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Early detection of photoreceptor dysfunction is essential for preventing vision loss due to retinal degenerative diseases, such as age-related macular degeneration (AMD) and inherited retinal degenerations (IRDs). Functional intrinsic optical signal (IOS) imaging promises a high-resolution method for objective optoretinography (ORG). Stimulus-evoked photoreceptor-IOS has been recently demonstrated in healthy animal and human retinas. The fast photoreceptor-IOS response was found to occur at the photoreceptor outer segment (OS) right after the onset of retinal stimulation. However, in vivo IOS response of photoreceptor dysfunctions is not yet validated, which is essential to measure the clinical usability of ORG measurement. In this study, we report in vivo IOS imaging of rod photoreceptor dysfunction in retinal degeneration 10 (rd10) mice. A custom-designed optical coherence tomography (OCT) was used for photoreceptor-IOS imaging. A significant attenuation of the photoreceptor-IOS was found in rd10 mice due to disorganized ultrastructure of the photoreceptor OSs, which appeared ahead of progressive rod cell death. Our experiments demonstrate that fast photoreceptor-IOS is highly sensitive to ultrastructural integrity of the photoreceptor OSs. We anticipate that quantitative imaging of fast photoreceptor-IOS will provide objective ORG measurement to advance the study and diagnosis of AMD, IRDs, and other retinal diseases that can cause photoreceptor dysfunctions.

Intrinsic optical signal (IOS) imaging promises a noninvasive method for objective assessment of retinal function. This study demonstrates concurrent optical coherence tomography (OCT) of amplitude-IOS and phase-IOS changes in human photoreceptors. A new procedure for differential-phase-mapping (DPM) is validated to enable depth-resolved phase-IOS imaging. Dynamic OCT revealed rapid amplitude-IOS and phase-IOS changes, which occur almost right away after the stimulus onset. These IOS changes were predominantly observed within the photoreceptor outer segment (OS), particularly two boundaries connecting to the inner segment and retinal pigment epithelium. The comparative analysis supports that both amplitude-IOS and phase-IOS attribute to transient OS morphological change associated with phototransduction activation in retinal photoreceptors. A simulation modeling is proposed to discuss the relationship between the photoreceptor OS length and phase-IOS changes.

PURPOSE To establish the factors responsible for attenuation of the optical coherence tomography (OCT) signal beneath the neurosensory detachment (NSD). METHODS We retrospectively reviewed 33 eyes with acute central serous chorioretinopathy. The thickness of the neurosensory retina, the thickness of the photoreceptor outer segment (PROS) layer, the height of the NSD, and the reflectivity of the underlying retinal pigment epithelium (RPE) were measured at selected points of B-scans exported from 6 × 6 mm OCT angiography protocols. The intensity of the flow signal was measured at the corresponding regions of the choriocapillaris slab. The correlation between the parameters of the NSD and both the reflectivity of underlying RPE and the intensity of the flow signal in the choriocapillaris was calculated. RESULTS Correlation coefficients between RPE reflectivity and neurosensory retinal thickness, PROS layer thickness, and NSD height were -0.32, -0.64, and -0.25, respectively (p

The article describes a clinical case of a 14-year old patient with RDH5 mutations (OMIM *601617) in patient with fundus albipunctatus (OMIM #136880) and characteristic biomarkers of this disease with previously described pathogenic variant of nucleotic sequence in exon 3 of the RDH5 gene (NM_002905.3:c.500GA), causing a missense change (p.Arg167His) in heterozygous state and previously not described pathogenic variant of nucleotic sequence in exon 5 of the RDH5 gene (NM_002905.3:c.838CT), leading to a missense change (p.Arg280Cys) in heterozygous state with characteristic biomarkers of the disease. Best-corrected visual acuity (BCVA) was 20/20. Nyctalopia was accompanied by reduced b-wave of scotopic (dark-adapted 0.01) ERG and decreased amplitude of a- and b-waves of maximum (dark-adapted 3) ERG. Decreased amplitude of the a- and b-waves of photopic (light-adapted 3) ERG and the amplitude of high-frequency (light-adapted 30 Hz) Flicker ERG shows the involvement of retinal cone system in the process. Fundus autofluorescence imaging of both eyes produced fuzzy and grainy images with slight hyperfluorescence of retinal flecks. Optical coherence tomography showed focal thickening centered in the photoreceptor outer segment corresponding to the multiple discrete albipunctate dots.В статье описывается клинический случай белоточечного глазного дна (fundus albipunctatus, OMIM #136880) с мутациями в гене RDH5 (OMIM *601617) в 3-м экзоне гена RDH5 (NM_002905.3:c.500GA), приводящий к образованию миссенс-замены (p.Arg167His) в гетерозиготном состоянии и не описанный ранее как патогенный вариант нуклеотидной последовательности в 5-м экзоне гена RDH5 (NM_002905.3:c.838CT), приводящий к образованию миссенс-замены (p.Arg280Cys) в гетерозиготном состоянии с характерными биомаркерами этого заболевания у пациента 14 лет. Острота зрения с коррекцией составила OU 1,0. Жалобы на ухудшение зрения в темноте сопровождались снижением амплитуды b-волны скотопической электроретинографии (ЭРГ) и a- и b-волн максимальной ЭРГ. Снижение амплитуды a- и b-волн фотопической ЭРГ и амплитуды высокочастотной ритмической ЭРГ на 30 Гц свидетельствовало о вовлечении в процесс колбочковой системы сетчатки. На аутофлюоресцентных изображениях глазного дна визуализировались нечеткие, зернистые, слегка гиперфлюоресцирующие очажки. На изображениях, полученных при оптической когерентной томографии, визуализировались фокальные утолщения с центром в наружных сегментах фоторецепторов, соответствующие множественным дискретным белоточечным пятнам.

The objective of this study is to verify the anatomic correlate of the second (2nd ) outer retina band in optical coherence tomography (OCT), and to demonstrate the potential of using intrinsic optical signal (IOS) imaging for concurrent optoretinography (ORG) of phototransduction activation and energy metabolism in stimulus activated retinal photoreceptors. A custom-designed OCT was employed for depth-resolved IOS imaging in mouse retina activated by a visible light flicker stimulation. The spatiotemporal properties of the IOS changes at the photoreceptor outer segment (OS) and inner segment (IS) were quantitatively evaluated. Rapid IOS change was observed at the OS almost right away, and the IOS at the IS was relatively slow. Comparative analysis indicates that the OS-IOS reflects transient OS deformation caused by the phototransduction activation, and IS-IOS might reflect the energy metabolism caused by mitochondria activation in retinal photoreceptors. The consistency of the distribution of the IS-IOS and the 2nd OCT band supports the IS ellipsoid (ISe), which has abundant mitochondria, as the signal source of the 2nd OCT band of the outer retina. This article is protected by copyright. All rights reserved.

PurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by thePNPLA2gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.MethodsMice in whichPNPLA2was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2silencing duplexes. POS were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.ResultsThe RPE of the cKO mice accumulated lipids as well as more abundant and larger rhodopsin particles compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and inPNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells.PNPLA2knock down also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor outer segment (POS) phagocytosis occurs as a daily peak, roughly about 1 hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2) lack a daily peak in POS phagocytosis. By qPCR analysis, we found that core clock genes were rhythmic over 24 hours in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time points preceding and during the peak of POS phagocytosis. In contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time point. Finally, based on STRING analysis, we found a group of interacting genes that potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b, and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.

Inherited retinal diseases (IRDs) were first classified clinically by history, ophthalmoscopic appearance, type of visual field defects, and electroretinography (ERG). ERGs isolating the two major photoreceptor types (rods and cones) showed some IRDs with greater cone than rod retinal dysfunction; others were the opposite. Within the cone-rod diseases, there can be phenotypic variability, which can be attributed to genetic heterogeneity and the variety of visual function mechanisms that are disrupted. Most cause symptoms from childhood or adolescence, although others can manifest later in life. Among the causative genes for cone-rod dystrophy (CORD) are those encoding molecules in phototransduction cascade activation and recovery processes, photoreceptor outer segment structure, the visual cycle and photoreceptor development. We review 11 genes known to cause cone-rod disease in the context of their roles in normal visual function and retinal structure. Knowledge of the pathobiology of these genetic diseases is beginning to pave paths to therapy.

Lipids play essential roles in maintaining cell structure and function by modulating membrane fluidity and cell signaling. The fatty acid elongase-4 (ELOVL4) protein, expressed in retina, brain, Meibomian glands, skin, testes and sperm, is an essential enzyme that mediates tissue-specific biosynthesis of both VLC-PUFA and VLC-saturated fatty acids (VLC-SFA). These fatty acids play critical roles in maintaining retina and brain function, neuroprotection, skin permeability barrier maintenance, and sperm function, among other important cellular processes. Mutations in ELOVL4 that affect biosynthesis of these fatty acids cause several distinct tissue-specific human disorders that include blindness, age-related cerebellar atrophy and ataxia, skin disorders, early-childhood seizures, mental retardation, and mortality, which underscores the essential roles of ELOVL4 products for life. However, the mechanisms by which one tissue makes VLC-PUFA and another makes VLC-SFA, and how these fatty acids exert their important functional roles in each tissue, remain unknown. This review summarizes research over that last decade that has contributed to our current understanding of the role of ELOVL4 and its products in cellular function. In the retina, VLC-PUFA and their bioactive “Elovanoids” are essential for retinal function. In the brain, VLC-SFA are enriched in synaptic vesicles and mediate neuronal signaling by determining the rate of neurotransmitter release essential for normal neuronal function. These findings point to ELOVL4 and its products as being essential for life. Therefore, mutations and/or age-related epigenetic modifications of fatty acid biosynthetic gene activity that affect VLC-SFA and VLC-PUFA biosynthesis contribute to age-related dysfunction of ELOVL4-expressing tissues., Graphical abstract

The retinal pigment epithelium (RPE) is a highly specialized monolayer of polarized, pigmented epithelial cells that resides between the vessels of the choriocapillaris and the neural retina. The RPE is essential for the maintenance and survival of overlying light-sensitive photoreceptors, as it participates in the formation of the outer blood-retinal barrier, phagocytosis, degradation of photoreceptor outer segment (POS) tips, maintenance of the retinoid cycle, and protection against light and oxidative stress. Autophagy is an evolutionarily conserved 'self-eating' process, designed to maintain cellular homeostasis. The daily autophagy demands in the RPE require precise gene regulation for the digestion and recycling of intracellular and POS components in lysosomes in response to light and stress conditions. In this review, we discuss selective autophagy and focus on the recent advances in our understanding of the mechanism of cell clearance in the RPE for visual function. Understanding how this catabolic process is regulated by both transcriptional and post-transcriptional mechanisms in the RPE will promote the recognition of pathological pathways in genetic disease and shed light on potential therapeutic strategies to treat visual impairments in patients with retinal disorders associated with lysosomal dysfunction.

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.

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to—and relieves inhibition of—PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9–1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor Outer Segment (POS) phagocytosis occurs as a daily peak, roughly about one hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2), lack a daily peak in POS phagocytosis. By qPCR analysis we found that core clock genes were rhythmic over 24h in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially, expressed genes between time-points preceding and during the peak of POS phagocytosis. By contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time-point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time-point. Finally, based on STRING analysis we found a group of interacting genes which potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.DeclarationsFundingThis project has been funded with support from the NeuroTime Erasmus+ grant (European Union), Rotterdamse Stichting Blindenbelangen (Netherlands), Nelly Reef fund (Netherlands), Stichting voor Ooglijders (Netherlands), Stichting tot Verbetering van het Lot der Blinden (Netherlands) and Retina France (France).Conflicts of interest/Competing interestsThe authors declare no competing interests.Availability of data and materialData supporting the conclusions of this article are included within the article and are available from the corresponding authors on reasonable request.Code availabilityThe R code for analysis is available from the corresponding authors on reasonable request.Ethics approvalAll experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on Use of Animals in Ophthalmic and Vision Research, as well as with the European Union Directive (2010/63/EU).Consent to participateNot applicableConsent for publicationAll authors read and approve of the contents of this manuscript.Author contributionsN.M. performed experiments, analysis, prepared figures, wrote the manuscript and obtained funding. O.A.-H.H. performed experiments, data analysis, prepared figures and obtained funding. P.D.M. and A.J. performed bioinformatics analysis and edited the manuscript. U.B., J.B.t.B. and C.S. provided technical assistance, performed experiments, prepared figures and edited the manuscript. D.H., A.A.B. and M.-P.F.-S. conceptualized and directed the project, obtained funding, provided resources, performed analysis and edited the manuscript.

Metabolic syndrome, a condition involving obesity and hypertension, increases the risk of aging-associated diseases such as age-related macular degeneration (AMD). Here, we demonstrated that high-fat diet (HFD)-fed mice accumulated oxidized low-density lipoprotein (ox-LDL) in macrophages through the renin–angiotensin system (RAS). The ox-LDL-loaded macrophages were responsible for visual impairment in HFD mice along with a disorder of the retinal pigment epithelium (RPE), which is required for photoreceptor outer segment renewal. RAS repressed ELAVL1, which reduced PPARγ, impeding ABCA1 induction to levels that are sufficient to excrete overloaded cholesterol within the macrophages. The ox-LDL-loaded macrophages expressed inflammatory cytokines and attacked the RPE. An antihypertensive drug, angiotensin II type 1 receptor (AT1R) blocker, resolved the decompensation of lipid metabolism in the macrophages and reversed the RPE condition and visual function in HFD mice. AT1R signaling could be a future therapeutic target for macrophage-associated aging diseases, such as AMD., Nagai et al. show that mice fed high-fat diet (HFD) accumulate oxidized low-density lipoprotein in macrophages through the renin–angiotensin system, which impairs visual function. They find that angiotensin II type 1 receptor (AT1R) improves the visual function of HFD mice, suggesting AT1R signaling as a potential therapeutic target for age-related macular degeneration.

The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment., Graphical Abstract, Highlights • The unique proteome of the photoreceptor outer segment plasma membrane is identified. • TMEM67, TMEM237, and embigin are novel unique components of this membrane. • Embigin in this membrane is associated with the monocarboxylate transporter MCT1. • The photoreceptor outer segment likely facilitates lactate transport., In Brief The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment.

The pupil-aligned orientation of retinal photoreceptors is dynamically phototropic, but different between outer and inner segment., Almost a century ago, Stiles and Crawford reported that the human eye is more sensitive to light entering through the pupil center than through its periphery (Stiles-Crawford effect). This psychophysical phenomenon, later found to correlate with photoreceptor orientation toward the pupil, was dynamically phototropic, adjustable within days to an eccentrically displaced pupil. For decades, this phototropism has been speculated to involve coordinated movements of the rectilinear photoreceptor outer and inner segments. We report here that, unexpectedly, the murine photoreceptor outer segment has a seemingly light-independent orientation, but the inner segment’s orientation undergoes light-dependent movement, giving rise to nonrectilinear outer and inner segments in adult mice born and reared in darkness. Light during an early critical period (~P0 to P8), however, largely sets the correct photoreceptor orientation permanently afterward. Unexpectedly, abolishing rod and cone phototransductions did not mimic darkness in early life, suggesting photosignaling extrinsic to rods and cones is involved.

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.

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models, one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn&minus, /&minus, and Prph2Y141C/+/Rtbdn&minus, retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

Most of the ATP to satisfy the energetic demands of the cell is produced by the F 1 F o -ATP synthase (ATP synthase) which can also function outside the mitochondria. Active oxidative phosphorylation (OxPhos) was shown to operate in the photoreceptor outer segment, myelin sheath, exosomes, microvesicles, cell plasma membranes and platelets. The mitochondria would possess the exclusive ability to assemble the OxPhos molecular machinery so to share it with the endoplasmic reticulum (ER) and eventually export the ability to aerobically synthesize ATP in true extra-mitochondrial districts. The ER lipid rafts expressing OxPhos components is indicative of the close contact of the two organelles, bearing different evolutionary origins, to maximize the OxPhos efficiency, exiting in molecular transfer from the mitochondria to the ER. This implies that its malfunctioning could trigger a generalized oxidative stress. This is consistent with the most recent interpretations of the evolutionary symbiotic process whose necessary prerequisite appears to be the presence of the internal membrane system inside the eukaryote precursor, of probable archaeal origin allowing the engulfing of the α-proteobacterial precursor of mitochondria. The process of OxPhos in myelin is here studied in depth. A model is provided contemplating the biface arrangement of the nanomotor ATP synthase in the myelin sheath.

PURPOSE: To longitudinally assess the effect of anti-vascular endothelial growth factor (VEGF) treatment on ellipsoid zone (EZ) integrity, subretinal hyperreflective material (SHRM), and the sub-retinal pigment epithelium (sub-RPE) compartment in eyes with neovascular age-related macular degeneration (nAMD). DESIGN: This study was a post hoc analysis of the OSPREY clinical trial, a prospective, double-masked, phase 2 study comparing brolucizumab 6 mg with aflibercept 2 mg over 56 weeks. PARTICIPANTS: Participants with treatment-naïve nAMD at the initiation of the trial were included in the analysis. METHODS: Eyes were evaluated with spectral domain optical coherence tomography (SD-OCT) at 4-week intervals in the OSPREY trial (n=81). SD-OCT scans collected from each visit were automatically. segmented using a proprietary, machine learning enabled higher-order feature-extraction platform for retinal layer, SHRM, and sub-RPE boundary lines, which were evaluated and corrected as needed by masked trained graders. The current analysis focused only on subjects evaluated with Cirrus (Zeiss) platform, n=28). MAIN OUTCOME MEASURES: Outcome measures included change from baseline in EZ-RPE volume, EZ-RPE central subfield thickness (CST), total EZ attenuation, SHRM volume, SHRM CST, and total sub-RPE volume. The correlation between each of these measures and best corrected visual acuity (BCVA) at each visit was evaluated. RESULTS: EZ-RPE volume and EZ-RPE CST showed significant increases, and total EZ attenuation, SHRM volume, SHRM CST, and total sub-RPE volume showed significant decreases from baseline at each visit from Week 4–56 (P

Photoreceptor disease results in irreparable vision loss and blindness, which has a dramatic impact on quality of life. Pathogenic mutations in RP1L1 lead to photoreceptor degenerations such as occult macular dystrophy and retinitis pigmentosa. RP1L1 is a component of the photoreceptor axoneme, the backbone structure of the photoreceptor&rsquo, s light-sensing outer segment. We generated an rp1l1 zebrafish mutant using CRISPR/Cas9 genome editing. Mutant animals had progressive photoreceptor functional defects as determined by electrophysiological assessment. Optical coherence tomography showed gaps in the photoreceptor layer, disrupted photoreceptor mosaics, and thinner retinas. Mutant retinas had disorganized photoreceptor outer segments and lipid-rich subretinal drusenoid deposits between the photoreceptors and retinal pigment epithelium. Our mutant is a novel model of RP1L1-associated photoreceptor disease and the first zebrafish model of photoreceptor degeneration with reported subretinal drusenoid deposits, a feature of age-related macular degeneration.

FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161atm1b/tm1b, lacking the major exon #3 which was replaced by a construct that include LacZ under the expression of the Fam161a promoter. LacZ staining was evident in ganglion cells, inner and outer nuclear layers and inner and outer-segments of photoreceptors in KO mice. No immunofluorescence staining of Fam161a was evident in the KO retina. Visual acuity and electroretinographic (ERG) responses showed a gradual decrease between the ages of 1 and 8 months. Optical coherence tomography (OCT) showed thinning of the whole retina. Hypoautofluorescence and hyperautofluorescence pigments was observed in retinas of older mice. Histological analysis revealed a progressive degeneration of photoreceptors along time and high-resolution transmission electron microscopy (TEM) analysis showed that photoreceptor outer segment disks were disorganized in a perpendicular orientation and outer segment base was wider and shorter than in WT mice. Molecular degenerative markers, such as microglia and CALPAIN-2, appear already in a 1-month old KO retina. These results indicate that a homozygous Fam161a frameshift mutation affects retinal function and causes retinal degeneration. This model will be used for gene therapy treatment in the future.

Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders such as retinitis pigmentosa, cone-rod dystrophy, and juvenile macular dystrophy. The proteins encoded by these genes are hypothesized to regulate photoreceptor outer segment disc morphogenesis, but their functions remain unknown. We used CRISPR/Cas9 to generate prom1-, cdhr1-, and prom1 + cdhr1-null X. laevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in dysmorphic photoreceptors comprised of overgrown and disorganized disc membranes. Cones were more severely affected than rods; outer segments were elongated and fragmented, and ERG response was impaired. Autofluorescent deposits in the outer segment layer of aging prom1-null animals indicate that secondary toxic effects to the retina or RPE drive retinal degeneration for this mutation, instead of direct effects on outer segment disc morphogenesis. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or aligned vertically within the plasma membrane. Prom1 + cdhr1-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the membrane fusion event involved in disc sealing. Rather, they are necessary for higher-order organization of the nascent outer segment discs. Prom1 may align and reinforce interactions between the disc leading edges, a function more critical in cone photoreceptors for structural support. Cdhr1 may help to align nascent discs and maintain horizontal disc orientation prior to fusion.

The Musashi family of RNA-binding proteins is known for its role in stem-cell renewal and is a negative regulator of cell differentiation. Interestingly, in the retina, the Musashi proteins MSI1 and MSI2 are differentially expressed throughout the cycle of retinal development, with MSI2 protein displaying robust expression in the adult retinal tissue. In this study, we investigated the importance of Musashi proteins in the development and function of photoreceptor neurons in the retina. We generated a pan-retinal and rod photoreceptor neuron-specific conditional KO mouse lacking MSI1 and MSI2. Independent of the sex, photoreceptor neurons with simultaneous deletion of Msi1 and Msi2 were unable to respond to light and displayed severely disrupted photoreceptor outer segment morphology and ciliary defects. Mice lacking MSI1 and MSI2 in the retina exhibited neuronal degeneration, with complete loss of photoreceptors within 6 months. In concordance with our earlier studies that proposed a role for Musashi proteins in regulating alternative splicing, the loss of MSI1 and MSI2 prevented the use of photoreceptor-specific exons in transcripts critical for outer segment morphogenesis, ciliogenesis, and synaptic transmission. Overall, we demonstrate a critical role for Musashi proteins in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in stark contrast with the canonical function of these two proteins in the maintenance and renewal of stem cells.

The retinal pigment epithelium (RPE) is a highly specialised pigmented monolayer sandwiched between the choroid and the photoreceptors in the retina. Key functions of the RPE include transport of nutrients to the neural retina, removal of waste products and water from the retina to the blood, recycling of retinal chromophores, absorption of scattered light and phagocytosis of the tips of the photoreceptor outer segments. These functions place a considerable membrane trafficking burden on the RPE. In this Cell Science at a Glance article and the accompanying poster, we focus on RPE-specific adaptations of trafficking pathways. We outline mechanisms underlying the polarised expression of membrane proteins, melanosome biogenesis and movement, and endocytic trafficking, as well as photoreceptor outer segment phagocytosis and degradation. We also briefly discuss theories of how dysfunction in trafficking pathways contributes to retinal disease.

Purpose Close to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone-rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches. Methods The Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels. Results This CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas. Conclusions To our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in mertk, the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris. However, the early onset and very fast progression of degeneration in mutMerTK-RP remains unexplained. Here, we explored the role of microglia in the Royal College of Surgeons (RCS) rat model of mutMerTK-RP. We found elevated levels of inflammatory cytokines and CD68 microglia activation marker, and more ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia in RCS retina when compared to wild-type retina as early as postnatal day 14 (P14). Strikingly, renewal of photoreceptor outer segments in P14 wild-type rat retina is still immature with low levels of RPE phagocytosis implying that at this early age lack of this process in RCS rats is unlikely to distress photoreceptors. Although the total number of Iba-1 positive retinal microglia remains constant from P14 to P30, we observed increasing numbers of microglia in the outer retina from P20 implying migration to the outer retina before onset of photoreceptor cell death at ~P25. Iba-1 and CD68 levels also increase in the retina during this time period suggesting microglia activation. To determine whether microglia affect the degenerative process, we suppressed retinal microglia in vivo using tamoxifen or a combination of tamoxifen and liposomal clodronate. Treatments partly prevented elevation of Iba-1 and CD68 and relocalization of microglia. Moreover, treatments led to partial but significant retention of photoreceptor viability and photoreceptor function. We conclude that loss of the phagocytosis receptor MerTK causes microglia activation and relocalization in the retina before lack of RPE phagocytosis causes overt retinal degeneration, and that microglia activities accelerate loss of photoreceptors in mutMerTK-RP. These results suggest that therapies targeting microglia may delay onset and slow the progression of this blinding disease.

Significance The main cause for blindness in the elderly worldwide is age-related macular degeneration (AMD). What causes AMD remains unknown. The high metabolic demands of photoreceptors are thought to contribute to disease pathogenesis, yet whether photoreceptor metabolism differs in individuals with AMD has not been determined. Here, we show that photoreceptor metabolism does differ between diseased and nondiseased individuals. Mimicking the metabolic profile of diseased individuals in mouse resulted in the similar advanced pathologies as those that cause blindness in humans. A disease model with photoreceptors as a contributing factor explains also why AMD affects preferentially the macula; it is the region of highest photoreceptor density. The data might open new avenues to study the role of PRs in disease pathogenesis., Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. While the histopathology of the different disease stages is well characterized, the cause underlying the progression, from the early drusen stage to the advanced macular degeneration stage that leads to blindness, remains unknown. Here, we show that photoreceptors (PRs) of diseased individuals display increased expression of two key glycolytic genes, suggestive of a glucose shortage during disease. Mimicking aspects of this metabolic profile in PRs of wild-type mice by activation of the mammalian target of rapamycin complex 1 (mTORC1) caused early drusen-like pathologies, as well as advanced AMD-like pathologies. Mice with activated mTORC1 in PRs also displayed other early disease features, such as a delay in photoreceptor outer segment (POS) clearance and accumulation of lipofuscin in the retinal-pigmented epithelium (RPE) and of lipoproteins at the Bruch’s membrane (BrM), as well as changes in complement accumulation. Interestingly, formation of drusen-like deposits was dependent on activation of mTORC1 in cones. Both major types of advanced AMD pathologies, including geographic atrophy (GA) and neovascular pathologies, were also seen. Finally, activated mTORC1 in PRs resulted in a threefold reduction in di-docosahexaenoic acid (DHA)–containing phospholipid species. Feeding mice a DHA-enriched diet alleviated most pathologies. The data recapitulate many aspects of the human disease, suggesting that metabolic adaptations in photoreceptors could contribute to disease progression in AMD. Identifying the changes downstream of mTORC1 that lead to advanced pathologies in mouse might present new opportunities to study the role of PRs in AMD pathogenesis.

PURPOSE To quantitatively evaluate the photoreceptor structural changes in the fellow unaffected eyes of patients with unilateral central serous chorioretinopathy (CSC). METHODS This is a retrospective cross-sectional study. We analyzed data from patients with diagnosis of unilateral CSC, as based on clinical examination and multimodal imaging, who had structural optical coherence tomography obtained. An additional group of age-matched healthy patients was included for comparison. Main outcome measures were as follows: (1) the foveal photoreceptor outer segment lateral surface and (2) the foveal choroidal thickness. RESULTS One hundred and sixty fellow unaffected eyes of 160 unilateral CSC patients and 50 age-matched controls (50 eyes) were included. The mean ± SD age was 51.6 ± 11.1 years (range 28-80 years) in the unilateral CSC group and 52.8 ± 10.8 years (range 31-74 years) in the control group (P = 0.511). The foveal photoreceptor outer segment lateral surface was significantly increased in the unaffected eyes with CSC in the fellow eye (0.068 ± 0.007 mm2) as compared with control eyes (0.060 ± 0.005 mm2, P < 0.0001). The mean ± SD foveal choroidal thickness was 368.0 ± 105.7 µm in the unilateral CSC group and 302.9 ± 92.2 µm in control patients (P < 0.0001). In the Pearson correlation test, the photoreceptor outer segment lateral surface correlated with the choroidal thickness in the CSC group (R = 0.166, P = 0.016) but not in the control group (R = -0.025, P = 0.864). CONCLUSION Our results corroborate the hypothesis that retinal and choroidal changes affect both eyes of patients with acute/history of unilateral disease. These structural changes could be intended as an imaging evidence of reduced photoreceptor outer segment turnover secondary to retinal pigment epithelium and choroid dysfunction.

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.

To the Editor:ABCA4 (ATP-binding cassette, subfamily A, member 4) encodes a protein localized to photoreceptor outer segment disk margins that transports vitamin A derivatives necessary for the vis...

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.

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium (hRPE) cells. Blue light damage to the retina was induced in 10-month-old (10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase (PERK), autophagy marker light chain 3 (LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of a-wave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment (OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed. PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.

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.

Purpose We propose and evaluate a hybrid model composed of two convolutional neural networks (CNNs) with different architectures for automatic segmentation of retina layers in spectral domain optical coherence tomography (SD-OCT) B-scans of retinitis pigmentosa (RP). Methods The hybrid model consisted of a U-Net for initial semantic segmentation and a sliding-window (SW) CNN for refinement by correcting the segmentation errors of U-Net. The U-Net construction followed Ronneberger et al. (2015) with an input image size of 256 × 32. The SW model was similar to our previously reported approach. Training image patches were generated from 480 horizontal midline B-scans obtained from 220 patients with RP and 20 normal participants. Testing images were 160 midline B-scans from a separate group of 80 patients with RP. The Spectralis segmentation of B-scans was manually corrected for the boundaries of the inner limiting membrane, inner nuclear layer, ellipsoid zone (EZ), retinal pigment epithelium, and Bruch's membrane by one grader for the training set and two for the testing set. The trained U-Net and SW, as well as the hybrid model, were used to classify all pixels in the testing B-scans. Bland–Altman and correlation analyses were conducted to compare layer boundary lines, EZ width, and photoreceptor outer segment (OS) length and area determined by the models to those by human graders. Results The mean times to classify a B-scan image were 0.3, 65.7, and 2.4 seconds for U-Net, SW, and the hybrid model, respectively. The mean ± SD accuracies to segment retinal layers were 90.8% ± 4.8% and 90.7% ± 4.0% for U-Net and SW, respectively. The hybrid model improved mean ± SD accuracy to 91.5% ± 4.8% (P < 0.039 vs. U-Net), resulting in an improvement in layer boundary segmentation as revealed by Bland–Altman analyses. EZ width, OS length, and OS area measured by the models were highly correlated with those measured by the human graders (r > 0.95 for EZ width; r > 0.83 for OS length; r > 0.97 for OS area; P < 0.05). The hybrid model further improved the performance of measuring retinal layer thickness by correcting misclassification of retinal layers from U-Net. Conclusions While the performances of U-Net and the SW model were comparable in delineating various retinal layers, U-Net was much faster than the SW model to segment B-scan images. The hybrid model that combines the two improves automatic retinal layer segmentation from OCT images in RP. Translational Relevance A hybrid deep machine learning model composed of CNNs with different architectures can be more effective than either model separately for automatic analysis of SD-OCT scan images, which is becoming increasingly necessary with current high-resolution, high-density volume scans.

The KCNJ13 gene encodes the inwardly rectifying potassium channel, Kir7.1. Mutations in this gene cause childhood blindness, in which the a- and b-wave responses of electroretinogram (ERG) are abolished. The ERG a-wave is the light-induced hyperpolarization of retinal photoreceptors, and the b-wave is the depolarization of ON-bipolar cells. The Kir7.1 channel is localized to the apical aspects of retinal pigment epithelium (RPE) cells and contributes to a delayed c-wave response. We sought to understand why a defect in an RPE ion-channel result in abnormal electrophysiology at the level of the retinal neurons. We have established the expression of Kir7.1 channels in the mouse RPE. ERGs recorded after mice Kir7.1 suppression by shRNA, or by blocking with VU590, showed reduced a-, b- and c-wave amplitudes. In contrast, the Kir7.1 blocker had no effect on the ex-vivo isolated mouse retina ERG where the RPE is not attached to the isolated retina preparation. Finally, we confirmed the specificity of VU590 action by inhibition of native mouse RPE Kir7.1 current in patch-clamp experiment. We propose that mutant RPE Kir7.1 channels contribute directly to the abnormal ERG associated with blindness via alterations in sub-retinal space K+ homeostasis in the vicinity of the photoreceptor outer segment.

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

AIM: To investigate the roles of PKC-α/ezrin signals in phagocytosis crisis of retinal pigment epithelium (RPE) cells in light damage model. METHODS: Light induced mice RPE injury model was established by continuously irradiating cool white light at different exposure time (0, 4, 8h light intensity: 4.18×10-6 J/cm2). In vitro, human ARPE-19 cells treated with the doses and intensity (1.57×10-6 J/cm2) of laser irradiation. Histology analysis was evaluated by hematoxylin and eosin (HE) staining. In vivo RPE phagocytosis was quantified by measuring the accumulation of photoreceptor outer segments in the sub-retinal space. In vitro RPE phagocytosis was assessed by calculating the relative fluorescence intensity of FITC-labeled microspheres in ARPE-19 cells. To further investigate the molecular mechanism, the activation of PKC-α/ezrin signal was evaluated by Western blot in vivo and in vitro. RESULTS: HE staining revealed that the thickness of outer nuclear layer decreased significantly after 4 and 8h light exposure. By immunostaining with rhodopsin, a significant greater accumulation of photoreceptor outer segment was noticed after light injury. In vitro, light injured RPE cells had less phagocytic activity in a dose dependent manner than that of the normal control (P