Selective Block of Upregulated Kv1.3 Potassium Channels in ON-Bipolar Cells of the Blind Retina Enhances Optogenetically Restored Signaling.

Loss of photoreceptors in retinal degenerative diseases also impacts the inner retina: bipolar cell dendrites retract, neurons rewire, and protein expression changes. ON-bipolar cells (OBCs) represent an attractive target for optogenetic vision restoration. However, the above-described maladaptations may negatively impact the quality of restored vision. To investigate this question, we employed human post-mortem retinas and transgenic rd1_Opto-mGluR6 mice expressing the optogenetic construct Opto-mGluR6 in OBCs and carrying the retinal degeneration rd1 mutation. We found significant changes in delayed rectifier potassium channel expression in OBCs of degenerative retinas. In particular, we found an increase in Kv1.3 expression already in early stages of degeneration. Immunohistochemistry localized Kv1.3 channels specifically to OBC axons. In whole-cell patch-clamp experiments, OBCs in the degenerated murine retina were less responsive, which could be reversed by application of the specific Kv1.3 antagonist Psora-4. Notably, Kv1.3 block significantly increased the amplitude and kinetics of Opto-mGluR6-mediated light responses in OBCs of the blind retina and increased the signal-to-noise ratio of light-triggered responses in retinal ganglion cells. We propose that reduction in Kv1.3 activity in the degenerated retina, either by pharmacological block or by KCNA3 gene silencing, could improve the quality of restored vision.

Extended functional rescue following AAV gene therapy in a canine model of LRIT3-congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a group of inherited retinal diseases in which either rod-to-ON-bipolar cell (ON-BC) signaling, or rod function is affected leading to impaired vision under low light conditions. One type of CSNB is associated with defects in genes (NYX, GRM6, TRPM1, GPR179, and LRIT3) involved in the mGluR6 signaling cascade at the ON-BC dendritic tips. We have previously characterized a canine model of LRIT3-CSNB and demonstrated short-term safety and efficacy of an ON-BC targeting AAV-LRIT3 (AAVK9#4-shGRM6-cLRIT3-WPRE) gene therapy. Herein, we demonstrate long-term functional recovery and molecular restoration following subretinal injection of the ON-BC targeting AAV-LRIT3 vector in all eight treated eyes for up to 32 months. Following subretinal administration of the therapeutic vector, expression of the LRIT3 transgene, as well as restoration of mGluR6 signaling cascade member TRPM1, were confirmed in the outer plexiform layer (OPL) of the treated area. However, further investigation of the transgene LRIT3 transcript expression by RNA in situ hybridization (RNA-ISH) revealed off-target expression in non-BCs including the photoreceptors, inner nuclear, and ganglion cell layers, despite the use of a mutant AAVK9#4 capsid and an improved mGluR6 promoter designed to specifically transduce and promote expression in ON-BCs. While the long-term therapeutic potential of AAVK9#4-shGRM6-cLRIT3-WPRE is promising, we highlight the necessity for further optimization of AAV-LRIT3 therapy in the canine CSNB model prior to its clinical application.

Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.

SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.

Mice Lacking Gpr179 with Complete Congenital Stationary Night Blindness Are a Good Model for Myopia.

Mutations in GPR179 are one of the most common causes of autosomal recessive complete congenital stationary night blindness (cCSNB). This retinal disease is characterized in patients by impaired dim and night vision, associated with other ocular symptoms, including high myopia. cCSNB is caused by a complete loss of signal transmission from photoreceptors to ON-bipolar cells. In this study, we hypothesized that the lack of Gpr179 and the subsequent impaired ON-pathway could lead to myopic features in a mouse model of cCSNB. Using ultra performance liquid chromatography, we show that adult Gpr179-/- mice have a significant decrease in both retinal dopamine and 3,4-dihydroxyphenylacetic acid, compared to Gpr179+/+ mice. This alteration of the dopaminergic system is thought to be correlated with an increased susceptibility to lens-induced myopia but does not affect the natural refractive development. Altogether, our data added a novel myopia model, which could be used to identify therapeutic interventions.

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

Functional Availability of ON-Bipolar Cells in the Degenerated Retina: Timing and Longevity of an Optogenetic Gene Therapy.

Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.

A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency.

Mutations in GPR179 lead to autosomal recessive complete congenital stationary night blindness (cCSNB). This condition represents a signal transmission defect from the photoreceptors to the ON-bipolar cells. To confirm the phenotype, better understand the pathogenic mechanism in vivo, and provide a model for therapeutic approaches, a Gpr179 knock-out mouse model was genetically and functionally characterized. We confirmed that the insertion of a neo/lac Z cassette in intron 1 of Gpr179 disrupts the same gene. Spectral domain optical coherence tomography reveals no obvious retinal structure abnormalities. Gpr179 knock-out mice exhibit a so-called no-b-wave (nob) phenotype with severely reduced b-wave amplitudes in the electroretinogram. Optomotor tests reveal decreased optomotor responses under scotopic conditions. Consistent with the genetic disruption of Gpr179, GPR179 is absent at the dendritic tips of ON-bipolar cells. While proteins of the same signal transmission cascade (GRM6, LRIT3, and TRPM1) are correctly localized, other proteins (RGS7, RGS11, and GNB5) known to regulate GRM6 are absent at the dendritic tips of ON-bipolar cells. These results add a new model of cCSNB, which is important to better understand the role of GPR179, its implication in patients with cCSNB, and its use for the development of therapies.

Multielectrode Array Recording of Mouse Retinas Transplanted with Stem Cell-Derived Retinal Sheets.

Retinal multielectrode array (MEA) recording allows us to examine the action potentials of retinal ganglion cells and field potentials of photoreceptors and bipolar cells. In addition to studying the retinal circuitry, it has become one of the standard examination tools for the characterization of stem cell-derived retinal transplantation in degenerated retinas. Besides the detection of responses to simple light stimulation, it is also necessary to consider the spatial correlation of the graft and the electrodes, in order to unbiasedly reveal the locally reconstructed retinal circuitry after transplantation. Here, we introduce our newly developed protocol of MEA recording and analysis that may serve as a standard for evaluating transplanted retinas.

Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors.

Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify unique physiological properties of individual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft-members of the heparan sulfate proteoglycan (HSPG) family-associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the pre-synaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals.

Molecular and Biochemical Aspects of the Retina on Refraction.

Mutant mouse models with specific visual pathway defects offer an advantage to comprehensively investigate the role of specific pathways/neurons involved in refractive development. In this review, we will focus on recent studies using mouse models that have provided insight into retinal pathways and neurotransmitters controlling refractive development. Specifically, we will examine the contributions of rod and cone photoreceptors and the ON and OFF retinal pathways to visually driven eye growth with emphasis on dopaminergic mechanisms.

Properties and functions of TRPM1 channels in the dendritic tips of retinal ON-bipolar cells.

An increase in light intensity induces a depolarization in retinal ON-bipolar cells via a reduced glutamate release from presynaptic photoreceptor cells. The underlying transduction cascade in the dendritic tips of ON-bipolar cells involves mGluR6 glutamate receptors signaling to TRPM1 proteins that are an indispensable part of the transduction channel. Several other proteins are recognized to participate in the transduction machinery. Deficiency in many of these leads to congenital stationary night blindness, because rod bipolar cells, a subgroup of ON-bipolar cells, constitute the main route for sensory information under scotopic conditions. Here, we review the current knowledge about TRPM1 ion channels and how their activity is regulated within the postsynaptic compartment of ON-bipolar cells. The functional properties of TRPM1 channels in the dendritic compartment are not well understood as they differ substantially from those of recombinant TRPM1 channels. Critical evaluation of possible explanations of these discrepancies indicates that some key components of this transduction pathway might still not be known. The continued exploration of this pathway will yield further clinically useful insights.