scholarly journals Acute RhoA/Rho Kinase Inhibition is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1927
Author(s):  
Yingyu Mao ◽  
Silvia C. Finnemann
Keyword(s):  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Wild Type ◽  
Phagocytic Capacity ◽  
Photoreceptor Outer Segment ◽  
Rpe Cells ◽  
Rhoa Activity ◽  
Phagocytic Cups

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

scholarly journals Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking αvβ5 Integrin

2004 ◽  
Vol 200 (12) ◽  
pp. 1539-1545 ◽  
Author(s):  
Emeline F. Nandrot ◽  
Yoonhee Kim ◽  
Scott E. Brodie ◽  
Xiaozhu Huang ◽  
Dean Sheppard ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Early Morning ◽  
Wild Type ◽  
Photoreceptor Outer Segment ◽  
Rpe Cells ◽  
Age Related

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor αvβ5 for particle binding. Here, we tested RPE phagocytosis and retinal function in β5 integrin–deficient mice, which specifically lack αvβ5 receptors. Retinal photoresponses severely declined with age in β5−/− mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. β5−/− RPE in culture failed to take up isolated photoreceptor particles. β5−/− RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in β5−/− retina. These results demonstrate an essential role for αvβ5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the β5−/− integrin mouse strain as a new animal model of age-related retinal dysfunction.

scholarly journals CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Hua Zhong ◽  
Yiyun Chen ◽  
Yumei Li ◽  
Rui Chen ◽  
Graeme Mardon
Keyword(s):  
Gene Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Cells ◽  
Mutant Mice ◽  
Wild Type ◽  
Cell Degeneration ◽  
Rpe Cells ◽  
Mosaic Expression

Abstract The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Here, we utilized the CRISPR-Cas9 system to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, mutations of which are thought to cause Leber congenital amaurosis (LCA), an early-onset form of blindness. We found that complete loss of Kcnj13 is likely postnatal lethal. Among surviving F0-generation mice examined, 80% show mosaic KCNJ13 expression in the retinal pigment epithelium (RPE). Mosaic expression correlates with decreased response to light and photoreceptor degeneration, indicating that Kcnj13 mutant mice mimic human KCNJ13-related LCA disease. Importantly, mosaic animals enable us to directly compare Kcnj13 mutant and wild-type RPE cells in the same eye. We found that RPE cells lacking KCNJ13 protein still survive but overlying photoreceptors exhibit cell degeneration. At the same time, wild-type RPE cells can rescue neighboring photoreceptor cells that overlie mutant RPE cells. These results suggest that KCNJ13 expression is required for RPE cells to maintain photoreceptor survival. Moreover, we show that CRISPR-Cas9 engineered mosaicism can be used to rapidly test candidate gene function in vivo.

In Vivo Multimodal Retinal Imaging Correlates With Granular-specific Changes in the Organelles of Retinal Pigment Epithelium of a Murine Model of Stargardt Disease

2021 ◽  
Author(s):  
Ratheesh K. Meleppat ◽  
Kaitryn E. Ronning ◽  
Sarah J. Karlen ◽  
Marie E. Burns ◽  
Edward N. Pugh ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Stargardt Disease ◽  
Rpe Cells ◽  
Age Related ◽  
Lipofuscin Granules

Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of granular pigmented organelles found in the retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin granules in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study the disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4-/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess the melanosome density in vivo, whereas the AF images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize the lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased number of melanosomes in the RPE of Abca4-/- mice and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo in the two mouse strains.

scholarly journals In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ratheesh K. Meleppat ◽  
Kaitryn E. Ronning ◽  
Sarah J. Karlen ◽  
Marie E. Burns ◽  
Edward N. Pugh ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Stargardt Disease ◽  
Rpe Cells ◽  
Age Related ◽  
Lipofuscin Granules

AbstractMelanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4−/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4−/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.

scholarly journals Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 50-60
Author(s):  
Veronica Rodriguez Fernandez ◽  
Giovanni Casini ◽  
Fabrizio Bruschi
Keyword(s):  
Ex Vivo ◽  
Cell Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Treatment Strategies ◽  
Experimental Models ◽  
Ocular Toxoplasmosis ◽  
Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

2017 ◽  
Vol 117 (04) ◽  
pp. 750-757
Author(s):  
Xin Jia ◽  
Chen Zhao ◽  
Qishan Chen ◽  
Yuxiang Du ◽  
Lijuan Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinal Pigment Epithelium ◽  
Cell Survival ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelium Cell ◽  
Photoreceptor Cells ◽  
Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

scholarly journals Isolation of Bovine Retinal Pigment Epithelial Cells Using Adhesion to Agarose: Demonstration of Cellular and Regional Heterogeneity

2003 ◽  
Vol 51 (1) ◽  
pp. 121-124 ◽  
Author(s):  
Eleonore Fröhlich ◽  
Elke Maier ◽  
Christian Klessen
Keyword(s):  
Enzymatic Activity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Histochemical Staining ◽  
Retinal Pigment Epithelial Cells ◽  
Isolated Cells ◽  
Regional Heterogeneity ◽  
Rpe Cells ◽  
A New Technique

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

scholarly journals Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-derived Factor Receptor (PEDF-R)

2021 ◽  
Author(s):  
Jeanee Bullock ◽  
Federica Polato ◽  
Mones Abu-Asab ◽  
Alexandra Bernardo-Colón ◽  
Elma Aflaki ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Phospholipase A2 ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Outer Segment ◽  
Western Blots ◽  
Rpe Cells ◽  
Photoreceptor Outer Segments ◽  
Transmission Electron ◽  
Bromoenol Lactone

AbstractPurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, 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 which PNPLA2 was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing 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 in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knock 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.

scholarly journals Iron is neurotoxic in retinal detachment and transferrin confers neuroprotection

2019 ◽  
Vol 5 (1) ◽  
pp. eaau9940 ◽  
Author(s):  
Alejandra Daruich ◽  
Quentin Le Rouzic ◽  
Laurent Jonet ◽  
Marie-Christine Naud ◽  
Laura Kowalczuk ◽  
...  
Keyword(s):  
Retinal Detachment ◽  
Vision Loss ◽  
Ex Vivo ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Subretinal Fluid ◽  
Predictive Marker ◽  
Visual Recovery ◽  
Neuroprotective Effects

In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

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