Mitophagy, Ferritinophagy and Ferroptosis in Retinal Pigment Epithelial Cells Under High Glucose Conditions: Implications for Diabetic Retinopathy and Age-Related Retinal Diseases

Abstract

Diabetic retinopathy (DR) is a devastating disease leading to blindness among majority of working adults around the globe. Nonetheless, an effective treatment or cure for the disease is still to be achieved. This is because the cellular and molecular mechanisms of DR are complex and not fully understood yet. In this article, we describe how high glucose induced TXNIP upregulation and associated redox stress may cause mitochondrial dysfunction, mitophagy, ferritinophagy (iron release by autophagy) and lysosome destabilization. Labile irons react with hydrogen peroxide (H2O2) to generate hydroxyl radicals (.OH) by the Fenton reaction and cause membrane phospholipid peroxidation due to reduction in glutathione (GSH) level and glutathione peroxidase 4 (GPX4) activity, which cause ferroptosis, a recently identified non-apoptotic cell death mechanism. We used in this study a retinal pigment epithelial cell line, ARPE- 19 and exposed it to high glucose in in vitro cultures to highlight some of the intricacies of these cellular processes, which may be relevant to the pathogenesis of DR and age-related retinal neurodegenerative diseases, such as age-related macular degeneration, AMD.

Keywords: Diabetic retinopathy; Ferritinophagy; Ferroptosis; Mitophagy; Oxidative stress; RPE; TXNIP.

Figure 1:

High glucose mediates aberrant redox and tight junction protein expression in APRE-19. ARPE- 19 cells were maintained in DMEM/F-12 medium containing 2% serum with antibiotics containing 5.5mM glucose (LG) or 25mM glucose (HG) for 5 days like those described previously (33). Western blotting and QPCR detected protein and mRNA levels. Data are presented as SEM+/−SE and p value of <0.05 is considered significant when compared between LG and HG using Student’s t-test. (A-B) TXNIP level is increased in HG compared to LG (p<0.04) while that SOD1 and Trx1 are down regulated significantly. Tubulin is used as a control protein. (C-E) Protein and mRNA levels of ZO-1 also decrease in HG compared to LG. (F) Furthermore, ZO-1 immunostaining (red) is also disrupted at the plasma membrane under HG (yellow arrows) when compared to LG. Furthermore, addition of 2μM azaserine (an inhibitor of TXNIP, ref. 25) in the last 24h of the experiment restores ZO-1 plasma membrane staining in HG. A representative of n=3 is shown here.

Figure 2:

High glucose induces mitophagic flux and lysosome enlargement in ARPE-19 cells. Transduction of mt-Keima and LAMP1-mCherry bearing adenovirus vectors in APRE-19 cells were described previously [33]. Mt-Keima and LAMP1-mCherry vectors were transfected together in ARPE-19 cells and incubated with LG (5.5mM) or HG (25mM glucose) for 5 days, then the cells were fixed, mounted on DAPI containing mounting medium to stain nuclei and imaged with a Zeiss Confocal microscopy at 630x magnification. The images were analyzed by Zen 3.0 blue software and compile in Adobe Photoshop. Under these experimental conditions, mt-Keima emits green both in mitochondria and lysosomes while LAMP1-mCherry emits red in lysosomes. With HG treatment, lysosome sizes are ~1.5- to 2-folds larger than in LG (mCherry, white arrows in insets) while there are also more yellow mito-lysosomes in HG versus LG (green and red combination, arrowheads in insets). A representative of three experiments is shown here.

Figure 3:

Ferritinophagy and ferroptosis induction by HG and iron-sulfate in ARPE-19. (A) ARPE-19 cells were cultured in LG (1) or HG (2) for 5 days and LG (3) or HG (4) 5 days with 0.5mM hydrogen peroxide (H2O2) in the last 24h of experiment, cells harvested and proteins (30mg) were detected by Western blotting as previously described [33,34]. Cytosolic aconitase (Aco1), ferritinophagy adaptor - nuclear receptor coactivator 4 (NCOA4), and ferritin light chain (Ferritin L) are reduced in HG and H2O2 indicating ferritinophagy induction. HG increases LC3BI, however, H2O2 reduces both LC3BI and II. The blot is a representative n=2. (B) Cell death assay by LDH leakage was performed as described before [34]. Fifty microliters of the medium were assayed for the LDH activity using a commercial LDH assay kit from Pierce (Cat# 88953), according to Manufacturer’s instructions. Incubation of ARPE-19 cells with 1mM FeSO4 increases LDH activity ~5 folds (p<0.05) in media from HG than from LG. Preincubation with ferrostatin 1 (Fer-1, a ferroptosis inhibitor) 2h before adding FeSO4 and present throughout the duration of the experiment, reduces LDH leakage significantly, suggesting ferroptotic cell death in ARPE-19. (C) Similarly, iron chelator deferasirox (25μM DFX), a combination of ALOX-5 inhibitor Zileuton (Zil, 100nM) and DFX (25μM), antioxidant Selenium (Se, 160nM) or a combination of 100nM Se and 50μM DFX reduces LDH activity significantly in ARPE-19 cells, indicating that ferroptosis mechanism may involve various signaling pathways. One-way ANOVA and Bonferroni post hoc test determined differences among means in multiple sets of experiments. Data are presented as SEM+/−SE and p value of <0.05 is considered significant, *<0.01 and ***<0.0001.

Figure 4:

Relationship among Mitophagy, Ferritinophagy and Ferroptosis under oxidative stress. Hyperglycemia-induced TXNIP upregulation and oxidative/nitrosative stress cause mitochondrial damage, ROS generation and mitophagic flux to lysosomes for degradation. A feedback mechanism to replenish mitochondrial mass and bioenergetics may activate ferritinophagy, an autophagic process of degradation and release of free iron Fe²⁺ from ferritin cages in lysosomes for using in the synthesis of mitochondrial Iron-sulfur cluster and heme moeity. However, the released ferrous iron also reacts with H2O2 via Fenton reaction to generate highly reactive hydroxyl radicals (.OH), and biproducts of hydroxide (OH−) and ferric iron (Fe³⁺). These .OH radicals react with cell membranes rich in polyunsaturated fatty acids (PUFA) to mediate lipid peroxidation (PUFA-OOH), including the mitochondria, lysosome and plasma membranes. PUFA-OOH in membranes disturb fluidity and membrane integrity. Free Fe²⁺ may also activate arachidonic 5-lipoxygenase (ALOX5), which generates lipid peroxides (L- OOH). The only enzyme that can remove and prevent lipid peroxidation is GPX4, a selenocysteine protein, using 2 GSH molecules. However, under oxidative stress, GSH level and biosynthesis are down-regulated leading to accumulation of oxidized GSSG. Extracellular cystine (di-cysteine) is transported into cells through plasma membrane glutamate-cystine exchanger (xCT). Down-regulation of xCT, which occurs in DR, will limit cystine import and, therefore, cysteine available for GSH synthesis. Under these conditions, iron-dependent PUFA-OOH due to inhibition of GPX4 activity causes cell death by ferroptosis, a recently identified non-apoptotic cell death. Ferroptosis itself induces an inflammatory response, which assembles the NLRP3 inflammasome, activating caspase-1, IL- 1β/IL-18 and gasdermin-D to cause pyroptotic cell death as well. These events, if unchecked, eventually will sustain a chronic low-grade inflammation, oxidative stress and premature retinal cell death leading to disease initiation and progression of DR and age-related retinal diseases. [Image created with BioRender.com.].