Targeted Deletion of Sphingosine‐1‐Phosphate Receptor Type 1 (S1PR1) Reveals a Predominant Functional Localization of S1PR1 in Astrocytes in the Spinal Cord

Abstract

Sphingosine-1-phosphate (S1P) is a tightly regulated bioactive lysolipid that acts as the endogenous ligand for G-protein coupled S1P receptors (S1PR) types 1-5. The S1PR system has been targeted for immune modulation, but research indicates its potential as a target for pain relief. FTY720 is a S1PR immunomodulatory prodrug that is phosphorylated in vivo to its active form, FTY720-phosphate (FTY-P), which activates S1PR 1, 3, 4, and 5. FTY720 is currently FDA approved for the treatment of Multiple Sclerosis, but preclinical evidence has indicated both S1P and FTY720 produce antinociception in models of acute and neuropathic pain. Results with S1PR1 selective agonists and antagonists suggest S1PR ligands most likely act to alleviate pain specifically through S1PR1. Here we used conditional gene deletion to determine the cell type(s) mediating S1P-stimulated G-protein activation in the spinal cord, measured using agonist-stimulated [35S]GTPγS binding. Deletion of S1PR1 in S1PR1-floxed mice expressing cre recombinase under control of the nestin promoter, which deletes S1PR1 from neurons, astrocytes, and oligodendroglia, showed a profound decrease in G-protein activation stimulated by S1P (by ~85%) or the S1PR1 agonist SEW2871 (>95%) in the spinal cord, suggesting the majority of S1PR activity in spinal cord is mediated by S1PR1. We next explored the extent to which S1PR1-mediated G-protein activation in the lumbar spinal cord of male and female mice is specifically localized in astrocytes. The lumbar spinal cord is thought to be a major point of transduction of pain signals from nerve-injury models of neuropathic pain in rodents. S1PR1-floxed mice were crossed with cre recombinase under the control of a glial fibrillary acidic protein (GFAP)-promotor. In this model, there was a ~75% loss in G-protein activation by S1P in the astrocyte specific conditional knock out (cKO) compared to the control mice, whereas there was a near complete (>95%) loss of SEW2871-stimulated G-protein activation in these cKO mice. No sex differences in S1PR-stimulated G-protein activation or the effect of S1PR1 deletion was observed. This loss of S1PR1-stimulated G-protein activation in the lumbar spinal cord upon astrocyte specific deletion of S1PR1 suggests S1PR1 activity is mainly localized in astrocytes within the lumbar spinal cord. These results aid in the understanding of S1PR1 localization and supports recently reported evidence for a role for S1PR1 in astrocytes in FTY720-responsive neuropathic pain models. Studies are also ongoing to determine the functional localization of S1PR1 in astrocytes in brain regions associated with pain perception and regulation.