小眼畸形相关转录因子在视网膜色素上皮细胞中的功能及其在视网膜退行性变性中的机制研究

The retina plays critical roles in light reception and image formation and hence is crucial for vision. Microphthalmia-associated transcription factor (MITF) plays important functions in a variety of cell types including retinal pigment epithelial cells (RPE). Mutations in Mitf such as microphthalmia-vga9 (Mitfmi-vga9) not only cause deafness and loss of pigmentation in the skin, but also results in microphthalmia, abnormal development of RPE and retinal degeneration. Although MITF expression in RPE cells and plays important roles during RPE development, in structural integrity and in function, we still do not know whether MITF is involved in the regulation of cellular events including visual cycle and neurotrophin production in RPE cells and whether Mitf regulating differentiated genes in RPE are related to retinal degeneration. In this proposal we will investigate how the mutation in MITF using microphthalmia-vga9 (Mitfmi-vga9) as a model results in mice with retinal degeneration, one of the leading causes of blindness in the world. Recently, our preliminary data suggest that MITF does not affect RPE formation, but affects RPE cell differefiation. We further show that MITF is ivoloved in regulating expressions of the visual cycle- and neurotrophin-related genes in RPE. In order to further understand the functional roles of MITF in RPE cells and dissect of the mechanisms of Mitf defect-associated retinal degeneration, we will perform various experiments including in situ hybridaization, ChIP-Seq, ChIP-PCR and luciferase reporter gene assay to investigate how MITF regulates expressions of the visual cycle- and neurotrophin-related genes in vivo and its related moleculau and cellular mechanisms. We also attempt to utilize co-cultures of RPE-Netina and RPE implantation to examine whether RPE plays any functional roles in the Mitf defect-associated retinal degeneration. At last, we will try to use subretinal injections of candidate molecules and proteins of the visual cycle and neurotrophins into the eyes of the Mitf knockout mices for analyzing the functional roles of the candidate factors in the retinal degeneration and repair in vivo. We expect that this proposal will dissect how the transcription factor MITF-downstream target genes-network of downstream effectors-RPE cell fuction-photorecptor cells are involved in retinal degeneration. In addition, we except that this project will further understand what functional roles of MITF play in protecting photoreceptor cells in the neural retina. We think that the outcomes from this project will provide insights to deep understanding of the molecular and cellular mechanisms of the photoreceptor damage and repair in the visual neural system and knowledge and experimental support for the function restrictions of the neural damage and the effective treatments of the retinal degeneration diseases.

小眼畸形相关转录因子(MITF)在视网膜色素上皮细胞(RPE)发育和视网膜变性中起着关键作用，但其调控机理未明。根据国内外的研究现状，我们假设其可通过：MITF→下游靶基因→下游效应分子网络→RPE细胞功能→感光细胞，这一途径调控RPE细胞的功能并导致视网膜病变。本项目拟在前期工作基础上，利用MITF功能缺失小鼠和RPE特异性Mitf过表达转基因小鼠，并运用原位分子杂交技术、ChIP-Seq、荧光素酶报告检测对MITF调控的候选视循环和神经营养因子的分子机制进行研究；继而对RPE在MITF功能缺失小鼠视网膜病变过程中的功能作用进行解析；最后通过在体注射候选视循环与神经营养因子，探讨其在MITF功能缺失导致视网膜变性过程中的功能和机制。这一原创性研究将进一步阐明MITF在RPE细胞功能和视网膜退行性病变中的调控分子机理，为寻求治疗RPE细胞异常相关致盲疾病的方法提供前瞻性思路和转化医学依据。

小眼畸形相关转录因子(MITF)在视网膜色素上皮细胞(RPE)发育和视网膜变性中起着关键作用，但其调控机制未明。本项目对其在RPE细胞中的功能及其在视网膜变性中的机制进行了研究。我们在前期工作基础上，利用MITF功能缺失小鼠以及多种现代生命医学研究手段，通过四年的科学实验和观察，在获得了大量的实验数据的基础上，证明了本项目提出的假设：MITF→下游靶基因→下游效应分子网络→RPE细胞功能→光感受器细胞；即MITF是通过上述途径来调控RPE细胞的功能并导致视网膜病变。通过这四年项目的实施，我们发现小眼畸形相关转录因子在RPE细胞中具有调控视循环、抗氧化、细胞增殖和神经营养因子等生物学功能及其调控的分子机制。我们的研究还揭示了视循环、神经营养因子等RPE细胞功能的异常有可能是小眼畸形相关转录因子功能丧失小鼠视网膜变性的主要原因。本项目研究的相关成果分别以SCI论文7篇发表在Progress in Retinal and Eye Research、Human Molecular Genetics 、Disease Models & Mechanisms、Pigment Cell & Melanoma Research 、Exp Eye Res等国际杂志上。这一原创性研究不仅是进一步阐明了MITF在调控 RPE细胞多种重要生物学功能的分子和细胞机制，加深了Mitf功能丧失小鼠视网膜变性机制的理解，进一步较为完整地阐明MITF在RPE细胞生物学与致病过程中的调控机理起到了重要的作用，并为寻求治疗RPE细胞异常相关致盲疾病的方法提供了重要的思路和转化医学依据。