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M. A. Mandal, J.-T. A. Tran, L. Zheng, R. S. Brush; Sphingolipid Signaling in Retinal Cell Death. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5189.
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Bio-active sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate (S1P) act as cellular second messengers in both autocrine and paracrine fashion and signal for cell death and survival. Ceramide-mediated photoreceptor cell death is recently being unraveled from various stress models and genetic mutations. Removal of cellular free ceramide rescued drosophila photoreceptors from cell death, and mutations in CERKL (Ceramide kinase like) gene cause RP-26 (Retinitis Pigmentosa 26). Ceramides have also been shown to be involved in retinal photoreceptor cell death from oxidative stress in vitro. On the other hand, S1P signals through sphingolipid G-protein-coupled receptors, also known as EDGs (endothelial differentiation genes). S1P signaling is involved in retinal angiogenesis and choroidal neovascularization. The purpose of this study was to determine the level and localization of bio-active sphingolipids and their receptors in the retina, and expression of their biosynthetic genes in normal and stressed retina or retinal cells.
Expression of ceramide and S1P metabolic genes were determined by qRT-PCR from the albino rat retina at different time points after exposure to intense light (2700 lux) for 1-6 h. Similarly, the expression profile of these genes was determined in H2O2-stressed 661W cells. Localization and up-regulation of retinal free ceramide and Sphingosine kinase (SK, which generates S1P from sphingosine) were determined by immunohistochemistry and Western blotting. Activity of neutral and acidic sphingomyelinase (SMase) enzyme was determined in both retinal and 661W samples.
SMase, which generates free ceramide from membrane sphigomyelin, rapidly activated after light exposure (within 1h) and returned to basal level quickly. It regained a higher level at 12h after the exposure. In H2O2-stressed 661W cells, increased SMase activity was observed within 10 min of treatment. Intense light stress significantly up-regulated the expression of S1P metabolic genes in the retina (Sphk1, Sgpp1 and Sgpl1)and also S1P receptors, Edg3 and Edg5. Ceramide biosynthetic genes, Spt2, Gcs, Ck, Lass4, and Lass2 were also up-regulated. Increased labeling of ceramide and SK was detected in the retina and RPE cells, respectively, after light-stress.
Alterations occured in the expression and activity of ceramide and S1P biosynthetic genes and proteins; increased labeling of ceramide in retina and SK in RPE indicate ceramide and S1P mediated cell death and differentiation pathway is involved in the light induced retinal degeneration.
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