Free Radical Research Oxidative stress in myelin sheath: The other ...

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Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability a

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S. Ravera , M. Bartolucci , P. Cuccarolo , E. Litamè , M. Illarcio , D. Calzia , P. Degan , A. a

Morelli & I. Panfoli

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Department of Pharmacy (DIFAR), Biochemistry Laboratory, University of Genova, Genova, Italy b

S. C. Mutagenesis, IRCCS AOU San Martino – IST (Istituto Nazionale per la Ricerca sul Cancro), Genova, Italy Published online: 12 Jun 2015.

Click for updates To cite this article: S. Ravera, M. Bartolucci, P. Cuccarolo, E. Litamè, M. Illarcio, D. Calzia, P. Degan, A. Morelli & I. Panfoli (2015) Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability, Free Radical Research, 49:9, 1156-1164, DOI: 10.3109/10715762.2015.1050962 To link to this article: http://dx.doi.org/10.3109/10715762.2015.1050962

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Free Radical Research, 2015; 49(9): 1156–1164 © 2015 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2015.1050962

ORIGINAL ARTICLE

Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability S. Ravera1*, M. Bartolucci1*, P. Cuccarolo2, E. Litamè1, M. Illarcio1, D. Calzia1, P. Degan2, A. Morelli1 & I. Panfoli1 of Pharmacy (DIFAR), Biochemistry Laboratory, University of Genova, Genova, Italy, and 2S. C. Mutagenesis, IRCCS AOU San Martino – IST (Istituto Nazionale per la Ricerca sul Cancro), Genova, Italy

Downloaded by [Istituto Giannina Gaslini], [Silvia Ravera] at 04:43 24 August 2015

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Abstract Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand. In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H2O2), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated. We report that isolated myelin produces high levels of MDA, 4-HNE, and H2O2, likely through the pathway composed by Complex I–III–IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation. Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS. Keywords: antioxidants, malondialdehyde, myelin energetic metabolism, OXPHOS, ROS production

Introduction Oxidative stress generation is a phenomenon involving all cellular structures [1]. It has been demonstrated that increased generation of reactive oxygen species (ROS) occurs in peroxisomes, plasma membrane, endoplasmic reticulum, and cytosol [2]. It is often assumed that, in physiological conditions, the aerobic energy production by oxidative phosphorylation (OXPHOS) in mitochondria is one of the main biological process leading to ROS generation [3,4]. The aerobic metabolism is essential to provide the necessary ATP for the cell [5], but, during the electron transfer along the Electron Transfer Chain (ETC), single electrons can escape and result in reduction of molecular oxygen, forming superoxide anion (O2•⫺) [6,7]. It is estimated that as much as 1% of all oxygen consumed may result in the formation of ROS such as O2•⫺, with the vast majority generated by the ETC [8]. Respiring Complex I and Complex III are considered the principal sites of O2•⫺ formation [9–11], even though lately Complex II has also been involved in ROS production [12]. After its formation, O2•⫺ can be reduced to hydrogen peroxide (H2O2), particularly dangerous for the cell, because

it can react with reduced metal ions, as Fe2⫹ or Cu⫹ (Fenton reaction), forming the fearsome hydroxyl radical [2]. Any form of ROS inside the cell can attack membrane polyunsaturated fatty acids causing the production of peroxides, or damage to structural proteins, enzymes, and nucleic acids, leading to loss of function and mutations, respectively [13,14]. For this reason, ROS production has often been associated with aging and several pathologies, included cancer [15]. As defense against free radicals, cell displays enzymatic and non-enzymatic detoxication mechanisms. The enzymatic detoxication mechanisms involve antioxidant enzymes, such as superoxide dismutase (SOD); catalase (CAT); and glutathione peroxidase (GPx), low-molecularweight antioxidants (vitamins E and C, glutathione, ubiquinone, beta-carotene, etc.), and adaptive mechanisms leading to antioxidant gene expression [16]. SOD converts O2•⫺ in O2 and H2O2, which, although not a radical, is still toxic. To remove H2O2, the cell displays CAT, when the levels are low, and GPx, when the production of H2O2 is higher [16]. Although it is known that mitochondria are the main site of oxidative metabolism, in the last years we have

*These authors have contributed equally to this work. Correspondence: Dr. Silvia Ravera, Biochemistry Laboratory, Department of Pharmacy, University of Genova, Viale Benedetto XV, 3 16132 Genova, Italy. Tel: ⫹ 39 0103538152. Fax:⫹ 39 0103538153. E-mail: [email protected] (Received date: 22 January 2015; Accepted date: 11 May 2015; Published online: 4 June 2015)


Evaluation of oxidative stress in myelin sheath

demonstrated that rod outer segment disks [17], isolated myelin [18–22], and plasma membrane of glioma cells [23] display ectopic functional OXPHOS, consuming oxygen to produce ATP. As far as myelin sheath is concerned, we have supposed that this aerobic metabolism supports the axonal energy demand [24]. This would be consistent with the hypothesis that myelin sheath plays an as yet unidentified trophic role [25] considering that myelin loss in demyelinating diseases leads to an axonal degeneration, the cause of the most severe symptoms in these pathologies [26]. Starting from the consideration that isolated myelin (IM) can efficiently conduct an aerobic metabolism [18,21] and that it is enriched in lipids [27], we have investigated oxidative stress in IM. In particular, the presence of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as markers of lipid peroxidation, as well as H2O2 production and the functional presence of SOD, CAT, and GPx, was evaluated. Oxidative stress occurrence in myelin was also assayed in the presence of uncoupling compounds or ETC Complex I inhibitors, simulating damage conditions, or after the addition of antioxidants.

Methods Reagents Salts, substrates, and all other chemicals (of analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein Molecular Weight or MW markers were from BioRad (Hercules, CA, USA). Ultrapure water (Milli-Q; Millipore, Billerica, MA, USA) was used throughout the study. Safety precautions were taken for chemical hazards in carrying out the experiments. Ampicillin (100 μg/ml) was used in all the solutions, and sterile experimental conditions were employed where appropriate. Antibodies Primary antibodies (Abs) were polyclonal goat anti-Cu–Zn SOD (SOD1) (sc-8637, Santa Cruz Biotechnology, CA, USA); polyclonal goat anti-Mn SOD (SOD2) (sc-30080, Santa Cruz Biotechnology, CA, USA). Horseradish peroxidase (HRP)-conjugated goat IgG Ab (Santa Cruz Biotechnology, CA, USA) was used for Western blot (WB) experiments.

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CA, USA). Step 1: 20 g of sample was homogenized with a Potter–Elvehjem homogenizer in 20 vol. (w/v) of 0.32 M sucrose in 2 mM ethylene glycol tetraacetic acid (EGTA). For each tube, 30 ml of homogenate was layered over l of 0.85 M sucrose in 2 mM EGTA and the tube was centrifuged at 75000 g for 30 min. This step was repeated twice. Step 2: Crude myelin layer at the interface was collected, and homogenized in water to a final volume of 60 ml. Suspension was centrifuged at 75000 g for 15 min. Step 3: Pellet was dispersed in a total volume of 60 ml of water and centrifuged at 12000 g for 10 min. The cloudy supernatant was discarded. Step 4: The loosely packed pellet was dispersed in water and centrifuged at 12000 g for 10 min. The myelin pellet was suspended in 30 ml of 0.32 M sucrose in 2 mM EGTA, and the suspension was layered onto 0.85 M sucrose in 2 mM EGTA and centrifuged at 75000 g for 30 min. Mitochondria-enriched fraction isolation Mitochondria are prepared from forebrain homogenate as follows: the forebrain was washed in phosphate-buffered saline (PBS) and homogenized in buffer containing 0.25 M sucrose, 0.15 M KCl, 10 mM Tris–HCl at pH: 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% bovine serum albumin (BSA). The homogenate was centrifuged at 800 g for 10 min; the supernatant was filtered and centrifuged at 12000 g for 15 min; the pellet was resuspended in buffer containing 0.25 M sucrose, 75 mM mannitol, 10 mM Tris–HCl at pH: 7.4, and 1 mM EDTA. Finally, the supernatant was centrifuged at 12000 g for 15 min and the mitochondria pellet was resuspended in a second buffer [29]. Oxygen consumption assay in IM Respiration was measured with an amperometric oxygen electrode in a closed chamber (1.7-ml volume), magnetically stirred at 37°C, essentially as previously described [18,20,21]. The sample (IM) was suspended in a medium containing 75 mM sucrose, 5 mM KH2PO4, 40 mM KCl, 0.5 mM EDTA, 3 mM MgCl2, and 30 mM Tris–HCl, at pH: 7.4. The sample was transferred to the chamber and, to measure the maximum respiration rate, several substrates were added at the following concentrations: 0.7 mM NADH, 5 mM pyruvate plus 2.5 mM malate, 20 mM succinate, 20 mM fumarate, 20 mM α-ketoglutarate, 20 mM β-hydroxybutyrate, 5 mM glucose, and 5 mM galactose.

Myelin isolation ATP synthesis assay in IM IM was obtained from bovine forebrain collected by a local slaughterhouse, immediately after the death of the animal. The method consists in a “floating up” sucrose gradient to reduce contaminants, according to Norton and Poduslo [28]. Protease inhibitor cocktail (Sigma-Aldrich), 50 μg/ml of 5-fluorouracil, and 20 μg/ml of ampicillin were present throughout isolation. Centrifugation was conducted in a Beckman FW-27 rotor (Beckman, Fullerton,

ATP synthesis was measured by the highly sensitive luciferin/luciferase method. Assays were conducted at 37°C for 2 min, by measuring formed ATP from added ADP. IM (20 μg of total protein) was added to incubation medium (1-ml final volume) containing 10 mM Tris–HCl (pH: 7.4), 50 mM KCl, 1 mM EGTA, 2 mM EDTA, 5 mM KH2PO4, 2 mM MgCl2, 0.6 mM ouabain, 0.040 mg/ml of


1158 S. Ravera et al. ampicillin and 0.2 mM adenylate kinase inhibitor di(adenosine-5’) pentaphosphate, and the metabolic substrate containing 0.7 mM NADH, 5 mM pyruvate plus 2.5 mM malate, 20 mM succinate, 20 mM fumarate, 20 mM α-ketoglutarate, 20 mM β-hydroxybutyrate, 5 mM glucose, and 5 mM galactose. Suspension was equilibrated for 10 min at 37°C and then ATP synthesis was induced by addition of 0.3 mM ADP. The ATP content was measured using the luciferin/ luciferase ATP bioluminescence assay kit CLSII (Roche, Basel, Switzerland), on a luminometer (Triathler, Bioscan, Washington, D.C.). When necessary, specific inhibitors or antioxidants were used during the incubation with NADH (10 μM oligomycin, 50 μM antimycin A, 50 μM resveratrol, 100 μM quercetin, or 100 μM curcumin plus 100 μM piperin). ATP standard solutions (Roche, Basel, Switzerland) in the concentration range 10 ⫺ 10–10 ⫺ 7 M were used for calibration [18,19]. Evaluation of lipid peroxidation in IM To assess lipid peroxidation in IM, MDA and 4-HNE levels were evaluated. For MDA, the thiobarbituric acid reactive substances (TBARS) assay [30] was employed, with minor modifications. This is based on the reaction of MDA, a breakdown product of lipid peroxides, with TBA. The TBARS solution containing 15% trichloroacetic acid (TCA) in 0.25 N HCl and 26 mM TBA. To evaluate the basal concentration of MDA, 600 μl of TBARS solution was added to 50 μg of total protein dissolved in 300 μl of milliQ water. The mix was incubated for 40 min at 100°C, then centrifuged at 14000 rpm for 2 min, and the supernatant was analyzed spectrophotometrically, at 532 nm. To estimate the lipid peroxidation in IM during the OXPHOS activity, IM was incubated for different times (0, 5, 10, 15, and 20 min) with 0.7 mM NADH or 20 mM succinate, to activate the pathways composed by Complexes I, III, and IV or Complexes II, III, and IV, respectively. After this step, the sample was centrifuged at 14000 rpm for 2 min, and pellet was resuspended in 300 μl of milliQ, before the addition of TBARS solution. When necessary, 20 μM rotenone, 50 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or 50 μM nigericin was added to evaluate the effect of the Complex I inhibitor and of two uncoupling molecules. To measure the effect of antioxidants on MDA production by IM, 100 μM reduced glutathione (GSH), 50 μM resveratrol, 100 μM quercetin, or 100 μM curcumin plus 100 μM piperin was used. Different MDA concentrations, 0.75, 1, and 2 μM, were used to obtain a standard curve. 4-HNE levels in IM were determined spectrophotometrically, at 586 nm, using the Biotech LPO-586 228 kit (Oxis International, Portland, OR, USA) [31]. As for MDA, 4-HNE was assayed after incubation with NADH and succinate at different time points, also when necessary in the presence of rotenone, FCCP, nigericin, and antioxidants.

Cytofluorimetric analysis ROS production by IM was quantified after staining with dihydrorhodamine 123 (DHR, Molecular Probes, Life Technologies, USA), a fluorescent probe used to evaluate the oxidative stress. IM was washed in PBS and resuspended in a buffer containing 10 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid or HEPES, 135 mM NaCl, and 5 mM CaCl2. The sample was incubated with 5 μM DHR for 20 min at 37°C, centrifuged, and the pellet was resuspended in a medium containing 50 mM Tris–HCl (pH: 7.4), 5 mM KCl, 1 mM EGTA, 5 mM MgCl2, and 0.6 mM ouabain. Immediately before the cytofluorimeter measures, respiring substrates (0.7 mM NADH and 20 mM succinate) were added. Flow cytometry was performed on a CyAn ADP cytometer (Beckman Coulter) equipped with three laser lamps. The plot of all physical parameters’ forward scatter (FSC) versus side scatter (SSC) was set to limits debris and aggregates. The results are reported as the percentage of sample relative to the relevant control, which display a fluorescence shift. Evaluation of H2O2 production in IM and mitochondria-enriched fractions H2O2 production was estimated using a luminometric assay. 50 μg of total protein was incubated for 10 min at 37°C in 50 mM Tris–HCl (pH: 7.4), 5 mM KCl, 1 mM EGTA, 5 mM MgCl2, and 0.6 mM ouabain, with the respiring substrates. For myelin, 5 mM pyruvate plus 2.5 mM malate, 0.7 mM NADH, 20 mM succinate, 20 mM β-hydroxybutyrate, 5 mM glucose, and 5 mM galactose were employed, while for mitochondria-enriched fractions only 5 mM pyruvate plus 2.5 mM malate or 20 mM succinate were used. In both cases, the sample was centrifuged and the supernatant was added to 10 μg/ml of HRP and 5 mM luminol. Concentration of H2O2 was measured using a calibration curve with H2O2 standard solutions between 10 and 40 μM. H2O2 production was measured in IM also in the presence of the following antioxidants: 100 μM GSH, 50 μM resveratrol, 100 μM quercetin, and 100 μM curcumin plus 100 μM piperin. Electrophoretic separation of IM and Semiquantitative WB Denaturing electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) was performed on IM using a Laemmli protocol [32] with minor modifications in a Mini-PROTEAN III (BioRad, Hercules, CA, USA) apparatus. Separating gel was a gradient gel from 4% to 16% w/v PAGE, containing 0.1% SDS, at pH: 8.8. Stacking gels contained 5% w/v PAGE and 0.1% SDS (at pH: 6.8). Samples were precipitated with 6% TCA and centrifuged at 14,000 rpm for 2 min in Eppendorf Centrifuge, in order to improve their solubilization [33]. Supernatant was discarded and pellet was resuspended in 10 mM Tris–HCl at pH: 7.4. 8% SDS w/v in 125 mM



Tris–HCl (pH: 6.8) and 1.25% v/v dithiothreitol or DTT and incubated for 15 min. Then, samples were boiled for 5 min and a second solution, containing 40% w/v sucrose and 0.008% w/v bromophenol blue, was added. Run was performed at 4°C, at 70 mA for each gel, for 120–150 min. Running buffer contained 0.05 M Tris (pH: 8.0), 0.4 M glycine, 1.8 mM EDTA, and 0.1% SDS. Total protein was estimated by Bradford method [34]. Electrophoretically separated samples were transferred onto nitrocellulose (NC) membranes by electroblotting, at 400 mA for 1 h in Tris–glycine buffer (50 mM Tris and 380 mM glycine) plus 20% (v/v) methanol, at 4°C. NC membranes were blocked in 5% BSA overnight, then washed in 0.15% PBS–Tween (PBST), and incubated with the specific Abs (over night at 4°C) diluted in PBST 0.15%: anti SOD1, 1:200; anti SOD2, 1:200. Secondary HRP-conjugated Abs were diluted in PBS 1:10000. After extensive washing with 0.15% PBST, binding of Abs was revealed by enhanced chemiluminescence detection system (ECL) (Roche). Blots were acquired by ChemiDoc (BioRad). Each band was converted by ChemiDoc into a densitometric trace allowing calculations of intensity and signals compared with the signal of whole protein pattern, expressing results as Relative Optical Density (R.O.D.) [18]. SOD, CAT, and GPx assays To evaluate SOD activity in IM and in mitochondriaenriched fractions, a spectrophotometric assay was carried out, following the conversion of nitroblue tetrazolium (NBT) to NBT-diformazan dye [35]. In the assay system, superoxide was generated by xanthine oxidase (XOD) during the conversion of xanthine to H2O2 and uric acid. Briefly, 100 μg of total proteins were added to a buffer containing 0.1 M glycine–NaOH (pH: 9), 3 mM xanthine, 3 mM EDTA, 0.15% BSA, and 0.75 mM NBT. After equilibration at 20°C for 10 min, the reaction was initiated by adding 6 mU of XOD and incubated further at 20°C for 20 min. The reaction was terminated by addition of 6 mM CuCl, and the absorbance at 560 nm was determined. To assess CAT and GPx activities, Catalase Activity Colorimetric/Fluorometric Assay Kit (cod: K773-100, BioVision Inc., Milpitas, CA, USA) and Glutathione Peroxidase

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Activity Colorimetric Assay Kit (cod: K762-100, BioVision Inc., Milpitas, CA, USA) were used, respectively. Statistical analysis GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego California USA, www.graphpad. com) was used to carry out statistical analysis. All data are reported as mean ⫾ standard deviation (SD). The Bonferroni post-hoc test was used to evaluate the difference between control and treatments.

Results OXPHOS activity in IM elicited by several respiring substrates Oxygen consumption and ATP synthesis were evaluated (Figure 1, Panels A and B, respectively) in IM in the presence of both typical and unconventional respiring substrates. Graphs show that IM conducts OXPHOS with the classical respiring substrates, that is, pyruvate plus malate or succinate and NADH, but displays the maximum oxidative metabolism in the presence of galactose. Notably, NADH does not appear to be a good respiring substrate for mitochondria-enriched fractions, because these do not contain channels to internalize NADH into the matrix. Figure 1, Panel C shows that ATP synthesis is sensitive to the common OXPHOS inhibitors (oligomycin and antimycin A), suggesting that the ATP production is really due to the presence of FoF1 ATP synthase in myelin. Panel H also reports that the putative IM ATP synthase is inhibited by antioxidant molecules (resveratrol, quercetin, and curcumin ⫹ piperin), as already reported by Hong and Pedersen for the mitochondrial enzyme [36]. The ETC assays, reported in Supplementary Table I (to be found online at http://informahealthcare.com/doi/abs/10.3109/10715762.2015.1050962), confirm that IM carry out an oxidative metabolism, using the mitochondrial machinery. Moreover, activities of Complexes I, III, and IV are sensitive to their specific inhibitors.

Figure 1. Functional expression of OXPHOS in IM in the presence of typical and unconventional respiring substrates. Panels A and B report the oxygen consumption and the ATP synthesis in IM, after the addition of several respiring substrates. Panel C shows the ATP synthesis after the addition of NADH in the presence of several inhibitors and antioxidant compounds. Data are reported as mean ⫾ SD, each panel is representative of at least ten experiments and * indicates a p ⬍ 0.001.

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Evaluation of the IM peroxidation status Considering that myelin is composed by 80% of lipids [27], mostly unsaturated, it can be presumed that the presence of a OXPHOS activity triggers a lipid peroxidation in myelin. To evaluate this possibility, MDA and 4-HNE were assayed, as reported in Figure 2, Panels A and D, respectively. Graphs show that IM displays basal levels of MDA and 4-HNE, which remain similar to basal level after the addiction of succinate, but increase after the addition of NADH. This may imply that Complex I is involved in the production of MDA and 4-HNE being the main ROS producer in the ETC [10]. Moreover, the MDA and 4-HNE levels induced by NADH increased in parallel with the time of incubation with the respiring substrate. Lipid peroxidation production after NADH induction was also assayed in the presence of rotenone, a specific inhibitor of Complex I, or two uncoupling molecules: FCCP and nigericin (Figure 2, Panels B and E). In all cases, MDA and 4-HNE levels increased with respect to the control (in the presence of NADH), suggesting that the lipid peroxidation is really due to the ectopic ETC. Figure 2, Panels C and F show that the addition of antioxidant compounds (resveratrol, quercetin, and curcumin ⫹ piperin) decrease the MDA and 4-HNE production in IM. Such results can be ascribed to their inhibitory effect on ATP synthase [36]. Such effect would result from a preventative instead of a

scavenging action. In fact, in coupled conditions, a partial reversible inhibition of ATP synthase by polyphenols can slow down the oxygen consumption by the ETC, thereby reducing ROS production [37]. To assess the oxidative status of IM, H2O2 (principally deriving from O2•⫺) was also evaluated. H2O2 production in IM was assayed by both cytofluorimetric assay, in the presence of DHR (Figure 3, Panels A–C), and luminometric assay (Figure 3, Panel E). Panels A and B show the fluorescent shift of DHR in IM incubated with NADH or succinate, respectively. Table in Panel C quantifies the fluorescent shift percentage. Oxidative stress increases in IM in the presence of both respiring substrates, but mainly in the presence of NADH, confirming that the pathway composed by Complex I–III–IV is the principal source of ROS. Cytofluorimetric data are confirmed by the H2O2 luminometric assay (Figure 3, Panel E). Also in this case, the highest amount of H2O2 is formed in the presence of NADH, even though other respiring substrates also promote the H2O2 production. Figure 3, Panel D reports a comparison of H2O2 production among IM and mitochondria-enriched fractions. Both samples produce H2O2 in the presence of pyruvate/malate or succinate, but in IM this production is considerably higher than in mitochondriaenriched fractions. To evaluate if IM contains an enzymatic antioxidant system, the functional presence of SOD, CAT, and GPx

Figure 2. Evaluation of MDA and 4-HNE production in IM, as lipid peroxidation markers. Panels A and D show the MDA and 4-HNE levels in IM after the incubation with NADH (light gray columns) or succinate (dark gray columns), as respiring substrate, for different time points. Data are reported as mean ⫾ SD, each panel is representative of at least five experiments and * indicates a p ⬍ 0.001 with respect to the basal levels. Panels B and E report the MDA and 4-HNE production in IM in the presence of rotenone, specific inhibitor of Complex I, and two uncoupling compounds (FCCP and nigericin). Data are reported as mean ⫾ SD, each panel is representative of at least five experiments and # indicates a p ⬍ 0.005 with respect to the sample incubated with only NADH. Panels C and F report the MDA and 4-HNE production in IM after the addition of NADH in the presence of GSH and several polyphenols (resveratrol, quercetin, and curcumin ⫹ piperin). Data are reported as mean ⫾ SD, each panel is representative of at least five experiments and ** indicates a p ⬍ 0.001 with respect to the sample incubated with only NADH.



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Figure 3. Evaluation of H2O2 production in IM. Panels A and B show the cytofluorimetric analysis of IM in the presence of DHR, the fluorescent probe suitable to quantify intracellular ROS production. Panel A reports the fluorescent shift for DHR in the presence of NADH. Panel B reports the fluorescent shift of DHR in the presence of succinate. The percentage of the fluorescence shift is reported in Panel C. Panel D reports a comparison of H2O2 production in mitochondria-enriched fraction with respect to IM. The luminometric evaluation of H2O2 production in IM was investigated in the presence of different respiring substrates (Panel E), rotenone, FCCP, and nigericin (Panel F), and antioxidants compounds (Panel G). Data reported in Panels C–G are expressed as mean ⫾ SD and * indicates a p ⬍ 0.001 and # indicates a p ⬍ 0.005, with respect to the sample treated with only NADH, in both cases. Each panel is representative of at least five experiments.

was investigated. Figure 4, Panels A and B report a WB analysis showing that IM expresses both the cytosolic and the mitochondrial SOD isoforms. Moreover, densitometric analysis (Panel C) shows that the signal intensity of mitochondrial SOD (SOD2) is higher in IM than in mitochondria. WB data are confirmed by the SOD activity assay (Panel D), in which the SOD activity was higher in IM than mitochondria. In Figure 4, Panels E and F, CAT and GPx activities are reported, respectively. Activities were higher in IM with respect to forebrain homogenate. These data, besides being confirmative that myelin displays an enzymatic antioxidant system, indicate an absence of mitochondrial contamination. In fact, it is unlikely that any

mitochondrial “contaminant” can be higher in IM than in mitochondria-enriched fractions or forebrain homogenate.

Discussion Data reported in this work show that the extramitochondrial OXPHOS activity in IM, which we have previously described [18–22], besides producing ATP can be a source of an oxidative stress in the sheath (Figures 2–4), due to the ETC expressed therein. This is not surprising, considering that IM contains the same ETC as mitochondria, and that the ETC is a major producer of ROS [2]. In fact, IM

Figure 4. Assay of SOD, CAT, and GPx activity in IM. Panels A and B show the semiquantitative WB analysis against SOD1 (cytosolic isoform) and SOD2 (mitochondrial isoform), respectively. The loaded samples are mitochondria-enriched fraction (Mit), lane 1; forebrain homogenate (FH), lane 2; crude myelin fraction (CM), lane 3; isolated myelin fraction (IM), lane 4. The values of densitometric analysis are shown in Panel C, expressed as R.O.D. Panel D reports the assay of total SOD activity in IM in comparison with mitochondria-enriched fraction and forebrain homogenate. Panels E and F show the activity assay of CAT and GPx in IM, comparing with the activity in forebrain homogenate. Each panel is representative of at least ten experiments and data in Panels C–F are expressed as mean ⫾ SD. * indicates a p ⬍ 0.005 and # indicates a p ⬍ 0.001, with respect to the forebrain homogenate.


1162 S. Ravera et al. expresses, for example, NADH-ubiquinone oxidoreductase subunit or ND4L (Supplementary Figure 1 Panel H to be found online at http://informahealthcare.com/doi/abs/10.3 109/10715762.2015.1050962) which is codified by mitochondrial DNA. Myelin is enriched in lipids that are prone to peroxidation in the presence of ROS. H2O2 is present in IM and its levels increase in the presence of several respiring substrates (Figure 3) especially with NADH. IM displays a basal level of both MDA and 4-HNE, classical lipid peroxidation markers whose concentration increases after incubation with NADH, but remains constant in the presence of succinate (Figure 2, Panels A and D). This fact is confirmative of a primary role for Complex I, the direct utilizer of NADH, in the production of ROS and consequent oxidative stress production in IM, as reported for mitochondria [2]. Therefore, it appears that oxidative stress represents an unsuspected constant danger for the stability of the sheath. Nevertheless, myelin appears to display a protection against the oxidative damage, represented by endogenous antioxidants such as SOD, CAT, and GPx, expressed in IM. Interestingly, the activity of CAT and GPx was higher in IM than in forebrain homogenate and mitochondriaenriched fractions (Figure 4, Panels B and C), suggesting that SOD is a pivotal enzyme to regulate the myelin energetic metabolism. The main product of SOD is H2O2; therefore, it is fundamental that IM also express catalase and GPx, to convert the peroxide hydrogen to water, completing the detoxification system. SOD, CAT, and GPx would be truly resident in myelin where they would play a pivotal role in the protection against the oxidative stress production. Notably, SOD2 is the mitochondrial SOD, and its expression is higher in IM than in mitochondriaenriched fractions (Figure 4). These data confirm that SOD2 is really resident in myelin and it is not due to a mitochondrial contamination, as also suggested by WB analyses showing that IM express SOD and the OXPHOS proteins, but not TIM and TOM, two mitochondrial proteins not involved in energetic metabolism (Supplementary Figure 1 to be found online at http://informahealthcare. com/doi/abs/10.3109/10715762.2015.1050962). However, in case of damage, oxidative stress in myelin could exceed the defenses, as shown by data in the presence of rotenone, the specific inhibitor of Complex I, or FCCP and nigericin, two uncoupling molecules, which were employed to mimic such damage conditions. An alteration of the OXPHOS coupling due to the addition of these molecules induced a major oxidative stress, increasing the production of MDA, 4-HNE, and H2O2 in IM when stimulated by NADH (Figures 2 and 3). Although rotenone is a Complex I inhibitor, it increases oxidative stress production because it interferes with the electron transfer from iron–sulfur centers to ubiquinone. A backwards electron pathway is created within the membrane, so that cellular oxygen is reduced to superoxide, producing ROS [38], increasing the lipid peroxidation. The same effect was observed in the presence of FCCP and nigericin, because, in uncoupled conditions, oxygen consumption increases dramatically, enhancing the oxidative stress

[39]. The sheath appear at risk of structural damage from uncoupling of the ETC. In these last years, research on antioxidants has dramatically increased. Among these molecules, GSH, resveratrol, quercetin, and curcumin plus piperin appear promising. MDA, 4-HNE, and H2O2 production in IM decreased in the presence of the cited antioxidant molecules, which were able to contrast the lipid peroxidation and ROS production in IM (Figure 2 and 3). This may be due to their inhibitory effect on FoF1 ATP synthase [36], which, in coupled conditions, would induce a slowdown of the ETC, with ROS production decline. The identification of myelin as a source of ROS may shed new light on their role in pathologic conditions of the nervous system. In fact, when the OXPHOS machinery is in a coupled status and the endogenous antioxidants work properly in the myelin sheath, oxidative stress is minimal. By contrast, it can be hypothesized that upon primary myelin sheath damage, as in demyelinating diseases, the ETC becomes uncoupled from the ATP synthesis, producing a great amount of ROS that the endogenous antioxidants cannot balance. This would create a selfperpetuating vicious circle leading to an increment in the oxidative stress and damage to the lipid-rich sheath which in turn promotes more ROS production by the uncoupled ETC [27]. Notably, oxidative stress is one of the major determinants of membrane alterations [40], contributing to the loss of cellular organization [41]. Moreover, oxidative stress itself can increase the cellular respiration and thus ROS generation, resulting in ATP depletion and cell death [39]. Some authors have observed that myelin sheath is highly vulnerable to oxidative stress [42,43]. In particular, the application of a source of superoxide radical directly in vitro to white matter from young adult human brain caused a change in the lipid phase of the myelin from a crystalline (ordered) state to a liquid crystalline (disordered) state [43]. This alteration was accompanied by a dramatic increase in the levels of lipid peroxidation products: MDA, a conjugated diene, and ethane [43]. Moreover, these changes in myelin sheath structure, due to an increment of oxidative stress, are similar to that which occurs in the course of natural aging [43], thus providing further substantiation for the notion that O2-. might be a major toxic agent associated with the aging process [44]. Interestingly, Qi et al. have observed that the neurodegeneration of optic nerve in optic neuritis is due to oxidative stress produced in the absence of inflammation [45]. The primary risk factors for Leber hereditary optic neuropathy (LHON), a disease with a complex etiology, are mutations in the mitochondrial genes encoding Complex I, the principal oxidative stress producer of the ETC [46]. Interestingly, it was shown that in LHON the mitochondrial antioxidant defenses are essential to cellular rescue [47]. Moreover, an increment in ROS production renders the retinal ganglion cells vulnerable to apoptotic cell death [46]. Oxidative stress arising from OXPHOS activity dysfunction is involved in the etiopathogenesis of a large number of optic nerve degenerative disorders, including autosomal dominant optic atrophy, toxic/nutritional optic



neuropathies, and glaucoma [48]. Also demyelinating diseases such as multiple sclerosis (MS) seem to be associated with enhanced oxidative stress. Wallberg et al. observed that myelin glycoproteins are modified in the presence of high level of MDA, leading to an increment in encephalitogenicity [49]. Griot et al. observed that ROS mediate a selective degeneration of oligodendrocytes [50], and play an important effector role in the encephalitis lesions. However, these authors ascribe the presence of ROS to their secretion from stimulated macrophages [50]. Other authors have observed that ROS are involved in the pathogenesis of MS and experimental allergic encephalomyelitis [51]. In this work, it was observed that the myelin phagocytosis by macrophages triggers the production of ROS, which, in their turn, plays a crucial role in the myelin phagocytosis [51]. By blocking the ROS production with NADPH oxidase inhibitors, the phagocytosis of myelin was prevented. Furthermore, scavenging of ROS with catalase (H2O2) or mannitol (OH⫺) decreased the phagocytosis of myelin by macrophages [51]. Data reported in this work suggest that a quote of oxidative stress production could depend directly on the myelin, when the OXPHOS machinery is altered or damaged. Based on our data, it can be hypothesized that—at least—part of the oxidative stress associated to optic neuritis and demyelinating diseases comes from the ectopic ETC expressed inside the myelin membranes, due to any impairment in its OXPHOS. In fact, it is possible to presume that an uncontrolled auto-determined increment in oxidative stress in myelin sheath could damage the sheath and amplify the inflammatory cascade. This in turn would recruit immune cells from the periphery, and activate resident microglia, compromising blood–brain barrier. An inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in the demyelination and free-radical-mediated tissue injury in the pathogenesis of MS [52]. In turn, an inflammatory environment in demyelinating lesions leads to the generation of free radicals as well as proinflammatory cytokines, which contribute to the development and progression of the demyelinating disease, as observed in MS [52]. Therefore, inflammation can lead to oxidative stress and vice versa, creating a self-perpetuating cycle.

Conclusion In conclusion, data reported herein demonstrate that the ectopic aerobic ATP production in myelin sheath is associated to the production of oxidative stress. This in turn, in pathological conditions, may render the sheath more vulnerable to damage. Data also show that oxidative stress in myelin can be prevented, at least partially, by endogenous detoxication mechanisms or by the use of antioxidants molecules. We believe that these data may provide a new avenue of experimental investigation to clarify the major causes of axonal damage in demyelinating diseases.

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Declaration of interest All authors have no conflicts of interest.This study was supported by a Grant from the “Fondazione Giuseppe Levi–Accademia Nazionale dei Lincei” for the research project entitled: “Produzione extramitocondriale di ATP in mielina: localizzazione dei complessi della catena respiratoria e possible ruolo nella degenerazione assonale in Sclerosi Multipla” and a Grant from the “Compagnia di San Paolo”–Neuroscience Program 2008, for the research project entitled: “Energetic metabolism in myelinated axon: a new trophic role of myelin sheath.”

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Supplementary material available online Supplementary materials and method, Table I, and Figure 1 to be found online at http://informahealthcare. com/doi/abs/10.3109/10715762.2015.1050962.

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