Sphingosine-1-phosphate receptor 1 activation in astrocytes contributes to neuropathic pain Zhoumou Chena, Timothy M. Doylea, Livio Luongob, Tally M. Largent-Milnesc, Luigino Antonio Giancottia, Grant Kolard, Silvia Squillacea,e, Serena Boccellab, John K. Walkera,f, Alexander Pendletonc, Sarah Spiegelg, William L. Neumannh, Todd W. Vanderahc, and Daniela Salveminia,1 aDepartment of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO 63104; bDepartment of Experimental Medicine, Division of Pharmacology, Università della Campania Luigi Vanvitelli, 81100 Naples, Italy; cDepartment of Pharmacology, University of Arizona College of Medicine, Tucson, AZ 85724; dDepartment of Pathology, Saint Louis University School of Medicine, St. Louis, MO 63104; eDepartment of Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, 00185 Rome, Italy; fDepartment of Chemistry, Saint Louis University, St. Louis, MO 63104; gDepartment of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298; and hDepartment of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University at Edwardsville, Edwardsville, IL 62026 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 28, 2019 (received for review December 3, 2018) Neuropathic pain afflicts millions of individuals and represents a discordant results from studies across models and types of neurop- major health problem for which there is limited effective and safe athies. This discordance presents a substantial barrier to cohesive therapy. Emerging literature links altered sphingolipid metabolism understanding of the function of S1PR subtypes in neuropathic pain, to nociceptive processing. However, the neuropharmacology of and thus the development of S1PR subtype-targeted strategies to sphingolipid signaling in the central nervous system in the context treat it. of chronic pain remains largely unexplored and controversial. We Emerging evidence implicates S1PR1 activation in the devel- now provide evidence that sphingosine-1-phosphate (S1P) gener- opment of chemotherapy (9, 10) and bone cancer-induced pain (11). ated in the dorsal horn of the spinal cord in response to nerve injury Moreover, intrathecal administration of a highly selective S1PR1 drives neuropathic pain by selectively activating the S1P receptor agonist, SEW2871 (12), caused the development of mechano- subtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharma- hypersensitivity in naïve animals (9). These findings suggest that cological inhibition of S1PR1 with multiple antagonists in distinct inhibiting S1PR1 with an antagonist would be beneficial to treating PHARMACOLOGY chemical classes, but not agonists, attenuated and even reversed neuropathic pain. However, studies in models of traumatic nerve neuropathic pain in rodents of both sexes and in two models of injury have attributed the analgesic effects of the S1PR modulator, traumatic nerve injury. These S1PR1 antagonists retained their FTY720 (Fingolimod) (13), to S1PR1 agonism (14) or combined ability to inhibit neuropathic pain during sustained drug adminis- tration, and their effects were independent of endogenous opioid S1PR1/S1P3 agonism (15). Several hypotheses have been offered for circuits. Moreover, mice with astrocyte-specific knockout of S1pr1 these apparently opposing mechanisms of action for S1PR1, in- did not develop neuropathic pain following nerve injury, thereby cluding differences in the neuropathic pain etiology (chemical toxicity identifying astrocytes as the primary cellular substrate of S1PR1 versus traumatic injury) (15). However, SEW2871 fails to attenuate activity. On a molecular level, the beneficial reductions in neuro- neuropathic pain arising from traumatic nerve injury (14, 15) or pathic pain resulting from S1PR1 inhibition were driven by interleu- kin 10 (IL-10), a potent neuroprotective and anti-inflammatory Significance cytokine. Collectively, our results provide fundamental neurobiolog- ical insights that identify the cellular and molecular mechanisms Understanding the cellular and molecular pathways of neuro- engaged by the S1PR1 axis in neuropathic pain and establish pathic pain is fundamental to discovering classes of non-opioid S1PR1 as a target for therapeutic intervention with S1PR1 antago- analgesics. Emerging evidence implicates altered sphingolipid nists as a class of nonnarcotic analgesics. metabolism and elevated levels of sphingosine-1-phosphate (S1P) in the spinal cord in the persistence of neuropathic pain, sphingosine-1-phosphate | S1P receptor subtype 1 | traumatic nerve but its neuropharmacology and pathways engaged are poorly injury-induced neuropathic pain | astrocytes | interleukin 10 understood. Using a multidisciplinary approach, we provide evi- dence that S1P drives neuropathic pain by selectively activating hronic neuropathic pain (1) constitutes a large unmet med- the S1P receptor subtype 1 (S1PR1) in astrocytes. Inhibiting S1PR1 Cical need affecting 15–30 million people in the USA with an attenuated and reversed neuropathic pain by engaging the annual economic burden of treatment that exceeds $600 billion potent anti-inflammatory cytokine, interleukin 10. Our find- (2). Neuropathic pain conditions are chronic, severe, debilitating, ings represent a paradigm shift in the understanding of S1PR1 and exceedingly difficult to treat (3). Opioids are widely used to signaling that has implications on the future development treat chronic pain but limited by severe side effects and strong of S1PR1 antagonists as a promising class of nonnarcotic abuse liability (4). Continued investigation into the molecular analgesics. underpinnings leading to neuropathic pain is essential for the Author contributions: T.W.V. and D.S. designed research; Z.C., T.M.D., L.L., T.M.L.-M., identification of nonnarcotic-based therapeutic approaches. L.A.G., G.K., S. Squillace, S.B., and A.P. performed research; J.K.W. and W.L.N. contributed Altered neuronal sphingolipid metabolism has been linked to new reagents/analytic tools; T.M.D., L.L., and T.M.L.-M. analyzed data; and T.M.D., L.L., clinically evident neuropathic pain (5, 6). Recent evidence from S. Spiegel, T.W.V., and D.S. wrote the paper. animal studies by our groups and others suggests that neuropathic Conflict of interest statement: D.S. is a cofounder of BioIntervene, Inc. that licensed re- pain arises from the dysregulation of sphingolipid metabolism in lated intellectual property from Saint Louis University. the dorsal horn of the spinal cord (DH-SC) and increased This article is a PNAS Direct Submission. sphingosine-1-phosphate (S1P) production (7). Release of S1P Published under the PNAS license. initiates autocrine or paracrine signaling by activating any of the 1To whom correspondence should be addressed. Email: [email protected]. five known G protein-coupled S1P receptor subtypes (S1PR1-5) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8). However, the precise role of S1P and the identity and function 1073/pnas.1820466116/-/DCSupplemental. of its primary receptor(s) in neuropathic pain are not clear due to Published online May 8, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1820466116 PNAS | May 21, 2019 | vol. 116 | no. 21 | 10557–10562 Downloaded by guest on October 1, 2021 chemotherapy-induced neuropathic pain (9). A recent study has hinted at the possibility of functional S1PR1 antagonism in the anti- allodynic effects of FTY720 following traumatic nerve injury (16), but selective S1PR1 antagonists and the cell substrate of this S1PR1 ac- tivity were not investigated. Targeting S1PR1 has clear benefits in treating neuropathic pain. However, resolving the mechanism of action of agents, such as FTY720, that target S1PR1 and identifying their cell substrate is crucial to understanding how S1P signaling via S1PR1 drives neuropathic pain. This could have immediate impact on the development of appropriate analgesic therapies that target S1PR1. For example, FTY720 is FDA approved for the treat- ment of relapsing–remitting multiple sclerosis (MS) and has a good safety profile (13), and second generation functional an- tagonists are in advanced clinical trials for other indications (17). Moreover, highly selective S1PR1 antagonists are now in ad- vanced preclinical studies for clinical application in various dis- ease states (17). Using a multidisciplinary genetic and pharmacological approach with multiple S1PR1 antagonists and agonists across traumatic nerve injurymodels,species,sexes,andlaboratories, we present evidence unequivocally establishing that S1P activation of S1PR1 signaling in astrocytes is required for the development and maintenance of traumatic nerve injury-induced neuropathic pain. Our findings pro- vide foundational evidence to support the development of S1PR1 antagonistsratherthanagonistsasaclassofnonnarcoticanalgesics. Results Inhibiting S1PR1 Attenuates and Reverses Neuropathic Pain States. Several competitive and functional S1PR1 antagonists were Fig. 1. S1PR1 antagonism, but not agonism, attenuated and reversed evaluated in two well-characterized rodent models of neuro- neuropathic pain states. (A) In mice with CCI, administration of TASP0277308 pathic pain: constriction of the sciatic nerve (CCI) (18) and (3 mg/kg, ▲; n = 5) or FTY720 (1 mg/kg, ▼; n = 5), but not SEW2871 (20 spared nerve injury (SNI) (19). The competitive S1PR1 an- mg/kg,