Microglia and S1PR1 Signaling in Multiple Sclerosis Associated Neuropathic Pain

项目摘要 神经性疼痛困扰着超过一半的多发性硬化症（MS）患者。目前MS的治疗方法 旨在延迟运动症状进展，但不解决MS相关的神经性疼痛 （MSNP）。这部分是由于缺乏对驱动MSNP的潜在机制的理解。的 MS的病理生理学包括促炎性小胶质细胞活化，其在实验性MS中重现。 MS的自身免疫性脑脊髓炎（EAE）小鼠模型。 敲除小胶质细胞特异性蛋白质可阻止或抑制已建立的神经病理性 神经性疼痛的非MS模型中的疼痛行为。这些研究表明，小胶质细胞激活介导 MSNP的行为特征。Fingolimod是FDA批准的MS药物，可减少异常性疼痛和痛觉过敏 在几种慢性疼痛的啮齿动物模型中，包括外周神经性疼痛;然而， 芬戈莫德在中枢神经性疼痛中的抗异常性疼痛作用，例如，MSNP，仍然未知。本实验室 报道，重复腹膜内给予芬戈莫德减弱了神经性疼痛样行为， EAE，并且这些作用可以被鞘氨醇-1-磷酸受体1（S1 PR 1）拮抗剂或 模拟S1 PR 1激动剂。我们的研究结果提出了芬戈莫德作为S1 PR 1激动剂的想法， EAE中的抗异常性疼痛作用。 芬戈莫德减少MS和原代细胞培养物中的小胶质细胞活化。因为小胶质细胞中Gi-GPCR的激活 抑制或阻断炎症，S1 PR 1是Gi-GPCR，我提出了芬戈莫德的总体假设， 导致S1 PR 1激活和随后的小胶质细胞抑制，这解释了其在EAE中的抗异常性疼痛作用。 具体目标1将检验脊髓小胶质细胞依赖性机制维持MSNP的假设。我会耗尽 整个中枢神经系统或脊髓中的小胶质细胞（目标1.1/1.2）并化学发生抑制脊髓小胶质细胞（目标1.3） EAE小鼠。我预测每种方法都能减轻EAE引起的异常性疼痛。具体目标2将测试 芬戈莫德的脊髓S1 PR 1激动剂作用通过抑制EAE逆转MSNP行为的假设 诱导促炎性小胶质细胞的活化。目的2.1预测鞘内注射芬戈莫德将 减少EAE诱导的异常性疼痛，并且这可以通过预先施用S1 PR 1拮抗剂来阻断。目的 2.2预测芬戈莫德将刺激脊髓G蛋白偶联（采用原位[35 S]GTPγS结合试验评估） 该受体被S1 PR 1拮抗剂阻断，并在EAE中增加，表明S1 PR 1依赖性机制， 与对照组相比，芬戈莫德在EAE中发挥更大的镇痛作用。目标2.3预测， S1 PR 1对小胶质细胞的抑制作用将阻止鞘内芬戈莫德和S1 PR 1激动剂在EAE中的抗异常性疼痛作用。 这表明芬戈莫德通过小胶质细胞S1 PR 1依赖性机制介导MSNP。

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.