Author Of 2 Presentations
P0357 - Mouse astrocytes exhibit agonist-induced functional S1P1 receptor antagonism (ID 1250)
Abstract
Background
Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P1) plays a key role in regulation of lymphocyte trafficking. In multiple sclerosis patients, S1P1 agonists, such as fingolimod or siponimod, inhibit the egress of pathogenic lymphocytes from lymph nodes and their infiltration into the central nervous system (CNS) via lymphocyte-expressed S1P1 receptor down-modulation, also known as S1P1-functional antagonism. However, there is no evidence of this phenomenon in cells of the CNS.
Objectives
To assess the presence of agonist-induced S1P1 down-modulation in astrocytes using a calcium (Ca2+) signaling assay.
Methods
Murine astrocytes (C8-D1A) were incubated overnight and then loaded with a probe (Fluo-4AM, a dye that becomes fluorescent upon Ca2+ binding) for 1 hour, followed by adenosine triphosphate (ATP; 10 µM) over 30 min to activate the Ca2+ pumps. The cells were then treated with various S1P1 agonists (S1P [natural ligand for S1P receptors], AUY954 [selective S1P1 agonist], fingolimod [S1P1,3,4,5 agonist] or siponimod [S1P1,5 agonist]) at different concentrations (0.0001 µM up to 30 µM) to construct dose-response curves using agonist-induced Ca2+ signaling, measured as an increase in the intracellular fluorescence (via a FLuorescent Imaging Plate Reader [FLIPR]). To investigate the S1P down-modulation, the astrocytes were pretreated overnight with S1P1 agonists (1 µM) prior to probe loading, ATP priming, and agonist stimulation.
Results
All of the tested S1P1 agonists increased intracellular Ca2+ influx in the astrocytes in a dose-dependent manner, with concentration inducing half-maximal effect (EC50) within the range of 2–5 nM. Similar results were obtained after overnight pretreatment with the natural ligand, S1P (S1P1,2,3,4,5 agonist), confirming that S1P does not induce down-modulation of its own receptors.However, cells pretreated overnight with AUY954 did not exhibit agonist-induced intracellular Ca2+ signaling, suggesting the down-modulation of the S1P1 receptors. Similar outcomes were observed upon pretreatment with either fingolimod or siponimod, indicating their role as functional S1P1 antagonists on the murine astrocytes.
Conclusions
To our knowledge, this is the first report of agonist-induced S1P1 down-modulation in the astrocytes. Additional investigations on other neuronal and glial cells are warranted to establish whether this is a generalized phenomenon in the CNS.
P0991 - Protective and Remyelinating Potential of Siponimod in a Xenopus Model of Demyelination and a Mouse Model of Experimental Autoimmune Encephalomyelitis (ID 1320)
Abstract
Background
Siponimod, a potent and highly selective sphingosine 1-phosphate receptor modulator, has recently been approved for treatment of relapsing forms of MS and active SPMS.
Objectives
To assess remyelination and neuroprotective potential of siponimod in a Xenopus remyelination and a mouse optic neuritis (EAEON) model using histological analysis and longitudinal visual system readouts.
Methods
We used a conditional demyelination transgenic Xenopus laevis model (MBP-GFP-NTR), in which oligodendrocyte apoptosis can be induced by metronidazole (MTZ) treatment. After MTZ withdrawal, remyelination was assessed with or without siponimod (0.1nM-1µM). In a pharmacokinetics study, brain siponimod levels were analysed. EAEON was induced in female C57BL/6J mice immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) and continuously fed with vehicle- or siponimod-loaded pellets at 10 or 30 mg/kg of food, either prophylactically or therapeutically, over 90 days. Sections of the optic nerve (Xenopus and mouse) were used to detect de- and remyelination, as well as inflammatory infiltrates. In mice, thickness of retinal layers and visual function were assessed by optical coherence tomography and optokinetic response, respectively. Circulating lymphocytes (flow-cytometry) and siponimod blood and brain levels were analysed at the end of the experiment.
Results
Treatment of demyelinated tadpoles with siponimod (1nM in swimming water) improved remyelination by a factor of 2.3±0.2 fold in comparison to control. The dose-response of siponimod efficiency to accelerate remyelination showed a bell-shaped curve with a maximum remyelination effect at concentrations ranging between 70-80 nM in tissues. In the EAEON mouse model, prophylactic siponimod treatments with 10 or 30 mg/kg attenuated the EAEON clinical scores by about 80% and 95%, respectively, and reduced the retinal neurodegeneration and the loss of visual function. Interestingly, therapeutic treatment starting at day 14 of EAEON had no impact on optic nerve immune cell infiltrates but resulted in increased myelin levels and protection of inner retinal layers also in a bell-shaped dose-response curve with significant protective effects only at the lower dose.
Conclusions
Our data suggest that while siponimod strongly impacts immune cells at higher concentrations (classical dose-response), its effects on remyelination and neuroaxonal survival are dose-dependent following the dynamics of a bell-shaped dose-response curve in both animal models.