Involvement of Sphingosine-1-Phosphate in Glutamate Secretion in Hippocampal Neurons

Abstract

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P 1 receptor at the puncta was activated during depolarization and that depolarization-induced S1P 1 receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na ؉ -channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.One of the remarkable features of the central nervous system (CNS) is its ability to integrate and store enormous information. Neuronal information is rapidly transferred through the chemical synapse to specialized regions of the postsynaptic plasma membrane, where neurotransmitter receptors are concentrated. Neurotransmitter secretion in the CNS shares many features with constitutive membrane trafficking (4, 19); however, it also exhibits several unique features, including storage of enormous amounts of information and plasticity, that indicate the presence of additional regulators. There is considerable interest in identifying such regulators that modulate the speed and potency of neurotransmitter release. Among various regulators sphingolipid metabolites such as sphingosine-1-phosphate (S1P) have recently attracted attention for their role in the regulation of neuronal function (9). S1P, a phosphorylated product of sphingosine catalyzed by sphingosine kinase (SK), has been implicated as an important lipid mediator acting both inside and outside the cells (26,33). Extracellular S1P binds to members of GTP-binding protein (G-protein)-coupled S1P receptor family (S1P 1-5 ), triggering diverse...

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes

“…We next tried to identify the Gi-coupled receptors on the CD63-positive MVEs. Our previous observation that PTX-sensitive S1P receptors were involved in vesicular trafficking and involved in glutamate release from internal vesicles in hippocampal neurons 41 prompted us to ask whether S1P receptors may be involved in the 'ongoing activation' of Gi-coupled receptors on the CD63-positive MVEs. To address this possibility, functional involvement of S1P receptors on these MVEs was assessed by FRET analysis using the Gi subunit pair in each S1P receptor subtype-depleted cells.…”

“…Human SphK2-GFP and murine S1P 1 -CFP were constructed as described previously 41,50 . Murine S1P 3 was amplified and cloned into pECFP-N1 (Clontech) and pEGFP-N1 (Clontech) for making CFP-or GFP-tagged S1P 3 , respectively.…”

“…FRAP analysis was performed using LSM 510 META with a  63 oil plan-apochromat objective as described previously 41 . Briefly, HeLa cells expressing SphK2-GFP and CD63-mCherry were pretreated with 10 mM nocodazole in Hanks' balanced salt solution (HBSS, Sigma Aldrich) containing 1 mM CaCl 2 and 5 mM HEPES buffer for 30 min.…”

Cellular Localization of Sphingosine-1-phosphate Receptor 1 Expression in the Human Central Nervous System

“…S1PRs are widely expressed in the central nervous system (CNS), with region-specific distributions (Toman and Spiegel 2002;Jaillard et al 2005;Ohuchi et al 2008;Chun and Hartung 2010). The S1P/S1PR signal has been shown to play an important role in neural development, regulation of neural stem cells and glial migration, astrocyte proliferation, protection against apoptosis, and, more recently, modulation of neuronal excitability and glutamatergic neurotransmission (Chun et al 2000;Toman and Spiegel 2002;Yamagata et al 2003;Harada et al 2004;Kajimoto et al 2007;Kimura et al 2007;Milstien et al 2007;Ohuchi et al 2008). However, in situ localization of S1PRs in the human CNS remains unclear because of the lack of specific antibodies against S1PRs; most findings have been obtained from experiments at the mRNA level (Brinkmann 2009).…”

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

“…On the other hand, several in vitro studies show that S1P can also increase neuronal functions. For example, S1P receptor activation augments glutamate secretion in hippocampal neurons (9) and elevated intracellular S1P concentrations enhance the spontaneous neurotransmitter release at neuromuscular junctions (10). Furthermore, intra-as well as extracellularly applied S1P facilitated the NGF-induced increases in the excitability of sensory neurons from dorsal root ganglia (DRG) (11,12).…”