Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4 -/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk -/- but not Abca4 -/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4 -/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4 -/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. Methods: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8+/− mice expressing ß-galactosidase. Aged Mfge8+/− and Mfge8−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. Results: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8−/− mice as compared to controls. Conclusions: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD

Follow-up Imaging of Disk Candidates from the Disk Detective Citizen Science Project: New Discoveries and False Positives in WISE Circumstellar Disk Surveys

The Disk Detective citizen science project aims to find new stars with excess 22 m emission from circumstellar dust in the All WISE data release from the Wide-field Infrared Survey Explorer. We evaluated 261 Disk Detective objects of interest with imaging with the Robo-AO adaptive optics instrument on the 1.5 m telescope at Palomar Observatory and with RetroCam on the 2.5 m du Pont Telescope at Las Campanas Observatory to search for background objects at 0 1512 separations from each target. Our analysis of these data leads us to reject 7% of targets. Combining this result with statistics from our online image classification efforts implies that at most7.9%0.2% of All WISE-selected infrared excesses are good disk candidates. Applying our false-positive rates to other surveys, we find that the infrared excess searches of McDonald et al. and Marton et al. all have false-positiverates >70%. Moreover, we find that all 13 disk candidates in Theissen & West with W4 signal-to-noise ratio >3are false positives. We present 244 disk candidates that have survived vetting by follow-up imaging. Of these,213 are newly identified disk systems. Twelve of these are candidate members of comoving pairs based on Gaia astrometry, supporting the hypothesis that warm dust is associated with binary systems. We also note the discovery of 22 m excess around two known members of the ScorpiusCentaurus association, and we identifyknown disk host WISEA J164540.79-310226.6 as a likely Sco-Cen member. Thirty of these disk candidates arecloser than 125 pc (including 26 debris disks), making them good targets for both direct-imaging exoplanetsearches

Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly

Follow-up Imaging of Disk Candidates from the Disk Detective Citizen Science Project: New Discoveries and False Positives in WISE Circumstellar Disk Surveys

The Disk Detective citizen science project aims to find new stars with excess 22 μm emission from circumstellar dust in the AllWISE data release from the Wide-field Infrared Survey Explorer. We evaluated 261 Disk Detective objects of interest with imaging with the Robo-AO adaptive optics instrument on the 1.5 m telescope at Palomar Observatory and with RetroCam on the 2.5 m du Pont Telescope at Las Campanas Observatory to search for background objects at 0.”15–12'' separations from each target. Our analysis of these data leads us to reject 7% of targets. Combining this result with statistics from our online image classification efforts implies that at most 7.9% ± 0.2% of AllWISE-selected infrared excesses are good disk candidates. Applying our false-positive rates to other surveys, we find that the infrared excess searches of McDonald et al. and Marton et al. all have false-positive rates >70%. Moreover, we find that all 13 disk candidates in Theissen & West with W4 signal-to-noise ratio >3 are false positives. We present 244 disk candidates that have survived vetting by follow-up imaging. Of these, 213 are newly identified disk systems. Twelve of these are candidate members of comoving pairs based on Gaia astrometry, supporting the hypothesis that warm dust is associated with binary systems. We also note the discovery of 22 μm excess around two known members of the Scorpius–Centaurus association, and we identify known disk host WISEA J164540.79-310226.6 as a likely Sco-Cen member. Thirty of these disk candidates are closer than ~125 pc (including 26 debris disks), making them good targets for both direct-imaging exoplanet searches

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization

Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs

Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders