Prominin 1 is crucial for the early development of photoreceptor outer segments.

Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision.

Genotypic and Phenotypic Characterization of a Cohort of Patients Affected by Rod Cyclic Nucleotide Channel-Associated Retinitis Pigmentosa.

INTRODUCTION: Retinitis pigmentosa (RP), a heterogeneous inherited retinal disorder causing gradual vision loss, affects over 1 million people worldwide. Pathogenic variants in CNGA1 and CNGB1 genes, respectively, accounting for 1% and 4% of cases, impact the cyclic nucleotide-gated channel in rod photoreceptor cells. The aim of this study was to describe and compare genotypic and clinical characteristics of a cohort of patients with CNGA1- or CNGB1-related RP and to explore potential genotype-phenotype correlations. METHODS: The following data from patients with CNGA1- or CNGB1-related RP, followed in five Italian inherited retinal degenerations services, were retrospectively collected: genetic variants in CNGA1 and CNGB1, best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, fundus photographs, and short-wavelength fundus autofluorescence (SW-AF) images. Comparisons and correlation analyses were performed by first dividing the cohort in two groups according to the gene responsible for the disease (CNGA1 and CNGB1 groups). In parallel, the whole cohort of RP patients was divided into two other groups, according to the expected impact of the variants at protein level (low and high group). RESULTS: In total, 29 patients were recruited, 11 with CNGA1- and 18 with CNGB1-related RP. In both CNGA1 and CNGB1, 5 novel variants in CNGA1 and 5 in CNGB1 were found. BCVA was comparable between CNGA1 and CNGB1 groups, as well as between low and high groups. CNGA1 group had a larger mean EZ width compared to CNGB1 group, albeit not statistically significant, while EZ width did not differ between low and high groups A statistically significant correlation between EZ width and BCVA as well as between EZ width and age were observed in the whole cohort of RP patients. Fundus photographs of all patients in the cohort showed classic RP pattern, and in SW-AF images an hyperautofluorescent ring was observed in 14/21 patients. CONCLUSION: Rod CNG channel-associated RP was demonstrated to be a slowly progressive disease in both CNGA1- and CNGB1-related forms, making it an ideal candidate for gene augmentation therapies.

Genetic manipulation of rod-cone differences in mouse retina.

Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.

Retinal metabolism displays evidence for uncoupling of glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle.

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilised 1H-NMR spectroscopy-based metabolomics, in situ enzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: (1) the mini-Krebs-cycle, fuelled by glutamine and branched chain amino acids, generating N-acetylaspartate; (2) the alanine-generating Cahill-cycle; (3) the lactate-releasing Cori-cycle. Moreover, the metabolomics data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.

Dark continuous noise from mutant G90D-rhodopsin predominantly underlies congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.

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.

NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

New Improved cGMP Analogues to Target Rod Photoreceptor Degeneration.

Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3',5'-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its RP-phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer SP-CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3.

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (Dnm1-3), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

Characterization of the development of the high-acuity area of the chick retina.

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.

Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Excessive tubulin glutamylation leads to progressive cone-rod dystrophy and loss of outer segment integrity.

Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors. The observed functional reduction was not due to cell death, levels, or the mislocalization of major phototransduction proteins. Instead, the increased tubulin glutamylation caused shortened photoreceptor axonemes and the formation of numerous abnormal membranous whorls that disrupted the integrity of photoreceptor outer segments (OS). Ultimately, excessive tubulin glutamylation led to the progressive loss of photoreceptors, affecting cones more severely than rods. Our results highlight the importance of maintaining tubulin glutamylation for normal photoreceptor function. Furthermore, we demonstrate that murine cone photoreceptors are more sensitive to disrupted tubulin glutamylation levels than rods, suggesting an essential role for axoneme in the structural integrity of the cone outer segment. This study provides valuable insights into the mechanisms of photoreceptor diseases linked to excessive tubulin glutamylation.

Abnormal α-Synuclein Aggregates Cause Synaptic- and Microcircuit-Specific Deficits in the Retinal Rod Pathway.

α-Synuclein (α-Syn) is a key determinator of Parkinson disease (PD) pathology, but synapse and microcircuit pathologies in the retina underlying visual dysfunction are poorly understood. Herein, histochemical and ultrastructural analyses and ophthalmologic measurements in old transgenic M83 PD model (mice aged 16 to 18 months) indicated that abnormal α-Syn aggregation in the outer plexiform layer (OPL) was associated with degeneration in the C-terminal binding protein 2 (CtBP2)+ ribbon synapses of photoreceptor terminals and protein kinase C alpha (PKCα)+ rod bipolar cell terminals, whereas α-Syn aggregates in the inner retina correlated with the reduction and degeneration of tyrosine hydroxylase- and parvalbumin-positive amacrine cells. Phosphorylated Ser129 α-synuclein expression was strikingly restricted in the OPL, with the most severe degenerations in the entire retina, including mitochondrial degeneration and loss of ribbon synapses in 16- to 18-month-old mice. These synapse- and microcircuit-specific deficits of the rod pathway at the CtBP2+ rod terminals and PKCα+ rod bipolar and amacrine cells were associated with attenuated a- and b-wave amplitudes and oscillatory potentials on the electroretinogram. They were also associated with the impairment of visual functions, including reduced contrast sensitivity and impairment of the middle range of spatial frequencies. Collectively, these findings demonstrate that α-Syn aggregates cause the synapse- and microcircuit-specific deficits of the rod pathway and the most severe damage to the OPL, providing the retinal synaptic and microcircuit basis for visual dysfunctions in PD.

Variants in UBAP1L lead to autosomal recessive rod-cone and cone-rod dystrophy.

PURPOSE: Progressive inherited retinal degenerations (IRDs) affecting rods and cones are clinically and genetically heterogeneous and can lead to blindness with limited therapeutic options. The major gene defects have been identified in subjects of European and Asian descent with only few reports of North African descent. METHODS: Genome, targeted next-generation, and Sanger sequencing was applied to cohort of ∼4000 IRDs cases. Expression analyses were performed including Chip-seq database analyses, on human-derived retinal organoids (ROs), retinal pigment epithelium cells, and zebrafish. Variants' pathogenicity was accessed using 3D-modeling and/or ROs. RESULTS: Here, we identified a novel gene defect with three distinct pathogenic variants in UBAP1L in 4 independent autosomal recessive IRD cases from Tunisia. UBAP1L is expressed in the retinal pigment epithelium and retina, specifically in rods and cones, in line with the phenotype. It encodes Ubiquitin-associated protein 1-like, containing a solenoid of overlapping ubiquitin-associated domain, predicted to interact with ubiquitin. In silico and in vitro studies, including 3D-modeling and ROs revealed that the solenoid of overlapping ubiquitin-associated domain is truncated and thus ubiquitin binding most likely abolished secondary to all variants identified herein. CONCLUSION: Biallelic UBAP1L variants are a novel cause of IRDs, most likely enriched in the North African population.

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function.

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, ß, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor-knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6.

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (GαT). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant GαT subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of GαT and the inhibitor vardenafil occupying the active sites on the PDEα and PDEß subunits. We proposed GαT-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody GαT binds to PDE6 in a similar manner as observed when the antibody is present, does GαT activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 GαT-PDE6 complexes form with either recombinant or retinal GαT in the absence of the GαT antibody. We show that GαT binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables GαT to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting GαT-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of GαT for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.

Hydrophobic interaction between the TM1 and H8 is essential for rhodopsin trafficking to vertebrate photoreceptor outer segments.

A major unsolved question in vertebrate photoreceptor biology is the mechanism of rhodopsin transport to the outer segment. In rhodopsin-like class A G protein-coupled receptors, hydrophobic interactions between C-terminal α-helix 8 (H8), and transmembrane α-helix-1 (TM1) have been shown to be important for transport to the plasma membrane, however whether this interaction is important for rhodopsin transport to ciliary rod outer segments is not known. We examined the crystal structures of vertebrate rhodopsins and class A G protein-coupled receptors and found a conserved network of predicted hydrophobic interactions. In Xenopus rhodopsin (xRho), this interaction corresponds to F313, L317, and L321 in H8 and M57, V61, and L68 in TM1. To evaluate the role of H8-TM1 hydrophobic interactions in rhodopsin transport, we expressed xRho-EGFP where hydrophobic residues were mutated in Xenopus rods and evaluated the efficiency of outer segment enrichment. We found that substituting L317 and M57 with hydrophilic residues had the strongest impact on xRho mislocalization. Substituting hydrophilic amino acids at positions L68, F313, and L321 also had a significant impact. Replacing L317 with M resulted in significant mislocalization, indicating that the hydrophobic interaction between residues 317 and 57 is exquisitely sensitive. The corresponding experiment in bovine rhodopsin expressed in HEK293 cells had a similar effect, showing that the H8-TM1 hydrophobic network is essential for rhodopsin transport in mammalian species. Thus, for the first time, we show that a hydrophobic interaction between H8 and TM1 is critical for efficient rhodopsin transport to the vertebrate photoreceptor ciliary outer segment.

Molecular basis of retinal remodeling in a zebrafish model of retinitis pigmentosa.

A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult zebrafish retina of wild type AB strain (WT) and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. Retinas from both female and male fish were pooled to generate each library, combining data from both sexes. We provide a benchmark atlas of retinal cell type transcriptomes in zebrafish and insight into how each retinal cell type is affected in the P23H model. Oxidative stress is found throughout the retina, with increases in reliance on oxidative metabolism and glycolysis in the affected rods as well as cones, bipolar cells, and retinal ganglion cells. There is also transcriptional evidence for widespread synaptic remodeling and enhancement of glutamatergic transmission in the inner retina. Notably, changes in circadian rhythm regulation are detected in cones, bipolar cells, and retinal pigmented epithelium. We also identify the transcriptomic signatures of retinal progenitor cells and newly formed rods essential for the regenerative process. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.