Abstract
Abstract: :
Purpose: Exposure to high levels of visible light induces apoptosis of photoreceptors. Caspases, a family of cystein proteases, are key enzymes for the regulation of apoptosis in a variety of systems. We tested the role of several caspases in light induced photoreceptor degeneration and focused on analysis of caspase–1 using a caspase–1 knockout mouse strain. Methods: 5–6 weeks old, dark–adapted Balb–C (WT), Casp–1 KO and c–fos KO mice were exposed to 1h/5000 or 2h/15000 lux of diffuse white light. With exception of light resistant c–fos KO where Rpe65–450Met variant has been used, the two other strains had Rpe65–450Leu genotype. Retinal degeneration was monitored qualitatively by light microscopy and quantitatively by measuring release of free nucleosomes using an ELISA kit. Expression of tested genes in isolated retinas was assessed by exponential RT–PCR, ELISA and Western blotting. Results: Intense visible light induced transcription of Casp–1, Il–1ß, TNF–alpha and HO–1 in WT mice with peak at 12 h after illumination. Casp–3, 6, 7, 8, 9 and 12 were expressed in the retina but their RNA and protein levels remained unchanged up to 24h postillumination. On the protein level, Casp–1 proenzyme (46 kDa) was induced starting around 6h post–illumination with increasing levels up to 96h. This was followed by appearance of the cleaved p37 form. Both, WT and Casp–1 KO animals showed increased RNA and protein expression levels of Il–1ß after light insult. This effect was absent in light resistant c–fos KO mouse strain. Although in WT animals TNF–alpha RNA expression was induced upon light exposure, TNF–alpha protein levels were not significantly changed. Lack of caspase–1 did not protect against light damage. Light exposure induced degeneration of photoreceptors and necrotic–like morphology of pigment epithelium in the Casp–1 KO animals. Interestingly when tested in WT, the pigment epithelium showed higher Casp–1 basal levels compared to the retina as judged by Western blotting. Conclusions: Caspase–1 is activated in light induced retinal degeneration. However, the lack of caspase–1 did not rescue retinal morphology. In fact, damage in pigment epithelial cells of these animals, in mice previously observed only after blue light exposure, suggests an important physiological function of the Casp–1 in the pigment epithelium. Such a function may be the proteolytic cleavage of as yet unidentified substrate not necessarily including Il–1ß, which obviously can be cleaved in the absence of Casp–1.
Keywords: apoptosis/cell death • gene/expression • photoreceptors