RETINAL DEGENERATION: MOLECULAR AND BIOCHEMICAL ASPECTS

视网膜变性：分子和生物化学方面

• 批准号: 6577662
• 负责人: MUAYYAD R AL-UBAIDI
• 金额: $ 36.63万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6577662
• 关键词: BCL2 gene /protein; apoptosis; biological signal transduction; gel electrophoresis; gene expression; gene mutation; laboratory mouse; mass spectrometry; matrix assisted laser desorption ionization; microarray technology; polymerase chain reaction; proteomics; retina degeneration; stress proteins

DESCRIPTION (provided by applicant): The identification of hundreds of mutations in over sixty retinal genes led to the generation of animal models, which were instrumental in establishing the relationship between the mutation and the disease phenotype. Currently, limited information exists to explain how a mutation leads to apoptosis. For apoptosis to take place an intrinsic or extrinsic signal must first be received by the cell followed by the activation of apoptotic executioners. Our hypothesis is that common early molecular events (apoptotic signals) precede the morphologic changes of photoreceptors (apoptotic execution). Our goal is to identify proteins that are modulated during these events. To identify apoptotic signals (Aim 1) we propose to use proteomics, differential display PCR (dd-PCR), and microarrays on the rd mouse, deltaI-255/256 transgenic model of isoleucine deletion at position 255 or 256 in opsin, Bouse transgenic mouse that over-expresses normal opsin, and SV40 T antigen transgenic mice. These models are chosen because, although they suffer from dysfunction resulting from the expression of different genes, synchronized apoptosis in all of them is initiated after P10 and completed by P21. As controls, we will use age matched wt mice and G90D (glycine to aspartic acid in opsin) transgenic model of non-degenerative congenital stationary night blindness. To identify early apoptotic signals, two-dimensional gels will be performed on retinas before any apparent morphologic changes (on P8) and the identity of informative protein spots will be revealed by characteristic peptide mass fingerprinting. Dd-PCR and cDNA arrays will be used to identify the transcripts of genes whose modulations are below proteomics levels of detection. We will also use proteomics to determine the identity and role in apoptosis of several potential stress proteins that are induced in retinas of transgenic mice expressing bcl-2 proto-oncogene (Aim 2). Finally, to elucidate the mechanism through which factors isolated in Aim 1 can initiate apoptosis, we will use protein arrays to identify prospective retinal apoptotic executioners in Aim 3. This research will help identify the principle genes controlling cell death regardless of the initial cellular insult. Uncovering these genes will lend itself to understanding the initiation and execution of retinal apoptosis in degenerative disorders and serves to enhance our understanding of normal age-related cell death.

描述（由申请人提供）：在超过60个视网膜基因中鉴定出数百个突变导致动物模型的产生，这些动物模型有助于建立突变与疾病表型之间的关系。目前，存在有限的信息来解释突变如何导致细胞凋亡。为了使凋亡发生，细胞必须首先接收内在或外在信号，然后激活凋亡执行者。我们的假设是，共同的早期分子事件（凋亡信号）先于光感受器的形态变化（凋亡执行）。我们的目标是确定在这些事件中被调节的蛋白质。为了鉴定凋亡信号（目的1），我们建议使用蛋白质组学、差异显示PCR（dd-PCR）和微阵列对rd小鼠、在视蛋白中的位置255或256处异亮氨酸缺失的deltaI-255/256转基因模型、过表达正常视蛋白的Bouse转基因小鼠和SV 40 T抗原转基因小鼠进行研究。选择这些模型是因为，尽管它们遭受由不同基因的表达引起的功能障碍，但它们中的所有模型的同步凋亡都在P10之后启动并在P21完成。作为对照，我们将使用年龄匹配的野生型小鼠和非变性先天性静止性夜盲症的G90 D（视蛋白中的甘氨酸至天冬氨酸）转基因模型。为了鉴定早期凋亡信号，将在任何明显的形态学变化之前（在P8上）对视网膜进行二维凝胶，并且将通过特征性肽质量指纹图谱揭示信息蛋白质点的身份。DD-PCR和cDNA阵列将被用来识别基因的转录本，其调节低于蛋白质组学检测水平。我们还将使用蛋白质组学来确定在表达bcl-2原癌基因（Aim 2）的转基因小鼠视网膜中诱导的几种潜在应激蛋白的身份和在细胞凋亡中的作用。最后，为了阐明在目标1中分离的因子可以启动凋亡的机制，我们将使用蛋白质阵列来识别目标3中的前瞻性视网膜凋亡执行者。 这项研究将有助于确定控制细胞死亡的主要基因，而不管最初的细胞损伤如何。揭示这些基因将有助于理解退行性疾病中视网膜细胞凋亡的启动和执行，并有助于增强我们对正常年龄相关细胞死亡的理解。