June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The role of TXNIP and redox imbalance in mitochondrial dysfunction and ferroptosis in retinal cells: Implications for diabetic retinopathy
Author Affiliations & Notes
  • Lalit P Singh
    OVAS, Wayne State Univ Sch of Med, Detroit, Michigan, United States
  • Thangal Yumnamcha
    OVAS, Wayne State Univ Sch of Med, Detroit, Michigan, United States
  • Ahmed S Ibraham
    OVAS, Wayne State Univ Sch of Med, Detroit, Michigan, United States
  • Takhellambam S Devi
    OVAS, Wayne State Univ Sch of Med, Detroit, Michigan, United States
  • Footnotes
    Commercial Relationships   Lalit Singh, None; Thangal Yumnamcha, None; Ahmed Ibraham, None; Takhellambam Devi, None
  • Footnotes
    Support   NIH/NEI R01 EY023992, NIH/NEI core grant P30EY004068, Research to Prevent Blindness, American Heart Association Grant 18CDA34080403
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 752. doi:
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      Lalit P Singh, Thangal Yumnamcha, Ahmed S Ibraham, Takhellambam S Devi; The role of TXNIP and redox imbalance in mitochondrial dysfunction and ferroptosis in retinal cells: Implications for diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2020;61(7):752.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To determine whether ferroptosis occurs in retinal cells under mitochondrial dysfunction and redox stress. Ferroptosis is a nonapoptotic cell death due to iron-dependent lipid peroxidation (LOOH) and glutathione peroxidase 4 (GPX4) inhibition.

Methods : Retinal cells, human retinal pigment epithelium (HRPE and APRE-19), Muller glia (rMC1) and cone photoreceptor (661W), were treated with high glucose (HG, 25 mM), H2O2, FeSO4 or RSL3, an inhibitor of GPX4 in culture. Cellular ROS, ATP levels and lactate dehydrogenase (LDH) leakage in media were measured to detect cellular stress and death. The mitophagy-lysosome pathway was monitored by mt-Keima, a mitophagy probe, and LAMP1-mcherry, a lysosomal membrane marker using confocal microscopy. Mitochondrial oxygen consumption rate (OCR) was determined by Seahorse XFe96 Analyzer. A p value of <0.05 was considered statistically significant in all analysis.

Results : TXNIP is significantly induced in all 3 retinal cell types under HG for 5 days compared to normal glucose (5.5 mM) causing oxidative stress, reduced ATP and mitophagic flux as red mt-Keima increases under HG. Mt-Keima emits green light in mitochondria (alkaline pH) while it emits red light in lysosomes (acidic pH). Similar observations are also seen after treatment with H2O2 suggesting oxidative stress may play a role in these events. Furthermore, we observe fewer but enlarged lysosomes suggesting lysosomal fusion. Under these conditions, OCR is reduced in APRE-19 indicating a metabolic switch to anaerobic glycolysis. Because mitochondria are major sources of iron in cells (containing FeS clusters, heme and mt-ferritin), enhanced mitophagy may release redox active free iron into lysosomes or to the cytosol. To examine if retinal cells undergo ferroptosis, we treated ARPE-19 cells with RSL3 or FeSO4 for 24h with or without ferrostatin-1 (ferroptosis inhibitor), or deteroxamine (iron chelator). LDH release (ferroptosis) is inhibited by ferrostatin-1, deferoxamine, lipoxygenase inhibitors and selenium, which increases GPX4. Similar observations are also seen with rMC1, but not in 661W.

Conclusions : TXNIP, oxidative stress and mitophagy-lysosomal axis dysregulation may cause free iron accumulation, membrane LOOH and ferroposis under sustained hyperglycemia. Iron chelators and mitochondria-targeted anti-oxidants may prevent ferroptosis and progression of DR.

This is a 2020 ARVO Annual Meeting abstract.

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