Alpha-1 catenin regulation of the mammalian blood-nerve barrier

Alpha-1 连环蛋白对哺乳动物血神经屏障的调节

• 批准号: 9978422
• 负责人: Eroboghene Ekamereno Ubogu
• 金额: $ 40.84万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978422
• 关键词: Address; Adult; Affect; Axon; Back; Binding; Biological; Biological Assay; Biophysics; Blinded; Blood-Nerve Barrier; CREB1 gene; Cell membrane; Chronic; Complex; Crush Injury; Cytoskeleton; Data; Dedications; Dextrans; Disease; EMS1 gene; Electrical Resistance; Electron Microscopy; Endothelial Cells; Endothelium; Ensure; Female; Functional disorder; Gene Silencing; Genes; Genetic Transcription; Goals; Health; Homeostasis; Horseradish Peroxidase; Hour; Human; In Vitro; Individual; Injury; Knockout Mice; Knowledge; Lead; MAPK Signaling Pathway Pathway; Mammals; Measures; Mediating; Membrane; Modeling; Molecular; Molecular Weight; Mus; Nerve Pain; Nociception; Outcome Measure; Pain Measurement; Paresthesia; Patients; Peripheral Nerves; Peripheral Nervous System Diseases; Peripheral nerve injury; Permeability; Pharmaceutical Preparations; Population; Process; Protein Tyrosine Kinase; Proteins; Publishing; Recombinants; Recovery; Reflex action; Regulation; Reproducibility; Resources; Role; Serum; Signal Transduction; Small Interfering RNA; Sodium; Structure; Symptoms; Tamoxifen; Tight Junctions; Time; Transgenic Mice; Transgenic Organisms; Translating; Western Blotting; Wild Type Mouse; Withdrawal; Work; alpha catenin; axon regeneration; base; chronic neuropathic pain; chronic pain; claudin 4; conditional knockout; design; experience; experimental group; experimental study; glial cell-line derived neurotrophic factor; healing; in vitro Assay; in vivo; injury recovery; kinase inhibitor; male; nerve injury; neurobehavioral; neurobehavioral test; neurotransmission; novel therapeutics; peripheral nerve regeneration; restoration; sciatic nerve; sex; solute; src-Family Kinases; transcription factor

Our long-term objective is to discover how the blood-nerve barrier (BNB) recovers after nerve injury. We have recently observed a relationship between loss of α1-catenin (CTNNA1), BNB dysfunction and chronic neuropathic pain in peripheral neuropathy. This project is designed to address a fundamental question: What is the role of CTNNA1 in BNB recovery and reduced nociception following peripheral nerve injury? Based on our published and exciting Preliminary Data, including murine sciatic nerve BNB permeability assays and reflexive neurobehavioral nociception tests in conditional GDNF transgenic wildtype and knockout mice following sciatic nerve crush injury, we propose the following hypotheses: GDNF (via RET-Tyrosine kinase/ Ras-MAPK signaling pathways) phosphorylates transcription factor CREB1 with resultant increase in CTNNA1, cortactin (CTTN) and tight junction protein claudin-4 (CLDN4) gene transcription. GDNF-RET- Tyrosine kinase also activates SRC kinase which phosphorylates membrane-bound CTTN that binds to CTNNA1. This induces CTNNA1 binding to CLDN4, resulting in tight junction formation. CTNNA1 is the critical adapter molecule that connects the tight junctional complex to the cytoskeleton during BNB recovery. This process restores BNB function and reduces nociception after peripheral nerve injury. In order to address this hypothesis, we will determine CTTN, phosphorylated CTTN, CTNNA1 and CLDN4 expression dynamics by western blot of confluent primary human endoneurial endothelial cell membrane extracts following injury with and without exogenous GDNF in vitro. We will subsequently inhibit specific gene transcription using commercially available siRNAs targeting CTTN, SRC kinase and CTNNA1 and determine effect on the GDNF-mediated BNB recovery via continuous transendothelial electrical resistance and solute permeability assays using low and high molecular weight fluoresceinated molecules. To validate the in vitro work and demonstrate a direct relationship between BNB recovery and reduced nociception after injury, we will perform sciatic nerve crush injury in tamoxifen-inducible microvascular-specific conditional CTTN and CTNNA1 transgenic knockout mice and block SRC kinase with Bosutinib in wildtype mice, with appropriate controls. We will quantify % permeable endoneurial microvessels to horseradish peroxidase by electron microscopy and perform reflexive neurobehavioral tests of nociception in a blinded manner, with appropriate controls and technical replicates to demonstrate scientific rigor, and data reproducibility and inference validity. Structural changes at the BNB are associated with chronic peripheral neuropathies, and we have observed a direct relationship with chronic neuropathic pain. Understanding mechanisms of BNB recovery after nerve injury could translate to novel therapies for chronic neuropathic pain achieved by restoring endoneurial homeostasis. BNB recovery could also provide a more conductive microenvironment for axonal regeneration.

我们的长期目标是发现神经损伤后血神经屏障(BNB)如何恢复。我们有 最近观察到α1-连环蛋白缺失、血脑屏障功能障碍与慢性阻塞性肺疾病 周围神经病的神经病理性疼痛。该项目旨在解决一个基本问题： CTNNA1在周围神经损伤后血脑屏障恢复和伤害性感受减少中的作用？ 基于我们发表的令人兴奋的初步数据，包括小鼠坐骨神经BNB通透性分析 条件性GDNF转基因野生型和敲除小鼠的反射性神经行为伤害性测试 在坐骨神经挤压伤后，我们提出了以下假设：GDNF(通过RET-酪氨酸激酶/ RAS-MAPK信号通路)使转录因子CREB1磷酸化，从而增加 CTNNA1、皮质肌动蛋白(CTTN)和紧密连接蛋白CLDN4(CLDN4)基因转录。GDNF-RET- 酪氨酸激酶还激活SRC激酶，SRC激酶使膜结合的CTTN磷酸化，结合到 CTNNA1.这诱导CTNNA1与CLDN4结合，导致紧密连接的形成。CTNNA1是 在BNB过程中连接紧密连接复合体和细胞骨架的关键适配分子 恢复。这一过程恢复了周围神经损伤后的BNB功能，减少了伤害性感受。 为了解决这一假设，我们将确定CTTN、磷酸化的CTTN、CTNNA1和CLDN4 原代人神经内皮细胞膜融合蛋白表达动态的蛋白质印迹分析 外源性和非外源性GDNF损伤后提取物的体外实验。我们随后将抑制特定的基因 使用商业上可获得的针对CTTN、SRC激酶和CTNNA1的siRNAs转录并确定 持续内皮电阻和溶质对GDNF介导的BNB恢复的影响 使用低分子和高分子荧光分子的通透性分析。为了验证体外实验 努力证明BNB恢复和受伤后伤害性感受减少之间的直接关系，我们将 在他莫昔芬诱导的微血管特异性条件性CTTN和CTNNA1中实施坐骨神经挤压损伤 转基因基因敲除小鼠，用博苏替尼阻断野生型小鼠的SRC激酶，并进行适当的对照。我们 将通过电子显微镜和电子显微镜对辣根过氧化物酶的渗透性微血管进行定量 以盲目的方式执行伤害性感觉的反射性神经行为测试，并设置适当的对照和 技术复制以证明科学的严谨性、数据的可重复性和推理的有效性。 BNB的结构变化与慢性周围神经病有关，我们观察到 与慢性神经病理性疼痛有直接关系。了解神经损伤后血脑屏障恢复的机制 损伤可转化为通过恢复神经内膜实现的慢性神经病理性疼痛的新疗法 动态平衡。BNB的恢复也为轴突再生提供了更好的传导微环境。