MAB3073 Sigma-AldrichAnti-G Protein Goα Antibody, clone 2A

Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell-specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.

Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

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 Ca(2+) uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca(2+) 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 Ca(2+) 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.

Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.

High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and β subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.

Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1(-/-) and Tpst2(-/-) mice, retinal function was compromised during early development. However, Tpst1(-/-) retinas became electrophysiologically normal by postnatal day 90 while Tpst2(-/-) mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina.

We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina.A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks.Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks.The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pH(i)) and chloride concentration ([Cl(-)](i)) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABA(A) receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pH(i) regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.