Characterization of Protein Kinase C in Photoreceptor Outer Segments

Abstract: Protein kinase C (PKC) has been implicated in regulating several proteins involved in phototransduction. This contribution characterizes the biochemical and immunological properties of PKC isozyme(s) in the photoreceptor outer segment. Activity measurements revealed that at least 85% of the PKC in this specialized compartment belongs to the subfamily of Ca²⁺-regulated (conventional) PKCs. Of the known Ca²⁺-dependent PKCs, only PKCα was immunodetected by western blot analysis of rod outer segment proteins. However, the ratio of immunoreactivity to enzyme activity for rod outer segment PKC was no more than 40% of that for brain PKC, using antibodies against conventional PKCs. Therefore, at least half the Ca²⁺/lipid-stimulated activity in rod outer segment preparations cannot be accounted for by the known isozymes, suggesting the presence of a previously uncharacterized isozyme. Despite extensive tests using a variety of antibodies against different domains of PKCα, PKCα could not be detected in rod outer segments by immunofluorescence of retinal sections. In summary, our data reveal that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s).     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina.     

Molecular Organization of Photoreceptor Membranes of Rod Outer Segments

A new model for the structure of rod outer segments is presented. This comprises an asymmetric membrane, comprising a lipid bilayer with rhodopsin in the hydrocarbon interior.     

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer''s solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco''s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10⁵ cells/cm². The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.     

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca²⁺), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca²⁺ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca²⁺ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (“slices”) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca²⁺ level. The protocol also allows “in-slice measurement” of absolute Ca²⁺ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca²⁺ signaling as well as the potential involvement of Ca²⁺ in photoreceptor death and retinal degeneration.     

LINC Complexes Mediate the Positioning of Cone Photoreceptor Nuclei in Mouse Retina

It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.     

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.     

Mechanistic Studies of ABCR, the ABC Transporter in Photoreceptor Outer Segments Responsible for Autosomal Recessive Stargardt Disease

ABCR is an ABC transporter that is found exclusively in vertebrate photoreceptor outer segments. Mutations in the human ABCR gene are responsible for autosomal recessive Stargardt disease, the most common cause of early onset macular degeneration. In this paper we review our recent work with purified and reconstituted ABCR derived from bovine retina and from cultured cells expressing wild type or site-directed mutants of human ABCR. These experiments implicate all-trans-retinal (or Schiff base adducts between all-trans-retinal and phosphatidylethanolamine) as the transport substrate, and they reveal asymmetric roles for the two nucleotide binding domains in the transport reaction. A model for the retinal transport reaction is presented which accounts for these experimental observations.     

A Novel Zebrafish Gene Expressed Specifically in the Photoreceptor Cells of the Retina

We have identified and characterized a novel protein from adult zebrafish retina, which we named ES1. Database search revealed that the ES1 gene has significant similarity to two genes with unknown functions: theEscherichia colisigma cross-reacting protein 27a (scrp27a) and the human KNP-I/GT335.In situhybridization and immunohistochemistry experiments showed that both ES1 mRNA and protein are expressed specifically in adult photoreceptor cells. ES1 seems to be a cytoplasmic protein. An ES1-like antigen was also detected in photoreceptor cells of goldfish with anti-ES1 antibodies. The retina specific expression and the evolutionary conservation suggest that ES1 protein may be important for maintaining normal retina structure and function.     

Ethanolamine Accumulation by Photoreceptor Cells of the Rabbit Retina

The rabbit retina accumulates ethanolamine by an overall process that has a high affinity for ethanolamine. This process is different from the choline uptake, since ethanolamine accumulation was unaffected by high choline concentrations. Autoradiography identified the major site of high-affinity uptake as the perinuclear region of the photoreceptor cells. Ethanolamine accumulated by the high-affinity uptake was not used for neurotransmission by photoreceptor cells but was used to synthesize phosphatidylethanolamine. However, only a small percentage of the accumulated ethanolamine was converted into phospholipid. The rate of phosphorylation may contribute to control of phospholipid synthesis, since choline kinase activity is much greater than ethanolamine kinase activity in the rabbit retina.     

The Photoreceptor Outer Segments of the River Lamprey (Lampreta fluviatilis). An Electron-, Fluorescence- and Light Microscopic Study

The outer segment of long and short photoreceptors in the retina of the river lamprey, Lampetra fluviatilis, were studied by light- and fluorescence microscopy together with some different electron microscopic methods. The outer segments show characteristics of both rods and cones and are suggested to represent intermediate kinds of photoreceptors.     

The Fine Structure of the Retina Studied with the Electron Microscope : IV. Morphogenesis of Outer Segments of Retinal Rods

The morphogenesis of the outer segments of retinal rods was studied mainly in the kitten before the opening of the eye, and the probable sequence of the morphogenetic stages is deduced. Since the development of retinal rods is not synchronous, the deductions were based on observations of many single and serial sections. One centriole extends ciliary tubules of about 0.5 µ long, in the growing primitive cilium. Beyond this length, each ciliary tubule becomes a row of small vesicles (called "ciliary vesicles" in this paper), which penetrate into the distal region of the cilium. Where the ciliary vesicles establish contact with the plasma membrane of the distal region of the cilium, more or less deep infoldings of the plasma membrane are observed. In the distal region can be seen rows of tubular or vesicular structures. A few of these membranous structures are continuous with the bottoms of the infoldings. At the following stage, the infoldings disappear and the ciliary vesicles lose contact with the distal plasma membrane. Nonetheless, the formation of the tubular structures continues in the distal region of the primitive outer segment. The tubular structures appear to be transformed into the primitive rod sacs by sidewise enlargement. At a subsequent time, presumably, these primitive rod sacs flatten and are rearranged into a position perpendicular to the long axis of the outer segment. The detailed structure of the basal body of the connecting cilium was also studied by means of serial sections.     

Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.     

Conservation of Docosahexaenoic Acid in Rod Outer Segments of Rat Retina During n-3 and n-6 Fatty Acid Deficiency

We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n-9), 20:4(n-6), and 20:5(n-3), respectively. The levels of these 20-carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n-3) with 22:5(n-6), as evidenced by a reduction of the 22:6(n-3)-22:6(n-3) molecular species and an increase in the 22:5(n-6)-22:6(n-3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n-3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n-3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22-carbon polyunsaturated fatty acids from the circulation.     

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.     

猫外层视网膜和脉络膜的年龄相关变化

取老年猫(12龄,2.5~3 kg)和青年猫(1~3龄,2~2.5 kg)各4只的视网膜,经4%多聚甲醛处理后用H.E染色以显示视网膜和脉络膜的结构。光学显微镜下观察感光细胞层、玻璃膜(Bruch’s membrane)结构的变化,计数色素上皮层(retinal pigment epithelium,RPE)细胞数、脉络膜毛细血管数,测量玻璃膜、脉络膜厚度,脉络膜毛细血管之间的距离。结果显示,与青年猫比较,老年猫视网膜感光细胞层结构杂乱;色素上皮细胞数显著下降;玻璃膜厚度无显著变化,出现较多碎片;脉络膜厚度明显变薄,脉络膜毛细血管数显著减小,脉络膜毛细血管之间的距离显著增大。推测老年猫脉络膜的退化可能是导致玻璃膜、色素上皮层的退化,进而导致感光细胞的功能衰退的重要原因。     

Graded Differential Photoreceptor Orientation: Ramifications for Ultramicrotomy of Retina

When sectioning small blocks of tissue from the retina of the eye, it is sometimes difficult to obtain sections which simultaneously cut squarely across the inner retinal layers and are on the long axis of the photoreceptors. This difficulty is, at least partially, due to the fact that the receptors tilt progressively relative to the tangent to the retinal curve at progressively more peripheral loci. Consideration of the graded differential orientation of the receptors indicates that, in order for the section to be simultaneously coaxial with the receptors and the inner retinal layers, the plane of the section must be parallel to and include the anterior-posterior axis of the eye, as in the case when the whole eye is sectioned through its center. It is illustrated that this criterion can be met for small blocks of retina if the block is excised along a parallel of the eye and the plane of section is perpendicular to the tangent to the retinal curve and the parallel. An approach which accomplishes this is described. Theoretical analysis suggests that distortion of apparent size of structures in the retina can become significant within a few degrees of the posterior pole if this condition in not met.