Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711
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Browsing Eye / Vision by Author "Liu, Xiaobin"
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Item Glutaredoxin 2 (Grx2) Protects Retinal Pigment Epithelial Cells from Oxidative Damage by Regulating Autophagy(2016-03-23) Liu, Xiaobin; Nguyen, Yen; Tran, Tyler; Wu, Hongli; Xavier, ChristyPurpose: Glutaredoxin 2 (Grx2) is an oxidoreductase present in the mitochondria where it protects the organelle from oxidative damage and maintains its redox homeostasis.The purpose of this study is to evaluate the cytoprotective effects of Grx2 in human retinal pigment epithelial (RPE) cells and characterize its potential function in regulating autophagy.Methods: Primary RPE cells were isolated from Grx2 knockout (KO) mice and treated with or without 400 µM H2O2 for 4 h. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a human Grx2 cDNA-containing plasmid (pCR3.1-hGrx2) or an empty vector pCR3.1. Cells were treated with or without 200 µM H2O2 for 16 h. Grx2 protein expression was detected by western blot analysis. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) and autophagy pathway proteins were measured by immunoblotting.Results: Primary RPE cells that lack Grx2 were more sensitive to oxidative damage. On the other hand, Grx2 overexpression protected RPE cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx2 were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control cells. PSSG accumulation was also attenuated by Grx2 overexpression with acute H2O2 challenge. Furthermore, protein levels of LC3II and Beclin-1, which are key molecules to initiate autophagy, were inhibited in Grx2 overexpressed cells with H2O2 treatment. Conversely, primary Grx2 KO RPE cells showed higher levels of LC3II and Beclin-1 under oxidative stress.Conclusion: Grx2 rescues RPE cells from lethal oxidative damage, possibly through alleviation of ROS-triggered autophagy and prevention of PSSG accumulation.Item Validate Grx2 gene knockout mice as a new model for age-related retinal degeneration(2016-03-23) Xavier, Christy; Liu, Yang; Chavala, Sai; Clark, Abbot F.; Pang, Iok-Hou; Wu, Hongli; Liu, XiaobinPurpose: Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. The poorly understood pathogenesis has greatly hindered our progress in therapeutic development. To address this shortcoming, this project was designed to examine how retinal redox dysregulation leads to AMD and characterize glutaredoxin 2 (Grx2), a mitochondrial thiol redox regulating enzyme, knockout mice as a new animal model for AMD. Methods: The retinal pigment epithelium (RPE) layers were isolated from healthy and AMD donor eyes. Grx2 protein levels were measured by Western blot analysis. Primary RPE cells were isolated from wild-type (WT) and Grx2 knockout (KO) mice for the in vitro study. The visual function of WT and Grx2 KO mice were examined by fundus photography and scotopic electroretinography (ERG). H&E staining was used for histological exams. RPE structural changes were assessed by immunostaining of tight junction protein ZO-1. Lipofuscin autofluorescence was examined on cryostatsections. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results: Grx2 protein level and enzyme activities were decreased by approximately 30% in AMD donor eyes. Primary RPE cells isolated from Grx2 KO mice were more sensitive to H2O2-induced oxidative damage than WT RPE cells. Grx2 KO mice developed age-dependent retinal degenerative pathology. By 12 months of age, Grx2 null mice showed ~50% decrease in a-wave and ~30% decline in b-wave amplitudes (n=8, P Conclusions: Grx2 plays a critical role in maintaining the mitochondrial redox homeostasis in the aging retina. Grx2 deficiency causes PSSG accumulation and sensitizes RPE cells to age-related oxidative damage, leading to RPE degeneration and photoreceptor damage. As a new animal model for AMD, Grx2 KO mice will provide new insights into the pathogenesis and therapeutics of AMD.