Novartis Pharma AG

Author Of 1 Presentation

Disease Modifying Therapies – Mechanism of Action Poster Presentation

P0357 - Mouse astrocytes exhibit agonist-induced functional S1P1 receptor antagonism (ID 1250)

Speakers
Presentation Number
P0357
Presentation Topic
Disease Modifying Therapies – Mechanism of Action

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.

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