Biomarker evidence of early vision and rod energy-linked pathophysiology benefits from very low dose DMSO in 5xFAD mice.

Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.

Mathematical model for rod outer segment dynamics during retinal detachment.

Retinal detachment (RD) is the separation of the neural layer from the retinal pigmented epithelium thereby preventing the supply of nutrients to the cells within the neural layer of the retina. In vertebrates, primary photoreceptor cells consisting of rods and cones undergo daily renewal of their outer segment through the addition of disc-like structures and shedding of these discs at their distal end. When the retina detaches, the outer segment of these cells begins to degenerate and, if surgical procedures for reattachment are not done promptly, the cells can die and lead to blindness. The precise effect of RD on the renewal process is not well understood. Additionally, a time frame within which reattachment of the retina can restore proper photoreceptor cell function is not known. Focusing on rod cells, we propose a mathematical model to clarify the influence of retinal detachment on the renewal process. Our model simulation and analysis suggest that RD stops or significantly reduces the formation of new discs and that an alternative removal mechanism is needed to explain the observed degeneration during RD. Sensitivity analysis of our model parameters points to the disc removal rate as the key regulator of the critical time within which retinal reattachment can restore proper photoreceptor cell function.

Light-evoked deformations in rod photoreceptors, pigment epithelium and subretinal space revealed by prolonged and multilayered optoretinography.

Phototransduction involves changes in concentration of ions and other solutes within photoreceptors and in subretinal space, which affect osmotic pressure and the associated water flow. Corresponding expansion and contraction of cellular layers can be imaged using optoretinography (ORG), based on phase-resolved optical coherence tomography (OCT). Until now, ORG could reliably detect only photoisomerization and phototransduction in photoreceptors, primarily in cones under bright stimuli. Here, by employing a phase-restoring subpixel motion correction algorithm, which enables imaging of the nanometer-scale tissue dynamics during minute-long recordings, and unsupervised learning of spatiotemporal patterns, we discover optical signatures of the other retinal structures' response to visual stimuli. These include inner and outer segments of rod photoreceptors, retinal pigment epithelium, and subretinal space in general. The high sensitivity of our technique enables detection of the retinal responses to dim stimuli: down to 0.01% bleach level, corresponding to natural levels of scotopic illumination. We also demonstrate that with a single flash, the optoretinogram can map retinal responses across a 12° field of view, potentially replacing multifocal electroretinography. This technique expands the diagnostic capabilities and practical applicability of optoretinography, providing an alternative to electroretinography, while combining structural and functional retinal imaging in the same OCT machine.

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.

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.

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.

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.

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.

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.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. The specialized downstream circuit for rod signalling, called the primary rod pathway, is well characterized in mammals, but circuitry for rod signalling in non-mammals is largely unknown. Here we demonstrate that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA sequencing, we identified two bipolar cell types in zebrafish that are related to mammalian rod bipolar cell (RBCs), the only bipolar type that directly carries rod signals from the outer to the inner retina in the primary rod pathway. By combining electrophysiology, histology and ultrastructural reconstruction of the zebrafish RBCs, we found that, similar to mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells postsynaptic to one RBC type is strikingly similar to that of mammalian RBCs and their amacrine partners, suggesting that the cell types and circuit design of the primary rod pathway emerged before the divergence of teleost fish and mammals. The second RBC type, which forms separate pathways, was either lost in mammals or emerged in fish.

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.

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.

Non-image-forming vision as measured through ipRGC-mediated pupil constriction is not modulated by covert visual attention.

In brightness, the pupil constricts, while in darkness, the pupil dilates; this is known as the pupillary light response (PLR). The PLR is driven by all photoreceptors: rods and cones, which contribute to image-forming vision, and intrinsically photosensitive retinal ganglion cells (ipRGCs), which mainly contribute to non-image-forming vision. Rods and cones cause immediate pupil constriction upon light exposure, whereas ipRGCs cause sustained constriction throughout light exposure. Recent studies have shown that covert attention modulated the initial PLR; however, it remains unclear whether the same holds for the sustained PLR. We tested this by leveraging ipRGCs' responsiveness to blue light, causing the most prominent sustained constriction. While replicating previous studies by showing that pupils constricted more when either directly looking at, or covertly attending to, bright as compared to dim stimuli (with the same color), we also found that the pupil constricted more when directly looking at blue as compared to red stimuli (with the same luminosity). Crucially, however, in two high-powered studies (n = 60), we did not find any pupil-size difference when covertly attending to blue as compared to red stimuli. This suggests that ipRGC-mediated pupil constriction, and possibly non-image-forming vision more generally, is not modulated by covert attention.

NUDC is critical for rod photoreceptor function, maintenance, and survival.

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC-/- ). Loss of NUDC in rods led to complete photoreceptor cell death at 6 weeks of age. By 3 weeks of age, rNudC-/- function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well-characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC-/- by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. The absence of NUDC also induced an inflammatory response in Müller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein-mediated protein trafficking in a postmitotic rod photoreceptor.

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.

Repeatability of Rod-Mediated Dark Adaptation Testing in Normal Aging and Early and Intermediate Age-Related Macular Degeneration.

PURPOSE: The vulnerability of rod photoreceptors in aging and early and intermediate age-related macular degeneration (AMD) has been well documented. Rod-mediated dark adaptation (RMDA) is a measure of the recovery of light sensitivity in rod photoreceptors following a bright light. Delays in RMDA during early and intermediate AMD have been widely reported. For RMDA's promise as an outcome for trials targeted at early and intermediate AMD to be realized, excellent test-retest reliability, its repeatability, must be established. METHODS: Test-retest performance in a commonly used RMDA test based on the rod intercept time metric (RIT) was evaluated in participants with early and intermediate AMD and with normal retinal aging with testing approximately 2 weeks apart. The test target was placed at 5° eccentricity superior to the foveal center, an area with maximal rod loss in aging and AMD. Disease severity was identified by a trained and masked grader of fundus photographs using both the AREDS 9-step and Beckman classification systems. Bland-Altman plots and intra-class correlation coefficients (ICC) evaluated repeatability. RESULTS: The analysis sample consisted of 37 older adults (mean age 76 years, standard deviation 5), with approximately one-third of the sample in each of three groups - normal aging, early AMD, and intermediate AMD. For the total sample, the ICC was 0.98. For individual AMD groups for both AREDS 9-step and Beckman classifications, the ICCs were also very high ranging from 0.82 to 0.99. CONCLUSION: We demonstrated that RMDA testing using the RIT metric has excellent repeatability when target location is at 5° in studying older adults from normal aging to intermediate AMD, suggesting the reliable use of this functional measure in trials.

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.

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.