Contributions
Abstract: EP1674
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Aims: Imbalance in the homeostasis of K+ ions contributes to the pathogenesis of multiple sclerosis (MS). 7-ketocholesterol (7KC) often found at increased levels in patients with MS is able to trigger numerous oligodendrocyte dysfunctions. We therefore studied the impact of 7KC on 158N murine oligodendrocytes, and determined its effect on the P2X7 ATP-dependent receptor channel, and on the Kv3.1 potential dependent-channel, which are involved in K+ homeostasis.
Methods: 158N murine oligodendrocytes were cultured with 7KC in the absence or in the presence of 4-aminopyridine (specific K+ channel blocker), diazoxide (ATP sensitive K+ channel activator), or glibenclamide (ATP sensitive K+ channel blocker). The activation of P2X7 was measured by flow cytometry with YOPRO1. Kv3.1b expression was determined by flow cytometry and western-blotting with a mouse monoclonal antibody (Sigma-Aldrich; ref: SAB5200030 (anti-kv3.1b)). The intracellular concentration of K+ ([K+]i) was determined by flame photometry and the ratiometric approach using the PBFI-AM fluorescence indicator. 7KC-induced cell death was evaluated by the crystal violet test as well as by mitochondrial depolarization and enhancement of the cytoplasmic membrane permeability measured by flow cytometry after staining with DiOC6(3) and propidium iodide, respectively. Overproduction of reactive oxygen species (ROS) was quantified with dihydroethidium.
Results: Positive correlations were found between P2X7 activation, Kv3.1b expression and intracellular K+ level, overproduction of ROS, loss of transmembrane mitochondrial potential and increased plasma membrane permeability in 158N cells. The correlations were determined with the Spearman correlation test: r was in the range of 0.8 and p ≤ 0.005.
Conclusion: Our data support that the lipid environment affects P2X7 activity and Kv3.1b channel expression and/or functionality, and that the subsequent rupture of K+ homeostasis as well as enhanced [K+]i is relied with oligodendrocytes damages. It is suggested that the ability to modulate K+ homeostasis with drugs capable to modulate ATP and voltage dependent K+ channels could constitute new pharmacological targets to prevent oligodendrocytes dysfunctions in multiple sclerosis.
Disclosure:
Maryem BEZINE, Thomas NURY, Rym BEN-KHALIFA, Anne VEJUX, Jérôme de SEZE, Mohamed EL-AYEB, Thibault MOREAU and Gérard LIZARD have nothing to disclose.
Abstract: EP1674
Type: ePoster
Abstract Category: Therapy - disease modifying - 27 Neuroprotection and Repair
Aims: Imbalance in the homeostasis of K+ ions contributes to the pathogenesis of multiple sclerosis (MS). 7-ketocholesterol (7KC) often found at increased levels in patients with MS is able to trigger numerous oligodendrocyte dysfunctions. We therefore studied the impact of 7KC on 158N murine oligodendrocytes, and determined its effect on the P2X7 ATP-dependent receptor channel, and on the Kv3.1 potential dependent-channel, which are involved in K+ homeostasis.
Methods: 158N murine oligodendrocytes were cultured with 7KC in the absence or in the presence of 4-aminopyridine (specific K+ channel blocker), diazoxide (ATP sensitive K+ channel activator), or glibenclamide (ATP sensitive K+ channel blocker). The activation of P2X7 was measured by flow cytometry with YOPRO1. Kv3.1b expression was determined by flow cytometry and western-blotting with a mouse monoclonal antibody (Sigma-Aldrich; ref: SAB5200030 (anti-kv3.1b)). The intracellular concentration of K+ ([K+]i) was determined by flame photometry and the ratiometric approach using the PBFI-AM fluorescence indicator. 7KC-induced cell death was evaluated by the crystal violet test as well as by mitochondrial depolarization and enhancement of the cytoplasmic membrane permeability measured by flow cytometry after staining with DiOC6(3) and propidium iodide, respectively. Overproduction of reactive oxygen species (ROS) was quantified with dihydroethidium.
Results: Positive correlations were found between P2X7 activation, Kv3.1b expression and intracellular K+ level, overproduction of ROS, loss of transmembrane mitochondrial potential and increased plasma membrane permeability in 158N cells. The correlations were determined with the Spearman correlation test: r was in the range of 0.8 and p ≤ 0.005.
Conclusion: Our data support that the lipid environment affects P2X7 activity and Kv3.1b channel expression and/or functionality, and that the subsequent rupture of K+ homeostasis as well as enhanced [K+]i is relied with oligodendrocytes damages. It is suggested that the ability to modulate K+ homeostasis with drugs capable to modulate ATP and voltage dependent K+ channels could constitute new pharmacological targets to prevent oligodendrocytes dysfunctions in multiple sclerosis.
Disclosure:
Maryem BEZINE, Thomas NURY, Rym BEN-KHALIFA, Anne VEJUX, Jérôme de SEZE, Mohamed EL-AYEB, Thibault MOREAU and Gérard LIZARD have nothing to disclose.