COBRE: PI 3-KINASE & ITS DOWNSTREAM TARGET BCL-XL IN RPE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我的实验室感兴趣的是磷脂酰肌醇(PI)循环中产生的脂质第二信使在光转导、视网膜变性和保护视网膜细胞免受凋亡中的作用。PI-3，4，5-P3是由PI-3激酶(PI3K)产生的一种信使，在细胞保护、细胞增殖、葡萄糖稳态、膜转运和细胞骨架重排等方面发挥重要作用。在神经细胞中，PI3K的激活被证明可以保护细胞免受应激诱导的细胞凋亡。然而，PI3K在视觉系统中的作用还不是很清楚。视网膜色素上皮(RPE)细胞是有丝分裂后的细胞，提供“血-视网膜屏障”，控制营养物质和废物在视网膜和血液之间的流动。这些细胞的死亡导致视杆细胞和视锥细胞的死亡，RPE功能受损可能是老年性黄斑变性的一个促成因素。自从Bl-x，， 在细胞毒应激条件下，PI3K下游靶基因在RPE细胞中高表达，可能在RPE细胞存活中起重要作用。我们推测PI3K和BOI-XL是RPE的主要生存因素。为了验证这一假设，我们将使用一种遗传方法来破坏RPE中的PI3K和Bclx(Bclxl)，并比较突变小鼠和野生型小鼠的功能、生化和结构表型。由于传统的PI3K(P85odp110O_)和Bclx基因敲除小鼠是新生或胚胎致死的，因此将使用Cre/Iox系统以RPE特有的方式产生PI3K和Bclx零突变的小鼠模型。因此，我建议1)建立和鉴定由RPE65启动子驱动的表达RPE特异性Cre的转基因小鼠；2)建立RPE特异性PI3K和/或Bcl-x缺失小鼠并验证PI3K和bcl-xl参与RPE细胞存活的假设；3)使用本研究产生的RPE特异性PI3K或Bcl-x缺失小鼠和ABCR缺失(RPE变性)小鼠，验证RPE细胞受损可能加剧含有PI3K和/或Bcl-x零突变的光感受器细胞的死亡的假说。在Cobre计划的背景下，该项目的一个主要目标是建立小鼠模型，该模型可用于未来的研究，以阐明先天性视网膜变性的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. My laboratory is interested in the roles of lipid second messengers generated in the phosphatidylinositol (PI) cycle in phototransduction, retinal degeneration, and protection of retinal cells from apoptosis. One messenger, PI-3,4,5-P3, generated by the PI-3 kinase (PI3K), plays an important role in cell protection, cell proliferation, glucose homeostasis, membrane trafficking and cytoskeletal rearrangement. In neuronal cells, activation of PI3K has been shown to protect the cells from stress-induced apoptosis. However, the functions of PI3K in visual systems are not well understood. Retinal pigment epithelial (RPE) cells are post-mitotic cells that provide the "blood-retinal barrier", and control the flow of nutrients and waste products between the retina and the blood. Death of these cells leads to death of rod and cone photoreceptor cells, and compromise in RPE function may be a contributing factor in age-related macular degeneration. Since Bcl-x,, a downstream target of PI3K is highly expressed in RPE cells under cytotoxic stress, it is likely that PI3K plays an important role in RPE cell survival. We hypothesize that PI3K and BOI-xL are major survivor factors in RPE. To test this hypothesis, we will use a genetic approach to disrupt PI3K and Bcl-x (BCl-xL) in RPE, and compare the functional, biochemical, and structural phenotypes of the mutant mice with those of wild type mice. Since the conventional PI3K(p85odp110o_) and Bcl-x knockout mice are neonatal or embryonic lethal, mouse models of PI3K and Bcl-x null mutation will be generated in a RPE specific fashion, using the Cre/Iox system. Therefore, I propose 1) to generate and characterize transgenic mice expressing RPE-specific Cre driven by RPE65 promoter; 2) to generate RPE-specific PI3K and/or Bcl-x null mice and test the hypothesis that PI3K and BCl-xL are involved in RPE cell survival, 3) to test the hypothesis that compromised RPE cells may exacerbate the death of photoreceptor cells with PI3K and/or Bcl-x null mutations using the RPE-specific PI3K, or Bcl-x null mice generated in this study and the ABCR null (RPE degeneration) mice. A major goal of this project in the context of the COBRE program is to establish mouse models that can be used in future studies to elucidate the mechanisms of inherent retinal degeneration.