Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study

Abstract

To evaluate the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro and in mouse models in-vivo. ARPE-19 cells, a human retinal pigment epithelial cell line, was exposed to 10 mM NaIO3 for 24 h, and the expression and localization of major iron modulating proteins was evaluated by Western blotting (WB) and immunostaining. Synthesis and maturation of cathepsin-D (cat-D), a lysosomal enzyme, was evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and WB, respectively. For in-vivo studies, C57BL/6 mice were injected with 40 mg/kg mouse body weight of NaIO3 intraperitoneally, and their retina was evaluated after 3 weeks as above. NaIO3 induced a 10-fold increase in ferritin in ARPE-19 cells, which co-localized with LC3II, an autophagosomal marker, and LAMP-1, a lysosomal marker. A similar increase in ferritin was noted in retinal lysates and retinal sections of NaIO3-injected mice by WB and immunostaining. Impaired synthesis and maturation of cat-D was also noted. Accumulated ferritin was loaded with iron, and released from retinal pigmented epithelial (RPE) cells in Perls’ and LAMP-1 positive vesicles. NaIO3 impairs lysosomal degradation of ferritin by decreasing the transcription and maturation of cat-D in RPE cells. Iron-loaded ferritin accumulates in lysosomes and is released in lysosomal membrane-enclosed vesicles to the extracellular milieu. Accumulation of ferritin in RPE cells and fusion of ferritin-containing vesicles with adjacent photoreceptor cells is likely to create an iron overload, compromising their viability. Moreover, reduced activity of cat-D is likely to promote accumulation of other cellular debris in lysosomal vesicles, contributing to AMD-like pathology.

Urolithin A promotes p62-dependent lysophagy to prevent acute retinal neurodegeneration

“…Following up on previous evidence [ 17 ], ProteoStat assay revealed a very severe accumulation of protein aggregates in the retina after SI treatment (Fig. 2 A), that was concomitant with an accumulation of LC3 + autophagosomes (Fig.…”

“…Sodium iodate (SI) is an oxidizing agent whose administration mimics the late stages of dry AMD, including RPE cell death, geographic atrophy and loss of visual function [ 16 ]. Previous evidence in the literature suggests that SI might inhibit macroautophagy [ 17 ] concomitant with mitochondrial dysfunction [ 18 ]. With this in mind, we tested whether the well-described mitophagy inducer Urolithin A (UA) [ 19 ] could have neuroprotective benefits in the SI model.…”

“…Recently, it was described that lipofuscin buildup can crystallize and damage the lysosomal membrane, leading to LMP, in a mouse model of impaired visual pigment processing ( Abca4 −/− Rdh8 −/− ) [ 35 ]. Previous research has also shown that iron-loaded ferritin accumulates inside the lysosomes of SI-treated ARPE-19 cells, leading to impaired hydrolase (Cathepsin D) maturation [ 17 ]. Our data highlights that SI similarly triggers severe LMP and upregulates the CLEAR network transcriptional program to stimulate lysosomal biogenesis, but is not enough to sustain cell homeostasis and ensure RPE survival.…”

Role of Oxidative Stress in Ocular Diseases: A Balancing Act

“…In hereditary disorders such as hemochromatosis, which involves systemic iron overload, iron accumulation has been observed in the retina in spite of an intact blood–retinal barrier (BRB), resulting in clinical signs of AMD [ 260 , 261 ]. Iron-mediated oxidative stress has been reported in a sodium iodate-induced dry AMD model, indicating the significant upregulation of ferritin and impaired cathepsin D synthesis and maturation, resulting in iron-loaded ferritin accumulation [ 262 ].…”