淀粉样蛋白β通过PU.1调控线粒体功能蛋白的启动子甲基化导致视网膜色素上皮细胞损伤机制研究

Amyloid β (Aβ), an important component of drusen, is one of the key pathogenic factors in retinal pigment epithelial cells (RPE) damage. Previous studies have found that RPE secretes senescence-associated inflammatory factors after Aβ deposition, but the regulatory mechanism is unclear. Proteomic analysis initially suggests that mitochondrial dysfunction is the key pathological mechanism of Aβ-mediated senescence-associated inflammatory activation in RPE. Based on this, this study intends, firstly, to use GSEA analysis to clarify that TCA rate-limiting enzyme and NOX are the key mediators to RPE mitochondrial dysfunction. Secondly, based on the result of biosynthesis analysis that the NOX and TCA rate-limiting enzyme promoter regions are enriched in the PU.1 binding site, the PU.1 related methylation site will be analyzed by DMR to further confirm the mechanism of the methylation regulated by PU.1. Finally, the expression of TCA rate-limiting enzyme and NOX will be examined by inhibiting PU.1. Through the above studies, the key role of TCA rate-limiting enzyme and NOX-mediated RPE mitochondrial dysfunction will be clarified and the mechanism by which PU.1 regulates the methylation levels of TCA rate-limiting enzymes and NOX promoters will be elucidated. This project will provide a new theoretical basis for improving the mechanism of Aβ-mediated inflammatory activation in RPE and finding effective therapeutic targets.

玻璃膜疣中重要成分淀粉样蛋白β（Aβ）是视网膜色素上皮细胞（RPE）损伤的关键致病因素之一。前期课题组研究发现Aβ沉积后RPE出现衰老相关炎症因子分泌表型，但调控机制不清。蛋白质组学分析初步提示线粒体功能障碍是Aβ介导RPE衰老相关炎症活化的关键病理机制，以此为基础，本课题拟首先采用GSEA分析，明确TCA限速酶及NOX是RPE线粒体功能障碍的关键“介导蛋白”；其次，在生信分析发现NOX及TCA限速酶启动子区均富集PU.1结合位点的基础上，通过DMR分析PU.1相关甲基化位点，明确PU.1调控上述区域甲基化水平的机制；最后通过抑制PU.1，观察TCA限速酶及NOX表达；通过上述研究，将明确TCA限速酶及NOX介导RPE线粒体功能障碍的关键作用，阐明PU.1调控TCA限速酶及NOX启动子甲基化水平的机制。课题的完成对于完善Aβ介导RPE衰老相关炎症活化机制，寻找有效治疗靶点提供新的理论依据。

项目背景：视网膜色素上皮（retinal pigment epithelium, RPE）衰老是年龄相关性黄斑变性（age related macular degeneration，AMD）致盲的基本病理机制。淀粉样蛋白（Aβ）沉积是AMD早期引起RPE细胞衰老的关键病理改变，RPE细胞衰老后产生衰老相关分泌表型（senescence-associated secretory phenotype）形成眼底炎性微环境，进一步引起RPE细胞级联损伤，加重感光细胞细胞损伤并促进脉络膜新生血管形成，最终导致视力不可逆下降。因此在AMD发生的早期，阻断RPE衰老的上游通路避免后续的级联损伤的发生是目前较理想的干预策略。.重要结果：PU.1转录调控线粒体NOX4-p22phox复合体及细胞质NOX2抑制mtROS及ROS表达改善RPE细胞衰老相关分泌表型.关键数据：本项目首先通过TMT蛋白组学明确Aβ沉积引起RPE细胞衰老的始动因素是线粒体损伤。其次通过基因集富集分析（Gene Set Enrichment Analysis，GSEA）及Reactome数据库筛选，初步发现定位于线粒体的NADPH氧化酶家族介导的线粒体ROS（mtROS）过量产生是RPE细胞衰老的关键分子机制。为了探索线粒体NADPH氧化酶的调控机制，我们构建了Aβ眼内沉积的动物模型，发现NAPDH氧化酶家族中定位于线粒体的NOX4-p22phox复合体是产生mtROS的最主要的蛋白酶复合体，两者蛋白表达均受到核转录因子PU.1的调控；最后为了明确可能的干预新靶点，我们分别敲除细胞及动物模型中的PU.1及NOX4，发现PU.1敲除后不仅可以抑制疾病早期NOX4介导的mtROS过量产生，其次也可以抑制后期NOX2介导的细胞质中ROS的过量产生，多靶点抑制改善RPE衰老相关分泌表型，较NOX4更为合适作为的上游干预靶点。.科学意义：该研究丰富了RPE细胞衰老的机制理论，为RPE细胞保护提供了新靶点。项目研究成果在Theranostics等期刊发表SCI论文2篇