Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.

Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.

beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Ultrastructural findings in solar retinopathy

This study documents the ultrastructural findings in a case of solar retinopathy, 6 days after sungazing. A malignant melanoma of the choroid was diagnosed in a 65-year-old man. On fundoscopy, the macula was normal. The patient agreed to stare at the sun prior to enucleation. A typical solar retinopathy developed, characterised by a small, reddish, sharply circumscribed depression in the foveal area. Structural examination of the fovea and parafovea revealed a spectrum of cone and rod outer segment changes including vesiculation and fragmentation of the photoreceptor lamellae and the presence of discrete 100-120 nm whorls within the disc membranes. Many photoreceptor cells, particularly the parafoveal rods, also demonstrated mitochondrial swelling and nuclear pyknosis. Scattered retinal pigment epithelial cells in the fovea and parafovea showed a degeneration characterised by loss of plasma membrane specialisations, swelling of the smooth endoplasmic reticulum and changes in the fine structure of the lipofuscin granules. The good visual prognosis in solar retinopathy was attributed to the resistance of the foveal cone cells to photochemical damage.

Early ultrastructural changes after low-dose X-irradiation in the retina of the rat

In this study Lister rats were given doses of X-rays ranging from 200-2,000 Rads to the retina of one eye, sacrificed at various time intervals between one hour and one month later and the irradiated eye processed for electron microscopy. The rod photoreceptor cells were by far the most radiosensitive cells in the retina, their outer segments showing distinctive membrane damage at one hour after 200 Rads of X-rays. Photoreceptor cell death was not seen at doses less than 1,000 Rads in the time period of the experiment. The retinal pigment epithelial (RPE) cells showed damage in the form of mitochondrial swelling but only in doses over 500 Rads. Retinal pigment epithelial cell loss did not occur under 2,000 Rads. The inner retinal neurones, glial elements and the retinal vasculature did not show any ill effects in the time period of this study.

An ultrastructural study of retinal photoreceptor degeneration associated with bronchial carcinoma

We studied both eyes of a 66-year-old man with retinal degeneration and oat cell carcinoma of the bronchus. Retinal degeneration was most marked peripheral to the parafovea where photoreceptor cells and their outer segments were absent. Within the parafovea, photoreceptor cells remained but rod outer segments were absent and cone outer segments were fragmented and disorganized. The retinal pigment epithelium contained many immature melanin granules within melanolysosomes, suggesting abnormal melanin synthesis and resorption. We suggest that a pharmacologically active substance resembling a hormone produced by the tumor increased melanin synthesis in the pigment epithelium and that the increased melanin content in these cells compromised their ability to phagocytose and maintain normal turnover of photoreceptor outer segments. We believe these changes led to photoreceptor outer segment loss and subsequent degeneration of the photoreceptor cells.

Projection structure of frog rhodopsin in two crystal forms.

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with a p2 lattice (a = 32 A, b = 83 A, gamma = 91 degrees) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger p22(1)2(1) lattices (a = 40 A, b = 146 A, gamma = 90 degrees). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-A resolution for the p22(1)2(1) form and to 6-A resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-A map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

Transcorneal Electrical Stimulation Shows Neuroprotective Effects in Retinas of Light-Exposed Rats

PURPOSE. To examine the effects of transcorneal electrical stimulation (TES) on retinal degeneration of light-exposed rats. METHODS. Thirty-three Sprague Dawley albino rats were divided into three groups: STIM (n = 15) received 60 minutes of TES, whereas SHAM (n = 15) received identical sham stimulation 2 hours before exposure to bright light with 16,000 lux; healthy animals (n = 3) served as controls for histology. At baseline and weekly for 3 consecutive weeks, dark-and light-adapted electroretinography was used to assess retinal function. Analysis of the response versus luminance function retrieved the parameters Vmax (saturation amplitude) and k (luminance to reach 1/2Vmax). Retinal morphology was assessed by histology (hematoxylin-eosin [HE] staining; TUNEL assay) and immunohistochemistry (rhodopsin staining). RESULTS. Vmax was higher in the STIM group compared with SHAM 1 week after light damage (mean intra-individual difference between groups 116.06 mu V; P = 0.046). The b-wave implicit time for the rod response (0.01 cd.s/m(2)) was lower in the STIM group compared with the SHAM group 2 weeks after light damage (mean intra-individual difference between groups 5.78 ms; P = 0.023); no other significant differences were found. Histological analyses showed photoreceptor cell death (TUNEL and HE) in SHAM, most pronounced in the superior hemiretina. STIM showed complete outer nuclear layer thickness preservation, reduced photoreceptor cell death, and preserved outer segment length compared with SHAM (HE and rhodopsin). CONCLUSIONS. This sham-controlled study shows that TES can protect retinal cells against mild light-induced degeneration in Sprague Dawley rats. These findings could help to establish TES as a treatment in human forms of retinal degenerative disease. (Invest Ophthalmol Vis Sci. 2012;53:5552-5561) DOI: 10.1167/iovs.12-10037

Intraflagellar transport in the developing and mature vertebrate retina

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia. In this study we analyzed the subcellular localization of IFT proteins in retinal cells by correlative high-resolution immunofluorescence and immunoelectron microscopy. The rod photoreceptor cell was used as a model system to analyze protein distribution in cilia. To date the expression of IFT proteins has been described in the ciliary region without deciphering the precise spatial and temporal subcellular localization of IFT proteins, which was the focus of my work. rnThe establishment of the pre-embedding immunoelectron method was an important first step for the present doctoral thesis. Results of this work reveal the differential localization of IFT20, IFT52, IFT57, IFT88, IFT140 in sub-ciliary compartments and also their presence in non-ciliary compartments of retinal photoreceptor cells. Furthermore, the localization of IFT20, IFT52 and IFT57 in dendritic processes of non-ciliated neurons indicates that IFT protein complexes also operate in non-ciliated cells and may participate in intracellular vesicle trafficking in eukaryotic cells in general.rnIn addition, we have investigated the involvement of IFT proteins in the ciliogenesis of vertebrate photoreceptor cilia. Electron microscopy analyses revealed six morphologically distinct stages. The first stages are characterized by electron dense centriolar satellites and a ciliary vesicle, while the formation of a ciliary shaft and of the light sensitive outer segment disks are features of the later stages. IFT proteins were expressed during all stages of photoreceptor cell development and found to be associated with the ciliary apparatus. In addition to the centriole and basal body IFT proteins are present in the photoreceptor cytoplasm, associated with centriolar satellites, post-Golgi vesicles and with the ciliary vesicle. Therewith the data provide an evidence for the involvement of IFT proteins during ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium of photoreceptor cells. Moreover, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis indicates roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella. rn

Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.