Membrane Biosynthesis in Normal and Dystrophic Retina

描述（由申请人提供）：为什么光感受器在表达诸如视紫红质的突变蛋白时死亡，以及为什么视锥细胞在不表达突变基因的情况下会死亡尚不清楚。在受精后的14天内视网膜发育成熟，视网膜上有丰富的视杆细胞和视锥细胞。我们建立了转基因黄斑田鼠视网膜变性模型。成年转基因蛙将转基因基因传给后代。6年来，我们培育了100多个转基因品系，发表了56只转基因动物的研究成果。我们的目的是：1)研究突出蛋白1在视杆细胞上的定位、功能及其突变的影响。pronin -1是一种只存在于视杆细胞基底盘的五aspan膜蛋白，其突变可导致人类视网膜变性。我们分离出了Xenopus pronin -1的cDNA，并构建了适合定位的GFP融合构建体。我们还制备了针对pronin的gst融合肽的抗体，并将利用抗体和融合肽进行下拉实验，分离出pronin的结合伙伴。将对蛋白质进行评估，以确定哪些结构域导致其不寻常的定位，并对其进行突变，以评估与人类突变的影响。2)通过表达rab8T22N突变体，建立了一种新的视网膜变性模型，该突变体在中央杆发育早期诱导细胞凋亡，阻断后高尔基膜与睫状体基部的对接，导致14 d后形成几乎全锥体的视网膜。我们将继续研究rab8的结合伙伴，以确定其在囊泡对接中的作用。3)我们正在启动光感受器中moesin的新研究，因为在心得安治疗的视网膜中，其定位的扰动已被证明与rab8T22N的作用相似。我们将使用moesin PERM和actin结合域片段，通过在转基因青蛙中表达片段来阻断其功能，以确定与内源性moesin竞争在体内的影响。4)我们将在我们的视网膜变性模型中研究抗凋亡药物可能改善视杆细胞早期死亡的视锥细胞死亡。由于在没有视杆细胞的情况下，视锥细胞甚至在蜕变后仍能存活，但最终会死亡，我们将寻找保存视锥细胞的药物，这可能为人类视网膜变性提供潜在的治疗方法。

DESCRIPTION (provided by applicant): Why photoreceptors die when expressing mutant proteins such as rhodopsin, and why cones should die in such instances when they do not express the mutated gene is still unclear. X. laevis frogs have abundant rods and cones in their retinas and develop a functioning retina within 14 days post-fertilization. We developed models of retinal degeneration in transgenic X. laevis. Adult transgenic frogs transmit the transgenes to offspring. We have generated more than 100 transgenic lines and published studies of 56 transgenic animals in 6 years. We aim 1) to evaluate the localization, function and impact of mutations of prominin-1 on rods. Prominin-1 is a pentaspan membrane protein localized exclusively to basal disks in rods and mutations of it cause retinal degeneration in humans. We have isolated the cDNA of Xenopus prominin-1 and made GFP fusion constructs that localize appropriately. We have also prepared antibodies to GST-fusion peptides of prominin and will use the antibodies and the fusion peptides in pull-down assays to isolate prominin's binding partners. The protein will be evaluated to determine which domains account for its unusual localization and it will be mutated to evaluate the impact by comparison with human mutations. 2) A new model of retinal degeneration has been created by expression of mutant rab8T22N which induces apoptosis early in the development of central rods, blocks docking of post-Golgi membranes to the base of the cilium and leads to formation of a nearly all-cone retina after 14 d. We will pursue the binding partners of rab8 in order to determine its role in vesicle docking. 3) We are initiating new studies of moesin in photoreceptors because perturbation of its localization has been shown in propranolol treated retinas similar to the effects of rab8T22N. We will use moesin PERM and actin binding domain fragments to block its function by expression of the fragments in transgenic frogs to determine the impact, in vivo, of the competition with endogenous moesin. 4) We will investigate the potential amelioration of cone death by antiapoptotic agents in our models of retinal degeneration in which the rods die early. Since the cones survive even beyond metamorphosis in the absence of rods, but eventually die, we will seek cone-preserving agents that may provide potential therapies in human retinal degenerations.