April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Protection of Retinal Ganglion Cells Against Hypoxia-Induced Cell Death by Peroxiredoxin 6, an Antioxidant
Author Affiliations & Notes
  • R. Tulsawani
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • N. Fatma
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • L. P. Suarez-Delgado
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • E. Kubo
    Ophthalmology, University of Fukui, Fukui, Japan
  • D. P. Singh
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3175. doi:
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      R. Tulsawani, N. Fatma, L. P. Suarez-Delgado, E. Kubo, D. P. Singh; Protection of Retinal Ganglion Cells Against Hypoxia-Induced Cell Death by Peroxiredoxin 6, an Antioxidant. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3175.

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

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Abstract

Purpose: : Hypoxia-induced insult and its sequelae have been implicated in progressive neurodegenerative diseases such as glaucoma. Recently hypoxia-induced generation of reactive oxygen species (ROS) and consequent activation of NF-ΚB has been shown to cause death of retinal ganglion cells (RGCs). Peroxiredoxin 6 (Prdx6) provides cellular protection against a variety of stressors. Using rat RGC-5 as a model, we investigated the cytoprotective efficacy of Prdx6 against hypoxia-induced cell death, and examined the regulation and regulatory role of Prdx6 in RGC-5 cells facing hypoxia.

Methods: : A construct containing a green fluorescent protein (GFP) linked to Prdx6 cDNA or its mutant at Cysteine(C) 47, redox-active site, was engineered, and was used to generate RGC-5 cells overexpressing Prdx6. These were cultured in DMEM containing 10% FBS. The cells were exposed to either 1% O2 (hypoxia) or normoxia for 24 to 48h. Cells overexpressing GFP-Prdx6 or its mutant were also treated with 0 to 500 uM of cobalt, a hypoxia-mimetic, for 24 to72 h to assess the protective ability of Prdx6. Cell viability was determined by MTS assay and apoptosis by DAPI and TUNEL assays. ROS expression was quantified with H2-DCFH-DA dye. Western blot and real-time PCR were used to monitor expression of Prdx6, NF-kB and β-actin.

Results: : RGC-5 cells overexpressing mutant Prdx6 cultured at 1% oxygen (hypoxia) revealed 50%-60% cell death after 48h of exposure, which was reduced to 26% to 32% when cells were overexpressed with Prdx6. Similarly, cells overexpressing Prdx6 showed reduced expression of ROS, and had resistance against CoCl2- induced death. The observed cell death was apoptotic, with higher levels of ROS and lower expression of Prdx6. Western analysis and real-time-PCR analysis showed upregulation of Prdx6 in RGCs exposed to hypoxia for 24h; expression levels of Prdx6 decreased after 48h. Also, cells exposed to hypoxia displayed activation of NF-kB, which was associated with the decrease in Prdx6 levels and RGC death. In contrast, cell death was significantly reduced and the levels of NF-ΚB deactivated in cells overexpressing Prdx6.

Conclusions: : Prdx6 was found to have neuroprotective effects against hypoxia-induced retinal cell damage, and these effects may be related to deactivation of NF-ΚB pathway by Prdx6. These findings may lead to a novel therapeutic strategy to reduce hypoxia-induced RGCs death.

Keywords: hypoxia • antioxidants • gene/expression 
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