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.     

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.     

Stability and Function of Mammalian Lethal Giant Larvae-1 Oncoprotein Are Regulated by the Scaffolding Protein RanBPM

The evolutionarily conserved lethal giant larvae (Lgl) tumor suppressor gene has an essential role in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. However, the precise molecular mechanism by which the Lgl carries out its function remains obscure. In the current study, we have identified Ran-binding protein M (RanBPM) as a novel binding partner of Mgl-1, a mammalian homolog of Drosophila tumor suppressor protein lethal (2) giant larvae (L(2)gl) by yeast two-hybrid screening. RanBPM seems to act as a scaffolding protein with a modulatory function with respect to Mgl-1. The Mgl-1 and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM resulted in inhibition of Mgl-1 degradation, and thereby extended the half-life of Mgl-1. Furthermore, the ability of Mgl-1 activity in cell migration and colony formation assay was enhanced by RanBPM. Taken together, our findings reveal that RanBPM plays a novel role in regulating Mgl-1 stability and contributes to its biological function as a tumor suppressor.     

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.     

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2^−/− mice the phagocytosis of outer segments was impaired, and in ANX A2^−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2^−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.     

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.     

Multiple Neuronal Connexins in the Mammalian Retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.     

Lrit1, a Retinal Transmembrane Protein,Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity

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Emerging Role of the Scaffolding Protein Dlg1 in Vesicle Trafficking

Discs large 1 (Dlg1) is a modular scaffolding protein implicated in the control of cell polarity through assembly of specific multiprotein complexes, including receptors, ion channels and signaling proteins, at specialized zones of the plasma membrane. Recent data have shown that in addition to these well‐known interaction partners, Dlg1 may also recruit components of the vesicle trafficking machinery either to the plasma membrane or to transport vesicles. Here, we discuss Dlg1 function in vesicle formation, targeting, tethering and fusion, in both the exocytotic and endocytotic pathways. These pathways contribute to cell functions as major and diverse as glutamatergic activity in the neurons, membrane homeostasis in Schwann cell myelination, insulin stimulation of glucose transport in adipocytes, or endothelial secretion of the hemostatic protein, von Willebrand factor (VWF).     

A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation

The mammalian Na⁺/H⁺ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.     

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.