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PURPOSE To quantitatively compare reflectivity and other morphological changes of the photoreceptors of normal eyes with amblyopic eyes using the longitudinal reflectance profile (LRP) on swept-source optical coherence tomography (SS-OCT) images in children and adolescents with unilateral high myopia. The relationships between OCT parameters and visual acuity were investigated. METHOD Twenty-six amblyopes with unilateral high myopia and 34 age-, axial length- and spherical equivalent-matched normal controls were recruited. All participants underwent SS-OCT and detailed ophthalmic investigations. The reflectivity of the outer retinal and photoreceptor outer segment layer thickness were quantified by LRP using ImageJ software. All parameters were measured at three selected regions: at the fovea, 1 mm nasal to the fovea and 1 mm temporal to the fovea. Differences between the groups were evaluated. RESULTS The mean choroidal thickness was thinner in amblyopic eyes compared with controls (165.19 ± 59.02 μm vs 214.97 ± 66.41 μm at the fovea; 128.77 ± 57.06 μm vs 161.54 ± 57.37 μm at 1 mm nasal to the fovea; 188.13 ± 59.51 μm vs 219.87 ± 61.78 μm at 1 mm temporal to the fovea, P

Mutations in thePRPH2gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened “disc” membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous “whorls” instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENTThe light-sensitive photoreceptor outer segment contains a stack of flattened “disc” membranes that are surrounded, or “enclosed,” by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

Unter „Biomarker“ versteht man „biologische Marker“, die objektiv auf den Gesundheitszustand hinweisen konnen. Neben funktionellen und zeitlichen Faktoren haben insbesondere bildgebende Biomarker in den letzten Jahren an Bedeutung gewonnen. Bei durchgreifenden und lamellaren Defekten der Makula wurden zahlreiche bildgebende Parameter beschrieben. Diese Erkenntnisse fuhrten fur beide Krankheitsbilder zu neuen Einteilungen, die sich auf wichtige „Biomarker“ dieser Krankheitsbilder und charakteristische Merkmale beschriebener Unterformen stutzen. Bildgebende Biomarker fur durchgreifende und lamellare Defekte der Makula werden im Hinblick auf die Klassifikation und ihre funktionelle prognostische Bedeutung beschrieben. Die Bedeutung dieser Biomarker wird dargestellt. Biomarker sind die Grundlage fur die aktuelle Klassifikation von durchgreifenden und lamellaren Makuladefekten. Biomarker dienen weiterhin zur prognostischen und therapeutischen Einschatzung und beeinflussen die operative Strategie. Zur Behandlung eines durchgreifenden Makulaforamens gibt es abhangig von der Foramengrose und anderen Biomarkern verschiedene operative Strategien. So erhoht die invertierte ILM(„internal limiting membrane“)-Flap-Technik die Verschlussrate bei grosen Makulaforamina. Zeigt ein Makulaschichtforamen eine deutliche Progredienz des Befundes, so ermoglicht eine fruhzeitige operative Intervention eine gute anatomische und funktionelle Prognose. Die multimodale Diagnostik liefert bei Makulaforamen und Makulaschichtforamen wichtige pra-, intra- und postoperative Merkmale, die als Biomarker fur die genaue Klassifikation, die Therapiestrategie sowie die Beurteilung der Prognose von herausragender Bedeutung sind.

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy., CLN3 disease is characterised by childhood-onset vision loss and premature death. Using patient-derived retinal cells, the authors show that CLN3 is required for retinal pigment epithelium (RPE) cell structure, microvilli and phagocytosis of photoreceptor outer segments that are essential for vision. They further suggest that gene-therapy targeting RPE cells can be effective for CLN3 disease.

Retinopathy is one of the most severe diabetes-related complications, and macular edema is the major cause of central vision loss in patients with diabetes mellitus. Significant progress has been made in recent years in optical coherence tomography and angiography technology. At the same time, various parameters have been attributed the role of biomarkers creating the frame for new monitoring and treatment strategies and offering new insights into the pathogenesis of diabetic retinopathy and diabetic macular edema. In this review, we gathered the results of studies that investigated various specific OCT (angiography) parameters in diabetic macular edema, such as central subfoveal thickness (CST), cube average thickness (CAT), cube volume (CV), choroidal thickness (CT), retinal nerve fiber layer (RNFL), retinal thickness at the fovea (RTF), subfoveal choroidal thickness (SFCT), central macular thickness (CMT), choroidal vascularity index (CVI), total macular volume (TMV), central choroid thickness (CCT), photoreceptor outer segment (PROS), perfused capillary density (PCD), foveal avascular zone (FAZ), subfoveal neuroretinal detachment (SND), hyperreflective foci (HF), disorganization of the inner retinal layers (DRIL), ellipsoid zone (EZ), inner segment/outer segment (IS/OS) junctions, vascular density (VD), deep capillary plexus (DCP), and superficial capillary plexus (SCP), in order to provide a synthesis of biomarkers that are currently used for the early diagnosis, assessment, monitoring, and outlining of prognosis.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3(f/f(iCre75)) and Stx3(f/f(CRX-Cre))] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis is an autosomal recessive disorder arising from SLC38A8 mutations. SLC38A8 is a putative glutamine transporter with strong expression within the photoreceptor layer in the retina. Previous studies have been limited due to lack of quantitative data on retinal development and nystagmus characteristics. In this multi-centre study, a custom-targeted next generation sequencing (NGS) gene panel was used to identify SLC38A8 mutations from a cohort of 511 nystagmus patients. We report 16 novel SLC38A8 mutations. The sixth transmembrane domain is most frequently disrupted by missense SLC38A8 mutations. Ninety percent of our cases were initially misdiagnosed as PAX6-related phenotype or ocular albinism prior to NGS. We characterized the retinal development in vivo in patients with SLC38A8 mutations using high-resolution optical coherence tomography. All patients had severe grades of arrested retinal development with lack of a foveal pit and no cone photoreceptor outer segment lengthening. Loss of foveal specialization features such as outer segment lengthening implies reduced foveal cone density, which contributes to reduced visual acuity. Unlike other disorders (such as albinism or PAX6 mutations) which exhibit a spectrum of foveal hypoplasia, SLC38A8 mutations have arrest of retinal development at an earlier stage resulting in a more under-developed retina and severe phenotype.

Purpose Ipilimumab is an immune checkpoint inhibitor that is now widely used for patients with metastatic malignant melanoma. However, this immune checkpoint inhibitor can induce related adverse events in various organs. Here, we report a case of bilateral drug (ipilimumab)-induced serous retinal detachment (SRD), and the characteristic features found during swept-source optical coherence tomography (SS-OCT). Case A 78-year-old man with metastatic melanoma received 4 cycles of dacarbazine, nimustine, vincristine, and interferon-beta starting in January 2014, followed by 47 cycles of nivolumab starting in September 2014. Treatment with ipilimumab was started in June 2017. After 2 cycles of ipilimumab, the patient noticed impairment of the visual field in both of his eyes (at day 22) and visited our department in August 2017. Best corrected visual acuity at this initial visit was 16/20 in both eyes, while a fundus examination revealed SRD and retinal pigment epithelial detachments in both eyes. When using the horizontal and vertical 12 mm B-scanned images of SS-OCT, we detected SRD accompanied by a widely distributed high reflection of the photoreceptor outer segment, including the area where the SRD appeared. In addition, the image also showed the interdigitation zone was indistinguishable. Fluorescein angiography showed little leakage in the maculae. During his course of follow-up examinations, there was an increase in the SRD at the beginning, after which it then gradually decreased. However, the obscurity of the interdigitation zone remained. Conclusion Ipilimumab may cause impairment of the outer retinal layer. We suggest that this is a presumed ipilimumab-induced SRD.

Purpose. To identify and correlate age-related changes in outer retinal layers’ thickness and choroidal thickness (CT) in the normal eyes using spectral-domain optical coherence tomography (SD-OCT) and to investigate factors affecting these changes. Study Design. Observational cross-sectional study. Subjects and Methods. We studied 125 healthy Egyptians between 20 and 79 years old. Patients were divided into 3 groups: group 1 (20–40 years), group 2 (40–60 years), and group 3 (>60 years). All patients had full ophthalmic examination. SD-OCT was done to measure the 9 ETDRS macular grid sectors of retinal pigment epithelium and photoreceptor outer segment (RPE-OS), outer nuclear layer and photoreceptor inner segment (ONL-IS), and choroidal thickness (CT) (by enhanced depth imaging). Results. RPE-OS was significantly thinner in group 3 than in the other 2 groups (central: P<0.001). Moreover, the 3 groups were significantly different from each other regarding the CT (central: P<0.001); significant thinning was noticed in the choroid with age. The 3 groups did not show significant difference concerning the ONL-IS thickness. RPE-OS and CT showed statistically significant negative correlation with age (central RPE-OS: r = −0 C.345, P<0.001, and central CT: r = −0.725, P<0.001) while ONL-IS showed statistically nonsignificant correlation with age (central ONL-IS: r = −0.08, P=0.376). Multiple regression analysis revealed that the most important determinant of central 1 mm RPE-OS thickness in this study was age (β = −0.087, P=0.010) rather than choroidal thinning (β = 0.001, P=0.879). Conclusion. RPE-OS layer thickness shows significant thinning with increasing age, and with decrease in CT, however, age is the most determinant factor of this thinning.

BACKGROUND: Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. METHODS: BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65 kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. RESULTS: The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7–14 days were positive for RPE65 (92.76–100%), CRALBP (95.21–100%) and OTX2 (94.88–100%), and after 30 days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. CONCLUSION: Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performance manner over a short period of time. These cells due to expressing the RPELC genes and markers can be used in cell replacement therapy for degenerative diseases including age-related macular degeneration as well as retinitis pigmentosa.

Retinitis pigmentosa (RP) (OMIM: 268000) is a rare, heterogeneous group of inherited ocular disorders that results in a progressive retinal degeneration.1, 2 The PRPH2 gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_000322.4","term_id":"194248075","term_text":"NM_000322.4"}}NM_000322.4) (OMIM: 179605), also known as RDS, AOFMD, AVMD, CACD2, DS, MDBS1, PRPH, rd2, RP7 and TSPAN22, is located on chromosome 6p21.1 with three exons spanning 26 395 bp length in human genome (GRCh38/hg38) that encodes a putative protein with 346 amino acids.3 The PRPH2 protein ({"type":"entrez-protein","attrs":{"text":"NP_000313.2","term_id":"118572596","term_text":"NP_000313.2"}}NP_000313.2) is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The majority of the members are cell‐surface proteins which were identified by the presence of four hydrophobic domains. The PRPH2 protein is a membrane‐associated glycoprotein, which is restricted to the area of photoreceptor outer segment discs.4 PRPH2 functions as an adhesion molecule by stabilization and compaction of outer segment discs. PRPH2 and ROM1 (OMIM: 180721) can be assembled into noncovalent tetramers (heterodimer) in vivo using disulphide bonds and higher order disulphide‐linked oligomers, thereby involving in photoreceptor disc morphogenesis.5 Mutations in the PRPH2 gene are involved with assorted blinding diseases of the retina, inducing degenerations in both central retinal and peripheral retinal.6, 7, 8 The relationships between the mutations in the PRPH2 gene and the resultant diseases in the patients are variable; making genotype/phenotype correlations different. PRPH2 mutation in RP patients and genotype/phenotype relationship have not been well described in the Chinese population.

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are in fact hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Retinal degeneration also occurs temporarily during the photoreceptor maturation but stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

To explore predisease biomarkers, which may help screen for the risk of age-related macular degeneration (AMD) at very early stages, macular pigment optical density (MPOD) and photoreceptor outer segment (PROS) length were analyzed. Thirty late AMD fellow eyes, which are at high risk and represent the predisease condition of AMD, were evaluated and compared with 30 age-matched control eyes without retinal diseases, there was no early AMD involvement in the AMD fellow eyes. MPOD was measured using MPS2&reg, (M.E. Technica Co. Ltd., Tokyo, Japan), and PROS length was measured based on optical coherence tomography images. MPOD levels and PROS length in the AMD fellow eyes were significantly lower and shorter, respectively, than in control eyes. MPOD and PROS length were positively correlated in control eyes (R = 0.386, p = 0.035) but not in AMD fellow eyes. Twenty (67%) AMD fellow eyes met the criteria of MPOD &lt, 0.65 and/or PROS length &lt, 35 &mu, m, while only five (17%) control eyes did. After adjusting for age and sex, AMD fellow eyes more frequently satisfied the definition (p &lt, 0.001, 95% confidence interval, 3.50&ndash, 60.4, odds ratio, 14.6). The combination of MPOD and PROS length may be a useful biomarker for screening predisease AMD patients, although further studies are required in this regard.

Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤ 2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and β-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. βA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.

Photoreceptor cells are specialized neurons with a sensory cilium carrying an elaborate membrane structure, the outer segment (OS). Inherited mutations in genes involved in ciliogenesis frequently result in OS malformation and blindness. ADP-ribosylation factor-like 2 (ARL2) has recently been implicated in OS formation through its association with Binder of ARL2 (BART or ARL2BP), a protein linked to inherited blinding disease. To test the role of ARL2 in vision we created a transgenic mouse model expressing a tagged-dominant active form of human ARL2 (ARL2-Q70L) under a rod-specific promoter. Transgenic ARL2-Q70L animals exhibit reduced photoreceptor cell function as early as post-natal day 16 and progressive rod degeneration. We attribute loss of photoreceptor function to the defective OS morphogenesis in the ARL2-Q70L transgenic model. ARL2-Q70L expression results in shortened inner and outer segments, shortened and mislocalized axonemes and cytoplasmic accumulation of rhodopsin. In conclusion, we show that ARL2-Q70L is crucial for photoreceptor neuron sensory cilium development. Future research will expand upon our hypothesis that ARL2-Q70L mutant interferes with microtubule maintenance and tubulin regulation resulting in impaired growth of the axoneme and elaboration of the photoreceptor outer segment.

Item does not contain fulltext Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an approximately 20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

There was a significant increase in the incidence of retinal degeneration in F344/N rats chronically exposed to Kava kava extract (KKE) in National Toxicology Program (NTP) bioassay. A retrospective evaluation of these rat retinas indicated a similar spatial and morphological alteration as seen in light-induced retinal degeneration in albino rats. Therefore, it was hypothesized that KKE has a potential to exacerbate the light-induced retinal degeneration. To investigate the early mechanism of retinal degeneration, we conducted a 90-day F344/N rat KKE gavage study at doses of 0 and 1.0 g/kg (dose which induced retinal degeneration in the 2-year NTP rat KKE bioassay). The morphological evaluation indicated reduced number of phagosomes in the retinal pigment epithelium (RPE) of the superior retina. Transcriptomic alterations related to retinal epithelial homeostasis and melatoninergic signaling were observed in microarray analysis. Phagocytosis of photoreceptor outer segment by the underlying RPE is essential to maintain the homeostasis of the photoreceptor layer and is regulated by melatonin signaling. Therefore, reduced photoreceptor outer segment disc shedding and subsequent lower number of phagosomes in the RPE and alterations in the melatonin pathway may have contributed to the increased incidences of retinal degeneration observed in F344/N rats in the 2-year KKE bioassay.

Stargardt macular dystrophy 3 (STGD3) is caused by dominant mutations in the ELOVL4 gene. Like other macular degenerations, pathogenesis within the retinal pigment epithelium (RPE) appears to contribute to the loss of photoreceptors from the central retina. However, the RPE does not express ELOVL4, suggesting photoreceptor cell loss in STGD3 occurs through two cell nonautonomous events: mutant photoreceptors first affect RPE cell pathogenesis, and then, second, RPE dysfunction leads to photoreceptor cell death. Here, we have investigated how the RPE pathology occurs, using a STGD3 mouse model in which mutant human ELOVL4 is expressed in the photoreceptors. We found that the mutant protein was aberrantly localized to the photoreceptor outer segment (POS), and that resulting POS phagosomes were degraded more slowly in the RPE. In cell culture, the mutant POSs are ingested by primary RPE cells normally, but the phagosomes are processed inefficiently, even by wild-type RPE. The mutant phagosomes excessively sequester RAB7A and dynein, and have impaired motility. We propose that the abnormal presence of ELOVL4 protein in POSs results in phagosomes that are defective in recruiting appropriate motor protein linkers, thus contributing to slower degradation because their altered motility results in slower basal migration and fewer productive encounters with endolysosomes. In the transgenic mouse retinas, the RPE accumulated abnormal-looking phagosomes and oxidative stress adducts; these pathological changes were followed by pathology in the neural retina. Our results indicate inefficient phagosome degradation as a key component of the first cell nonautonomous event underlying retinal degeneration due to mutant ELOVL4.

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

To describe early structural and functional retinal changes in a patient with Cohen syndrome.A 13-month-old Caucasian girl of Irish and Spanish ancestry was noted to have micrognathia and laryngomalacia at birth, which prompted a genetic evaluation that revealed biallelic deletions in COH1 (VPS13B) (a maternally inherited 60-kb deletion involving exons 26-32 and a paternally inherited 3.5-kb deletion within exon 17) consistent with Cohen syndrome. She underwent a complete ophthalmic examination, full-field flash electroretinography and retinal imaging with spectral domain optical coherence tomography.Central vision was central, steady, and maintained. There was bilateral myopic astigmatic refractive error. Fundus exam was notable for dark foveolar pigmentation, but no obvious abnormalities of either eye. Spectral domain optical coherence tomography cross sections through the fovea revealed a normal appearing photoreceptor outer nuclear layer but loss of the interdigitation signal between the photoreceptor outer segments and the apical retinal pigment epithelium. Retinoschisis involving the inner nuclear layer of both eyes and possible ganglion cell layer thinning were also noted. There was a detectable electroretinogram with similarly reduced amplitudes of rod- (white, 0.01 cd.s.mPhotoreceptor outer segment abnormalities and retinoschisis may represent the earliest structural retinal change detected by spectral domain optical coherence tomography in patients with Cohen syndrome, suggesting a complex pathophysiology with primary involvement of the photoreceptor cilium and disorganization of the structural integrity of the inner retina.

Purpose/Aim. The Ins2 (Akita) mouse is a spontaneous diabetic mouse model with a heterozygous mutation in the insulin 2 gene that results in sustained hyperglycemia. The purpose of the study was to assess global and local retinal layer thickness alterations in Akita mice by analysis of spectral domain optical coherence tomography (SD-OCT) images.Materials and Methods. SD-OCT imaging was performed in Akita and wild-type mice at 12 and 24 weeks of age. Inner retinal thickness (IRT), outer retinal thickness (ORT), total retinal thickness (TRT), and photoreceptor outer segment length (OSL) were measured. Mean global thickness values were compared between Akita and wild-type mice. Local thickness variations in Akita mice were assessed based on normative values in wild-type mice.Results. Akita mice had higher blood glucose levels and lower body weights (p<0.001). On average, IRT, ORT, and TRT were approximately 2% lower in Akita mice than in wild-type mice (p≤0.02). In Akita mice, the percent difference between retinal areas with thickness below and above normative values for IRT, ORT, and TRT was 22%, 32%, and 38%, respectively.Conclusions. These findings support the use of the Akita mouse model to study the retinal neurodegenerative effects of hyperglycemia.

Kimiko Shimizu,1 Yohei Hashimoto,1 Keiko Azuma,1 Yoko Nomura,1 Ryo Obata,1 Hidenori Takahashi,1,2 Yasuo Yanagi3,4 1Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo, 2Department of Ophthalmology, Jichi Medical University, Tochigi, Japan; 3Singapore Eye Research Institute, Singapore National Eye Center, 4Ophthalmology and Visual Sciences Program, Duke-NUS Medical School, National University of Singapore, Singapore Purpose: To evaluate the morphological changes in retinal microstructures following modified photodynamic therapy (PDT) for chronic central serous chorioretinopathy. Subjects and methods: Retrospective study of 21 consecutive eyes (age, 59±8.0 years [mean ± SD]) of 21 patients with chronic central serous chorioretinopathy. Inner foveal thickness (the distance between the internal limiting membrane and external limiting membrane), photoreceptor outer segment (PROS) length at the point without serous retinal detachment and choroidal thickness were assessed with enhanced depth imaging optical coherence tomography before treatment and at 1, 3 and 6 months after modified PDT. Results: Six eyes had half-dose and 15 eyes had half-fluence PDT. PROS length within the irradiated area was significantly decreased by 3.9% at 1 month compared with that before modified PDT (43.5±5.72 and 40.5±7.53, P=0.020 [paired t-test]) and returned to baseline length at 3 and 6 months (43.0±5.42 and 43.5±4.33 µm [mean ± SD]; P=0.53 and 1.00 [paired t-test]). PROS length of the nonirradiated area at 1, 3 and 6 months after modified PDT did not significantly differ from that before PDT (41.8±6.35, 41.4±5.33, 41.9±4.67 and 42.3±4.26 µm [mean ± SD]; P=0.66, 0.90 and 0.60, respectively [paired t-test]). There was no significant change in inner foveal thickness during the observation period. At all time points, choroidal thickness was significantly thinner than it was before PDT (P

Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc.

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but USH1 protein function in the retina is unclear. Schietroma et al. use Xenopus to model the deficiency in two USH1 proteins—protocadherin-15 and cadherin-23—and identify crucial roles for these molecules in shaping the photoreceptor outer segment., Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.

The membranes of retina photoreceptors have unique lipid composition. They contain a high concentration of polyunsaturated docosahexaenoic acid, with six double bonds, and are enriched in phosphatidylethanolamines. Based on their phospholipid composition and cholesterol content, membranes of photoreceptors can be divided into three types: plasma membrane, young disks membranes, and old disks membranes. High amount of docosahexaenoic acid, abundant illumination, and high respiratory demands make these membranes sensitive to oxidative stress and lipid peroxidation. Human retinas are not easily available for research, therefore most research is done on bovine retinas. However, to follow, in a controlled manner, the changes in membrane properties caused by different factors it seems advisable to apply carefully prepared models of photoreceptor membranes. Using synthetic lipids we prepared liposome models of three types of photoreceptor membranes, and by means of electron paramagnetic resonance spectroscopy and spin labeling technique we compared polarity and fluidity of those model membranes with the properties of membranes consisting of natural lipids extracted from photoreceptor outer segments of bovine retinas. Additionally, we studied the effect of oxidation on the membrane properties in the presence and in the absence of zeaxanthin, which is an antioxidant naturally present in the human retina. The results show that there are significant differences in polarity and fluidity between all investigated membranes, which reflect differences in their lipid composition. The properties of the membranes made of natural photoreceptor outer segment lipids are most similar to the ones of the models of old disks membranes. Oxidation did not change the membrane properties significantly; however, a slight ordering effect was observed in liposomes made of natural photoreceptor outer segment lipids and in the model of old disks membranes. Zeaxanthin affected polarity and fluidity mostly in the model of old disks membranes. The results show that by careful selection and appropriate proportions of lipid mixtures, it is possible to obtain synthetic membranes of the properties similar to the natural ones.

Visual signal transduction occurs on the surface of membrane discs stacked inside the ciliary outer segment of photoreceptor cells. Salinas et al. show that discs are formed from ciliary ectosomes whose release is blocked by the protein peripherin/RDS. This explains how photoreceptors transform their primary cilia into the light-sensing outer segment organelle., The primary cilium is a highly conserved organelle housing specialized molecules responsible for receiving and processing extracellular signals. A recently discovered property shared across many cilia is the ability to release small vesicles called ectosomes, which are used for exchanging protein and genetic material among cells. In this study, we report a novel role for ciliary ectosomes in building the elaborate photoreceptor outer segment filled with hundreds of tightly packed “disc” membranes. We demonstrate that the photoreceptor cilium has an innate ability to release massive amounts of ectosomes. However, this process is suppressed by the disc-specific protein peripherin, which enables retained ectosomes to be morphed into discs. This new function of peripherin is performed independently from its well-established role in maintaining the high curvature of disc edges, and each function is fulfilled by a separate part of peripherin’s molecule. Our findings explain how the outer segment structure evolved from the primary cilium to provide photoreceptor cells with vast membrane surfaces for efficient light capture.

Mutations in the peripherin-2 gene (PRPH2, also known as rds) cause a heterogeneous range of autosomal dominant retinal diseases. PRPH2 encodes a photoreceptor-specific tetraspanin protein, PRPH2, that is a main structural component of the photoreceptor outer segment. PRPH2 distributes to the rims of outer segment disc membranes as they undergo the process of disc membrane enclosure. Within these rims, PRPH2 exists in homo-oligomeric form or as a hetero-oligomer with another tetraspanin protein, ROM1. While complete loss of PRPH2 prevents photoreceptor outer segment formation, mutations affecting the state of its oligomerization, including C150S, C213Y and Y141C, produce outer segment structural defects. In this study, we addressed whether any of these mutations also affect disc enclosure. We employed recently developed methodology for ultrastructural analysis of the retina, involving tissue processing with tannic acid, to assess the status of disc enclosure in knockin mouse models bearing either one or two alleles of the C150S, C213Y and Y141C PRPH2 mutations. While varying degrees of outer segment structural abnormalities were observed in each of these mouse models, they contained both newly forming "open" discs and mature "enclosed" discs. These data demonstrate that normal PRPH2 oligomerization is not essential for photoreceptor disc enclosure.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.