Bovine Retina Research Articles

Neuroprotective Actions of Hydrogen Sulfide-Releasing Compounds in Isolated Bovine Retinae

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

Analysis of Intact In Vivo Peroxidized Phospholipids from Bovine Retina via LC-MS/MS and GC-MS/MS Using Autoxidized Retina Reference Standards.

Highly unsaturated fatty acids (HUFAs) are vulnerable to oxygen attack, thus making HUFA-rich, high metabolic rate/reactive oxygen species (ROS)-generating neurological tissue particularly susceptible to increased oxidative stress. Lipid oxidation is a putative early stage marker of neurodegenerative diseases, suggesting that reliable monitoring of oxidized neural lipids in vivo reveals early pathogenesis. Here, we present a novel methodology to detect and quantify intactin vivo ROS-driven peroxidized phospholipids (LPOx-PLs) in bovine retina extract. A protocol for preparing autoxidized pure phospholipids (PLs) and complex retinal extracts served as reference standards and was adapted to enable analytical parameter development. Fatty acid profiles of bovine retinas were first established with routine gas chromatography (GC) methods and used to customize mass spectrometry scanning for major HUFA-carrying PLs in the retinal extract. Targeted multiple reaction monitoring (MRM) scanning via triple quadrupole tandem mass spectrometry detected native (unoxidized) and oxidation-damaged PL regardless of the position of the O or O2 addition along the acyl chains and enabled quantification of relative signals from intact native and oxidized PL (5%-10% CV). MRM-triggered information-dependent acquisition (IDA) spectra confirmed the structure of peroxidized PLs, revealing that peroxidized species (+O-OH) dominated over single O-added species in vitro and in vivo. Positive identification and relative quantification are reported for 12 selected in vivo native and peroxidized phosphatidylcholines and phosphatidylethanolamines. These results enable future studies of the initial peroxidation due to toxins, genetics, or other initiating events influencing in vivo oxidation levels and potentially the effectiveness of strategies to mitigate this mechanism of action.

VEGF-A165a and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells

Exposure to VEGF-A165a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A165a and this was more pronounced at lower concentrations of VEGF-A165a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A165a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.

Photovoltaic, wireless wide‐field epiretinal prosthesis to treat retinitis pigmentosa

AbstractPurposeTo develop and evaluate a photovoltaic, wireless wide‐field epiretinal prosthesis for the treatment of retinitis pigmentosa.MethodsA mosaic array of thinned silicon‐based photodiodes with integrated thin‐film stimulation electrodes was fabricated with a flexible polyimide substrate film to form a film‐based miniaturized electronic system with wireless optical power and signal transmission and integrated electrostimulation. Manufactured implants were characterized with respect to their optoelectronic performance and biocompatibility following DIN EN ISO 10993.ResultsA 14 mm diameter prosthesis containing 1276 pixels with a maximum sensitivity at a near infrared wavelength of 905 nm and maximized stimulation current density 30–50 μm below the electrodes was developed for direct activation of retinal ganglion cells during epiretinal stimulation. Fabricated prostheses demonstrated mucosal tolerance and the preservation of both metabolic activity, proliferation and membrane integrity of human fibroblasts as well as the retinal functions of bovine retinas. Illumination of the prosthesis, which was placed epiretinally on an isolated perfused bovine retina, with infrared light resulted in electrophysiological recordings reminiscent of an a‐wave (hyperpolarization) and b‐wave (depolarization).ConclusionsA photovoltaic, wireless wide‐field epiretinal prosthesis for the treatment of retinitis pigmentosa using near infrared light for signal transmission was designed, manufactured and its biocompatibility and functionality demonstrated in vitro and ex vivo.

Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.

Identifying Ciliary Proteins in Mammalian Retinas using a Gentle Extraction Method.

Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood (Wheway et al., 2014). Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliopathy disease. This study shows our method can be used to isolate retinal primary cilia to obtain sufficient quantities of native protein samples.

The Metabolism of 2-arachidonoylglycerol in Rod Outer Segments Is Modulated by Proteins Involved in the Phototransduction Process.

We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPβS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.

The Bovine Ex Vivo Retina: A Versatile Model for Retinal Neuroscience.

The isolated ex vivo retina is the standard model in retinal physiology and neuroscience. During isolation, the retina is peeled from the retinal pigment epithelium (RPE), which plays a key role in the visual cycle. Here we introduce the choroid-attached bovine retina as an in vivo-like model for retinal physiology. We find that-in the bovine eye-the choroid and retina can be peeled from the sclera as a single thin sheet. Importantly, the retina remains tightly associated with the RPE, which is sandwiched between the retina and the choroid. Furthermore, bovine tissue is readily available and cheap, and there are no ethical concerns related to the use of animals solely for research purposes. We combine multi-electrode array and single-cell patch-clamp recordings to characterize light responses in the choroid-attached bovine ex vivo retina. We demonstrate robust and consistent light responses in choroid-attached preparations. Importantly, light responses adapt to different levels of background illumination and rapidly recover from photobleaching. The choroid-attached retina is also thin enough to permit targeted electrophysiological recording from individual retinal neurons using standard differential interference contrast microscopy. We also characterize light responses and membrane properties of bovine retinal ganglion cells and compare data obtained from bovine and murine retinas. The choroid-attached retinal model retains the advantages of the isolated retina but with an intact visual cycle and represents a useful tool to elucidate retinal physiology.

Mammalian Animal and Human Retinal Organ Culture as Pre-Clinical Model to Evaluate Oxidative Stress and Antioxidant Intraocular Therapeutics.

Oxidative stress (OS) is involved in the pathogenesis of retinal neurodegenerative diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) and an important target of therapeutic treatments. New therapeutics are tested in vivo despite limits in terms of transferability and ethical concerns. Retina cultures using human tissue can deliver critical information and significantly reduce the number of animal experiments along with increased transferability. We cultured up to 32 retina samples derived from one eye, analyzed the model's quality, induced OS, and tested the efficiency of antioxidative therapeutics. Bovine, porcine, rat, and human retinae were cultured in different experimental settings for 3-14 d. OS was induced by a high amount of glucose or hydrogen peroxide (H2O2) and treated with scutellarin, pigment epithelium-derived factor (PEDF), and/or granulocyte macrophage colony-stimulating factor (GM-CSF). The tissue morphology, cell viability, inflammation, and glutathione level were determined. The retina samples showed only moderate necrosis (23.83 ± 5.05 increased to 27.00 ± 1.66 AU PI-staining over 14 d) after 14 days in culture. OS was successfully induced (reduced ATP content of 288.3 ± 59.9 vs. 435.7 ± 166.8 nM ATP in the controls) and the antioxidants reduced OS-induced apoptosis (from 124.20 ± 51.09 to 60.80 ± 319.66 cells/image after the scutellarin treatment). Enhanced mammalian animal and human retina cultures enable reliable, highly transferable research on OS-triggered age-related diseases and pre-clinical testing during drug development.

Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.

ICG-mediated photodisruption of the inner limiting membrane enhances retinal drug delivery.

Many groundbreaking therapies for the treatment of blindness require delivery of biologics or cells to the inner retina by intravitreal injection. Unfortunately, the advancement of these therapies is greatly hampered by delivery difficulties where obstruction of the therapeutics at the inner limiting membrane (ILM) represents the dominant bottleneck. In this proof-of-principle study, we explore an innovative light-based approach to locally ablate the ILM in a minimally invasive and highly controlled manner, thus making the ILM more permeable for therapeutics. More specifically, we demonstrate that pulsed laser irradiation of ILM-bound indocyanine green (ICG), a clinically applied ILM dye, results in the formation of vapor nanobubbles which can disrupt the bovine ILM as well as the extraordinary thick human ILM. We have observed that this photodisruption allows for highly successful retinal delivery of model nanoparticles which are otherwise blocked by the intact ILM. Strikingly, this treatment is furthermore able of enhancing the efficacy of mRNA-loaded lipid nanoparticles within the bovine retina by a factor of 5. In conclusion, this study provides evidence for a light-based approach to overcome the ILM which has the potential to improve the efficacy of all retinal therapies hampered by this delivery barrier.

Impairment of the Retinal Endothelial Cell Barrier Induced by Long-Term Treatment with VEGF-A165 No Longer Depends on the Growth Factor's Presence.

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A165 depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a measure of permeability—was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A165 protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of β-catenin were only elevated on day two. Extracellular levels of VEGF-A—measured by ELISA—were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A165 to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled.

The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.

In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.

CNN-based CP-OCT sensor integrated with a subretinal injector for retinal boundary tracking and injection guidance.

.Significance: Subretinal injection is an effective way of delivering transplant genes and cells to treat many degenerative retinal diseases. However, the technique requires high-dexterity and microscale precision of experienced surgeons, who have to overcome the physiological hand tremor and limited visualization of the subretinal space.Aim: To automatically guide the axial motion of microsurgical tools (i.e., a subretinal injector) with microscale precision in real time using a fiber-optic common-path swept-source optical coherence tomography distal sensor.Approach: We propose, implement, and study real-time retinal boundary tracking of A-scan optical coherence tomography (OCT) images using a convolutional neural network (CNN) for automatic depth targeting of a selected retinal boundary for accurate subretinal injection guidance. A simplified 1D U-net is used for the retinal layer segmentation on A-scan OCT images. A Kalman filter, combining retinal boundary position measurement by CNN and velocity measurement by cross correlation between consecutive A-scan images, is applied to optimally estimate the retinal boundary position. Unwanted axial motions of the surgical tools are compensated by a piezoelectric linear motor based on the retinal boundary tracking.Results: CNN-based segmentation on A-scan OCT images achieves the mean unsigned error (MUE) of () using an ex vivo bovine retina model. GPU parallel computing allows real-time inference () and thus real-time retinal boundary tracking. Involuntary tremors, which include low-frequency draft in hundreds of micrometers and physiological tremors in tens of micrometers, are compensated effectively. The standard deviations of photoreceptor (PR) and choroid (CH) boundary positions get as low as when the depth targeting is activated.Conclusions: A CNN-based common-path OCT distal sensor successfully tracks retinal boundaries, especially the PR/CH boundary for subretinal injection, and automatically guides the tooltip’s axial position in real time. The microscale depth targeting accuracy of our system shows its promising possibility for clinical application.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R).

PurposeTo examine the contribution of pigment epithelium-derived factor receptor (PEDF-R) to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.MethodsMice in which PNPLA2 was conditionally knocked out (cKO) in the RPE were generated. Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POSs were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse–chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.ResultsThe RPE of the cKO mice accumulated lipids, as well as more abundant and larger rhodopsin particles, compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knockdown also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

Hydrogen sulfide is synthesized endogenously in both retinal artery and retina mostly via CSE

Hydrogen sulfide (H2S) is an important gasotransmitter expressed in various tissues of the organism, including the eye. It is known that H2S is localized especially in the retina and corneal layers in bovine eye. The enzymes that mediate H2S synthesis are 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Herein, we aimed to investigate the concentration levels and distribution profiles of these enzymes in bovine retina and retinal artery. Enzyme levels were measured by ELISA and distribution were determined by immunofluorescence microscopic analysis. Much higher concentrations of CBS and CSE have been detected in the retinal artery compared to the retina. In both tissues, particulary 3-MST was found at the lowest level while, CSE was determined to be the most abundant enzyme among the others. CBS distribution was shown in both endothelial and smooth muscle layers, while CSE was seen especially in the endothelial layer of the retinal artery. In the retina, CBS and CSE were expressed in cone-basil cells and retinal ganglion cells, while CSE was also present in bipolar cells. Our results indicated that H2S is synthesized endogenously in ocular tissues. The widespread expression of H2S synthesizing enzymes in the retina and retinal artery of the bovine eye, which has anatomical similarities with the human eye, may suggest a protective role for H2S against retinal vascular diseases as well as a regulatory role in the retinal vascular tone.

TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments.

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

Purification of a Src family tyrosine protein kinase from bovine retinas.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.