June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Protective role of glutathione peroxidase 4 in retinal ganglion cells
Author Affiliations & Notes
  • Takatoshi Uchida
    Department of Ophthalmology, University of Tokyo School of Medicine, Bunkyo CIty, Japan
    Reseach & Development Division, Senju Pharmaceutical, Kobe, Japan
  • Osamu Sakai
    Department of Ophthalmology, University of Tokyo School of Medicine, Bunkyo CIty, Japan
    Reseach & Development Division, Senju Pharmaceutical, Kobe, Japan
  • Takashi Ueta
    Department of Ophthalmology, University of Tokyo School of Medicine, Bunkyo CIty, Japan
  • Shiro Amano
    Inouye Eye Hospital, Chiyoda, Japan
    Miyata Eye Hospital, Miyakonojo, Japan
  • Footnotes
    Commercial Relationships Takatoshi Uchida, Senju Pharmaceutical co.,ltd. (E); Osamu Sakai, Senju Pharmaceutical co.,ltd. (E); Takashi Ueta, None; Shiro Amano, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3650. doi:
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    • Get Citation

      Takatoshi Uchida, Osamu Sakai, Takashi Ueta, Shiro Amano; Protective role of glutathione peroxidase 4 in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3650.

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

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Abstract

Purpose: Purpose: The purpose of the present study was to investigate whether glutathione peroxidase 4 (GPx4) could be an essential antioxidative enzyme in retinal ganglion cells.

Methods: Methods: In vitro experiments, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or control siRNA. Knockdown was confirmed by real-time RT-PCR and Western blot. Lipid peroxidation was evaluated by immunostaining of 4-hydroxy-2-nonenal (4-HNE). The cytotoxicity was assessed by LDH activity assay and Annexin V/propidium iodide (PI) staining. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (final concentration: 2 mM), and the cytotoxicity was evaluated by LDH activity assay. In vivo study, retinal ganglion cell damage was induced by intravitreal injection of 25 mM N-methyl-D-aspartate (NMDA, 2 mL/eye) in GPx4+/+ and GPx4+/ mice. Evaluation of lipid peroxidation (immunostaining of 4-HNE), apoptosis (TUNEL staining) and cell density in the ganglion cell layer (GCL) were performed at 12 hr, 1 day and 7 days after NMDA injection, respectively.

Results: Results: Knockdown of GPx4 significantly induced cytotoxicity and increased the level of lipid oxidation by 3.2 fold in R28 cells (p<0.01). The level of LDH activity was increased by GPx4 knockdown (13.9 fold, P<0.01). Moreover, knockdown of GPx4 enhanced glutamate-induced cytotoxicity determined by LDH activity (1.52 fold, P<0.01), though glutamate did not influence LDH activity of cells treated with control siRNA. In vivo study, GPx4+/− mice had higher levels of lipid peroxidation than GPx4+/+ mice in the retina treated with NMDA (1.26 fold, P<0.05). GPx4+/ mice had more TUNEL-positive cells induced by NMDA in GCL (1.45 fold, P<0.05). In addition, the cell density in GCL of GPx4+/ mice was significantly lower than that of GPx4+/+ mice after treatment with NMDA (19%, P<0.05).

Conclusions: Conclusion: These results suggested that GPx4 was essential for maintaining oxidative homeostasis and protecting from glutamate-induced cytotoxicity in retinal ganglion cells.

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