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      Drusen in patient-derived hiPSC-RPE models of macular dystrophies


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          Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, pharmacological treatments in these diseases are limited due to the lack of knowledge of underlying disease mechanisms, partly because appropriate human models to study AMD and related MDs are lacking. Furthermore, in the living human eye, the entire retina acts as a functional unit, making it difficult to investigate the specific contribution of a particular retinal cell type in the disease. Here, we established human models of multiple MDs, which demonstrated similar molecular and phenotypic manifestations within these diseases. Furthermore, we showed that dysfunction of an individual cell type, retinal pigment epithelium cells in the retina, is sufficient for the development of key pathological features in these MDs.


          Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby’s fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich “drusen-like” composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE–ECM interface.

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          Most cited references 47

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          Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.

          Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.
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            A role for local inflammation in the formation of drusen in the aging eye.

            The accumulation of numerous or confluent drusen, especially in the macula, is a significant risk factor for the development of age-related macular degeneration (AMD). Identifying the origin and molecular composition of these deposits, therefore, has been an important, yet elusive, objective for many decades. Recently, a more complete profile of the molecular composition of drusen has emerged. In this focused review, we discuss these new findings and their implications for the pathogenic events that give rise to drusen and AMD. Tissue specimens from one or both eyes of more than 400 human donors were examined by light, confocal or electron microscopy, in conjunction with antibodies to specific drusen-associated proteins, to help characterize the transitional events in drusen biogenesis. Quantification of messenger RNA from the retinal pigment epithelium (RPE)/choroid of donor eyes was used to determine if local ocular sources for drusen-associated molecules exist. The results indicate that cellular remnants and debris derived from degenerate RPE cells become sequestered between the RPE basal lamina and Bruch's membrane. We propose that this cellular debris constitutes a chronic inflammatory stimulus, and a potential "nucleation" site for drusen formation. The entrapped cellular debris then becomes the target of encapsulation by a variety of inflammatory mediators, some of which are contributed by the RPE and, perhaps, other local cell types; and some of which are extravasated from the choroidal circulation. The results support a role for local inflammation in drusen biogenesis, and suggest that it is analogous to the process that occurs in other age-related diseases, such as Alzheimer's disease and atherosclerosis, where accumulation of extracellular plaques and deposits elicits a local chronic inflammatory response that exacerbates the effects of primary pathogenic stimuli.
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              The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.

              During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals. Copyright 2009 Elsevier Ltd. All rights reserved.

                Author and article information

                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                26 September 2017
                6 September 2017
                : 114
                : 39
                : E8214-E8223
                aDepartment of Ophthalmology, University of Rochester , Rochester, NY 14642;
                bDepartment of Biomedical Genetics, University of Rochester , Rochester, NY 14642;
                c Centre for Eye Research Australia , East Melbourne, VIC 3002, Australia;
                d Royal Victorian Eye and Ear Hospital , East Melbourne, VIC 3002, Australia;
                eOphthalmology, Department of Surgery, The University of Melbourne , Parkville VIC 3010, Australia;
                f Lions Eye Institute , Nedlands WA 6009, Australia;
                gCentre for Ophthalmology and Visual Science, University of Western Australia , Perth WA 6009, Australia;
                hMenzies Institute for Medical Research, University of Tasmania , Hobart TAS 7005, Australia;
                iSchool of Medicine, University of Tasmania , Hobart TAS 7005, Australia;
                jDepartment of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles , CA 90095;
                kJules Stein Eye Institute, University of California, Los Angeles , CA 90095;
                lDepartment of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles , CA 90095;
                mMolecular Biology Institute, University of California, Los Angeles , CA 90095;
                nBrain Research Institute, University of California, Los Angeles , CA 90095;
                oCenter for Visual Science, University of Rochester , Rochester, NY 14642;
                pWaisman Center, University of Wisconsin , Madison, WI 53705;
                qMcPherson Eye Research Institute, University of Wisconsin , Madison, WI 53706;
                rDepartment of Ophthalmology and Visual Sciences, University of Wisconsin , Madison, WI 53706
                Author notes
                2To whom correspondence should be addressed. Email: ruchira_singh@ 123456urmc.rochester.edu .

                Edited by Stephen H. Tsang, Brown Glaucoma Laboratory, College of Physicians and Surgeons, Columbia University, New York, NY, and accepted by Editorial Board Member Jeremy Nathans August 9, 2017 (received for review June 13, 2017)

                Author contributions: C.A.G., S.D., A.P., A.W.H., and R.S. designed research; C.A.G., S.D., S.S.C.H., L.A.M., R.C.B.W., A.P., and A.W.H. performed research; L.R.L., R.H.G., D.A.M., M.M.C., D.M.G., A.P., A.W.H., and R.S. contributed new reagents/analytic tools; C.A.G., S.D., S.S.C.H., L.A.M., D.S.W., A.P., A.W.H., and R.S. analyzed data; C.A.G., S.D., S.S.C.H., R.H.G., D.A.M., M.M.C., A.P., A.W.H., and R.S. wrote the paper; and R.H.G. provided patient samples.

                1C.A.G. and S.D. contributed equally to this work.

                PMC5625924 PMC5625924 5625924 201710430
                Page count
                Pages: 10
                Funded by: BrightFocus Foundation 100006312
                Award ID: M2015267
                Funded by: Foundation Fighting Blindness (FFB) 100001116
                Award ID: TA-RM-0616-0702-UR
                Funded by: Research to Prevent Blindness (Individual Investigator Award)
                Award ID: RPB-Singh
                Funded by: Research to Prevent Blindness (Unrestricted grant)
                Award ID: RPB-Feldon
                Funded by: Knights Templar Eye Foundation (KTEF) 100001209
                Award ID: KTEF-Singh
                Funded by: Retina Research Foundation
                Award ID: RRF-Singh
                Funded by: Australian Research Council (ARC) 501100000923
                Award ID: ARC-Pebay
                Funded by: The Ophthalmic Research Institute of Australia (ORIA) 501100001108
                Award ID: ORIA-Hewitt
                Funded by: David Bryant Trust
                Award ID: DBT-Singh
                PNAS Plus
                Biological Sciences
                Cell Biology
                PNAS Plus


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