Noncanonical autophagy promotes the visual cycle.

Phagocytosis and degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components, but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 associated with single-membrane phagosomes containing engulfed POS in an Atg5-dependent manner that required Beclin1, but not the autophagy preinitiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.

Germline knockout of Nr2e3 protects photoreceptors in three distinct mouse models of retinal degeneration.

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targeting Nr2e3, a transcription factor required for the normal differentiation of rod photoreceptors. Nr2e3 knockout results in hybrid rod photoreceptors that express the full complement of rod genes, but also a subset of cone genes. We show that germline deletion of Nr2e3 potently protects rods in three mechanistically diverse mouse models of retinal degeneration caused by bright-light exposure (light damage), structural deficiency (rhodopsin-deficient Rho-/- mice), or abnormal phototransduction (phosphodiesterase-deficient rd10 mice). Nr2e3 knockout confers strong neuroprotective effects on rods without adverse effects on their gene expression, structure, or function. Furthermore, in all three degeneration models, prolongation of rod survival by Nr2e3 knockout leads to lasting preservation of cone morphology and function. These findings raise the possibility that upregulation of one or more cone genes in Nr2e3-deficient rods may be responsible for the neuroprotective effects we observe.

Acyl-CoA:wax alcohol acyltransferase 2 modulates the cone visual cycle in mouse retina.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

Preparative Production and Purification of Recombinant Human Cyclophilin A.

In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.

Phosphorylation at Serine 21 in G protein-coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors.

Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.

Photoreceptors in a mouse model of Leigh syndrome are capable of normal light-evoked signaling.

Mitochondrial dysfunction is an important cause of heritable vision loss. Mutations affecting mitochondrial bioenergetics may lead to isolated vision loss or life-threatening systemic disease, depending on a mutation's severity. Primary optic nerve atrophy resulting from death of retinal ganglion cells is the most prominent ocular manifestation of mitochondrial disease. However, dysfunction of other retinal cell types has also been described, sometimes leading to a loss of photoreceptors and retinal pigment epithelium that manifests clinically as pigmentary retinopathy. A popular mouse model of mitochondrial disease that lacks NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), a subunit of mitochondrial complex I, phenocopies many traits of the human disease Leigh syndrome, including the development of optic atrophy. It has also been reported that ndufs4-/- mice display diminished light responses at the level of photoreceptors or bipolar cells. By conducting electroretinography (ERG) recordings in live ndufs4-/- mice, we now demonstrate that this defect occurs at the level of retinal photoreceptors. We found that this deficit does not arise from retinal developmental anomalies, photoreceptor degeneration, or impaired regeneration of visual pigment. Strikingly, the impairment of ndufs4-/- photoreceptor function was not observed in ex vivo ERG recordings from isolated retinas, indicating that photoreceptors with complex I deficiency are intrinsically capable of normal signaling. The difference in electrophysiological phenotypes in vivo and ex vivo suggests that the energy deprivation associated with severe mitochondrial impairment in the outer retina renders ndufs4-/- photoreceptors unable to maintain the homeostatic conditions required to operate at their normal capacity.

Light deprivation reduces the severity of experimental diabetic retinopathy.

Illumination of the retina is a major determinant of energy expenditure by its neurons. However, it remains unclear whether light exposure significantly contributes to the pathophysiology of common retinal disease. Driven by the premise that light exposure reduces the metabolic demand of the retina, recent clinical trials failed to demonstrate a benefit for constant illumination in the treatment of diabetic retinopathy. Here, we instead ask whether light deprivation or blockade of visual transduction could modulate the severity of this common cause of blindness. We randomized adult mice with two different models of diabetic retinopathy to 1-3 months of complete dark housing. Unexpectedly, we find that diabetic mice exposed to short or prolonged light deprivation have reduced diabetes-induced retinal pathology, using measures of visual function, compared to control animals in standard lighting conditions. To corroborate these results, we performed assays of retinal vascular health in diabetic Gnat1-/- and Rpe65-/- mice, which lack phototransduction. Both mutants displayed less diabetes-associated retinal vascular disease compared to respective wild-type controls. Collectively, these results suggest that light-induced visual transduction promotes the development of diabetic retinopathy and implicate photoreceptors as an early source of visual pathology in diabetes.

Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Cone photoreceptors are essential for vision under moderate to high illuminance and allow color discrimination. Their fast dark adaptation rate and resistance to saturation are believed to depend in part on an intraretinal visual cycle that supplies 11- cis-retinaldehyde to cone opsins. Candidate enzymes of this pathway have been reported, but their physiologic contribution to cone photoresponses remains unknown. Here, we evaluate the role of a candidate retinol isomerase of this pathway, sphingolipid δ4 desaturase 1 (Des1). Single-cell RNA sequencing analysis revealed Des1 expression not only in Müller glia but also throughout the retina and in the retinal pigment epithelium. We assessed cone functional dependence on Müller cell-expressed Des1 through a conditional knockout approach. Floxed Des1 mice, on a guanine nucleotide-binding protein subunit α transducin 1 knockout ( Gnat1-/-) background to allow isolated recording of cone-driven photoresponses, were bred with platelet-derived growth factor receptor α (Pdgfrα)-Cre mice to delete Des1 in Müller cells. Conditional knockout of Des1 expression, as shown by tissue-selective Des1 gene recombination and reduced Des1 catalytic activity, caused no gross changes in the retinal structure and had no effect on cone sensitivity or dark adaptation but did slightly accelerate the rate of cone phototransduction termination. These results indicate that Des1 expression in Müller cells is not required for cone visual pigment regeneration in the mouse.-Kiser, P. D., Kolesnikov, A.V., Kiser, J. Z., Dong, Z., Chaurasia, B., Wang, L., Summers, S. A., Hoang, T., Blackshaw, S., Peachey, N. S., Kefalov, V. J., Palczewski, K. Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Dephosphorylation by protein phosphatase 2A regulates visual pigment regeneration and the dark adaptation of mammalian photoreceptors.

Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone photoreceptors arguably represent the best-understood example of such GPCR signaling. Their visual pigments (opsins) are activated by light, transduce the signal, and are then inactivated by a GPCR kinase and arrestin. Although pigment inactivation by phosphorylation is well understood, the enzyme(s) responsible for pigment dephosphorylation and the functional significance of this reaction remain unknown. Here, we show that protein phosphatase 2A (PP2A) acts as opsin phosphatase in both rods and cones. Elimination of PP2A substantially slows pigment dephosphorylation, visual chromophore recycling, and ultimately photoreceptor dark adaptation. These findings demonstrate that visual pigment dephosphorylation regulates the dark adaptation of photoreceptors and provide insights into the role of this reaction in GPCR signaling.

Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations.

Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.

P23H opsin knock-in mice reveal a novel step in retinal rod disc morphogenesis.

Retinal rod photoreceptor cells have double membrane discs located in their outer segments (ROS) that are continuously formed proximally from connecting cilia (CC) and phagocytized distally by the retinal pigmented epithelium. The major component of these rod discs, the light-sensitive visual pigment rhodopsin (Rho), consists of an opsin protein linked to 11-cis-retinal. The P23H mutation of rod opsin (P23H opsin) is the most common cause of human blinding autosomal dominant retinitis pigmentosa (adRP). A mouse model of adRP with this mutation (Rho(P23H/+)) shows low levels of P23H opsin protein, partial misalignment of discs and progressive retinal degeneration. However, the impact of mutant P23H opsin on the formation of abnormal discs is unclear and it is still unknown whether this mutant pigment can mediate phototransduction. Using transretinal ERG recordings, we demonstrate that P23H mutant Rho can trigger phototransduction but Rho(P23H/P23H) rods are ∼17 000-fold less sensitive to light than Rho(+/+) rods and produce abnormally fast photo-responses. By analyzing homozygous Rho(P23H/P23H) knock-in mice, we show that P23H opsin is transported to ciliary protrusions where it forms sagittally elongated discs. Transmission electron microscopy of postnatal day (PND) 14 Rho(P23H/+) mouse retina revealed disordered sagittally oriented discs before the onset of retinal degeneration. Surprisingly, we also observed smaller, immature sagittally oriented discs in PND14 Rho(+/)(-) and Rho(+/+) mice that were not seen in older animals. These findings provide fundamental insights into the pathogenesis of the P23H mutant opsin and reveal a novel early sagittally aligned disc formation step in normal ROS disc expansion.

Reprogramming of adult rod photoreceptors prevents retinal degeneration.

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Retinol dehydrogenase 8 and ATP-binding cassette transporter 4 modulate dark adaptation of M-cones in mammalian retina.

KEY POINTS: This study explores the molecular mechanisms that regulate the recycling of chromophore required for pigment regeneration in mammalian cones. We report that two chromophore binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter (ABCA4) accelerate the dark adaptation of cones, first, directly, by facilitating the processing of chromophore in cones, and second, indirectly, by accelerating the turnover of chromophore in rods, which is then recycled and delivered to both rods and cones. Preventing competition with the rods by knocking out rhodopsin accelerated cone dark adaptation, demonstrating the interplay between rod and cone pigment regeneration driven by the retinal pigment epithelium (RPE). This novel interdependence of rod and cone pigment regeneration should be considered when developing therapies targeting the recycling of chromophore for rods, and evaluating residual cone function should be a critical test for such regimens targeting the RPE. ABSTRACT: Rapid recycling of visual chromophore and regeneration of the visual pigment are critical for the continuous function of mammalian cone photoreceptors in daylight vision. However, the molecular mechanisms modulating the supply of visual chromophore to cones have remained unclear. Here we explored the roles of two chromophore-binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter 4 (ABCA4), in dark adaptation of mammalian cones. We report that young adult RDH8/ABCA4-deficient mice have normal M-cone morphology but reduced visual acuity and photoresponse amplitudes. Notably, the deletion of RDH8 and ABCA4 suppressed the dark adaptation of M-cones driven by both the intraretinal visual cycle and the retinal pigmented epithelium (RPE) visual cycle. This delay can be caused by two separate mechanisms: direct involvement of RDH8 and ABCA4 in cone chromophore processing, and an indirect effect from the delayed recycling of chromophore by the RPE due to its slow release from RDH8/ABCA4-deficient rods. Intriguingly, our data suggest that RDH8 could also contribute to the oxidation of cis-retinoids in cones, a key reaction of the retina visual cycle. Finally, we dissected the roles of rod photoreceptors and RPE for dark adaptation of M-cones. We found that rods suppress, whereas RPE promotes, cone dark adaptation. Thus, therapeutic approaches targeting the RPE visual cycle could have adverse effects on the function of cones, making the evaluation of residual cone function a critical test for regimens targeting the RPE.

Cone phosphodiesterase-6α' restores rod function and confers distinct physiological properties in the rod phosphodiesterase-6ß-deficient rd10 mouse.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the vertebrate phototransduction pathway in rods and cones. Rod PDE6 catalytic core is composed of two distinct subunits, PDE6α and PDE6ß, whereas two identical PDE6α' subunits form the cone PDE6 catalytic core. It is not known whether this difference in PDE6 catalytic subunit identity contributes to the functional differences between rods and cones. To address this question, we expressed cone PDE6α' in the photoreceptor cells of the retinal degeneration 10 (rd10) mouse that carries a mutation in rod PDEß subunit. We show that adeno-associated virus-mediated subretinal delivery of PDE6α' rescues rod electroretinogram responses and preserves retinal structure, indicating that cone PDE6α' can couple effectively to the rod phototransduction pathway. We also show that restoration of light sensitivity in rd10 rods is attributable to assembly of PDE6α' with rod PDE6γ. Single-cell recordings revealed that, surprisingly, rods expressing cone PDE6α' are twofold more sensitive to light than wild-type rods, most likely because of the slower shutoff of their light responses. Unlike in wild-type rods, the response kinetics in PDE6α'-treated rd10 rods accelerated with increasing flash intensity, indicating a possible direct feedback modulation of cone PDE6α' activity. Together, these results demonstrate that cone PDE6α' can functionally substitute for rod PDEαß in vivo, conferring treated rods with distinct physiological properties.

Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors.

G-protein ß subunits perform essential neuronal functions as part of G-protein ßγ and Gß5-regulators of G-protein signaling (RGS) complexes. Both Gßγ and Gß5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesis in vivo, we deleted the Phlp1 gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gßγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gß5 and RGS9-1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstrate in vivo that PhLP1 is required for the folding and assembly of both Gßγ and Gß5-RGS9.

Polyunsaturated Fatty Acid - mediated Cellular Rejuvenation for Reversing Age-related Vision Decline.

The retina is uniquely enriched in polyunsaturated fatty acids (PUFAs), which are primarily localized in cell membranes, where they govern membrane biophysical properties such as diffusion, permeability, domain formation, and curvature generation. During aging, alterations in lipid metabolism lead to reduced content of very long-chain PUFAs (VLC-PUFAs) in the retina, and this decline is associated with normal age-related visual decline and pathological age-related macular degeneration (AMD). ELOVL2 (Elongation of very-long-chain fatty acids-like 2) encodes a transmembrane protein that produces precursors to docosahexaenoic acid (DHA) and VLC-PUFAs, and methylation level of its promoter is currently the best predictor of chronological age. Here, we show that mice lacking ELOVL2-specific enzymatic activity (Elovl2 C234W ) have impaired contrast sensitivity and slower rod response recovery following bright light exposure. Intravitreal supplementation with the direct product of ELOVL2, 24:5n-3, in aged animals significantly improved visual function and reduced accumulation of ApoE, HTRA1 and complement proteins in sub-RPE deposits. At the molecular level, the gene expression pattern observed in retinas supplemented with 24:5n-3 exhibited a partial rejuvenation profile, including decreased expression of aging-related genes and a transcriptomic signature of younger retina. Finally, we present the first human genetic data showing significant association of several variants in the human ELOVL2 locus with the onset of intermediate AMD, underlying the translational significance of our findings. In sum, our study identifies novel therapeutic opportunities and defines ELOVL2 as a promising target for interventions aimed at preventing age-related vision loss.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

Investigating the Role of Rhodopsin F45L Mutation in Mouse Rod Photoreceptor Signaling and Survival.

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.