Select Page

RESEARCH ARTICLE

PDF logo-blue 30

Effect of Etazolate on ROS Production after tBHP-Induced Oxidative Stress in Oligodendroglial 158N Cell Line

Elena Chierto, Giulia Cristinziano, Francesca Sapone, Delphine Meffre, and MehrnazJafarian-Tehrani 

INSERM UMR 1124, Université Paris Descartes–Université de Paris, Faculté des Sciences Fondamentales et Biomédicales, 45 rue des Saints-Pères, Paris, France 

Correspondence: mehrnaz.jafarian@parisdescartes.fr (M.J-T.) 

ChiertoE et al. Reactive Oxygen Species 9(25):22–35, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.807

(Received: October 16, 2019; Revised: November 17, 2019; Accepted: November 18, 2019) 

ABSTRACT|Oligodendrocytes are the key cells for the myelin synthesis in the central nervous system (CNS). They are sensitive to oxidative stress because of their lipid-rich membranes and their limited antioxidant defense against an excessive production of reactive oxygen species (ROS). Hence, antioxidant strategies are required for myelin protection. We have previously shown that etazolate has neuroprotective and remyelinating activities, and we hypothesized that part of etazolate effect is due to its antioxidant activity. The aim of our study was to test the antioxidant potential of etazolate, in 158N oligodendroglial cell line subjected to tert-butyl hydroperoxide (tBHP) in vitro. We developed a model of tBHP-induced oxidative stress in 158N oligodendroglial cell line and used WST-1 assay and FACS analysis to evaluate cell viability and ROS production. tBHP caused cell death and ROS production in 158N oligodendroglial cells, and etazolate did not protect cells at the concentration range of 0.02 to 200 µM. At the neuroprotective and remyelinating concentration of 2 µM, etazolate did not reduce ROS production, while N-acetylcysteine (NAC), a potent antioxidant compound, counteracted tBHP effects. In conclusion, etazolate does not exert an antioxidant activity in vitro, and the previously reported antioxidant activity of etazolate in vivo might be related to an indirect effect. 

KEYWORDS|N-Acetylcysteine; Antioxidants; tert-Butyl hydroperoxide; Etazolate; Flow cytometry; Oligodendrocytes; 158N oligodendroglial cell; Oxidative stress; Reactive oxygen species 

ABBREVIATIONS |tBHP, tert-butyl hydroperoxide; CAT, catalase; CM-H2DCFDA, carboxy-dichlorodihydrofluorescein diacetate; CNS, central nervous system; DCF, dichlorofluorescein; FACS, fluorescence-activated cell sorting; GSH, the reduced form of glutathione; GSSG, glutathione disulfide; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; PDE, phosphodiesterase; PI, propidium iodide; ROS, reactive oxygen species; SOD, superoxide dismutase; TBI, traumatic brain injury 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. 158N Oligodendroglial Cell Line Culture

2.2. In Vitro Model of tBHP-Induced Oxidative Stress

2.3. Pharmacological Treatments

2.3.1. NAC Treatment against tBHP-Induced Oxidative Stress

2.3.2. Etazolate Treatment against tBHP-Induced Oxidative Stress

2.4. Cell Viability Assay by WST-1

2.5. Flow Cytometric Analysis (FACS)

2.6. Statistical Analysis

  1. Results

3.1. Response of 158N Cell Line to tBHP-Induced Oxidative Stress: Effect of Etazolate and NAC

3.2. NAC Treatment Counteracted tBHP-Induced Oxidative Stress in the158N Cell Line

3.3.Etazolate Did Not Decrease the Intracellular ROS Production Induced by tBHP

  1. Discussion
  2. Conclusion

1. INTRODUCTION 

The central nervous system (CNS) is the part of the nervous system that includes brain and spinal cord and it is composed of grey and white matter. The grey matter consists mainly of neuronal bodies and dendrites, while the white matter consists mainly of axons that are mostly myelinated. CNS in general has a relatively limited potential for anaerobic metabolism which makes it especially sensitive to hypoxia and oxidative stress [1]. In particular, it has been shown that the brain tissue is more sensitive to oxidative stress, in comparison to the spinal cord or the peripheral nervous system [2, 3]. The most important effect of reactive oxygen species (ROS) damage is reported to be in oligodendrocytes, which are the key cells for myelin synthesis in the CNS. 

Oligodendrocytes are among the most sensitive cells to oxidative stress; they can be easily damaged by both hypoxia and ROS, especially during their terminal differentiation phase and while generating myelin sheaths. Oligodendrocyte precursor cells (OPCs) are more sensitive to oxidative stress than mature oligodendrocytes [4, 5]; the mature cells are better protected by a higher level of glutathione [6] and by the switch from an oxidative to a glycolytic metabolism [7]. Nonetheless, if antioxidant systems are not properly regulated, oligodendrocytes are left vulnerable to oxidative and free radical damage [8]. Overall, oxidative stress is a critical player in the pathophysiology of demyelination as reported in different studies [4, 9–15]. 

There are different in vitro models for reproducing an oxidative stress [16, 17]. A fundamental condition is that the redox balance has to be pushed in favor of the pro-oxidant systems. The ROS production can be stimulated by different methods, for example, exposing cells to γ-irradiation, to elevated oxygen tension (hyperoxia), to extracellular superoxide (O2˙ˉ) and H2O2, or by using free radical-generating compound, such as paraquat, menadione, and tert-butyl hydroperoxide (tBHP). We are particularly interested in tBHP, a membrane-permeant oxidant compound that generates free radicals resulting in lipid peroxidation and depletion of intracellular glutathione [18, 19]. For this reason, tBHP has been extensively used as an oxidative stress-inducing agent to study the effect of free radicals in different cell types and regions of the brain [20–23]. Low doses of tBHP cause mostly apoptosis, while higher concentrations lead to both necrosis and apoptosis in the brain [24, 25]. In fact, French and collaborators reported an arrest of oligodendrocyte maturation at the low doses of tBHP [26]. These results suggest that oxidative stress directly interferes with the program of oligodendrocyte differentiation impairing myelin protection and repair after demyelination. Hence, therapeutic antioxidant strategies are required for white matter protection. 

It has been shown that etazolate, a pyrazolopyridine derivative compound, promotes neuroprotection in vitro [27], and after experimental traumatic brain injury (TBI) [28, 29]. We have recently shown that etazolate enhances remyelination and oligodendrocyte maturation after experimental demyelination [30]. Interestingly, the same compound was described to exert an antioxidant effect in vivo in experimental models of depression attenuating the increase of oxidative markers in the brain [31, 32]. Chronic etazolate treatment also restored the altered oxidative-nitrosative stress following experimental TBI [33] and in a model of Parkinson’s disease [34]. We therefore hypothesized that the remyelinating effect of etazolate may be in part due to its antioxidant effect. However, it is not known yet if the observed beneficial effects of etazolate are due to a direct or indirect antioxidant effect. 

In this study, we investigated the potential antioxidant effect of etazolate in an experimental model of oxidative stress in vitro. We developed a model of tBHP-induced oxidative stress using the 158N oligodendroglial cell line and we assessed cell viability and ROS production. The effects of etazolate, particularly at the neuroprotective and remyelinating concentration, and N-acetylcysteine (NAC), a potent antioxidant [35, 36], were analyzed on cell death and ROS production. While NAC reduced these two parameters, etazolate did not protect cells from oxidative stress suggesting that the antioxidant potential of etazolate described previously is not due to a direct antioxidant activity.

2. MATERIALS AND METHODS 

2.1. 158N Oligodendroglial Cell Line Culture 

The 158N oligodendroglial cell line, which preserves oligodendrocyte characteristics and expresses myelin proteins [37–39], was kindly provided by Dr. S.M. Ghandour (CNRS UMR 7357, Strasbourg, France). Cells were maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 5% heat inactivated fetal bovine serum (HI-FBS, Gibco, NYC, NY, USA), L-glutamine 2 mM, sodium pyruvate 1 mM, penicillin (0.1 U/ml)/streptomycin (0.1 μg/ml) and 0.5 μg/ml fungizone. Cells were grown at 37°C in a humidified atmosphere of 5% CO2. For fluorescence-activated cell sorting (FACS) analysis, cells were seeded at a density of 8 × 105 cells/well in medium with 5% FBS. The medium was changed after 48 h of culture with fresh medium containing 1% FBS. After 72 h of culture in total, cells were trypsinized and they underwent the FACS protocol. For cell viability (WST-1) analysis, cells were seeded in 96-well microplates at a density of 5 × 103 cells/well in medium with 5% FBS. After 72 h of culture, tBHP was added to the cells in medium with 1% FBS, and the plate was incubated for 2 h in a humidified atmosphere (37°C, 5% CO2). To evaluate tBHP cytotoxicity, a concentration-effect study (10, 20, 50, and 100 µM) has been performed. 

2.2. In Vitro Model of tBHP-Induced Oxidative Stress 

The compound tBHP (458139, Sigma-Aldrich, St. Louis, MO, USA) was used as oxidative stress-inducing agent in 158N cell line [26]. For WST-1 analysis, 10, 20, 50, or 100 µM tBHP was added to cells and incubated for 2 h, while for FACS analysis, a higher concentration of tBHP (100 µM) was added to cells and incubated for 30 min in a humidified atmosphere (37°C, 5% CO2). In order to validate the model, the cell viability and ROS production were evaluated following treatment with 5 mM NAC (A7250, Sigma-Aldrich) treatment. Once the model was validated, we tested the potential antioxidant effect of etazolate (0438, Tocris, Bristol, UK). 

2.3. Pharmacological Treatments 

2.3.1. NAC Treatment against tBHP-Induced Oxidative Stress 

For evaluation of the effect of NAC on ROS production and cell viability by FACS analysis, cells were trypsinized and 5 mM NAC was added to cells in suspension in 0.1 M phosphate-buffered saline (PBS, Ca2+ and Mg2+ free) and incubated for 1 h. After 30 min following cell suspension, 100 µM tBHP was added in the same tube for further incubation (30 min at 37°C, 5% CO2). 

2.3.2. Etazolate Treatment against tBHP-Induced Oxidative Stress 

For evaluation of the effect of etazolate on ROS production and cell viability by FACS analysis, 2 µM etazolate was tested using three different protocols: (a) protocol with 1 h etazolate incubation—cells were trypsinized and etazolate was added and incubated for 1 h. After 30 min, 100 µM tBHP was added and incubated for another 30 min; (b) protocol with 24 h of etazolate pre-treatment—etazolate pre-treatment started on adherent cells, 24 h before the trypsinization of cells; (c) protocol with 24 h of pre-treatment and 1 h of etazolate incubation—etazolate pre-treatment started on adherent cells, 24 h before the trypsinization of cells. Then, cells were trypsinized and etazolate was added to cells in suspension in 0.1 M PBS (Ca2+ and Mg2+ free) for 1 h. After 30 min, 100 µM tBHP was added (for 30 min at 37°C, 5% CO2). 

2.4. Cell Viability Assay by WST-1 

WST-1 assay (11644807001, Roche, Basel, Switzerland) is based on the enzymatic cleavage of the tetrazolium salt WST-1 to formazan by cellular dehydrogenases present in viable cells. After incubation, medium including tBHP was removed and cell viability evaluation was performed according to the manufacturer’s protocolwhich was provided with the assay kit. 

2.5. Flow Cytometric Analysis (FACS) 

158N cells were trypsinized and collected for analysis with a BD FACSCanto™ II flow cytometer. For each analysis, 10,000 cells were counted. We considered only the population of cells devoid of aggregates and debris. In order to evaluate cell viability and ROS production, a double staining with propidium iodide (PI) and carboxy-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA) was performed [40]. Cells were incubated with 1.25 µM of CM-H2DCFDA probe for 30 min at 37°C in the dark. Excess probe was washed out and cells were suspended in PBS (Ca2+ and Mg2+ free) and incubated for 10 min with 1 µg/mm of PI at room temperature. DCF fluorescence was recorded in the FL-1 (530/30 nm) channel together with PI fluorescence in FL-2 (585/42 nm). 

2.6. Statistical Analysis 

One-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used for comparison among multiple groups when data followed a normal distribution. Otherwise, the Kruskal-Wallis with Dunn’s post-hoc test was performed, which does not require the assumption of normal distributions. To test for normality, the d’Agostino–Pearson test was used. The criterion for statistical significance was p < 0.05 (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Data are expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed with the software GraphPad Prism6 (San Diego, CA, USA).

3. RESULTS 

The aim of this study was to assess the potential antioxidant effect of etazolate on oligodendrocytes using the 158N cell line following an experimental model of oxidative stress in vitro. We first developed an in vitromodel of tBHP-induced oxidative stress. 

3.1. Response of 158N Cell Line to tBHP-Induced Oxidative Stress: Effect of Etazolate and NAC 

We treated the 158N cell line with different concentrations of tBHP (10, 20, 50, and 100 µM) for 2 h. As shown in the Figure 1A, cells were susceptible to tBHP with a significant cytotoxicity observed at the highest concentration. Cell viability was in fact reduced by 60% at 100 mM tBHP. Etazolate (0.2 and 2 mM) did not show any beneficial effect on tBHP-induced cytotoxicity, whereas NAC treatment showed a marked increase in cell viability (Figure 1). Etazolate at concentrations up to 200 µM (concentration range of 0.02, 0.2, 2, 20, and 200 µM) did not protect against tBHP-induced cytotoxicity (data not shown).

 

FIGURE 1. Validation of a model of oxidative stress induced by tBHP in the 158N cell line and effects of etazolate and NAC on cell toxicity induced by tBHP. Cells were incubated with different concentrations of tBHP (10, 20, 50, and 100 µM) for 2 h in the absence or presence of etazolate (0.2 and 2 µM) or NAC (5 mM).  Then medium was removed, and cell viability was evaluated by WST-1 assay. Cells were treated with tBHP at concentrations of 10, 20, 50 and 100 µM (a). Etazolate was tested at different concentrations (0.2 and 2 µM) in cells treated with tBHP at 20 (b), 50 (c), and 100 µM (d). NAC was tested in cells treated with tBHP at 50 µM (e). Results are expressed as percentage of cell viability compared to that of the vehicle group. Values are the mean ± SEM of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. 

3.2. NAC Treatment Counteracted tBHP-Induced Oxidative Stress in the 158N Cell Line 

To test the effect of NAC and etazolate on ROS production, we used FACS analysis. The concentration of 100 µM tBHP was chosen to induce ROS production in 158N cells. This method requires the preparation of cells in suspension in contrary to the use of adherent cells to study tBHP cytotoxicity via WST-1 assay. 

The ROS production was evaluated by assessing redox sensitive intracellular conversion of H2DCFDA to DCF via FACS (Figure 2). tBHP induced a 20-fold increase in the DCF fluorescence compared to control group (p < 0.0001) and NAC was able to reduce tBHP-induced ROS production by decreasing the DCF fluorescence by 50% compared to tBHP control (p < 0.01) (Figure 2c). In our experimental oxidative stress condition, NAC treatment showed a protective effect indicating that our protocol and analysis are appropriate for studying the potential antioxidant effect of etazolate in the 158N cell line.

 

FIGURE 2. NAC decreased the level of intracellular ROS in the 158N cell line in a model of tBHP-induced oxidative stress. Treatment with NAC at 5 mM was performed for 1 h in cells in suspension. Thirty min prior to analysis, 100 µM tBHP was added. The intracellular ROS level was detected by DCFH-DA fluorescent probe. (a) Representative FACS plots of DCF-stained (FITC channel) and PI-stained (PE channel) cells were obtained from control (Ctrl), tBHP-treated (tBHP), and tBHP/NAC-treated (tBHP + NAC) cells. (b) Representative FACS analysis of the mean fluorescence intensity (MFI) of DCF measured on DCF+/PIˉ cells. (c) Quantitative analysis of the DCF fluorescence expressed as geometric mean. (d) Quantitative analysis of the percentage of PI+ cells. All results are expressed as fold change compared to control group. Values are the mean ± SEM of five experiments. **, p < 0.01; ****, p < 0.0001 compared to control group; ##, p < 0.01 compared to tBHP group. 

Cell death was also evaluated by the presence of PI+ cells through FACS. In control, tBHP-treated, or tBHP/NAC-treated cells, a low number of PI+ cells were present, excluding tBHP-induced cell death in these conditions analyzed by FACS (Figure 2d) probably due to the short period of tBHP incubation with 158N cells in suspension. 

3.3. Etazolate Did Not Decrease the Intracellular ROS Production Induced by tBHP 

We used FACS to assess ROS production after tBHP-induced oxidative stress in order to better analyse the potential antioxidant effect of etazolate (Figure 3). The concentration of 2 µM was chosen, given that etazolate at this concentration is able to exert neuroprotective and remyelinating effect [27, 30]. Treatment with tBHP induced a 20-fold increase in the DCF fluorescence compared to control group (p < 0.001) and etazolate was devoid of any effect neither in pre-treatment protocol nor without pre-treatment protocol (Figure 3c).

 

FIGURE 3. Effect of etazolate on the cell death and the level of intracellular ROS in the 158N cell line after tBHP-induced oxidative stress. Etazolate at 2 µM was tested using three different protocols: (i) etazolate in pre-treatment was added 24 h before the addition of tBHP (100 µM tBHP + 2 µM Etaz, pre); (ii) etazolate in pre-treatment was added 24 h before the addition of tBHP and during the 1 h tBHP incubation (100 µM tBHP + 2 µM Etaz, pre + 1 h); and (iii) etazolate was added during the 1 h tBHP incubation (100 µM tBHP + 2 µM Etaz, 1 h). The intracellular ROS level was detected by DCFH-DA fluorescent probe. (a) Representative FACS plots of DCF-stained (FITC channel) and PI-stained (PE channel) cells were obtained from control (Ctrl), tBHP-treated (tBHP) and tBHP/etazolate-treated cells with the three tested protocols. (b) Representative FACS analysis of the mean fluorescence intensity (MFI) of DCF measured on DCF+/PIˉcells. (c) Quantitative analysis of the DCF fluorescence expressed as geometric mean. (d) Quantitative analysis of the percentage of PI+ cells. Results are expressed as the fold change compared to that of the control group. Values are the mean ± SEM of five independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control. 

Cell death was also assessed by the presence of PI+ cells. In control, tBHP-treated, or tBHP/etazolate-treated cells, the number of PI+ cells remained the same (Figure 3d), thus excluding cell death in all conditions analyzed by FACS, as already observed above (Figure 2d). In addition, etazolate by itself did not exert a significant effect on cell viability and ROS production in both protocols without tBHP (Figure 4).

 

FIGURE 4. Effect of etazolate on the level of intracellular ROS in the 158N cell line without tBHP treatment. Etazolate at 2 µM was tested using two different protocols: (i) etazolate in pre-treatment was added 24 h before the trypsination of cells (Etaz 2 µM, pre) and (ii) etazolate was added on cells in suspension for 1 h before the FACS analysis (Etaz 2 µM, 1 h). The intracellular ROS level was detected by DCFH-DA fluorescent probe. (a) Representative FACS plots of DCF-stained (FITC channel) and PI-stained (PE channel) cells were obtained from control (Ctrl) and etazolate-treated cells with the two tested protocols. (b) Representative FACS analysis of the mean fluorescence intensity (MFI) of DCF measured on DCF+/PIˉ cells. (c) Quantitative analysis of the DCF fluorescence expressed as geometric mean. (d) Quantitative analysis of the percentage of PI+ cells. Results are expressed as the fold change compared to that of the control group. Values are the mean ± SEM of five experiments.

4. DISCUSSION 

Oxidative stress is detrimental for myelin protection and repair after demyelination, requiring the necessity of therapeutic antioxidant strategies for white matter protection. The aim of this study was to test the antioxidant effect of etazolate on isolated oligodendrocytes, and we clearly showed that etazolate was devoid of any effect in a model of tBHP-induced oxidative stress in vitro in spite of promising antioxidant effects described in vivo. 

Etazolate, a pyrazolopyridine derivative compound, is known to have multiple mechanisms of action, including being (i) a positive allosteric modulator of the GABAA receptor, (ii) an adenosine receptor antagonist, (iii) a phosphodiesterase (PDE) inhibitor selective for the PDE4 isoform, and (iv) an α-secretase activator [27, 41–43]. Etazolate was previously shown, in part by our team, to exert neuroprotective and remyelinating effects and to be anti-inflammatory and antioxidant in vivo [28–34, 44–47]. In particular, Jindal and colleagues demonstrated that etazolate has an antioxidant effect in an in vivo model of chronic unpredictable stress depression in mice attenuating the increase of oxidative markers in the brain, particularly, the lipid peroxidation and the nitrosative level. In the same model, etazolate treatment normalized the decreased level of the reduced form of glutathione (GSH) and the decreased activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in the brain of depressed animals [31]. The etazolate antioxidant effect was also described in a model of olfactory bulbectomy-induced depression in mice [32] and in an impact accelerated TBI model [33]. A recent study also showed that etazolate treatment normalized the ratio of GSH to glutathione disulfide (GSSG) and the activity of glutathione peroxidase (GPx) in the hippocampus, and it could prevent motor dysfunction, depressive-like behavior, and learning and memory deficits in a rat model of Parkinson’s disease [34]. The same group also demonstrated that etazolate can prevent oxidative stress and histones acetylation in a rat model of post-traumatic stress disorder [48]. 

Despite the aforementioned promising results in vivo, the antioxidant effect of etazolate has never been tested directly on oligodendrocytes, which are cells particularly vulnerable to oxidative stress. In fact, oligodendrocytes are one of the cell types with the highest metabolism in the brain, since proper myelination requires large amounts of oxygen and adenosine triphosphate (ATP). Myelination requires that oligodendrocytes first construct and then maintain an extensive plasma membrane, and for this, a vast supply of both precursor molecules and oxidative substrates is required [5]. A high metabolism, with high-energy consumption, means also large amounts of toxic by-products, such as ROS. Oligodendrocytes are also highly sensitive to oxidative stress because of their lipid-rich membranes where unsaturated long-chain fatty acids are unusually concentrated [26]. In addition to their relatively poor antioxidant production, oligodendrocytes are even more susceptible to oxidative stress due to the need of iron as a cofactor for myelin production [49, 50]. They are the most iron-loaded cells of the CNS and the intracellular accumulation of iron can stimulate the conversion of hydrogen peroxide to hydroxyl radicals—the most reactive free radicals [51, 52]. Therefore, oligodendrocytes can be easily damaged and they are particularly sensitive to both hypoxia and oxidative stress, especially during their terminal differentiation phase and while generating myelin sheaths. During the process of myelin production and remyelination, the peroxisomal fraction of oligodendrocytes increases the volume and density, in accordance with metabolic requirements, thus representing an important independent ROS source [53–55]. 

In order to evaluate the antioxidant potential of etazolate, we first developed an in vitro model of oxidative stress induced by tBHP in the 158N oligodendroglial cell line. tBHP is a membrane-permeant oxidant compound extensively used as an oxidative stress-inducing agent in different cell types and tissues [20–23, 25, 26, 56]. As expected, tBHP was able to increase intracellular ROS (FACS analysis) and induce cell death (WST-1 assay) in the 158N cell line in which NAC treatment was able to reverse both ROS production and cytotoxicity. NAC is known to be a powerful antioxidant [57, 58]. It acts as a scavenger of free radicals and it is considered a treatment option for different disorders resulting from generation of free oxygen radicals [57]. It showed positive effects in in vivo models of diseases characterized by white matter alterations, such as experimental autoimmune encephalomyelitis [59], ischemia-induced neonatal white matter injury [60], and traumatic brain injury [61]. 

Our results showed that etazolate was devoid of any antioxidant activity in our model. In fact, etazolate was not able to reduce ROS production at 2 µM, a concentration able to exert neuroprotective and remyelinating effects [27, 30]. Our data clearly show the discrepancy between our in vitro results and in vivo reports. It remained to investigate if the antioxidant effect of etazolate observed in vivo (0.5 mg/kg/day and 1 mg/kg/day) [31–34] is a direct or an indirect effect through etazolate’s anti-inflammatory activity via PDE4 inhibition. In fact, a recent study showed that another PDE4 inhibitor, FCPR16, at the concentrations of 12.5–50 μM protects SH-SY5Y cells against oxidative stress reducing ROS production in vitro [62]. 

In our in vitro model, etazolate was tested only on isolated oligodendrocytes excluding the in vivo systemic response. Moreover, the potential antioxidant activity of etazolate has been tested in a model where tBHP induces a remarkable production of ROS (20-fold increase). It is possible that ROS production in this model is too high to be reversed by etazolate. Nevertheless, NAC is able to counteract tBHP-induced ROS production in our model. As a future direction, it could be interesting to test the effect of etazolate on different oxidative stress parameters, including the activity of the antioxidant enzymes SOD and CAT and the levels of intracellular GSH/GSSG.

5. CONCLUSION 

In our model of tBHP-induced oxidative stress using the 158N oligodendroglial cell line, etazolate does not exert an antioxidant activity in vitro. The previously reported antioxidant activity of etazolate in vivo might be related to an indirect effect. 

ACKNOWLEDGMENTS 

This work was supported by the non-profit organization “Fondation des GueulesCassées” (grants to M.J-T.), Paris Descartes University and by INSERM (Institut National de la Santé Et de la Recherche Médicale). E.C. is a recipient of PhD fellowship from French Ministry of Research. The authors acknowledge the assistance provided by different facilities (molecular biology and FACS facilities) from the Faculty of Basic and Biomedical Sciences (Paris Descartes University), particularly Dr. Stéphanie Dupuy for flow cytometry. The authors acknowledge also Dr. Said Ghandour (CNRS UMR 7357) for providing the 158N oligodendroglial cell line, and Dr. Julien Grenier (INSERM UMR-S 1124) for the cell line maintenance. Regarding author contributions, M.J-T. and E.C. designed the study; E.C., G.C., and F.S. performed experiments; E.C., D.M., and M.J-T. analyzed and discussed data; E.C. and M.J-T. wrote the main manuscript; E.C. prepared all figures; M.J-T., D.M., and E.C. revised the manuscript. All authors reviewed and approved the manuscript. The authors declare no conflicts of interest. 

REFERENCES 

  1. Yokoyama H, Yano R, Aoki E, Kato H, Araki T. Comparative pharmacological study of free radical scavenger, nitric oxide synthase inhibitor, nitric oxide synthase activator and cyclooxygenase inhibitor against MPTP neurotoxicity in mice. Metab Brain Dis 2008;23:335–49. doi:10.1007/s11011-008-9096-3.
  2. Macco R, Pelizzoni I, Consonni A, Vitali I, Giacalone G, Martinelli Boneschi F, et al. Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation. J Neuroinflammation 2013;10:130. doi:10.1186/1742-2094-10-130.
  3. Schnell L, Fearn S, Klassen H, Schwab ME, Perry VH. Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 1999;11:3648–58.
  4. Ljubisavljevic S. Oxidative Stress and Neurobiology of Demyelination. Mol Neurobiol 2016;53:744–58. doi:10.1007/s12035-014-9041-x.
  5. Roth AD, Núñez MT. Oligodendrocytes: functioning in a delicate balance between high metabolic requirements and oxidative damage. Adv Exp Med Biol 2016;949:167–81.doi:10.1007/978-3-319-40764-7_8.
  6. Back SA, Gan X, Li Y, Rosenberg PA, Volpe JJ. Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion. J Neurosci 1998;18:6241–53.
  7. Funfschilling U, Supplie LM, Mahad D, Boretius S, Saab AS, Edgar J, et al. Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 2012;485:517–21. doi:10.1038/nature11007.
  8. Li J, Baud O, Vartanian T, Volpe JJ, Rosenberg PA. Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci USA 2005;102:9936–41.
  9. Haider L. Inflammation, iron, energy failure, and oxidative stress in the pathogenesis of multiple sclerosis. Oxid Med Cell Longev 2015;2015:725370. doi:10.1155/2015/725370.
  10. Hamilton RT, Bhattacharya A, Walsh ME, Shi Y, Wei R, Zhang Y, et al. Elevated protein carbonylation, and misfolding in sciatic nerve from db/db and Sod1-/- mice: plausible link between oxidative stress and demyelination. PloS One 2013;8:e65725. doi:10.1371/journal.pone.0065725.
  11. Lee D-H, Gold R, Linker RA. Mechanisms of oxidative damage in multiple sclerosis and neurodegenerative diseases: therapeutic modulation via fumaric acid esters. Int J Mol Sci 2012;13:11783–11803. doi:10.3390/ijms130911783.
  12. Ljubisavljevic S, Stojanovic I. Neuroinflammation and demyelination from the point of nitrosative stress as a new target for neuroprotection. Rev Neurosci 2015;26:49–73. doi:10.1515/revneuro-2014-0060.
  13. di Penta A, Moreno B, Reix S, Fernandez-Diez B, Villanueva M, Errea O, et al. Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation. PLoS One 2013;8:e54722. doi:10.1371/journal.pone.0054722.
  14. Shi H, Hu X, Leak RK, Shi Y, An C, Suenaga J, et al. Demyelination as a rational therapeutic target for ischemic or traumatic brain injury. Exp Neurol 2015;272:17–25. doi:10.1016/j.expneurol.2015.03.017.
  15. Vladimirova O, O’Connor J, Cahill A, Alder H, Butunoi C, Kalman B. Oxidative damage to DNA in plaques of MS brains. Mult Scler 1998;4:413–8.
  16. Feitosa CM, da Silva Oliveira GL, Cavalcante A do N, Chaves SKM, Rai M. Determination of parameters of oxidative stress in vitro models of neurodegenerative diseases: a review. Curr Clin Pharmacol 2018;13(2):100-9.doi:10.2174/1574884713666180301091612.
  17. Gille JJ, Joenje H. Cell culture models for oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia. Mutat Res 1992;275:405–14.
  18. Adams JD, Wang B, Klaidman LK, LeBel CP, Odunze IN, Shah D. New aspects of brain oxidative stress induced by tert-butylhydroperoxide. Free Radic Biol Med 1993;15:195–202.
  19. Adams JD, Mukherjee SK, Klaidman LK, Chang ML, Yasharel R. Apoptosis and oxidative stress in the aging brain. Ann N Y Acad Sci 1996;786:135–51.
  20. He Y, Jackman NA, Thorn TL, Vought VE, Hewett SJ. Interleukin-1β protects astrocytes against oxidant-induced injury via an NF-κB-dependent upregulation of glutathione synthesis: IL-1β increases gsh and protects astrocytes. Glia 2015;63:1568–80. doi:10.1002/glia.22828.
  21. Hughes RH, Silva VA, Ahmed I, Shreiber DI, Morrison B. Neuroprotection by genipin against reactive oxygen and reactive nitrogen species-mediated injury in organotypic hippocampal slice cultures. Brain Res 2014;1543:308–14. doi:10.1016/j.brainres.2013.11.020.
  22. Pan X-D, Zhu Y-G, Lin N, Zhang J, Ye Q-Y, Huang H-P, et al. Microglial phagocytosis induced by fibrillar β-amyloid is attenuated by oligomeric β-amyloid: implications for Alzheimer’s disease. Mol Neurodegener 2011;6:45. doi:10.1186/1750-1326-6-45.
  23. Peterson B, Stovall K, Monian P, Franklin JL, Cummings BS. Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury. Chem Biol Interact 2008;174:163–76. doi:10.1016/j.cbi.2008.05.028.
  24. Mukherjee SK, Adams JD. The effects of aging and neurodegeneration on apoptosis-associated DNA fragmentation and the benefits of nicotinamide. Mol Chem Neuropathol 1997;32:59–74.
  25. Mukherjee SK, Yasharel R, Klaidman LK, Hutchin TP, Adams JD. Apoptosis and DNA fragmentation as induced by tertiary butylhydroperoxide in the brain. Brain Res Bull 1995;38:595–604.
  26. French HM, Reid M, Mamontov P, Simmons RA, Grinspan JB. Oxidative stress disrupts oligodendrocyte maturation. J Neurosci Res 2009;87:3076–87. doi:10.1002/jnr.22139.
  27. Marcade M, Bourdin J, Loiseau N, Peillon H, Rayer A, Drouin D, et al. Etazolate, a neuroprotective drug linking GABAA receptor pharmacology to amyloid precursor protein processing. J Neurochem 2008;106:392–404. doi:10.1111/j.1471-4159.2008.05396.x.
  28. Siopi E, Llufriu-Dabén G, Cho AH, Vidal-Lletjós S, Plotkine M, Marchand-Leroux C, et al. Etazolate, an α-secretase activator, reduces neuroinflammation and offers persistent neuroprotection following traumatic brain injury in mice. Neuropharmacology 2013;67:183–192. doi:10.1016/j.neuropharm.2012.11.009.
  29. Llufriu-Dabén G, Meffre D, Massaad C, Jafarian-Tehrani M. A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging. J Neurosci Methods 2019;311:385–93. doi:10.1016/j.jneumeth.2018.09.023.
  30. Llufriu-Dabén G, Carrete A, Chierto E, Mailleux J, Camand E, Simon A, et al. Targeting demyelination via α-secretases promoting sAPPα release to enhance remyelination in central nervous system. Neurobiol Dis 2018;109:11–24. doi:10.1016/j.nbd.2017.09.008.
  31. Jindal A, Mahesh R, Bhatt S. Etazolate, a phosphodiesterase 4 inhibitor reverses chronic unpredictable mild stress-induced depression-like behavior and brain oxidative damage. Pharmacol Biochem Behav 2013;105: 63–70. doi:10.1016/j.pbb.2013.01.020.
  32. Jindal A, Mahesh R, Bhatt S. Etazolate, a phosphodiesterase-4 enzyme inhibitor produces antidepressant-like effects by blocking the behavioral, biochemical, neurobiological deficits and histological abnormalities in hippocampus region caused by olfactory bulbectomy. Psychopharmacology (Berl) 2015;232:623–37. doi:10.1007/s00213-014-3705-0.
  33. Jindal A, Mahesh R, Bhatt S, Pandey D. Molecular modifications by regulating cAMP signaling and oxidant-antioxidant defence mechanisms, produce antidepressant-like effect: a possible mechanism of etazolate aftermaths of impact accelerated traumatic brain injury in rat model. Neurochem Int 2017;111:3–11. doi:10.1016/j.neuint.2016.12.004.
  34. Alzoubi KH, Mokhemer E, Abuirmeileh AN. Beneficial effect of etazolate on depression-like behavior and, learning, and memory impairment in a model of Parkinson’s disease. Behav Brain Res 2018;350:109–15. doi:10.1016/j.bbr.2018.05.004.
  35. Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 1989;6:593–7.
  36. Samuni Y, Goldstein S, Dean OM, Berk M. The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta 2013;1830:4117–29. doi:10.1016/j.bbagen.2013.04.016.
  37. Feutz AC, Pham-Dinh D, Allinquant B, Miehe M, Ghandour MS. An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway. Glia 2001;34:241–52.
  38. Ghandour MS, Feutz A-C, Jalabi W, Taleb O, Bessert D, Cypher M, et al. Trafficking of PLP/DM20 and cAMP signaling in immortalized jimpy oligodendrocytes. Glia 2002;40:300–11. doi:10.1002/glia.10122.
  39. Chierto E, Simon A, Castoldi F, Meffre D, Cristinziano G, Sapone F, et al. Mechanical stretch of high magnitude provokes axonal injury, elongation of paranodal junctions, and signaling alterations in oligodendrocytes. Mol Neurobiol 2018; 56(6):4231-48. doi:10.1007/s12035-018-1372-6.
  40. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983;130:1910–7.
  41. Barnes DM, White WF, Dichter MA. Etazolate (SQ20009): electrophysiology and effects on [3H]flunitrazepam binding in cultured cortical neurons. J Neurosci 1983;3:762–72.
  42. Chasin M, Harris DN, Phillips MB, Hess SM. 1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009): a potent new inhibitor of cyclic 3’,5’-nucleotide phosphodiesterases. Biochem Pharmacol 1972;21:2443–50.
  43. Daly JW, Hong O, Padgett WL, Shamim MT, Jacobson KA, Ukena D. Non-xanthine heterocycles: activity as antagonists of A1- and A2-adenosine receptors. Biochem Pharmacol 1988;37:655–64.
  44. Désiré L, Blondiaux E, Carrière J, Haddad R, Sol O, Fehlbaum-Beurdeley P, et al. Blood transcriptomic biomarkers of Alzheimer’s disease patients treated with EHT 0202. J Alzheimers Dis 2013;34:469–83. doi:10.3233/JAD-121501.
  45. Drott J, Desire L, Drouin D, Pando M, Haun F. Etazolate improves performance in a foraging and homing task in aged rats. Eur J Pharmacol 2010;634:95–100. doi:10.1016/j.ejphar.2010.02.036.
  46. Guo J, Lin P, Zhao X, Zhang J, Wei X, Wang Q, et al. Etazolate abrogates the lipopolysaccharide (LPS)-induced downregulation of the cAMP/pCREB/BDNF signaling, neuroinflammatory response and depressive-like behavior in mice. Neuroscience 2014;263:1–14. doi:10.1016/j.neuroscience.2014.01.008.
  47. Alzoubi KH, Al Subeh ZY, Khabour OF. Evaluating the protective effect of etazolate on memory impairment, anxiety- and depression-like behaviors induced by post traumatic stress disorder. Brain Res Bull 2017;135:185–92. doi:10.1016/j.brainresbull.2017.10.012.
  48. Alzoubi KH, Al Subeh ZY, Khabour OF. Molecular targets for the interactive effect of etazolate during post-traumatic stress disorder: role of oxidative stress, BDNF and histones. Behav Brain Res 2019;369:111930. doi:10.1016/j.bbr.2019.111930.
  49. Cheepsunthorn P, Palmer C, Connor JR. Cellular distribution of ferritin subunits in postnatal rat brain. J Comp Neurol 1998;400:73–86.
  50. Thorburne SK, Juurlink BH. Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress. J Neurochem 1996;67:1014–22.
  51. Braughler JM, Duncan LA, Chase RL. The involvement of iron in lipid peroxidation: importance of ferric to ferrous ratios in initiation. J Biol Chem 1986;261:10282–9.
  52. Juurlink BH. Response of glial cells to ischemia: roles of reactive oxygen species and glutathione. Neurosci Biobehav Rev 1997;21:151–66.
  53. Singh I, Paintlia AS, Khan M, Stanislaus R, Paintlia MK, Haq E, et al. Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment. Brain Res 2004;1022:1–11. doi:10.1016/j.brainres.2004.06.059.
  54. Sullivan GM, Mierzwa AJ, Kijpaisalratana N, Tang H, Wang Y, Song S-K, et al. Oligodendrocyte lineage and subventricular zone response to traumatic axonal injury in the corpus callosum. J Neuropathol Exp Neurol 2013;72:1106–25. doi:10.1097/NEN.0000000000000009.
  55. Zeis T, Graumann U, Reynolds R, Schaeren-Wiemers N. Normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection. Brain J Neurol 2008;131:288–303. doi:10.1093/brain/awm291.
  56. Lu D, Xu A, Mai H, Zhao J, Zhang C, Qi R, et al. The synergistic effects of heat shock protein 70 and ginsenoside Rg1 against tert-butyl hydroperoxide damage model in vitro. Oxid Med Cell Longev 2015;2015:437127. doi:10.1155/2015/437127.
  57. Mokhtari V, Afsharian P, Shahhoseini M, Kalantar SM, Moini A. A review on various uses of N-acetyl cysteine. Cell J 2017;19:11–7. doi: 10.22074/cellj.2016.4872.
  58. Shimamoto K, Hayashi H, Taniai E, Morita R, Imaoka M, Ishii Y, et al. Antioxidant N-acetyl-L-cysteine (NAC) supplementation reduces reactive oxygen species (ROS)-mediated hepatocellular tumor promotion of indole-3-carbinol (I3C) in rats. J Toxicol Sci 2011;36:775–86. doi:10.2131/jts.36.775.
  59. Ljubisavljevic S, Stojanovic I, Pavlovic D, Milojkovic M, Vojinovic S, Sokolovic D, et al. Correlation of nitric oxide levels in the cerebellum and spinal cord of experimental autoimmune encephalomyelitis rats with clinical symptoms. Acta Neurobiol Exp (Wars). 2012;72:33–9.
  60. Nance E, Porambo M, Zhang F, Mishra MK, Buelow M, Getzenberg R, et al. Systemic dendrimer-drug treatment of ischemia-induced neonatal white matter injury. J Control Release 2015;214:112–20. doi:10.1016/j.jconrel.2015.07.009.
  61. Abdel Baki SG, Schwab B, Haber M, Fenton AA, Bergold PJ. Minocycline synergizes with N-acetylcysteine and improves cognition and memory following traumatic brain injury in rats. PloS One 2010;5:e12490. doi:10.1371/journal.pone.0012490.
  62. Zhong J, Yu H, Huang C, Zhong Q, Chen Y, Xie J, et al. Inhibition of phosphodiesterase 4 by FCPR16 protects SH-SY5Y cells against MPP+-induced decline of mitochondrial membrane potential and oxidative stress. Redox Biol 2018;16:47–58. doi:10.1016/j.redox.2018.02.008.