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

溶酶体机械敏感离子通道在疼痛中的作用

• 批准号: 10580905
• 负责人: JILL C FEHRENBACHER
• 金额: $ 43.59万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-28 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10580905
• 关键词: Afferent Neurons; American; Behavior; Cations; Cell membrane; Cells; Chronic; Data; Exocytosis; Goals; Hydro-Lyases; Immunohistochemistry; In Vitro; Ion Channel; Ions; Lead; Lysosomes; Measures; Mechanics; Mediating; Membrane; Methods; Modeling; Molecular; Mus; Neurons; Nociception; Nociceptors; Opiate Addiction; Pain; Patients; Peripheral; Peripheral Nerves; Peripheral nerve injury; Pharmacology; Positioning Attribute; Quality of life; Reporting; Research; Role; Sensory; Skin; Spinal Cord; Spinal Ganglia; Stains; Swelling; Testing; Tissues; Up-Regulation; Virus; Western Blotting; Work; cell type; chronic pain; chronic pain management; chronic painful condition; immunocytochemistry; in vivo; knock-down; multiple data types; non-opioid analgesic; novel; opioid overdose; opioid overuse; overexpression; oxaliplatin; pain behavior; pain model; pain reduction; painful neuropathy; particle; patch clamp; sciatic nerve; selective expression; side effect; trafficking; treatment strategy

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.

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，了解 慢性疼痛的潜在机制和治疗方法仍然有限。本研究的长期目标 目的是确定治疗神经性疼痛的主要离子通道机制和药理学靶点。 拟议研究的目标是探索溶酶体离子通道 (Tmem63A) 在介导 DRG 神经元的功能性电流以及与周围神经损伤相关的神经性疼痛。中央 该提议的假设是 Tmem63A 在非溶酶体中介导机械敏感离子电流 肽能伤害性 DRG 神经元，并对与外周相关的神经性疼痛有积极贡献 神经损伤。当前项目提出了两个具体目标：1）测试 Tmem63A 介导 溶酶体中的机械敏感离子电流，并促进非肽能的溶酶体外周转运 伤害性 DRG 神经元； 2) 测试 Tmem63A 的功能性上调是否有助于神经病变 与周围神经损伤相关的疼痛。在目标 1 中，小鼠 DRG 神经元将被解离。游离的 DRG 神经元将受到病毒介导的 Tmem63A 敲低或过表达。细胞类型- Tmem63A 的特异性、亚细胞特异性表达和机械敏感电流将在 使用单细胞 PCR、免疫细胞化学和全溶酶体膜片钳对四组 DRG 神经元进行了研究。 此外，还将通过免疫细胞化学研究溶酶体在这些 DRG 神经元组中的定位。在 目标2，将使用小鼠体内重复奥沙利铂治疗模型（或慢性奥沙利铂治疗）， 并将评估感官行为（机械、热和冷）。病毒介导的体内敲低 将在周围神经元中进行 Tmem63A。 Tmem63A的表达将在多个 使用 qPCR、蛋白质印迹和免疫组织化学方法检测感觉组织。溶酶体定位为 通过免疫组织化学检查。 Tmem63A 的功能电流将以游离状态测量 DRG 神经元。奥沙利铂对Tmem63A表达和功能的影响，溶酶体定位 研究过。此外，Tmem63A 敲低对奥沙利铂诱导的神经性疼痛行为的影响 将被研究。据我们所知，溶酶体特异性离子通道或溶酶体机械力的作用 敏感离子通道在疼痛中的作用尚未见报道。因此，当前的研究有潜力 建立溶酶体离子通道作为治疗多种疼痛病症的潜在靶标。