p27(Kip1) and Retinal Attachment

描述（由申请人提供）：拟议的研究的总体目标是确定正常视网膜附着的机制，目的是开发更好的方法来恢复神经视网膜与其支持视网膜色素上皮（RPE）之间的相互作用，从而改善视网膜脱离导致的视觉功能丧失。关于rpe -神经视网膜界面的结构成分是如何产生的，就特定的信号事件及其在发育过程中的激活顺序而言，我们知之甚少。拟议的实验专门关注上皮的作用，并将利用缺乏细胞周期调节蛋白p27（Kip1）的小鼠品系。除了视网膜增生缺陷和视网膜发育不良的有限病灶外，这些动物还表现出大面积的视网膜脱离，明显表现为光感受器外节和RPE微绒毛之间的指间性缺失。Aim 1将验证p27（Kip1）在rpe -神经视网膜界面的建立和维持中发挥作用的假设，该假设独立于其参与上皮细胞周期调节。在这些实验中，两株敲入小鼠将使用既定的光学和电子显微镜方法进行检查。第一株（p27CK-）在细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）的p27（Kip1）结合位点上含有点突变，导致一种蛋白质不能作为细胞周期抑制剂，但仍保留其细胞骨架调节特性。在第二种菌株（p27S10A）中，调节p27（Kip1）蛋白向细胞质输出的主要磷酸化位点被改变，产生一种蛋白，该蛋白可以在细胞核内发挥其作为细胞增殖负调节因子的功能，但不能影响肌动蛋白细胞骨架。p27（S10A）突变导致视网膜脱离的可能性，以及p27CK-挽救p27（Kip1）基因消融导致的脱离的可能性将被检验。最后，通过将p27（Kip1）缺失的小鼠与缺乏细胞周期调节基因cyclin D1的小鼠杂交，研究视网膜脱离的可能性是由于RPE细胞或核密度本身的增加。目的2将检验我们在p27（Kip1）缺失小鼠中观察到的视网膜脱离是否至少部分是由于上皮内特异性蛋白质功能的丧失。为了验证这一点，将实施条件基因靶向的Cre-lox系统，以选择性地消融RPE细胞中的p27（Kip1）基因。

DESCRIPTION (provided by applicant): The proposed research has as its overall goal to define mechanisms that underlie normal retinal attachment, with the aim that better methods of restoring the interaction between the neural retina and its supporting retinal pigment epithelium (RPE) may be developed, and thus loss of visual function resulting from retinal detachment ameliorated. Little is known about how the structural components of the RPE-neural retina interface are generated, in terms of specific signaling events and their sequence of activation in development. The proposed experiments focus specifically on the role of the epithelium, and will make use of a strain of mice that is deficient in the cell cycle regulatory protein p27(Kip1). Aside from retinal proliferative defects and limited foci of retinal dysplasia, these animals exhibit broad areas of retinal detachment, evident as a loss of interdigitation between photoreceptor outer segments and RPE microvilli. Aim 1 will test the hypothesis that p27(Kip1) plays a role in establishment and maintenance of the RPE-neural retina interface that is independent of its involvement in epithelial cell cycle regulation. For these experiments, two strains of knock-in mice will be examined using established light and electron microscopic methods. The first strain (p27CK-) contains point mutations in the p27(Kip1) binding sites for cyclins and cyclin-dependent kinase (CDK), resulting in a protein that is unable to act as a cell cycle inhibitor, but nevertheless retains its cytoskeleton regulatory properties. In the second strain (p27S10A), the major phosphorylation site regulating p27(Kip1) protein export to the cytoplasm has been altered, yielding a protein that can carry out its function as a negative regulator of cell proliferation within the nucleus but is unable to influence the actin cytoskeleton. The possibility that the p27(S10A) mutation causes the induction of retinal detachment, and that p27CK- rescues detachment resulting from p27(Kip1) gene ablation will be examined. Finally, the possibility that retinal detachment is due to increased RPE cellular or nuclear density per se will be examined by crossing p27(Kip1)-null mice with mice lacking the positive cell cycle regulatory gene cyclin D1. Aim 2 will examine whether the retinal detachment we observe in p27(Kip1)-null mice results, at least in part, from a loss of protein function specifically within the epithelium. To test this, the Cre-lox system for conditional gene targeting will be implemented to ablate the p27(Kip1) gene selectively in RPE cells.