Fn14, non-canonical NF-kappaB and downstream signaling in neuropathic pain

背景：持续的阿片类药物流行部分是由于合法使用阿片类药物， 神经性疼痛使用非成瘾性药物控制疼痛的替代途径是一个主要目标， 神经性疼痛研究。周围神经损伤后，细胞因子和趋化因子表达上调 在中枢，它们在机制上有助于神经性疼痛的发病机制。反应性星形胶质细胞 背角表现出慢性激活促炎分泌（CAPS）表型，其特征在于 许多因子的分泌，包括白细胞介素-6（IL-6）、趋化因子C-C基序配体2（CCL 2）、趋化因子 C-X-C基序配体1（CXCL 1）和神经生长因子（NGF）。PNI后星形胶质细胞CAPS表型 通过神经元趋化因子受体促进炎症和神经元过度活化，导致 神经性疼痛已知典型的NF-κB信号传导起着至关重要的作用，但令人惊讶的是，尽管其 在涉及大多数器官的许多炎症性疾病中的重要性，包括大脑，非 先前在PNI中还没有鉴定出通过p52：RelB异二聚体的典型NF-κB信号传导。新工作 我们实验室的研究证实了一个新的发现，即肿瘤坏死引起的非经典NF-κB信号转导， 凋亡因子样弱诱导因子（TWEAK）/成纤维细胞生长因子诱导因子14（Fn 14）轴是突出的 PNI后，特别是在背角星形胶质细胞，并可能负责转录表达的Sur 1- Trpm 4在这些细胞中。此外，我们的初步数据表明，胶质细胞Sur 1-Trpm 4在 调节IL-6、CCL 2和CXCL 1的表达。我们的中心假设是，在背角星形胶质细胞中， PNI后，TWEAK诱导的非经典NF-κB信号传导是Sur 1-Trpm 4表达的上游调节因子， 而Sur 1-Trpm 4又调节下游效应物IL-6、CCL 2和CXCL 1的表达， 其促进慢性神经炎症、神经元过度活化和神经性疼痛。 说明：该项目有三个机械目标。在目标1中，遗传（Tweak-/-，Fn 14-/-，p100-/-， Abcc 8-/-，整体或GFAP-特异性或GFAP-和-位点特异性）和药理学（抗TWEAK抗体， 将进行格列本脲）实验以确定非经典NF-κB和Sur 1在 坐骨神经中表达GFAP的胶质细胞。与背角在两种神经病理性疼痛模型中：坐骨神经痛。套入坐骨神经 n.暴露于HIV/gp 120蛋白。在目标2中，我们将扩展染色质的体内和体外数据 免疫沉淀法确定非经典NF-κB在功能性Sur 1-Trpm 4表达中的作用 渠道在目标3中，我们将证实并扩展体内和体外数据，以确定Sur 1- Trpm 4调节IL-6、CCL 2和CXCL 1的Ca 2 +/钙调神经磷酸酶依赖性转录。这个项目是第一个 研究背角和坐骨神经非经典NF-κB的表达。神经胶质在神经病理性疼痛，并将有助于确定新的 使用非成瘾性药物改善神经性疼痛的药物靶点。

BACKGROUND: The ongoing opioid epidemic is driven in part by legitimate use of opioids prescribed for neuropathic pain. Targeting alternative pathways for pain control using non-addicting drugs is a major goal of neuropathic pain research. After peripheral nerve injury (PNI), cytokines and chemokines are upregulated centrally, where they contribute mechanistically to the pathogenesis of neuropathic pain. Reactive astrocytes in the dorsal horn exhibit a chronically activated, pro-inflammatory secretory (CAPS) phenotype characterized by secretion of numerous factors, including interleukin-6 (IL-6), chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 1 (CXCL1) and nerve growth factor (NGF). The post-PNI astrocytic CAPS phenotype contributes to both inflammation and neuronal hyperactivation via neuronal chemokine receptors, leading to neuropathic pain. Canonical NF-κB signaling is known to play a crucial role, but surprisingly, despite its documented importance in numerous inflammatory conditions involving most organs including the brain, non- canonical NF-κB signaling via the p52:RelB heterodimer has not been identified previously in PNI. New work from our laboratory demonstrates the novel finding that non-canonical NF-κB signaling by the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) / fibroblast growth factor-inducible 14 (Fn14) axis is prominent after PNI, especially in dorsal horn astrocytes, and may be responsible for transcriptional expression of Sur1- Trpm4 in these cells. Moreover, our preliminary data suggest that glial Sur1-Trpm4 plays a crucial role in regulating the expression of IL-6, CCL2 and CXCL1. Our central hypothesis is that, in dorsal horn astrocytes post-PNI, TWEAK-induced non-canonical NF-κB signaling is an upstream regulator of Sur1-Trpm4 expression, and that Sur1-Trpm4, in turn, regulates the expression of the downstream effectors, IL-6, CCL2 and CXCL1, which promote chronic neuroinflammation, neuronal hyperactivation and neuropathic pain. DESCRIPTION: This project has three mechanistic aims. In Aim 1, genetic (Tweak–/–, Fn14–/–, p100–/–, Abcc8–/–, either global or GFAP-specific or GFAP-&-site-specific) and pharmacological (anti-TWEAK antibody, glibenclamide) experiments will be carried out to determine the role of non-canonical NF-κB and Sur1 in GFAP-expressing glia in sciatic n. vs. dorsal horn in two neuropathic pain models: sciatic n. cuffing and sciatic n. exposure to HIV/gp120 protein. In Aim 2, we will expand upon in vivo and in vitro data from chromatin immunoprecipitation to establish the role of non-canonical NF-κB in the expression of functional Sur1-Trpm4 channels. In Aim 3, we will corroborate and expand upon in vivo and in vitro data to establish the role of Sur1- Trpm4 in regulating Ca2+/calcineurin-dependent transcription of IL-6, CCL2 and CXCL1. This project is the first to study non-canonical NF-κB in dorsal horn vs. sciatic n. glia in neuropathic pain, and will help identify novel druggable targets to ameliorate neuropathic pain using non-addictive drugs.