Expression of the Blue-Light Receptor Cryptochrome in the Human Retina

PURPOSE. To analyze the patterns of expression of the cryptochromes, CRY1 and CRY2, in the human retina and to correlate expression of these putative blue-light receptors with nonvisual photoreceptor localization. METHODS. CRY1 and CRY2 mRNA expression was analyzed in 4-mm diameter punches of macula and midperipheral human retina by quantitative RT-PCR. CRY2 protein expression was examined by immunohistochemistry in cross sections of human retina, and its subcellular localization was determined by immunoblot analysis of fractionated human retinal extracts. RESULTS. CRY2 mRNA was 11 times more abundant than CRY1 throughout adult human retina. CRY2 immunoreactivity was detected in most cells in the ganglion cell layer (GCL) and in a subset of cells in the inner nuclear layer (INL) in both the macula and periphery. Immunoperoxidase staining further revealed that CRY2 was localized throughout the cytoplasm of cells in the GCL as well as within nuclei. This intracellular localization of CRY2 was confirmed by immunoblot analysis of fractionated human retinal extracts. CONCLUSIONS. Photopigments governing circadian photoreception have been localized to the inner retina. The relative abundance of CRY2 transcripts, coupled with CRY2 localization to the inner retina, supports a photoreceptive role for CRY2 in human retina. Furthermore, the discovery that CRY2 is also localized within the cytoplasm of some cells in the GCL, suggests it may perform a function separate from its known nuclear role in the transcriptional feedback loop underlying the molecular circadian clock

Expression of the blue light receptor cryptochrome in the human retina. Investigative Ophthalmology and Visual Science

PURPOSE. To analyze the patterns of expression of the cryptochromes, CRY1 and CRY2, in the human retina and to correlate expression of these putative blue-light receptors with nonvisual photoreceptor localization. METHODS. CRY1 and CRY2 mRNA expression was analyzed in 4-mm diameter punches of macula and midperipheral human retina by quantitative RT-PCR. CRY2 protein expression was examined by immunohistochemistry in cross sections of human retina, and its subcellular localization was determined by immunoblot analysis of fractionated human retinal extracts. RESULTS. CRY2 mRNA was 11 times more abundant than CRY1 throughout adult human retina. CRY2 immunoreactivity was detected in most cells in the ganglion cell layer (GCL) and in a subset of cells in the inner nuclear layer (INL) in both the macula and periphery. Immunoperoxidase staining further revealed that CRY2 was localized throughout the cytoplasm of cells in the GCL as well as within nuclei. This intracellular localization of CRY2 was confirmed by immunoblot analysis of fractionated human retinal extracts. CONCLUSIONS. Photopigments governing circadian photoreception have been localized to the inner retina. The relative abundance of CRY2 transcripts, coupled with CRY2 localization to the inner retina, supports a photoreceptive role for CRY2 in human retina. Furthermore, the discovery that CRY2 is also localized within the cytoplasm of some cells in the GCL, suggests it may perform a function separate from its known nuclear role in the transcriptional feedback loop underlying the molecular circadian clock. (Invest Ophthalmol Vis Sci. 2003; 44:4515-4521

Retinal Degenerative DiseasesMechanisms and Experimental Therapy /

LXI, 862 p. 188 illus., 187 illus. in color.onlin

Human RPE expression of cell survival factors. Invest Ophthalmol Vis Sci.

PURPOSE. To determine basal and tumor necrosis factor (TNF)-␣-regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-B. METHODS. Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-B or ␤-galactosidase and treated with TNF-␣ for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively. RESULTS. Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1␤-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-␣ upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-␣ in a time-dependent manner, whereas c-IAP1, survivin, Bcl-x L , and TRAF-2 protein levels were not influenced by TNF-␣ treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-␣ treatment. Overexpression of mutant IB blocked TNF-␣-induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-x L proteins were localized diffusely in RPE cytoplasm. CONCLUSIONS. Multiple RPE cell survival factors are expressed by human RPE cells. TNF-␣ regulates expression of some of these factors in an NF-B-dependent manner, whereas others are not influenced by NF-B. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). (Invest Ophthalmol Vis Sci. 2005;46: 1755-176