Extracellular cyclic GMP and its derivatives GMP and guanosine protect from oxidative glutamate toxicity

Neurochemistry International 62 (2013) 610–619

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Extracellular cyclic GMP and its derivatives GMP and guanosine protect from oxidative glutamate toxicity Philipp Albrecht a, Nadine Henke a, Mai-Ly Tran Tien a, Andrea Issberner a, Imane Bouchachia a, Pamela Maher b, Jan Lewerenz c, Axel Methner a,d,⇑ a

Department of Neurology, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA University of Ulm, Department of Neurology, Germany d Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn2), Johannes Gutenberg University Medical Center Mainz, Department of Neurology, Langenbeckstr. 1, D-55131 Mainz, Germany b c

a r t i c l e

i n f o

Article history: Available online 26 January 2013 Keywords: cGMP GMP Guanosine Oxidative stress Neuroprotection

a b s t r a c t Cell death in response to oxidative stress plays a role in a variety of neurodegenerative diseases and can be studied in detail in the neuronal cell line HT22, where extracellular glutamate causes glutathione depletion by inhibition of the glutamate/cystine antiporter system xc, elevation of reactive oxygen species and eventually programmed cell death caused by cytotoxic calcium influx. Using this paradigm, we screened 54 putative extracellular peptide or small molecule ligands for effects on cell death and identified extracellular cyclic guanosine monophosphate (cGMP) as a protective substance. Extracellular cGMP was protective, whereas the cell-permeable cGMP analog 8-pCPT-cGMP or the inhibition of cGMP degradation by phosphodiesterases was toxic. Interestingly, metabolites GMP and guanosine were even more protective than cGMP and the inhibition of the conversion of GMP to guanosine attenuated its effect, suggesting that GMP offers protection through its conversion to guanosine. Guanosine increased system xc activity and cellular glutathione levels in the presence of glutamate, which can be explained by transcriptional upregulation of xCT, the functional subunit of system xc. However, guanosine also provided protection when added late in the cell death cascade and significantly reduced the number of calcium peaking cells, which was most likely not mediated by transcriptional mechanisms. We observed no changes in the classical protective pathways such as phosphorylation of Akt, ERK1/2 or induction of Nrf2 or ATF4. We conclude that extracellular guanosine protects against endogenous oxidative stress by two probably independent mechanisms involving system xc induction and inhibition of cytotoxic calcium influx. Ó 2013 Elsevier Ltd. All rights reserved.

Abbreviations: ARE, antioxidant response element; BCA, bicinchoninic acid; BSA, bovine serum albumin; cDNA, complementary deoxyribonucleic acid; cGMP, cyclic guanosine monophosphate; CTB, cell titer blue; DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; eGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GMP, guanosine monophosphate; GPCR, G-protein coupled receptor; GSH, glutathione; GUA, guanosine; HBSS, Hank’s buffered salt solution; HEPES, N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid); HPRT, hypoxanthine-guanine phosphoribosyltransferase; mRNA, messenger ribonucleic acid; IBMX, 3-isobutyl-1-methylxanthine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NADPH, reduced b-nicotinamide adenine dinucleotide phosphate; Nrf2, nuclear factor erythroid 2-related factor 2; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PDE5, phosphodiesterase 5; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SSA, sulfosalicylic acid. ⇑ Corresponding author at: Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn2), Johannes Gutenberg University Medical Center Mainz, Department of Neurology, Langenbeckstr. 1, D-55131 Mainz, Germany. Tel.: +49 6131 17 2695; fax: +49 6131 17 5967. E-mail address: [email protected] (A. Methner). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.01.019

1. Introduction Oxidative stress plays a role in acute and chronic neurodegenerative diseases such as ischemic stroke, epilepsy, Alzheimer’s-, Huntington’s- and Parkinson’s diseases (Browne and Beal, 2006; Costello and Delanty, 2004; Ilieva et al., 2007; Steiner et al., 2006; Wiedau-Pazos et al., 1996; Wong and Crack, 2008) and can be studied in detail using oxidative glutamate toxicity in the hippocampal cell line HT22. In this paradigm, extracellular glutamate inhibits cystine uptake via the cystine/glutamate antiporter system xc(Albrecht et al., 2010, 2012). Cysteine, the reduced form of cystine, is required for synthesis of glutathione (GSH), the most important antioxidant in the brain (Dringen, 2000; Schulz et al., 2000). System xc inhibition therefore results in GSH depletion leading to an exponential increase in reactive oxygen species (ROS) that mostly originate from mitochondrial complex I activity (Tan et al., 1998). After approximately 6 h of glutamate exposure,

P. Albrecht et al. / Neurochemistry International 62 (2013) 610–619

the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15- LOX; EC 1.13.11.33) is activated and generates 12- and 15- hydroxyeicosatetraenoic acids (Li et al., 1997b) that directly damage mitochondria and cause mitochondrial depolarization and increased ROS production (Pallast et al., 2009). The eicosanoids produced by 12LOX are also activators of soluble guanylate cyclases and thereby increase the concentration of intracellular cyclic guanosine monophosphate (cGMP), resulting in a detrimental influx of calcium at the end of the cell death cascade through a cGMP-dependent calcium channel (Li et al., 1997a) which most probably corresponds to the plasma membrane Ca2+ channel Orai1 (Henke et al., in press). Cell death by this series of events is also called oxytosis and is distinct from classical apoptosis. We and others have used this paradigm extensively to identify novel neuroprotective substances (Albrecht et al., 2010, 2012; Lewerenz et al., 2009; Maher, 2006), proteins (Dittmer et al., 2008) and signal transduction pathways (Lewerenz et al., 2003; Lewerenz and Maher, 2009; Lewerenz et al., 2009, 2012; Tan et al., 2001b). In the current work, we investigated the effects of a library of 54 putative ligands or stimulators of G protein-coupled receptors on cell death caused by oxidative glutamate toxicity and identified extracellular cGMP as a protective substance. As intracellular cGMP production is induced during oxidative glutamate toxicity and considered to be detrimental, we hypothesized that toxic intracellular cGMP is released by dying cells to protect the surrounding cells in a paracrine manner. Others, however, have found that the inhibition of phosphodiesterases that degrade cGMP provided protection in different models of oxidative stress (Choi et al., 2007; Li et al., 1997a; Montoliu et al., 1999; Nakamizo et al., 2003). To clarify these apparently contradictory results, we studied the effects of cGMP and its degradation products GMP and guanosine (GUA) on oxidative glutamate toxicity and elucidated their mechanism of action. 2. Experimental procedures 2.1. Materials Tissue culture dishes were from Greiner BIO-ONE and NUNC; fetal calf serum (FCS) was obtained from Hyclone; L-glutamine, penicillin/streptomycin, 5-(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate, acetyl ester (CM–H2DCFDA), high-glucose Dulbecco’s modified Eagle medium (DMEM), Alpha-MEM, trypsin/EDTA and TriZol reagent for RNA purification and fluorescent secondary antibodies were from Invitrogen; acivicin, bovine serum albumin (BSA), L-cystine, L-glutamate, N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid) (HEPES), glutathione reductase, sulfosalicylic acid (SSA), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), homocysteate, b-mercaptoethanol (b-ME), NADPH, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cGMP, GMP, GUA, zaprinast, LY294002 and 8-pCPT-cGMP were from Sigma–Aldrich. L-[3H]-glutamate was obtained from Perkin Elmer NEN. Oligonucleotides were synthesized by MWG Biotech AG. Celltiter blue (CTB) was from Promega. The library of 54 putative ligands or stimulators of G protein-coupled receptors was a kind gift from Chica Schaller (Zentrum für Molekulare Neurobiologie, ZMNH, University Clinic Hamburg Eppendorf, Hamburg, Germany). All other chemicals were obtained from Merck Eurolabs. 2.2. Cell culture and viability assays Viability assays of HT22 cells and immature primary neuronal cortical cultures were performed as previously described (Albrecht et al., 2012; Lewerenz et al., 2009). HT22 cells were seeded in 96well plates at a density of 5  103 cells per well, with the indicated

611

concentrations of the substances listed in table 1 or vehicle. For further analysis of their protective activity, cGMP, GMP and GUA were used at concentrations of 0.1 mM; N-acetylcysteine (NAC) as the positive control at a concentration of 2 mM; and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 at a concentration of 10 lM. After 24 h, glutamate, 8-pCPT-cGMP or zaprinast were added at the indicated concentrations to induce cell death. 24 h later again, cell viability was assessed by the CTB (Promega) or MTT assay. 105 primary cortical neurons derived from E14 rats were directly seeded onto PLL-coated 96-well plates and treated with vehicle or 0.1 mM of cGMP, GMP or GUA followed by glutamate 24 h later. Again, viability was quantified another 24 h later by the CTB assay. 2.3. Quantification of total GSH 3  105 HT22 cells were seeded in 60 mm-dishes with or without 0.1 mM cGMP, GMP or GUA. After 24 h, medium with the indicated concentrations of glutamate was added for 8 h. Cells were harvested, processed and total GSH was measured enzymatically as described previously (Albrecht et al., 2012; Maher and Hanneken, 2005). 2.4. cGMP ELISA HT22 cells were seeded in 6-well plates at a density of 1.65  105 cells per well, grown for 24 h and treated with 5 mM glutamate or vehicle for 8 h. Intracellular and extracellular cGMP was then measured using the BIOTRAK cGMP enzyme immunoassay kit (Amersham) according to the manufacturer’s instructions. 2.5. Measurement of system xc activity For measurement of system xc activity, 2.5  104 HT22 cells were plated onto 24-well plates in the presence of vehicle or 0.1 mM cGMP, GMP or GUA. Cells were grown for 24 h. System xc activity was then measured as homocysteate-sensitive sodium-independent 3H-glutamate uptake, as described previously (Lewerenz and Maher, 2009). 2.6. Quantitative real-time PCR RNA extraction, reverse transcription and quantitative real-time PCR were performed as previously described (Albrecht et al., 2012; Lewerenz et al., 2009), using Fam/Dark-quencher probes from the Universal Probe Library™ (Roche) or individually designed Fam/ Tamra probes (MWG). Beta-actin and HPRT served as endogenous control genes and showed no differential expression after incubation with GUA. Primer and probe sequences can be obtained from the authors. 2.7. Cell fractionation, SDS–PAGE and immunoblotting HT22 cells were plated at a density of 8.8  105 cells per 10 cm dish. After 24 h, the cells were treated with 100 lM cGMP, GMP or GUA for 10 min, 2 h or 24 h. For cell fractionation, cells were rinsed twice in ice-cold, Tris-buffered saline (TBS)scraped into an ice-cold nuclear fractionation buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na3VO4, 1x protease inhibitor cocktail (Sigma–Aldrich) and 1x phosphatase inhibitor cocktail) and incubated on ice for 15 min. NP40 was then added at a final concentration of 0.6%, the cells were vortexed and their nuclei pelleted by centrifugation. The supernatant was collected as cytosolic/membrane fraction. Nuclear proteins were extracted from the pellet by sonication in the nuclear fractionation

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Table 1 cGMP and hemorphin protect from oxidative glutamate toxicity. The difference between vehicle- and verum-treated cells in percent is depicted ± the standard error of the mean. Significant differences between vehicle- and verum treatment is indicated by an asterisk and bold letters (p < 0.05, two tailed t-test). Abbreviations are as follows: BAM22 = Bovine adrenal medulla 22, EEP = ethanolic extract of propolis, NeurokA = neurokinin A, GAL = glactosidase, GRP = gastrin-releasing peptide, LHRH = luteinizing hormone-releasing hormone, LPA = lysophosphatidic acid, LPC = lipocortin, cAMP = Cyclic adenosine monophosphate, FMLF = formyl-methionyl-leucyl phenylalanine, GHRP6 = Growth hormone releasing hexapeptide, HOE140 = peptide bradykinin B2 receptor antagonist Hoe 140, Mif-1 = Pro-Leu-Gly-NH(2) tripeptide, NPF = neuropeptide F, PAMP = proadrenomedullin N-terminal 20 peptide, SRIF = somatotropin release inhibiting factor, TRH = thyrotropin releasing hormone, UDP = uridine diphosphate, UDPGlc = uridine-diphosphate-glucose, UDP-Glc-Nac = uridine-diphosphate-glucose-N-acetylcysteine. Difference between vehicle-and verum treated cells in% Concentration 1 (high)

Concentration 2 (low)

Name

Veh.

2.5 mM Glu

5 mM Glu

Veh.

2.5 mM Glu

5 mM Glu

Protective effects BAM22 (1 lM, 10 nM) ß-Endorphin (100 nM, 10 nM) cGMP (0.1 mM, 10 lM) Chemerin (1 lM, 100 nM) EEP (1 lM, 100 nM) Endomorphin (1 lM, 100 nM) Hemorphin (1 lM, 0,1 lM) Motilin (100 nM, 10 nM) NeurokA (1 lM, 100 nM) Relaxin (100 nM, 10 nM) Urotensin (100 nM, 10 nM) Valorphin (1 lM, 100 nM)

0.7 0.5 1.0 1.1 0.2 1.9 0.5 0.2 0.2 0.3 0.4 0.8

9.0 (±1.9) 11.1 (±0.6) 30.3 (±1.5)⁄ 5.3 (±0.4) 7.9 (±0.8) 12.2 (±1.3) 19.1 (±0.2)⁄ 8.0 (±1.5) 6.2 (±0.0) 7.1 (±0.2) 8.5 (±0.3) 6.4 (±1.3)

2,8 (±0,5) 11.0 (±1.7) 9.5 (±1.8) 0.2 (±0.2) 5.9 (±0,0) 1.5 (±1.4) 6.3 (±1.0) 0.1 (±0.5) 0.2 (±0.1) 1.5 (±1.3) 0.9 (±1.3) 9.7 (±0.6)

0.8 1.0 0.1 0.5 0.4 0.7 0.1 0.9 1.9 -0.4 0.3 1.2

0.2 (±1.9) 10.8 (±1.1) 17.6 (±2.2) 0.6 (±0.4) 6,1 (±1,4) 2.5 (±0.7) 12.8 (±0.4) 4.0 (±1.0) 1.9 (±0.5) 3.6 (±2.0) 3.4 (±1.5) 3.6 (±1.8)

1.7 (±0.6) 9.2 (±1.9) 4.1 (±1.8) 1.3 (±0.5) 5,1 (±0,7) 1.0 (±1.0) 2.1 (±0.9) 1.7 (±0.4) 2.7 (±0.7) 2.7 (±0.8) 0.2 (±1.7) 1.6 (±0.9)

Toxic effects Amylin (10 nM,1 nM) GAL (1 lM, 100 nM) GRP (1 lM, 100 nM) Kallidin (1 lM,100 nM) LHRH II (1 lM, 100 nM) LPA (10 lM, 1 lM) LPC (1 lM, 100 nM) NeuromedinU (1 lM, 100 nM) Prokinetin (100 nM, 10 nM)

0.7 1.4 0.3 1.5 0.3 1.8 0.6 2.5 0.6

16.1 (±0.3) 13.9 (±1.1) 16.2 (±1.9) 5.7 (±1.4) 13.3 (±1.6) 7.6 (±1.1) 5.1 (±1.6) 4.0 (±0.4) 2.06 (±0.5)

3.1 (±0.7) 3.0 (±0.3) 17 (±1.7) 5.4 (±0.9) 17.5 (±0.7) 8.1 (±0.7) 0.2 (±0.2) 8.6 (±0.4) 12.4 (±0.1)

2.1 0.0 0.1

6.9 (±0.4) 6.2 (±1.4) 12.4 (±1.0)

1.3 (±0.1) 2.5 (±0.4) 9.8 (±1.8)

1.1

2.0 (±0.1)

12.9 (±0.4)

0.1 1.2 1.4

0.7 (±1.5) 2.3 (±1.4) 0.3 (±2.9)

0.5 (±0.9) 8.2 (±1.3) 13.9 (±1.9)

No effects Amyloid b (1 lM, 1 nM) Apelin (1 lM, 10 nM) cAMP (1 lM, 100 nM) Leu-Enkephalin (1 lM, 100 nM) FMLF (100 nM, 10 nM) GAL1–16 (1 lM, 1 nM) GastrinTetrapeptid (1 lM,10 nM) GHRP-6 (1 lM,1 nM) HOE140 (1 lM, 100 nM) Leu-Enkephalin (1 lM) LHRH (1 lM) LHRH1–5 (1 lM, 100 nM) MIF-1 (10 lM, 1 lM) Morphiceptin (1 lM, 100nM) Neuromedin B (1 lM, 100 nM) Nocistatin (1 lM, 100 nM) NPF (100 nM, 10 nM) PAMP (100 nM, 0,1 pM) Progesteron (1 lM, 100 nM) Gastrin (1 lM, 100 nM) Secreton (100 nM, 10 nM) Somatostatin (1 lM, 0.1 nM) SRIF (1 lM, 100 nM) Substance K (1 lM, 100 nM) Substance P (1 lM, 100 nM) TRH (100 nM, 10 nM) UDP (1 lM, 1 nM) UDP-Glc (100 lM, 10 lM) UDP-Glc-Nac (1 lM, 1 nM)

2.4 1.8 0.3 2.0 1.7 2.7 2.1 0.9 2.9 2.4 1.4 1.4 2.1 0.2 0.2 2.1 1.4 1.5 0.9 1.1 0.4 0.4 1.8 1.0 2.0 1.5 1.0 1.9 0.5

3.0 (±2.4) 0.5 (±0.3) 1.4 (±3.6) 4.6 (±3.2) 4.8 (±4.7) 3.1 (±3.1) 1.6 (±3.7) 3.02 (±2.3) 6.8 (±3.5) 6.6 (±3.7) 0.1 (±3.1) 0.3 (±0.4) 7.3 (±1.1) 1.1 (±1.6) 4.6 (±0.7) 0.1 (±2.3) 4.6 (±1.2) 3.5 (±1.8) 4.2 (±1.7) 0.2 (1.4) 0.1 (±0.7) 0.3 (±0.4) 2.3 (±1.27) 1.1 (±1.18) 0.42 (±1.62) 3.73 (±1.63) 0.32 (±0.6) 0.39 (±0.85) 0.54 (±0.63)

1.0 (±1.2) 2.5 (±0.3) 2.1 (±0.5) 2.9 (±0.6) 1.9 (±1.4) 0.5 (±1.5) 2.8 (±0.3) 1.5 (±1.2) 3.1 (±1.0) 1.3 (±3.2) 6 (±0.8) 0.3 (±0.1) 0.3 (±1.8) 2 (±1.5) 4.8 (±1.8) 0.02 (±0.3) 3.9 (±1.3) 7.3 (±1.6) 1 (±1.1) 0.7 (±0.9) 0.1 (±0.4) 1.6 (±0.2) 2.9 (±0.7) 0.1 (±1.5) 1.91 (±0.3) 2.29 (±2.2) 0.5 (±1.0) 0.79 (±0.6) 0.84 (±0.6)

1.1 2.3 2.8 2.8 0.2 0.1 0.3 2.3 0.5

0.1 (±3.7) 1.9 (±1.5) 3.5 (±0.2) 2.5 (±2.8) 6.2 (±2.9) 2.8 (±0.8) 1.3 (±0.3) 3.8 (±0.8) 0.3 (±2.9)

0.6 (±4.1) 2.6 (±0.9) 4.5 (±2.9) 4.3 (±1.0) 2.9 (±0.5) 0.6 (±0.2) 2.8 (±0.9) 2.9 (±0.1) 0.1 (±0.5)

0 1.1 1.9 1.0 1.6 3.7 0.4 1.6 1.0 0.4 0.0 3.0 2.8 0.1 1.1 1.2 0.6 0.7

0.0 (±0.5) 2.3 (±1.6) 0.2 (±2.8) 1.1 (±2.5) 0.1 (±1.6) 1.9 (±0.1) 0.5 ±(0.6) 0.1 (±0.4) 2.5 (±1.7) 0.5 (±0.0) 1.2 (±1.0) 1.2 (±0.8) 0 (±1.3) 0.0 (1.2) 1.2 (0.3) 0.2 (±1.7) 1.2 (±0.4) 1.4 (±0.6)

0.2 (±0.4) 0.8 (±0.8) 2.4 (±2.3) 5.5 (±1.4) 0.0 (±1.8) 4.3 (±0.7) 2.6 (±0.3) 2.7 (±1.1) 1.2 (±0.6) 0.7 (±0.2) 1.0 (±1.2) 2.8 (±0.8) 1.8 (±0.3) 0.8 (±0.6) 1.2 (±0.7) 0.5 (±1.1) 1.3 (±0.3) 1.5 (±0.1)

buffer and the extracts were cleared by additional centrifugation. Protein in the different fractions was quantified by the bicinchoninic acid method (Pierce) and adjusted to equal concentrations. SDS–PAGE and Western blotting was performed as described previously (Lewerenz and Maher, 2009). For cytosolic extracts, rabbit anti-ERK (#4695) at 1/1000 and anti-phospho-ERK (#9101) at 1/ 5000, mouse anti-phospho-Ser473-Akt (#4051, 1/1000) and rabbit anti-total Akt (#9272, 1/1000), rabbit anti-phospho-Ser21/9-

GSK3a/b (#9331, 1/1000) and rabbit anti-total GSK-3b (#9315, 1/ 1000) were used, all from Cell Signaling. Nrf2 (#sc-13032, 1/500) and ATF4 (#sc-200, 1/500) were detected in nuclear extracts using rabbit polyclonal antibodies purchased from Santa Cruz Biotechnology. Histones detected by Ponceau-S staining served a loading control. Ponceau S-stained membranes and autoradiographs were scanned using a Bio-Rad GS800 scanner. Band density was measured using the manufacturer’s software.

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2.8. Quantification of intracellular calcium GCaMP5 was a kind gift from Loren Looger (Howard Hughes Medical Institute, Ashburn) that was subcloned using EcoRI and NotI into the IRES-RFP containing vector PB531A-1 (System Biosciences). Cells were transfected with GCaMP5 in 6-well plates using Attractene (Qiagen) according to the manufacture’s protocol and 24 h later transferred to 96-well imaging plates (BD Biosciences) at a density of 5  103 cells per well. For calcium imaging, phenol-red containing medium was replaced by colorless DMEM. 2 h before measurement, 25 mM glutamate or vehicle was added together with cGMP, GMP or GUA; control wells were supplemented with vehicle alone. The plate was incubated in a BD Pathway 855 high-content microscope at 37° with 5% CO2 and 95% air. Cytosolic calcium rise was monitored by GCaMP5 relative to RFP fluorescence; pictures were taken every 15 min. For analysis, the maximal ratio was divided by the average ratio and a threshold was defined by the mean of vehicle-treated cells. Each cell above that threshold was counted as Ca2+-peak positive. 2.9. Statistical analysis Data were summarized as mean ± SEM and the statistical significance assessed using a two-tailed t-test or analysis of variance (ANOVA) and the Tukey’s or Dunnett’s Multiple Comparison Test as indicated. For comparison of cell death curves, the area under the curve of the normalized graphs was calculated and the different groups compared using ANOVA and Dunnett’s post hoc test. p values < 0.05 were considered significant. 3. Results 3.1. Extracellular cGMP and hemorphin protect from oxidative glutamate toxicity We investigated the effects of a library of 54 putative ligands or stimulators of G protein-coupled receptors (a kind gift from Chica

35 30

cGMP

hemorphin

Schaller, ZMNH, Hamburg, Germany) on cell death caused by oxidative glutamate toxicity. The cells were plated in the presence of two different concentrations of each ligand (table 1) and treated 24 h later, for 24 h, with 2.5 or 5 mM glutamate. Relative survival was then measured using the CTB assay. Only two substances increased survival over an arbitrary threshold of 15% and had a statistically significant positive effect on cell survival against 2.5 mM glutamate (p < 0.05, ANOVA, Dunnett’s post hoc multiple comparison test), whereas none of them protected against 5 mM glutamate. Hemorphin, an endogenous fragment of the ß-globin chain, increased survival by 13% (0.1 lM) and 19% (1 lM), respectively. The presumed intracellular second messenger cGMP increased survival by 18% (0.1 mM) and 30% (1 mM) respectively (Fig. 1). Forskolin, neuropeptide Y, insulin-like growth factor 1 (IGF-1), dynorphin and endomorphin also stimulated cell proliferation and were not studied further. Several compounds also increased susceptibility to oxidative stress; all screen results are depicted in full detail in Table 1. We conclude that extracellular cGMP and hemorphin protect against oxidative glutamate toxicity. Because of its detrimental intracellular role (Li et al., 1997a, b), we decided to further focus on cGMP to dissect its apparently more complicated role in endogenous oxidative stress.

3.2. Extracellular cGMP is protective but intracellular cGMP or inhibition of cGMP degradation is detrimental Addition of cGMP to the cell culture medium protected cells against oxidative glutamate toxicity in a concentration-dependent manner, with significant protective effects at cGMP concentrations of 0.1 and 1 mM (p < 0.05, area under the curve compared by ANOVA with Dunnett’s post hoc test, Fig. 2A). On the other hand and in line with the results of Ishige et al., the cell-permeable and stable cGMP analog 8-pCPT-cGMP was toxic to HT22 cells with an IC50 of 1.2 mM even without glutamate (Fig. 2B). (Ishige et al., 2001) Similarly, increasing intracellular cGMP through inhibition of the cGMP-specific phosphodiesterase five with the selective inhibitor zaprinast induced cell death (Fig. 2C). These results sug-

5 mM glutamate 2.5 mM glutamate Vehicle

Viability (% of ctrl)

25 20 15 10 5 0 -5 -10

-20

Aβ Amylin Apelin BAM22 β-endorphin cAMP cGMP Chemerin EEP Endomorphin FMLF GAL GAL1-16 GastrinTetrapeptid GHRP-6 GRP Hemorphin HOE14B Kallidin Leu-Enkephalin Leu-enkephalin LHRH LHRH1-5 LHRHII LPA LPC MIF-1 Morphiceptin Motilin NeurokA NeuromedinB NeuromedinU Nocistatin NPF PAMP Progesteron Prokinetin Relaxin rGastron Secreton Somatostatin SRIF SubstanzK SubstanzP TRH UDP UDP-Glc UDP-Glc-Nac Urotensin

-15

Fig. 1. Extracellular cGMP protects from oxidative glutamate toxicity. 5000 HT22 cells were seeded into 96-well plates with vehicle or the indicated substances at the concentrations listed in Table 1. 24 h later, cells were treated with 5 mM glutamate (black), 2.5 mM glutamate (grey) or vehicle (white). Viability was quantified 24 h later by the CTB assay. The difference of viability for cells treated with the compounds was calculated to cells treated with vehicle only (Ctrl) and the change of viability is depicted as empty bars. The difference between the cell viability of cells treated with the compounds and either 2.5 mM (grey) or 5 mM glutamate (black) and the viability with the respective glutamate concentrations alone was calculated in percent. The bar graphs represent the mean of two independent experiments performed in triplicates. Treatment with cGMP and hemorphin differed significantly from vehicle (p < 0.05, one-way ANOVA with Dunnett’s post hoc test).

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50 25

B

0

50 25

0

5

D

100

cGMP (pM)

Viability (% of ctrl)

C

1 2 3 4 Glutamate (mM)

75 50 25 0

0.5 1 1.5 2 2.5 8-pCPT-cGMP (mM)

200

100 200 300 400 Zaprinast (µM)

100

75

75

50

50

25

25 0 0

0 8 Glutamate exposure (h)

Fig. 2. Extracellular cGMP is protective but intracellular cGMP or inhibition of cGMP degradation is detrimental. 5000 HT22 cells were seeded into 96-well plates and treated either with (A) vehicle or the indicated concentrations of cGMP followed by glutamate 24 h later; (B) the cell-permeable and more stable cGMPanalog 8-pCPT-cGMP; or (C) the phosphodiesterase five-inhibitor zaprinast. Viability was quantified 24 h later by the CTB assay. Graphs represent mean ± SEM of three independent experiments, each performed in triplicates. Significant differences between vehicle- and verum-treatment are indicated by asterisks (⁄p < 0.05, area under the curve compared by ANOVA with Dunnett’s post hoc test). (D) Oxidative glutamate toxicity increases the concentration of cGMP in the intracellular- and extracellular spaces. 165,000 cells were seeded in 6-well plates and after 24 h, 5 mM of glutamate was added for 8 h. Intracellular (cells) and extracellular (sup.) cGMP concentrations were measured by ELISA. The bar graphs represent the mean ± SEM of two independent experiments, each performed in triplicates.

gest that extracellular cGMP is protective, whereas intracellular cGMP is detrimental. 3.3. Extracellular glutamate induces cGMP production and transport to the extracellular space To test the hypothesis that intracellular cGMP is transported to the extracellular space where it might exert its protective effect on the surrounding cells in a paracrine manner, we quantified cGMP within the cell and in the supernatant of cells after 8 h of treatment with glutamate, when most cells are committed to die during the following 24 h but are still viable. We found approximately 2-fold more extracellular than intracellular cGMP in the cells not treated with glutamate, indicating that cGMP is indeed transported out of the cell. Addition of glutamate increased both intracellular and extracellular cGMP levels, however this was not statistically significant (Fig. 2D), suggesting that the extracellular cGMP might be rapidly degraded and that this is influenced or even increased by glutamate treatment. 3.4. The metabolites of cGMP, GMP and Guanosine are even more protective than cGMP We therefore analyzed the protective properties of guanosine monophosphate (GMP) and guanosine (GUA), which are both degradation products of cGMP (Francis et al., 2011; Saute et al., 2006). As shown in Fig. 3A, both substances proved to be even more protective against glutamate than cGMP, for which the protective activity was only small and not very robust. Furthermore, it must be stated that compared to the direct antioxidant NAC, the protection brought about by cGMP and its degradation products was less

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Fig. 3. The cGMP metabolites GMP and guanosine are even more protective than cGMP. (A) 5  103 HT22 cells or (B) 1  105 primary cortical cultures derived from E14 rats were directly seeded onto 96-well plates and treated with vehicle or 0.1 mM of cGMP, GMP, GUA or 2 mM NAC as the positive control (only in A) followed by glutamate 24 h later. Viability was quantified another 24 h later by the CTB assay. Significant differences between vehicle- and verum-treated cells are indicated by asterisks (⁄p < 0.05, area under the curve compared by ANOVA with Dunnett’s post hoc test). (C) 5000 HT22 cells were seeded into 96-well plates and treated with vehicle or 0.1 mM GMP or GUA in the presence of 200 lM of the ectodiphosphatase inhibitor alpha-ß-methyleneadenosine 50 -diphosphate (Inh). Glutamate in the indicated concentrations was added 24 h later and viability quantitated 24 h later again by the CTB assay. All graphs represent mean ± SEM of three independent experiments, each performed in triplicates, and significant differences between vehicle- and verum-treated cells are indicated by asterisks (⁄p < 0.05, area under the curve compared by ANOVA with Dunnett’s post hoc test).

pronounced (Fig. 3A). GUA was the only degradation product capable of also protecting immature primary neuronal cortical cultures that do not yet express ionotropic glutamate receptors and succumb to glutamate via the same mechanisms as described for the HT22 cells (Murphy et al., 1989) (Fig. 3B). Interestingly, the ecto-diphosphatase inhibitor alpha-ß-methyleneadenosine 50 diphosphate (AOPCP), which inhibits the conversion of GMP to GUA, attenuated the protection mediated by GMP but had no effect on GUA, suggesting that GMP needs to be converted to GUA to protect (Fig. 3C).

3.5. The protective effect of cGMP and metabolites is not mediated by the PI3K/Akt pathway or ERK1/2 phosphorylation In hippocampal slice cultures, GUA has been reported to protect against in vitro ischemia using oxygen glucose deprivation (DalCim et al., 2011). Here, GUA provided protection via activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway (Dal-Cim et al., 2011). We therefore investigated whether the protective activity of cGMP, GMP and guanosine would be inhibited by coincubation with 10 lM of the well-established PI3K inhibitor LY294002, but observed no effect of LY294002 on the protective activity of the three agents (Fig. 4A). We also found no significant changes in the relative phosphorylation of the serine/threonine kinase Akt or glycogen synthase kinase 3b (GSK3b), both downstream markers of PI3K activity, although GMP and GUA showed

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Fig. 4. Protective effect of cGMP derivatives is not mediated by PI3K or Akt phosphorylation. (A) 5  103 HT22 cells were seeded into 96-well plates and treated with vehicle or 0.1 mM cGMP, GMP or guanosine with additional treatment by the PI3-kinase inhibitor LY294002 (10 lM) or vehicle followed by 1.25 mM glutamate 24 h later. Viability was quantified 24 h later by the CTB assay. Bar graphs represent the mean ± SEM of three independent experiments, each performed in triplicates. (B and C) 5  105 HT22 cells were seeded onto 10 cm dishes and treated after 24 h with 0.1 mM cGMP, GMP and GUA for the indicated time. The cells were harvested and cytosolic extracts were separated by SDS–PAGE, blotted and hybridized with antibodies against (B) phosphorylated Akt (pAkt) and phosphorylated GSK3b (pGSK3b), which also recognized pGSK3a, or (C) with antibodies against phosphorylated ERK1/2. Total Akt and GSK3b or total ERK1/2 served as loading controls, respectively. Representative blots of two independent experiments are shown. Autoradiographs were quantified by densitometry and the results of the two independent experiments are shown below (all p > 0.05, ANOVA with Dunnett’s post hoc test).

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Fig. 5. Effect of cGMP derivatives on cellular GSH levels and system xc activity. (A) Cells were seeded in 6-well plates with or without 0.1 mM cGMP, GMP or guanosine at a density of 165.000 cells per well. After 24 h, cells were treated with 2.5 mM glutamate or vehicle and 8 h later GSH was measured enzymatically, normalized to protein and the amount present in vehicle-treated cells. (B) 25  105 HT22 cells were plated onto 24-well plates in the presence of vehicle or 0.1 mM cGMP, GMP or GUA. Cells were grown for 24 h before system xc activity was measured as homocysteate-sensitive 3H-glutamate uptake (⁄p < 0.05, ANOVA with Dunnett’s post hoc test). (C) Cells were seeded in 6-well plates with or without 0.1 mM cGMP, GMP or guanosine at a density of 165.000 cells per well. After 24 h, cells were harvested and relative mRNA abundance was assessed by quantitative real-time PCR. The bar graph shows the pooled results of 3 independent experiments performed in triplicates (⁄p < 0.05, t-test). (D) HT22 cells were plated at a density of 8.8  105 cells per 10 cm dish. After 24 h, the cells were treated with 0.1 mM cGMP, GMP or GUA for 24 h before cells were harvested and immunoblotting of the nuclear fraction was performed. The bar graphs show the densitometric analysis of the results of 3 independent immunoblots (all p > 0.05, ANOVA with Dunnett’s post test).

a slight, non-specific tendency to induce a short-lived increase in Akt phosphorylation (Fig. 4B). Others have shown that the protective actions of GUA are mediated through activation of the MAPK/ ERK pathway (Tarozzi et al., 2010; Thauerer et al., 2012; Tomaselli

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xc

3.6. cGMP derivatives induce system activity and increase intracellular GSH in the presence of glutamate During oxidative glutamate toxicity in HT22 cells, the cystine/ glutamate antiporter system xc is blocked by the high concentrations of extracellular glutamate resulting in a subsequent GSH depletion due to lack of cystine. Thus, we quantified intracellular GSH in HT22 cells after 8 h of glutamate treatment with the nucleosides or vehicle. Indeed, cGMP, GMP and GUA increased intracellular GSH in the presence of 2.5 glutamate 2-, 4.3- and 4.6-fold, respectively (Fig. 5A, p < 0.05 ANOVA with Dunnett’s post test). A similar pattern was observed when system xc activity was measured as homocysteate-sensitive 3H-glutamate uptake. Again, GUA had the most prominent effect with a 136% increase that was statistically significant (Fig. 5B, p < 0.05 ANOVA with Dunnett’s post test). In line with this small but significant increase in activity, quantitative real-time PCR revealed a 1.2-fold upregulation of xCT, the functional subunit of system xc (Fig. 5C, p < 0.05 t-test). However, the nuclear factors Nrf2 and ATF4, which regulate the expression of xCT and thereby upregulate system xc activity (Lewerenz and Maher, 2009), were not increased in nuclear fractions after incubation with GUA (Fig. 5C), suggesting that the effect

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et al., 2005), however, we found no activation of ERK phosphorylation in HT22 cells in response to cGMP, GMP or GUA treatment (Fig. 4C). Thus, it is rather unlikely that the nucleosides protect against glutamate by activation of either of these two neuroprotective pathways.

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whereas cGMP had no effect (Fig. 6A). Substances such as the calcium channel blocker cobalt chloride (Tan et al., 1998) and dopamine D4 receptor agonists (Ishige et al., 2001) that protect against oxidative glutamate toxicity when added this late after glutamate addition have been repeatedly reported to inhibit the cGMP-dependent calcium influx. If the inhibition of the cGMPdependent Ca2+ influx was the major protective mechanism of cGMP derivatives in HT22 cells, they should protect against direct activation of this Ca2+ influx when it is induced by treatment with cell-permeable stable cGMP analogs (Li et al., 1997a). Indeed, GMP and particularly GUA robustly protected HT22 cells against treatment with the stable and cell-permeable cGMP analog 8-pCPTcGMP (Fig. 6B). Moreover, a similar effect was observed when the phosphodiesterase-5 inhibitor zaprinast was used to increase cellular cGMP and induce cells death (Fig. 6C). 3.8. GUA inhibits the final toxic Ca2+ influx during oxidative glutamate toxicity To test whether the capacity of GUA to protect against glutamate really involves inhibition of the toxic Ca2+ influx, we transfected HT22 cells with the Ca2+ sensor GCaMP5. This sensor exhibits a Ca2+-dependent fluorescence signal, which we normalized to the fluorescence of a monomeric red fluorescing protein (mRFP) that was expressed from the same vector as a control. The GCaMP5/ mRFP ratio was quantified in an automated high-content imaging microscope every 15 min from 2 h until 15 h after addition of glutamate. Using this method, we were able to demonstrate a Ca2+ overload occurring as early as 2 h after glutamate addition and starting to gain momentum at 7 h. Some cells showed an increased Ca2+ content for hours, whereas others reverted to baseline within minutes before disintegrating completely, making it difficult to quantitate the Ca2+ overload at a specific time point (Fig. 7A). To analyze this overload, we therefore divided the maximal GCaMP5/mRFP ratio by the average ratio of the same cell and used the average value obtained from vehicle treated cells as threshold. Cells with values above this threshold were considered as Ca2+peak positive. This revealed that only guanosine-treated cells had a significant reduction in peaking cells over the whole time course (Fig. 7B). 4. Discussion We identified extracellular cGMP and even more so its metabolites GMP and GUA as being protective compounds in the paradigm of oxidative glutamate toxicity. Oxidative glutamate toxicity is an excellent model for studying neuronal cell death in response to endogenous oxidative stress as ROS are not applied externally as in other models of oxidative stress-induced cell death such as hydrogen peroxide, but generated endogenously within the cell (Albrecht et al., 2012). The results obtained in the HT22 cell line have been reproduced in primary cultures (Albrecht et al., 2012; Lewerenz et al., 2009; van Leyen et al., 2008) and the protective substances identified with this model have shown efficacy in animal models for various diseases including stroke, Alzheimer’s disease (Lipski et al., 2007; Maher et al., 2007) and multiple sclerosis (Melzer et al., 2008). Montoliu and colleagues have reported protection from glutamate toxicity by extracellular cGMP in mature cerebellar neurons while the cell permeable 8-Br-cGMP was toxic (Montoliu et al., 1999). In their study, extracellular cGMP already provided protection at nanomolar concentrations and the observed protection was stronger than in our study, where 0.1 cGMP did not protect from high concentrations of 5 mM glutamate and the protection was not very robust. Interestingly, in Montoliu’s study the phosphodi-

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esterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and zaprinast had protective effects. This finding might be explained by an export of cGMP from these cells as in this study the intracellular cGMP concentration remained constant while the extracellular concentration went up. In contrast with Montoliu’s findings, the selective phosphodiesterase five inhibitor zaprinast had toxic effects in the HT22 cells used in our study. In line with this, we observed no increase of extracellular cGMP upon treatment with 100 lM zaprinast (not shown), presumably due to a less active export. The exact mode of action of cGMP during oxidative stress is, however, only poorly understood. During oxidative glutamate toxicity, the levels of intracellular cGMP increase (Li et al., 1997a) and should accumulate in the extracellular space because cGMP is rapidly transported to the outside through members of the multidrug resistance protein family of transporters (Sager, 2004) or after cell lysis. There, extracellular cGMP is probably rapidly degraded and exerts protective effects in a concentration-dependent manner via its metabolites. This is important as it means that during pathological conditions involving oxidative stress, cGMP can be released to protect surrounding cells and prevent further damage. Increasing cGMP transport might be a possible rationale for antioxidative stress therapy. Cellular cGMP is generated from guanosine triphosphate (GTP) by guanylate cyclases, serves as a second messenger and is readily degraded to GMP by phosphodiesterases (Francis et al., 2011). GMP is then either phosphorylated to GTP or degraded to GUA (Saute et al., 2006). We have demonstrated that the degradation products of cGMP, GMP and GUA have even stronger protective properties than cGMP and that inhibition of GMP conversion to GUA attenuates its protective capacity, although the effect of GMP itself in these experiments was rather weak. This might be of interested for any further studies on the protective effects of cGMP and GMP as the observed effects may at least in part be mediated by their degradation product, GUA. The variability of the protective capacity of cGMP observed in our study was possibly due to differences in the conversion to GUA. In addition, the fact that cGMP did not protect from high concentrations of 5 mM glutamate may be explained by a reduced conversion to GUA, particularly as GUA did provide protection from these concentrations. GUA was reported to protect against staurosporine- and amyloid-beta toxicity by activation of PI3K, Akt and ERK (Di Iorio et al., 2004; Tarozzi et al., 2010) and purine nucleosides such as GUA, adenosine and inosine were reported to protect against hypoxia by activation of ERK (Thauerer et al., 2010; Tomaselli et al., 2005). We found no evidence of the involvement of PI3K or its downstream kinases Akt and GSK3b, or of the ERK pathway in the protective mechanism of cGMP or its degradation products in the HT22 cells. We did, however, observe a small but significant increase in the activity of system xc and an overexpression of its functional subunit, xCT, on mRNA level after incubation with GUA, indicating a transcriptional mechanism. We did not observe an increase of the nuclear factors Nrf2 or ATF4, which upregulate xCT, in nuclear fractions after incubation with GUA, probably because the amount of nuclear translocation was too small to be detectable by immunoblotting or because xCT and system xc are upregulated by other, yet unknown mechanisms. In any case, these effects were rather small compared to the protective capacities of GUA, suggesting additional mechanisms. Furthermore, the kinetics of GUA- and GMP-mediated protection in oxidative glutamate toxicity with protective effects seen even at 6 h after glutamate addition strongly argue against a transcriptional mechanism as the sole mode of action of GUA in our cells because this is typically not effective at such a late time point (Albrecht et al., 2012; Lewerenz et al., 2009). We conclude that GMP and GUA do increase system xc activity by transcriptional mechanisms involving xCT, leading

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to elevated GSH levels and protection from oxidative stress, although the protection time course and small degree of regulation suggest additional non-transcriptional mechanisms. Interestingly, GMP and GUA even served to protect against the toxic effects brought about by intracellular cGMP because they dampened the toxic effects of the cell-permeable and more stable 8-pCPT-cGMP as well as an inhibition of cGMP degradation by zaprinast. These toxic effects of intracellular cGMP are most likely due to a cGMP-dependent calcium influx (Henke et al., 2012) hat is apparently inhibited by GMP and GUA. We therefore analyzed cellular calcium levels and found a decrease of calcium peaking cells during oxidative glutamate toxicity following treatment with guanosine. This finding might simply be due to the strong protection by GUA but the observation that GUA can be added at a late stage in the cell death process and still provide protection strongly suggests that GUA has a direct effect on the Ca2+ homeostasis, which may account for a significant part of its protective capacities. These late effects during oxidative glutamate toxicity suggest that extracellular GUA may block the Orai1 Ca2+ channel that is opened by intracellular cGMP and causes the final toxic Ca2+ influx before cell death (Henke et al., 2012). In summary, we conclude that part of the protective activity of extracellular cGMP is mediated by its degradation products, GMP and GUA, which have dual effects in our cell culture model. They inhibit the toxic Ca2+ influx during oxidative glutamate toxicity and activate system xc in an Nrf2- and ATF4-independent manner, leading to increased GSH levels under glutamate stress. Acknowledgements This work was funded by the Stiftung für Altersforschung der Heinrich-Heine Universität Düsseldorf Grant J-10-16-53 to Philipp Albrecht and the Dr. Kurt und Irmgard Meister-Stiftung to Axel Methner. The authors thank Christie Dietz for language revisions. References Albrecht, P., Lewerenz, J., Dittmer, S., Noack, R., Maher, P., Methner, A., 2010. Mechanisms of oxidative glutamate toxicity: the glutamate/cystine antiporter system xc as a neuroprotective drug target. CNS Neurol. Disord. Drug Targets 9, 373–382. Albrecht, P., Bouchachia, I., Zimmermann, C., Hofstetter, H.H., Kovacs, Z., Henke, N., Lisak, D., Issberner, A., Lewerenz, J., Maher, P., Goebels, N., Quasthoff, K., Mausberg, A.K., Hartung, H.P., Methner, A., 2012. Effects of dimethyl fumarate on neuroprotection and immunomodulation. J. Neuroinflammation 9, 163. Browne, S.E., Beal, M.F., 2006. Oxidative damage in Huntington’s disease pathogenesis. Antioxid. Redox Signaling 8, 2061–2073. Choi, J.H., Kim, D.H., Yun, I.J., Chang, J.H., Chun, B.G., Choi, S.H., 2007. Zaprinast inhibits hydrogen peroxide-induced lysosomal destabilization and cell death in astrocytes. Eur. J. Pharmacol. 571, 106–115. Costello, D.J., Delanty, N., 2004. Oxidative injury in epilepsy: potential for antioxidant therapy? Expert Rev. Neurother. 4, 541–553. Dal-Cim, T., Martins, W.C., Santos, A.R., Tasca, C.I., 2011. Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca(2)+-activated K+ channels, phosphatidilinositol-3 kinase/ protein kinase B pathway activation and glutamate uptake. Neuroscience 183, 212–220. Di Iorio, P., Ballerini, P., Traversa, U., Nicoletti, F., D’Alimonte, I., Kleywegt, S., Werstiuk, E.S., Rathbone, M.P., Caciagli, F., Ciccarelli, R., 2004. The anti apoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes. Glia 46, 356–368. Dittmer, S., Sahin, M., Pantlen, A., Saxena, A., Toutzaris, D., Pina, A.L., Geerts, A., Golz, S., Methner, A., 2008. The constitutively active orphan G-protein-coupled receptor GPR39 protects from cell death by increasing secretion of pigment epithelium-derived growth factor. J. Biol. Chem. 283, 7074–7081. Dringen, R., 2000. Glutathione metabolism and oxidative stress in neurodegeneration. Eur. J. Biochem. 267, 4903. Francis, S.H., Blount, M.A., Corbin, J.D., 2011. Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol. Rev. 91, 651–690. Henke, N., Albrecht, P., Pfeiffer, A., Toutzaris, D., Zanger, K., Methner, A., 2012. Stromal interaction molecule 1 (STIM1) is involved in the regulation of mitochondrial shape and bioenergetics and plays a role in oxidative stress. J. Biol. Chem. 287, 42042–42052.

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