Randa Sghaier

Thomas Nury

Valerio Leoni

Claudio Caccia

Jean-Paul Pais De Barros

Ameur Cherif

Anne Vejux

Thibault Moreau

Khalifa Limem

Mohammad Samadi

Jean-Paul Pais

D E Barros

John J Mackrill

Ahmed Slaheddine Masmoudi

Gérard Lizard
email: gerard.lizard@u-bourgogne.fr

Amira Zarrouk
email: zarroukamira@gmail.com

Dimethyl fumarate and monomethyl fumarate attenuate oxidative stress and mitochondrial alterations leading to oxiapoptophagy in 158N murine oligodendrocytes treated with 7β-hydroxycholesterol

Keywords: dimethyl fumarate, monomethyl fumarate, 7β-hydroxycholesterol, 158N cells, oxidative stress, mitochondria, peroxisome, oxiapoptophagy, apoptosis, autophagy, lipid profile

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Oxidative stress is characterized by an imbalance between excessive production of reactive oxygen species (ROS) and the capacity of antioxidant defenses to attenuate damage caused by free radicals [START_REF] Betteridge | What is oxidative stress?[END_REF] Oxidative stress is amplified in a variety of neurodegenerative, chronic inflammatory, vascular and neurodegenerative diseases. Thus, several studies have carefully characterized the relationships between oxidative stress and demyelinating and non demyelinating neurodegenerative diseases [START_REF] Sayre | Oxidative stress and neurotoxicity[END_REF][START_REF] Zarrouk | Involvement of oxysterols in age-related diseases and ageing processes[END_REF].

Oxidative injury can be involved in brain lesions via the induction of protein and lipid oxidation [START_REF] Fischer | NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury[END_REF], or mitochondrial dysfunction [START_REF] Campbell | Mitochondria as crucial players in demyelinated axons: lessons from neuropathology and experimental demyelination[END_REF]. In the presence of oxidative stress, the high content of cholesterol in the brain makes it vulnerable to oxidation, which favors the generation of deleterious compounds, named oxysterols (cholesterol oxide products), in the central nerve system (CNS) [START_REF] Zarrouk | Lipid Biomarkers in Alzheimer's Disease[END_REF]. Some of these oxysterols (mainly those oxidized on C7, such as 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-OHC), mostly generated by the autoxidation of cholesterol Huntington's disease [START_REF] Kreilaus | Evidence for altered cholesterol metabolism in H untington's disease post mortem brain tissue[END_REF] or Alzheimer's disease (AD) [START_REF] Testa | Changes in brain oxysterols at different stages of Alzheimer's disease: Their involvement in neuroinflammation[END_REF]. Aβ peptide, a toxic peptide characteristic of AD, was shown to participate in the production of 7β-OHC [START_REF] Nelson | Oxidation of cholesterol by amyloid precursor protein and β-amyloid peptide[END_REF]. It is also known that 7KC can be converted to 7β-OHC [14] by 11β-hydroxyxteroid dehydrogenase type 1 (11β-HSD1) [START_REF] Odermatt | 11β-Hydroxysteroid dehydrogenase 1: Regeneration of active glucocorticoids is only part of the story[END_REF]. In humans, the 11β-HSD1 is also able to catalyse the conversion of 7β-OHC into 7KC [START_REF] Larsson | In vivo interconversion of 7β-hydroxycholesterol and 7ketocholesterol, potential surrogate markers for oxidative stress[END_REF]. As 7β-OHC can be formed from 7KC and vice versa, the deleterious effects of both are closely related. It is therefore important to consider the activities of 7KC and 7β-OHC since these two cytotoxic oxysterols are involved in neurodegeneration [START_REF] Zarrouk | Involvement of oxysterols in age-related diseases and ageing processes[END_REF]. Currently, it has been demonstrated in different cell types from various species [START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF][START_REF] Zarrouk | Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22: 6 n-3)[END_REF][START_REF] Clarion | 7β-Hydroxycholesterol-induced energy stress leads to sequential opposing signaling responses and to death of C6 glioblastoma cells[END_REF][START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF], that 7β-OHC induces a more or less pronounced apoptotic mode of cell death associated with oxidative stress. Later, a complex cell death process defined as oxiapoptophagy ((OXIdative stress, APOPTOsis, autoPHAGY)) was described on microglial cells and oligodendrocytes [START_REF] Nury | Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7βhydroxycholesterol-, or 24 (S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22: 6 n-3)[END_REF][START_REF] Nury | 7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD[END_REF]. This particular type of cell death involving oxidative stress, mitochondrial dysfunction, and induction of apoptosis and autophagy, which are hallmarks of neurodegenerative diseases, was observed in 7β-OHC-treated murine oligodendrocytes 158N [START_REF] Nury | Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7βhydroxycholesterol-, or 24 (S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22: 6 n-3)[END_REF]. The cellular model used in the present study (murine oligodendrocytes 158N treated with 7β-OHC) is therefore an interesting tool for identifying molecules targeting major cell alterations (oxidative stress and mitochondrial dysfunction leading to cell death) associated with demyelination, since oligodendrocytes are myelin synthesizing cells.

To prevent demyelination, different molecules have been tested for their neuroprotective capacities such as dimethyl fumarate (DMF), the active component of Tecfidera (Biogen) also named BG-12 [START_REF] Werdenberg | Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters[END_REF]. DMF is a fumaric acid ester which has immunomodulatory, anti-inflammatory, and antioxidant proprieties [START_REF] Lin | The antiinflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1[END_REF][START_REF] Scannevin | Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway[END_REF][START_REF] Huang | Dimethyl fumarate modulates antioxidant and lipid metabolism in oligodendrocytes[END_REF]. This small drug has been approved as an oral therapeutic agent for the treatment of patients with relapsing-remitting MS [START_REF] Havrdova | Oral BG-12 (dimethyl fumarate) for relapsing-remitting multiple sclerosis: a review of DEFINE and CONFIRM: Evaluation of: Gold R[END_REF][START_REF] Xu | Dimethyl fumarate for multiple sclerosis[END_REF]. Many studies have provided that the pharmacological effect of DMF is due to its bioactive metabolite, monomethyl fumarate (MMF) [START_REF] Werdenberg | Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters[END_REF][START_REF] Gillard | DMF, but not other fumarates, inhibits NF-κB activity in vitro in an Nrf2-independent manner[END_REF]. This metabolite has beneficial effects on neuroinflammation, neurodegeneration and oxidative stress [START_REF] Scannevin | Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway[END_REF][START_REF] Linker | Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway[END_REF]. It is supposed that DMF and MMF act by the activation of Nrf2 (nuclear factor E2-related factor2) pathway, through the induction of antioxidant protein expression [START_REF] Lin | The antiinflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1[END_REF]. Under physiological conditions, Nrf2 is sequestered in the cytoplasm through its interaction with the repressor protein 'kelch-like ECHassociated protein 1' (Keap1). Treatment with compounds, like DMF, results in the conformational modifications of cysteine residues on Keap1 [START_REF] Linker | Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway[END_REF], which allows activation and nuclear translocation of Nrf2 to promote protective genes such as heme-oxygenase-1 [START_REF] Lin | The antiinflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1[END_REF] and NADPH-quinone oxidoreductase-1 [START_REF] Linker | Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway[END_REF].

In the present study, we evaluated the cytoprotective effects of DMF and MMF on 7β-OHC-treated murine oligodendrocytes 158N. We asked whether DMF and MMF were able to prevent or reduce 7β-OHC-induced cytotoxicity (oxidative stress, mitochondrial dysfunction, modification of lipid profile, apoptosis and autophagy) accompanying demyelination in MS. In addition, as 158N cells express major myelin proteins, proteolipid protein (PLP) and myelin basic protein (MBP) [START_REF] Baarine | Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes[END_REF], which are major compounds of myelin (a proteolipidic complex which wraps around the axons and is essential for the transmission of nerve impulse) [START_REF] Baumann | Biology of oligodendrocyte and myelin in the mammalian central nervous system[END_REF], the effects of 7β-OHC in the presence or absence DMF and MMF on PLP and MBP expression were also evaluated.

Material and methods

Cell culture and treatments

Murine oligodendrocyte cells (158N) [START_REF] Baarine | Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes[END_REF] were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Amboise, France) containing 5% (v/v) heat-inactivated fetal bovine serum (FBS) (Dutscher, Brumath, France) and 1% antibiotics (penicillin, streptomycin) (Dutscher). Cells were incubated in a humidified atmosphere (5% CO2, 95% air) at 37°C and passaged twice a week.

The stock solutions of 7β-hydroxycholesterol (7β-OHC) (Sigma-Aldrich, St Quentin Fallavier, France) was prepared at 800 µg/mL (2 mM) as previously described [START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF] and stored at 4°C. Dimethyl fumarate (DMF) (ref: 242926, Sigma-Aldrich) and monomethyl fumarate (MMF) (ref: 651419; Sigma-Aldrich) stock solutions, were prepared at 50 mM in DMSO (Sigma-Aldrich). α-Tocopherol (the major component of Vitamin E; ref: T3251, Sigma-Aldrich) solution was prepared at 80 mM as previously described [START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF].

In the different experimental conditions, murine oligodendrocytes 158N were seeded either in Petri dishes (30,000 cells/cm 2 , 100 mm diameter), or in six-well plates (2.4 x 10 5 cells per well). After 24 h of culture, and for an additional 24 h period of time, DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM) (used as a positive control for cytoprotection) were introduced in the culture medium 2 h before the addition of 7β-OHC (50 µM). The choice of the concentrations of 7β-OHC (50 µM) as well as of DMF (25 µM) and MMF (25 µM) was based on a study by Ragot et al. [START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF] and on the doseeffect relationship of 7β-OHC (1, 6.25, 12.5, 25, 50, 100 and 200 µM) (Supplementary Fig. 1) and of DMF or MMF in the presence or absence of 7β-OHC (Supplementary Fig. 2). With 7β-OHC, the concentration of 50 µM corresponds to the 50 % inhibiting concentration on 158N cells (Supplementary Fig. 1). In vitro, it is important to underline that only a part of oxysterols (1-35% depending on the cell type considered) accumulates in the cells [START_REF] Zarrouk | Involvement of oxysterols in age-related diseases and ageing processes[END_REF][START_REF] Sghaier | Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes[END_REF]., It has been established that the median level of 7β-OHC was 1.11 µg/mL (2.8 µM) and 1.43 µg/mL (3.6 µM) in the cerebrospinal fluid of normal subjects and of patients with multiple sclerosis, respectively [START_REF] Leoni | Levels of 7-oxocholesterol in cerebrospinal fluid are more than one thousand times lower than reported in multiple sclerosis[END_REF]. Few data are available on the intracellular level of oxysterols in nerve cells. However, when the oxysterols such as 7β-OHC are used in vitro at µM concentrations (50 µM in the present study), the estimated intracellular levels of this compound are in the range of those found in the frontal and occipital cortex of patients with Alzheimer'disease [START_REF] Testa | Changes in brain oxysterols at different stages of Alzheimer's disease: Their involvement in neuroinflammation[END_REF]. Additional experiments were also performed to assess the effect of DMF and MMF in the recovery of 7β-OHC (50 µM)-induced cell death. In these experiments, after 24 h of culture, 158N cells were incubated with 7β-OHC for 6 and 10 h before the addition of DMF or MMF, or α-tocopherol. The cells were further incubated with 7β-OHC in combination with DMF, MMF or αtocopherol for an additional 14 or 18 h.

Evaluation of cell morphology by phase contrast microscopy

After treatment of 158N cells with or without 7β-OHC (50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM), cell morphology, cell density and cell detachment (loss of cell adhesion) were evaluated by phase contrast microscopy using an inverted-phase contrast microscope (Axiovert 40 CFL, Zeiss, Jena, Germany). Images were obtained with a camera (Axiocam ICm 1, Zeiss).

Quantification of adherent cells with crystal violet staining

Adherent cells were estimated by staining with crystal violet (Sigma-Aldrich). After treatment of 158N cells, adherent cells were stained with crystal violet for 5 min, rinsed three times with water. Crystal violet was then dissolved with 0.1 % sodium citrate (Sigma-Aldrich) in 50% ethanol (Carlo-Erba, Val de Reuil, France) and the optical density per well was read at 570 nm with a microplate reader (Tecan Sunrise, Tecan, Lyon, France).

Evaluation of cell viability with the fluorescein diacetate (FDA) assay

The FDA assay evaluates the ability of living cells to transform the FDA to fluorescein after cleavage by plasma membrane esterases

Cell counting of viable cells with trypan blue

Adherent and non-adherent cells were collected by trypsinization and resuspended in culture medium.

The total number of viable cells was determined with a Mallasez counting chamber under a light microscope in the presence of trypan blue (v/v), which stains dead cells in blue.

Evaluation of plasma membrane permeability and cell death by staining with propidium iodide

Propidium iodide (PI) was used to quantify plasma membrane permeability and cell death [START_REF] Lizard | Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis[END_REF]. At the end of reatment with or without 7β-OHC (50 µM)) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). 158N cells (adherent and non-adherent cells) collected by trypsinization, were stained with 1 µg/mL of PI (5 min at 37°C) and then immediately analyzed with a Galaxy flow cytometer (Partec, Münster, Germany). Fluorescence was collected through a 590/10 bandpass filter on a Galaxy flow cytometer (Partec): 10,000 cells were acquired. Data were analyzed with Flomax (Partec) or FlowJo (Tree Star Inc.) software.

Mitochondrial analysis

Measurement of mitochondrial succinate dehydrogenase activity with the MTT assay

Mitochondrial activity and/or cell proliferation were evaluated with an MTT based assay. Only metabolically active cells can transform MTT into formazan by mitochondrial succinate dehydrogenase. MTT data was interpreted by comparison with results obtained by cell counting in the presence of trypan blue. The MTT assay was carried as described previously [START_REF] Zarrouk | Majdoub, 7βhydroxycholesterol-induced cell death, oxidative stress, and fatty acid metabolism dysfunctions attenuated with sea urchin egg oil[END_REF]. The absorbance was read at 570 nm with a microplate reader (TECAN Sunrise, Tecan, Lyon, France).

Measurement of mitochondrial transmembrane potential with DiOC6(3)

The mitochondrial transmembrane potential (∆Ψm) was assessed by flow cytometry with the cationic lipophilic dye 3,3'-dihexyloxacarbocyanine iodide DiOC6(3) (Thermo Fisher Scientific, Courtaboeuf, France). At the end of treatment, adherent and non-adherent cells were pooled, incubated with DiOC6(3) (40 nM, 15 min, 37°C) and then analyzed with a Galaxy flow cytometer (Partec) [START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF][START_REF] Zarrouk | Attenuation of 7-ketocholesterol-induced overproduction of reactive oxygen species, apoptosis, and autophagy by dimethyl fumarate on 158N murine oligodendrocytes[END_REF].

The loss of transmembrane mitochondrial potential (ΔΨm) is indicated by a decreased of the green fluorescence collected through a 520 ± 10 nm band pass filter. For each sample, 10,000 cells were acquired and data were analyzed with Flomax (Partec) or FlowJo (Tree Star Inc.) software.

Measurement of mitochondrial mass with Mitotracker Red

Mitotracker Red (Thermo Fisher Scientific) was used to evaluate mitochondrial mass [START_REF] Zarrouk | Induction of mitochondrial changes associated with oxidative stress on very long chain fatty acids (C22: 0, C24: 0, or C26: 0)-treated human neuronal cells (SK-NB-E), Oxidative medicine and cellular longevity[END_REF]. Mitotracker Red stock solution was prepared at 1 mM in DMSO. Adherent and non-adherent cells were pooled, washed with DMEM without FBS and then incubated for 30 min at 37°C in a Mitotraker Red solution (100 nM), which was prepared in DMEM without FBS. The fluorescent signals were collected through a 580 ± 20 nm band pass filter on a Galaxy flow cytometer (Partec). Data were analyzed with Flomax software (Partec).

Measurement of mitochondrial reactive oxygen species production with MitoSOX-Red

Mitochondrial production of superoxide anion anion (O2 •-) was quantified by flow cytometry after staining with MitoSOX Red (Thermo Fisher Scientific). This positively charged probe accumulates in the mitochondria [START_REF] Zarrouk | Induction of mitochondrial changes associated with oxidative stress on very long chain fatty acids (C22: 0, C24: 0, or C26: 0)-treated human neuronal cells (SK-NB-E), Oxidative medicine and cellular longevity[END_REF] and exhibits an orange/red fluorescence (λEx Max = 510 ; λEm Max= 580 nm).

MitoSOX Red stock solution was prepared at 5 mM in PBS and used at 5 μM in the cells. After cell treatments, adherent and non-adherent cells were pooled by trypsinization and incubated with MitoSOX Red for 15 min at 37°C. The fluorescent signals were collected through a 580 ± 20 nm band pass filter on a Galaxy flow cytometer (Partec). For each sample, 10,000 cells were acquired. Data were analyzed with Flomax software (Partec).

Measurement of mitochondrial cadiolipin levels

The characterisation and quantification of cardiolipins was conducted by liquid chromatography tandem-mass spectrometry (LC-MS/MS) on an Agilent 6460 QqQ (Agilent Technologies, Santa Clara, CA, USA). Total lipid was extracted from cell pellet as described previously by Folch et al. [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF], then dissolved in 100 μL of chloroform/methanol/water mixture. About 1 to 8 µL of extacted lipid were used for lipidomic analysis. Lipid standards and chemicals of the highest grade available were from Avanti Polar Lipids (Coger SAS, Paris, France) and Sigma-Aldrich, respectively. LC-MS/MS quality grade solvents were from Thermo Fischer Scientific. Extracted lipids for cardiolipin analysis were determined as described previously by Vial et al. [38].

Mass spectrometry analysis of sterols, fatty acids phospholipids and organic acids

Total lipid was extracted by the Folch's method [37], and fatty acids, sterols and organic acids were quantified as described previously [START_REF] Sghaier | Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes[END_REF][START_REF] Leoni | Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with α-tocopherol: Impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors[END_REF][START_REF] Leoni | Metabolic consequences of mitochondrial coenzyme A deficiency in patients with PANK2 mutations[END_REF]. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Clarus 600D (Perkin Elmer, USA). The GC was equipped with an Elite column (30 m x 0.32 mm id x 0.25 mm film; Perkin Elmer) and injection was performed in splitless mode using helium (1 mL/min) as carrier gas. The temperature program was as follows: initial temperature of 80°C was held for 1 min, followed by a linear ramp of 10°C/min to 220°C, 20°C/min to of 280°C and 5°C/min up to 290°C, which was held for 10 min. The mass spectrometer operates in full mass scan or selected ion-monitoring mode. Peak integration was performed manually, and metabolites were quantified from selected-ion monitoring analyses against internal standards using standard curves for all the compounds [39, 40]. For phospholipid analysis, total lipid was also extracted by the Folch's method [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF]; phosphatidylcholine and sphingomyelin were quantified as described previously [START_REF] Ragot | Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes[END_REF].

Evaluation of oxidative stress

Analysis of antioxidant enzymes activities: catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD)

Catalase (CAT) activity was determined by photometric measurement of H2O2 consumption at 240 nm. 158N cells were trypsinized, washed with PBS, and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.5% Nonidet NP40, 0.5% Na deoxycholate, 0.1% SDS, 2 mM EDTA and 50 mM NaF) in the presence of 1/25 complete protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA) for 30 min on ice. Cell lysates were collected after centrifugation at 12,000 g for 20 min at 4°C. The reaction was initiated by the addition of Tris-HCL (1 M, pH 7.4), and H2O2 (400 mM), andthe amount of H2O2 remaining after 2 min was measured spectrophotometrically (240 nm), using a microplate reader (Tecan Infinite M 200 Pro).

Glutathione peroxydase (GPx) activity was quantified as previously described [START_REF] Flohé | Assays of glutathione peroxidase[END_REF]. 158N cells were lysed by sonication and centrifuged at 20,000 g for 30 min at 4°C. Cell lysates were incubated for 5 min at 25°C with 0.1 mM reduced glutathione (GSH) and phosphate buffer saline (50 mM, pH 7.8).

The reaction mixture was initiated by addition of H2O2 and stopped by the incubation with 1% trichlorric acid, for 30 min at 4°C, and then centrifuged at 1,000g for 10 min. The absorbance was measured at 412 nm (Lambda 25 Spectrophotometer, Perkin Elmer, Villebon sur Yvette, France). Superoxide dismutase (SOD) activity was quantified using the method of Misra and Fridovich [42].

This colorimetric assay, evaluates the ability of SOD to inhibit the Cytochrome c-reduction, induced by superoxide anions produced by xanthine oxidase enzyme [START_REF] Zarrouk | Majdoub, 7βhydroxycholesterol-induced cell death, oxidative stress, and fatty acid metabolism dysfunctions attenuated with sea urchin egg oil[END_REF]. To this end, cell lysates were added to potassium phosphate buffer (0.05 M, pH 7), and 50 µM xanthine (ref 69-89-6, Sigma-Aldrich).

Then 0.01U of xanthine oxidase (ref 9002-17-9, Sigma-Aldrich) and 5 µM of cytochrome c were added. Absorbance was measured at 550 nm at 20 min intervals for at least five reading. Antioxidant enzyme activities were expressed relative to the protein content determined with the Bradford method.

Measurement of thiol (SH group) levels

Measurement of thiols (SH group) was conducted according to the method of Flaure & Lafon [START_REF] Faure | Measurement of plasma sulfhydryl and carbonyl groups as a possible indicator of protein oxidation, Analysis of free radicals in biological systems[END_REF].

Cellular lysates were mixed with 5-5'DiThiobs-(2-NitroBenzoic acid) (DTNB), and incubated in dark for 15 min. The absorbance was measured at 415 nm on a Lambda 25 Spectrophotometer (Perkin Elmer).

Measurement of lipid peroxidation products: conjugated dienes (CDs) and malondialdehyde (MDA)

Conjugated dienes (CDs) are the products of polyunsaturated fatty acid peroxidation. The level of CDs was determined with the method of Esterbauer et al. [START_REF] Esterbauer | Continuous monitoring of in vztro oxidation of human low density lipoprotein[END_REF]. Malondialdehyde (MDA) is a lipid peroxidation product. It is an alkylating agent capable of reacting with biologic macromolecules. The level of MDA was evaluated as previously described [START_REF] Yoshioka | Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood[END_REF]. The experimental procedures on 158N cells were performed as described previously [START_REF] Sghaier | Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes[END_REF].

Measurement of carbonylated proteins (CPs) level

Similarly than lipids, proteins can also be oxidized. The measurement of carbonylated proteins (CPs) level was conducted as described by Oliver et al. [48]. This method, which is based on the reaction of 2, 4-dinitrophenylhydrazine (DNPH) with CP groups to form protein hydrazones, was realized as previously described [START_REF] Sghaier | Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes[END_REF].

Cell death characterization

Evaluation of cell death by the measurement of lactate dehydrogenase activity

The culture medium of 158N cells treated with or without 7β-OHC (50 µM) in combination with with DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM) were collected, and centrifuged to eliminate non adherent cells. Lactate dehydrogenase (LDH) activity was quantified using a commercial kit (#ab102526, Abcam, Paris, France). A microplate reader (TECAN Infinite M 200 Pro) was used to measure LDH activity at a wavelength of 450 nm in a kinetic mode at 25°C for 30-60 min.

Characterization of cell death by the evaluation of nuclear morphology after staining with

Giemsa or Hoechst 33342

Normal and apoptotic versus necrotic cells can be identified by nuclear morphology evaluated after staining with Giemsa and/or Hoechst 33342 as described previously [START_REF] Lizard | Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis[END_REF]. Apoptotic cells were characterized by condensed and/or fragmented nuclei whereas normal cells have regular and round nuclei. A total of 300 cells per sample were counted to determine the percentage of apoptotic cells.

Measurement of lysosomal destabilization with Acridine Orange

Acridine orange (AO) was used to evaluate lysosomal integrity [49] and or to quantify autophagic vesicular (acidic vesicular) formation [50-52]. AO stock solution was prepared at 1 mg/mL in DMSO and used at 1 µg/mL. After 15 min of incubation at 37°C, cells were washed, and resuspended in PBS for flow cytometry analysis. The fluorescence of AO was collected using a 630 nm longpass filter and measured on a logarithmic scale. Ten thousand cells were acquired on a Galaxy flow cytometer (Partec). Data were analyzed with Flomax (Partec) or FlowJo (Tree Star Inc.) software.

Polyacrylamide gel electrophoresis and Western blotting

Adherent and floating cells were harvested and lysed for 30 min on ice in a RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.5% Nonidet NP40, 0.5% Na deoxycholate, 0.1% SDS, 2 mM EDTA and 50 mM NaF) containing a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The cell debris were eliminated by centrifugation (20 min, 10,000 g), and the supernatant was collected. The protein concentrations were measured using bicinchoninic acid reagent (Sigma Aldrich). Seventy micrograms of protein were diluted in a loading buffer (125 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromophenol blue), separated on a SDS-PAGE gel, and transferred onto a nitrocellulose membrane (Thermo Fischer Scientific).

Nonspecific binding sites were blocked by incubation with 5% milk powder in PBST (PBS, 0.1% Tween 20, pH 7.2) and the membranes were incubated overnight at 4°C with the primary antibody diluted in 5% milk in PBST. For apoptosis and autophagy analysis, the antibodies used were directed against caspase-3 (Ozyme / Cell Signaling (# 9662) (for detection of the endogenous level of full length caspase-3 (35 kDa) and its large cleavage product (17 kDa)), and LC3-I/II (#L8918, Sigma Aldrich) (detecting LC3-I (18 kDa) and LC3-II (16 kDa)), respectively; all antibodies were rabbit polyclonal antibodies; they were used at a final dilution of 1/1000. For myelin protein analysis (proteolipid protein (PLP); myelin basic potein (MBP)), PLP expression was determined with a rabbit polyclonal antibody (PLP; Novus, NB10074503) and MBP expression with a mouse monoclonal antibody (MBP; Millipore, MAB381). An antibody directed against β-actin (mouse monoclonal antibody (#A2228, Sigma Aldrich)) was used at a final concentration of 1/10,000. Membranes were washed three times for 5 min, with PBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit (for caspase-3, LC3-I/-II, and PLP) diluted at 1/5,000 in 1% milk powder in PBST or anti-mouse antibody against MBP or β-actin, diluted at 1/5,000 in 1% milk powder in PBST. The membranes were then washed and antibody binding revealed using an enhanced chemiluminescence detection kit (Supersignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific) and Chemidoc XRS+ (Bio-Rad, Marnes la Coquette, France). Band intensity was determined with Image Lab software (Bio-Rad). The expression of caspase-3 (uncleaved and cleaved) and the ratio LC3-II / LC3-I were calculated with Image Lab software (Bio-Rad). The expression of caspase-3 and the level of cleaved caspase-3 were normalized versus β-actin, and expressed as normalized expression versus control (untreated cells). The ratio LC3-II / LC3-I was calculated for each assay.

Evaluation of the activation of PKA, PKC and MEK / ERK signalling pathways in 7β-

hydroxycholesterol-treated 158N cells

To evaluate the signalling pathways activated during 7β-OHC-induced cell death, different inhibitors were used. To this end, 158N cells were seeded in 24 wells at 60,000 cells / well. After 24 h of culture, 158N cells were cultured in the presence or absence of 7β-OHC (50 µM) with or without different inhibitors such as H89 (20 µM, Sigma-Aldrich), U73122 (1 µM, Sigma-Aldrich), chelerythrine (1 µM, Sigma-Aldrich), and U0126 (20 µM, Calbiochem, San Diego, CA, USA), which inhibit protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC) and MEK, respectively. These inhibitors were choosen for the following reasons: cAMP level leading to PKA activation is increased during during 7β-OHC-induced cell death; a cytosoloic Ca 2+ increase contributing to PLC activation is observed during 7β-OHC-induced cell death; and 7β-OHC-induced cell death is associated with inflammatory cytokines overproduction as well as enhanced expression of integrins via the activation of the nuclear factor κB (NF-κB) involving PKC and MEK-ERK signalling pathways [START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF][START_REF] Clarion | 7β-Hydroxycholesterol-induced energy stress leads to sequential opposing signaling responses and to death of C6 glioblastoma cells[END_REF]. These compounds were introduced in culture medium 30 min before the 7β-OHC. Stock solutions of these inhibitors were prepared as follow: the H89 stock solution was prepared in distilled water at 1 mM, while U0126 (0.1 mM), U73122 (0.1 mM) and chelerythrine (1 mM) stock solutions were prepared in DMSO. At the end of the treatments, an FDA assay was carried out as described previously.

Quantification of 11β-hydroxyxteroid dehydrogenase type 1 (11β-HSD1) and 11βhydroxyxteroid dehydrogenase type 2 (11β-HSD2) by RT-qPCR

11β-HSD1 is an intracellular enzyme that catalyzes interconversion of cortisone in cortisol [START_REF] Ah Kilgour | 11β-Hydroxysteroid dehydrogenase activity in the brain does not contribute to systemic interconversion of cortisol and cortisone in healthy men[END_REF]. 11β-HSD1 also catalyses the conversion of 7KC to 7β-OHC and reciprocally [START_REF] Wamil | 7-oxysterols modulate glucocorticoid activity in adipocytes through competition for 11beta-hydroxysteroid dehydrogenase type[END_REF][START_REF] Mitić | 11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto-and 7hydroxy-oxysterols in vivo[END_REF]. However, the conversion of 7β-OHC to 7KC can also be catalyzed by 11β-HSD2 [START_REF] Griffiths | Oxysterol research: a brief review[END_REF]. Total mRNA from 158N cells (and microglial BV-2 cells used as positive control [START_REF] Nury | 7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD[END_REF]) were extracted and purified using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). Total mRNA concentration was measured with TrayCell (Hellma, Paris, France). The purity of nucleic acids was controlled by the ratio of absorbance at 260 nm and 280 nm (ratios of 1.8 -2.2 were considered satisfactory). One microgram of total mRNA was used for reverse transcription with the iScript cDNA Synthesis Kit (Bio-Rad) according to the following protocol: 5 min at 25°C, 20 min at 46°C, 5 min at 95°C. cDNA was amplified using the FG Power SYBR Green (Thermo Fischer Scientific). All PCR reactions were performed on an Applied Biosystem Step One plus QPCR machine (Life Science Technologies). The primer sequence of 11β-HSD1 was:

11β-HSD1: forward 5′-actcagacctcgctgtctct-3′ and reverse 5′-gcttgcagagtagggagcaa-3' 11β-HSD2: forward 5′-ggttgtgacactggttttggc-3′ and reverse 5′-agaacacggctgatgtcctct-3'

Thermal cycling conditions were as follows: activation of DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s, followed by a melting curve analysis to test for the absence of non-specific products. Gene expression was quantified using cycle to threshold (Ct) values and normalized by the 36B4 reference gene (forward 5′gcgacctggaagtccaacta-3′ and reverse 5′-atctgcttggagcccacat-3′). The quantitative expressions of 11β-HSD1 and 11β-HSD2 were determined as fold induction above the control.

Statistical analysis

Statistical analyses were performed using the Statview Software (SAS Institut Inc, NC, USA) using a one way or a two way analysis of variance (ANOVA) followed by Student's t-test. Data shown are the mean ± standard deviation (SD) of two or three independent experiments, usually conducted in triplicate. Data were considered statistically significant at a P-value of 0.05 or less.

Results

Effects of dimethyl fumarate and monomethyl fumarate on the adhesion and morphology of 158N cells

158N cells treated with 7β-OHC are a relevant model to evaluate the relationship between oxidative stress, apoptosis and autophagy (oxiapoptophagy); to specify the role played by mitochondria, in this process; and to identify natural or synthetic molecules capable of preventing the toxic effects of 7β-OHC associated with major age-related and neurodegenerative diseases. To evaluate the cytoprotective activity of DMF and MMF on 7β-OHC-treated 158N cells, these cells were cultured for 24 h with or without 7β-OHC (50 µM, 24 h) in the presence or absence of DMF and MMF, used at 25 µM. αtocopherol (400 µM) was choosen as positive cytoprotective control. The effects of DMF and MMF were evaluated compared to untreated cells (control) and vehicle-treated cells (ethanol 0.6% or DMSO 0.05%).

Based on the observations performed by phase contrast microscopy, morphological changes were identified under treatment with 7β-OHC: a pronounced increase of the number of round and floating cells, indicating of loss of cell adhesion and an induction of cell death, was observed; whereas only few non adherent cells were present in control and vehicle-treated cells (Fig 1A).Of note, when 7β-OHC was associated with DMF, MMF or α-tocopherol, the loss of cell adhesion was prevented and an important decreases of the number of non adherent cells was observed. (Fig 1A).

The impact of DMF and MMF on cell adhesion was confirmed by the crystal violet assay. Under treatment with 7β-OHC and compared to control and vehicle-treated cells, a significant decrease of adherent cells was revealed (Fig. 1B). When 7β-OHC was associated with DMF and MMF the percentage of adherent cells was significantly increased: DMF and MMF increased by 30-32% the percentage of adherent cells, respectively. This effect is comparable to those observed with αtocopherol (Fig. 1B).

Effects of dimethyl fumarate and monomethyl fumarate on plasma membrane integrity and cell viability

The impact of DMF and MMF on 7β-OHC-induced loss of plasma membrane integrity and on cell death induction was determined on 158N cells. To this end, 158N cells were stained either with fluoresceine diacetate (FDA), propidium iodide (PI) or trypan blue, and in addition LDH was measured in the culture medium.

After staining with FDA, a significant decrease of FDA positive cells (FDA+ cells) was observed in 7β-OHC-treated cells compared to the corresponding vehicle and to the control (Fig. 2A). With the trypan blue assay, a significant decrease of the total number of living cells was simultaneously observed with 7β-OHC. Compared to 7β-OHC, co-treatment with DMF or MMF induced a significant increase of almost 40% of (FDA+ cells) as well as an increase of the total number of living cells evaluated after staining with trypan blue. More pronounced cytoprotective effects were seen with αtocopherol (Fig. 2A-B).

After staining with PI, in the presence of 7β-OHC, more than 70% of propidium iodide positive cells (PI +) were observed, indicating an increased permeability of the plasma membrane to PI which stains cells with damaged plasma membranes, often corresponding to dead cells (Fig. 2C). The increase of permeability to PI was strongly attenuated by DMF and MMF (Fig. 2C).

In addition, the quantification of LDH acitivity in the culture medium, which is a criteria of cell death, showed an increased LDH activity in 7β-OHC-treated cells (Fig. 2D). This increase was strongly attenuated by DMF and MMF; in the culture medium of (7β-OHC + DMF) or (7β-OHC + MMF), the LDH activity was only slightly higher than in control and vehicle-treated cells (Fig. 2D).

Of note, the ability of DMF and MMF to prevent the decrease of FDA+ cells and of living cells, as well as the increase of PI+ cells was similar to that observed with α-tocopherol (Fig. 2).

Effects of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced oxidative stress

To determine the ability of DMF and MMF to prevent 7β-OHC-induced oxidative stress, we studied the impact of these fumarate esters on the production of reactive oxygen species (ROS: O2 •-, H2O2), antioxidant enzyme (SOD, CAT, GPx) activities, and macromolecule oxidation products (MDA, carbonylated proteins) as well as on the level of SH-groups and conjugated dienes (CDs).

The measurement of ROS production was evaluated by flow cytometry after staining with DHE and DHR123. The highest percentage of DHE and DHR123 positive cells was observed when 158N cells were exposed to 7β-OHC compared to control and vehicle-treated cells. This increase was significantly inhibited when 7β-OHC was associated with DMF and MMF (Fig. 3). The most important attenuation of 7β-OHC-induced ROS overproduction was observed with α-tocopherol used as positive control anti-oxidant molecule (Fig. 3).

Under treatment with 7β-OHC, a significant increase of GPx, SOD, and CAT activities was observed compared to control and vehicle-treated cells (Fig. 4A-C). Compared to 7β-OHC-treated cells, GPx, SOD and CAT activities were significantly increased when 7β-OHC was associated with DMF or MMF, and the most important increase was observed with DMF (Fig. 4A-C). In the presence of αtocopherol, the effects were similar to those of DMF and MMF. On the otherhand, a significant decrease in the level of SH-groups was observed in 7β-OHC-treatd cells compared to control and vehicle-treated cells (Fig. 4D).Of note, the level of SH-groups was almost normalized, and in the range of that measured in control and vehicle-treated cells, when 7β-OHC was associated with DMF, MMF and α-tocopherol (Fig. 4D). With biochemical colorimetric methods of analysis, allowing evaluation of lipid and protein oxidation products, a significant increase of CDs, MDA, and carbonylated proteins levels (CPs) which are biomarkers of lipid and protein oxidation, was observed when 158N cells were exposed to 7β-OHC compared to control and vehicle-treated cells (Fig. 5A-C). Interestingly, this increase was significantly attenuated when 7β-OHC was associated with DMF and MMF (Fig. 5A-C).

Similar effects than DMF and MMF were found in the presence of α-tocopherol (Fig. 5).

Effects of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced mitochondrial damage and peroxisomal changes

As mitochondrial damage is induced by 7β-OHC in 158N cells, the impact of DMF and MMF at the mitochondrial level was studied. To this end, succinate dehydrogenase activity, transmembrane mitochondrial potential (∆Ψm), mitochondrial mass, superoxide anions (O2 •-) production, and cardiolipin levels were evaluated.

The MTT assay, used to measure succinate dehydrogenase activity, showed that the ratio [(% MTT positive cells in the assay) / (% MTT positive cells in the control)] was significantly reduced with 7β-OHC compared to control and ethanol-treated cells (Fig. 6A). When 7β-OHC was associated with DMF and MMF this ratio was significantly increased. A similar effect was observed with αtocopherol. These data showed that DMF and its main metabolite MMF prevent the decrease of succinate dehydrogenase activity which participates in both the TCA cycle and the electron transport chain at the mitochondrial level. These data lead us to examine the effect of DMF and MMF on 7β-OHC-induced loss of transmembrane mitochondrial potential (∆Ψm) on 158N cells by staining with DiOC6(3). A high value (33%) of DiOC6(3) negative cells, indicating a decrease in ∆Ψm (cells with depolarized mitochondria) was detected under treatment with 7β-OHC (Fig. 6B). The percentage of DiOC6(3) negative cells was strongly reduced when 158N cells were co-treated with DMF, MMF or αtocopherol (Fig. 6B). Another fluorochrome (MitoTracker Red) was used to evaluate the mitochondrial mass. Upon staining with Mitotracker Red, an increase in the mean fluorescence intensity (MFI) of this dye was observed when the cells were treated with 7β-OHC compared to control and vehicle-treated cells (Fig. 6C). This increase was significantly inhibited by DMF and MMF, as well as by α-tocopherol (Fig 6C).

Due to the important role played by cardiolipins, which are essential mitochondrial phospholipids for electron transport, oxidative phosphorylation and energy production [57, 58], it was important to determine the effect of 7β-OHC on the cardiolipin content. No difference in cadiolipin content was observed between untreated cells (control) and vehicle-treated cells (Fig. 6D). The highest levels of cardiolipins was found with DMF and MMF (Fig. 6D). Total cardiolipin content was significantly decreased in 7β-OHC-treated cells compared to control and vehicles-treated cells (Fig. 6D), When 7β-OHC was associated with DMF and MMF, this decrease was counteracted and the cardiolipin values observed were in the range of that observed in control and vehicle-treated cells (Fig. 6D). The same observation was made with α-tocopherol (Fig. 6D).

In addition, flow cytometric analyses were performed with MitoSOX allowing the measurement of O2 •-production at the mitochondrial level (MitoSOX positive cells). In cells incubated with 7β-OHC compared control and to vehicle-treated cells an overproduction of O2 •-was observed. This overproduction of O2 •-was counteracted by DMF, MMF and α-tocopherol (Fig. 6E).

As mitochondria and peroxisome are tightly connected organelles [59], the effects of 7β-OHC on the peroxisome was evaluated by transmission electron microscopy (Fig. 7). Whereas round peroxisomes were mainly observed in control cells, several peroxisomes with irregular shapes, which were often larger or smaller than in the control, were detected in 7β-OHC-treated cells (Fig. 7). Several mitochondria, which were smaller than in control cells, were also frequently observed in 7β-OHCtreated cells (Fig. 7). Of note, these modifications of the peroxisomal and mitochondrial morphologies were prevented when 7β-OHC was associated with DMF (Fig. 7).

Altogether, our data demonstrate that 7β-OHC induced several types of mitochondrial damage, and that DMF and MMF have strong cytoprotective effects at the mitochondrial level.

Impact of 7β-hydroxycholesterol, dimethyl fumarate, and monomethyl fumarate on cell metabolism

Effects of 7β-hydroxycholesterol, dimethyl fumarate, and monomethyl fumarate on glycolysis and TCA cycle

TCA cycle, also known as Krebs cycle, is the metabolic pathway used to release stored energy through the oxidation of acetyl-CoA, to provide the reducing agent NADH and precursors of certain amino acids. Thic cycle takes place in the matrix of the mitochondria. Thus, we considered pertinent to understand the impact of 7β-OHC, DMF, MMF and 7β-OHC associated with DMF or MMF on the TCA cycle. Lactate dehydrogenase (LDH) converts two pyruvates into 2 lactate molecules, using NADH as a coenzyme so that the cell can continue to perform glycolysis by regenerating NAD + . The effect of 7β-OHC, DMF, MMF and 7β-OHC associated with DMF or MMF on these metabolic pathways was also evaluated. When exposed to 7β-OHC, a significant increase in lactic acid (lactate) levels was observed (220.59 ± 2.50 ng/million cells) compared to vehicle -treated cells (EtOH 0.6%: 122.64 ± 3.53 ng/million cells); this increase in lactic acid was significantly reduced, under treatment with DMF and MMF in the presence of 7β-OHC (Table 1). However, a marked and significant decreases in pyruvic acid (pyruvate), succinic acid (succinate), fumaric acid (fumarate), malic acid (malate) and citric acid (citrate) levels were observed in 7β-OHC-treated 158N cells compared to vehicle; this decrease was attenuated when the cells were simultaneously treated with 7β-OHC and DMF or MMF (Table 1).

Effects of 7β-hydroxycholesterol, dimethyl fumarate, and monomethyl fumarate on cholesterol metabolism

In the presence of 7β-OHC, the cholesterol level was significantly decreased compared to vehicletreated cells (Table 2). This reduction of the cholesterol level was still observed when the cells were simultaneously treated with 7β-OHC and MMF (Table 2). Compared to 7β-OHC-treated cells, the cholesterol level was significantly increased, when the cells were co-treated with 7β-OHC and DMF (Table 2). When cholesterol precursors were measured after treatment with 7β-OHC, significant reduced levels of lathosterol, desmosterol, and lanosterol were observed compared to control and vehicle (EtOH 0.6%)-treated cells (Table 2). While lanosterol level was normalized with DMF and MMF, a most important decrease of other cholesterol precursors levels was detected (Table 2). In addition, cholesterol auto-oxidation products (triol, ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-OHC)) were measured in the same conditions. In 158N cells treated with 7β-OHC, important levels of this oxysterol were found supporting an accumulation and/or an interaction of this compounds with the cells (Table 2). After 24 h of incubation with 7β-OHC, the levels of this oxysterol present in the cells (when 158N cells were only cultured in the presence of 7β-OHC) represents 1% of the quantity of 7β-OHC introduced in the culture medium (Table 2). This accumulation is lower than those reported with 7KC and 27-hydroxycholesterol which represents 10-20% of the quantity of oxysterols initially introduced in the culture medium [START_REF] Zarrouk | Involvement of oxysterols in age-related diseases and ageing processes[END_REF]. It is noteworthy that in the plasma of X-ALD patients, the concentration of 7β-OHC can reach 2.5 µM [START_REF] Nury | 7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD[END_REF]. In the presence of 7β-OHC, in agreement with the ability of 7β-OHC to stimulate the oxidative stress, higher levels of triol and 7KC were also observed compared to control cells and vehicle-treated cells; this increase was significantly reduced when 7β-OHC was associated with DMF or MMF (Table 2). Despite the levels of triol and 7KC being significantly reduced, when 7β-OHC was cultured in the presence of DMF or MMF, the levels of 7β-OHC remained increased, and were even higher than in cells only treated with 7β-OHC (Table 2). Therefore, DMF and MMF inhibit the signaling pathways leading to 7β-OHC-induced cytotoxicity without preventing the cellular accumulation of this oxysterol. In 158N cells, the Ct value of the gene HSD11B1 encoding for the enzyme 11β-HSD1 (Ct (HSD11B1) = 36.5 ± 1.0) was high whereas the Ct value of the reference gene 36B4 was low (Ct (36B4) = 18.4 ± 0.1) supporting a good expression of 36B4 in 158N cells (Supplementary Fig. 3). In 158N cells, the gene HSD11B1 encoding for the enzyme 11β-HSD1 can be considered as non expressed since multiple peaks were observed on the melt curve demonstrating a non specific amplification (Supplementary Fig. 3).

However, on microglial BV-2 cells, used as positive control, the Ct value of the gene HSD11B1 encoding for 11β-HSD1 was higher (Ct (HSD11B1) = 30.2 ± 0.6) than in 158N cells (Ct (HSD11B1) = 36.5 ± 1.0); as a melt curve with homogeneous peaks was observed, this supports that BV-2 express the gene of 11β-HSD1 and that the primer sequences used are appropriated (Supplementary Fig. 3). According to these data, our results do not support a conversion of 7KC in 7β-OHC, and reciprocally via 11β-HSD1, in 158N cells. Since 7β-OHC can be converted to 7KC by the enzyme 11β-HSD2, the expression of the HSD11B2 gene encoding this enzyme was quantified on both 158N (Ct=26.0 ± 0.6) and BV-2 (Ct=29.3 ± 0.3) (Supplementary Fig. 4). The Ct values and the appearance of the melt curves are in favor of an expression of the HSD11B2 gene in these cells. This suggests that in 158N cells, 7β-OHC could be converted to 7KC, especially when the cells are co-treated with 7β-OHC associated with DMF or MMF. The expression of the HSD11B2 gene, which is decreased in the presence of 7β-OHC, evokes that of the control when the cells are cultured in the presence of 7β-OHC associated with DMF or MMF (Supplementary Fig. 4).

Effects of 7β-hydroxycholesterol, dimethyl fumarate and monomethyl fumarate on the cellular fatty acid profile

In order to identify the effects of 7β-OHC, DMF, MMF, and 7β-OHC with or without DMF and MMF on fatty acid metabolism, fatty acid profiles were determined.

As shown in Table 3, 7β-OHC induced a significant decrease inthe total long chain saturated fatty acids (∑SFA (C<22)) compared to vehicle-treated cells. A significant decrease in myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and arachidic acid (C20:0) levels was observed in 7β-OHC-treated 158N cells compared to the vehicle-treated cells. However, under treatment with 7β-OHC, the total very long chain saturated fatty acid ((∑VLCSFA C≥22) level was 3 times higher than in the vehicle treated cells. Thus, behenic acid (C22:0), tetracosanoic acid (C24:0), and cerotic acid (C26:0) levels were significantly enhanced in 7β-OHC-treated cells compared to vehicle. When 158N cells were co-tretaed with MMF and 7β-OHC, all SFA levels were in the range of untreated and vehicle-treated cells (Table 3). However, when 158N cells were co-treated with DMF and 7β-OHC, only C18:0, C22:0 and C26:0 levels were normalized (Table 3).

As shown in Table 4, 7β-OHC also induced a significant decrease in the total mono-unsaturated fatty acid (∑MUFA) compared to vehicle-treated cells. Thus, myristoleic acid (C14:1 n-5), sapienic acid (C16:1 n-10), palmitoleic acid (C16:1 n-7), eicinoneic acid (C20:1 n-9), and eicisenoic acid (C20:1 n-7) levels were significantly decreased in 158N treated with 7β-OHC compared to vehicle-treated cells; however, under the same conditions, very long chain monounsaturated fatty acids such as nervonic acid (C24:1 n-9) and hexacosaenoic acid (C26:1 n-9) levels were enhanced (Table 4). MMF and DMF had almost similar effects on the MUFA profile when used 2 h prior the incubation with 7β-OHC. In fact, all the MUFA levels came closer to those of the untreated cells or vehicle treated cells, with the exception of C16:1 n-10 or n-9 and C16:1 n-7, of which levels were higher even than control and vehicle treated cells. Interestingly when added alone, these molecules (DMF and MMF) and in particular MMF were able to significantly enhance levels of several MUFA compared to the corresponding vehicle.

As shown in Table 5, 7β-OHC induced a significant decrease of all polyunsaturated fatty acid (PUFA) levels compared to vehicle-treated cells; thus, total PUFA content was reduced by about half in the cells. When DMF and MMF were used alone on 158N cells, linoleic acid (C18:2 n-6), arachidonic acid (AA; C20:4 n-6), and docosahexaenoic acid (DHA; C22:6 n-3) were enhanced, compared to corresponding to the vehicle. Co-treatment of 7β-OHC with DMF and MMFinduced a correction of all PUFA levels which are in the range of vehicle treated cells (Table 5), with the expection of (C20:4 n-6) and eicosapentaenoic acid (EPA; C20:5 n-3), which remained lower than in vehicle-and 7β-OHCtreated cells.

Furthermore, Δ4-desaturase (C22:6 n-3/C22:5 n-3), Δ8-desaturase (C20:3 n-6 /C20:2 n-6), Δ9desaturase (C18:1 n-9/ C18:0), andelongase activity index (C22:5 n-3 /C20:5 n-3) activity index were significantly increased in 7β-OHC-treated cells (Table 5). Compared to 7β-OHC, these indices were significantly reduced when the cells were co-treated with 7β-OHC associated with DMF and MMF.

The elongase (C18:0/C16:0) activity index was higher in 7β-OHC cells comparatively to (7β-OHC + DMF); however, it was reduced in (7β-OHC + MMF) compared to 7β-OHC (Table 5).

Our results demonstrated the impact of 7β-OHC on fatty acid metabolism, which could be at least in part, due to mitochondrial damage, since this organelle plays important roles in fatty acid anabolism and catabolism. Our data also demonstrate that DMF and MMF prevent fatty acidsmetabolic disorders in 7β-OHC-treated cells.

Effects of 7β-hydroxycholesterol, dimethyl fumarate and monomethyl fumarate on sphingomyelin and phosphatidylcholine content

The effects of 7β-OHC, DMF, MMF, and 7β-OHC with or without DMF orMMF on the cell content in sphingomyelin (SM) and phosphatidylcholine (PC) were determined by GC/MS. In the presence of 7β-OHC, significant decreases in SM and PC were observed comparatively to control (untreated cells), vehicle-treated cells (EtOH 0.6% or DMSO 0.05%), α-tocopherol, DMF and MMF (Fig. 8). Whereas α-tocopherol either failed to, or had a minor effect in preventing the 7β-OHC-induced decreased of SM and PC, respectively, DMF was more efficient than MMF at attenuating the decrease of SM and PC under treatment with 7β-OHC (Fig. 8).

Effects of 7β-hydroxycholesterol with or without dimethyl fumarate and monomethyl fumarate on apoptosis and autophagy

The oxidative stress induced by 7β-OHC in 158N cells was associated with characteristic features of apoptotosis and autophagy, in agreement with previous studies showing that 7β-OHC triggers an oxiapoptophagic mode of cell death on 158N cells [8] (Fig. 9). Apoptosis is morphologically characterized by the presence of cells with condensed and/or fragmented nuclei (Fig. 9A) and by an induction of cleaved caspase-3 (Fig. 10). The occurrence of autophagy is supported by an enhancement of the percentage of acridine orange (AO)-positive cells (Fig. 9B), which could correspond to the presence of cells with large and/or numerous autophagic vesicles, as well as by an activation of LC3-I into LC3-II leading to an enhancement of the [(LC3-II) / (LC3-I)] ratio (Fig. 10).

Of note, DMF and MMF were able to counteract 7β-OHC-induced apoptosis and autophagy. Thus, in (7β-OHC + DMF)-and (7β-OHC + MMF)-treated cells, the percentage of apoptotic cells was strongly and significantly decreased, and the cleavage of caspase-3 was also reduced; in addition, the percentage of AO positive cells was also strongly decreased and the activation of LC3-I into LC3-II was diminished (Fig. 10). As little is known on the signaling pathway leading to 7β-OHC-induced cell death, various inhibitors were used: H89 (20 µM), U73122 (1 µM), chelerythrine (1 µM), and U0126 (20 µM), which inhibit protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC) and MEK, respectively. Undetectable or slight inhibition of cell death evaluated with the FDA assay was observed when 7β-OHC was associated with U73122 and H89, respectively (Fig. 11). However, 7β-OHC-induced cell death was markedly reduced in the presence of chelerythrine and U0126 (Fig. 11) supporting an involvement of PKC and MEK in the deleterious effects of 7β-OHC in 158N cells.

Effect of dimethyl fumarate and monomethyl fumarate on the recovery of 7βhydroxycholesterol-induced cell death

We also studied the effect of DMF (25 µM) and MMF (25 µM) on 7β-OHC (50 µM)-induced cell death when DMF and MMF were added after 7β-OHC. With the FDA assay, the ability of αtocopherol (400 µM) to prevent 7β-OHC-induced cell death was the most efficient when it was introduced in the culture medium 6 h after 7β-OHC; whereas significant, the cytoprotection found when α-tocopherol was introduced 10 h after 7β-OHC, was very slight (Supplementary Fig. 5). Based on these data, the effects of DMF and MMF were evaluated when they were added 6 h after 7β-OHC;of note, under these conditions, data obtained by cell counting with trypan blue, and nuclear staining with Hoechst 33342 show that DMF and MMF prevent 7β-OHC-induced cell death and apoptosis (Fig. 12). Therefore, DMF and MMF also have cytoprotective effects on 7β-OHC-induced cell death when they are added after this oxysterol.

Analysis of myelin proteins expression (PLP, MBP) in 7β-hydroxycholesterol-treated murine oligodendrocytes 158N without or with dimethyl fumarate and monomethyl fumarate

Myelin proteins such as proteolipid protein (PLP) and myelin basic protein (MBP) correspond to 50 and 30% of myelin proteins, respectively [30], and are expressed by 158N cells [START_REF] Baarine | Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes[END_REF]. Therefore, we determined whether PLP and MBP expression was affected by 7β-OHC (50 µM), and the impact of DMF (25 µM), MMF (25 µM) and α-tocopherol (400 µM; used as cytoprotective agent) on these proteins.

Compared to control cells, PLP expression was either similar or slightly lower in vehicles-treated cells (supplementary Fig. 6); it was similar in, α-tocopherol-, DMF-and MMF-treated cells (supplementary Fig. 6). However, PLP expression was higher in 7β-OHC-, (7β-OHC + αtocopherol)-, and (7β-OHC + MMF)-treated cells (supplementary Fig. 6). In the mouse, MBP is present under 2 major (around 18.5 and 14 kDa) and two minor (around 21.5 and 17 kDa) forms [START_REF] At Campagnoni | Expression of myelin protein genes in the developing brain[END_REF].

In 158N cells, the 21 kDa (minor form) and 18 kDa (major form) were easily detected (supplementary Fig. 6). Compared to control cells, MBP expression was enhanced by DMF (supplementary Fig. 6). MBP expression was also higher in 7β-OHC-, (7β-OHC + α-tocopherol), (7β-OHC + DMF), and (7β-OHC + MMF)-treated cells (supplementary Fig. 6). Thus, an increase in the expression of PLP and MBP is observed with 7β-OHC; the increase in PLP is not corrected by αtocopherol, and DMF but normalized with MMF; the increase in MBP observed with 7β-OHC is slightly and non significantly accentuated in the presence of α-tocopherol (supplementary Fig. 6).

DMF is more efficient than MMF to reduce the overexpression of PLP, mainly the 18 kDa (supplementary Fig. 6).

Discussion

Oxidative stress and mitochondrial dysfunction are involved in numerous neurodegenerative diseases (Biogen), is used to treat the relapsing-remitting form of MS. In this study, 158N murine cells were used since they present several characteristics of differentiated oligodendrocytes, which are myelin producing cells in the CNS and consequenty essential for an efficient transmission of the nervous impulse. In addition, 158N cells express major myelin proteins (PLP, MBP) [START_REF] Baarine | Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes[END_REF]. Therefore, the effects of 7β-OHC with or without DMF and MMF on PLP and MBP expression was also evaluated.

Our data show important cytoprotective effects of DMF and MMF on 7β-OHC-treated 158N cells, and demonstrate the ability of DMF and MMF to prevent 7β-OHC-induced oxiapoptophagy.

In 158N cells, 7β-OHC exhibits cytotoxic effects. An increased percentage of floating round cells and a decreased of adherent cells associated with a loss of membrane integrity were revealed, suggesting an alteration of membrane constituents associated with cell death. These alterations could be explained by Redox disequilibrum induced by 7β-OHC, which could affect the different cellular compartments as a consequence of lipid peroxidation. Indeed, an overproduction of ROS and of mitochondrial O2 •- associated with an enhancement of antioxidant enzyme activities (SOD, catalase, GPx), as well as lipid and protein oxidized derivatives were revealed under treatment with 7β-OHC. The enhanced activity of SOD evocates the data obtained by Yuan et al., who showed that human macrophages exposed to 7KC have increased levels of MnSOD mRNA [START_REF] Yuan | Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products[END_REF]. The increased levels of GPx, catalase, and SOD activities, were considered as a cellular reaction involved in the defense against the free radicals. The overproduction of ROS could favor the increase of SOD, catalase and GPx activities in a cascade of events to reduce oxidative stress. The disruption of Redox equilibrium observed under treatment with 7β-OHC, in agreement with previous studies [8, 19, 34, 64], is also associated with an increase of lipid and protein oxidation (MDA, CDs and CPs) which could be used as biomarkers of oxidative stress in diseases associated with increased levels of this oxysterol.

In 158N cells exposed to 7β-OHC, as observed under treatment with 7KC [39], the overproduction of ROS was associated with a loss of transmembrane mitochondrial potential (∆Ψm). Thus, we were interested in the characterization of mitochondrial functions in the presence of 7β-OHC. In agreement with previous studies, we found that 7β-OHC is an inducer of cell death associated with mitochondrial dysfunction, including morphological, functional and metabolic mitochondrial alterations [8]. This includes: a loss of succinate dehydrogenase activity, a loss of ∆Ψm, an increase of mitochondrial mass, and an overproduction of O2 •-at the mitochondria level. These findings are in accordance with studies conducted on various cell types from different species [START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF][START_REF] Zarrouk | Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22: 6 n-3)[END_REF]. The increase of mitochondrial mass could be an adaptative response of the mitochondria to prevent the loss of transmembrane mitochondrial potential and preserve mitochondrial metabolism [START_REF] Zahm | Chronology of cellular alterations during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells: Analysis by time lapse-video microscopy and conventional fluorescence microscopy[END_REF]. It has been reported that an increase of mitochondrial mass was correlated to the hyperactivity of the mitochondrial complex IV in the axons of patients with MS [66, 67]. As it is known, the main mitochondrial function is to control respiratory chain in order to produce energy in the form of ATP via TCA cycle which is primordial for numerous cellular functions such as carbohydrates, proteins and lipids metabolism [START_REF] Pierrel | Metal Ion availability in mitochondria[END_REF]. Here, we remarked an enhancement of cellular lactate (lactic acid), associated with a decrease of pyruvate level in the presence of 7β-OHC. This suggests a defect of carbohydrate metabolism and in particular of glycolysis. The accumulation of lactic acid could result from pyruvate conversion to regenerate NAD + used to restore glycolysis. Since pyruvate is the precursor of acetyl-CoA, the decreased amount of pyruvate could be at the origin of TCA cycle impairment. The alteration of the TCA cycle is supported by the reduced concentrations of some organic acids of the TCA cycle, citrate, fumarate, succinate, and malate in the presence of 7β-OHC. TCA cycle impairment can lead to oxidative phosphorylation alteration, and consequently to mitochondrial failure [START_REF] Leoni | Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with α-tocopherol: Impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors[END_REF]. On the other hand, 7β-OHC induces a decrease in the cardiolipin levels. Similar observations were made in158N exposed to 7KC [START_REF] Nury | Induction of peroxisomal changes in oligodendrocytes treated with 7-ketocholesterol: Attenuation by αtocopherol[END_REF].

Cardiolipins are mitochondrial phospholipids synthesized and located in the inner mitochondrial membrane, and are involved in various mitochondrial functions and in bioenergetics [START_REF] Paradies | Functional role of cardiolipin in mitochondrial bioenergetics[END_REF]. The loss of cardiolipin content in mitochondria may also reflect biochemical modifications of mitochondrial membrane. It is suggested that the observed decrease of cardiolipin content could be consequence of their increased hydrolysis by endogenous phospholipases, and/or by a decreased de novo synthesis resulting from impaired function of the enzymes involved in their synthesis, or by a decreased bioavailability of cardiolipin precursors. During cell death, release of cardiolipins outside the mitochondria and outside the cells is also possible. These data underline the major role of 7β-OHC in mitochondrial dysfunction.

Given the important role of lipids, including cholesterol in myelin structure and brain health [70], it was important to evaluate the impact of 7β-OHC on cholesterol metabolism via the quantification of some of its precursors and derivatives. Under treatment with 7β-OHC, reduced levels of total cholesterol and its precursors, lathosterol, lanosterol, and desmosterol were observed, arguing in favor a defect on cholesterol synthesis. Our results support previous findings reported on different cellular lines treated with 24(S)-hydroxycholesterol or 7KC: these studies have shown that cholesterol synthesis alteration was associated with other events such as mitochondrial damage and cell death [START_REF] Leoni | Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with α-tocopherol: Impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors[END_REF]. As our study reveals oxidative stress along with severe mitochondrial dysfunctions, under treatment with 7β-OHC, this could explain even in part, the cellular accumulation of 7KC formed by an oxidation of cholesterol induced by ROS overproduction.

Our results support the idea that the alteration of cholesterol synthesis, lead to cholesterol homeostasis disturbance, which in oxidative stress conditions might be associated with increased levels of some oxysterols. In addition, it has been reported that oxysterols can disturb lipid homeostasis through the activation of transcription factors. They can act on sterol responsive element binding proteins (SREBPs), which activate several genes involved in cholesterol, triglycerides, phospholipids, and fatty acid synthesis [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF]. In addition, fatty acids, which are components of phospholipids, are essential constituents in the structure and functions of myelin sheath [START_REF] Morell | Characteristic composition of myelin[END_REF]. Thus, it was important to evaluate both the fatty acid profile and the phospholipid content (sphingomyelin, phosphatidylcholine) when 158N cells were exposed to 7β-OHC. In this condition, a reduction of saturated fatty acids as myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), structural elements of membrane phospholipids, as well as of phosphatidylcholine and sphingomyelin were observed. In addition, an accumulation of total and very long chain saturated fatty acids (VLCSFAs) has been remarked under treatment with 7β-OHC, which points towards peroxisomal defect, since β-oxidation of VLCSFAs leading to their degradation takes place in the peroxisome [START_REF] Wanders | Biochemistry of mammalian peroxisomes revisited[END_REF]. Thus, as reported with 7KC, the treatment with 7β-OHC also induces peroxisomal dysfunction According to previous findings on different cell lines, 7β-OHC was considered as a strongly cytotoxic oxysterol. So, we intended to characterize 7β-OHC-induced cell death. Our data show that 7β-OHCinduced cell death triggers caspase-3 activation associated with fragmented and/or condensed nuclei. This supports that 7β-OHC induces an apoptotic mode of cell death. Moreover, our data confirm that 7β-OHC also triggers autophagy. Indeed, a higher percentage of cells with altered lysosomes was observed. These data underline that 7β-OHC-induced cell death is characterized by lysosomal dysfunction [78] which could favour mitochondrial alteration [START_REF] Chwieralski | Cathepsin-regulated apoptosis[END_REF]. Furthermore, a conversion of LC3-I to LC3-II, and an increase of (LC3-II / LC3-I) ratio, which are characteristics of autophagy, were detected demonstrating that 7β-OHC simultaneously triggers apoptosis and autophagy, and induces an oxiapoptophagic process [START_REF] Nury | Improved synthesis and in vitro evaluation of the cytotoxic profile of oxysterols oxidized at C4 (4α-and 4β-hydroxycholesterol) and C7 (7-ketocholesterol, 7α-and 7β-hydroxycholesterol) on cells of the central nervous system[END_REF]. Altogether, our data highlight the major role played by 7β-OHC in mitochondrial dysfunction, oxidative stress induction, cellular metabolism alteration and cell death induction which are common features observed during neurodegeneration. In addition, our study brings new information on the signalling pathway induced during 7β-OHC-induced cell death and supports the hypothesis of an activation of the PKC / p38 / MEK signalling pathway [START_REF] Clarion | 7β-Hydroxycholesterol-induced energy stress leads to sequential opposing signaling responses and to death of C6 glioblastoma cells[END_REF]. When 158N cells were simultaneously treated with DMF or MMF and 7β-OHC, our data demonstrate that DMF, a therapeutic agent (prescribed under the name of Tecfidera used for the treatment of relapsing remitting MS), as well as MMF, which is its major metabolite, known to have antioxidant and neuroprotective activities are able to prevent 7β-OHC-induced cytotoxicity [START_REF] Lin | The antiinflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1[END_REF][START_REF] Scannevin | Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway[END_REF]. Under these conditions, we observed an attenuation of cell detachment and a preservation of plasma membrane integrity. It has also been shown that DMF induces an increase of antioxidant molecules such as glutathione (GSH), carnitine, and ascorbic acid above basal levels [START_REF] Huang | Dimethyl fumarate modulates antioxidant and lipid metabolism in oligodendrocytes[END_REF]. These molecules, especially ascorbic acid, are able to maintain membrane integrity in a pro-oxidative environment by preventing lipid peroxidation [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate[END_REF]. In our study, the ability of DMF to increase the levels of SH-groups suggests that this compound might favor this mechanism. It may act through the activation of Nrf2 pathway [START_REF] Nguyen | The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress[END_REF]. Thus, it was of interest to evaluate the part taken by DMF and its major metabolite MMF on Redox status. We therefore determine the ability of DMF and MMF to inhibit the overproduction of ROS, to increase the antioxidant enzyme activities (SOD, catalase, GPx), to increase SH-groups level, and to reduce lipid peroxidation and protein oxidation products. Overall, the cytotoxic effects induced by 7β-OHC on 158N cells were strongly attenuated by DMF and MMF. In line with these findings, on human oligodendrocytes (MO3.13 cells), it has been reported that DMF prevent H2O2 overproduction- with mitochondrial dysfunction, the restoration of their content argues in favor of the ability of DMF and MMF to prevent and restore mitochondrial dysfunction. This conclusion is reinforced by the ability of DMF and MMF to prevent TCA cycle alterations. These results underline that DMF and MMF influence the activities of mitochondrial enzymes including those of the TCA cycle. Pyruvate levels were also increased associated with a reduction of lactate after preatreatment with DMF or MMF: this argues in favour a recovery on carbohydrate metabolism and especially glycolysis.

It is also well known that the myelin sheath is composed of approximately 70-80% of lipids [START_REF] Morell | Characteristic composition of myelin[END_REF]. The synthesis and incorporation of phospholipids, sphingolipids, and cholesterol are critical for its development and maintenance. Based on data demonstrating neuroprotection of DMF, we postulated that this molecule and its metabolite (MMF) might increase the synthesis of lipids, which are major components of myelin sheath. Of note, DMF and MMF were able to prevent 7β-OHC-induced modification of cholesterol synthesis through the restoration of the content of cholesterol precursors.

The formation of oxidized cholesterol derivatives (mainly 7KC) resulting chiefly from cholesterol autoxidation was also reduced in the presence of DMF or MMF. However, DMF and MMF were unable to prevent the intracellular accumulation of 7β-OHC. As the expression of the gene HSD11B1 encoding for the enzyme 11β-HSD1 is considered unlikely, a conversion of 7β-OHC to 7KC by this enzyme, is excluded. On the other hand, as the gene HSD11B2 encoding for the enzyme 11β-HSD2 catalyzing the conversion of 7β-OHC to 7KC is expressed, this may explain the persistent presence of 7KC despite the powerful antioxidant effects of DMF and MMF. In addition, we report the ability of DMF and MMF to normalize the fatty acid profile. The reduction of VLCSFAs levels observed when 7β-OHC was associated with DMF and MMF support that DMF and MMF not only tend to normalize mitochondrial activity, but also peroxisomal activity.Of note, DMF and MMF were also able to attenuate the decrease of phospholipids (sphingomyelin, phosphatidylcholine) which are important component of myelin. Indeed, whereas the molar ratio of cholesterol, phospholipids and glycosphingolipids in most membranes is in the order of 25%:65%:10%, the molar ratios in myelin are in the range of 40%:40%:20% [START_REF] O'brien | Stability of the myelin membrane[END_REF]. As DMF and MMF contribute to restore a normal lipid profile, concerning myelin associated-lipids (cholesterol, phospholipids), it was important to determine how major myelin proteins such as proteolipid protein (PLP) and myelin basic protein (MBP), which correspond to 50 and 30% of myelin proteins, respectively [START_REF] Baumann | Biology of oligodendrocyte and myelin in the mammalian central nervous system[END_REF], and which are expressed by the 158N cells [29], were affected by 7β-OHC and what was the impact of DMF and MMF on these proteins.

Noteworthy, the increased expression of PLP was normalized with MMF, and the increase expression of MBP observed with 7β-OHC was more or less attenuated by DMF and MMF.

Finally, DMF and MMF are also able to counteract 7β-OHC-induced apoptosis as well as autophagy.

At the moment, on 158N cells, similar cytoprotective effects were only observed with α-tocopherol, DHA, and biotin which allow attenuation of 7β-OHC-induced oxiapoptophagy [START_REF] Nury | Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7βhydroxycholesterol-, or 24 (S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22: 6 n-3)[END_REF][START_REF] Sghaier | Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes[END_REF].

Conclusion

Our data obtained on oligodendrocytes 158N demonstrate that 7β-OHC induces ROS overproduction subsequently leading to increased antioxidant enzyme activity, and oxidation of cellular macromolecules. In addition, 7β-OHC triggers important mitochondrial dysfunctions: decreases of ΔΨm, increases of mitochondrial mass, decreased cardiolipin content and alteration of the TCA cycle, which can influence the cellular metabolism, especially lipid metabolism. These different alterations contribute to cell death through oxiapoptophagy. The different events associated with oxiapoptophagy, revealed from data obtained in the present study and in previous studies, are summarized in Supplementary Fig. 7. Our data also show that DMF and MMF attenuate 7β-OHC-induced oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and cell death, which are hallmarks of neurodegenerative diseases. As 7β-OHC is associated with neurodegeneration, the ability of DMF and MMF to counteract the toxicity of this oxysterol reinforces the interest of DMF for the treatment of neurodegenerative diseases as well as of other diseases associated with increased levels of 7β-OHC [START_REF] Zarrouk | Involvement of oxysterols in age-related diseases and ageing processes[END_REF][START_REF] Vejux | Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis[END_REF]. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF), (7β-OHC + MMF) or (7β-OHC + αtocopherol)-treated cells; * P≤0.05. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%). 25µM) or α-tocopherol (400 µM) was determined on cell viability and plasma membrane integrity with complementary criteria: plasma membrane integrity was measured with the FDA assay (A), plasma membrane permeability was measured after staining with propidium iodide (PI) (% of PI positive cells) (B), the impact on cell viability was evaluated by the counting of living cells after staining with trypan blue (dead cells are blue whereas living cells are not) (C) as well as by the measurement LDH activity in the culture medium (D). Data shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05.

Figure legends

Significance of the differences between 7β-OHC-treated cells and (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + α-tocopherol)-treated cells; * P≤0.05. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%). or α-tocopherol (400 µM). ROS overproduction was measured by flow cytometry; ROS overproduction, including superoxide anion (O2 •-), was evaluated with DHE (% DHE positive cells) (A), and with DHR123, which takes into account hydrogen peroxide (H2O2) overproduction (% DHR 123 positive cells) (B). Data shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized. Significance of the difference between vehicle (ethanol (EtOH) 0.6%) and 7β-OHC-treated cells; # p≤0.05 or less.

Significance of the differences between 7β-OHC-treated cells and (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + α-tocopherol)-treated cells;* P≤0.05. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%). Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF), (7β-OHC + MMF)-or (7β-OHC + α-toco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%). shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + α-toco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%). on human retinal pigment epithelial cells (ARPE-19), large myelin figures (evocating reticulophagy) were observed. On ARPE-19 cells, a link between lysosome and cell death was also established. In 158N cells, the complex mode of cell death induced by 7β-OHC (oxiapoptophagy) is characterized by a dephosphorylation of PKB / Akt, an activation of GSK3, and by a reduced expression of Bcl-2; altogether these events contribute to mitochondrial depolarization leading to caspase-3 activation, PARP degradation and internucleosomal DNA fragmentation. Moreover, 7β-OHC promotes the conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a criteria of autophagy. Altogether, these data establish that 7β-OHC is a potent inducer of oxiapoptophagy through the concomittent activation of several signalling pathways involved in oxidative stress, apoptosis and autophagy.

  [7]) are known to amplify oxidative stress and mitochondrial damage which contributes to cell death [8]. In humans, 7KC and 7β-OHC levels were increased in the plasma, cerebrospinal fluid (CSF) or postmortem brain tissues of patients with neurodegenerative diseases, including multiple sclerosis (MS) [9], Parkinson's disease [10],

  [START_REF] Jones | An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide[END_REF].After 24 h of treatment with or without 7β-OHC (50 µM) in the presence or absence of DMF or MMF (25 µM), 158N cells were incubated for 5 min at 37°C with 50 µM FDA (Sigma-Aldrich), and then lysed with 10 mM of Tris-HCl solution containing 1% sodium dodecyl sulfate (SDS, Sigma-Aldrich). Fluorescence intensity of fluorescein (λEx Max: 485 nm, λEm Max: 528 nm) was measured with a TECAN fluorescence microplate reader (Tecan Infinite M 200 Pro, Lyon, France).

2. 9 . 3 .

 93 Measurement of reactive oxygen species (ROS) overproduction with dihydroethidium and dihydrorhodamine 123 ROS overproduction, including superoxide anion (O2 •-), was quantified by flow cytometry after staining with dihydroethidium (DHE; Thermo Fisher Scientific) used at 2 µM. Once in the cell, DHE is rapidly oxidized and exhibits an orange fluorescence (λEx Max= 488 nm; λEm Max= 575 nm) [44]. ROS overproduction, including hydrogen peroxide (H2O2), was also detected with dihydrorhodamine 123 (DHR 123, Cliniscience, Nanterre, France) used at 6 µM, which is oxidized to Rhodamine 123 (RH123) (λEx Max = 505 nm; λEm Max = 529 nm) [45]. After 24 h of treatment, adherent and nonadherent cells were stained with DHE or DHR123 and incubated at 37°C for 15 min. Fluorescent signals were collected with 590 ± 20 nm (DHE) and 520 ± 20 nm (DHR 123) band pass filters, respectively. Ten thousand cells were acquired from each sample. Data were analyzed with Flomax (Partec) or FlowJo (Tree Star Inc.) software.

[ 61 ]

 61 . These dysfunctions favor lipid peroxidation leading to increased levels of 7-ketocholesterol (7KC) and 7β-OHC [7, 19]. These oxysterols are found at significantly elevated levels in the brain, CSF and / or plasma of patients with AD [3], MS [9] as well as in patients with X-linked adrenoleukodystrophy [20]. These modifications could play a critical role in the evolution of neurodegenerative diseases since these oxysterols are able to modify numerous cellular functions [3]. Indeed, it has been reported that 7β-OHC as well as 7KC, known as indicators of oxidative stress [62], contribute to disruption of Redox homeostasis and are potent inducers of inflammation and cell death in different cell types of the CNS [3, 16]. This simultaneous induction of cell death associated with oxidative stress, and presenting apoptotic and autophagic criteria, has been described in oligodendrocytes and microglial cells treated with 7KC, 7β-OHC and 24S-hydroxycholesterol, and was defined as oxiapoptophagy [8]. To prevent neurodegenerative diseases associated with these molecules, it is therefore important to better know their signaling pathways to develop therapeutic strategies to oppose their deleterious effects. Currently, the ability of several molecules to attenuate oxysterols-induced oxiapoptophagy was mainly studied on oligodendrocytes 158N and the most powerful compound identified at the moment is α-tocopherol [8, 17, 19, 39]. In the present study, DMF and its main metabolite, MMF, have been chosen to counteract 7β-OHC-induced oxidative stress, mitochondrial damage and cell death given their ability to prevent oxidative stress via upregulation of anti-oxidative mechanisms [63]. DMF, marketed under the name of Tecfidera

  [69]. Furthermore, it has been demonstrated that the VLCSFAs are able to induce cell death, mitochondrial impairment, and oxidative stress in nerve cells [74], which could aggravate the cytotoxic effect of 7β-OHC. Moreover, MUFA and PUFA levels were also reduced, which is in accordance with other studies, conducted on 158N cells in the presence of 7β-OHC [34]. It is also well established that MUFA and PUFA are indispensable for neuronal membrane fluidity [75], and have neuroprotective, anti-inflammatory, and antioxidant properties [76, 77].

  induced cell death, and enhances antioxidant molecule levels [24]. In the presence of 7β-OHC, treatment with DMF and MMF favors the recovery of mitochondrial function through the restoration of succinate dehydrogenase activity, and mitochondrial transmembrane potential. Furthermore, DMF and MMF are able to counteract mitochondrial O2 •-overproduction, supporting a role of DMF and MMF on the control of cellular Redox potential [82]. In addition, DMF and MMF were able to attenuate the increase of mitochondrial mass corresponding either to an increase of the number of mitochondria and/or of their size [34]. Moreover, DMF and MMF normalize cardiolipin levels. Since a loss of cardiolipin content or alterations in the composition of these molecules have been associated

Fig. 1 :

 1 Fig. 1: Effect of 7β-OHC, dimethyl fumarate and monomethyl fumarate on cell growth and cell adhesion. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). The effects on cell growth and cell adhesion were determined by phase contrast microscopy (A) and crystal violet staining (B). Data shown are mean ± SD of three independent experiments performed in triplicate.

Fig. 2 :

 2 Fig. 2: Effect of 7β-OHC, dimethyl fumarate and monomethyl fumarate on cell viability and plasma membrane integrity. 158N cells were cultured for 24 h with or without 7βhydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). The effect of 7β-OHC (50 µM) with or without DMF (25 µM), MMF (25

Fig. 3 :

 3 Fig. 3: Effect of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced reactive oxygen species (ROS) overproduction. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC; 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM),

Fig. 4 :

 4 Fig. 4: Effect of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced oxidative stress. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). The effects on antioxidant enzyme activities were determined by colorimetric assays with the measurement of glutathione peroxidase (GPx) activity (A) superoxide dismutase (SOD) activity (B) and catalase (CAT) activity (C) as well as by the measurement of thiol-SH group (D). Data shown are mean ± SD of three independent experiments conducted in triplicate. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF), (7β-OHC + MMF) or (7β-OHC + α-toco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%).

Fig. 5 :

 5 Fig. 5: Effect of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced lipid peroxidation and carbonylated protein formation. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). The effects on lipid peroxidation product formation were evaluated by the measurement of CDs (A), and MDA (B) levels. The impact on protein oxidation was evaluated by the measurement of carbonylated protein (CPs) levels (C). Data shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's ttest was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF)-, (7β-OHC + MMF) or (7β-OHC + α-toco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%).

Fig. 6 :

 6 Fig. 6: Evaluation of the effect of dimethyl fumarate and monomethyl fumarate on 7βhydroxycholesterol-induced mitochondrial damage. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). Under these conditions, the effects succinate deshydrogenase activity were evaluated with the MTT test (A). The transmembrane mitochondrial potential (∆Ψm) was measured by flow cytometry after staining with DiOC6(3) (B). The mitochondrial mass was determined by flow cytometry after staining with MitoTracker Red (C). Cardiolipin levels were determined by GC-MS (D) and mitochondrial superoxide anion (O2 •-) production was evaluated by flow cytometry after staining with MitoSOX (E). Data shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized.

Fig. 7 :

 7 Fig. 7: Ultrastructural characterization by transmission electron microscopy of mitochondria and peroxisomes on 158N murine oligodendrocytes incubated with or without 7βhydroxycholesterol in the presence or absence of dimethyl fumarate (DMF). 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 μM) in the presence or absence of DMF (25 μM). Ultrastructural aspects of mitochondria and peroxisomes in untreated (control) (A), DMF (B), 7β-OHC (C), and (7β-OHC + DMF) (D)-treated 158N cells.In the presence of 7β-OHC, several peroxisomes and mitochondria smaller than in the control were observed (C); these differences were no longer observed when 7β-OHC was associated with DMF (D). One experiment was performed.

Fig. 8 :

 8 Fig. 8: Phospholipid (sphingomyelin, phospatidylcholine) content in 158N cells exposed to 7βhydroxycholesterol with or without dimethyl fumarate or monomethyl fumarate. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). Under these conditions, the quantity of sphingomyelin (SM) and phospatidylcholine (PC) per 10 6 cells was determined by GC/MS. Data

Fig. 9 :

 9 Fig. 9: Effect of dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced morphological nuclear changes characteristic of apoptosis and lysosomal modifications evocating autophagy. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). Under these conditions, the quantification of the percentage of apoptotic cells was evaluated by nuclear morphologic criteria after staining with Giemsa and Hoechst 33342; apoptotic cells are characterized by condensed and/or fragmented nuclei whereas normal cells have round and regular nuclei (A). The impact of 7β-OHC (50 µM) with or without DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM)on the lysosome, which is involved in the autophagic process, was determined by flow cytometry after staining with acridine orange (AO); under these conditions, the percentage of AO positive cells was quantified (B). Data shown are mean ± SD of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH) 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + αtoco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (Ethanol 0.6%), and vehicle (DMSO 0.05%).

Fig. 10 :

 10 Fig. 10: Western blotting analysis of the effects dimethyl fumarate and monomethyl fumarate on 7β-hydroxycholesterol-induced apoptosis and autophagy on 158N murine oligodendrocytes. 158N cells were cultured for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF (25 µM), MMF (25 µM) or α-tocopherol (400 µM). Apoptosis and autophagy were characterized by Western blotting with appropriate antibodies raised against uncleaved and cleaved caspase-3 and LC3-I / LC3-II, respectively. Autophagy is characterized by an enhanced (LC3-II / LC3-I) ratio. Data shown are characteristic of at least three independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significance of the differences between 7β-OHC-treated cells and its vehicle (Ethanol (EtOH, 0.6%); # P≤0.05 or less. Significance of the differences between 7β-OHC-treated cells, (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + αtoco)-treated cells; * P≤0.05 or less. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%).

Fig. 11 :

 11 Fig. 11: Evaluation of protein kinase A (PKA), phospholipase C (PLC)/protein kinase C (PKC) and MEK / ERK signaling pathways in 7β-OHC-induced cell death on 158N cells. Murine oligodendrocyte 158N cells previously cultured for 24 h were further cultured for 24 h with or without 7β-OHC (50 µM) in the presence or absence of different inhibitors, H89, U0126, U73122 and chelerythrine, introduced 30 min before the addition of 7β-OHC. Data shown are mean ± SD of two independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized. No significant difference was observed between control (untreated cells), vehicle (ethanol (EtOH): 0.6%)-treated cells and inhibitors (H89, U0126, U73122 or chelerythrine)-treated cells. Significance of the differences between 7β-OHC-treated cell and vehicle (EtOH 0.6%); # P≤0.05 or less. Significant of the differences between 7β-OHC-treated cells and (7β-OHC + (H89, U0126, U73122, or chelerythrine))-treated cells: * P≤0.05.

Fig. 12 :Supplementary Fig. 1 :

 121 Fig. 12: Effects of dimethyl fumarate and mono methyl fumarate on the recovery of 7βhydroxycholesterol-induced cell death. In these experiments, after 24 h of culture, 158N cells were incubated with 7β-OHC (50 µM) for 6 h before the addition of DMF (25 µM), MMF (25 µM), or αtocopherol (400 µM). The cells were subsequently incubated with 7β-OHC (50 µM) associated with DMF, MMF or α-tocopherol for an 18 h additional period of time. A: total number of living cells evaluated after staining with trypan blue; B: percentage of cells with apoptotic nuclei determined after staining with Hoechst 33342. Data shown are mean ± standard deviation (SD) of three independent experiments performed in triplicate. A two way ANOVA followed by a Student's t-test was realized.Significant differences between 7β-OHC-treated cells and the vehicle (ethanol (EtOH, 0.6%): # P≤0.05. Significant differences between 7β-OHC-treated cells and (7β-OHC + DMF)-, (7β-OHC + MMF)-or (7β-OHC + α-tocopherol)-treated cells; * P≤0.05. No significant differences were observed between control (untreated cells), vehicle (EtOH 0.6%), and vehicle (DMSO 0.05%).

Fig. 12 ;

 12 Fig. 1; Sghaier R et al

Table 1 : Profile of organic acids in 158N cells exposed to 7β-hydroxycholesterol with or without dimethyl fumarate (DMF) or monomethyl fumarate (MMF)

 1 Concentrations are expressed as ng/million cells. Results are expressed as mean ± SD of three independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significant difference between vehicle (EtOH 0.6%) and 7β-OHC-treated cells is indicated by #; P < 0.05; significant difference between 7β-OHC and (7β-OHC + DMF) or (7β-OHC + MMF) is indicated by *; P < 0.05. No significant differences are observed between Control, EtOH and DMSO. 7β-hydroxycholesterol: 7β-OHC.

	Sghaier R et al.

Table 2 : Profiles of cholesterol, cholesterol precursors (lathosterol, desmosterol, lanosterol) and oxysterols (triol, 7-ketocholesterol, 7β- hydroxycholesterol) in 158N cells exposed to7β-hydroxycholesterol with or without dimethyl fumarate (DMF) or monomethyl fumarate (MMF)

 2

	Concentrations are expressed as ng/million cells.
	Results are expressed as mean ± SD of three independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significant difference between vehicle
	(EtOH 0.6%) and 7β-OHC-treated cells is indicated by #; P < 0.05; significant difference between 7β-OHC and (7β-OHC + DMF) or (7β-OHC + MMF) is indicated by *; P
	< 0.05. No significant differences are observed between Control, EtOH and DMSO.
	7β-hydroxycholesterol: 7β-OHC.

Sghaier R et al.

OHC (50 µM) + DMF (25 µM)

	2.56 ± 0.05 *	5.33 ± 0.14 *	6.20 ± 0.07*	0.66 ± 0.03 *	0.86 ± 0.01 *	5.72 ± 0.39 *	470.12 ± 9.95 *

7β-OHC (50 µM) + MMF (25 µM)

	2.16 ± 0.12	6.72 ± 0.34 *	5.08 ± 0.21 *	0.32 ± 0.00*	0.88 ± 0.29 *	9.33 ± 0.47 *	426.67 ± 6.19 *

Table 3 : Profile of saturated fatty acids in 158N cells exposed to 7β-hydroxycholesterol withor without dimethyl fumarate (DMF) or monomethyl fumarate (MMF)

 3 Concentrations are expressed as ng/million cells.Results are expressed as mean ± SD of three independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significant difference between vehicle (EtOH 0.6%) and 7β-OHC-treated cells is indicated by #; P < 0.05; significant difference between 7β-OHC and (7β-OHC + DMF) or (7β-OHC + MMF) is indicated by *; P < 0.05; significant difference between vehicle (DMSO 0.05%) and DMF or MMF treated cells is indicated by ¥; P < 0.05. No significant differences are observed between Control, EtOH and DMSO. Sum of very long chain saturated fatty acids; 7β-hydroxycholesterol: 7β-OHC.

	Sghaier R et al.

∑ SFA: Sum of saturated fatty acids; ∑ VLCSFA:

Table 4 : Profile of mono-unsaturated fatty acids profile in 158N cells exposed to 7β-hydroxycholesterol with or without dimethyl fumarate (DMF) and monomethyl fumarate (MMF)

 4 Concentrations are expressed as ng/million cells.Results are expressed as mean ± SD of 3 independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significant difference between vehicle (EtOH 0.6%) and 7β-OHC-treated cells is indicated by #; P < 0.05; significant difference between 7β-OHC and (7β-OHC + DMF) or (7β-OHC + MMF) is indicated by *; P < 0.05; significant difference between vehicle (DMSO 0.05%) and DMF or MMF treated cells is indicated by ¥; P < 0.05. No significant differences are observed between Control, EtOH and DMSO. ∑MUFA: Sum of mono-saturated fatty acids; 7β-hydroxycholesterol: 7β-OHC.

								7β-OHC (50 µM)	7β-OHC (50 µM)
		Control	EtOH (0.6%)	DMSO (0.05%)	DMF (25 µM)	MMF (25 µM)	7β-OHC (50 µM)	+	+
								DMF (25 µM)	MMF (25 µM)
	C14:1 n-5	2.19 ± 0.00	2.10 ± 0.02	1.53 ± 0.22	2.22 ± 0.16 ¥	3.29 ± 0.16 ¥	1.61 ± 0.09 #	1.82 ± 0.05 *	1.71 ± 0.20
	C16:1 n-10 or n-9	231.31 ± 6.96	230.16 ± 9.14	175.46 ± 3.55	176.83 ± 13.98	241.36 ± 6.80 ¥	207.41 ± 6.36 #	274.92 ± 6.32 *	276.70 ± 14.62 *
	C16:1 n-7	454.04 ± 26.71	420.16 ± 28.80	352.91 ± 10.09	400.29 ± 4.27 ¥	553.16 ± 11.81 ¥	313.62 ± 11.98 #	373.66 ± 2.06 *	545.29 ± 4.92 *
	C18:1 n-9	2239.11 ± 16.15 2035.46 ± 48.32	1812.39 ± 30.35	2053.93 ± 47.61 ¥ 2487.96 ± 8.81 ¥	1970.72 ± 36.33	1746.52 ± 0.25 *	1759.29 ± 0.74 *
	C18:1 n-7	502.24 ± 8.92	464.92 ± 46.02	448.74 ± 28.15	546.50 ± 18.31 ¥	718.05 ± 51.47 ¥	403.71 ± 2.75 #	452.47 ± 31.14 *	439.80 ± 1.16 *
	C20:1 n-9	62.63 ± 3.25	54.37 ± 0.23	56.61 ± 2.99	91.13 ± 8.80 ¥	84.90 ± 4.67 ¥	16.82 ± 0.11 #	55.57 ± 0.17 *	55.03 ± 1.27 *
	C20:1 n-7	32.03 ± 0.04	34.63 ± 0.47	35.26 ± 0.16	24.77 ± 2.21 ¥	27.13 ± 0.48 ¥	5.65 ± 1.00 #	12.83 ± 0.45 *	14.74 ± 0.40 *
	C22:1 n-9	2.37 ± 0.05	2.34 ± 0.04	2.00 ± 0.06	2.96 ± 0.14 ¥	2.85 ± 0.25 ¥	2.29 ± 0.16	3.06 ± 0.20 *	2.25 ± 0.11
	C24:1 n-9	12.58 ± 0.53	13.01 ± 0.01	14.61 ± 0.51	22.37 ± 0.58 ¥	24.19 ± 1.78 ¥	32.71 ± 0.96 #	23.52 ± 1.44 *	23.99 ± 2.85 *
	C26:1 n-9	0.59 ± 0.03	0.62 ± 0.00	0.57 ± 0.04	1.25 ± 0.05 ¥	1.32 ± 0.05 ¥	2.27 ± 0.08 #	1.30 ± 0.02 *	1.64 ± 0.03 *
	∑MUFA	3539.09 ± 47.48 3257.77 ± 17.64	2900.09 ± 13.39	3322.26 ± 78.18 ¥ 4144.22 ± 55.05¥	2956.82 ± 13.18 #	2945.67 ± 20.57	3120.44 ± 22.97 *

Sghaier R et al.

Table 5 : Profile of polyunsaturated fatty acids in 158N cells exposed to 7β-hydroxycholesterol with or without dimethyl fumarate (DMF) or monomethyl fumarate (MMF)

 5 Concentrations are expressed as ng/million cells. Results are expressed as mean ± SD of three independent experiments. A two way ANOVA followed by a Student's t-test was realized. Significant difference between vehicle (EtOH 0.6%) and 7β-OHC-treated cells is indicated by #; P < 0.05; significant difference between 7β-OHC and (7β-OHC + DMF) or (7β-OHC + MMF) is indicated by *; P < 0.05; significant difference between vehicle (DMSO 0.05%) and DMF or MMF treated cells is indicated by ¥; P < 0.05. No significant differences are observed between Control, EtOH and DMSO.

								7β-OHC (50 µM)	7β-OHC (50 µM)
		Control	EtOH (0.6%)	DMSO (0.05%)	DMF (25 µM)	MMF (25 µM)	7β-OHC (50 µM)	+	+
								DMF (25 µM)	MMF (25 µM)
	C18:2 n-6	32.66 ± 2.10	28.06 ± 0.94	29.72 ± 0.41	36.30 ± 1.14 ¥	36.77 ± 3.82 ¥	23.14 ± 1.48 #	26.58 ± 1.44 *	26.33 ± 1.95
	C20:4 n-6 (AA)	314.84 ± 6.38	277.13 ± 2.48	269.37 ± 1.78	338.36 ± 44.67 ¥	317.85 ± 15.25 ¥	203.91 ± 2.73 #	185.39 ± 5.99 *	195.29 ± 13.48
	C20:5 n-3 (EPA)	450.26 ± 2.05	400.48 ± 22.37	451.25 ± 13.07	200.83 ± 5.67 ¥	203.16 ± 10.21 ¥	127.58 ± 0.95 #	100.79 ± 8.65 *	114.83 ± 5.05 *
	C20:3 n-6	87.23 ± 7.07	84.27 ± 2.34	67.61 ± 0.67	81.62 ± 2.39 ¥	89.69 ±1.34 ¥	65.15 ± 2.24 #	81.93 ± 2.81 *	72.33 ± 3.41 *
	C20:2 n-6	52.96 ± 2.27	47.51 ± 0.10	51.64 ± 1.70	32.50 ± 0.25 ¥	36.01 ± 4.04 ¥	15.72 ± 2.19 #	17.71 ± 1.38*	34.50 ± 1.08 *
	C22:6 n-3 (DHA)	95.82 ± 2.21	82.47 ± 0.90	79.76 ± 1.66	98.43 ± 5.62 ¥	97.45 ± 4.70 ¥	70.85 ± 0.86 #	70.65 ± 0.46	76.97 ± 3.81 *
	C22:5 n-3 (DPA)	92.21 ± 1.70	90.63 ± 3.51	79.05 ± 1.42	93.39 ± 5.68 ¥	89.42 ± 2.41 ¥	63.52 ± 0.87 #	76.81 ± 0.17 *	75.06 ± 0.15 *
	∑PUFA	1588.33 ± 14.33	1373.67 ± 29.60 1512.94 ± 4.59	1318.22 ± 34.65 ¥	870.35 ± 39.09 ¥	872.40 ± 22.61 #	562.57 ± 0.47 *	628.02 ± 1.89 *
	∑PUFA (n-3)	638.29 ± 5.97	573.58 ± 26.79	610.05 ± 9.99	392.65 ± 5.60 ¥	390.04 ± 17.32 ¥	261.95 ± 0.95 #	248.25 ± 8.02 *	266.85 ± 8.71
	∑PUFA (n-6)	487.70 ± 5.07	436.96 ± 1.18	418.35 ± 1.16	488.78 ± 43.66 ¥	480.31 ± 21.77 ¥	307.91 ± 4.25 #	311.61 ± 6.01	328.45 ± 17.76
	∑PUFA n-3/∑PUFA n-6	1.31 ± 0.00	1.31 ± 0.06	1.46 ± 0.03	0.81 ± 0.06 ¥	0.81 ± 0.00 ¥	0.85 ± 0.01 #	0.80 ± 0.04	0.82 ± 0.07
	Δ 4 : C22:6 n-3/C22 :5 n-3	1.04 ± 0.00	0.91 ± 0.03	1.01 ± 0.00	1.06 ± 0.12	1.09 ± 0.02 ¥	1.12 ± 0.03 #	0.92 ± 0.00 *	1.03 ± 0.05 *
	Δ 5: C20:4 n-6 /C20:3 n-6	3.64 ± 0.37	3.29 ± 0.12	3.98 ± 0.01	4.16 ± 0.67	3.55 ± 0.22 ¥	3.13 ± 0.07	2.27 ± 0.15 *	2.70 ± 0.06 *
	Δ 8: C20:3 n-6 /C20 :2 n-6	1.64 ± 0.06	1.77 ± 0.05	1.31 ± 0.06	2.51 ± 0.09 ¥	2.53 ± 0.32 ¥	4.25 ± 0.73 #	4.67 ± 0.52	2.10 ± 0.16 *
	Δ 9: C16:1 n-7/ C16:0	0.19 ± 0.01	0.18 ± 0.02	0.16 ± 0.00	0.21 ± 0.00 ¥	0.24 ± 0.00 ¥	0.18 ± 0.00	0.23 ± 0.00 *	0.21 ± 0.00 *
	Δ 9: C18:1 n-9/ c18:0	1.81 ± 0.02	1.74 ± 0.07	1.92 ± 0.05	2.35 ± 0.01 ¥	1.86 ± 0.02	2.73 ± 0.01 #	2.17 ± 0.02 *	2.12 ± 0.01 *
	Elongase: 22:5 n-3 /C20:5 n-3	0.20 ± 0.00	0.23 ± 0.00	0.18 ± 0.01	0.47 ± 0.04 ¥	0.44 ± 0.01 ¥	0.50 ± 0.00 #	0.77 ± 0.07 *	0.65 ± 0.03 *
	Elongase : C18:0/C16:0	0.53 ± 0.00	0.51 ± 0.02	0.42 ± 0.01	0.45 ± 0.02 ¥	0.58 ± 0.01 ¥	0.42 ± 0.02 #	0.49 ± 0.00 *	0.32 ± 0.00 *

∑PUFA: sum of polyunsaturated fatty acids; Δ 4: delta 4 desaturase; Δ 5: delta 5 desaturase; Δ 8: delta 8 desaturase; Δ 9: delta 9 desaturase; 7β-hydroxycholesterol: 7β-OHC.

7β-hydroxycholesterol Neuroprotective properties of DMF and MMF (158N oligodendrocytes) Oxiapoptophagy

• Oxidative stress • Mitochondrial dysfunctions / apoptosis • Autophagy Modifications of lipid profile (sterols, fatty acids) SGHAIER R et al. Graphical Abstract

Acknowledgments: This work was presented as a poster at the 8 th ENOR Symposium "Oxysterols and Sterols: from Lipidomics to Food Sciences", September 20-21, 2018, University of Bologna, Bologna, Italy (https://www.oxysterols.net/). This work was supported by grants from: Univ. Bourgogne (Dijon, France), Univ. Monastir (Monastir, Tunisia), and Univ. Manouba (Tunis, Tunisia). We acknowledge Dr Delphine Meffre (Inserm UMR 1124, Paris, France) for her valuable advice on the choice of anti-PLP and and anti-MBP antibodies, and for the conditions of analysis of these proteins by Western blotting.

Conflict of interest:

The authors have no conflict of interest to declare. Supplementary Fig. 2: Evaluation of the optimal concentration of DMF and MMF preventing 7β-hydroxycholesterol-induced cytotoxicity on murine oligodendrocytes 158N. 158N cells previously cultured for 24 h were further incubated for 24 h with or without 7β-hydroxycholesterol (7β-OHC, 50 µM) in the presence or absence of DMF or MMF (1, 12.5, 25, 50 and 100 µM). In these conditions, the cytotoxicity was determined with the MTT test. Vehicles: ethanol (EtOH, 0.6%) or DMSO (0.002, 0.025, 0.05, 0.1, 0.2%). Data shown are mean ± SD of three independent experiments realized in triplicate. A two way ANOVA followed by a Student's t-test was realized. No significant differences were observed between control (untreated cells), and vehicle (EtOH or DMSO). Significant differences between 7β-OHC-treated cells and the vehicle (EtOH: 0.6%): # P≤0.05. Significant determined with the FDA test when biotin or α-tocopherol were added after 7β-OHC (6 and 10 h). The most efficient time to prevent 7β-OHC-induced cell death when α-tocopherol and biotin were introduced in the culture medium after 7β-OHC was 6 h. Data shown are mean ± SD (Two independent experiments realized in triplicate). A two way ANOVA followed by a Student's t-test was realized. No significant differences were observed between control (untreated cells), and vehicle (ethanol (EtOH) 0.6% or DMSO 0.05%). Significant differences between treated cells and the vehicle (EtOH: 0.6%): # P≤0.05. Significant differences between 7β-OHC-treated cells and (7β-OHC + DMF), (malondialdehyde (MDA), conjugated dienes (CDs)) and of carbonylated proteins (CPs) which can further contribute to cell death. Important impact on the mitochondria was also observed whatever the cells are considered. In human monocytic THP-1 and U937 cells, a down-regulation of Bcl-2 expression was also detected as well as an activation of the pro-apoptotic proteins (Bid, Bax), associated with a release of cytochrome c and an activation of caspase-9, caspase-8, caspase-3 and caspase-7. In U937 cells, 7β-OHC also induced an increase in cytosolic Ca 2+ concentration, associated with a decrease of Akt activation and a mitochondrial release of various proteins such as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease-G (Endo-G), associated with caspase-3, -7, -8, and -9 activation, Bid cleavage and poly(ADP-ribose)polymerase (PARP) degradation. In C6 glioblastoma cells, 7β-OHC induces apoptosis through the decrease of ERK signalling, the transient PI3K / Akt activation, the loss of GSK3β activation and the activation of p38. The data obtained in the present study on 158N cells support that p38 activation could be triggered by PKC. On U937 cells as well as