O-GlcNAc糖基化修饰RBPJ调控线粒体功能蛋白介导的代谢重编程在感光细胞死亡中的作用及机制研究

视网膜脱离(RD)后感光细胞线粒体功能障碍是其死亡级联信号启动的关键枢纽。申请人前期发现RD后感光细胞缺血缺氧、发生能量代谢异常，表现为乳酸/丙酮酸比值升高，代谢过程主要富集于线粒体TCA和糖酵解途径，初步提示感光细胞发生代谢重编程极有可能是线粒体功能障碍始动因素。据此本课题拟首先研究代谢重编程表型，明确其引起线粒体功能障碍；其次探究线粒体PDHC及TCA限速酶是代谢重编程的关键介导蛋白；进一步采用酶促标记法证明O-GlcNAc糖基化修饰RBPJ是调控代谢重编程的上游核心机制；最后通过干预RBPJ特异位点糖基化，观察PDHC及TCA限速酶表达并验证线粒体功能障碍缓解、细胞存活，从而明确糖基化调控RBPJ转录抑制PDHC及TCA限速酶表达，是引起线粒体功能障碍并导致感光细胞死亡的关键机制。研究结果将完善感光细胞代谢理论，为建立通过调控代谢重编程恢复代谢稳态新策略，保护感光细胞提供理论依据。

Mitochondrial dysfunction of photoreceptor cells after retinal detachment (RD) is the key hub of initiation of death cascade signal. The applicant found that the energy metabolism of photoreceptor cells was abnormal after RD, which showed that the ratio of lactate to pyruvate increased, and the metabolic process was enriched in mitochondrial TCA and glycolytic pathway, which was related to the abnormal expression of pyruvate dehydrogenase complex PDHC and TCA rate-limiting enzyme. It was speculated that metabolic reprogramming of photoreceptor cells might be the initial factor of mitochondrial dysfunction. Therefore, this topic plans to study the phenotype of metabolic reprogramming injury first, and clarify the mitochondrial dysfunction caused by it; Secondly, it is explored that mitochondrial PDHC and TCA rate-limiting enzymes are the key mediating proteins of metabolic reprogramming. Furthermore, enzymatic labeling method was used to prove that O-GlcNAc glycosylation modified RBPJ is the upstream core mechanism of regulating metabolic reprogramming. Finally, by interfering with the glycosylation of RBPJ specific sites, the expression levels of PDHC and TCA rate-limiting enzymes and phenotype of mitochondrial dysfunction was verified, and it was clear that the regulation of RBPJ transcription by glycosylation inhibited the expression of PDHC and TCA rate-limiting enzymes, which was the key mechanism of mitochondrial dysfunction and photoreceptor death. The research results will improve the metabolism theory of photoreceptor cells, and provide theoretical basis for establishing a new strategy to restore metabolic homeostasis by regulating metabolic reprogramming and protecting photoreceptor cells.