In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

Decoding Race and Age-Related Macular Degeneration: GPR 143 Activity Is the Key.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world. Caucasians are eightfold more likely to develop AMD than any other race, indicating a racial bias in AMD incidence which is unexplained. We hypothesize that pigmentation of the retinal pigment epithelium (RPE) and choroid protects from AMD and underlies this peculiar racial bias. We investigated GPR143, a receptor in the pigmentation pathway, which is activated by a melanin synthesis by-product, l-dopa. In this model, greater pigmentation leads to greater l-dopa production and, in turn, greater GPR143 signaling. GPR143 activity upregulates PEDF and downregulates both VEGF and exosomes; all of which reduce the angiogenic potential in the retina. Moreover, we demonstrate that GPR143 signaling enhances the digestion of shed photoreceptor outer segments. Together, our data suggests a central role for GPR143 signaling in RPE-photoreceptor interaction which is critical to healthy vision.

The Retinal Pigment Epithelium: Cells That Know the Beat!

The retinal pigment epithelium (RPE) ensures different functions crucial for photoreceptor survival, and thus for vision, such as photoreceptor outer segments (POS) phagocytosis and retinal adhesion. Both follow a circadian rhythm with an activity peak occurring respectively 1.5-2 and 3.5 h after light onset. Interestingly, we showed that two rodent models, ß5-/- and Prpf31+/- mice, display distinct alterations in both functions leading to different phenotypes. Indeed, the phagocytic peak totally disappears in ß5 knockout mice but is attenuated and shifted in Prpf31+/- mice. Conversely, the retinal adhesion peak only attenuated in ß5-/- mice is lost in Prpf31+/- mice. These distinct alterations have different consequences on retinal homeostasis proportional to the observed defects: ß5-/- mice progressively lose vision and accumulate RPE lipofuscin deposits, while Prpf31+/- mice develop RPE metabolic dysfunctions and gradual structural modifications indicative of cellular stress. Hence, animal models are useful to understand the importance of the proper regulation of these functions.

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Transient Retention of Photoreceptor Outer Segments in Matrigel-Embedded Retinal Organoids.

Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main retinal cell types under defined culture conditions. ROs show several highly specialized retinal features, including the outgrowth of photoreceptor outer segments (OSs). In vivo, the photoreceptor OSs are enveloped and maintained by protrusions of retinal pigment epithelium (RPE) cells, the so-called apical microvilli, while ROs fail to recapitulate this critical interaction in culture development. Here, we define specific co-culture conditions aiming to compensate for the missing physical proximity of RPE and OSs in RO development. Accordingly, functional RPE cells and ROs were differentiated simultaneously from the same iPSC clone, the former resulting in byproduct RPE or bRPE cells. While some co-culture approaches indicated a temporary functional interaction between bRPE and RO photoreceptors, they did not improve the photoreceptor histoarchitecture. In contrast, embedding ROs in a basement membrane extract without bRPE cells showed a robust improvement in the rate of photoreceptor OS retention. RO embedding is a quick and easy method that greatly enhances the preservation of photoreceptor OSs, an important structure for modelling retinal diseases with the involvement of photoreceptors.

Retinal Proteomics of a Mouse Model of Dystroglycanopathies Reveals Molecular Alterations in Photoreceptors.

Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for α-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, here we used comparative mass spectrometry-based proteomics and immunofluorescence microscopy and investigated the changes in the retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to the effects related to impaired α-DG O-mannosylation, we observed morphological alterations in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins, and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.

Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.

The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

Granulosa cells provide elimination of apoptotic oocytes through unconventional autophagy-assisted phagocytosis.

STUDY QUESTION: Do human granulosa cells (GCs) ingest and destroy apoptotic oocytes? SUMMARY ANSWER: Somatic GCs ingest and destroy apoptotic oocytes and other apoptotic substrates through unconventional autophagy-assisted phagocytosis. WHAT IS KNOWN ALREADY: Most (99%) ovarian germ cells undergo apoptosis through follicular atresia. The mode of cleaning of atretic follicles from the ovary is unclear. Ovarian GCs share striking similarities with testicular Sertoli cells with respect to their origin and function. Somatic Sertoli cells are responsible for the elimination of apoptotic spermatogenic cells through unconventional autophagy-assisted phagocytosis. STUDY DESIGN, SIZE, DURATION: Human GCs were tested for the ability to ingest and destroy the apoptotic oocytes and other apoptotic substrates. A systemic study of the main phagocytosis steps has been performed at different time points after loading of apoptotic substrates into the GC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of GC retrieved following controlled ovarian stimulation of five women for IVF/ICSI and a human granulosa KGN cell line were incubated with different apoptotic substrates: oocytes which underwent spontaneous apoptosis during the cultivation of immature germ cells for IVF/ICSI; apoptotic KGN cells; and apoptotic membranes from rat retinas. Cultured GC were analyzed for the presence of specific molecular markers characteristic of different steps of phagocytic and autophagy machineries by immunocytochemistry, confocal microscopy, transmission electron microscopy and western blotting, before and after loading with apoptotic substrates. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation of human GC with apoptotic substrates resulted in their translocation in cell cytoplasm, concomitant with activation of the phagocytosis receptor c-mer proto-oncogene tyrosine kinase MERTK (P < 0.001), clumping of motor molecule myosin II, recruitment of autophagy proteins: autophagy-related protein 5 (ATG5), autophagy-related protein 6 (Beclin1) and the rise of a membrane form of microtubule-associated protein 1 light chain 3 (LC3-II) protein. Ingestion of apoptotic substrates was accompanied by increased expression of the lysosomal protease Cathepsin D (P < 0.001), and a rise of lysosomes in the GCs, as assessed by different techniques. The level of autophagy adaptor, sequestosome 1/p62 (p62) protein remained unchanged. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of patients described here is limited. Also the dependence of phagocytosis on reproductive hormone status of patients should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Removal of apoptotic oocytes by surrounding GC seems likely to be a physiological mechanism involved in follicular atresia. Proper functioning of this mechanism may be a new strategy for the treatment of ovarian dysfunctions associated with an imbalance in content of germ cells in the ovaries, such as premature ovarian failure and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Rennes Metropole (AIS 2015) and Agence de BioMédecine. This work was supported by funding from Université de Rennes1, Institut National de la Santé et de la Recherche Médicale (INSERM) and CHU de Rennes. A.B. is funded in part by the program Actions Concertées Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology. This work is supported by the Agence Nationale de la Recherche Grants ANR-17-CE14-0038 and ANR-10-LABX-73. The authors declare no competing interests.

Prolonged ocular exposure leads to retinal lesions in mice.

Retinal lesions in the posterior pole of laboratory mice occur due to native, developmental abnormalities or as a consequence of environmental or experimental conditions. In this study, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Following experimental preparation induction procedures (EPIP) involving general anesthesia, mydriasis/cycloplegia, and topical anesthesia to the cornea, two ocular recovery conditions (protected and unprotected) were tested within two different animal recovery chambers (open or closed). The anterior and posterior poles were evaluated for the development of retinal lesions using digital color photography, scanning laser ophthalmoscopy, and spectral-domain optical coherence during anesthesia recovery and up to 2.5 months thereafter. In some mice, electroretinograms, histological and immunohistological evaluations were performed to assess functional and structural changes that accompanied the retinal lesions detected by in vivo imaging. Our data suggests that prolonged ocular surface exposure to circulating ambient room air leads to significant anterior and posterior segment ocular complications. The most abundant, semi-reversible complication observed was the development of lesions in the outer retina, which had a 90% probability of occurring after 45 min of exposure. The lesions mostly resolved short-term, but functional and imaging evidence suggest that some perturbations to the outer retina may persist one or more months following initial development.

No Difference Between Age-Matched Male and Female C57BL/6J Mice in Photopic and Scotopic Electroretinogram a- and b-Wave Amplitudes or in Peak Diurnal Outer Segment Phagocytosis by the Retinal Pigment Epithelium.

Mice provide informative models of enormous utility for eye research. Sex as biological variable must be considered when conducting studies exploring mouse models. To determine if sex confounds neural retina or retinal pigment epithelium (RPE) activity in wild-type C57BL/6J mice, we compared male and female mice with respect to retinal light response and RPE phagocytosis. We tested 2-month-old mice at peak fertility and 12-month-old mice past fertility. Retinal function was assessed by quantifying a- and b-wave amplitudes of photopic and scotopic electroretinograms (ERGs). These experiments did not reveal differences between male and female mice at either age. As expected from earlier studies, 12-month-old mice showed reduced light responses compared to 2-month-old mice, but age-related decline was identical for male and female mice. RPE functionality was assessed by quantifying RPE phagosome content 1 h after light onset in mice 2 months of age, an age of maturity of the process of outer segment turnover that includes RPE phagocytosis. These experiments did not reveal differences in RPE phagocytosis between male and female mice. Altogether, male and female C57BL/6J mice do not differ in retinal light response and peak RPE phagocytic activity. Retinal activity is impaired with age to the same extent in male and female mice. Our results justify testing mixed-sex mouse cohorts in studies on outer segment renewal and RPE phagocytosis and illustrate the importance of careful consideration of cohort age.

A Novel Approach for Integrating AF-SLO and SDOCT Imaging Data Demonstrates the Ability to Identify Early Retinal Abnormalities in Mutant Mice and Evaluate the Effects of Genetic and Pharmacological Manipulation.

Noninvasive ocular imaging platforms are undeniably useful in identifying retinal abnormalities. The purpose of this study was to investigate a novel method for integrating information acquired from two independent imaging platforms, AF-SLO and SDOCT, in order to demonstrate retinal perturbations as a result of genetic or pharmacological manipulation. Two cohorts of mice were investigated, Nyx nob and C57BL/6 J. In Nyx nob mice, SLO revealed an atypical but variable amount of autofluorescent foci (AFF); SDOCT showed altered photoreceptor outer segment architecture. Naïve Nyx nob had significantly more AFF than C57BL/6 J, suggesting that Nyx nob have some predisposition for developing AFF. Interestingly, both findings were significantly ameliorated in diabetic Nyx nob mice as compared to the controls. These data were incorporated into a novel analysis plot comparing AF-SLO and SDOCT results. The integration of the qualitative changes and accompanying quantitative analysis approach described herein provide a sensitive means for detecting whether a mouse model is susceptible to degeneration before other hallmark indicators are observed.

Opposite Roles of MerTK Ligands Gas6 and Protein S During Retinal Phagocytosis.

MerTK is required for photoreceptor outer segment (POS) phagocytosis by retinal pigment epithelial (RPE) cells, a diurnal function essential for vision maintenance. In vivo, MerTK is stimulated at the time of the phagocytic peak through an intracellular signaling pathway. However, MerTK ligands Gas6 and Protein S are expressed in both RPE cells and photoreceptors, and at least one of them required for phagocytosis to occur. Still, their exact role in the retina was not clear until recently. This review combines results from different studies to shed the light on a tissue-specific regulation of MerTK function by its ligands. Indeed, with opposite effects on RPE phagocytosis and changes in their expression levels around the time of POS uptake, Gas6 and Protein S may contribute to the tight control of the acute phagocytic peak in the retina.

Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 µm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments.

In photoreceptors, the assembly of signaling molecules into macromolecular complexes is important for phototransduction and maintaining the structural integrity of rod outer segments (ROSs). However, the molecular composition and formation of these complexes are poorly understood. Using immunoprecipitation and mass spectrometry, 4.1G was identified as a new interacting partner for the cyclic-nucleotide gated (CNG) channels in ROSs. 4.1G is a widely expressed multifunctional protein that plays a role in the assembly and stability of membrane protein complexes. Multiple splice variants of 4.1G were cloned from bovine retina. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with peripherin-2-CNG channel complex. A combination of truncation studies and domain-binding assays demonstrated that CNG channels selectively interacted with 4.1G through their FERM and CTD domains. Using immunofluorescence, labeling of 4.1G was seen to be punctate and partially colocalized with CNG channels in the ROS. Our studies indicate that 4.1G interacts with a subset of CNG channels in the ROS and implicate this protein-protein interaction in organizing the spatial arrangement of CNG channels in the plasma membrane of outer segments.

Photopigment self-screening and the determination of macular pigment absorbance using heterochromatic flicker photometry.

PURPOSE: Heterochromatic flicker photometry (HFP) is commonly used to determine macular pigment optical density (MPOD). Since HFP in this application is a locus comparison method, an identical relative spectral response at each locus is required for a perfect measure. We know this requirement cannot be strictly true since the optical density of photopigments increases as the foveal center is approached. Thus, the self-screening effect would result in an underestimate of MPOD. An earlier study concluded that the underestimate is on the order of 30%. We examined this issue by manipulating photopigment optical density, and consequently the degree of selfscreening. METHODS: A continuously exposed, 470 nm, background bleached cone photopigments over a range from 0 to 80%. MPOD was determined 10' and 30' from the foveal center. Two subjects were used in the main experiment. Five additional subjects were studied with just the 0% and 80% bleach levels. Spectral measures were obtained at 0% and 70% bleach levels for the two primary subjects. RESULTS: Subjects in the main experiment showed MPOD estimates that increased with increasing bleaching. The effect, however, was small: one observer's MPOD increased 0.08 and 0.02 for the 10' and 30' loci, respectively; the other observer's values were 0.04 and 0.01 for the same loci. Comparable values were obtained for the other five subjects using the 0% and 80% bleach conditions. Spectral measures were consistent with the findings of the main experiment. CONCLUSIONS: When self-screening is nearly abolished (80% bleach), a relatively small underestimation is revealed for the unbleached state. For the 1° target we show about 2-3% underestimation. Our 20' target reveals a larger underestimate (8-9%), consistent with longer photoreceptor outer-segments nearer the foveal center. We conclude that HFP yields values essentially independent of self-screening for targets of 1° diameter or greater. Smaller targets are less than 10% underestimated for near-zero bleach conditions.

Do different spectral domain OCT hardwares measure the same? Comparison of retinal thickness using third-party software.

PURPOSE: Spectral-domain optical coherence tomographies (OCTs) from different companies do not give identical retinal thicknesses. The purpose of this study was to evaluate if differences in thickness when using a spectral domain Cirrus OCT or a Heidelberg Spectralis are due to hardware differences, or if they are caused by the segmentation algorithms. METHODS: Thirty-seven healthy eyes were examined within the same session with a Cirrus OCT and a Spectralis OCT, the latter using averaged B-scans. Scans from similar positions and passing the fovea were analyzed by custom-made software. Thickness was analyzed at the fovea, the central 1-mm line and the 6-mm line. RESULTS: When Cirrus and Spectralis scans were analyzed with the same software, the retinal thickness at the foveal center was 225.92 µm (SD 17.0) using the Cirrus and 228.70 µm (SD 18.4) using the Spectralis; the difference of 2.78 µm was not significant (p = 0.055). For the central 1 mm, the difference was 1.78 µm (p = 0.0414), and for all points out to 6 mm, the Spectralis retinal thickness was also significantly larger than the Cirrus thickness (p = 0.0052), though the mean difference was only 1.85 µm. Also for the RPE_OScomplex, Spectralis measured a greater thickness than did Cirrus, with a mean of 3.32 µm (p < 0.0001) for all points. CONCLUSION: The retinal thicknesses from the Cirrus and from the Spectralis differed by 14 µm with the standard software of the instruments, and by less than 3 µm when analyzed with the same custom-made software, indicating that the major differences between the two SD-OCT systems are due to differences in their built-in software algorithms.

Microtubule-associated protein tau in bovine retinal photoreceptor rod outer segments: comparison with brain tau.

Recent studies have suggested a possible involvement of abnormal tau in some retinal degenerative diseases. The common view in these studies is that these retinal diseases share the mechanism of tau-mediated degenerative diseases in brain and that information about these brain diseases may be directly applied to explain these retinal diseases. Here we collectively examine this view by revealing three basic characteristics of tau in the rod outer segment (ROS) of bovine retinal photoreceptors, i.e., its isoforms, its phosphorylation mode and its interaction with microtubules, and by comparing them with those of brain tau. We find that ROS contains at least four isoforms: three are identical to those in brain and one is unique in ROS. All ROS isoforms, like brain isoforms, are modified with multiple phosphate molecules; however, ROS isoforms show their own specific phosphorylation pattern, and these phosphorylation patterns appear not to be identical to those of brain tau. Interestingly, some ROS isoforms, under the normal conditions, are phosphorylated at the sites identical to those in Alzheimer's patient isoforms. Surprisingly, a large portion of ROS isoforms tightly associates with a membranous component(s) other than microtubules, and this association is independent of their phosphorylation states. These observations strongly suggest that tau plays various roles in ROS and that some of these functions may not be comparable to those of brain tau. We believe that knowledge about tau in the entire retinal network and/or its individual cells are also essential for elucidation of tau-mediated retinal diseases, if any.

New findings in ATP supply in rod outer segments: insights for retinopathies.

BACKGROUND INFORMATION: The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. RESULTS: OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F1F0-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O2) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. CONCLUSIONS: Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O2. The ability of OS to manipulate O2 may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.