Vascular biology of the human blood-nerve barrier

描述（由申请人提供）：我们的长期目标是研究人类血神经屏障的血管生物学。具体来说，我们感兴趣的是研究GDNF及其信号通路在损伤后恢复血神经屏障功能中的作用。该项目旨在解决一个基本问题：GDNF对于周围神经损伤和变性后血神经屏障的恢复是否足够和必要？基于我们激动人心的新初步数据，我们提出以下假设：GDNF充分恢复损伤后的人类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间隙。这种情况在体外没有显著增强粘附物和紧密连接相关蛋白的表达。GDNF在体外通过ret -酪氨酸激酶/ Ras-MAP激酶信号通路充分恢复人神经内皮细胞损伤后紧密连接屏障功能。使用一种新的条件GDNF敲除小鼠模型，我们假设GDNF对于坐骨神经挤压损伤后血神经屏障功能的早期和持续恢复至关重要。GDNF在体外以剂量依赖的方式充分恢复血清停药后的人血神经屏障抵抗。我们的初步数据特别暗示ret -酪氨酸激酶/ Ras-MAP激酶信号通路在体外。在GDNF治疗血清缺失的血神经屏障后，f -肌动蛋白微丝的膜定位和更连续的细胞间粘附和紧密连接发生。与未处理的体外损伤血神经屏障相比，我们未观察到¿-catenin， VE cadherin, ZO-1， occludin和claudin-5的表达或claudin-5酪氨酸磷酸化的显著变化。为了解决这些假设，我们将通过完善的内皮屏障实验分析来证实GDNF在恢复血神经屏障功能方面的充分作用。同样，我们将确定ret -酪氨酸激酶/ Ras-MAP激酶信号通路是体外血清停药后gdnf介导效应的原因，在上述实验分析中使用特定的细胞渗透性抑制剂。最后，我们将利用一种新型的他莫昔芬诱导的条件GDNF敲除小鼠品系，在体内评估GDNF在恢复周围神经损伤后血神经屏障功能方面的作用。我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专门研究人类血神经屏障在健康和疾病中的生物学提供了理论依据。血神经屏障功能的改变与许多周围神经病变和神经性疼痛有关。通过GDNF恢复血神经屏障功能可以加速从这些普遍的、致残的医疗问题中恢复。

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.