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.
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