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。多发性硬化症的当前治疗方法 旨在延缓运动症状的进展，但不能解决MS相关的神经病理性疼痛 (MSNP)。这在一定程度上是由于对推动MSNP的潜在机制缺乏了解。这个 MS的病理生理包括促炎性小胶质细胞激活，在实验中概括了这一点 自身免疫性脑脊髓炎(EAE)小鼠模型的药物抑制和遗传 小胶质细胞特异性蛋白的敲除可防止或抑制已建立的神经病的发展 神经病理性疼痛非多发性硬化症模型的疼痛行为。这些研究表明，小胶质细胞的激活在 MSNP的行为体征。Fingolimod是FDA批准的多发性硬化症药物，可减少痛觉过敏和痛觉过敏 在几种慢性疼痛的啮齿动物模型中，包括周围神经病理性疼痛；然而，部位和机制 Fingolimod在中枢神经病理性疼痛(如MSNP)中的抗痛觉过敏作用尚不清楚。我们的实验室 报道称，重复给药可减轻小鼠神经病理性痛样行为。 这些作用可被鞘氨醇-1-磷酸受体1(S1PR1)拮抗剂或 模拟S1PR1激动剂。我们的结果提出了这样一种想法，即Fingolimod在S1PR1起激动剂的作用，从而引发 EAE的抗过敏性作用。 Fingolimod减少MS和原代细胞培养中的小胶质细胞激活。因为小胶质细胞中GI-GPCR的激活 抑制或阻断炎症，而S1PR1是一种GI-GPCR，我提出了Fingolimod的总体假设 导致S1PR1激活和随后的小胶质细胞抑制，这解释了其在EAE中的抗痛觉异常作用。 具体目标1将检验脊髓小胶质细胞依赖机制维持MSNP的假设。我会耗尽的 整个中枢或脊髓的小胶质细胞(目标1.1/1.2)和化学发生抑制脊髓小胶质细胞(目标1.3) 在EAE小鼠身上。我预测，每一种方法都会减轻EAE引起的痛觉过敏。《特定目标2》将测试 脊髓S1PR1激动剂Fingolimod通过抑制EAE逆转MSNP行为的假说 诱导促炎性小胶质细胞活化。Aim 2.1预测鞘内注射Fingolimod将 减少EAE引起的痛觉异常，这可以通过预先给予S1PR1拮抗剂来阻断。目标 预测Fingolimod将刺激脊髓G蛋白偶联(用原位[35S]GTPγS结合试验进行评估) 它被S1PR1拮抗剂阻断，并在EAE中增加，表明S1PR1依赖机制 与对照组相比，Fingolimod可以在EAE中发挥更大的止痛作用。AIM 2.3预测淘汰赛 S1PR1对小胶质细胞的作用可阻断鞘内注射Fingolimod和S1PR1激动剂对EAE的抗痛觉过敏作用。 这可能表明Fingolimod通过小胶质细胞S1PR1依赖的机制介导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.