Prevention of Inherited Retinal Diseases by Therapeutic Rare Earth Nanoparticles

目前已有近200个基因和染色体位点被确认为导致某种形式的遗传性视网膜变性。不管主要的突变或原因如何，所有这些疾病都被认为共享一个重大的共同事件。这是由于活性氧物种(ROS)的慢性或急性升高而造成的“氧化应激”。抗氧化剂和抗氧化酶的过度表达已被证明可以抑制许多视网膜疾病的进展。无机氧化铈纳米颗粒(Nanoceria)是一种模拟过氧化氢酶和超氧化物歧化酶活性的抗氧化剂，通过催化清除ROS，已被证明可以预防过氧化诱导的大鼠失明。我们认为纳米氧化钙类似于治疗失明的阿司匹林，因为它们不会治愈主要缺陷，但会降低疾病的严重程度。 我们假设，由于ROS代表了许多遗传性致盲疾病常见的结节，它们也代表了一个“阿喀琉斯的脚跟”，可以用清除ROS的纳米鞭毛虫来特别靶向。作为这一策略的一部分，纳米氧化钙将在这项研究中用于抑制小鼠品系Tubby中光感受器的遗传性程序性死亡，Tubby是Usher综合征的表型模型。具体目标1将测试这样的假设，即纳米氧化钙颗粒将破坏ROS，并通过减少视网膜神经元的氧化损伤，将抑制由粗大突变引起的视网膜退化。纳米氧化钙将与萝卜硫素(特定目标2)或人类硫氧还蛋白转基因过表达(特定目标3)协同作用，为筒状视网膜中的光感受器细胞提供增强和长期的保护的假设也将得到测试。我们的初步和公布的数据支持这些疗法中的每一种在胖鼠身上单独使用时的有效性。 纳米氧化钙在胖乎乎的小鼠身上单独发挥作用的机制，以及与萝卜硫素或TRX转基因结合使用的机制，将通过以下方法进行鉴定。视网膜中的超氧阴离子自由基将用羟乙胺法测定，而H20 2将用2‘，T-二氯二氢化钠测定. 荧光素-二乙酸酯。ROS活性产物包括丙烯醛、硝基酪氨酸和8-羟基-2-脱氧鸟苷的抗体将显示ROS引起的损伤。我们将通过蛋白质印迹、基因芯片和实时定量聚合酶链式反应来分析纳米陶瓷对神经保护通路的影响。 在苏木素和伊红染色的视网膜切片上，使用明亮的视野显微镜进行定量组织学，将用于评估光感受器细胞的形态保存。视网膜功能将通过视网膜电描记术来确定。我们目前拥有大量的胖鼠和Trx胖鼠，并将能够在两年内完成特定的目标。我们的目标的成功实现应该与大多数形式的小鼠遗传性失明直接相关。这种纳米陶瓷有望在人类身上发挥作用，就像它们在其他哺乳动物身上一样，因此应该可以保护人类的视力，防止人类失明。

There are now almost 200 genes and chromosomal loci which have been identified as causing some form of inherited retinal degeneration. Irrespective of the primary mutation or cause, all of these diseases are thought to share a major common event. This is "oxidative stress" as a result of a chronic or acute rise in Reactive Oxygen Species (ROS). Antioxidants and over-expression of antioxidant enzymes have been shown to inhibit the progression of many retinal diseases. Inorganic cerium oxide nanoparticles (nanoceria) are antioxidants which mimic the activities of catalase and super oxide dismutase by catalytically scavenging ROS and have been shown to prevent peroxide induced blindness in rats. We think of the nanoceria as being analogous to an aspirin for blindness in that they won't cure the primary defect but will decrease the severity of the disease. We have hypothesized, that because ROS represent a node common to many inherited blinding diseases, they also represent an "Achilles' heel" which can be specifically targeted using ROS-scavenging nanoceria. As a component of that strategy, nanoceria will be used in this study to inhibit the inherited programmed death of photoreceptors in a mouse strain, tubby, which is a phenotypic model for Usher's Syndrome. Specific Aim 1 will test the hypothesis that the nanoceria particles will destroy ROS and, by decreasing oxidative damage in retinal neurons, will inhibit retinal degeneration induced by the tubby mutation. The hypothesis that nanoceria will act synergistically with sulphoraphane (Specific Aim 2) or with the overexpression of the human thioredoxin transgene (Specific Aim 3) to provide enhanced and prolonged protection of the photoreceptor cells in the tubby retina will also be tested. Our preliminary and published data support the effectiveness of each of these therapies when used alone in the tubby mouse. The mechanisms by which the nanoceria function in the tubby mouse alone, and in combination with sulphoraphane, or the Trx transgene, will be identified by the following methods. Superoxide radicals in the retina will be assessed using a hydroxyethidine assay whereas H20 2 will be assayed with 2',T-dichlorodihydro- fluorescein-diacetate. ROS-induced damage will be visualized with antibodies against products of ROS activity including acrolein, nitrotyrosine and 8-hydroxy-2-deoxy-guanosine. The effects of the nanoceria on neuroprotective pathways will be analyzed by Western blots, cDNA micro arrays, and Real Time-PCR. Quantitative histology, using bright field microscopy on hematoxylin and eosin stained retinal sections, will be used to evaluate the morphological preservation of photoreceptor cells. Retinal function will be determined by electroretinography. We currently have large colonies of both the tubby and the Trx-tubby mice and will be able to complete the specific aims within two years. The successful achievement of our objectives should be directly relevant to most forms of inherited blindness in mice. The nanoceria are expected to function in humans as they do in other mammals and therefore should preserve vision and prevent blindness in humans.