Membrane Biosynthesis in Normal and Dystrophic Retina

描述(申请人提供)：为什么光感受器在表达视紫红质等突变蛋白时死亡，以及为什么视锥细胞在不表达突变基因的情况下死亡仍不清楚。青蛙的视网膜中有大量的视杆和视锥，并在受精后14天内发育出功能正常的视网膜。我们建立了转基因莱氏X.laevis的视网膜变性模型。成年转基因青蛙将转基因基因传递给后代。我们已经培育了100多个转基因品系，并在6年内发表了56只转基因动物的研究。我们的目的1)评估突起蛋白-1突变在视杆细胞中的定位、功能和影响。Prominin-1是一种五聚体膜蛋白，仅定位于视杆基底盘，它的突变会导致人类视网膜变性。我们已经分离了非洲爪哇突起蛋白-1的cDNA，并构建了定位正确的GFP融合构建体。我们还制备了针对重要蛋白的GST融合多肽的抗体，并将在下拉试验中使用这些抗体和融合多肽来分离重要蛋白的结合伙伴。将对该蛋白质进行评估，以确定其不寻常定位的原因是哪些结构域，并将对其进行突变，以通过与人类突变的比较来评估其影响。2)通过表达突变型Rab8T22N基因建立了一种新的视网膜变性模型，该突变基因在中心杆发育早期诱导细胞凋亡，阻断高尔基体后膜与纤毛基底部的对接，并在14d后形成几乎全锥的视网膜，我们将追踪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，因为在普萘洛尔治疗的视网膜中，已经显示出其定位的扰动，类似于Rab8T22N的影响。我们将使用moesin perm和肌动蛋白结合结构域片段通过在转基因青蛙中表达这些片段来阻断其功能，以确定在体内与内源性moesin竞争的影响。4)在视杆细胞早期死亡的视网膜变性模型中，我们将研究抗凋亡药物对视锥细胞死亡的潜在改善作用。由于视锥细胞在没有视杆的情况下甚至可以在变态后存活，但最终会死亡，因此我们将寻找可能为人类视网膜退化提供潜在治疗方法的视锥细胞保存剂。

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.