGAM1-treated group, the distance traveled was significantly decreased compared to that in the vehicle or control-PGAM1-treated group (59.22% and 61.01%, respectively). In addition, numerous NeuN immunoreactive neurons (61.24% of the control group) were observed in the hippocampal CA1 region compared to that in the vehicle or control-PGAM1-treated group (Fig. 3A and B). 3.4.2. Reactive gliosis In the control group, GFAP immunoreactive astrocytes and Iba-1 immunoreactive microglia were abundantly observed in the stratum oriens and stratum radiatum of CA1 region. They had little cytoplasm and had thin and long processes. In the vehicle-treated group, GFAP immunoreactive astrocytes and Iba-1 immunoreactive microglia had hypertrophied cytoplasm with thickened processes and GFAP and Iba-1 immunoreactivities were significantly increased in the hippocampal CA1 region to 244.94% and 282.96% of the control group, respectively. In this group, they were mainly observed in the stratum oriens and stratum radiatum, while Iba-1 immunoreactive microglia was also observed in the stratum pyramidale. In the control-PGAM1-treated group, the morphology and distribution pattern of GFAP immunoreactive astrocytes and Iba-1 immunoreactive microglia were similar to those in the vehicle-treated group. In the Tat-PGAM1-treated group, the morphology of GFAP immunoreactive astrocytes and Iba-1 immunoreactive microglia was similar to the morphology of those in the control group, although some of them had hypertrophied cytoplasm (Fig. 3C). 5

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Fig. 3. Effect of control-PGAM1 and Tat-PGAM1 proteins on ischemic damage in gerbils. (A) Spontaneous motor activity measured in gerbils one day before and after ischemia in the sham-operated (control) group, and the Tat peptide (vehicle)-treated, control-PGAM1-treated, and Tat-PGAM1-treated groups (n = 5 per group; ap < 0.05, significantly different from the control group; bp < 0.05, significantly different from the vehicle-treated group; cp < 0.05, significantly different from the control-PGAM1-treated group). Data are expressed as mean ± S.E.M. (B) NeuN immunohistochemical staining in the hippocampus of the control, vehicle, control-PGAM1, and Tat-PGAM1-treated groups at 4 days after ischemia. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. The percentage of NeuNimmunoreactive neurons vs. control group, per section in all the groups is also shown (n = 5 per group; ap < 0.05, significantly different from the control group; b p < 0.05, significantly different from the vehicle-treated group; cp < 0.05, significantly different from the control-PGAM1-treated group). Data are expressed as mean ± S.E.M. (C) Immunohistochemistry for GFAP and Iba-1 in the CA1 region of the control, vehicle, control-PGAM1, and Tat-PGAM1-treated groups at 4 days after ischemia. Scale bar = 50 μm (B and C). Relative optical densities (ROD) are expressed as a percentage of the value of GFAP and Iba-1 immunoreactivity in the hippocampal CA1 region of control group per section, respectively (n = 5 per group; ap < 0.05, significantly different from the control group; bp < 0.05, significantly different from the vehicle-treated group; cp < 0.05, significantly different from the control-PGAM1-treated group). Data are expressed as mean ± S.E.M.

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Fig. 4. Effects of control-PGAM1 and Tat-PGAM1 on SDH activity, ATP content, pH, lactate, and HNE levels in the gerbil hippocampus at various time points after ischemia (n = 6 per group; ap < 0.05, significantly different from the control group; bp < 0.05, significantly different from the vehicle-treated group; cp < 0.05, significantly different from the control-PGAM1-treated group). Data are expressed as mean ± S.E.M.

protein into HT22 cells was confirmed by Western blot analysis for polyhistidine; no band was observed in control-PGAM1-treated cells. In addition, treatment with Tat-PGAM1 significantly reduced H2O2-induced ROS formation, DNA damage, and cell death in HT22 cells, while control-PGAM showed no positive effects. This result is supported by a previous study, which showed that PGAM1 knockdown significantly changed the levels of proteins related to DNA damage-induced apoptosis. In addition, PGAM1 is one of the essential elements in the homologous recombination-mediated repair of double-strand DNA breaks (Qu et al., 2017). Cerebral ischemia induces motor activity in gerbils and this hyperactivity continues for several days (Karasawa et al., 1994; Yamamoto et al., 2001; Yoo et al., 2019). In the present study, we observed the locomotor activity and the neurons by NeuN immunohistochemistry at 1 and 4 days after ischemia, respectively. Vehicle (Tat peptide)-treated group showed increased locomotor activity and a decrease in the numbers of NeuN immunoreactive neurons within the hippocampal CA1 region. No difference in the locomotor activity was observed between control-PGAM1 and vehicle-treated group, although more NeuN immunoreactive neurons were observed in the control-PGAM1-treated group. Administration of Tat-PGAM1 significantly ameliorated the ischemia-induced hyperactivity and neuronal death in the hippocampal CA1 region at 1 and 4 days after ischemia, respectively. This result suggests that the control-PGAM1 could not cross the blood-brain barrier because in gerbil ischemic model, it has been demonstrated that IgG leakage is seen 2 days after ischemia, but extravasation of Evans blue dye cannot be detected at any time point after ischemia (Ahn et al., 2018). This is the first study to show the neuroprotective potential of Tat-PGAM1 against ischemic damage in gerbil hippocampus. To elucidate the possible neuroprotective mechanisms of TatPGAM1 against ischemic damage, we measured the pH, ATP, and HNE levels as well as SDH activity in hippocampal homogenates at various time points after ischemia. SDH is one of the major enzymes of the tricarboxylic acid cycle and its activity was significantly increased in

significantly lower in the Tat-PGAM1-treated group at 15 min and 6 h after ischemia as compared to that in the vehicle- and control-PGAM1treated groups. HNE levels were significantly increased in the vehicle-treated group when compared to those in the control group at 15 min after ischemia, but other groups did not show any significant changes. Thereafter, HNE levels in the vehicle-treated group dramatically increased at 6 h after ischemia and decreased at 2 days after ischemia. However, the levels were significantly higher in the vehicle-treated group than in the control group. In the control-PGAM1 and Tat-PGAM1-treated groups, a similar pattern of HNE changes was observed when compared to that in the vehicle-treated group. However, the HNE levels were significantly lower in Tat-PGAM1-treated group as compared to that in the vehicletreated group at 6 h and 2 days after ischemia (Fig. 4). 4. Discussion PGAM is a glycolytic enzyme that coordinates energy production; PGAM1 levels have been shown to be higher in the serum of patients with cerebral infarction (Takahashi et al., 1998). PGAM1 protects the cells from DNA damaging agents and promotes the repair of the doublestrand DNA breaks (Qu et al., 2017). In addition, PGAM1 expression is reduced in the hippocampus of Alzheimer's disease patients, animal models of Alzheimer's disease, and aged mice (Bigl et al., 1999; DíezVives et al., 2009; Iwangoff et al., 1980; Sultana et al., 2006). In addition, suppression of PGAM1 gene expression induces apoptosis in spermatogenic cells (Zhao et al., 2019), while Tat-PGAM1 treatment induces proliferation and neuroblast differentiation in the mouse dentate gyrus (Jung et al., 2019). In the present study, we constructed a Tat-PGAM1 fusion protein to investigate the roles of PGAM1 in oxidative damage induced by H2O2 in HT22 cells and in ischemic damage in gerbil hippocampus. We used Tat-fused PGAM1 since it has been shown that Tat-fused cargo can easily cross cell membranes and the blood-brain barrier (Dietz and Bähr, 2005). Time- and dose-dependent uptake of Tat-PGAM1 fusion 7

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the hippocampus. These results suggest that Tat-PGAM1 could be a useful therapeutic agent to reduce the oxidative damage in ischemia.

the hippocampal homogenates at 4 and 7 days after ischemia. This result is supported by a previous study that showed that SDH activity was significantly increased at 2 and 7 days after 7 min of ischemia in gerbils (Shcherbak et al., 2013), although we did not observe any statistical significance in SDH activity at 2 days after ischemia. This discrepancy may be associated with the occlusion time; longer occlusion causes more cell damage in the hippocampus. Treatment with TatPGAM1, and not with control-PGAM1, significantly ameliorated the increase in SDH activity in the hippocampus as compared to the vehicle-treated group. Inhibition of SDH reduces neuronal cell death in a rat model of cardiac arrest (Xu et al., 2018). In addition, ebselen treatment prevents the toxicity and enhanced SDH activity induced by 3-nitropropionic acid in rats (Wilhelm et al., 2014). Intracellular and interstitial pH levels are tightly regulated in the brain at ~7.04 and ~7.3, respectively (Roos and Boron, 1981). However, anaerobic glycolysis during ischemia may disturb H+ homeostasis and the resulting lactic acidosis causes neuronal damage after ischemia (Plum, 1983). In the present study, the pH levels were seen to drop to 6.360 and 6.325 in the hippocampal homogenates of vehicle and control-PGAM1-treated groups, respectively. Treatment with Tat-PGAM1 significantly ameliorated the ischemia-induced pH decline in the hippocampal homogenates. This result is consistent with previous studies, which showed a decrease in the intracellular and extracellular pH levels in ischemic animals (Hansen and Olsen, 1980; Kraig et al., 1986). Transient forebrain ischemia in gerbils gradually decreased the ATP content in the hippocampus to about half of the baseline value at 7 days after ischemia/reperfusion. Similar results have been reported in a previous study, which showed that ATP content was gradually reduced to 25–40% of the baseline value at 7 days after ischemia (Kimura et al., 2002), although another study in gerbils showed that ATP was completely depleted in the brain during a 5 min cerebral ischemia (Munekata and Hossman, 1987). Administration of Tat-PGAM1 maintained ATP content to 78% of the baseline value at 7 days after ischemia/reperfusion. L-Lactate is a common metabolite between glycolysis and oxidative phosphorylation (Smith et al., 2003). However, lactate has been shown to have neuroprotective effects against oxygen and glucose deprivation in hippocampal slices and ischemia/reperfusion induced by middle cerebral artery occlusion in mice (Berthet et al., 2009). High doses of lactate, however, show toxic effects in hippocampal slices after oxygen and glucose deprivation (Berthet et al., 2009). This result suggests that a moderate increase of lactate plays important roles in neuroprotection against ischemic damage. In the present study, we observed significant increases in lactate levels in the hippocampus at 15 min and 6 h after ischemia and significantly reductions in the Tat-PGAM1-treated group. Lipid peroxidation in the hippocampus, as assessed by HNE levels, was significantly increased at 6 h after ischemia in the vehicle and control-PGAM1-treated groups. This result is consistent with previous studies, which showed that HNE levels were significantly increased in the hippocampus at early time points after ischemia/reperfusion (Wang et al., 2006; Yoo et al., 2014). In the present study, we observed that HNE levels were higher in the vehicle (Tat peptide)-treated group than in the control-PGAM1-treated group. However, it could not be concluded that control-PGAM1 reduced the lipid peroxidation at 6 h after ischemia/reperfusion because Tat peptide increases the oxidative stress in neurons (Haughey et al., 2004) and causes neurocognitive disorders (Kim et al., 2015; Louboutin and Strayer, 2014). Treatment with TatPGAM1 significantly mitigated the ischemia-induced lipid peroxidation in the hippocampus at 6 h and 2 days after ischemia.

Declaration of competing interest The authors declare that there is no financial conflict of interests to publish these results. Acknowledgements This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF-2019R1A2C1005440 to In Koo Hwang and KRF-2018R1A2B6001941 to Dae Won Kim). In addition, this work was supported by the Research Institute for Veterinary Science, Seoul National University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.neuint.2019.104631. References Ahn, J.H., Chen, B.H., Park, J.H., Shin, B.N., Lee, T.K., Cho, J.H., Lee, J.C., Park, J.R., Yang, S.R., Ryoo, S., Shin, M.C., Cho, J.H., Kang, I.J., Lee, C.H., Hwang, I.K., Kim, Y.M., Won, M.H., 2018. Early IV-injected human dermis-derived mesenchymal stem cells after transient global cerebral ischemia do not pass through damaged bloodbrain barrier. J. Tissue Eng. Regenerat. Med. 12, 1646–1657. Betrán, E., Wang, W., Jin, L., Long, M., 2002. Evolution of the phosphoglycerate mutase processed gene in human and chimpanzee revealing the origin of a new primate gene. Mol. Biol. Evol. 19, 654–663. Bigl, M., Brückner, M.K., Arendt, T., Bigl, V., Eschrich, K., 1999. Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease. J. Neural Transm. 106, 499–511. Babcock, A.M., Baker, D.A., Lovec, R., 1993. Locomotor activity in the ischemic gerbil. Brain Res. 625, 351–354. Berthet, C., Lei, H., Thevenet, J., Gruetter, R., Magistretti, P.J., Hirt, L., 2009. Neuroprotective role of lactate after cerebral ischemia. J. Cereb. Blood Flow Metab. 29, 1780–1789. Butterfield, D.A., Reed, T., Newman, S.F., Sultana, R., 2007. Roles of amyloid beta-peptide-associated oxidative stress and brain protein modifications in the pathogenesis of Alzheimer's disease and mild cognitive impairment. Free Radic. Biol. Med. 43, 658–677. Datta, A., Jingru, Q., Khor, T.H., Teo, M.T., Heese, K., Sze, S.K., 2011. Quantitative neuroproteomics of an in vivo rodent model of focal cerebral ischemia/reperfusion injury reveals a temporal regulation of novel pathophysiological molecular markers. J. Proteome Res. 10, 5199–5213. Dietz, G.P., Bähr, M., 2005. Peptide-enhanced cellular internalization of proteins in neuroscience. Brain Res. Bull. 68, 103–114. Díez-Vives, C., Gay, M., García-Matas, S., Comellas, F., Carrascal, M., Abian, J., OrtegaAznar, A., Cristòfol, R., Sanfeliu, C., 2009. Proteomic study of neuron and astrocyte cultures from senescence-accelerated mouse SAMP8 reveals degenerative changes. J. Neurochem. 111, 945–955. Djuricic, B.M., Paschen, W., Bosma, H.J., Hossmann, K.A., 1983. Biochemical changes during graded brain ischemia in gerbils. Part 1. Global biochemical alterations. J. Neurol. Sci. 58, 25–36. Durany, N., Joseph, J., Cruz-Sánchez, F.F., Carreras, J., 1997. Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and creatine kinase activity and isoenzymes in human brain tumours. Br. J. Canc. 76, 1139–1149. Gao, H., Yu, B., Yan, Y., Shen, J., Zhao, S., Zhu, J., Qin, W., Gao, Y., 2013. Correlation of expression levels of ANXA2, PGAM1, and CALR with glioma grade and prognosis. J. Neurosurg. 118, 846–853. González-Cinca, N., Rivera, F., Carreras, J., Climent, F., 2003. Effects of hypoxia and thyroid hormone on mRNA levels and activity of phosphoglycerate mutase in rabbit tissues. Horm. Res. 59, 16–20. Hansen, A.J., Olsen, C.E., 1980. Brain extracellular space during spreading depression and ischemia. Acta Physiol. Scand. 108, 355–365. Hansen, A.J., Zeuthen, T., 1981. Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex. Acta Physiol. Scand. 113, 437–445. Hansen, A.J., 1985. Effect of anoxia on ion distribution in the brain. Physiol. Rev. 65, 101–148. Harris, R.J., Symon, L., Branston, N.M., Bayhan, M., 1981. Changes in extracellular calcium activity in cerebral ischaemia. J. Cereb. Blood Flow Metab. 1, 203–209. Haughey, N.J., Cutler, R.G., Tamara, A., McArthur, J.C., Vargas, D.L., Pardo, C.A., Turchan, J., Nath, A., Mattson, M.P., 2004. Perturbation of sphingolipid metabolism and ceramide production in HIV-dementia. Ann. Neurol. 55, 257–267. Hayashi, T., Abe, K., 2004. Ischemic neuronal cell death and organellae damage. Neurol.

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