Photoreceptor Structure Research Articles

Morphology of the normative human cone photoreceptor mosaic and a publicly available adaptive optics montage repository.

Adaptive optics ophthalmoscopy has enabled visualization of the in vivo human photoreceptor mosaic in health, disease and its treatment. Despite this, the clinical utility of the imaging technology has been limited by a lack of automated analysis techniques capable of accurately quantifying photoreceptor structure and a lack of an available normative image database. Here, we present a fully automated algorithm for estimating cone spacing and density over a complete adaptive optics montage along with a database of normative images and cone densities. We imaged the cone mosaics surrounding the fovea and along the horizontal and vertical meridians of fifty normal-sighted controls with a custom-built, multimodal adaptive optics scanning light ophthalmoscope. Cone spacing was automatically measured in the frequency domain and spacing measurements were converted to estimates of cone density at all locations across the montage. Consistent with previous reports, cone density measurements were highest near fovea (152,906 ± 53,209 cones/mm2) and decreased exponentially with eccentricity. A 2.5-fold variation was found in cone density estimates at 0.1mm, this variation decreased to 1.75-fold at 1mm. We provide all images, mosaic quantifications, and automated software open source. This database will aid investigators in translating adaptive optics ophthalmoscopy to clinical applications.

Safety and efficacy of CRISPR-mediated genome ablation of VEGFA as a treatment for choroidal neovascularization in nonhuman primate eyes

CRISPR-based genome editing enables permanent suppression of angiogenic factors like vascular endothelial growth factor (VEGF) as potential treatment for choroidal neovascularization (CNV) – a major cause of blindness in age-related macular degeneration. We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and guide RNAs (gRNAs) to target conserved sequences in VEGFA across mouse, rhesus macaque, and human, with successful suppression of VEGF and laser-induced CNV in mice. Here, we advanced the platform to nonhuman primates and found that subretinal AAV8-SpCas9 with gRNAs targeting VEGFA may reduce VEGF and CNV severity as compared to SpCas9 without gRNAs. However, all eyes that received AAV8-SpCas9 regardless of gRNA presence developed subfoveal deposits, concentric macular rings, and outer retinal disruption that worsened at higher dose. Immunohistochemistry showed subfoveal accumulation of retinal pigment epithelial cells, collagen, and vimentin, disrupted photoreceptor structure, and retinal glial and microglial activation. Subretinal AAV8-SpCas9 triggered aqueous elevations in CCL2, but minimal systemic humoral or cellular responses against AAV8, SpCas9, or GFP reporter. Our findings suggest that CRISPR-mediated VEGFA ablation in NHP eyes may suppress VEGF and CNV, but can also lead to unexpected subretinal fibrosis, photoreceptor damage, and retinal inflammation despite minimal systemic immune responses.

Age-related retinal degeneration resulting from the deletion of Shp2 tyrosine phosphatase in photoreceptor neurons

Shp2, a critical SH2-domain-containing tyrosine phosphatase, is essential for cellular regulation and implicated in metabolic disruptions, obesity, diabetes, Noonan syndrome, LEOPARD syndrome, and cancers. This study focuses on Shp2 in rod photoreceptor cells, revealing its enrichment, particularly in rods. Deletion of Shp2 in rods leads to age-dependent photoreceptor degeneration. Shp2 targets occludin (OCLN), a tight junction protein, and its deletion reduces OCLN expression in the retina and retinal pigment epithelium (RPE). The isolation of actively translating mRNAs from rods lacking Shp2, followed by RNA sequencing, reveals alterations in cell cycle regulation. Additionally, altered retinal metabolism is observed in retinal cells lacking Shp2. Our studies indicate that Shp2 is crucial for maintaining the structure and function of photoreceptors.

Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

Intensity-based optoretinography reveals sub-clinical deficits in cone function in retinitis pigmentosa.

Clinical tools have been widely used in the diagnosis, description, and monitoring the progression of retinitis pigmentosa (RP); however, many of these methods have inherently low sensitivity and specificity, and significant photoreceptor disruption can occur before RP progression has clinically manifest. Adaptive optics scanning light ophthalmoscopy (AOSLO) has shown promise as a powerful tool for assessing photoreceptor disruption both structurally and functionally due to its increased resolution. Here we assess photoreceptor structure and function at the cellular level through AOSLO by acquiring intensity based optoretinography (iORG) in 15 individuals with no reported retinal pathology and 7 individuals with a prior clinical diagnosis of RP. Photoreceptor structure was quantified by calculating cone nearest neighbor distance (NND) across different retinal eccentricities from the AOSLO images. Cone outer segment length was measured across different retinal eccentricities using optical coherence tomography (OCT) derived longitudinal reflectivity profiles (LRPs). Finally, iORG measures of photoreceptor function were compared to retinal sensitivity as measured using the macular integrity assessment (MAIA) microperimeter. Broadly, participants with RP exhibited increasing cone nearest neighbor distances and decreasing cone outer segment length as a function of retinal eccentricity, consistent with prior reports for both controls and individuals with RP. Nearly all individuals with RP had reduced iORG amplitudes for all retinal eccentricities when compared to the control cohort, and the reduction was greater in eccentricities further from the fovea. Comparing iORG amplitudes to MAIA retinal sensitivity, we found that the iORG was more sensitive to early changes in photoreceptor function whereas MAIA was more sensitive to later stages of disease. This highlights the utility of iORG as a method to detect sub-clinical deficits in cone function in all stages of disease progression and supports the future use of iORG for identifying cells that are candidates for cellular based therapies.

Adaptive optics retinal imaging in patients with usher syndrome.

To determine the structure of the cone photoreceptor mosaic in the macula in eyes with retinitis pigmentosa related to Usher syndrome using adaptive optics fundus (AO) imaging and to correlate these findings with those of the standard clinical diagnostics. Ten patients with a genetically confirmed retinitis pigmentosa in Usher syndrome due to biallelic variants in MYO7A or USH2A were enrolled in the study. All patients underwent a complete ophthalmological examination including best corrected visual acuity (BCVA), spectral-domain optical coherence tomography (SD-OCT) with fundus autofluorescence photography (FAF), full-field (ffERG) and multifocal electroretinography (mfERG) and Adaptive Optics Flood Illuminated Ophthalmoscopy (AO, rtx1™, Imagine Eyes, Orsay, France). The cone density was assessed centrally and at each 0.5 degree horizontally and vertically from 1-4 degree of eccentricity. In the AO images, photoreceptor cell death was visualized as a disruption of the cone mosaic and low cone density. In the early stage of the disease, cones were still visible in the fovea, whereas outside the fovea a loss of cones was recognizable by blurry, dark patches. The blurry patches corresponded to the parafoveal hypofluorescent ring in the FAF images and the beginning loss of the IS/OS line and external limiting membrane in the SD-OCT images. FfERGs were non-recordable in 7 patients and reduced in 3. The mfERG was reduced in all patients and correlated significantly (p <0.001) with the cone density. The kinetic visual field area, measured with III4e and I4e, did not correlate with the cone density. The structure of the photoreceptors in Usher syndrome patients were detectable by AO fundus imaging. The approach of using high-resolution technique to assess the photoreceptor structure complements the established clinical examinations and allows a more sensitive monitoring of early stages of retinitis pigmentosa in Usher syndrome.

Cell-cell interaction in the pathogenesis of inherited retinal diseases.

The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.

Light and electron microscopic observations on retinal neurons of red-tail shark (Epalzeorhynchos bicolor H. M. Smith, 1931).

The structure of photoreceptors (PR) and the arrangement of neurons in the retina of red-tail shark were investigated using light and electron microscopy. The PR showed a mosaic arrangement and included double cones, single cones (SC), and single rods. Most cones occur as SC. The ratio between the number of cones and rods was 3:1.39 (±0.29). The rods were tall that reached the pigmented epithelium. The outer plexiform layer (OPL) showed a complex synaptic connection between the horizontal and photoreceptor terminals that were surrounded by Müller cell processes. Electron microscopy showed that the OPL possessed both cone pedicles and rod spherules. Each rod spherule consisted of a single synaptic ribbon within the invaginating terminal endings of the horizontal cell (hc) processes. In contrast, the cone pedicles possessed many synaptic ribbons within their junctional complexes. The inner nuclear layer consisted of bipolar, amacrine, Müller cells, and hc. Müller cells possessed intermediate filaments and cell processes that can reach the outer limiting membrane and form connections with each other by desmosomes. The ganglion cells were large multipolar cells with a spherical nucleus and Nissl' bodies in their cytoplasm. The presence of different types of cones arranged in a mosaic pattern in the retina of this species favors the spatial resolution of visual objects. RESEARCH HIGHLIGHTS: This is the first study demonstrating the structure and arrangement of retinal neurons of red-tail shark using light and electron microscopy. The current study showed the presence of different types of cones arranged in a mosaic pattern that may favor the spatial resolution of visual objects in this species. The bipolar, amacrine, Müller, and horizontal cells could be demonstrated.

Lycium barbarum glycopeptide (wolfberry extract) slows N-methyl-N-nitrosourea-induced degradation of photoreceptors.

JOURNAL/nrgr/04.03/01300535-202410000-00030/figure1/v/2024-02-06T055622Z/r/image-tiff Photoreceptor cell degeneration leads to blindness, for which there is currently no effective treatment. Our previous studies have shown that Lycium barbarum (L. barbarum) polysaccharide (LBP) protects degenerated photoreceptors in rd1, a transgenic mouse model of retinitis pigmentosa. L. barbarum glycopeptide (LbGP) is an immunoreactive glycoprotein extracted from LBP. In this study, we investigated the potential protective effect of LbGP on a chemically induced photoreceptor-degenerative mouse model. Wild-type mice received the following: oral administration of LbGP as a protective pre-treatment on days 1-7; intraperitoneal administration of 40 mg/kg N-methyl-N-nitrosourea to induce photoreceptor injury on day 7; and continuation of orally administered LbGP on days 8-14. Treatment with LbGP increased photoreceptor survival and improved the structure of photoreceptors, retinal photoresponse, and visual behaviors of mice with photoreceptor degeneration. LbGP was also found to partially inhibit the activation of microglia in N-methyl-N-nitrosourea-injured retinas and significantly decreased the expression of two pro-inflammatory cytokines. In conclusion, LbGP effectively slowed the rate of photoreceptor degeneration in N-methyl-N-nitrosourea-injured mice, possibly through an anti-inflammatory mechanism, and has potential as a candidate drug for the clinical treatment of photoreceptor degeneration.

Intravitreal Injection of ZYAN1 Restored Autophagy and Alleviated Oxidative Stress in Degenerating Retina via the HIF-1α/BNIP3 Pathway.

Mitochondrial autophagy plays a contributary role in the pathogenesis of retina degeneration (RD). ZYAN1 is a novel proline hydroxylase domain (PHD) inhibitor that can enhance the expression of hypoxia-inducible factor 1-alpha (HIF-1α). This study investigated whether ZYAN1 could alleviate progressive photoreceptor loss and oxidative damage in a pharmacologically induced RD model via the modulation of mitophagy. ZYAN1 was injected into the vitreous body of the RD model, and the retinal autophagy level was analyzed. The therapeutic effects of ZYAN1 were evaluated via a function examination, a morphological assay, in situ reactive oxygen species (ROS) detection, and an immunofluorescence assay. It was shown that the thickness of the outer nuclear layer (ONL) increased significantly, and visual function was efficiently preserved via ZYAN1 treatment. The mitochondria structure of photoreceptors was more complete in the ZYAN1-treated mice, and the number of autophagosomes also increased significantly. Membrane disc shedding and ROS overproduction were alleviated after ZYAN1 treatment, and the axonal cilia were more structurally intact. A Western blot analysis showed that the expression levels of the autophagy-related proteins LC3-B, Beclin-1, and ATG5 increased significantly after ZYAN1 treatment, while the expression of P62 was down-regulated. Moreover, the expression levels of HIF-1α and BNIP3 were up-regulated after ZYAN1 treatment. Therefore, an intravitreal injection of ZYAN1 can act as part of the pharmacologic strategy to modulate mitophagy and alleviate oxidative stress in RD. These findings enrich our knowledge of RD pathology and provide insights for the discovery of a therapeutic molecule.

Outstanding women scientists who have broadened the knowledge on biological photoreceptors

Short biographical sketches are given of women born before 1955 who have contributed to our knowledge on the function, structure, and molecular basis of biological photoreceptors, both energy converters and photosensors.Graphical abstract

Ultrastructural Characteristics and Synaptic Connectivity of Photoreceptors in the Simplex Retina of Little Skate (Leucoraja erinacea).

The retinas of the vast majority of vertebrate species are termed "duplex," that is, they contain both rod and cone photoreceptor neurons in different ratios. The retina of little skate (Leucoraja erinacea) is a rarity among vertebrates because it contains only a single photoreceptor cell type and is thus "simplex." This unique retina provides us with an important comparative model and an exciting opportunity to study retinal circuitry within the context of a visual system with a single photoreceptor cell type. What is perhaps even more intriguing is the fact that the Leucoraja retina is able use that single photoreceptor cell type to function under both scotopic and photopic ranges of illumination. Although some ultrastructural characteristics of skate photoreceptors have been examined previously, leading to a general description of them as "rods" largely based on outer segment (OS) morphology and rhodopsin expression, a detailed study of the fine anatomy of the entire cell and its synaptic connectivity is still lacking. To address this gap in knowledge, we performed serial block-face electron microscopy imaging and examined the structure of skate photoreceptors and their postsynaptic partners. We find that skate photoreceptors exhibit unusual ultrastructural characteristics that are either common to rods or cones in other vertebrates (e.g., outer segment architecture, synaptic ribbon number, terminal extensions), or are somewhere in between those of a typical vertebrate rod or cone (e.g., number of invaginating contacts, clustering of multiple ribbons over a single synaptic invagination). We suggest that some of the ultrastructural characteristics we observe may play a role in the ability of the skate retina to function across scotopic and photopic ranges of illumination. Our findings have the potential to reveal as yet undescribed principles of vertebrate retinal design.

The tectonic complex regulates membrane protein composition in the photoreceptor cilium

The primary cilium is a signaling organelle with a unique membrane composition maintained by a diffusional barrier residing at the transition zone. Many transition zone proteins, such as the tectonic complex, are linked to preserving ciliary composition but the mechanism remains unknown. To understand tectonic’s role, we generate a photoreceptor-specific Tctn1 knockout mouse. Loss of Tctn1 results in the absence of the entire tectonic complex and associated MKS proteins yet has minimal effects on the transition zone structure of rod photoreceptors. We find that the protein composition of the photoreceptor cilium is disrupted as non-resident membrane proteins accumulate in the cilium over time, ultimately resulting in photoreceptor degeneration. We further show that fluorescent rhodopsin moves faster through the transition zone in photoreceptors lacking tectonic, which suggests that the tectonic complex acts as a physical barrier to slow down membrane protein diffusion in the photoreceptor transition zone to ensure proper removal of non-resident membrane proteins.

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2−/− and Pnpla2+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.

Assessment of outer retinal thickness and function in mice after experimental optic nerve trauma

BackgroundOptic nerve trauma caused by crush injury is frequently used for investigating experimental treatments that protect retinal ganglion cells (RGCs) and induce axonal regrowth. Retaining outer retinal light responses is essential for therapeutic rescue of RGCs after injury. However, whether optic nerve crush also damages the structure or function of photoreceptors has not been systematically investigated. In this study, we investigated whether outer retinal thickness and visual function are altered by optic nerve crush in the mouse.MethodsWildtype mice underwent optic nerve crush and intravitreal injection of a control solution in one eye with the fellow eye remaining uninjured. Two weeks after injury, the thickness of the ganglion cell region (GCL to IPL) and photoreceptor layer (bottom of the OPL to top of the RPE) were measured using OCT. Retinal function was assessed using flash ERGs. Immunodetection of RGCs was performed on retinal cryosections and RGCs and ONL nuclei rows were counted. Multiple comparison analyses were conducted using Analysis of Variance (ANOVA) with Tukey’s post hoc test and P values less than 0.05 were considered statistically significant.ResultsOptic nerve crush injury induced RGC death as expected, demonstrated by thinning of the ganglion cell region and RGC loss. In contrast, outer retinal thickness, photopic and scotopic a-wave and b-wave amplitudes and photoreceptor nuclei counts, were equivalent between injured and uninjured eyes.ConclusionsSecondary degeneration of the outer retina was not detected after optic nerve injury in the presence of significant RGC death, suggesting that the retina has the capacity to compartmentalize damage. These findings also indicate that experimental treatments to preserve the GCL and rescue vision using this optic nerve injury model would not require additional strategies to preserve the ONL.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

Photoreceptor Function and Structure in Autosomal Dominant Vitelliform Macular Dystrophy Caused by BEST1 Mutations.

The purpose of this study was to evaluate rod and cone function and outer retinal structure within macular lesions, and surrounding extralesional areas of patients with autosomal dominant Best vitelliform macular dystrophy caused by BEST1 mutations. Seventeen patients from seven families were examined with dark- and light-adapted chromatic perimetry and optical coherence tomography. Subsets of patients had long-term follow-up (14-22 years, n = 6) and dark-adaptation kinetics measured (n = 5). Within central lesions with large serous retinal detachments, rod sensitivity was severely reduced but visual acuity and cone sensitivity were relatively retained. In surrounding extralesional areas, there was a mild but detectable widening of the subretinal space in some patients and some retinal areas. Available evidence was consistent with subretinal widening causing slower dark-adaptation kinetics. Over long-term follow-up, some eyes showed formation of de novo satellite lesions at retinal locations that years previously demonstrated subretinal widening. A subclinical abnormality consisting of a retina-wide mild thickening of the outer nuclear layer was evident in many patients and thickening increased in the subset of patients with long-term follow-up. Outcome measures for future clinical trials should include evaluations of rod sensitivity within central lesions and quantitative measures of outer retinal structure in normal-appearing regions surrounding the lesions.

Adaptive optics scanning laser ophthalmoscopy characterization of cone photoreceptor reflectivity perturbation by drusen and subretinal drusenoid deposits

AbstractPurpose: To examine the structure of photoreceptors around drusen and subretinal drusenoid deposits (SDD) in patients with age‐related macular degeneration (AMD) using multimodal imaging, including adaptive optics scanning laser ophthalmoscopy (AOSLO).Methods: Thirty‐four (n = 34) patients with early to intermediate AMD were enrolled and classified into 3 groups based on predominant lesion type. Group 1 included 20 eyes of 15 subjects with medium‐large soft drusen (63–1000 μm diameter), group 2 had 15 eyes of 10 subjects with cuticular drusen (&lt;63 μm), and group 3 had 14 eyes of 9 subjects with SDD. Lesion presence was ascertained by colour fundus photography, near‐infrared reflectance, blue reflectance, autofluorescence, and spectral‐domain optical coherence tomography (SD‐OCT). SDD were classified with a 3‐stage OCT‐based grading system. In addition, the reflectivity and packing mosaic of cone photoreceptors were assessed with AOSLO.Results: AOSLO revealed characteristic photoreceptor reflectivity perturbation by different types of AMD's extracellular lesions. Around large soft drusen, the photoreceptor mosaic was invisible at the edge and visible on the top. The photoreceptors in the retinae with cuticular drusen were largely maintained as a contiguous mosaic but with reduced reflectivity over the lesions. Photoreceptors surrounding SDD showed reduced reflectivity associated with the SDD stage. The en face appearance of the photoreceptor mosaic was consistent with its cross‐sectional structure as rendered by SD‐OCT.Conclusions: The dramatic photoreceptor reflectivity perturbation associated with SDD indicates that SDD may impose a more direct and severe impact on overlying photoreceptors than drusen, suggesting that the retinal function in eyes with SDD may be more severely impaired.@@@@

Photoreceptor function and structure in retinal degenerations caused by biallelic BEST1 mutations

The only approved retinal gene therapy is for biallelic RPE65 mutations which cause a recessive retinopathy with a primary molecular defect located at the retinal pigment epithelium (RPE). For a distinct recessive RPE disease caused by biallelic BEST1 mutations, a pre-clinical proof-of-concept for gene therapy has been demonstrated in canine eyes. The current study was undertaken to consider potential outcome measures for a BEST1 clinical trial in patients demonstrating a classic autosomal recessive bestrophinopathy (ARB) phenotype. Spatial distribution of retinal structure showed a wide expanse of abnormalities including large intraretinal cysts, shallow serous retinal detachments, abnormalities of inner and outer segments, and an unusual prominence of the external limiting membrane. Surrounding the central macula extending from 7 to 30 deg eccentricity, outer nuclear layer was thicker than expected from a cone only retina and implied survival of many rod photoreceptors. Co-localized however, were large losses of rod sensitivity despite preserved cone sensitivities. The dissociation of rod function from rod structure observed, supports a large treatment potential in the paramacular region for biallelic bestrophinopathies.

The medaka mutant deficient in eyes shut homolog exhibits opsin transport defects and enhanced autophagy in retinal photoreceptors.

Eyes shut homolog (EYS) encodes a proteoglycan and the human mutation causes retinitis pigmentosa type 25 (RP25) with progressive retinal degeneration. RP25 most frequently affects autosomal recessive RP patients with many ethnic backgrounds. Although studies using RP models have facilitated the development of therapeutic medications, Eys has been lost in rodent model animals. Here we examined the roles for Eys in the maintenance of photoreceptor structure and function by generating eys-null medaka fish using the CRISPR-Cas9 system. Medaka EYS protein was present near the connecting cilium of wild-type photoreceptors, while it was absent from the eys-/- retina. The mutant larvae exhibited a reduced visual motor response compared with wild-type. In contrast to reported eys-deficient zebrafish at the similar stage, no retinal cell death was detected in the 8-month post-hatching (8-mph) medaka eys mutant. Immunohistochemistry showed a significant reduction in the length of cone outer segments (OSs), retention of OS proteins in the inner segments of photoreceptors, and abnormal filamentous actin network at the base of cone OSs in the mutant retina by 8 mph. Electron microscopy revealed aberrant structure of calyceal processes, numerous vesiculation and lamellar interruptions, and autophagosomes in the eys-mutant cone photoreceptors. In situ hybridization showed an autophagy component gene, gabarap, was ectopically expressed in the eys-null retina. These results suggest eys is required for regeneration of OS, especially of cone photoreceptors, and transport of OS proteins by regulating actin filaments. Enhanced autophagy may delay the progression of retinal degeneration when lacking EYS in the medaka retina.