Combined Proteomic and Functional Analysis of Sensory Neuron Plasticity

描述（由申请人提供）： 本阶段I（CEBRA R21）应用的目标是开发一种实验范式，用于对定义的感觉神经元子集进行综合蛋白质组学和功能分析。将使用流式细胞术鉴定和分选感觉神经元的亚组。分选的神经元将被处理用于使用蛋白质组学方法的大规模表达分析或用于功能分析，如Ca2+成像。在一个长期的第二阶段项目中，这种范式将被应用于慢性疼痛条件下的感觉神经元可塑性的研究。目前的项目将使用细胞因子肿瘤坏死因子α（TNF α）及其在神经性疼痛中的作用作为模型系统，用于开发这种创新的实验范式及其验证，用于感觉神经元可塑性的研究。以下具体目的将阐明TNF α通过TNF受体介导的对感觉神经元蛋白表达和功能的影响而有助于神经损伤诱导的可塑性的假设：1）使用流式细胞术检查在正常和神经病条件下急性分离的感觉神经元中TNF受体的表达。这些实验将建立通过流式细胞术分析和分选感觉神经元的方案。2)在表达TNF受体的细胞中，鉴定神经损伤后其表达受TNF α调节的蛋白质。这些实验将采用尖端的蛋白质组学方法来分析流式细胞仪分选的感觉神经元。3)确定表达TNF受体的感觉神经元的TNF α治疗是否调节前伤害感受和镇痛信号传导。这些实验将为流式细胞仪分选的感觉神经元的功能分析开发程序。总之，本申请中的研究将扩展我们对TNFa在神经性疼痛中的作用的理解，同时开发一种分析感觉神经元可塑性的创新方法。从长远来看，与感觉神经元的特定亚群中的慢性疼痛的特定条件相关的可塑性的表征将导致更有选择性的治疗干预措施的发展，以避免常用的镇痛药，如阿片类药物的不良副作用。

DESCRIPTION (provided by applicant): The goal of the present Stage I (CEBRA R21) application is to develop an experimental paradigm for integrated proteomic and functional analysis of defined subsets of sensory neurons. Subsets of sensory neurons will be identified and sorted using flow cytometry. The sorted neurons will be processed for largescale expression analysis using proteomic approaches or for functional analysis such as Ca2+ imaging. In a long-term Stage II project, this paradigm will be applied to studies of sensory neuron plasticity under conditions of chronic pain. The current project will use the cytokine Tumor Necrosis Factor alpha (TNFa) and its role in neuropathic pain as a model system for the development of this innovative experimental paradigm and its validation for the study of sensory neuron plasticity. The following specific aims will address the hypothesis that TNFa contributes to nerve injury-induced plasticity through TNF receptor-mediated effects on sensory neuron protein expression and function: 1) Examine the expression of TNF receptors in acutely dissociated sensory neurons under normal and neuropathic conditions using flow cytometry. These experiments will establish protocols for analysis and sorting of sensory neurons by flow cytometry. 2) In cells expressing TNF receptors, identify proteins whose expression is modulated by TNFa after nerve injury. These experiments will adapt cutting-edge proteomic approaches to analysis of flow cytometry-sorted sensory neurons. 3) Determine if TNFa treatment of sensory neurons expressing TNF receptors modulates pronociceptive and analgesic signaling. These experiments will develop procedures for functional analysis in flow cytometrysorted sensory neurons. In summary, the studies in this application will extend our understanding of the role of TNFa in neuropathic pain, while developing an innovative approach to the analysis of sensory neuron plasticity. In the long term, characterization of the plasticity associated with specific conditions of chronic pain in specific subsets of sensory neurons will lead to development of more selective therapeutic interventions that avoid the unwanted side effects of commonly used analgesics such as opiates.