Contributions
Abstract: EP1676
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Aims: Mitochondrial dysfunctions and oxidative stress are involved in multiple sclerosis (MS) associated with lipid peroxidation leading to increased levels of 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-OHC). These compounds induce oxidative stress, inflammation and cell death. Therefore, it is important to identify natural and synthetic compounds to counteract their side effects. So, the eventual protective effects of dimethylfumarate (DMF) and biotin (Vit B8), used for the treatment of MS, and of monomethylfumarate (MMF), the main metabolite of DMF, were evaluated on 7KC- and 7β-OHC-treated oligodendrocytes.
Methods: Murine oligodendrocytes 158N were exposed to 7KC or 7β-OHC (50 µM, 24 h) without or with DMF, MMF (25 µM) or biotin (10-100 nM). The activities of 7KC and/or 7β-OHC without or with DMF, MMF and biotin were evaluated by complementary methods: phase contrast microscopy, crystal violet test (quantification of adherent cells), MTT test (measurement of succinate dehydrogenase activity), flow cytometry (measurement of the mitochondrial potential (ΔΨm), O2●- and H2O2 production, with DiOC6(3), dihydroethidine and dihydrorhodamine 123, respectively; measurement of plasmic membrane integrity with propidium iodide (PI) and fluoresceine diacetate (FDA)). Apoptosis and autophagy were evaluated with Hoechst 33342 and acridine orange, respectively, and by Western blotting with specific antibodies against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II.
Results: The cytotoxic effects of 7KC were attenuated by DMF and MMF: cell growth inhibition, loss of cell adhesion, decrease of ΔΨm, O2●- and H2O2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cell death: the number of non-adherent cells is decreased as well as the number of cells with damaged membranes and/or dead cells identified with PI and FDA; the number of cells with depolarized mitochondria is reduced, and the decrease of mitochondrial succinate dehydrogenase activity is attenuated. The side effects of 7KC and 7β-OHC were significantly reduced (Mann Whitney test, P< 0.05) with DMF, MMF and biotin.
Conclusion: Major side effects of MS, oxidative stress and mitochondrial dysfunctions leading to cell death of oligodendrocytes, are attenuated by DMF, MMF and Biotin. These data support that these molecules could be of interest to prevent oligodendrocyte dysfunctions in MS.
Disclosure:
Randa SGHAIER, Amira ZARROUK, Thomas NURY, Anne VEJUX, Ahmed Slaheddine MASMOUDI, Thibault MOREAU and Gérard LIZARD have nothing to diclose
Abstract: EP1676
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Aims: Mitochondrial dysfunctions and oxidative stress are involved in multiple sclerosis (MS) associated with lipid peroxidation leading to increased levels of 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-OHC). These compounds induce oxidative stress, inflammation and cell death. Therefore, it is important to identify natural and synthetic compounds to counteract their side effects. So, the eventual protective effects of dimethylfumarate (DMF) and biotin (Vit B8), used for the treatment of MS, and of monomethylfumarate (MMF), the main metabolite of DMF, were evaluated on 7KC- and 7β-OHC-treated oligodendrocytes.
Methods: Murine oligodendrocytes 158N were exposed to 7KC or 7β-OHC (50 µM, 24 h) without or with DMF, MMF (25 µM) or biotin (10-100 nM). The activities of 7KC and/or 7β-OHC without or with DMF, MMF and biotin were evaluated by complementary methods: phase contrast microscopy, crystal violet test (quantification of adherent cells), MTT test (measurement of succinate dehydrogenase activity), flow cytometry (measurement of the mitochondrial potential (ΔΨm), O2●- and H2O2 production, with DiOC6(3), dihydroethidine and dihydrorhodamine 123, respectively; measurement of plasmic membrane integrity with propidium iodide (PI) and fluoresceine diacetate (FDA)). Apoptosis and autophagy were evaluated with Hoechst 33342 and acridine orange, respectively, and by Western blotting with specific antibodies against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II.
Results: The cytotoxic effects of 7KC were attenuated by DMF and MMF: cell growth inhibition, loss of cell adhesion, decrease of ΔΨm, O2●- and H2O2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cell death: the number of non-adherent cells is decreased as well as the number of cells with damaged membranes and/or dead cells identified with PI and FDA; the number of cells with depolarized mitochondria is reduced, and the decrease of mitochondrial succinate dehydrogenase activity is attenuated. The side effects of 7KC and 7β-OHC were significantly reduced (Mann Whitney test, P< 0.05) with DMF, MMF and biotin.
Conclusion: Major side effects of MS, oxidative stress and mitochondrial dysfunctions leading to cell death of oligodendrocytes, are attenuated by DMF, MMF and Biotin. These data support that these molecules could be of interest to prevent oligodendrocyte dysfunctions in MS.
Disclosure:
Randa SGHAIER, Amira ZARROUK, Thomas NURY, Anne VEJUX, Ahmed Slaheddine MASMOUDI, Thibault MOREAU and Gérard LIZARD have nothing to diclose