Contributions
Abstract: EP1617
Type: Poster Sessions
Abstract Category: Therapy - Neuroprotection and Repair
Background: The sphingosine-1-phosphate (S1P) receptor subtype 5 (S1P5), mainly expressed on oligodendrocytes and brain endothelial cells, influences neuronal myelination processes and integrity of the blood-brain barrier. Further, evidence suggest this receptor as a potential therapeutic target to prevent and/or reverse the progressive demyelination that causes multiple sclerosis (MS). However, it is not clear whether such pro-myelination effects are triggered by S1P5 signaling or rather by S1P5 down-modulation (i.e. internalisation and degradation) as seen with the S1P1-mediated effects on lymphocytes.
Objective: The study assessed the ability of established S1P1/5 agonists (fingolimod, siponimod and ozanimod) to induce down-modulation versus selective human (h) S1P1 (AUY954) and hS1P5 (UC-42-WP04) receptor agonists.
Methods: Chinese Hamster Ovary cells overexpressing hS1P1 or hS1P5 receptors were tagged at the N-terminal (extracellular). The hS1P1 (Myc tag), mu-S1P5 (Myc tag) or hS1P5 (FLAG tag) receptors were generated using an anti-Myc/FLAG-epitope antibody and incubated for 1‒3 hours with an agonist (0.0001-1 µM). Receptor internalisation was measured using a flow cytometry assay, which quantified the levels of surface expression of tagged-receptors. In this assay, a reduction in fluorescence indicated disappearance of the tagged receptor from the cell surface.
Results: Under such experimental conditions, no S1P5 down-modulation was observed in response to fingolimod, siponimod or ozanimod up to 1 µM. As expected, all agonists down-modulated the S1P1 receptor internalisation in a dose-dependent manner, with doses inducing mean effective concentration of 50% (EC50) and 90% (EC90) within the 1-5 nM range and 10-70 nM range, respectively. Moreover, no reappearance of S1P1 receptor was observed within 3 hours after washout, confirming long term down-modulation. In comparison, S1P and AUY954 down-modulated the S1P1 but not the S1P5 receptors, whereas UC-42-WP04 had no impact on both S1P1 and S1P5 receptors.
Conclusion: These observations suggest that, in contrast to the S1P1 receptor, the S1P5 receptor is not subjected to agonist-induced down-modulation. Consequently, S1P5 downstream signaling should be considered when studying the direct pro-myelination and neuroprotective effects of S1P1/5 selective agonists, such as siponimod.
Disclosure: This study was funded by Novartis Pharma AG, Basel, Switzerland. All authors are employees of Novartis Pharma AG.
Abstract: EP1617
Type: Poster Sessions
Abstract Category: Therapy - Neuroprotection and Repair
Background: The sphingosine-1-phosphate (S1P) receptor subtype 5 (S1P5), mainly expressed on oligodendrocytes and brain endothelial cells, influences neuronal myelination processes and integrity of the blood-brain barrier. Further, evidence suggest this receptor as a potential therapeutic target to prevent and/or reverse the progressive demyelination that causes multiple sclerosis (MS). However, it is not clear whether such pro-myelination effects are triggered by S1P5 signaling or rather by S1P5 down-modulation (i.e. internalisation and degradation) as seen with the S1P1-mediated effects on lymphocytes.
Objective: The study assessed the ability of established S1P1/5 agonists (fingolimod, siponimod and ozanimod) to induce down-modulation versus selective human (h) S1P1 (AUY954) and hS1P5 (UC-42-WP04) receptor agonists.
Methods: Chinese Hamster Ovary cells overexpressing hS1P1 or hS1P5 receptors were tagged at the N-terminal (extracellular). The hS1P1 (Myc tag), mu-S1P5 (Myc tag) or hS1P5 (FLAG tag) receptors were generated using an anti-Myc/FLAG-epitope antibody and incubated for 1‒3 hours with an agonist (0.0001-1 µM). Receptor internalisation was measured using a flow cytometry assay, which quantified the levels of surface expression of tagged-receptors. In this assay, a reduction in fluorescence indicated disappearance of the tagged receptor from the cell surface.
Results: Under such experimental conditions, no S1P5 down-modulation was observed in response to fingolimod, siponimod or ozanimod up to 1 µM. As expected, all agonists down-modulated the S1P1 receptor internalisation in a dose-dependent manner, with doses inducing mean effective concentration of 50% (EC50) and 90% (EC90) within the 1-5 nM range and 10-70 nM range, respectively. Moreover, no reappearance of S1P1 receptor was observed within 3 hours after washout, confirming long term down-modulation. In comparison, S1P and AUY954 down-modulated the S1P1 but not the S1P5 receptors, whereas UC-42-WP04 had no impact on both S1P1 and S1P5 receptors.
Conclusion: These observations suggest that, in contrast to the S1P1 receptor, the S1P5 receptor is not subjected to agonist-induced down-modulation. Consequently, S1P5 downstream signaling should be considered when studying the direct pro-myelination and neuroprotective effects of S1P1/5 selective agonists, such as siponimod.
Disclosure: This study was funded by Novartis Pharma AG, Basel, Switzerland. All authors are employees of Novartis Pharma AG.