从RGMa/BMP/YAP通路探讨多发性硬化血脑屏障通透性

The disruption of the blood brain barrier（BBB）permeability is one of the earliest and indispensable pathological changes in multiple sclerosis（MS）. However, clinical therapeutics targeted BBB permeability to treat MS are not readily available, as the pathophysiological mechanism underlying the BBB permeability has yet to be elucidated. Recent studies have revealed that repulsive guidance molecule a (RGMa) plays a significant role in the onset of MS, notably through mediating axonal inhibition and modulation of inflammatory responses and immune activity. Our experimentation with animal models following Evans Blue staining revealed that inhibition of RGMa prevented leakage of the BBB. This data indicates that RGMa may regulate the integrity of the neurovascular unit within the BBB and moderate its permeability. We hypothesize RGMa’s effect on the permeability of the BBB, is a result of its action with endothelial BMP/BMP receptors, mediating downstream activity of the Yes-associated protein (YAP). This project aims to explore correlations in expression of RGMa with loss of BBB integrity in MS patients, to examine its role in modulating BBB permeability and to delineate the mechanism of RGMa interaction with the BMP/YAP pathway. Several experimental methods will be performed, including application of RGMa knockouts, recombinant adenovirus transfection, molecular imaging, and chromatin immunoprecipitation, which shall be performed in vitro, in vivo and in clinical experiments. Cumulatively, this study will aid in elucidating a novel biological function of RGMa in regulating BBB integrity, and of MS pathology, whilst additionally exploring a novel therapeutic strategy to treating MS.

血脑屏障（BBB）通透性改变是多发性硬化（MS）最早期的病理变化之一，也是MS发病的必要条件，但因其机制未明而使BBB靶向治疗临床应用受限。排斥性导向分子RGMa是参与MS轴突抑制、炎性浸润和免疫激活的重要分子，但具体作用机制尚未阐明。我们在动物模型研究发现抑制RGMa表达显著减少脑组织伊文思蓝染色渗出，说明RGMa参与BBB通透性损伤。结合文献我们推测RGMa很可能通过内皮细胞BMP/BMP受体通路抑制YAP激活而调控BBB通透性。本研究拟利用基因敲除、腺病毒转染、分子影像学、ChIP等研究手段，从细胞、动物、临床三个水平详细探讨MS患者RGMa水平与BBB通透性的相关性，全面揭示RGMa对EAE小鼠BBB通透性的功能影响，深入阐明RGMa通过BMP/BMP受体/YAP信号通路调控BBB通透性的分子机制。研究有助深化RGMa的生物学功能和BBB损伤的病理机制，为MS治疗提供思路和依据。

血脑屏障（Blood-Brain Barrier，BBB）通透性改变是多发性硬化（Multiple Sclerosis, MS）最早期的病理变化之一，也是MS发病的必要条件，但导致BBB通透性改变的具体机制不明。Repulsive guidance molecule A（RGMa）是参与MS轴突抑制、炎性浸润和免疫激活的重要分子。本研究旨在探讨RGMa通过调控BBB通透性改变参与MS病理过程及分子机制。首先收集急性期MS患者的血样，通过ELISA检测血清RGMa表达水平，结果示MS患者RGMa表达水平较对照组明显升高。进而在EAE C57/BL6小鼠模型中，通过蛋白印迹实验、免疫荧光等技术，发现脑和脊髓组织RGMa表达随病情加重逐渐升高，与BBB破坏程度呈一致趋势，同时伴随通路蛋白骨形态发生蛋白（BMP）、骨形态发生蛋白受体（BMPR）表达水平升高，Yes 相关蛋白（YAP）及BBB通透性相关蛋白ZO-1表达降低。结果提示RGMa可能通过BMP/BMPR/YAP通路调控BBB通透性，导致BBB破坏。最后在人脑微血管内皮细胞（HBMECs）模型中进一步证实，抑制RGMa导致BMP、BMPR表达水平降低，而ZO-1表达水平升高，内皮细胞通透性降低。说明RGMa通过BMP/BMPR通路参与BBB的破坏。. 结论：血清RGMa水平可以作为早期判断MS神经功能损伤严重程度的生物学标记物。RGMa通过BMP/BMPR/YAP通路调控血脑屏障损伤，结果进一步揭示了RGMa的生物学功能及其通过血脑屏障通透性参与MS发病的分子机制，为以BBB通透性为靶点的临床治疗方案提供一些思路和依据。