Faculty Bibliography

Apoptosis is a regulated mode of single cell death that involves gene expression in many instances and occurs under physiological and pathological conditions in a large variety of systems. We briefly summarize major features of apoptosis in general and describe the occurrence of apoptosis in the retina in different situations that comprise animal models of retinitis pigmentosa, light-induced lesions, histogenesis during development, and others. Apoptosis can be separated into several phases: the induction by a multitude of stimuli, the effector phase in which the apoptotic signal is transmitted to the cellular death machinery, the excecution period when proteolytic cascades are activated, and the phagocytic removal of cellular remnants. Control mechanisms for retinal apoptosis are only beginning to be clarified. Potential apoptotic signal transducers were investigated in our laboratory, including metabolites of arachidonic acid and downstream mediators of signaling molecules such as transcription factors. Work in our laboratory revealed an essential role of the immediate-early gene product c-Fos in light-induced apoptosis. c-Fos is a member of the AP-1 family of transcription factors and, together with other members of this family, it may regulate apoptosis in the central nervous system. Expression of the c-fos gene in the retina can be evoked by light exposure and follows a diurnal rhythm. Future studies will have to clarify how light can control the expression of specific genes, and specifically, the role of c-fos and other genes of retinal apoptosis including potential target genes and signaling pathways.

BACKGROUND:Apoptosis is a gene-regulated mode of cell death which gains increasing importance in retinal pathologies such as retinitis pigmentosa, retinal detachment and proliferative vitreoretinopathy. A better understanding of the regulation of apoptosis could imply the means to reduce photoreceptor cell death and thereby provide therapeutic strategies to influence the time course of retinal diseases. Previous studies in our laboratory demonstrated that light induces apoptosis in the rat retina in vivo as a function of light dose. In several cell systems, oxidative stress including oxygenated metabolites of arachidonic acid (AA) was found to evoke apoptosis. We have observed a light-elicited release of AA and the subsequent formation of its metabolites in the rat retina. Therefore, AA and its metabolites appeared to be suitable candidates for the induction of apoptosis during light exposure. MATERIALS AND METHODS/METHODS:Isolated rat retinas were incubated for 60, 120 and 180 min, respectively, with and without the addition of 30 mumol 5S-hydroperoxyeicosatetraenoic acid (5-HPETE). Retinas were then processed for light- and electron microscopy and examined for the morphological signs of apoptosis. The rate of apoptosis in the outer nuclear layer was assessed quantitatively. RESULTS:5S-HPETE induces apoptosis of photoreceptors in the rat retina in vitro. Quantitative analysis revealed a significant increase in the rate of apoptosis of 5S-HPETE-treated retinas when compared to untreated controls. CONCLUSION/CONCLUSIONS:Arachidonic acid metabolites released upon light exposure may represent messenger candidates for apoptosis in the retina.

PURPOSE/OBJECTIVE:Cell death by apoptosis is essential for normal development and tissue homeostasis, and it is involved also in a variety of pathologic processes. Apoptosis is the final common pathway of photoreceptor cell death in retinal dystrophies and degeneration. So far, little is known about genes regulating apoptosis in the retina. The tumor-suppressor gene product p53 is a potent regulator of apoptosis in numerous systems. However, p53-independent apoptotic pathways also have been described. In this study the authors investigated the role of p53 in the light-induced apoptosis of retinal photoreceptors using mice lacking p53. METHODS:Free-moving p53-/- and p53+/+ mice were dark adapted and were exposed to 8,500 or 15,000 lux of diffuse, cool, white fluorescent light for 2 hours. Animals were killed before and immediately after light exposure or at 12 hours in darkness after light exposure. Eyes were enucleated and processed for light and electron microscopy and histochemistry (TdT-dUTP terminal nick-end labeling method). Isolated retinas were subjected to the extraction of total retinal DNA. Electroretinogram (ERG) recordings were performed at all time points. RESULTS:Morphologic, biochemical, histochemical, and ERG analysis showed that the retinas of untreated p53-/- mice and wild-type control mice were structurally and functionally indistinguishable. After exposure to diffuse white fluorescent light, light-induced photoreceptor cell death was analyzed and was found to be the same in both groups of mice. CONCLUSIONS:These data suggest that light-induced apoptosis of photoreceptors is independent of functional p53.

Death of retinal photoreceptors by apoptosis is observed under many physiological and pathological conditions such as histogenesis, retinal dystrophies and light-induced photoreceptor degeneration. To date, little is known about regulatory mechanisms for apoptosis in the retina. The tumor suppressor gene p53 is a regulator of apoptosis in a number of systems, however, p53-independent apoptosis has also been described. We have therefore investigated whether the lack of p53 influences the dark-adapted ERG in C57BL/6 p53-/- mice compared to p53+/+ control littermates under physiological (regular light-dark cycle) conditions. We also recorded ERGs at 12 to 14 h in darkness following diffuse bright light exposure to 8,000 or 15,000 lux for 2 h. ERG analysis over a range of 6 logarithmic units of light intensity revealed normal and virtually identical a-, b-, c-waves and oscillatory potentials in dark-adapted p53+/+ and p53-/- mice. After exposure to diffuse white fluorescent light strong decreases of all ERG components were found to be very similar in both genotypes. These data support the notion that the p53 protein is neither essential for normal retinal function nor for processes involved in light-induced depression of the ERG in mice.

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.

Apoptotic cell death in the retina was recently demonstrated in animal models of the hereditary human retinal dystrophy known as retinitis pigmentosa. Although recent evidence indicates that the proto-oncogene c-fos is a mediator of apoptosis, its precise role is unclear. In fact, under some conditions, c-fos may even protect against apoptotic cell death. In the retina, c-fos is physiologically expressed in a diurnal manner and is inducible by light. We previously observed a light-elicited, dose-dependent apoptotic response in rat photoreceptors. To determine whether c-fos is involved in the light-induced apoptotic pathway we have used control mice and mice lacking c-fos. We found that following dark adaptation and two hours of light exposure both groups of animals exhibited only a few apoptotic cells. However, at 12 and 24 additional hours after light exposure, apoptosis increased dramatically in controls but was virtually absent in those mice lacking c-fos. Therefore, c-fos is essential for light-induced apoptosis of photoreceptors. Notably, c-fos is continuously upregulated concomitant with apoptotic photoreceptor death in our system and in animal models of retinitis pigmentosa (Agarwal, N. et al., Invest. Ophthalmol. Vis.Sci. Suppl. 36, S638 and Rich, K.A. et al., Invest. Ophthalmol. Vis. Sci. Suppl. 35, 1833). Inhibition of c-fos expression might therefore represent a novel therapeutic strategy to retard the time course of retinal dystrophies and light-induced retinal degeneration.