Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons

The signaling lipid sphingosine 1-phosphate regulates mechanical pain

Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds

Genetic Evidence for Involvement of Neuronally Expressed S1P1 Receptor in Nociceptor Sensitization and Inflammatory Pain

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in "cation-free" solution and blocked by the nonspecific Cl(-) channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P(3) receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P(3)(-/-) mice, whereas in control S1P(1) floxed (S1P(1)(fl/fl)) mice and mice with a nociceptor-specific deletion of S1P(1)(-/-) receptor (SNS-S1P(1)(-/-)), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl(-) conductance that is largely mediated by S1P(3) receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.María Camprubí-Robles, Norbert Mair, Manfred Andratsch, Camilla Benetti, Dimitra Beroukas, Roman Rukwied ... et al

Reduced thermal hypersensitivity in S1P₁^−/−mice.

<p>(A) Injection of the S1P₁ agonist SEW2871 induced a significant transient decrease in paw withdrawal latencies in S1P₁^fl/fl (n = 9) which was significantly less pronounced than in SNS-S1P₁^−/− mice (n = 10, *p<0.05; ANOVA). (B, C) While only a minor reduction of paw withdrawal latencies was observed in both mouse strains with local low dose S1P injection, we observed a significant decrease in paw withdrawal latencies in S1P₁^fl/fl mice (n = 7) which was similar to wt. In SNS-S1P₁^−/− mice the degree of hypersensitivity was significantly ameliorated in comparison to S1P₁^fl/fl mice (n = 9, *p<0.05, ** p<0.01; ANOVA). (D) CFA (20 µl) injection into the plantar hindpaw induced a pronounced decrease of PWL which was significantly attenuated in S1P₁-Cre mice (p<0.05, n = 4; ANOVA). (E) Paw swelling was similar in SNS-S1P₁^−/− and S1P₁^fl/fl mice (n = 4).</p

S1P-induced sensitization of heat pain behavior, nociceptor neuron discharge <i>in vitro</i> and heat-activated ionic currents.

<p>(A) Injection of S1P into the paw skin (5 µl of a 100 µM S1P solution in PBS) but not vehicle (n = 10, n.s.) induced a significant transient drop in paw withdrawal latencies (PWL) in response to heat stimulation from 10.15±0.63 to 3.62±0.24 s (n = 10, *p<0.05; ANOVA). Heat sensitization fully recovered to baseline within three hours. (B) Discharge activity of single primary nociceptive neurons <i>in vitro</i> significantly increased from 2.03±0.39 before (black columns) to 3.21±0.50 Imp/s (grey columns) after the receptive fields of the fibers were exposed to 1 µM S1P for 5 min (n = 11, p<0.05; Wilcoxon matched pairs test). (C) After conditioning stimulation with S1P, the heat-induced current of a dorsal root ganglion neuron exhibited increased peak amplitudes and was activated at a lower temperature compared with control. (D) Temperature-current plots of four neurons stimulated with a ramp-shaped heat stimulus with a linear rise of temperature from room temperature to 50°C before (open circles) and after conditioning stimulation with S1P (filled circles, threshold temperature).</p

Primer pairs used.

<p>Primer pairs used.</p

Expression of S1P₁ receptors in sensory neurons.

<p>(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P₁, S1P₂ and S1P₃ mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P₄ and S1P₅ mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P₁ was present in neurons and intraganglionic capillaries (arrowhead). S1P₁-IR was colocalized with immunoreactivity for peripherin, whereas S1P₁-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P₁ receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P₁-IR positive neurons revealed that S1P₁-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P₁-IR+ neurons had diameters >20 µm. (F) Expression of S1P₁ immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.</p