Cellular and molecular mechanisms of retinoic acid-mediated blood-retinal barrier regulation

项目总结 血-视网膜屏障(BRB)调节分子从血液到视网膜的运动， 保护视网膜神经组织免受潜在有害分子的伤害，维护视网膜 动态平衡。视网膜色素变性与糖尿病视网膜病变等眼部疾病有关 和年龄相关的黄斑水肿。BRB由两个内部屏障组成，由微血管形成 内皮细胞，以及由RPE形成的外部屏障。这些细胞之间的紧密连接是 对于屏障功能来说是必不可少的。然而，调节紧密连接完整性的分子机制 BRB以及BRB破裂对视网膜形态和视功能的影响还不完全 明白了。斑马鱼(Danio Rerio)是研究BRB发病机制的理想模型 故障和维护由于其快速的体外视觉发展和广泛的可获得性 一系列遗传工具。我们最近确定维甲酸(RA)是维生素A的代谢物， 在维护BRB方面起着至关重要的作用。斑马鱼幼体RA信号的中断和 使用泛维A酸受体抑制剂(BMS493)的成年人会导致BRB分解，中断 紧密连接蛋白在视网膜血管系统和RPE中的表达，并降低视力。 初步的RNA测序和差异表达基因的通径分析表明 MTOR信号通路在鱼的视网膜中显著上调 RA信号与未经处理的鱼相比。此外，对mTOR信号转导的抑制作用 雷帕霉素足以恢复RA抑制剂处理的幼虫BRB的完整性。因此，我们建议 RA通过与mTOR通路的串扰维持BRB，以及RA抑制剂诱导的BRB 故障会导致视网膜损伤和视觉功能障碍。我们将描述RA的角色和 通过评估mTOR激活模式在BRB维持中的mTOR信号通路 内部和外部BRB，并确定BMS493涉及的上游和下游监管机构- 诱导的BRB击穿。我们还将确定视网膜中哪些细胞对RA抑制物有贡献- 诱导BRB分解，并表征视网膜细胞的变化作为反应。 了解RA介导的BRB维持的细胞和分子机制 BRB破裂后视网膜损伤和视力丧失的进展将是至关重要的 确定预防由于BRB中断而导致的视力丧失的治疗方法。

PROJECT SUMMARY The blood-retinal barrier (BRB) mediates movement of molecules from the blood to the retina, protecting the retinal neural tissue from potentially harmful molecules and maintaining retinal homeostasis. Breakdown of the BRB is associated with ocular diseases such as diabetic retinopathy and age-related macular edema. The BRB consists of both an inner barrier, formed by microvascular endothelial cells, and an outer barrier, formed by the RPE. Tight junctions between these cells are essential to barrier function. However, the molecular mechanisms regulating tight junction integrity in the BRB and the effects of BRB breakdown on retinal morphology and visual function are not fully understood. The zebrafish (Danio Rerio) is an ideal model to investigate the mechanisms of BRB breakdown and maintenance due to its rapid ex vivo visual development and the availability of a wide array of genetic tools. We have recently determined that retinoic acid (RA), a metabolite of Vitamin A, plays a critical role in the maintenance of the BRB. Disruption of RA signaling in zebrafish larvae and adults with a pan-retinoic acid receptor inhibitor (BMS493) results in BRB breakdown, disrupted expression of tight junction proteins in the retinal vasculature and RPE, and decreased visual acuity. Preliminary RNA sequencing and pathway analysis of differentially expressed genes indicates that the mTOR signaling pathway is significantly upregulated in retinas of fish treated with an inhibitor of RA signaling compared to untreated fish. Additionally, inhibition of mTOR signaling by treatment with rapamycin is sufficient to restore BRB integrity in RA-inhibitor-treated larvae. Therefore, we propose that RA maintains the BRB via crosstalk with the mTOR pathway and that RA-inhibitor-induced BRB breakdown leads to retinal damage and visual dysfunction. We will characterize the role of RA and the mTOR signaling pathway in BRB maintenance by assessing the pattern of mTOR activation in the inner and outer BRB and identifying the upstream and downstream regulators involved in BMS493- induced BRB breakdown. We will also determine which cells in the retina contribute to RA-inhibitor- induced BRB breakdown and characterize the cellular changes in the retina in response. Understanding the cellular and molecular mechanisms involved in RA-mediated BRB maintenance and the progression of retinal damage and vision loss following BRB breakdown will be critical to identify therapeutic approaches for preventing vision loss due to BRB disruption.