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突变蛋白如视紫红质时死亡，以及为什么视锥细胞在不表达突变基因的情况下会死亡仍然不清楚。X.非洲蛙的视网膜中有丰富的视杆细胞和视锥细胞，并在受精后14天内发育出功能性视网膜。我们开发了转基因X染色体视网膜变性模型。光滑。成年转基因青蛙将转基因传递给后代。我们已经产生了100多个转基因品系，并在6年内发表了56个转基因动物的研究。我们的目的是1）评估视杆细胞上的Bcl-in-1突变的定位、功能和影响。Prominin-1是一种仅定位于视杆细胞基底盘的五跨膜蛋白，其突变可导致人类视网膜变性。我们已经分离出非洲爪蟾的cDNA-1和GFP融合构建体，适当的本地化。我们还制备了抗GST融合肽的抗体，并将在下拉测定中使用抗体和融合肽来分离抗GST融合肽的结合伴侣。将对蛋白质进行评价，以确定哪些结构域导致其不寻常的定位，并对其进行突变，以通过与人类突变进行比较来评价影响。2)通过表达突变体rab 8 T22 N已经创建了一种新的视网膜变性模型，该突变体rab 8 T22 N在中央杆发育的早期诱导细胞凋亡，阻断高尔基体后膜与纤毛基部的对接，并导致14 d后形成几乎全锥视网膜。我们将追踪rab 8的结合伙伴，以确定其在囊泡对接中的作用。3)我们正在启动新的研究膜突蛋白在光感受器，因为其本地化的扰动已被证明在普萘洛尔处理的视网膜类似的作用rab 8 T22 N。我们将使用膜突蛋白PERM和肌动蛋白结合结构域片段，通过在转基因青蛙中表达片段来阻断其功能，以确定在体内与内源性膜突蛋白竞争的影响。4)我们将在我们的视网膜变性模型中研究抗凋亡药物对视锥细胞死亡的潜在改善作用，在视网膜变性模型中，视杆细胞死亡较早。由于视锥细胞即使在没有视杆细胞的情况下也能存活，但最终会死亡，因此我们将寻求可能为人类视网膜变性提供潜在治疗的视锥细胞保护剂。

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.