Proteomic changes in the photoreceptor outer segment upon intense light exposure.

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic (18)O labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.

Light-induced changes in protein nitration in photoreceptor rod outer segments.

PURPOSE: Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS). METHODS: Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase (NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis. RESULTS: Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure. CONCLUSIONS: The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.

Expression of neurturin, glial cell line-derived neurotrophic factor, and their receptor components in light-induced retinal degeneration.

PURPOSE: Dysregulation of neurturin (NTN) expression has been linked to photoreceptor apoptosis in a mouse model of inherited retinal degeneration. To investigate the extent to which any such dysregulation depends on the nature of the apoptotic trigger, the expression of NTN, glial cell line-derived neurotrophic factor (GDNF), and their corresponding receptor components were compared in a rat model of light-induced retinal degeneration. METHODS: Retinal expression of NTN, GDNF, their corresponding receptors GFRalpha-2 and -1, the transmembrane receptor tyrosine kinase (Ret), and cSrc-p60, a member of the cytoplasmic protein-tyrosine kinases family, were analyzed by Western blot analysis and immunocytochemistry in cyclic light- and dark-reared rats in the presence and absence of intense light exposure. RESULTS: All components for NTN-mediated signaling activation are present in rat photoreceptors and retinal pigment epithelium, the cells primarily affected by light-induced damage. The expression levels of GDNF, its receptor components, and NTN, were not affected by light-induced stress. However, GFRalpha-2 expression strikingly increased with the extent of retinal damage, especially at the photoreceptors, in contrast to decreased levels that were observed previously in an inherited degeneration model. CONCLUSIONS: The present study indicates that the expression of receptors of the GDNF family is independently regulated in normal and light-damaged rat retina, and in conjunction with previous work, suggests that the pattern of modulation of these genes during photoreceptor degeneration is determined by the nature of the apoptotic trigger. Such differential responses to different modes of retinal degeneration may reflect influences of the neurotrophic system on photoreceptor survival or in the regulation of neuronal plasticity.

Intense light exposure changes the crystallin content in retina.

Toward a better understanding of light-induced photoreceptor damage, the crystallin content of rat retina was examined following intense light exposure. Nine crystallin species were identified by mass spectrometric analysis of rat retina fractionated by 2D gel electrophoresis. The Coomassie blue staining intensity of all crystallin 2D gel components was 2- to 3-fold greater in light exposed than in control retinas. Following light exposure, anti-alphaB-crystallin immunoreactivity was increased in rod outer segments and retinal pigment epithelium. These findings support a possible role for crystallins in protecting photoreceptors from light damage.

A morphometric study of light-induced damage in transgenic rat models of retinitis pigmentosa.

PURPOSE: To determine relative susceptibility to, and regional variation of, light-induced retinal damage in two rhodopsin-mutant rat models of retinitis pigmentosa, using slow- and fast-degenerating lines. METHODS: Transgenic S334ter (lines 4 and 9) and P23H (lines 2 and 3) rats were reared in dim cyclic light or darkness and then exposed to intense green light for 1 to 8 hours. Sections along the vertical meridian were collected for retinal morphology and photoreceptor morphometry 2 weeks later. Unexposed transgenic and normal Sprague-Dawley rats served as the control. Mean outer segment lengths and outer nuclear layer thicknesses were analyzed as a function of position along the vertical meridian and as averages across that vector. RESULTS: Rapidly degenerating S334ter-4 retinas, reared in dim cyclic light, exhibited no light-induced damage, whereas retinas in the other sublines sustained damage within a sensitive region in the superior hemisphere. Light-induced damage always involved loss of outer segment membrane and photoreceptors. In some cases, the retinal pigment epithelium and inner nuclear layer were also affected. Potentiation of light-induced damage by dark-rearing was increased by at least a factor of three, and in some sublines the sensitive region was enlarged to include the entire vertical meridian. CONCLUSIONS: A complex pattern of light-induced damage outcomes was identified in S334ter (sublines 4 and 9) and P23H (sublines 2 and 3) rats. The relative susceptibilities of each subline to damage by light were different, even within the same transgene, but consistent factors included a sensitive region in the superior hemisphere and potentiation by dark-rearing.

Susceptibility to retinal light damage in transgenic rats with rhodopsin mutations.

PURPOSE: To determine relative light-induced retinal damage susceptibility in transgenic rats expressing mutations in the N- or C-terminal region of rhodopsin. METHODS: Heterozygous transgenic rats, including P23H sublines 2 and 3 and S334ter sublines 4 and 9, were reared in dim cyclic light or in darkness before visible light exposure starting at various times of the day or night. Before exposure to light, some rats were given the synthetic antioxidant dimethylthiourea (DMTU). At various times after intense light treatment, rats were killed for determinations of rhodopsin and retinal DNA recovery, DNA fragmentation patterns, and Northern blot analysis of retinal heme oxygenase (HO)-1 and interphotoreceptor retinol binding protein (IRBP). Rod outer segments (ROSs) were isolated for Western blot analysis of rhodopsin using N- and C- terminal-specific monoclonal antibodies. RESULTS: All rats incurred greater photoreceptor cell damage from exposure to light starting at 1 AM than from exposure at 5 PM. Among cyclic-light-reared rats, P23H line 3 animals were more susceptible to light-induced damage than P23H line 2 animals. S334ter rats exhibited retinal light damage profiles similar to those in normal rats. Dark-rearing potentiated retinal damage by light. However, dark-rearing alone prolonged photoreceptor cell life in P23H rats, but had no such effect in S334ter animals. DMTU pretreatment was effective in preventing or reducing light-induced retinal damage in all transgenic rats. S334ter rat ROSs contained the truncated form of rhodopsin. Intense light exposure resulted in DNA ladders typical of apoptotic cell death and the simultaneous induction of retinal HO-1 mRNA and reduced expression of IRBP. CONCLUSIONS: Light-induced retinal damage in transgenic rats depends on the time of day of exposure to light, prior light-or dark-rearing environment, and the relative level of transgene expression. Retinal light damage leads to apoptotic visual cell loss and appears to result from oxidative stress. These results suggest that reduced environmental lighting and/or antioxidant treatment may delay retinal degenerations arising from rhodopsin mutations.

Evidence that light modulates protein nitration in rat retina.

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.

Evidence for a circadian rhythm of susceptibility to retinal light damage.

This study investigated a possible circadian rhythm of light damage susceptibility in photoreceptors of both cyclic light-reared and dark-reared rats. A single exposure to intense green light was administered, beginning either in the early light period, the late light period or the dark period. In some animals exposed in the dark period, the synthetic antioxidant dimethylthiourea was administered before or after the onset of intense light exposure. Retinas were examined either immediately after exposure or after 2 weeks of recovery in darkness. Rod outer segment length and outer nuclear layer thickness measurements were used to assess light damage, along with qualitative analysis of swelling and disruption of the outer retinal layers. In all animals, retinal light damage was the most severe when intense light exposure began during the dark period. However, this severe damage was significantly reduced by pretreatment with the antioxidant. In a separate set of unexposed animals, fluctuations in plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations followed the same time course, regardless of the light regime during rearing. Our data support the notion of a circadian rhythm of light damage susceptibility that peaks in the dark period and yet can be modulated by the exogenous administration of an antioxidant.