Light induced and circadian effects on retinal photoreceptor cell crystallins.

Crystallins in the retina may serve a chaperone-like protective function. In this study we measured mRNA levels for alpha-, beta- and gamma-crystallins in rat retinas following treatment with potentially damaging levels of light. We also determined crystallin protein patterns in photoreceptor cell rod outer segments (ROSs) isolated from rats exposed to intense light. Weanling albino rats were maintained in a dim cyclic light environment or in darkness for 40days. At P60 animals were treated with intense visible light, for as long as 8h, beginning at various times of the day or night. Retinas were excised immediately after light treatment and used for quantitative RT-PCR, or to prepare ROSs for western analysis. Some eyes were frozen in OCT for crystallin immunohistochemistry. Intense light exposure led to increases in mRNA expression for all retinal crystallins and to changes in ROS crystallin immunoreactivity. These light-induced changes were found to depend on the time of day that exposure started, duration of light treatment and previous light rearing history. We suggest that crystallin synthesis in retina exhibits a dependence on both light stress and circadian rhythm and that within photoreceptor cells crystallins appear to migrate in a light-independent, circadian fashion.

Genetic, age and light mediated effects on crystallin protein expression in the retina.

To probe for possible relationships between retinal crystallins and retinal degenerations, protein expression was compared in normal Sprague-Dawley rats, treated or not with intense light, Royal College of Surgeons (RCS) rats and transgenic rats expressing rhodopsin mutations. Rats were reared in dim cyclic light for 21-75 days. Photoreceptor cell DNA levels were determined at various ages to assess the rates of visual cell loss. 1D- and 2D-gel electrophoresis was used to profile retinal protein expression. Crystallins were identified by western analysis and by tandem mass spectrometry. In normal rat retinas, alpha, beta and gamma crystallins were present, although alphaA- and gamma-crystallins exhibited some increase with age. As measured by DNA levels, the rate of genetically induced photoreceptor cell loss was greater in rats with faster degenerating retinas (RCS, S334-ter Line 4, P23H Line 3) than in rats with slower degenerating retinas (S334-ter Line 9, P23H Line 2). In genetic models of retinal degeneration increased levels of immunoreactivity for all crystallins, especially alphaA-insert, correlated with the different rates of photoreceptor loss. In the light induced degeneration model alphaA-insert was unchanged, truncated alphaB-crystallin levels were increased and gamma-crystallins were greatly reduced. In the RCS rat retina 16 different crystallins were identified. Our data suggests that an increase in crystallin expression occurs during various retinal degenerations and that the increases may be related to the severity, type and onset of retinal degeneration.

Coordinated changes in classes of ribosomal protein gene expression is associated with light-induced retinal degeneration.

PURPOSE: To identify genes with altered expression levels in the degenerating retina in a light-induced retinal degeneration (LIRD) model. METHODS: Adult Sprague-Dawley rats were exposed to intense green light for 4 hours. After this treatment, the retinas were excised, RNA was extracted, and a cDNA library was prepared. The cDNA library was differentially cross-screened with probes representing 0-hour and 4-hour light-exposed rat retina. Transcripts with altered expression levels were sequenced and expression was confirmed by Northern blot analysis. Gene-specific primers were designed and used to examine the expression levels of other genes involved in protein synthesis. Promoter sequences of the ribosomal-binding protein (Rbp) genes were analyzed for transcription-binding sites. RESULTS: Of the 10,000 clones that were initially screened, 41 exhibited altered expression levels. Six of these corresponded to five known Rbp genes. Six additional Rbp genes were also examined. In total, 9 of 11 Rbp genes exhibited an increase in expression levels in response to a 4-hour light exposure. In contrast, the transcript levels of elongation factor 1alpha1 and 18S rRNA did not increase. The most abundant transcription factor-binding sites conserved in the promoter regions of all Rbp genes examined in this study include AP-1, Oct-1, V-myb, USF, Pax-4, and the FOX family of transcription factors. CONCLUSIONS: The results indicate that light-induced retinal degeneration (LIRD) is associated with increased expression of specific Rbp genes. These Rbp genes may be involved in mediating visual cell loss in LIRD through a translational or an extraribosomal mechanism.

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.

Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats.

PURPOSE: To determine whether dietary-induced alterations in the long-chain polyunsaturated fatty acid content of retinal rod outer segments (ROS) of P23H rats, a transgenic model of retinitis pigmentosa (RP), prolongs photoreceptor cell life. METHODS: Heterozygous P23H and normal Sprague-Dawley rats were fed a standard house diet or a diet deficient in 18:3n-3. Diet-deficient rats were given supplements of either linseed oil (high in 18:3n-3) or fish oil (high in 20:5n-3). ROS fatty acid profiles and serum fatty acids were determined by gas chromatography. Serum cholesterol was evaluated by HPLC. Retinal damage was assessed by measuring whole-retina rhodopsin and DNA content before and after exposure to high-intensity light. RESULTS: The retinas of 60 day old, cyclic-light-reared, P23H transgenic rats contained 50% of the rhodopsin and 75% of the DNA content found in control Sprague-Dawley rats. Eight hours of intense light had little effect on the rhodopsin or DNA content in the Sprague-Dawley rats, but resulted in rhodopsin and DNA losses of nearly 70%, compared to controls, in P23H animals fed either a standard or an 18:3n-3-deficient diet. Supplementation with linseed oil resulted in small, statistically insignificant, increases in the rhodopsin and DNA losses, which occurred after exposure to intense light, in P23H transgenics. In unexposed animals, supplementation with linseed oil or fish oil had no effect on either rhodopsin or DNA levels in P23H rats or in Sprague-Dawley controls. On standard diet, the ROS 22:6n-3 (DHA) content in P23H rats was lower than that of control animals. DHA decreased in both groups when an 18:3-deficient diet was fed. The reduction was greater in controls than in P23H transgenics, but a concomitant increase in 22:5n-6 was nearly the same in both groups. Supplementation of the 18:3-deficient diet with linseed oil or fish oil in P23H rats resulted in a ROS fatty acid profile comparable to that of Sprague-Dawley rats raised on a standard diet. Serum DHA and 22:5n-6 levels were low in both groups. No significant differences in serum cholesterol were observed as a function of genotype or diet. CONCLUSIONS: Heterozygous P23H rats are capable of forming ROS DHA from dietary fatty acid precursors found in linseed oil (18:3n-3) or fish oil (20:5n-3). Under all dietary conditions, P23H transgenics are highly susceptible to retinal damage from exposure to intense light. Although levels of DHA in the ROS of P23H rats could be altered by dietary manipulation, only small changes in photoreceptor cell survival, as measured by whole-retina rhodopsin and DNA content, were observed. The lower-than-normal levels of ROS DHA may reflect an adaptive, possibly protective, mechanism in the P23H transgenic rat model of RP.