The role of a lysosomal mechano-sensitive ion channel in pain

项目摘要/摘要： 慢性疼痛降低了数百万美国人的生活质量和工作能力。目前，对 慢性疼痛的潜在机制和治疗方法仍然有限。这项研究的长期目标是 确定治疗神经病理性疼痛的主要离子通道机制和药理靶点。 这项研究的目的是探索溶酶体离子通道(Tem63A)在介导 背根节神经元的功能电流，以及与周围神经损伤相关的神经病理性疼痛。中环 这一设想的假设是，Tem63A介导了一种机械敏感的离子电流，这种电流发生在非小鼠的溶酶体内。 肽能伤害性背根神经节神经元与周围神经病理性疼痛 神经损伤。在本项目中提出了两个具体的目标：1)测试Tem63A中介 溶酶体中的机械敏感离子电流，并促进溶酶体外周运输非肽能 伤害性背根神经节神经元；以及2)测试Tem63A的功能上调在神经病变中的作用 与周围神经损伤相关的疼痛。在目标1中，小鼠背根神经节神经元将被分离。已解除关联 DRG神经元将受到病毒介导的基因敲除或Tem63A过表达的影响。细胞类型- Tem63A的特异性、亚细胞特异性表达和机械敏感电流将在 应用单细胞聚合酶链式反应、免疫细胞化学和全溶酶体膜片钳技术对四组背根神经节神经元进行检测。 此外，还将通过免疫细胞化学研究溶酶体在这些DRG神经元群中的定位。在……里面 目的2，采用小鼠体内奥沙利铂重复治疗模型(或慢性奥沙利铂治疗)， 并将评估感觉行为(机械、热和冷)。一种病毒介导的体内基因敲除 Tem63A将在外周神经元中进行。将在多种情况下检测到Tem63A的表达 感觉组织采用定量聚合酶链式反应、免疫印迹和免疫组织化学方法。溶酶体定位将是 免疫组织化学检测。Tem63A的功能电流将在解离状态下进行测量 背根神经节神经元。奥沙利铂对Tem63A表达和功能的影响 学习。此外，Tem63A基因敲除对奥沙利铂诱导的神经病理性疼痛行为的影响 将会被研究。据我们所知，溶酶体特异性离子通道或溶酶体机制的作用。 疼痛中的敏感离子通道尚未见报道。因此，目前的研究有可能 建立溶酶体离子通道作为治疗多种疼痛状况的潜在靶点。

Project Summary/Abstract: Chronic pain reduces quality of life and work capability in millions of Americans. Currently, understanding of the underlying mechanisms and treatments for chronic pain are still limited. The long-term goal of this research is to identify major ion channel mechanisms and pharmacological targets for the treatment of neuropathic pain. The goal of the proposed research is to explore the role of a lysosomal ion channel (Tmem63A) in mediating functional currents in DRG neurons, and neuropathic pain associated with peripheral nerve injury. The central hypothesis of this proposal is that Tmem63A mediates a mechano-sensitive ion current in lysosomes of non- peptidergic nociceptive DRG neurons and positively contributes to neuropathic pain associated with peripheral nerve injury. Two specific aims are proposed in the current project: 1) Test that Tmem63A mediates a mechano-sensitive ion current in lysosomes, and facilitates lysosomal peripheral trafficking in non-peptidergic nociceptive DRG neurons; and 2) Test that functional up-regulation of Tmem63A contributes to neuropathic pain associated with peripheral nerve injury. In Aim 1, mouse DRG neurons will be dissociated. Dissociated DRG neurons will be subjected to virus-mediated knockdown or overexpression of Tmem63A. The cell type- specific, and subcellular-specific expression and mechano-sensitive currents of Tmem63A will be studied in four groups of DRG neurons using single-cell PCR, immunocytochemistry, and whole-lysosome patch clamp. Additional, lysosome positioning in these DRG neuron groups will also be studied by immunocytochemistry. In Aim 2, an in vivo repeated oxaliplatin treatment model (or chronic oxaliplatin treatment) of mouse will be used, and sensory behaviors (mechanical, heat and cold) will be assessed. A virus-mediated in vivo knockdown of Tmem63A in peripheral neurons will be conducted. The expression of Tmem63A will be detected in multiple sensory tissues using qPCR, Western blot, and immunohistochemistry methods. Lysosomal positioning will be examined by immunohistochemistry. The functional currents of Tmem63A will be measured in dissociated DRG neurons. The effects of oxaliplatin on expression and function of Tmem63A, lysosomal positioning will be studied. Moreover, the effects of Tmem63A knockdown on the oxaliplatin-induced neuropathic pain behaviors will be studied. To our knowledge, the roles of lysosomal-specific ion channels, or lysosomal mechano- sensitive ion channels in pain have not been reported. Therefore, the current study has the potential to establish lysosomal ion channels as potential targets for the treatment of multiple pain conditions.