Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells

Disease Models & Mechanisms · 26 authors, 7 centres

AI SUMMARY

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POPULATIONHuman induced pluripotent stem cell-derived RPE (iPS-RPE) cells and liver-specific hepcidin knockout (LS-Hepc^KO^) mice

INTERVENTIONChronic iron overload (FeSO4 at 50 or 100 μM for 6-9 weeks in vitro; genetic iron overload in LS-Hepc^KO^ mice in vivo)

COMPARISONUntreated iPS-RPE cells (in vitro) and age-matched control mice (AlbCre^−^, Hepc^f/f^ or wild-type) (in vivo)

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TL;DR

This study demonstrates that iron-induced oxidative stress causes lysosomal dysfunction, lysosomal membrane permeabilization (LMP), and ceramide accumulation in retinal pigment epithelial (RPE) cells, both in vitro and in a mouse model. These findings suggest a mechanism by which oxidative stress contributes to age-related macular degeneration (AMD) pathology, highlighting lysosomal impairment as a key event. The clinical significance lies in identifying potential therapeutic targets, such as reducing iron levels or preventing lipid peroxidation, to slow AMD progression.

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**Background:** Oxidative stress is implicated in age-related macular degeneration (AMD), but the mechanisms are unclear. Iron accumulates in the retinal pigment epithelium (RPE) of AMD patients and can catalyze reactive oxygen species (ROS) production via the Fenton reaction, leading to lipid peroxidation. The RPE is crucial for photoreceptor health, and its lysosomes are essential for digesting phagocytosed photoreceptor outer segments. Lysosomal dysfunction is thought to contribute to AMD. This study investigates the impact of iron-induced oxidative stress on RPE lysosomal function and its role in AMD pathology.

**Methods:** Human iPS-RPE cells were cultured with 50 or 100 μM FeSO4 for 6-9 weeks to model chronic iron overload. Lysosomal number (LysoTracker), proteolysis (DQ-BSA), pH (LysoSensor), and enzyme activities (cathepsin D, cathepsin B, glucosylceramidase, acid sphingomyelinase, lysosomal acid lipase) were measured. In vivo, liver-specific hepcidin knockout (LS-Hepc^KO^) mice, which develop systemic iron overload and RPE iron accumulation, were studied at 12-16 months. RPE morphology was assessed by optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (cSLO), electron microscopy (EM), and immunohistochemistry (IHC) for lysosomal markers (Lamp1, cathepsin D), LMP marker (galectin-3), lipid peroxidation products (4-HNE, CEP), and advanced glycation end products (methylglyoxal). Proteomics and lipidomics/metabolomics were performed on isolated RPE.

**Key Results:** In iPS-RPE cells, iron loading for 6 weeks significantly increased LysoTracker-positive puncta per cell (p<0.01) and decreased DQ-BSA-positive puncta per cell (p<0.01), indicating lysosomal accumulation and impaired proteolysis. Lysosomal pH was decreased (p<0.05). Cathepsin D activity was unchanged, but cathepsin B activity increased (p<0.05). Acid sphingomyelinase and lysosomal acid lipase activities decreased (p<0.05 and p<0.01, respectively), while glucosylceramidase activity was unchanged. In LS-Hepc^KO^ mice, OCT showed RPE hypertrophy and thinning of photoreceptor layers. EM revealed RPE cells packed with electron-dense, single-membrane-bound inclusions identified as lysosomes. IHC showed strong Lamp1 and cathepsin D labeling, with cathepsin D often in the cytoplasm, suggesting LMP. Galectin-3 colocalized with Lamp1 in many RPE cells, confirming LMP. Western blot of isolated RPE showed increased pro-cathepsin D, mature cathepsin D (ratio 6 times higher than controls), Lamp1 (4-fold increase), cathepsin B (17-fold increase), LC3B-II (4-fold increase), and p62 (4-fold increase), indicating lysosomal accumulation and impaired autophagy. Proteomics identified 2500 differentially expressed proteins (adjusted p<0.05), with lysosomal proteins highly enriched (e.g., cathepsin D, Lamp1, CD63). Lipidomics showed significant accumulation of ceramides (4-20 fold increase), dihydroceramides (4-14 fold), lactosylceramides (6-14 fold), lysophosphatidylcholines (LPCs), and docosahexaenoic acid (DHA) in LS-Hepc^KO^ RPE, while A2E was depleted. IHC demonstrated increased 4-HNE, CEP, and methylglyoxal in LS-Hepc^KO^ RPE, often colocalizing with Lamp1. At 16 months, LS-Hepc^KO^ mice showed RPE cell death and AMD-like morphologies including atrophy, sloughed, bilaminar, and intraretinal RPE cells.

**Clinical Implications:** This study provides a mechanistic link between iron-induced oxidative stress and RPE dysfunction in AMD. Iron overload leads to lysosomal accumulation, impaired lysosomal enzyme activity (particularly acid sphingomyelinase and lysosomal acid lipase), LMP, and accumulation of toxic lipids (ceramides) and lipid peroxidation products. These events ultimately cause RPE cell death and AMD-like pathology. The findings suggest that reducing iron levels (e.g., through chelation or dietary changes), inhibiting lipid peroxidation (e.g., with deuterated DHA), or enhancing lysosomal function could be therapeutic strategies for AMD. The study also highlights the potential role of ceramide metabolism and LMP as targets for intervention.

PICO

PPOPULATION

Human induced pluripotent stem cell-derived RPE (iPS-RPE) cells and liver-specific hepcidin knockout (LS-Hepc^KO^) mice

IINTERVENTION

Chronic iron overload (FeSO4 at 50 or 100 μM for 6-9 weeks in vitro; genetic iron overload in LS-Hepc^KO^ mice in vivo)

OOUTCOME

Lysosomal accumulation, lysosomal enzyme activity, lysosomal membrane permeabilization (LMP), ceramide accumulation, lipid peroxidation, RPE cell death, and AMD-like morphologies

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

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ORIGINAL PAPER

PMC10399446

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells

Disease Models & Mechanisms

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ABSTRACT

ABSTRACT Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc ( Hamp ) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes. Keywords: Oxidative stress, Aging, Retina, Age-related macular degeneration, Lysosome

CITATION EXPORT

Kevin R Zhang, Connor S R Jankowski, Rayna Marshall et al.. Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells. Disease Models & Mechanisms. (2023). https://doi.org/10.1242/dmm.050066

AUTHORS

26 · 7 CENTRES

Kevin R Zhang, Connor S R Jankowski, Rayna Marshall, Rohini Nair, Néstor Más Gómez, Ahab Alnemri, Yingrui Liu, Elizabeth Erler, Julia Ferrante, Ying Song, Brent A Bell, Bailey H Baumann, Jacob Sterling, Brandon Anderson, Sierra Foshe, Jennifer Roof, Hossein Fazelinia, Lynn A Spruce, Jen-Zen Chuang, Ching-Hwa Sung, Anuradha Dhingra, Kathleen Boesze-Battaglia, Venkata R M Chavali, Joshua D Rabinowitz, Claire H Mitchell, Joshua L Dunaief

1F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA
2Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA
3Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
4Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
5CHOP-PENN Proteomics Core Facility, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA
6Department of Ophthalmology, Weill Cornell Medicine, New York, NY 10065, USA
7Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY 10065, USA