Role of Interleukin-1 Receptor Clustering in Neuropathic Pain Signaling

IL-1 受体簇在神经病理性疼痛信号转导中的作用

• 批准号: 10043097
• 负责人: Parijat Sengupta
• 金额: $ 15.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043097
• 关键词: Affect; Animal Model; Antibodies; Attenuated; Binding; Biochemical; Biological Models; Brain; Calcium; Calcium Signaling; Cell Culture Techniques; Cell Line; Cell Surface Receptors; Cell model; Cells; Cerebrospinal Fluid; Complex; Data; DsRed; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Gene Silencing; Genes; Goals; Green Fluorescent Proteins; Hyperalgesia; Hypersensitivity; IL1R1 gene; Immune; Inflammation; Inflammatory; Injury; Interleukin-1; Interleukin-1 Receptors; Lead; Link; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Microscopic; Molecular; Mus; Nervous System Trauma; Nervous system structure; Neuraxis; Neurons; Neuropathy; PTGS2 gene; Pain; Pathway interactions; Patients; Perception; Peripheral; Peripheral nerve injury; Photons; Play; Population; Process; Protein Isoforms; Proteins; Rattus; Regulation; Research; Rest; Role; Signal Pathway; Signal Transduction; Signaling Protein; Spectrum Analysis; Stimulus; Structure; Substance P; Symptoms; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Tumor Necrosis Factor Receptor; allodynia; anakinra; caveolin 1; chronic neuropathic pain; cyclooxygenase 2; cytokine; design; experimental study; improved; inflammatory neuropathic pain; interleukin-1 receptor type I; intravenous injection; monomer; nerve injury; new therapeutic target; novel; pain model; pain perception; pain processing; pain sensation; pain sensitivity; pain signal; painful neuropathy; protein protein interaction; receptor; recruit; red fluorescent protein; response; single molecule; spontaneous pain; standard care; stimulus sensitivity; stoichiometry; transmission process

Summary How (and if) pain signaling is different in the central nervous system (CNS) compared to the rest of the body remains an open question. Nervous system inflammation and injury often lead to neuropathic pain (NP) where sensitivity of stimuli is greatly exaggerated or harmless stimuli are perceived as painful. Many patients suffering from these conditions do not respond to the standard treatment paradigms that are currently in use. This is primarily due to a lack of comprehensive understanding of the molecular mechanisms underlying transmission and processing of pain that limits treatment options. The pro-inflammatory cytokine, Interleukin-1 (IL-1) is known to facilitate pain sensitivity in various inflammatory neuropathic conditions in animal models of pain. IL-1 is capable of inducing multiple intracellular signaling cascades that ultimately leads to the expression of multiple inflammatory genes and sensation of pain. IL-1 signaling initiates when IL-1 binds to its cell surface receptor (IL-1R1) and its accessory protein (AcP). Recently a new accessory protein to IL-1 receptor (AcPb) has been identified that is only expressed in central nervous system (CNS). AcPb attenuates some, but not all, events in the IL-1 signaling pathway. This proposal aims to test a novel CNS-specific mechanism for regulation of IL-1 induced neuropathic pain signaling that is, hypothetically, regulated via clustering stoichiometry of IL-1 receptor and its two accessory proteins. We propose to experimentally verify existence of this regulatory mechanism. IL1R1, AcP and AcPb tagged with fluorescent proteins will be cloned and expressed in a model cell line and primary neurons for these experiments. We will employ advanced microscopic live cell measurements technologies, such as, Förster resonance energy transfer (FRET) to measure average distance (and molecular interaction) between two proteins, fluorescence correlation spectroscopy (FCS) to measure protein mobility (and association), and photon counting histogram (PCH) analysis to measure molecular brightness (and aggregation number) of the protein clusters. Additionally, calcium signaling and both mRNA and protein levels of cyclooxygenase-2 (COX- 2) and substance-P (Sub-P) will be measured to study pain-related functional aspects of IL-1 signaling. The long term goal of this research is to reconstruct multiple protein-protein interaction steps involved in IL-1R signaling pathway associated with imflammatory NP in live cells. Inflammatory neuropathic pain is a debelitating condition affecting many lives. A detailed molecular mechanism describing IL-1 in pain processing and perception, and how it is different in the CNS, may eventually lead to discovery of new drug targets for designing therapeutics for treatment options that are nonexistent today.

总结 与身体其他部位相比，中枢神经系统（CNS）中的疼痛信号如何（以及是否）不同 仍然是个悬而未决的问题。神经系统炎症和损伤通常导致神经性疼痛（NP），其中 刺激的敏感性被大大夸大，或者无害的刺激被认为是痛苦的。许多患者遭受 从这些条件不响应于目前使用的标准治疗范例。这是 主要是由于缺乏对传播的分子机制的全面了解 和疼痛的处理限制了治疗方案。 已知促炎性细胞因子白细胞介素-1 β（IL-1）促进各种疼痛中的疼痛敏感性。 炎症性神经病性病症的动物模型中的疼痛。IL-1能够诱导多种细胞内 最终导致多种炎症基因表达和炎症反应的信号级联反应。 痛苦当IL-1与其细胞表面受体（IL-1 R1）及其辅助蛋白结合时，IL-1信号传导开始 （AcP）。最近，已经鉴定了一种新的IL-1受体辅助蛋白（AcPb），其仅表达于 中枢神经系统（CNS）。AcPb减弱IL-1信号通路中的一些但不是全部事件。这 一项提案旨在测试一种新的CNS特异性机制，用于调节IL-1诱导的神经病理性疼痛信号传导 即，假设通过IL-1受体及其两种辅助蛋白的聚类化学计量来调节。 我们建议通过实验验证这种调节机制的存在。IL 1 R1、AcP和AcPb标记 与荧光蛋白将克隆和表达在一个模型细胞系和原代神经元为这些 实验我们将采用先进的显微活细胞测量技术，如Förster 共振能量转移（FRET）测量两个分子之间的平均距离（和分子相互作用） 蛋白质、荧光相关光谱（FCS）来测量蛋白质迁移率（和结合），以及 光子计数直方图（PCH）分析，以测量分子的亮度（和聚集数） 蛋白质簇此外，钙信号传导和环氧化酶-2（考克斯-2）的mRNA和蛋白质水平均与细胞凋亡有关。 2)和物质-P（Sub-P）将被测量以研究IL-1信号传导的疼痛相关功能方面。 本研究的长期目标是重建IL-1 R参与的多个蛋白质-蛋白质相互作用步骤 与活细胞中炎性NP相关的信号通路。炎症性神经性疼痛是一种 影响许多生命的衰弱状况。IL-1参与疼痛过程的分子机制 和感知，以及它在中枢神经系统中是如何不同的，最终可能导致发现新的药物靶点， 设计治疗方案来治疗目前还不存在的疾病。