Inner Segment Research Articles

A Frameshift Mutation inRPGRExon ORF15 Causes Photoreceptor Degeneration and Inner Retina Remodeling in a Model of X-Linked Retinitis Pigmentosa

To characterize the course of retinal disease in X-linked progressive retinal atrophy 2 (XLPRA2), a canine model of early onset X-linked retinitis pigmentosa (XLRP) caused by a two-nucleotide microdeletion in RPGR ORF15. The retinas of 25 XLPRA2-affected dogs (age range, 2-40.6 weeks) and age-matched control subjects were collected, fixed, and embedded in epoxy resin for morphologic evaluation or in optimal cutting temperature (OCT) medium for TUNEL assay and immunohistochemistry. Cell-specific antibodies were used to examine changes in rods and cones and to evaluate the effects of the primary photoreceptor degeneration on inner retinal cells. Abnormal development of photoreceptors was recognizable as early as 3.9 weeks of age. Outer segment (OS) misalignment was followed by their disorganization and fragmentation. Reduction in length and broadening of rod and cone inner segments (IS) was next observed, followed by the focal loss of rod and cone IS at later time points. The proportion of dying photoreceptors peaked at approximately 6 to 7 weeks of age and was significantly reduced after 12 weeks. In addition to rod and cone opsin mislocalization, there was early rod neurite sprouting, retraction of rod bipolar cell dendrites, and increased Müller cell reactivity. Later in the course of the disease, changes were also noted in horizontal cells and amacrine cells. XLPRA2 is an early-onset model of XLRP that is morphologically characterized by abnormal photoreceptor maturation followed by progressive rod-cone degeneration and early inner retina remodeling. The results suggest that therapeutic strategies for this retinal degeneration should target not solely photoreceptor cells but also inner retinal neurons.

Neuroglobin and Cytoglobin: Oxygen-Binding Proteins in Retinal Neurons

The goal of this study was to describe the detailed localization of the novel oxygen-binding molecules, neuroglobin (Ngb) and cytoglobin (Cygb), in mammalian retinas and to determine whether Ngb and Cygb are neuronal or glial proteins in the retina. Antibodies directed against Ngb and Cygb were used to examine their patterns of distribution in normal canine retinas. Immunoblot analysis was performed to verify antibody specificity and the presence of Ngb and Cygb in canine tissues. Double-labeling immunohistochemistry was performed with the Ngb and Cygb antibodies along with antibodies against neuronal (MAP-2, class III beta-tubulin (TUJ1), PKCalpha, and calretinin) and glial antigens (vimentin and CRALBP). Tissue sections were analyzed with light and confocal microscopy. Ngb and Cygb proteins were observed in different retinal cells. Cygb (but not Ngb) was also present in canine kidney, liver, lung, and heart tissue. Immunohistochemical analysis of canine retinas demonstrated Ngb immunoreactivity (IR) in the ganglion cell layer (GCL), inner (INL) and outer (ONL) nuclear layers, inner (IPL) and outer plexiform (OPL) layers, photoreceptor inner segments (IS), and retinal pigment epithelium (RPE). Ngb IR was localized within retinal neurons, but not in glia. Cygb IR was found in neurons and their processes in the GCL, IPL, INL, and OPL and within the RPE, but not in glia. Ngb and Cygb are widely distributed in retinal neurons and RPE, but not in glial cells of the canine retina. Their structure and distribution is suggestive of a possible role in oxygen transport in the mammalian retina.

Transducin Activation State Controls Its Light-dependent Translocation in Rod Photoreceptors

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.

Light-Dependent Redistribution of Arrestin in Vertebrate Rods Is an Energy-Independent Process Governed by Protein-Protein Interactions

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old transgenic pigs in which almost all rods had died, compared to faint staining in two 10-month-old control pig retinas. In 22- and 24-month-old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

An ultramicroscopic study on the distribution of Müller cell processes in the outer retinal layers of the zebrafish

The zebrafish, Brachydanio rerio, is a good model for studying the development of various organs. We have assayed the distribution pattern of Müller cell processes in zebrafish retinas by electron microscopy. In the outer nuclear (ON) layer, multiple layers of Müller cell processes were present along both sides of the large pyramidal endings of the synaptic terminals. We found that the inner segments (ISs) of the zebrafish photoreceptors (PRs), including the cones, double cones and rods, were arranged in different planes, and that the Müller cell processes formed multilayered sheaths around virtually all PR compartments except their outer segments. Thus, Müller cell processes beyond the outer limiting membrane (OLM) are more easily observed in zebrafish retina than in the retinas of other species. To our knowledge, this is the first study to show the exact ultrastructural distribution of Müller cell processes around the OLM and the PR layer in zebrafish retina.

Comparison of ultrahigh- and standard-resolution optical coherence tomography for imaging macular hole pathology and repair

Purpose To compare ultrahigh-resolution optical coherence tomography (UHR-OCT) technology to a standard-resolution OCT instrument for the imaging of macular hole pathology and repair; to identify situations where UHR-OCT provides additional information on disease morphology, pathogenesis, and management; and to use UHR-OCT as a baseline for improving the interpretation of the standard-resolution images. Design Observational and interventional case series. Participants Twenty-nine eyes of 24 patients clinically diagnosed with macular hole in at least one eye. Methods A UHR-OCT system has been developed and employed in a tertiary-care ophthalmology clinic. Using a femtosecond laser as the low-coherence light source, this new UHR-OCT system can achieve an unprecedented 3-μm axial resolution for retinal OCT imaging. Comparative imaging was performed with UHR-OCT and standard 10-μm resolution OCT in 29 eyes of 24 patients with various stages of macular holes. Imaging was also performed on a subset of the population before and after macular hole surgery. Main outcome measures Ultrahigh- and standard-resolution cross-sectional OCT images of macular hole pathologies. Results Both UHR-OCT and standard-resolution OCT exhibited comparable performance in differentiating various stages of macular holes. The UHR-OCT provided improved imaging of finer intraretinal structures, such as the external limiting membrane and photoreceptor inner segment (IS) and outer segment (OS), and identification of the anatomy of successful surgical repair. The improved resolution of UHR-OCT enabled imaging of previously unidentified changes in photoreceptor morphology associated with macular hole pathology and postoperative repair. Visualization of the junction between the photoreceptor IS and OS was found to be an important indicator of photoreceptor integrity for both standard-resolution and UHR-OCT images. Conclusions Ultrahigh-resolution optical coherence tomography improves the visualization of the macular hole architectural morphology. The increased resolution of UHR-OCT enables the visualization of photoreceptor morphology associated with macular holes. This promises to lead to a better understanding of the pathogenesis of macular holes, the causes of visual loss secondary to macular holes, the timing of surgical repair, and the evaluation of postsurgical outcome. Ultrahigh-resolution optical coherence tomography imaging of macular holes that correspond to known alterations in retinal morphology can be used to interpret retinal morphology in UHR-OCT images. Comparisons of UHR-OCT images with standard-resolution OCT images can establish a baseline for the better interpretation of clinical standard-resolution OCT images. The ability to visualize photoreceptors and their integrity or impairment is an indicator of macular hole progression and surgical outcome.

Arrestin migrates in photoreceptors in response to light: a study of arrestin localization using an arrestin-GFP fusion protein in transgenic frogs

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45–75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25–2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.

Connexin 40 expression in bovine and rat retinas.

Retinal neurons are extensively coupled through gap junction intercellular channels, but few connexin subtypes have been identified in mammalian retinal neurons. Based on previous findings that retinal gap junctional coupling is modulated by both dopamine and nitric oxide, presumably through connexin phosphorylation, we examined whether the connexin phosphoprotein subtype, connexin 40 (Cx40), was expressed in mammalian retinas. Immunostaining of rat and bovine retinas using Cx40-specific antibodies from two independent sources showed punctate staining between cells in the outer nuclear layer (ONL) and a sublayer of cells within the inner nuclear layer (INL). In addition, sparse punctate staining was detected in the ganglion cell/axon fiber layers (GCL/AFL). No punctate staining was observed in the outer (OS) or inner segment (IS) layers, and rarely in the outer plexiform layer (OPL) or inner plexiform layer (IPL). Double immunostaining of bovine retinas with antibodies to G(o), which stains bipolar cells, and to Cx40, showed little overlap, suggesting these bipolar cells do not express Cx40. Western blot analysis of alkaline-extracted bovine retinal membranes revealed Cx40 immunopositive bands of about 40 kD (monomer) and 80 kD (dimer). In both locations (monomer and dimer), the bands appeared as doublets, and their immunoreactivity was abolished when the antibody was pre-adsorbed with immunogenic Cx40 peptide. The doublet at 40 kD co-migrated with an immunopositive doublet present in heart membranes. Treatment with alkaline phosphatase altered the banding pattern of Cx40. The results suggest that the connexin phosphoprotein subtype, Cx40, is expressed within the neural layers of the mammalian retina.

Experimental retinal detachment in the cone-dominant ground squirrel retina: morphology and basic immunocytochemistry.

The cellular responses of the cone-dominant ground squirrel retina to retinal detachment were examined and compared to those in rod-dominant species. Retinal detachments were made in California ground squirrels. The retinas were prepared for light, electron, and confocal microscopy. Tissue sections were labeled with antibodies to cone opsins, rod opsin, glial fibrillary acidic protein (GFAP), vimentin, synaptophysin, cytochrome oxidase, and calbindin D 28K. Wax sections were probed with the MIB-1 antibody to detect proliferating cells. By 10 h postdetachment many photoreceptor cells in the ground squirrel already show structural signs of apoptosis. At 1 day many photoreceptors have collapsed inner segments (IS), yet others still have short stacks of outer segment discs. At 3 days there is a marked increase in the number of dying photoreceptors. Rod and medium-/long-wavelength opsins are redistributed in the cell membrane to their synaptic terminals. At 7 days photoreceptor cell death has slowed. Some regions of the outer nuclear layer (ONL) have few photoreceptor somata. IS remnants are rare on surviving photoreceptors. At 28 days these trends are even more dramatic. Retinal pigmented epithelium (RPE) cells do not expand into the subretinal space. The outer limiting membrane (OLM) appears flat and uninterrupted. Müller cells remain remarkably unreactive; they show essentially no proliferation, only negligible hypertrophy, and there is no increase in their expression of GFAP or vimentin. Horizontal cells show no dendritic sprouting in response to detachment. The speed and extent of photoreceptor degeneration in response to detachment is greater in ground squirrel than in cat retina-only a small number of rods and cones survive at 28 days of detachment. Moreover, the almost total lack of Müller cell and RPE reactivity in the ground squirrel retina is a significant difference from results in other species.

Retina-derived fetuin (RDF): Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot

PURPOSE. This study was undertaken to determine the pres-ence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. METHODS. Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. RESULTS. Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, in-ner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve pro-cesses were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicat-ing that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot con-firmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. CONCLUSIONS. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cys-teine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

The differential osmoregulation and localization of taurine transporter mRNA and Na +/ myo-inositol cotransporter mRNA in rat eyes

We studied the cellular localization and osmotic regulation of taurine transporter (TauT) mRNA in the rat eyes using in situ hybridization. TauT mRNA signals were expressed in the ciliary body, and the outer part of the inner nuclear layer (INL), the outer nuclear layer (ONL) and the inner segment (IS) of the adult rat retina. Chronic hypernatrema, induced by gavaging with 1 ml/100 g body weight of 5% NaCl every other day for 7 days, markedly increased in TauT mRNA in the retina compared with control rats. However, there was little change in TauT mRNA in the eyes in acute hypernatremic state that is induced by single injection of high concentration of NaCl. On the contrary, acute hypernatremic rats displayed markedly elevated Na +/ myo-inositol cotransporter (SMIT) mRNA in the retina and the iris-ciliary body and the lens epithelium. Under chronic hypernatremic conditions, there was no significant increase in SMIT mRNA in rat eyes. These findings suggest that TauT mRNA is osmotically regulated in vivo to protect retinal neuronal function, especially against chronic hypernatremic conditions, in contrast to rapid up-regulation of SMIT mRNA in acute hypernatremic rats.

Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors.

The immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3-amino-9-ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor outer segment (OS) layer (0.061 +/- 0.004; average +/- S.E.M.) was less than that of the inner segment (IS) layer (0.138 +/- 0.011). In light-adapted control retinas, OS labeling density (0.139 +/- 0.007) exceeded IS labeling density (0.095 +/- 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 +/- 0.006 (OS) and 0.183 +/- 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 +/- 0.004 (OS) and 0.182 +/- 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed to 3 min or 13 min of bright light and then immediately fixed.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and ERG findings in progressive rod‐cone dystrophy in a litter of Labrador Retrievers

Early ultrastructural findings of a progressive photoreceptor dystrophy and corresponding ERG findings are reported in 3 Labrador Retrievers from a litter of 7 pups bred from 2 dogs clinically and electroretinographically affected with generalized progressive retinal dystrophy. The pups were euthanized at 5, 11 and 15 months post partum. The most prominent ultrastructural finding was photoreceptor dystrophy. At 5 months the outer nuclear layer (ONL) consisted of 8-10 layers and seemed reduced in thickness, pyknotic nuclei were seen in this layer. The receptor outer segments (OS) were short and swollen. Some disorientation of OS discs occurred. In the 11-months specimen 7-8 ONL layers were identified. Overall thinning of the neuro-retina had occurred and fewer receptors compared to the 5-months specimen were present. By 15 months the ONL was further reduced to about 4 layers. Enlarged internuclear spaces were present in the ONL as well as around inner segments (IS). Phagocytic cells were frequent among remains of OS. The pigment epithelium appeared normal. The dark adapted ERG b-wave amplitudes and photopic 30 Hz flicker responses were low in comparison to controls of the same breed, and decreased with age. The condition represents a progressive rod-cone dystrophy which shares similarities with primary receptor dystrophy in man such as retinitis pigmentosa.

Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats

In this study retinas of pink-eyed 26 day-old RCS dystrophic rats were injected with normal, melanotic retinal pigment epithelial (RPE) cells into the subretinal space. The areas containing underlying photoreceptor cells were subsequently investigated by electron microscopy at 60 days and light microscopy immunocytochemistry at 60 and 120 days. Retinal regions containing viable RPE-cell grafts exhibited melanin-contained RPE cell transplants attached to Bruch's membrane and containing shed rod outer segments (ROS). Also, the ROS of these photoreceptor cells exhibited a normal-appearing structural relationship with apical membrane projections of these RPE cells. The debris zone, prominent in retinas of 60 day-old RCS dystrophic rats, was much reduced in RPE-cell grafted regions. By at least 3 months after RPE transplantation, immunostaining for Na +,K +-ATPase was demonstrated along inner segments (IS) of photoreceptor cells in grafted regions, as was also shown in control retinas, but not in non-grafted regions. Opsin immunostaining in RPE-cell grafted regions and in control retinas was detected along ROS and rod inner segments (RIS) and at the periphery of cell bodies in the outer nuclear layer. This study has shown that transplanted normal RPE cells in retinas of RCS dystrophic rats appear to have normal functional and structural characteristics at least three months after transplantation. Furthermore, the rescued PRC's exhibited a normal distribution of and immunostaining density for the membrane-bound enzyme Na +,K +-ATPase and the photopigment opsin.