To investigate whether excessive glucocorticoid (GC) exposure disrupts retinal pigment epithelial (RPE) homeostasis through glucocorticoid receptor (GR)-dependent autophagy hyperactivation.

Human retinal pigment epithelial cell line (ARPE-19 cells) were exposed to GC (0–100 μmol/L, 48h). GR subcellular localization was analyzed via mitochondrial fractionation and immunofluorescence. Functional assessments included barrier integrity [zonula occludens-1 (ZO-1) immunostaining], phagocytic capacity (FluoSpheres^TM-labeled photoreceptor outer segment uptake), and ultrastructural analysis (transmission electron microscopy, TEM). Autophagic flux was quantified using mCherry-green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (LC3) B reporters. Molecular mechanisms were probed through AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway activity and autophagic flux assessed by LC3-Ⅱ levels. GR dependency was confirmed using the antagonist RU486, and cell-type specificity was assessed using mouse photoreceptor (661W) cells.

GC induced GR upregulation and mitochondrial translocation in a dose- and time-dependent manner, correlating with pathological autophagy activation. Key findings included: 1) tight junction disruption, evidenced by ZO-1 fragmentation, 2) reduction in phagocytic uptake efficiency, 3) mitochondrial shrinkage and autolysosome accumulation (TEM), 4) altered AMPK/mTOR signaling (p-AMPK up, p-mTOR down) associated with autophagosome formation, 5) elevated LC3-Ⅱ levels confirming excessive autophagic flux. Critically, all GC-induced effects, including GR upregulation, signaling changes, and increased autophagy, were abolished by GR antagonism with RU486.

This study shows that GR‑dependent GC exposure induces autophagic flux with ultrastructural evidence suggestive of mitochondria‑targeted autophagy and alters AMPK/mTOR signaling, correlating with RPE barrier collapse and phagocytic failure, a possible link to steroid‑associated central serous chorioretinopathy (CSC).