Subcellular localization of sphingosine 1-phosphate receptors in synapses of the mouse cortex

Sphingosine 1-phosphate (S1P) has pleiotropic biological functions in the regulation of proliferation, survival, migration, inflammation or angiogenesis. S1P acts as intracellular second messenger, as well as extracellular receptor ligand via five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation or apoptosis, excitatory neurotransmission and neuroglia activation, and S1P metabolism alterations have been associated to neurodegenerative disorders. Interestingly, an agonist targeting S1PR1,3,4,5 (FTY720) shows neuroprotective properties through mechanisms that are not fully unveiled, but might include the control of neuroinflammation, vascular deterioration and synaptic dysfunction. The subcellular distribution of S1PRs in nerve terminals is hitherto unknown. The present study aimed at determining the synaptic localisation of S1PRs in the cortex of adult male mice. Synaptosomes were purified from mouse cortex homogenates using a sucrose density gradient centrifugation, and further fractioned into pre-, post- and extrasynaptic zones using a series of pH shifts that allow successive solubilisation of synaptic components (Phillips et al., Neuron 32:63, 2001). Western blot analysis of the obtained fractions, as well as total protein extracts from cortex revealed that S1PR1 is present in similar amounts in total extracts, synaptosomes and the extrasynaptic fraction, but absent from the pre- and postsynaptic fractions. S1PR2 was ubiquitously distributed, showing 3-fold higher levels in the presynaptic zone than the post- (P<0.05) and extrasynaptic (P<0.05) fractions. Similarly, S1PR4 was also distributed across all synaptic fractions but 4-fold more enriched presynaptically. S1PR3 and S1PR5 were not efficiently detected by immunoblotting in synaptosomes and synaptic fractions. Altogether, these results point towards S1PR2 and S1PR4 being particularly well poised to directly modulate synaptic transmission and plasticity upon S1P activation.