Protective effect of the octadecaneuropeptide on hydrogen peroxide-induced oxidative stress and cell death in cultured rat astrocytes

Authors

  • Yosra Hamdi,

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
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    • These authors contributed equally to this study.

  • Olfa Masmoudi-Kouki,

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
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    • These authors contributed equally to this study.

  • Hadhemi Kaddour,

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
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  • Feten Belhadj,

    1. Biological Engineering Unit 99UR09-26, National Institute of Applied Sciences and Technology, Tunis, Tunisia
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  • Pierrick Gandolfo,

    1. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    2. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    3. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • David Vaudry,

    1. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    2. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    3. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • Meherzia Mokni,

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
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  • Jérôme Leprince,

    1. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    2. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    3. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • Raya Hachem,

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
    2. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    3. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    4. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • Hubert Vaudry,

    1. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    2. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    3. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • Marie-Christine Tonon,

    1. Inserm U413/U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
    2. International Associated Laboratory Samuel de Champlain, University of Rouen, Mont-Saint-Aignan, France
    3. European Institute for Peptide Research (IFRMP 23), Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), University of Rouen, Mont-Saint-Aignan, France
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  • Mohamed Amri

    1. Laboratory of Functional Neurophysiology and Pathology, Research Unit and 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, Tunis, Tunisia
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Address correspondence and reprint requests to Pr Mohamed Amri, Laboratory of Functional Neurophysiology and Pathology, 00/UR/08-01, Department of Biological Sciences, Faculty of Science of Tunis, University Tunis El Manar, 2092 Tunis, Tunisia. E-mail: mohamed.amri@fst.rnu.tn and Dr Hubert Vaudry, Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, International Associated Laboratory Samuel de Champlain, Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr

Abstract

J. Neurochem. (2011) 118, 416–428.

Abstract

Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H2O2)-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H2O2 for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H2O2 was associated with morphological modifications that were characteristic of apoptotic cell death. H2O2-treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H2O2. This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H2O2-evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.

Abbreviations used
AD

Alzheimer’s disease

DBI

diazepam-binding inhibitor

DCF

2′,7′-dichlorofluorescein

DPPH

2,2-diphenyl-1-picrylhydrazyl

H2O2

hydrogen peroxide

JC-1

5,5″,6,6″-tetrachloro-1,1″,3,3″-tetraethylbenzimidazolylcarbocyanine iodide

LDH

lactate dehydrogenase

ODN

octadecaneuropeptide DBI33-50

PBS

phosphate-buffered saline

ROS

reactive oxygen species

SOD

superoxide dismutase

TEAC

Trolox equivalent antioxidant capacity

The octadecaneuropeptide (ODN) has been originally isolated from the rat brain and characterized as an endogenous ligand of benzodiazepine receptors (Guidotti et al. 1983). ODN is generated by proteolytic cleavage of an 87-amino acid polypeptide precursor called diazepam-binding inhibitor (DBI) whose gene is mainly expressed in glial cells in the central nervous system (Burgi et al. 1999; Compère et al. 2006). DBI and its processing products are collectively designated by the term endozepines (Tonon et al. 2006). It was initially reported that the endozepines ODN and DBI act as inverse agonists of central-type benzodiazepine receptors (Ferrero et al. 1984). Subsequently, DBI was found to interact also with peripheral-type benzodiazepine receptors (Papadopoulos et al. 1991). More recently, it has been shown that DBI-derived peptides can activate a metabotropic receptor positively coupled to phospholipase C (Patte et al. 1995; Leprince et al. 2001; Marino et al. 2003).

The sequence of the DBI cDNA has now been characterized in several mammalian species and it has been found that the primary structure of ODN has been strongly conserved during evolution (Tonon et al. 2006), suggesting that this peptide plays important biological functions. Indeed, behavioral studies have shown that ODN, acting through central-type benzodiazepine receptors, increases aggressive interactions (Kavaliers and Hirst 1986), induces anxiety and proconflict behavior (De Mateos-Verchere et al. 1998), shortens pentobarbital-induced sleeping time (Dong et al. 1999) and reduces drinking (Manabe et al. 2001) in rodents. Beside, ODN acting through its metabotropic receptor, exerts a potent anorexigenic effect (De Mateos-Verchere et al. 2001; Do Régo et al. 2007). At the cellular level, ODN increases intracellular calcium in cultured rat astrocytes (Gandolfo et al. 1997), stimulates glial cell proliferation (Gandolfo et al. 1999), activates neurosteroid biosynthesis (Do-Rego et al. 2001) and modulates the expression of neuropeptides that control feeding behavior (Compère et al. 2003, 2005). These observations indicate that ODN acts both as an autocrine factor regulating glial cell activity and as a gliotransmitter modulating neurotransmission.

There is now clear evidence that the number of reactive astrocytes and the expression of benzodiazepine receptors increase after various nervous system insults such as ischemia, stroke, neuroinflammatory processes and neurodegenerative diseases (Veiga et al. 2007; Chen and Guilarte 2008). In addition, recent reports indicate that benzodiazepines can prevent brain injury induced by neurotoxic chemicals, ischemia and neurological disorders (Ricci et al. 2007; Corbett et al. 2008). Altogether, these observations suggest that ODN, acting as an endogenous ligand for benzodiazepine receptors, may exert a protective effect on astroglial cells during oxidative stress.

Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death observed in several pathological processes including cerebral ischemia, brain tumor and neurodegenerative diseases (Lotharius et al. 2005; Resende et al. 2008). Astrocytes contain high levels of antioxidant molecules such as vitamins E and C and the antioxidant enzymes Mn- and Cu,Zn-superoxide dismutases (Mn- and Cu,Zn-SOD), catalase and glutathione peroxidase, which play a major neuroprotective role against the deleterious effects of superoxides (Lotharius et al. 2005; Resende et al. 2008). Besides, astrocytes exert neuroprotective effects by providing neurons with substrates for antioxidants notably glutathione (Elekes et al. 1996). Although astrocytes are generally less susceptible to oxidative injury than neurons, there is strong evidence that oxidative stress also alters astrocyte functions (Chen et al. 2001; Choi et al. 2007). In particular, glial cells are extremely vulnerable to hydrogen peroxide (H2O2) and astrocytic apoptosis is observed in brain injuries caused by trauma and ischemia (Takuma et al. 2004; Giffard and Swanson 2005). Reciprocally, cultured astrocytes derived from Cu,Zn-SOD-over-expressing transgenic mice exhibit increased resistance to oxidative stress induced by menadione or oxygen-glucose deprivation (Chen et al. 2001). In situ, astrocytes likely contribute to the overall protection observed during and after ischemia in Cu,Zn-SOD-over-expressing animals (Chen et al. 2001; Manni and Oury 2007). Therefore, protection of astrocytes from oxidative assault appears essential to maintain cerebral antioxidant competence and to prevent neuronal damage as well as to facilitate neuronal recovery.

It has been reported that, during the prenatal period, which corresponds to intense astrocyte generation, high concentrations of DBI mRNA and DBI-derived peptides occur in the rat brain (Malagon et al. 1993; Burgi et al. 1999). On the other hand, the concentration of endozepines in brain and in cerebrospinal fluid is increased in various pathological conditions including Alzheimer’s disease (AD) (Ferrero et al. 1988; Ferrarese et al. 1990). We have recently shown that β-amyloid peptide induces activation of endozepine biosynthesis and release, suggesting that over production of ODN may contribute to astrocyte proliferation associated with AD (Tokay et al. 2005, 2008). Finally, endozepines increase [3H]thymidine incorporation in cultured rat astrocytes (Gandolfo et al. 1999). Altogether, these observations suggest that endozepines may act as neurotrophic factors regulating proliferation and/or survival of astroglial cells under injury conditions. In order to test the hypothesis of a glioprotective action of ODN, we have analyzed the effects of the peptide on several oxidative stress-related parameters in H2O2-treated astrocytes.

Materials and methods

Animals

Animals were handled in accordance with the ethical committee of Tunis University for care and use of animals in conformity with NIH guidelines.

Chemicals

Dulbecco’s modified Eagle’s medium, F12 culture medium, D(+)-glucose, l-glutamine, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, foetal bovine serum and the antibiotic-antimycotic solution were obtained from Gibco (Invitrogen, Grand Island, NY, USA). Fluorescein diacetate-acetoxymethyl ester, Triton X-100, trypsin-EDTA, insulin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and sodium cyanide (NaCN), 3-aminotriazole and Trolox were purchased from Sigma Aldrich (St. Louis, MO, USA). Lactate dehydrogenase (LDH; EC 1.1.1.27) assay kit was commercialized by Bio-Maghreb (Ariana, Tunisia). 5-6-Chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and the mitochondrial potential sensor 5,5″,6,6″-tetrachloro-1,1″,3,3″-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained from Molecular Probes (Eugene, OR, USA). Apo-ONE Homogeneous Caspase-3/7 assay kit was supplied by Promega (Charbonnières, France). Bovine liver catalase and dl-epinephrine were kindly provided by Pr. F. Limam (National Science and Technology Park, Borj Cedria, Tunisia). Rat ODN (QATVGDVNTDRPGLLDLK) was synthesized by using the standard Fmoc procedure, as previously described (Leprince et al. 2001). All other reagents were of A grade purity.

Cell culture

Secondary cultures of rat cortical astrocytes were prepared as previously described (Brown and Mohn 1999) with minor modifications. Briefly, cerebral hemispheres from 1- or 2-day-old Wistar rats were collected in Dulbecco’s modified Eagle’s medium/F12 (2 : 1; v/v) culture medium supplemented with 2 mM glutamine, 1% insulin, 5 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 0.4% glucose and 1% of the antibiotic-antimycotic solution. The tissues were dissociated mechanically with a syringe equipped with a 1-mm gauge needle, and filtered through a 100-μm sieve (Falcon, Franklin Lakes, NJ, USA). Dissociated cells were resuspended in culture medium supplemented with 10% foetal bovine serum, plated in 175-cm2 flask (Greiner Bio-one GmbH, Frickenhausen, Germany) and incubated at 37°C in a 5% CO2/95% O2 atmosphere. When cultures were confluent, astrocytes were isolated by shaking overnight the flasks with an orbital agitator. Adhesive cells were detached by trypsination and pre-plated for 5 min to discard contaminating microglial cells. Then, the non-adhering astrocytes were harvested and plated on 35-mm Petri dishes at a density of 0.3 × 106 cells/mL. The cells were incubated at 37°C in a humid atmosphere (5% CO2). After 5 days (DIV5), more than 99% of cells were labeled with antibodies against glial fibrillary acidic protein (Castel et al. 2006). All experiments were performed on 5- to 7-day-old secondary cultures.

Measurement of cell cytotoxicity

DIV5–6 cells were incubated at 37°C with fresh serum-free medium in the absence or presence of test substances. At the end of the incubation, the cytotoxicity of H2O2 on astrocytes was determined by measurement of LDH activity in culture medium. The amount of LDH released into medium was measured by LDH assay kit (Bio-Maghreb) according to the manufacturer’s instructions. The results were expressed as a percentage of total LDH release after cell lysis with 1% Triton X-100 in phosphate-buffered saline (PBS, 0.1 M, pH 7.4).

Measurement of cell survival

Cultured cells were incubated at 37°C for 1 h with fresh serum-free culture medium in the absence or presence of H2O2 with or without ODN. Then, astrocytes were incubated for 8 min with 15 μg/mL fluorescein diacetate-acetoxymethyl ester, rinsed twice with PBS and lysed with a Tris/HCl solution containing 1% sodium dodecyl sulfate. Fluorescence was measured with excitation at 485 nm and emission at 538 nm using a FL800TBI fluorescence microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Measurement of intracellular reactive oxygen species formation

Reactive oxygen species were detected by measuring the fluorescence of 2′,7′-dichlorofluorescein (DCF) which is derived from the deacetylation and oxidation of the non-fluorescent compound DCFH2-DA. Cells seeded into 24-well plates were first incubated with 10 μM cell-permeant DCFH2-DA in serum-free loading medium for 30 min at 37°C, washed twice with PBS, and then exposed to H2O2 with or without ODN. Fluorescence was measured with excitation at 485 nm and emission at 538 nm using a FL800TBI fluorescence microplate reader (Bio-Tek Instruments).

Measurement of mitochondrial activity

Mitochondrial membrane potential was quantified using the JC-1 probe. Cells seeded into 24-well plates were subjected to various treatments, incubated in the presence of the JC-1 probe (10 μg/mL) at 37°C for 15 min and then washed twice with PBS. In healthy astrocytes, the intact membrane potential allows the lipophilic dye JC-1 to enter into the mitochondria where it accumulates and aggregates producing an intense orange signal. In dead cells, where the mitochondrial membrane potential collapses, the monomeric JC-1 remains cytosolic and stains the cell in green. Fluorescence was measured with excitations at 485 (monomer) and 535 nm (aggregates), and emissions at 530 (green) and 590 nm (orange), respectively.

Measurement of caspase 3 activity

Cultured astrocytes were incubated at 37°C for 1 h with fresh serum-free culture medium in the absence or presence of H2O2 with or without ODN. At the end of the incubation, cells were washed twice and caspase 3 activity was measured with a caspase 3 assay system (Apo-ONE Homogeneous Caspase-3/7 kit, Promega). Briefly, 100 μL of the cell suspension was incubated with 100 μL of kit buffer and caspase substrate in 96-well plates. The caspase activity was calculated from the slope of the fluorescence measured every 15 min for 3 h with excitation at 485 nm and emission at 530 nm, and expressed as a percentage of the control.

Measurement of antioxidant enzyme activities

Cultured cells were incubated at 37°C for 1 h with fresh serum-free medium in the absence or presence of test substances. At the end of the incubation, culture medium was removed, cells were washed twice with PBS and then homogenized in the same solution at 4°C. Cells were harvested by centrifugation (350 g, 4°C, 10 min) and the cell pellet was resuspended in 50 μL of ice-cold lysing buffer containing 50 mM Tris–HCl (pH 8), 10 mM EDTA, 100 μM phenylmethyl-sulfonylfluoride and 1% Triton X-100. Samples were then centrifuged (16 000 g, 20 min, 4°C) and supernatants were finally stored at −20°C until enzyme activity determinations.

The activity of SOD was measured using a spectrophotometric assay which consists in measuring epinephrine autoxidation induced by superoxide anion. Samples, prepared as described above, were incubated for 3 min with a mixture containing bovine catalase (0.4 U/μL), epinephrine (5 mg/mL) and Na2CO3/NaHCO3 buffer (62.5 mM, pH 10.2). The oxidation of epinephrine was measured at 480 nm with a Bio-Rad spectrophotometer (Bio-Rad Laboratories, Philadelphia, PA, USA).

Proteins (60 μg) contained in cellular extracts were resolved on 12% polyacrylamide gel electrophoresis and detection of SOD activity was revealed by staining gels for 30 min in the dark with a mixture containing 2.5 mM nitroblue tetrazolium, 28 mM N,N,N″,N″-tetramethyl-ethylenediamine and 28 mM riboflavin.

Catalase activity was determined on the basis of the decrease of H2O2. Samples, prepared as described above, were mixed with 30 mM H2O2 in PBS. The disappearance of H2O2 was measured at 240 nm for 180 s at 30-s intervals. Catalase activity was calculated using the extinction coefficient of 40/mM/cm for H2O2.

Proteins (80 μg) contained in cellular extracts were resolved on 7% polyacrylamide gel electrophoresis. Gels were first incubated for 30 min in 3.27 mM H2O2 and detection of catalase activity was revealed by staining gels with a mixture containing 1% (w/v) potassium ferricyanide and 1% (w/v) ferric chloride.

Radical scavenging and antioxidant activity assays

The radical scavenging capacity and the antioxidant activity of ODN were evaluated by using the DPPH method and a Trolox equivalent antioxidant capacity (TEAC) assay, respectively. Ascorbic acid was used as a reference compound. Briefly, ODN or ascorbic acid solution was mixed with 10−10 M DPPH ethanolic solution (1 : 1). After a 30-min incubation in the dark at 25°C, the absorbance of the mixture was measured at 517 nm. The antioxidant activity of ODN was expressed as mM TEAC, using Trolox (0.05–1 × 10−3 M) as standard, and compared to that of ascorbic acid.

Measurement of SOD and catalase mRNA

Cultured cells were incubated at 37°C for 5 min with fresh serum-free medium in the absence or presence of ODN. At the end of the incubation, the culture medium was removed and the cells were washed twice with PBS. Total RNA was extracted by the guanidine thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987) using Tri reagent. Approximately 1 μg of total RNA was reverse transcribed by ImPROM-IITM reverse transcriptase (50 U/μL, Promega) using random hexanucleotides (50 μM) as primers. Quantitative-PCR was performed on 15 ng of total cDNA using 1× SYBR Green universal PCR Master mix (Applied Biosystem, Courtaboeuf, France) containing dNTPs, MgCl2, AmpliTaq Gold DNA polymerase, forward (5′-CCTTCTTGTTCTGCAACCTGCTA-3′) and reverse (5′-CCGGACTCTCCGGTATCTGA-3′) SOD primers or forward (5′-CCACAGTCGCTGGAGAGTCA-3′) and reverse (5′-GTTTCCCACAAGGTCCCAGTT-3′) catalase primers (300 nM each; Proligo, Paris, France). cDNA was first heated at 50°C for 2 min and 95°C for 10 min, followed by 40 reaction cycles at 95°C for 15 s and 60°C for 1 min, using the ABI Prism 7000 sequence detection system (Applied Biosystem). The amount of SOD and catalase cDNA in each sample was calculated by the comparative threshold cycle (Ct) method and expressed as 2exp(−ΔΔCt) using glyceraldehyde-3-phosphate dehydrogenase as an internal control.

Statistical analysis

Data are presented as the mean ± SEM from three independent experiments performed in quadruplicate or quintuplicate. Statistical analysis of the data was performed by using Student’s t-test and anova, followed by Bonferroni’s test. A p-value of 0.05 or less was considered as statistically significant.

Results

Effect of ODN on H2O2-induced astrocyte death

Incubation of astrocytes with graded concentrations of H2O2 induced a dose-dependent increase of LDH levels in the culture medium. Addition of ODN (10−10 M) to the culture medium totally suppressed the effect of moderate concentrations of H2O2 (100–400 μM) and markedly attenuated the effect of higher concentrations of H2O2 (500 and 800 μM) on LDH release (Fig. 1).

Figure 1.

 Effect of ODN on H2O2-induced release of lactate dehydrogenase from cultured rat astrocytes. Cells were pre-incubated for 10 min in the absence or presence of 10−10 M ODN and then incubated for 1 h with medium alone (□) or with graded concentrations of H2O2 (100–800 μM) in the absence (•) or presence of ODN (bsl00001). The results are expressed as percentages of lactate dehydrogenase (LDH) released in Triton-lysed cells, quantified by using an LDH activity kit assay. Each value is the mean (± SEM) calculated from at least 10 different wells from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01; ***< 0.001 and NS not statistically different from control. #< 0.05; ###< 0.001 vs. H2O2-treated cells.

Examination of cultures by phase-contrast microscopy revealed that ODN-treated cells displayed a flat polygonal morphology similar to that of untreated-astrocytes (Fig. 2a and d), while, H2O2 induced cell shrinkage and the appearance of long thin processes (Fig. 2b and c). Pre-treatment of cells with ODN (10−10 M) totally prevented the morphological changes induced by 300 μM H2O2 (Fig. 2e) but was unable to suppress the effect of 500 μM H2O2 (Fig. 2f).

Figure 2.

 Phase-contrast images illustrating the effect of ODN on H2O2-induced changes in morphology of cultured rat astrocytes. Cells were pre-incubated for 10 min in the absence (a–c) or presence of 10−10 M ODN (d–f), and then incubated for 1 h with 300 μM (b and e) or 500 μM H2O2 (c and f). Scale bar = 50 μm.

Effect of ODN on H2O2-induced intracellular ROS accumulation

To examine whether ODN could also block H2O2-induced intracellular ROS accumulation, astrocytes were labeled with CMH2DCFDA which forms the fluorescent DCF compound upon oxidation with ROS. Incubation of cultured astrocytes with graded concentrations of H2O2 (100–800 μM) for 1 h, induced a dose-dependent increase in DCF fluorescence intensity (Fig. 3). Addition of ODN (10−10 M) to the incubation medium totally blocked the effect of moderate concentrations of H2O2 (from 100 to 300 μM) and partially reversed the effect of higher concentrations of H2O2 (500 and 800 μM) on DCF formation. In contrast, ODN by itself was unable to produce DCF from DCFH2-DA in the absence of cells (Fig. 3, inset).

Figure 3.

 Effect of ODN on H2O2-induced intracellular accumulation of ROS in cultured rat astrocytes. Cells were pre-incubated for 10 min in the absence or presence of 10−10 M ODN, and then incubated for 1 h with medium alone, ODN or with H2O2 (100–800 μM) without or with ODN. Cellular ROS were detected by measuring the fluorescence of 2′,7′-dichlorofluorescein (DCF), and the results are expressed as a percentage of fluorescence with respect to that of control cells (F0). Each value is the mean (± SEM) calculated from at least four different wells from five independent cultures. anova followed by the Bonferroni’s test. **< 0.01; ***< 0.001 and NS not statistically different from control; #< 0.05; ##< 0.01; ###< 0.001 vs. H2O2-treated cells and ns not statistically different from H2O2 alone. Inset, DCF fluorescence intensity, in the absence of cells, in wells containing medium with CMH2DCFDA alone (M) or with 10−10 M ODN (ODN). Each value is the mean (± SEM) calculated from at least six different wells. anova followed by the Bonferroni’s test. NS, not statistically different from medium alone.

Effect of ODN on H2O2-induced alteration of mitochondrial activity and activation of caspase 3

Considering the major effect of ROS in the permeabilization of the mitochondrial outer membrane, we examined the effect of ODN on the integrity of mitochondria by visualizing the membrane potential using the fluorescent ratiometric probe JC-1. Treatment of astrocytes with graded concentrations of H2O2 (100–800 μM) for 1 h, induced a dose-dependent decrease of the red signal (590 nm); instead, green fluorescence (530 nm) was observed in cell bodies, indicating that the mitochondrial integrity was severely altered by H2O2 (Fig. 4). Incubation of astrocytes with ODN (10−10 M) had no effect on the 590/530 nm fluorescence ratio, but totally suppressed the deleterious effects of moderate concentrations of H2O2 (from 100 to 400 μM) on mitochondrial membrane potential (Fig. 4). At higher concentrations of H2O2 (500 and 800 μM), however, ODN was unable to prevent the decrease of mitochondrial activity. H2O2-induced reduction of membrane potential was associated with an increase of caspase 3 activity (Fig. 5). Addition of ODN (10−10 M) in the incubation medium totally suppressed the stimulatory effect of low concentrations (100–300 μM) of H2O2 on caspase 3 activity and markedly reduced the effects of higher concentrations (Fig. 5).

Figure 4.

 Effect of ODN on H2O2-induced alteration of mitochondrial membrane potential in cultured rat astrocytes. Cells were pre-incubated for 10 min in the absence or presence of 10−10 M ODN, and then incubated for 1 h with medium alone, ODN or with H2O2 (100–800 μM) without or with ODN. Mitochondrial transmembrane potential was assessed by using the JC-1 probe, and the ratio of fluorescence emissions 590 nm vs. 530 nm was measured as an index of the mitochondrial activity. The results are expressed as percentage of control. Each value is the mean (± SEM) calculated from at least four different wells from three independent cultures. anova followed by the Bonferroni’s test. **< 0.01; ***< 0.001 and NS not statistically different from control; ##< 0.01 vs. H2O2-treated cells; ns not statistically different from H2O2 alone.

Figure 5.

 Effect of ODN on caspase 3 activation induced by H2O2 in cultured rat astrocytes. Cells were pre-incubated for 10 min in the absence or presence of 10−10 M ODN, and then incubated for 1 h with medium alone or with graded concentrations of H2O2 (100–500 μM) in the absence or in presence of ODN. Caspase 3 activity was measured by caspase substrate, Z-DEVD-rhodamine 110, fluorescence. The results are expressed as percentages of the control value. Each value is the mean (± SEM) calculated from four different wells from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01; ***< 0.001 and NS not statistically different from control.

Effect of ODN on antioxidant enzyme activities in cultured astrocytes

To further explore the mechanism involved in the protective action of ODN we monitored the activities of the two antioxidant enzymes SOD and catalase in astrocytes. Exposure of astrocytes to graded concentrations of ODN (10−14 M to 10−10 M) for 10 min, resulted in a dose-dependent increase in SOD activity (Fig. 6a). The half-maximum effect of ODN was observed at a concentration of 1.7 × 10−12 M and the maximum stimulation of SOD activity (< 0.001) was obtained at a concentration of 10−10 M. ODN also induced a concentration-dependent stimulation of catalase activity, with an EC50 value of 1.4 × 10−12 M (Fig. 6b). To exclude that ODN could by itself possess free radical scavenging activity, and thus could inhibit oxidation reaction occurring in SOD and catalase assays, we measured the possible antioxidant activity of ODN. The DPPH scavenging potential of ODN, for concentrations ranging from 10−12 M to 10−6 M, was similar to that of the culture medium alone and largely lower than that of ascorbic acid (95.20 ± 1.50%), a strong free radical scavenger compound (Fig. 6c). Similarly, ODN, at all concentrations tested, was devoid of antioxidant activity in the TEAC assay (Fig. 6c).

Figure 6.

 Effect of graded concentrations of ODN on SOD and catalase activities in cultured rat astrocytes. Cells were incubated for 10 min with increasing concentrations of ODN (10−14 M to 10−10 M). SOD activity was determined by measurement of epinephrine autoxidation induced by superoxide anion, and catalase activity by the decrease of H2O2. The results are expressed as a percentage of SOD (a) or catalase (b) activity with respect to control. Each value is the mean (± SEM) calculated from at least four different dishes from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; ***< 0.001 and NS not statistically different from control. Mean basal SOD and catalase activities in these experiments were 22.4 ± 1.7 and 0.93 ± 0.1 U/mg protein. (c) The capacity of medium alone (M), ODN (10−12 M to 10−6 M) and ascorbic acid (Asc, 10−10 M; used as antioxidant reference compound) to scavenge the DPPH radical (absorbance measurement) is expressed as a percentage of DPPH radical scavenging activity and Trolox equivalent antioxidant capacity (TEAC). Each value is the mean (± SEM) calculated from two different wells from three independent experiments.

Time-course experiments revealed that ODN (10−10 M) significantly enhanced SOD and catalase activities within 2 min and reached a maximum effect after 10 min and 20 min of incubation, respectively (Fig. 7a and b). Thereafter, the stimulatory effect of ODN on SOD and catalase activities gradually declined and vanished after 90 and 45 min, respectively, after the onset of ODN administration. Addition of a novel dose of ODN (10−10 M) after 10 min (SOD) or 20 min (catalase) of incubation restored the stimulatory effect of ODN on SOD and catalase activities (Fig. 7). Quantitative-PCR experiments showed that treatment of cultured astrocytes for 5 min with ODN (10−10 M) provoked an increase of SOD and catalase mRNA levels (Fig. 7, insets).

Figure 7.

 Time-course of the effects of ODN on SOD and catalase activities and mRNA levels in cultured rat astrocytes. Cells were incubated in the absence or presence of ODN (10−10 M) for the times indicated. After 10 (a) or 20 min (b) of incubation, a refill of ODN (10−10 M) was added in the incubation medium (arrows). SOD activity was determined by measurement of epinephrine autoxidation induced by superoxide anion, and catalase activity by the decrease of H2O2. The results are expressed as a percentage of SOD (a) or catalase (b) activity with respect to control mediums. Each value is the mean (± SEM) of at least four different dishes from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01; ***< 0.001 and NS not statistically different from control. Mean basal SOD and catalase activities in these experiments were 18.5 ± 1.08 and 0.89 ± 0.13 U/mg protein. Inset, effect of ODN on SOD and catalase mRNA levels in cultured rat astrocytes. Cells were incubated for 5 min in the absence or presence of 10−10 M ODN. Each value, expressed as a percentage of the control, is the mean (± SEM) of at least four different dishes from three independent cultures. Data were corrected using the glyceraldehyde-3-phosphate dehydrogenase signal as an internal control. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01; ***< 0.001 and NS not statistically different from control.

Incubation of astrocytes with graded concentrations of H2O2 (100–800 μM; 1 h) induced a dose-dependent decrease of both SOD and catalase activities. As illustrated in Fig. 8, ODN alone, at a concentration of 10−10 M, provoked a significant increase in SOD and catalase activities. At all concentrations of H2O2 tested, ODN restored SOD and catalase activities above control levels (Fig. 8a and b). Addition of 2 × 10−3M NaCN, a SOD inhibitor, significantly reduced basal enzymatic activity and totally suppressed the stimulatory effects of ODN (Fig. 8c). In a very similar manner, exposure of cells to 10−2 M 3-aminotriazole, a specific catalase inhibitor, strongly decreased basal and ODN-enhanced catalase activity (Fig. 8d). Exposure of astrocytes to 2 × 10−3 M NaCN and 10−2 M 3-aminotriazole, which did not affect cell survival by themselves, totally blocked the protective effect of ODN against H2O2-induced cell death (Fig. 9a). The capacity of NaCN and 3-aminotriazole to block SOD and catalase activities has been confirmed by enzyme activity gel assay (Fig. 9b). Finally, we have examined whether ODN could exert a protective effect on ROS-independent apoptotic cell death. Exposure of cultured astrocytes to acidosis (pH 6.8) for 1 h did not increase the production of ROS (data not shown) but reduced by ∼ 40% the proportion surviving cells. Addition of 10−10 M ODN in the incubation medium was unable to abrogate acidosis-induced astrocyte death (Fig. 9c).

Figure 8.

 Effect of ODN and H2O2 on SOD and catalase activities in cultured rat astrocytes. (a, b) Cells were incubated in the absence or presence of H2O2 (100–800 μM) for 1 h, and 10−10 M ODN was added to the incubation medium during the last 10 min of incubation. (c, d) Cells were pre-incubated for 10 min or 3 h in the absence or presence of the SOD inhibitor NaCN (2 × 10−3 M, c) or the catalase inhibitor 3-aminotriazole (AZT, 10−2 M, d), then incubated in the absence or presence of H2O2 (300 μM) for 1 h, and 10−10 M ODN was added to the incubation medium during the last 10 min of incubation. SOD activity was determined by measurement of epinephrine autoxidation induced by superoxide anion, and catalase activity by the decrease of H2O2. The results are expressed as a percentage of SOD (a, c) or catalase (b, d) activity with respect to control mediums. Each value is the mean (± SEM) of at least four different dishes from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01, ***< 0.001 and NS not statistically different from control. ##< 0.01; ###< 0.001 vs. H2O2-treated cells. +++< 0.001 vs. NaCN- or AZT-treated cells. Mean basal SOD and catalase activities in these experiments were 17.8 ± 0.25 and 0.62 ± 0.28 U/mg protein.

Figure 9.

 Effect of the SOD inhibitor NaCN and the catalase inhibitor 3-aminotriazole on the protective action of ODN against H2O2-induced astrocyte cell death. (a) Cells were pre-incubated for 10 min or 3 h in the absence or presence of the SOD inhibitor NaCN (2 × 10−3 M) or the catalase inhibitor 3-aminotriazole (AZT, 10−2 M), then incubated for 10 min in the absence or presence of 10−10 M ODN, and for an additional hour without or with H2O2 (300 μM). (b) SOD and catalase activities were detected by enzyme activity gel assay in extracts of astrocytes incubated in the absence (lanes 1 and 3, respectively) or presence of 2 × 10−3 M NaCN (lane 2) or 10−2 M AZT (lane 4). (c) Effect of ODN on acidosis-induced astrocyte cell death. Cells were incubated for 1 h in the absence or presence of 10−10 M ODN in acidic medium (pH 6.8). Cell survival was quantified by measuring fluorescein diacetate fluorescence intensity, and the results are expressed as percentages of control. Each value is the mean (± SEM) of at least four wells from three independent cultures. anova followed by the Bonferroni’s test. *< 0.05; **< 0.01; ***< 0.001 and NS not statistically different from the control; ns not statistically different from acidosis. ###< 0.001 vs. H2O2-treated cells. +++< 0.001 vs. NaCN-treated cells. §§§< 0.001 vs. AZT-treated cells.

Discussion

It is clearly established that oxidative stress causes apoptosis in different cell types, notably in astroglial cells (Giffard and Swanson 2005; Feeney et al. 2008). We have recently found that the endozepine ODN induces a concentration-dependent increase of the number of astrocytes in primary culture (Tokay et al. 2005), suggesting that ODN may be involved in the control of survival or/and cell proliferation. The present study demonstrates for the first time that ODN exerts a protective effect on H2O2 injured astrocytes and activates the antioxidant system of astroglial cells.

In agreement with previous reports (Huang and Philbert 1995; Dringen and Hamprecht 1997), we observed that exposure of cultured astrocytes to H2O2 at concentrations below 100 μM has little effect on astrocyte viability in culture. The insensitivity of astrocytes in this range of concentrations may be ascribed to their high hydrogen peroxidase activity and their ability to clearing H2O2 from culture medium (Dringen and Hamprecht 1997; Ferrero-Gutierrez et al. 2008). In contrast, at higher concentrations (200–800 μM), H2O2 provoked astrocyte death which exhibited the characteristic features of apoptosis, that is, cell shrinkage. Recent reports have provided evidence that astroglial cells can be affected, in terms of viability and functionality, by an insurmountable oxidative stress (Ferrero-Gutierrez et al. 2008; Park et al. 2009). Indeed, astrocytic apoptosis is observed in brain injuries caused by trauma and ischemia (Takuma et al. 2004). The present data suggest that ODN, which is specifically produced by glial cells in the brain (Tonon et al. 1990; Compère et al. 2006), may act as an autocrine factor to reduce the sensitivity of astrocytes to H2O2.

We next determined the mechanisms involved in the protective effect of ODN against H2O2-induced astrocyte cell death. H2O2 can easily cross cell membranes and reach different subcellular compartments (Pedroso et al. 2009). Inside the cell and in the presence of transition metal ions such as Fe2+ or Cu+, H2O2 can be converted into the highly reactive hydroxyl radical (OH°). Thus, an excess of H2O2 induces imbalance in ROS generation, impairs cellular antioxidant defenses, leading finally to the cell death, as previously shown by others in various cell types including astroglial cells (Bi et al. 2008; Dai et al. 2010; Kaczara et al. 2010). Thus, in agreement with previous reports, we found that H2O2 increases in a concentration-dependent manner the production of ROS in cultured rat astrocytes. The present study reveals that pre-treatment of cells with ODN significantly attenuates ROS formation which is likely responsible for a reduction of cell death induced by H2O2. In agreement with this observation, we have shown that the H2O2-generating enzyme system, glucose and glucose oxidase, induced an increase of the production of ROS and a reduction of the proportion of surviving cells, and that these effects were totally abrogated by addition of 10−10 M ODN in the incubation medium (data not shown).

Although our understanding of the precise signaling pathways that trigger apoptosis of astrocytes is still fragmentary, it is widely accepted that ROS can cause cell death by multiple mechanisms including damage of mitochondria leading to a decrease of ATP production (Whittemore et al. 1995), collapse of the mitochondrial potential and formation of mitochondrial permeability transition pore (Copin et al. 2001). Mitochondrial dysfunction causes activation of caspases, the effectors of apoptotic cell death (Wu et al. 2007). Measurement of mitochondria activity with the membrane potential-sensitive probe JC-1 revealed that treatment with H2O2 resulted in a concentration-dependent decrease of the proportion of active mitochondria, and that treatment of the cells with ODN prevented the deleterious effect of H2O2. Collectively, these data strongly suggest that ODN promotes astrocyte survival by decreasing the amount of ROS, which in turn preserves mitochondrial activity. In support of this hypothesis, it has been shown that the neuropeptide pituitary adenylate cyclase-activating polypeptide, which inhibits the deleterious effect of H2O2 on mitochondrial membrane potential, markedly reduces cell apoptosis in cultured cerebellar granule neurons (Vaudry et al. 2002).

It is widely accepted that permeabilization of the mitochondrial outer membrane, yielding to leakage of cytochrome C, is one of the initiator pathways leading to activation of caspases (Wu et al. 2007). We have thus investigated the possible effect of ODN on H2O2-induced caspase 3 activation in cultured astrocytes. In agreement with previous reports (Juknat et al. 2005; Shin et al. 2009), we observed that H2O2 causes a massive increase of caspase 3 activity. Pre-treatment of astrocytes with a subnanomolar concentration of ODN totally suppressed caspase 3 activation induced by H2O2, indicating that the protective effect of ODN can be accounted for by an inhibition of caspase 3 activity, probably resulting from oxidative stress suppression.

Astroglial cells possess an array of cellular defense systems, including antioxidant enzymes, that is, SOD, glutathione peroxidase and catalase, to prevent damage caused by ROS. Several lines of evidence indicate that the protective effect of ODN against H2O2-induced cell death could be mediated through activation of the antioxidant enzyme system. As previously shown (Sokolova et al. 2001; Lopez et al. 2007), high concentrations of H2O2 provoked astrocyte death probably in part through reduction of SOD and catalase activities. Here, we demonstrate for the first time that the gliopeptide ODN is able to stimulate both SOD and catalase activities in astrocytes. Kinetic studies revealed that ODN provokes a rapid and transient stimulation of SOD and catalase activities with a maximal effect occurring respectively 10 and 20 min after the onset of treatment. Thereafter, the action of ODN gradually declined and vanished. It has been previously reported that cultured astrocytes release proteolytic enzymes which actively degrade various neuropeptides, including somatostatin and neurotensin (Mentlein and Dahms 1994; Masmoudi et al. 2005). Therefore, the attenuation of the stimulatory effect of ODN on antioxidant enzyme activities could be accounted for by inactivation of the peptide by proteolytic enzymes released in the culture medium. In support of this hypothesis, refill of ODN 10 min (SOD) and 20 min (catalase) after the onset of peptide administration restored the stimulatory effect of ODN on both SOD and catalase activity.

The mechanism involved in ODN-induced increase of SOD and catalase activity is currently unknown. It has been demonstrated that dephosphorylation of SOD and catalase in a calcium- and protein kinase C-dependent manner is associated with an increase of the activity of these two enzymes (Hopper et al. 2006; Kumar et al. 2010). It has also been shown that expression of SOD in human tumoral cell lines (Kim et al. 2007) and in cultured glial cells (Huang et al. 2001) depends of protein kinase C activation. In glial cells, the increase of SOD mRNA induced by hypoxia reaches a maximum at 5 min (Huang et al. 2001). Here, we show that subnanomolar concentrations of ODN markedly increased SOD and catalase mRNA levels within 5 min, in cultured astroglial cells. We have previously shown that ODN provokes rapid calcium mobilization from inositol triphosphate (IP3)-sensitive pools in rat astrocytes (Patte et al. 1995; Leprince et al. 2001). Thus, the stimulatory effect of ODN on SOD and catalase activities could be ascribed to a rapid increase in SOD and catalase expression, and/or intracellular calcium increase inducing SOD/catalase activation through a dephosphorylation process.

Pre-treatment of astrocytes with subnanomolar concentrations of ODN markedly reduced the inhibitory effect of H2O2 on SOD and catalase activities, that might be responsible, at least in part, for the glioprotective action of ODN on H2O2-induced cell death. The present study reveals that exposure of astrocytes to SOD and catalase inhibitors, NaCN and 3-aminotriazole, respectively abrogated the effect of ODN on H2O2-evoked inhibition of SOD and catalase activities. In addition, the two blockers, which did not affect cell viability by themselves, suppressed the protective effect of ODN against H2O2-induced astrocyte apoptosis. Similarly, data from other groups indicate that reduction of the antioxidant enzyme activities is not associated with a pronounced decrease of cell viability but antagonizes the effects of protective molecules (Smith et al. 2007; Gaspar et al. 2008). Consistent with these observations, over-expression of Cu,Zn- and Mn-SOD has been found to be protective in transgenic mouse models of ischemia and AD (Dumont et al. 2009) and reduction of SOD activity is associated with an exacerbation of oxidative damages in a mouse AD model (Schuessel et al. 2005). Besides, in vitro studies have confirmed that astrocytes isolated from Cu,Zn-SOD transgenic mice exhibit an increased resistance to oxidative stress induced by menadione- or oxygen-glucose deprivation (Chen et al. 2001; Wang et al. 2005). Altogether, these data indicate that the protective effect of ODN against H2O2-induced cell death in astrocytes is attributable to activation of the antioxidant enzymes that act as scavengers of H2O2 and ROS. The observation that ODN was unable to protect astrocytes against acidosis-induced ROS-independent apoptotic death provides further evidence that the protective effect of ODN on astrocyte death can be ascribed to the ability of the peptide to reduce ROS production.

The protective effect of ODN against H2O2-induced oxidative stress and cell death might have a physiopathological significance in patients suffering of degenerative disorders of the central nervous system, including AD and Parkinson’s disease, and in pathological conditions such as ischemia. Clinical studies have shown that the concentration of endozepines is elevated in the cerebrospinal fluid of patients with AD and Parkinson’s disease (Ferrarese et al. 1990). Along these lines, we have recently reported that exposure of cultured astrocytes to beta-amyloid peptide, the main constituent of senile plaques in AD brain, stimulates the biosynthesis and release of ODN-like peptide (Tokay et al. 2005, 2008), suggesting that up-regulation of endozepine production observed in neurodegenerative disorders may be involved in the protection of glial cells. Moreover, astrocytes contain high levels of antioxidants enzymes and play an important role in antioxidative processes in the brain (Peuchen et al. 1997). Nevertheless, despite their high antioxidative activities, astrocytes cannot survive and protect neurons under insurmountable oxidative stress (Takuma et al. 2004; Giffard and Swanson 2005). Thus, enhanced expression of the endozepine ODN, which acts as a protective agent on astrocytes from oxidative assault, might delay neuronal damages in various pathological conditions involving oxidative neurodegeneration. Concurrently, we have recently found that supernatants from astrocytes treated with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), at a concentration (10−9 M) which induces ODN-like peptide production but had no neuroprotective action by itself, prevented H2O2-induced death of cultured cerebellar granule cells (Masmoudi-Kouki et al. 2011).

In conclusion, the present study demonstrates that the endozepine ODN exerts a potent protective action against apoptosis induced by oxidative stress in astrocytes. The anti-apoptotic effect of ODN is attributable, at least in part, to activation of endogenous antioxidant systems and reduction of ROS formation which preserve mitochondrial functions and prevent caspase 3 activation.

Acknowledgements

The authors wish to thank Mr Samir Elbahi for skilful technical assistance. Y.H. and H. K. were recipients of fellowships from the University of Tunis El Manar and a France-Tunisia exchange programs CMCU-Utique and Inserm-DGRS. D.V. and H.V. are Affiliated Professors at the INRS − Institut Armand-Frappier. This study was supported by the Research Unit 00-UR-08-01, an Inserm-DGRS program (to M.A. and M.C.T.), Inserm (U982), the European Institute for Peptide Research (IFRMP23) and the Region Haute-Normandie.

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