title: Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina.
abstract: OBJECTIVE
  The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina.
  
  
  METHODS
  For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4+/+ and GPx4+/- mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection.
  
  
  RESULTS
  GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4+/- mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4+/+ mice (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 19% lower than that in GPx4+/+ mice after treatment with NMDA (P < 0.05).
  
  
  CONCLUSIONS
  These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.
authors.affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan; Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Sakai
authors.affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan; Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan.
authors.forename: Murilo F
authors.initials: MF
authors.lastname: Roggia
authors.affiliation: School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Imai
authors.affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueta
authors.affiliation: Inouye Eye Hospital, Tokyo, Japan; Miyata Eye Hospital, Miyazaki, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Amano
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000633:toxicity*; D005979:Glutathione Peroxidase / Q000502:physiology*; D007124:Immunoenzyme Techniques; D015227:Lipid Peroxidation / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018384:Oxidative Stress / Q000187:drug effects*; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D018698:Glutamic Acid; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase