WRIGHT STATE UNIVERSITY
Daniel T. Organisciak Ph.D.
OLERF Report, July 1, 2010 – July 1, 2011
The rate of vision loss in age-related macular degeneration (AMD) and in retinitis pigmentosa (RP) exceeds the modest rate of visual cell loss in people unaffected by ocular disease. Genetic inheritance, combined with a high light environment appears to underlie the rapid loss of photoreceptors in both AMD and RP, but the molecular steps involved in the process are not known. We have studied interactions between retinal genes and light environment in a transgenic rat model of RP. This preclinical laboratory animal model was created to mimic the same genetic mutation found in the most prevalent form of autosomal dominant RP. The mutation results from a substituted amino acid in the visual pigment rhodopsin, consisting of a histidine in place of the normal amino acid proline (P23H). Importantly, the P23H animal model exhibits a high rate of age-related visual cell loss and an enhanced sensitivity to light, characteristics found in RP patients with the P23H rhodopsin mutation. One marker of oxidative damage in ocular tissues is a reaction product between the omega 3 polyunsaturated fatty acid DHA and retinal proteins. This reaction results in carboxyethylpyrrole (CEP) protein adducts which can be detected by an antibody specific for CEP. Increased levels of CEP adducted proteins have been found in drusen from AMD patients and we have detected elevated CEP reactivity in retinas from rats exposed to intense light. Accordingly, we proposed that CEP levels might also be elevated in retinal tissues from P23H rats and that light might enhance accumulation of this oxidation product. The aims of our proposal were: (1) to determine how light environment and DHA impacts the level of CEP adducted proteins in the P23H rat retina. (2) to determine the effects of genetics in P23H rats by comparing heterozygous and homozygous offspring for CEP reactivity and for crystallin protein expression.
To determine CEP levels in retinal proteins we used gel electrophoresis followed by western analysis of CEP immunoreactivity using an antibody that specifically detects CEP. Because DHA oxidation is the only fatty acid source of CEP forming protein adducts, we also measured DHA levels in rod outer segments (ROS). Within the retina rhodopsin with a normal amino acid sequence as well as the mutated rhodopsin present in P23H are located in the membranes of ROS. We found elevated levels of CEP reactivity in retinas and ROS from P23H rats reared in dim cyclic light after exposure to intense light for 2 or 4 hours. As measured by rhodopsin and retinal DNA, the loss of photoreceptors in P23H rats following a 4 hour light exposure was over 50%, whereas no photoreceptor cell loss occurred in normal rats (Sprague-Dawley) which are unaffected by the P23H mutation. In mutated P23H rats reared in darkness the loss of photoreceptors occurred with only 1 or 3 hours of intense light. Surprisingly, when we measured DHA levels the amount in ROS from P23H rats was only about 34% of the total compared to over 45% in normal rats. This means that 1 out of every 3 ROS fatty acids in the P23H rat is DHA while in normal animals DHA is present at a higher concentration, almost 1 out of 2 ROS fatty acids. Accordingly, although normal rat ROS have more DHA, their photoreceptors are undamaged by light that caused massive damage in P23H rats. The finding that CEP reactivity in P23H retina is also enhanced by short duration light exposure indicates that light induced oxidation plays a major role in this animal model of retinal degeneration.
Using the same type of gel electrophoresis based analysis of protein immunoreactivity (western analysis) we determined CEP adduct levels in the retinas of rats heterozygous and homozygous for the P23H rhodopsin gene. Heterozygous (+/-) inheritance of P23H results in rod cell degeneration, because P23H is an autosomal dominant disease. Homozygous (-/-) inheritance, in which two copies of the mutated P23H gene are present, causes an acceleration of the condition and a more rapid loss of photoreceptor cells. As expected, the -/- P23H rat retinas had a higher level of CEP reactivity than found in +/- rats. When we used antibodies to detect opsin (rhodopsin minus vitamin A) we found that higher levels were initially present in ROS from 18-24 day old -/- rats than in +/- animals. However, when we extracted and measured the actual photopigment rhodopsin, the levels were lower in -/- animals than found in +/- rats. Because opsin is the protein form of rhodopsin without vitamin A, we suggest that over expression of opsin occurs in -/- rats, but that vision is compromised because vitamin A is missing. This results in additional, perhaps oxidative, stress in retinas of -/- rats and leads to higher CEP reactivity in those retinas. Similarly, a group of small stress proteins known as crystallins and in particular alpha-crystallins are elevated in -/- P23H rats when compared to +/- animals. Taken together these results tell us that inheritance influences the rate of retinal degeneration in our animal model of RP. Furthermore, it appears that oxidation plays a role in accelerating the loss of vision in these animals.