Expression and localization of CERKL in the mammalian retina, its response to light-stress, and relationship with NeuroD1 gene.

Mutations in the Ceramide kinase like (CERKL) gene are associated with retinitis pigmentosa (RP26) and cone-rod dystrophy. CERKL is homologous to Ceramide kinase (CERK), and its function is still unknown. The purpose of this study was to test the expression and distribution of this gene and its protein in rat and in mouse tissues, in light-stressed rat retinas and in the retinas of NeuroD1 knock-out mice to understand the role of CERKL in the retina. Expression of Cerkl and Cerk mRNA was determined by quantitative RT-PCR. Expression of the protein was determined by Western blotting with anti-CERKL antibody. Localization of the protein was determined by using immunofluorescence microscopy. With qRT-PCR, we revealed that the relative mRNA expression of Cerkl was the highest in the retina among all the rat tissue tested; it was >10-fold higher than in the brain. On the other hand, Cerk has ubiquitous expression and its relative abundance is >2 fold of Cerkl in the retina. Cerkl was expressed minimally in the developing mouse eyes and reached a peak at retinal maturity at 2 months. Western blots of retinal tissues revealed two major CERKL protein bands: 59 kDa (C1) and 37 kDa (C2). However, only C2 CERKL was found in the rat retinal rod outer segment (ROS) at level of that was not changed in light vs. dark adaptation. In the light-stressed retina, expression of Cerkl mRNA increased significantly, which was reflected in only on C2 CERKL protein. The CERKL protein localized prominently to the ganglion cells, inner nuclear layers (INL), retinal pigment epithelial (RPE) cells, and photoreceptor inner segments in the retinal sections. Nuclear localization of CERKL was not affected in RPE, INL and the ganglion cell layers in the light-stressed retina; however, the perinuclear and outer segment locations appear to be altered. In the NeuroD1 knock-out mouse retina, the expression of Cerkl mRNA and protein decreased and that decrease also pertains to C2 CERKL. In conclusion, the retina had the highest level of Cerkl mRNA and protein expression, which reached its maximum in the adult retina; CERKL localized to ROS and RPE cells and the light-adaptation did not change the level of CERKL in ROS; light-stress induced Cerkl expression in the retina; and its expression decreased in NeuroD1 knock-out retina. Thus, CERKL may be important for the stress responses and protection of photoreceptor cells.

Pattern of expression of p53, its family members, and regulators during early ocular development and in the post-mitotic retina.

PURPOSE: Because of its role in cell cycle regulation and apoptosis, p53 may be involved in maintaining the post-mitotic state of the adult eye. To shed light on the role of p53 in retinal development and maintenance, this study investigated the pattern of expression of p53, its family members, and its regulators during the development of the mouse eye. METHODS: Relative quantitative real-time PCR (qRT-PCR) was used to determine the steady-state levels of target transcripts in RNA extracted from wild-type mouse whole eyes or retinas between embryonic day (E) 15 and post-natal day (P) 30. Immunoblotting was used to compare the steady-state levels of the protein to that of the transcript. RESULTS: Transcript and protein levels for p53 in the eye were highest at E17 and E18, respectively. However, both p53 transcript and protein levels dropped precipitously thereafter, and no protein was detected on immunoblots after P3. Expression patterns of p63, p73, Mdm2, Mdm4, and Yy1 did not follow that of p53. Immunohistochemistry analysis of the developing eye showed that both p53 and Mdm2 are abundantly expressed at E18 in all layers of the retinal neuroblast. CONCLUSIONS: Downregulation of p53 in the post-mitotic retina suggests that, although p53 may be involved in ocular and retinal development, it may play a minimal role in healthy adult retinal function.

X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice.

PURPOSE: To examine an immortalized mouse retinal cell line (661W) for markers characteristic of photoreceptor cells and validate its photoreceptor origin. METHODS: The 661W cells were cloned from retinal tumors of a transgenic mouse line that expresses the simian virus (SV) 40 T antigen under control of the human interphotoreceptor retinol-binding protein (IRBP) promoter. Morphologic, immunocytochemical, and immunoblot analyses were performed to characterize these cells. Total cellular protein was used for immunoblot analysis of various photoreceptor-specific proteins. RESULTS: 661W cells grew as a monolayer and exhibited processes characteristic of neuronal cells. Immunoblot analysis showed that 661W cells expressed SV40 T antigen, blue and green cone pigments, transducin, and cone arrestin. Immunocytochemical detection of blue and green opsins showed distribution throughout the cell, the nucleus included. However, these cells did not express rod-specific antigens, such as opsin and arrestin or rod- and cone-specific proteins such as phosducin, peripherin/rds, and ROM1. Furthermore, the cells did not express RPE65, a cone- and RPE-cell-specific protein. CONCLUSIONS: 661W cells demonstrate cellular and biochemical characteristics exhibited by cone photoreceptor cells. These cells also resemble neuronal cells with their spindlelike processes and should serve as a useful alternative in vitro model for the study of cone photoreceptor cell biology and associated diseases.