Citation: Ng, E.S.Y.; Kady, N.; Hu, J.; Dave, A.; Jiang, Z.; Pei, J.; Gorin, M.B.; Matynia, A.; Radu, R.A. Membrane Attack Complex Mediates Retinal Pigment Epithelium Cell Death in Stargardt Macular Degeneration. Cells 2022, 11, 3462. https://doi.org/10.3390/ cells11213462 Academic Editor: Hossein Ameri Received: 8 October 2022 Accepted: 30 October 2022 Published: 2 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Membrane Attack Complex Mediates Retinal Pigment Epithelium Cell Death in Stargardt Macular Degeneration Eunice Sze Yin Ng 1,2,† , Nermin Kady 1,3,4,† , Jane Hu 1 , Arpita Dave 1 , Zhichun Jiang 1 , Jacqueline Pei 1 , Michael B. Gorin 1 , Anna Matynia 1 and Roxana A. Radu 1, * 1 UCLA Stein Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, University of California at Los Angeles, CA 90095, USA 2 Molecular Cellular and Integrative Physiology Interdepartmental Program, University of California, Los Angeles, CA 90095, USA 3 Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, Ann Arbor, MI 48109, USA 4 Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt * Correspondence: radu@jsei.ucla.edu † These authors contributed equally to this work. Abstract: Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE cells derived via induced pluripotent stem-cell from a molecularly and clinically diagnosed STGD1 patient exhibited reduced ABCA4 protein and diminished activity compared to a normal subject. Consequently, STGD1 RPE cells accumulated intracellular autofluorescence- lipofuscin and displayed increased complement C3 activity. The level of C3 inversely correlated with the level of CD46, an early negative regulator of the complement cascade. Persistent complement dysregulation led to deposition of the membrane attack complex on the surface of RPE cells, decrease in transepithelial resistance, and subsequent cell death. These findings are strong evidence of complement-mediated RPE cell damage in STGD1, in the absence of photoreceptors, caused by reduced CD46 regulatory protein. Keywords: recessive Stargardt disease (STGD1); retinal pigment epithelium (RPE); complement system; bisretinoid-lipofuscin; “disease-in-a-dish”; retinoids; macular degeneration; ABCA4 1. Introduction Accumulation of toxic bisretinoid-lipofuscin material in the cells of the retinal pigment epithelium (RPE) is an age-dependent process, accelerated by loss or dysfunction of the ATP binding cassette subfamily A member 4 (ABCA4) protein. Mutations in the ABCA4 gene are responsible for recessive Stargardt disease (STGD1), a juvenile maculopathy that shares many clinical and pathologic features with dry-form of age-related macular degener- ation (AMD) [1,2]. Other mutations in ABCA4 cause cone-rod dystrophy in approximately one-third of cases and serve as rare susceptibility loci for AMD [3,4]. None of these ABCA4- mediated blinding diseases are currently treatable. ABCA4 is a flippase for retinaldehyde conjugated to phosphatidylethanolamine (N-retinylidene-phosphatidylethanolamine, or N-ret-PE) to promote clearance of retinaldehyde and prevent the formation of retinalde- hyde dimers (bisretinoids) [5,6]. Recently, the ABCA4 was shown to be expressed in RPE cells, in addition to photoreceptors [7,8]. ABCA4 in RPE endo-lysosomal membranes is responsible for retinaldehyde recycling during proteolysis of visual pigments following the daily phagocytosis of photoreceptor outer segments (OS). The hypothesized function of ABCA4 in RPE cells was evaluated in vivo using a transgenic mouse line on the Abca4 −/− Cells 2022, 11, 3462. https://doi.org/10.3390/cells11213462 https://www.mdpi.com/journal/cells