Progress Report: Age related retinal degenerations, whether of genetic origin or arising from environmental stress, dramatically decrease personal productivity and negatively impact quality of life. To better understand these conditions research directed at developing animal models of retinal disease and treatments designed to slow or prevent vision loss is essential. Toward this goal we have found that antioxidants prevent light induced retinal damage and that the retina possesses its own internal clock which also prevents retinal degeneration in animals exposed to light during normal daylight hours. However, when light exposure occurs during the usual nighttime hours the retina is predisposed to damage. The question then is what proteins are different during the day and night which can lead to retinal damage in one case, or prevent it from occurring in the other? To address this question we have studied retinal protein levels in different rat models of retinal degeneration. In animals undergoing genetic retinal degenerations we find increased levels of a group of small protective proteins called crystallins. Thus we hypothesized that we might see the same changes in crystallin levels in our light induced degeneration model. Because this type of retinal degeneration is triggered by light’s effects on the protein rhodopsin in photoreceptor cell rod outer segments (ROS), we determined ROS crystallin levels at various times of the day and night (aim 1). For aim 2 we administered an antioxidant to rats first and then determined its effects on ROS crystallins.

Aim 1: We used a technique called western analysis to determine how ROS crystallin levels were affected by time of day. ROS were prepared from the retinas of rats at 4 hour intervals around the clock. The times chosen were 1, 5 and 9 am and 1, 5 and 9 pm. Retinal ROS proteins were then separated by electrical charge differences on gels by the technique of electrophoresis. Following electrophoresis the proteins were electrically transferred to a membrane for western analysis. Although all of the ROS proteins were transferred we focused on crystallins by using antibodies that detect them and not other proteins. The three groups of crystallins (alpha, beta and gamma) were then detected sequentially with antibodies specific for each group and their relative concentrations determined by differences in staining intensity. Three groups of 4 animals each were used to prepare ROS for electrophoresis and western analysis.

Analysis of alpha, beta and gamma crystallins all yielded the same relative findings. When samples were prepared from rats at 9 pm and 1 am there was little evidence of ROS crystallin proteins. These times are during the normal night time period for our rats. However, starting at 5 am there was a dramatic increase in all three classes, a response that lasted through the day e.g. 9 am, 1 pm and 5 pm. Although 5 am is still in the dark period, 9 am and 1and 5 pm are all during the period when our rats are in their light period. To be sure the effect we saw was not caused by the light itself all animals were kept in darkness before the experiment began. Because these animals were kept in darkness for 16 hours before sacrifice we conclude that the result was not caused by light, but by the normal anticipation of daylight. This type of time dependent change is a circadian response, which routinely occurs in most animals and humans on a 24 hour cycle. The finding is significant in that, intense light exposure of rats during the normal daylight period does not lead to retinal damage and vision loss. However, when animals are exposed to light during the night e.g. 9 pm and 1 am, they incur massive visual cell destruction. Potentially this degree of photoreceptor cell loss is due to lower levels of protective ROS crystallins at night and higher levels during the day.

Aim 2: Vitamins containing antioxidants are a part of many normal diets and are often recommended for patients with certain age related vision problems. Because antioxidants can reduce or eliminate light damage to photoreceptor cells we treated rats with a synthetic antioxidant 1 hour before light exposure beginning at 1 am. One am is the most sensitive period for damage in the light exposed retina. The animals were then either exposed to intense light for 8 hours or kept in darkness for the same period. In both untreated rats and in those given the antioxidant dimethylthiourea (DMTU), ROS crystallins were at undetectable at 1 am. The stress of light exposure beginning at 1 am led to elevated ROS crystallin levels at 9 am. This occurred in both types of animals, but the changes were modest. As expected from our findings in aim 1, both DMTU treated and untreated rats kept in darkness for the same 8 hour period had higher crystallin levels. However, the increase in crystallins was dramatic for the antioxidant treated animals and only very modest for the rats not given the antioxidant. We conclude that antioxidant treatment caused an induction in ROS crystallin proteins during the 8 hour post treatment period. This may partially account for their protective effect against light induced retinal damage, but suggests that multiple treatments would be required. Such an effect may be an example of a beneficial side effect of prolonged drug treatment in contrast to the usual side effects which often limit the use of therapeutic drugs.

Directions for Future Research: Retinal photoreceptor cell ROS contain an unusually high level of unsaturated fatty acids, the most prominent of which is docosahexaenoic acid (DHA). This omega 3 fatty acid is 22 carbons in length and has 6 carbon-carbon double bonds. It accounts for a much as 50% of the total fatty acids found in ROS lipids. In addition, the retina has a very high oxygen tension and a high photon flux during light. Because DHA is a polyunsaturated fatty acid it is quite susceptible to oxidation, especially under the conditions present in the retina. DHA oxidation products are able to react with proteins to form covalent adducts, which could be detrimental to visual cells. The chemical name of one such DHA oxidation product is carboxyethylpyrrole (CEP). This oxidatively modified fatty acid derivative forms CEP lipid-protein adducts which can be detected by the western technique described earlier. Recently we obtained an antibody which is specific for CEP linked to proteins and we now propose to study these products. Our hypothesis is that CEP lipid-protein adducts formed in ROS will be toxic to the photoreceptor cell. Toward a better understanding of CEP oxidation products, we propose to use western analysis to detect their presence in ROS and to establish base line levels in rats under normal light- dark rearing conditions (aim 1). Because light can be toxic to visual cells we will determine its effects on the formation CEP protein adducts in ROS using different intensities and durations of light treatment (aim 2).

The significance of research into DHA oxidation resides in the fact that a clinical trial is currently underway to determine the effects of dietary DHA on the progression of AMD. Whether dietary supplementation with DHA proves to be beneficial or not, we need to know more about the role of DHA in the eye, its potential oxidation products and whether these can be toxic in visual cells.

Presentations: Association for Research in Vision and Ophthalmology Meetings.

D.T.Organisciak, R.M.Darrow, L.S.Barsalou. Translocation of Crystallin Proteins in Photoreceptor Cells during Day and Night. ARVO Annual Meeting, Ft. Lauderdale, FL. A 4656 (2007).

M.Miyagi, V.Palamalai, D. Hajkova, K.C.S.Rao, R.M.Darrow, D.T.Organisciak. Comparative Proteome Analysis of Light Exposed and Unexposed Photoreceptor Rod Outer Segments. ARVO Annual Meeting, Ft.Lauderdale, FL. A588 (2007).

D.T.Organisciak, R.M.Darrow, L.S.Barsalou, K.Renganathan, J.S.Crabb, J.W.Crabb. Carboxyethylpyrrole Modified Proteins in Rat ROS and Retinal Pigment Epithelium. ARVO Ocular Cell and Molecular Biology Meeting, Sarasota, FL. A 28 (2007).

Publications:

R.E.Marc, B.W.Jones, C.B.Watt, F.Vazquez-Chona, D.K.Vaughan, D.T.Organisciak. Extreme Retinal Remodeling Triggered by Light Damage: Implications for Age Related Macular Degeneration. Molecular Vision 14: 782-806 (2008).

Aim 1: From DNA measurements we determined that the rate of retinal photoreceptor cell loss was greatest in RCS rats, followed by the fast sub lines of P23H and S334ter animals (P23H line 3 and S334ter line 4) and then the slower degenerating sub lines of these transgenic RP models (P23H line 2 and S334ter line 9). In each case, the expression of retinal crystallins increased before the onset of DNA loss, indicating that their expression occurred in anticipation of genetic stress. In normal control rats neither, a loss of retinal DNA nor a marked increase in crystallins occurred during the 60 day period studied.

To determine the effects of genetic inheritance on crystallin protein expression we used a technique called electrophoresis, with detection of retinal crystallin levels by an antibody technique called western analysis. Initially we used 1D electrophoresis to detect alpha A- and alpha B- crystallins in retinal protein extracts from animals at various ages. The results show that in normal rats alpha crystallin levels are very low, while in RCS rats these were markedly elevated at 30 days of age. Our analysis also shows that P23H line 3 animals had very high alpha crystallin levels after 50 days, while the slower degenerating line 2 rats expressed only slightly elevated levels. Basically the same findings applied to the fast and slow degenerating sub lines of S334ter rats. We confirmed the changes in crystallin protein levels by 2D gel electrophoresis and extended those findings by using antibodies against beta- and gamma- crystallins. The effects seen for alpha crystallins were also true for the other crystallins. Taken together, our results suggest that increased crystallin expression occurs in various forms of retinal degeneration and that the increases are related to the onset and severity of the degeneration.

Aim 2: Using the added stress of intense environmental light, we found that only 2 hours of exposure resulted in a massive loss of visual cells in P23H rats (sub line 3). Normal rats required much longer times to exhibit extensive photoreceptor loss. The loss of visual cells was determined by measuring the loss of rhodopsin and retinal DNA in these animals. We also found that retinal crystallins were modified by intense light induced reactions, including phosphorylations and truncations, but that these alterations occurred within 2 hours in P23H rats and only after a minimum of 8 hours in the normal animals. Thus, the alterations in retinal crystallins caused by light stress are accelerated by imposing that stress on a genetic background predisposing P23H animals to vision loss.

Directions for Future Research: Crystallins are thought to provide protection to tissue proteins by serving as molecular chaperones or by interrupting the effects of environmental stress on their denaturation. The well known effects of intense light on vision loss, initiated by rhodopsin within photoreceptor rod outer segments (ROS), and recent findings on crystallins suggest that high ROS crystallin levels may protect photoreceptor cells. Our working hypothesis is that by elevating retinal crystallin levels before intense light stress we will reduce the loss of photoreceptors and vision. Retinal light damage has a circadian component in which the retina is susceptible to light at night and refractory to damage during the day. To address this difference in light damage susceptibility we propose to determine the levels of ROS crystallins at various times of the day and night (aim1).

Likewise, antioxidants reduce or prevent the effects of intense light on photoreceptor loss, primarily by reducing the levels of damaging reactive oxygen species. Our hypothesis here is that antioxidants may also elevate ROS crystallins which will serve as a beneficial side effect to help protect photoreceptors. Accordingly, we propose to use antioxidant treatments in rats to determine if ROS crystallin levels can be elevated prior to light exposure (aim2).

Presentations: At the Association for Research in Vision and Ophthalmology Meeting.

R.M. Kelln, M.A. Chrenek, R. Darrow, D.T. Organisciak, V. Vasireddy, R. Ayyagari, P. Wong. Cellular Localization of 1363, an Uncharacterized Gene Identified in a Screen for Differentially Expressed Light – Induced Retinal Degeneration Genes in the Rat. Arvo Annual Meeting, Fort Lauderdale, Fl. (2006)

M. Miyagi, V. Palamalai, R.M. Darrow, D.T. Organisciak. Comparative Proteome Analysis of Light Exposed and Unexposed Photoreceptor Rod Outer Segments. Arvo Annual Meeting, Fort Lauderdale, Fl. (2006)

D.T. Organisciak, R.M. Darrow, L.S. Barsalou, B. McDonald, P. Wong. Circadian and Stress Induced Changes in Rod Outer Segment Crystallins. Arvo Annual Meeting, Fort Lauderdale, Fl. (2006)

M. Chrenek, A.C. Ziesel, R.M. Darrow, L. Barsalou, D.T. Organisciak, P.Wong. Differential Expression of Cope During Light – Induced Retinal Damage in Rats. Arvo Annual Meeting, Fort Lauderdale, Fl. (2006)

 

Publications:

T. Duncan, B. Wiggert, N. Wittaker, R. Darrow, D.T. Organisciak. Effect of Visible Light on the Retinoids in Normal and P23H-3 Transgenic Rat Retina: Analysis of a Novel Retinoic Acid Derivative Present in P23H-3 Retina. Photochem. Photobiol. 82:741-745 (2006)

M. Sun, S.C. Finnemann, M. Febbraio, L. Shan, S.P. Annangudi, E.A. Podrez, R. Darrow, D.T. Organisciak, R.G. Salomon, R.L. Silverstein, S.L. Hazen. Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: A Potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions. J. Biol. Chem. 281:4222-4230 (2006)

D.T. Organisciak, R. Darrow, Xiaorong Gu, L. Barsalou, J. Crabb. Genetic, Age and Light Mediated Effects on Crystallin Protein Expression in the Retina. Photochem. Photobiol. 82:1088-1096 (2006)

V. Palamalai, R.Darrow, D.T. Organisciak, M. Miyagi. Light-induced changes in protein nitration in photoreceptor rod outer segments. Molecular Vision. 12:1543-1551 (2006)

B.W. Jones, R.E. Marc, C.B. Watt, D.K. Vaughan, D.T. Organisciak. Neural Plasticity Revealed by Light-Induced Photoreceptor Lesions. Advances in Experimental Medicine and Biology. Eds. J.G. Hollyfield, R.E. Anderson, M.M. LaVail. Vol. 572 Springer, N.Y. p405-410 (2006)

R. Grewal, D. Organisciak, P. Wong. Factors Underlying Circadian Dependent Susceptibility to Light Induced Retinal Damage. Advances in Experimental Medicine and Biology. Eds. J.G. Hollyfield, R.E. Anderson, M.M. LaVail. Vol. 572 Springer, N.Y. p411-416 (2006)