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Roxana A Radu, Nermin Kady, Michael B Gorin, Anna Matynia, Jane G Hu; Membrane attack complex induces RPE cell death in Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2994.
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Mutations in the ABCA4 gene are responsible for recessive Stargardt (STGD1), a central blinding disease similar to AMD. STGD1 patients and Abca4-/- mouse model exhibit deposition of bisretinoids in the retinal pigment epithelium (RPE) and photoreceptor degeneration. Bisretinoid-mediated complement activation was reported in cultured RPE cells and detected in the RPE of Abca4-/- mice and STGD1 donor eye. Here, we used induced pluripotent stem cell (iPSC) derived RPE from a STGD1 patient to investigate the role of complement in disease pathogenesis.
STGD1 patient, with two ABCA4 mutations on different alleles: (1) c.3386G>T; p.Arg1129Leu and (2) c.[5461-10T>C;5603A>T]; p.[Thr1821Aspfs*6,Thr1821Valfs*13;(Asn1868Ile)], and control (no ABCA4 mutations) fibroblasts were reprogrammed into iPSCs and differentiated to RPE cells using standard protocols. iPSC-RPE cells were grown on filters and analyzed at 3- and 12-mo by confocal and electron microscopy. Cell count and height were determined by morphometric analysis. Transepithelial resistance (TER) was measured to evaluate the RPE monolayer integrity.Immunohistochemistry was performed on the flatmount of iPSC-RPE cells with antibodies against ZO1, 4-hydroxynonenal (4HNE), complement negative regulators (CD46 and CD59), C3/C3b/iC3b, C3aR, and membrane attack complex (MAC).
At 3-mo, control and STGD1 iPSC-RPE cells displayed normal morphology with silimar ZO1 staining, TER measurements, and cell count. Over time, STGD1 iPSC-RPE cells accumulated autofluoscence at ~1.2- and ~1.6-fold higher levels vs control at 3- and 12-mo respectively. 4HNE immunostaining was also enhanced in STGD1 RPE cells. While levels of C3aR and CD59 were similar, CD46 levels were reduced by half in the RPE of STGD1 vs control. In contrast, C3/C3b/iC3b and MAC levels were ~1.5-fold higher in the RPE of STGD1 vs control. At 12-mo, STGD1 RPE cells height and number were reduced by ~30% vs control. Changes in morphology correlated with ~80% reduction of TER in STGD1 vs control RPE, consistent with cell membranes disruption.
Using STGD1 patient iPSC-derived RPE cells, we provide evidence of complement dysregulation triggered by the buildup of autofluorescence material. Additionally, ongoing complement activation led to MAC-mediated cellular death in 12-mo cultured STGD1 iPSC-RPE cells. These findings strongly support a common inflammatory etiology of AMD and STGD1 maculopathy.
This is a 2021 ARVO Annual Meeting abstract.
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