Microglia and S1PR1 Signaling in Multiple Sclerosis Associated Neuropathic Pain

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Project Summary Neuropathic pain afflicts well over half of people living with multiple sclerosis (MS). Current treatments for MS were designed to delay motor symptom progression, but do not address MS associated neuropathic pain (MSNP). This is due in part to a lack of understanding about the underlying mechanisms that drive MSNP. The pathophysiology of MS includes proinflammatory microglial activation that is recapitulated in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Both pharmacological inhibition and genetic knockout of microglia-specific proteins prevented the development of or suppressed established neuropathic pain behaviors in non-MS models of neuropathic pain. These studies indicate that microglia activation mediates the behavioral signs of MSNP. Fingolimod is an FDA approved MS drug that reduces allodynia and hyperalgesia in several rodent models of chronic pain, including peripheral neuropathic pain; however, the site and mechanism of antiallodynic action of fingolimod in central neuropathic pain, e.g., MSNP, remains unknown. Our laboratory reported that repeated administration of intraperitoneal fingolimod attenuated neuropathic pain-like behaviors in EAE, and these effects could be blocked by sphingosine-1-phosphate receptor 1 (S1PR1) antagonists or mimicked S1PR1 agonists. Our results raised the idea that fingolimod acts as an agonist at S1PR1 to elicit antiallodynic effects in EAE. Fingolimod reduces microglial activation in MS and primary cell culture. Because Gi-GPCR activation in microglia inhibits or blocks inflammation, and S1PR1 is a Gi-GPCR, I propose the overall hypothesis that fingolimod leads to S1PR1 activation and subsequent microglial inhibition that explains its anti-allodynic effects in EAE. Specific Aim 1 will test the hypothesis that spinal microglia-dependent mechanisms maintain MSNP. I will deplete microglia in the whole CNS or spinal cord (Aim 1.1/1.2) and chemogenetically inhibit spinal microglia (Aim 1.3) in EAE mice. I predict that each approach will attenuate EAE-induced allodynia. Specific Aim 2 will test the hypothesis that the spinal S1PR1 agonist actions of fingolimod reverse MSNP behavior through inhibition of EAE induced activation of pro-inflammatory microglia. Aim 2.1 predicts that intrathecal injection of fingolimod will reduce EAE-induced allodynia, and that this can be blocked by pre-administration of S1PR1 antagonists. Aim 2.2 predicts fingolimod will stimulate spinal G-protein coupling (assessed with in situ [35S]GTPγS binding assays) that is blocked with S1PR1 antagonists and increased in EAE, indicating an S1PR1 dependent mechanism that allows fingolimod to exert greater analgesic actions in EAE compared to controls. Aim 2.3 predicts that knockout of S1PR1 on microglia will prevent the antiallodynic effects of intrathecal fingolimod and S1PR1 agonists in EAE. This would indicate that fingolimod mediates MSNP through a microglial S1PR1-dependent mechanism.