Research Project
10298716
ABSTRACT Retinitis pigmentosa (RP) is a progressive form of retinal degeneration caused by at least 3,000 mutations in 70 different genes that result in the death of rods, followed by cones, and ultimately blindness. Although oxidative stress and inflammation are observed in RP, early molecular mechanisms in disease progression are unknown. We propose to compare a genetic mouse model of RP, rd10, with wild type mice to determine key pathways that suggest therapeutic targets for treating retinal degeneration utilizing a novel strategy of broad-spectrum metabolomics integrated with proteomics and mitochondrial dysfunction analysis. The rd10 mouse has a mutation in the ? subunit of phosphodiesterase 6 (PDE6), which hydrolyzes cGMP in response to light as part of the photoresponse. Mutations in this gene cause recessive RP in humans. Our recent untargeted metabolomics study found abnormal increases in the levels of several deoxypyrimidines and deoxypurines, as well as a key metabolite in the Coenzyme A synthesis pathway, in rd10 compared to wild type mice. These changes were observed at P18, which is when cell death and reduced photoreceptor function are first detected. We hypothesize that the observed changes in the metabolome in rd10 mice are due to changes in the activities of the related proteins/enzymes. To identify changes in the metabolome that occur before reduced photoreceptor function and cell death are detected, we propose to analyze mice at the earlier timepoint, P14. In Specific Aim 1, we will use broad spectrum (untargeted) at P14 and targeted metabolomics at both P14 and P18. In Specific Aim 2, untargeted proteomics will be used to identify proteins/enzymes in membrane, soluble and mitochondrial fractions that may be linked to the disease process. This will be followed by targeted evaluation of proteins that are related to our metabolomic results in SA1 using computer analysis to integrate the metabolomic and proteomic data for developing an early mechanistic disease profile. Specific Aim 3 will evaluate mitochondrial oxygen consumption rate (OCR) at P14, based on our preliminary data demonstrating a reduced OCR in rd10 mice as early as P16 and P18, suggesting that mitochondrial dysfunction is an early event in this model of RP. We will also test this hypothesis by performing evaluations of mitochondrial health such as mitochondrial dynamics (fusion and fission) and the integrity of the mitochondrial structure using Serial Block Face Scanning Electron Microscopy (SBFSEM). Abnormal mitochondrial structure or function identified in this aim will be integrated with the metabolomic and proteomic information from SA1 and SA2. For all specific aims, dark-reared and light-reared mice will be compared, since light is known to be an exacerbating factor for RP caused by the rd10 mutation. Based on a report that oxidative stress occurs at different time points and is differentially localized in male and female rd10 mice, we will also include gender as a variable. The ultimate goal of this proposal is to identify potential therapeutic targets in key pathways early in retinal degeneration.
