CERKL突变引起自噬缺陷导致视网膜色素变性的机制研究

CERKL（Ceramide kinase-like）is an important gene, which mutated is associated with retinitis pigmentosa. However, the physiological roles of CERKL in retina and the molecular mechanism by which CERKL mutations causing RP remains unknown. Supported by the last NSFC grant, we generated the first zebrafish model with CERKL knockout by using TALEN technique. The phagocytosis of outer segments (OSs) by the retinal pigment epithelium (RPE) is impaired in the CERKL-/- zebrafish. We also found that the level of autophagy in the retinae of CERKL-/- zebrafish was significantly decreased, and express of SIRT1 protein was down-regulated. These results suggested that CERKL may play an important role in autophagy and phagocytosis, and loss of this function of CERKL in zebrafish may lead to apoptosis of rod and cone. This proposal is aimed to investigate the specific pathways that CERKL regulated autophagy and phagocytosis via SIRT1. We will using CERKL-/- zebrafish and ARPE-19 cell-line with CERKL deficiency, to reveal the molecular mechanism by which CERKL regulates SIRT1. This study will analyze the signal pathway that CERKL mutations regulate phagocytosis through autophagy and eventually lead to photoreceptor apoptosis. Our study may not only uncover the molecular mechanism of CERKL mutation causing retinal degeneration, but also understand the novel roles of CERKL in retina physiology, and help to provide evidences for developing effective strategy for RP prevention and treatment in the future.

CERKL是一个重要的视网膜色素变性致病基因，但该基因在视网膜中的生化功能及其突变导致RP发病的分子机制尚不清楚。在上一个NSFC项目的支持下，我们成功构建了CERKL敲除的斑马鱼模型，首次发现CERKL敲除破坏RPE对感光细胞外节的吞噬；检测CERKL-/-斑马鱼还发现视网膜细胞的自噬能力下降，自噬相关蛋白SIRT1的蛋白水平显著降低，提示CERKL突变可能通过影响视网膜细胞自噬进而影响吞噬而导致RP。本项目将在前期工作的基础上，应用CERKL-/-斑马鱼模型结合细胞生物学实验，分析CERKL突变通过SIRT1调控自噬和吞噬并最终导致感光细胞凋亡的信号通路，揭示CERKL调控SIRT1的分子机制，从而阐明CERKL突变导致视网膜变性的分子机制。本项目如进展顺利，可能发现CERKL在视网膜中新的生理功能，揭示RP发病新的致病机理，为视网膜色素变性的预防和治疗打下扎实的基础。

CERKL 基因是一个重要的视网膜变性致病基因，其突变可引起视网膜色素变性和视锥视杆营养不良，但其突变引起视网膜变性的病理机制还不清楚。在国家自然科学基金项目的支持下，我们应用遗传学、细胞生物学、生物化学、生物信息学方法，结合转录组和代谢组学，以及模式生物为手段，对CERKL在视网膜中的生理功能进行了深入系统研究，取得重要研究成果：1，世界上首次成功构建了CERKL基因突变引起视网膜色素变性的动物模型,确定CERKL基因突变导致斑马鱼视网膜色素变性。2，应用CERKL-/-斑马鱼模型，发现CERKL通过与SIRT1相互作用并调控SIRT1的磷酸化，进而在视网膜细胞自噬中发挥作用；3，揭示CERKL/SIRT1/靶蛋白乙酰化/自噬的信号途径，阐明CERKL突变通过引起视网膜细胞自噬缺陷导致视网膜色素变性的致病机理；4，首次发现CERKL参与视网膜细胞脂类代谢，揭示CERKL突变导致视网膜变性新的致病机制。这些重要研究成果的取得，不仅阐明了CERKL新的生理功能和视网膜变性新的分子病理机制，也为发展针对该基因突变引起的视网膜色素变性的预防和治疗方法打下了扎实的基础。