Anuradha Dhingra

Axons and dendrites originate from neuroepithelial-like processes of retinal bipolar cells.

The cellular mechanisms underlying axogenesis and dendritogenesis are not completely understood. The axons and dendrites of retinal bipolar cells, which contact their synaptic partners within specific laminae in the inner and outer retina, provide a good system for exploring these issues. Using transgenic mice expressing enhanced green fluorescent protein (GFP) in a subset of bipolar cells, we determined that axonal and dendritic arbors of these interneurons develop directly from apical and basal processes attached to the outer and inner limiting membranes, respectively. Selective stabilization of processes contributed to stratification of axonal and dendritic arbors within the appropriate synaptic layer. This unusual mode of axogenesis and dendritogenesis from neuroepithelial-like processes may act to preserve neighbor-neighbor relationships in synaptic wiring between the outer and inner retina.

Evaluation of the 17-kDa Prenyl-binding Protein as a Regulatory Protein for Phototransduction in Retinal Photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEδ) tightly bound. This protein, here termed PrBP/δ, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/δ in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/δ in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/δ was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/δ. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/δ can solubilize PDE6 and prevent its activation by transducin. PrBP/δ also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/δ content of purified ROS reveals insufficient amounts of PrBP/δ (<0.1 PrBP/δ per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/δ in frog and bovine retina shows greatest PrBP/δ immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/δ labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/δ as a PDE6 subunit and implicate PrBP/δ in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.

Probing Neurochemical Structure and Function of Retinal ON Bipolar Cells with a Transgenic Mouse

Retinal ON bipolar cells make up about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G‐protein Go1, and closing an unidentified cation channel. To facilitate investigation of ON bipolar cells, we here report on the production of a transgenic mouse (Grm6‐GFP) in which enhanced green fluorescent protein (EGFP), under control of mGluR6 promoter, was expressed in all and only ON bipolar cells. We used the mouse to determine density of ON bipolar cells, which in central retina was 29,600 cells/mm2. We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. With this library, we determined expression of 27 genes of interest. We obtained positive transcripts for Go interactors: regulators of G‐protein signaling (RGS), Ret‐RGS1 (a variant of RGS20), RGS16, RGS7, purkinje cell protein 2 (PCP2, also called L7 or GPSM4), synembryn (RIC‐8), LGN (GPSM2), RAP1GAP, and Gβ5; cGMP modulators: guanylyl cyclase (GC) 1α1, GC1β1, phosphodiesterase (PDE) 1C, and PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, and sperm‐specific cation channels CatSper 2–4. The following transcripts were not found in our library: AGS3 (GPSM1), RGS10, RGS19 (GAIP), calbindin, GC1α2, GC1β2, PDE5, PDE2A, amiloride‐sensitive sodium channel ACCN4, and CatSper1. We then localized Kir2.4 to several cell types and showed that, in ON bipolar cells, the channel concentrates in their dendritic tips. The channels and modulators found in ON bipolar cells likely shape their light response. Additional uses of the Grm6‐GFP mouse are also discussed. J. Comp. Neurol. 510:484–496, 2008.

A Retinal-Specific Regulator of G-Protein Signaling Interacts with Gαo and Accelerates an Expressed Metabotropic Glutamate Receptor 6 Cascade

Go is the most abundant G-protein in the brain, but its regulators are essentially unknown. In retina, Gαo1 is obligatory in mediating the metabotropic glutamate receptor 6 (mGluR6)-initiated ON response. To identify the interactors of Go, we conducted a yeast two-hybrid screen with constituitively active Gαo as a bait. The screen frequently identified a regulator of G-protein signaling (RGS), Ret-RGS1, the interaction of which we confirmed by coimmunoprecipitation with Gαo in transfected cells and in retina. Ret-RGS1 localized to the dendritic tips of ON bipolar neurons, along with mGluR6 and Gαo1. When Ret-RGS1 was coexpressed in Xenopus oocytes with mGluR6, Gαo1, and a GIRK (G-protein-gated inwardly rectifying K+) channel, it accelerated the deactivation of the channel response to glutamate in a concentration-dependent manner. Because light onset suppresses glutamate release from photoreceptors onto the ON bipolar dendrites, Ret-RGS1 should accelerate the rising phase of the light response of the ON bipolar cell. This would tend to match its kinetics to that of the OFF bipolar that arises directly from ligand-gated channels.

Autoantibodies in Melanoma-Associated Retinopathy Target TRPM1 Cation Channels of Retinal ON Bipolar Cells

Melanoma-associated retinopathy (MAR) is characterized by night blindness, photopsias, and a selective reduction of the electroretinogram b-wave. In certain cases, the serum contains autoantibodies that react with ON bipolar cells, but the target of these autoantibodies has not been identified. Here we show that the primary target of autoantibodies produced in MAR patients with reduced b-wave is the TRPM1 cation channel, the newly identified transduction channel in ON bipolar cells. Sera from two well characterized MAR patients, but not from a control subject, stained human embryonic kidney cells transfected with the TRPM1 gene, and Western blots probed with these MAR sera showed the expected band size (∼180 kDa). Staining of mouse and primate retina with MAR sera revealed immunoreactivity in all types of ON bipolar cells. Similar to staining for TRPM1, staining with the MAR sera was strong in dendritic tips and somas and was weak or absent in axon terminals. This staining colocalized with GFP in Grm6-GFP transgenic mice, where GFP is expressed in all and only ON bipolar cells, and also colocalized with Gαo, a marker for all types of ON bipolar cells. The staining in ON bipolar cells was confirmed to be specific to TRPM1 because MAR serum did not stain these cells in a Trpm1−/− mouse. Evidence suggests that the recognized epitope is likely intracellular, and the sera can be internalized by retinal cells. We conclude that the vision of at least some patients with MAR is compromised due to autoantibody-mediated inactivation of the TRPM1 channel.

Gβ3 Is Required for Normal Light ON Responses and Synaptic Maintenance

Heterotrimeric G-proteins, comprising Gα and Gβγ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor-based signaling complexes. A G-protein β subunit, Gβ3, plays a critical role in several physiological processes, as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express Gβ3, and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptors signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that Gβ3 participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. Gβ3 deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, Gβ3 deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, Gβ3 may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca2+ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca2+ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi/Go proteins, did not affect basal or mGluR6‐induced Ca2+ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.

Cones Respond to Light in the Absence of Transducin β Subunit

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gβ3 and Gγt2 subunits. The function of Gβγ in this cascade has not been examined. Here, we investigate the role of Gβ3 by assessing cone structure and function in Gβ3-null mouse (Gnb3−/−). We found that Gβ3 is required for the normal expression of its partners, because in the Gnb3−/− cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3−/− cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gβ subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3−/− cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gβ3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.

Differential function of Gγ13 in rod bipolar and ON cone bipolar cells

The G‐protein‐mediated metabotropic glutamate receptor 6 (mGluR6) signalling cascade is critical for light responses in all retinal ON bipolar cells. The present study evaluates the role of G‐protein subunit Gγ13 in ON bipolar cells by deleting the mouse Gng13 gene. Gγ13 is a partner of Gβ3 in all types of retinal ON bipolar cells, and contributes to generating the light response. Gγ13 also contributes to maintenance because, in its absence, the light response decreases with age. Three independent functionality assessments (i.e. light response as measured by electroretinogram b‐waves, localization of GTPase activating proteins and deterioration of the light response with age) show that the contribution of Gγ13 is much greater in rod bipolar cells than in ON cone bipolar cells. Our data also suggest that the contribution of Gγ13 to coupling mGluR6 to its effector is smaller than its contribution to maintaining GTPase activating protein localization.

mGluR6 Transcripts in Non-neuronal Tissues

To study mGluR6 expression, the authors investigated two transgenic mouse lines that express enhanced green fluorescent protein (GFP) under control of mGluR6 promoter. In retina, GFP was expressed exclusively in all ON bipolar cell types, either uniformly across all cells of this class (line 5) or in a mosaic (patchy) fashion (line 1). In brain, GFP was found in certain cortical areas, superior colliculus, axons of the corpus callosum, accessory olfactory bulb, and cells of the subcommissural organ. Outside the nervous system, GFP was seen in the corneal endothelium, testis, the kidney’s medulla, collecting ducts and parietal layer that surround the glomeruli, and B lymphocytes. Furthermore, RT-PCR showed that most tissues that expressed GFP in the transgenic mouse also transcribed two splice variants of mGluR6 in the wild-type mouse. The alternate variant was lacking exon 8, predicting a protein product of 545 amino acids that lacks the 7-transmembrane domains of the receptor. In cornea, immunostaining for mGluR6 gave strong staining in the endothelium, and this was stronger in wild-type than in mGluR6-null mice. Furthermore, calcium imaging with Fura-2 showed that application of L-AP4, an agonist for group III metabotropic glutamate receptors including mGluR6, elevated calcium in endothelial cells.