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

描述（由申请人提供）：年龄相关性黄斑变性（AMD）是美国老年人的一种主要致盲性疾病，虽然VEGF抑制剂通常阻止疾病的晚期渗出阶段，但对于早期萎缩型AMD尚无有效治疗方法。对视网膜色素上皮（RPE）的氧化损伤通过降低这些细胞的功能并通过刺激导致疾病的病理学标志、视网膜的局部萎缩和脉络膜新血管形成的炎性级联反应而促成AMD。损伤RPE的活性氧（ROS）的一个来源是RPE细胞本身的线粒体。为了确定线粒体是否是体内RPE中ROS的重要来源，将使用含有SOD 2等位基因的小鼠系在小鼠的RPE中特异性地缺失编码保护酶MnSOD的SOD 2基因，所述等位基因的两侧是loxP位点和Cre重组酶的RPE特异性表达。我们计划了三组实验：（1）我们将使用电生理学（ERG）、高分辨率结构分析（SD-OCT）和行为分析（Optomotry）对活体小鼠视网膜变性的时间过程进行真实的实时监测。在死后样本中，我们将检查RPE、布鲁赫膜和神经视网膜的形态学变化，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SOD 2的缺失，以确定哪种方法更好地建立人类地图状萎缩模型。(2)我们将通过在携带转录因子Nrl的基因中的纯合突变的小鼠中缺失S 0 D2来增加线粒体氧化应激。这些小鼠具有仅视锥细胞的视网膜，其可能比正常小鼠视网膜更类似于富含视锥细胞的人类黄斑。通过这种方法，我们希望了解为什么中央视网膜比周边视网膜对氧化应激更敏感。(3)我们将尝试通过升高一组抗氧化酶，包括血红素加氧酶-1、谷胱甘肽转移酶和NAD（P）H：醌氧化还原酶1来对抗线粒体氧化应激。这些酶的这些基因含有共同的序列组分，表示为抗氧化剂序列元件的“ARE”。我们将以两种方式刺激这种抗氧化反应通过病毒传递一种刺激这种反应的基因和口服一种激活这种途径的新药。我们希望这些方法可能会导致萎缩型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.