RETINAL DEGENERATION: MOLECULAR AND BIOCHEMICAL ASPECTS

描述（由申请人提供）：对60多个视网膜基因的数百个突变的鉴定导致了动物模型的产生，这有助于建立突变与疾病表型之间的关系。目前，解释突变如何导致细胞凋亡的信息有限。细胞要发生凋亡，必须首先接收到一个内在或外在的信号，然后激活凋亡的刽子手。我们的假设是，常见的早期分子事件（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。为了识别凋亡信号（目的1），我们建议使用蛋白质组学、差分显示PCR （dd-PCR）和微阵列技术对rd小鼠、眼蛋白255或256位异亮氨酸缺失的deli -255/256转基因模型、过表达正常视蛋白的house转基因小鼠和SV40 T抗原转基因小鼠进行检测。选择这些模型的原因是，尽管它们因不同基因的表达而出现功能障碍，但它们都在P10之后开始同步凋亡，并在P21完成。作为对照，我们将使用年龄匹配的wt小鼠和G90D（视蛋白中甘氨酸到天冬氨酸）转基因非退行性先天性静止性夜盲症模型。为了识别早期凋亡信号，在视网膜出现任何明显的形态学变化之前（在P8上）进行二维凝胶检测，并通过特征肽质量指纹图谱揭示信息蛋白斑点的身份。Dd-PCR和cDNA阵列将用于鉴定其调节低于蛋白质组学检测水平的基因转录本。我们还将使用蛋白质组学来确定表达bcl-2原癌基因的转基因小鼠视网膜中诱导的几种潜在应激蛋白的身份及其在细胞凋亡中的作用（目的2）。最后，为了阐明在Aim 1中分离的因子启动细胞凋亡的机制，我们将使用蛋白质阵列识别Aim 3中潜在的视网膜凋亡刽子手。

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.