Vascular biology of the human blood-nerve barrier

描述(申请人提供)：我们的长期目标是研究人类血-神经屏障的血管生物学。具体地说，我们感兴趣的是研究GDNF及其信号通路在损伤后血神经屏障功能恢复中的作用。该项目旨在解决一个基本问题：周围神经损伤和变性后血-神经屏障的恢复，GDNF是否充分和必要？基于我们令人兴奋的新的初步数据，我们提出以下假设：GDNF能够充分恢复损伤后的人神经内皮细胞紧密连接功能，主要是通过细胞骨架重组，导致更连续的细胞间接触和更少的细胞间隙。这在体外没有显著增强黏附和紧密连接相关蛋白的表达。GDNF可通过RET-酪氨酸激酶/RAS-MAP激酶信号通路在体外充分恢复损伤后人神经内皮细胞紧密连接屏障功能。利用一种新的条件性GDNF基因敲除小鼠模型，我们假设GDNF对于坐骨神经挤压伤后血-神经屏障功能的早期和持续恢复是必不可少的。GDNF能以剂量依赖的方式在体外恢复血清撤除后的人血神经屏障阻力。我们的初步数据在体外特别涉及RET-酪氨酸激酶/RAS-MAP激酶信号通路。在无血清血-神经屏障的GDNF治疗后，F-肌动蛋白微丝和更多连续的细胞间黏附和紧密连接发生了膜定位。与未经处理的血-神经屏障相比，我们在体外没有观察到连接素、VE钙粘蛋白、ZO-1、occludin和claudin-5的表达以及claudin-5酪氨酸磷酸化的变化。为了解决这些假设，我们将使用成熟的内皮屏障实验来证实GDNF在恢复血-神经屏障功能方面的充分作用。同样，我们将在上述实验中使用特定的细胞通透性抑制剂，在体外确定RET-酪氨酸激酶/Ras-MAP激酶信号通路对GDNF介导的血清撤除效应负责。最后，我们将使用一种新的三苯氧胺诱导的条件性GDNF基因敲除小鼠品系，在体内评估GDNF在周围神经损伤后血-神经屏障功能恢复方面的作用。人们对人类血-神经屏障的血管生物学知之甚少。血管内皮细胞之间的表型和功能差异为在健康和疾病中具体研究人类血神经屏障的生物学提供了理论基础。血-神经屏障功能的改变与许多周围神经病和神经病理性疼痛有关。使用GDNF恢复血-神经屏障功能可以加速从这些普遍存在的、致残的医学问题中恢复过来。 公共卫生相关性：我们工作的目的是发现一种名为胶质细胞衍生神经营养因子(GDNF)的分子如何在损伤后恢复周围神经的血-神经屏障功能。周围神经疾病在美国影响着2000多万人，在全球范围内影响着数百万人。通过研究GDNF如何帮助恢复血-神经屏障功能，我们将获得重要的新知识，从而能够更好地治疗周围神经病和神经病理性疼痛。这可以显著改善患者及其家人的生活，并减轻国内和国际医疗保健系统的财政负担。

DESCRIPTION (provided by applicant): Our long-term objective is to study the vascular biology of the human blood-nerve barrier. Specifically, we are interested in studying the role of GDNF and its signaling pathways in restoring blood-nerve barrier function following injury. This project is designed to address a fundamental question: Is GDNF sufficient and essential to the recovery of the blood-nerve barrier following peripheral nerve injury and degeneration? Based on our exciting new preliminary data, we propose the following hypothesis: GDNF sufficiently restores human endoneurial endothelial cell tight junction function following injury primarily by cytoskeletal reorganization that results in more continuous intercellular contacts and fewer intercellular gaps. This occurs without significantly enhancing adherens and tight junction associated protein expression in vitro. GDNF sufficiently restores human endoneurial endothelial cell tight junction barrier function following injury via RET-Tyrosine kinase/ Ras-MAP kinase signaling pathways in vitro. Using a novel conditional GDNF knockout mouse model, we hypothesize that GDNF is essential for the early and persistent restoration of blood-nerve barrier function following sciatic nerve crush injury. GDNF sufficiently restored human blood-nerve barrier resistance following serum withdrawal in vitro in a dose-dependent manner. Our preliminary data specifically implicates the RET-Tyrosine kinase/ Ras-MAP kinase signaling pathways in vitro. Membrane localization of F-actin microfilaments and more continuous intercellular adherens and tight junctions occurred following GDNF treatment of the serum-deprived blood-nerve barrier. We did not observe significant changes in ¿-catenin, VE cadherin, ZO-1, occludin and claudin-5 expression or alterations in claudin-5 tyrosine phosphorylation compared to the untreated injured blood-nerve barrier in vitro. In order to address these hypotheses, we will confirm the sufficient effect of GDNF in restoring blood-nerve barrier function using well-established endothelial barrier experimental assays. Similarly, we will determine that the RET-Tyrosine kinase/ Ras-MAP kinase signaling pathway is responsible for the GDNF-mediated effect following serum withdrawal in vitro, using specific cell-permeable inhibitors in the above experimental assays. Finally, we will evaluate effect of GDNF in restoring blood-nerve barrier function following peripheral nerve injury in vivo using a novel tamoxifen-inducible conditional GDNF knockout mouse strain. Very little is known about the vascular biology of the human blood-nerve barrier. Phenotypic and functional differences between vascular endothelial cells provide the rationale to specifically study the biology of the human blood-nerve barrier in health and disease. Alternations in blood-nerve barrier function have been implicated in many peripheral neuropathies and neuropathic pain. Restoration of blood-nerve barrier function by GDNF could accelerate recovery from these prevalent, disabling medical problems. PUBLIC HEALTH RELEVANCE: The purpose of our work is to discover how a molecule called glial cell-derived neurotrophic factor (GDNF) may restore blood-nerve barrier function in peripheral nerves following injury. Peripheral neuropathies affect over 20 million people in the United States and millions more worldwide. By studying how GDNF helps restore blood-nerve barrier function, we will obtain important new knowledge that can lead to better treatments for peripheral neuropathies and neuropathic pain. This could significantly improve the lives of patients and their families, and reduce the financial burden on the health care system nationally and internationally.