Mitochondrial Oxidative Stress in the Retinal Pigment Epithelium as a Model for A

描述(申请人提供)：年龄相关性黄斑变性(AMD)是这个国家老年人的一种主要致盲疾病，虽然血管内皮生长因子抑制剂通常阻止疾病的晚期渗出期，但对于早期萎缩的AMD没有有效的治疗方法。视网膜色素上皮(RPE)的氧化损伤通过降低这些细胞的功能并通过刺激炎症级联反应导致疾病的病理特征、视网膜局限性萎缩和脉络膜新生血管，从而导致AMD。RPE细胞本身的线粒体是RPE损伤活性氧簇(ROS)的来源之一。为了确定线粒体是否是活体RPE中ROS的重要来源，将在小鼠的RPE中特异性地删除编码保护酶MnSOD的SOD2基因，使用一个含有SOD2等位基因的小鼠系，该等位基因两侧有loxP位点和RPE特异性表达的Cre重组酶。我们计划进行三组实验：(1)使用电生理学(ERG)、高分辨率结构分析(SD-OCT)和行为分析(Optomotry)实时监测活体小鼠视网膜变性的时间进程。在身体标本中，我们将检查RPE、Bruchs膜和神经视网膜的形态变化，并测量RPE中脂褐素的积累。我们将在新生小鼠和成年小鼠中诱导SOD2的缺失，以确定哪种方法更好地模拟人类地理萎缩。(2)我们将在携带转录因子NRL基因纯合突变的小鼠中通过缺失SOD2来增加线粒体氧化应激。这些小鼠只有一个视锥视网膜，可能比正常的小鼠视网膜更像富含视锥的人类黄斑。通过这种方式，我们希望了解为什么中央视网膜比外围视网膜对氧化应激更敏感。(3)我们将试图通过上调一系列抗氧化酶来对抗线粒体的氧化应激，这些抗氧化酶包括血红素加氧酶-1、谷胱甘肽转移酶和NAD(P)H：苯醌氧化还原酶1。这些酶的这些基因包含一个共同的序列成分，表示为抗氧化序列元件。我们将通过两种方式刺激这种抗氧化反应--通过病毒传递刺激反应的基因和通过口服激活这一途径的新药。我们希望这些方法可以导致对萎缩型AMD的治疗。

DESCRIPTION (provided by applicant): Age Related Macular Degeneration (AMD) is a major blinding disease of the elderly in this country and, while VEGF inhibitors often stem the late, exudative stages of the disease, there is no effective therapy for the earlier, atrophic form of AMD. Oxidative damage to the retinal pigment epithelium (RPE) contributes to AMD by reducing the function of these cells and by stimulating an inflammatory cascade that leads to the pathologic hallmarks of the disease, localized atrophy of the retina and choroidal neovascularization. One source of the RPE- damaging reactive oxygen species (ROS) are the mitochondria of the RPE cells themselves. To determine whether mitochondria are an important source of ROS in the RPE in vivo, the SOD2 gene, encoding the protective enzyme MnSOD, will be deleted specifically in the RPE of mice, using a mouse line containing an allele of SOD2 flanked by loxP sites and RPE-specific expression of Cre recombinase. We plan 3 sets of experiments: (1) We will monitor the time course of retinal degeneration in real time in living mice using electrophysiology (ERG), high resolution structural analysis (SD-OCT) and behavioral analysis (Optomotry). In post mortem samples, we will examine morphological changes to the RPE, Bruch's membrane and the neural retina and to measure accumulation of lipofuscin in the RPE. We will induce the deletion of SOD2 both in neonatal and in adult mice to determine which approach better models human geographic atrophy. (2) We will increase mitochondrial oxidative stress by deletion of SOD2 in mice bearing a homozygous mutation in the gene for the transcription factor Nrl. These mice have a cone-only retina which may resemble the cone-rich human macula better than the normal mouse retina. By this means we hope to learn why the central retina is more sensitive to oxidative stress than the peripheral retina. (3) We will attempt to counteract mitochondrial oxidative stress by elevating a set of antioxidant enzymes, including heme oxygenase-1, glutathione transferases, and NAD(P)H:quinone oxidoreductase 1. These genes for these enzymes contain a common sequence component denoted "ARE" for antioxidant sequence element. We will stimulate this antioxidant response in two ways-by viral delivery of a gene that stimulates the response and by oral delivery of a novel drug that activates this pathway. We hope that these approaches may lead to a therapy for the atrophic form of AMD.