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Top search choices8. Floaters 9. Optic Nerve 12. Vision Rehabilitation of Infants
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Wright Sate University
Daniel T. Organisciak Ph.D.
Progress Report: In ocular diseases such as age related macular degeneration (AMD) and retinitis pigmentosa (RP) the rate of photoreceptor death and vision loss is accelerated, relative to the loss of retinal photoreceptors that normally occurs during aging. We now know that visual cell loss in both AMD and RP has a strong genetic component and we have a strong suspicion that over time high levels of environmental light can further accelerate that rate of loss. We also know that factors such as diet may influence the onset and rate of vision loss, but the long term relationships between genetic inheritance and light environment or diet is difficult to study in humans. In this regard, light conditions and diet can be well controlled in laboratory animals, allowing for pre-clinical testing of gene/environment interactions and for the initial development of potential therapeutic treatments for disease. We have studied fatty acid derivatives formed from the highly unsaturated dietary omega-3 fatty acid DHA, a 22 carbon fatty acid containing 6 carbon-carbon double bonds. DHA forms a potentially protective compound called neuroprotectin D-1, which functions primarily in the retinal pigment epithelium (RPE) and a potentially toxic derivative, carboxyethylpyrrole (CEP) which may impact retinal function. Currently an AREDS clinical trial involving DHA supplements is underway to determine if dietary DHA can actually slow the rate vision loss in AMD patients. Whether DHA proves to be protective or not, characterizing its metabolism can provide important information. Aside from the potential benefits of DHA supplementation, its oxidation leads specifically to the formation of CEP, a seven carbon fragment derived from DHA breakdown. Once formed, CEP can react with nearby proteins to form CEP-protein adducts, potentially inactivating those proteins. Using the techniques of gel electrophoresis and western analysis with anti-CEP antibodies we have detected these derivatives in rat retina. We found that intense light exposure increases the level of CEP formation and, by using mass spectroscopy, we have identified some of the CEP-protein adducts that were formed. The aims of our proposal were: (1) to determine the identities of CEP reactive proteins in the retina and the RPE. (2) To determine if an antioxidant can prevent the oxidative breakdown of DHA.
Aim 1: To identify retinal proteins that reacted with CEP we used 2 D gel electrophoresis followed by western analysis, using an anti-CEP antibody, and mass spectroscopy. The rationale was that 1D gel electrophoresis does not completely separate proteins while 2 D electrophoresis, which involves separations based on both protein size and electrical charge, is much better. After western detection, individual CEP positive proteins were isolated from the 2D gels and digested with an enzyme named trypsin. The peptide fragments that were formed were then sent to a colleague for mass spectroscopic identification of the original proteins. We found that a few structural proteins were modified, but a surprisingly large number of metabolic enzymes reacted with CEP. Some of these enzymes are known to be involved in glycolysis, a process that yields energy for cellular function. Not convinced, we used a different antibody, which detects a different oxidized protein derivative named nitrotyrosine. Many of the same retinal proteins were also found to contain oxidatively modified tyrosine. This dual approach indicates that some retinal proteins are particularly susceptible to oxidative modification and that their oxidation may, in turn, affect energy production in photoreceptors. Confirming this, we found many of the same CEP modified proteins in rod outer segments (ROS), an integral part of retinal photoreceptor cells. In the RPE, the number of CEP modified proteins was fewer and these were largely different than in retina. However, aldolase, a glycolytic enzyme, and complement factor-4 appeared to be modified. Interestingly, an amino acid mutation(s) in one of the cellular complement system proteins is a major genetic marker associated with an increased risk of AMD.
Aim 2: To determine if an antioxidant could prevent the appearance of CEP adducted proteins we treated rats with dimethylthiourea (DMTU) 1X IP at 500 mg/kg and then exposed them to intense visible light. Following light treatment retinas were excised and gel electrophoresis/western analysis conducted. The rationale for these experiments was based on earlier studies in which we found that intense light resulted in decreased levels of DHA in ROS, while the synthetic antioxidant DMTU reduced that loss and completely prevented the visual cell degeneration normally seen after light exposure. Contrary to our expectations, DMTU did not appreciably diminish CEP reactive proteins in retina or RPE. We were able to detect small decreases, but these changes were inconsistent and certainly not significant. At the same time DMTU did diminish the expression of a protein marker of oxidative stress, heme oxygenase I (HO-I). Next we used a natural antioxidant, ascorbic acid, at the same high concentration. Once again, there were no major changes in CEP reactive proteins in retina or RPE. Finally we tried a more powerful phenolic antioxidant and were able to see decreased CEP reactivity, along with decreased expression of the HO-I stress protein. We conclude that the concentration of oxygen in retina and RPE is so high that antioxidants are often unable to totally interrupt its ability to react with unsaturated fatty acids or proteins. This tells us that oxidation is a constant and on-going process in light and in darkness and that neither retina nor the RPE is significantly affected in the short term. Although retina may appear to function normally, even as CEP-protein adducts are continuously formed, ROS tips are shed on a daily basis and taken up by RPE. Over time therefore, CEP modified proteins can be expected to accumulate in RPE increasing the potential for adverse reactions and compromised function.
Directions for Future Research: An increasing body of evidence indicates that oxidative stress is part of the etiology of ocular diseases such as AMD and RP. Although rodent animal models of human ocular disease do not capture all of the characteristics of disease, a transgenic rat model has been developed which mimics the principle characteristics of rod cell loss found in RP. This model has a proline to histidine amino acid mutation (P23H) in the visual pigment rhodopsin which is identical to the most prevalent form of autosomal dominant RP in humans. One feature of the P23H form of RP is that people who work in high light environments tend to lose vision more rapidly than patients typically working in lower light environments. Another is that patients that inherit two abnormal genes (homozygous) exhibit an earlier onset of disease and a more rapid decline in vision than those having a single abnormal gene (heterozygous). The P23H rat is also highly sensitive to light, rapidly losing vision following light treatment and homozygous animals lose their vision more rapidly than heterozygous animals. We will take advantage of these characteristics in the proposed studies. Our hypothesis is that oxidative stress is part of the pathology of visual cell loss in P23H rats and that increased levels of oxidative products will be detected in the retinas of these animals. We will test this by comparing visual cell loss and CEP reactivity in retinas from rats in a dim light environment, darkness and after exposure to intense visible light.
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W. R. Bryan Diabetic Eye Disease Research Fund 2008 OLERF Annual Report (PDF file) |
