Sur1-Trpm4 regulation of the pro-inflammatory astrocytic secretome in EAE

背景：CNS中的先天性免疫应答在多发性骨髓瘤的疾病进展中起关键作用。 硬化症（MS）/实验性自身免疫性脑脊髓炎（EAE）。星形胶质细胞、小胶质细胞和促炎因子 单核细胞积极促进神经变性、脱髓鞘和瘢痕形成，但治疗策略 靶向神经胶质细胞仍然难以捉摸。EAE中的反应性星形胶质细胞（这里简称为“EAE-星形胶质细胞”）在EAE中的表达。 脊髓分泌许多促炎因子，单独抑制每种因子是有益 在EAE。尽管单独针对促炎因子可能是有益的，但更有希望的策略 可能是更广泛地靶向EAE-星形胶质细胞的促炎分泌蛋白。这个想法来自于 已发表的关于口服MS药物和鞘氨醇-1-磷酸（S1 P）受体调节剂芬戈莫德的研究， 其显示在芬戈莫德存在下星形胶质细胞活化产生分泌蛋白质组， 煽动性我们实验室最近发现Sur 1-Trpm 4在MS/ EAE中的病理参与。在慢性 在EAE的早期阶段，Sur 1-Trpm 4通道主要由反应性星形胶质细胞（EAE-1）强烈上调。 星形胶质细胞），与整体抑制SUR 1减少分泌的几个促炎性细胞因子， 细胞因子/趋化因子，并减少临床症状、脱髓鞘和轴突损失。新实验 扩展了这项工作，揭示了Sur 1-Trpm 4和S1 P信号传导之间的关键相互作用， EAE-星形胶质细胞。初步数据显示：（i）在EAE中，Sur 1-Trpm 4、S1 P1和S1 P3受体高度共表达， 定位于EAE-星形胶质细胞;（ii）在EAE中，编码Sur 1的Abcc 8的星形胶质细胞特异性缺失， 与芬戈莫德相似的分泌蛋白质组;（iii）在培养的星形胶质细胞中，Sur 1-Trpm 4通道位于 和所需的，S1 P信号;（iv）在培养的星形胶质细胞，Sur 1-Trpm 4通道调节转录 多种促炎因子的作用我们的中心假设是，Sur 1-Trpm 4通道是必需的， S1 P介导的信号传导使促炎性星形胶质细胞分泌组永久存在并导致疾病 MS/ EAE的维持/进展。 描述：该项目有三个机制目标，都集中在EAE中的Sur 1-Trpm 4。在目标1中，我们 髓鞘少突胶质细胞糖蛋白（MOG）35-55 EAE在基因缺失雄性和雌性小鼠中研究 在EAE诱导后不同时间启动的星形胶质细胞中的Sur 1，以确定星形胶质细胞Sur 1在EAE诱导后不同时间启动的星形胶质细胞中的作用。 EAE的不同阶段。在目标2中，将使用分子和膜片钳实验来证明 Sur 1-Trpm 4在调节活化星形胶质细胞分泌组的S1 P信号传导中的作用。在目标3中， 本实验旨在研究Sur 1-Trpm 4对活化T细胞核因子的调节作用 在活化的星形胶质细胞中，NFAT介导的细胞因子/趋化因子的转录。这些研究将有助于 对星形胶质细胞中Sur 1-Trpm 4和S1 P信号传导之间的分子相互作用的新机制见解， 从而揭示MS的新治疗靶点。

BACKGROUND: The innate immune response in the CNS plays a critical role in disease progression in multiple sclerosis (MS)/ experimental autoimmune encephalomyelitis (EAE). Astrocytes, microglia, and pro-inflammatory monocytes actively promote neurodegeneration, demyelination and scar formation, but therapeutic strategies targeting glial cells remain elusive. Reactive astrocytes in EAE (here called the “EAE-astrocytes” for short) in the spinal cord secrete numerous pro-inflammatory factors, with inhibition of each factor individually being beneficial in EAE. Whereas targeting pro-inflammatory factors individually may be beneficial, a more promising strategy may be to target the pro-inflammatory secretome of EAE-astrocytes more broadly. This idea emerges from published work with the oral MS drug and sphingosine-1-phosphate (S1P) receptor modulator, fingolimod, in which it was shown that astrocyte activation in the presence of fingolimod, yields a secretome that is less pro- inflammatory. Our lab recently showed pathological involvement of Sur1-Trpm4 in MS/ EAE. During the chronic phase of EAE, Sur1-Trpm4 channels are robustly upregulated predominantly by reactive astrocytes (EAE- astrocytes), with global suppression of SUR1 reducing secretion of several pro-inflammatory cytokines/chemokines, and reducing clinical symptoms, demyelination and axonal loss. New experiments have expanded on this work, revealing what may be a critical interaction between Sur1-Trpm4 and S1P signaling in EAE-astrocytes. Preliminary data show that: (i) in EAE, Sur1-Trpm4, S1P1 and S1P3 receptors are highly co- localized in EAE-astrocytes; (ii) in EAE, astrocyte-specific deletion of Abcc8, which encodes Sur1, yields a secretome similar to that with fingolimod; (iii) in cultured astrocytes, Sur1-Trpm4 channels are downstream of, and are required for, S1P signaling; (iv) in cultured astrocytes, Sur1-Trpm4 channels regulate the transcription of numerous pro-inflammatory factors. Our central hypothesis is that Sur1-Trpm4 channels are required for the S1P-mediated signaling that perpetuates the pro-inflammatory astrocytic secretome and contributes to disease maintenance/progression in MS/ EAE. DESCRIPTION: This project has three mechanistic aims, all focused on Sur1-Trpm4 in EAE. In Aim 1, we will study myelin oligodendrocyte glycoprotein (MOG) 35–55 EAE in male and female mice with genetic deletion of Sur1 in astrocytes, initiated at different times after EAE induction, to establish the role of astrocytic Sur1 at different stages of EAE. In Aim 2, molecular and patch clamp experiments will be used to demonstrate the role of Sur1-Trpm4 in S1P signaling that regulates the secretome of activated astrocytes. In Aim 3, molecular experiments will be used to characterize the role of Sur1-Trpm4 in regulating nuclear factor of activated T-cell (NFAT)-mediated transcription of cytokines/chemokines in activated astrocytes. These studies will contribute new mechanistic insights into the molecular interactions between Sur1-Trpm4 and S1P signaling in astrocytes, and thereby reveal new treatment targets for MS.