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Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina.

Sakai O, Uchida T, Roggia MF, Imai H, Ueta T, Amano S - PLoS ONE (2015)

Bottom Line: 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).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).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan; Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.

ABSTRACT

Purpose: 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).

Conclusion: These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

No MeSH data available.


Related in: MedlinePlus

TUNEL staining in the retina of GPx4+/− and GPx4+/+ mice treated with NMDA or vehicle.(A) TUNEL staining of the retina after NMDA or vehicle treatment. Scale bar, 30 μm. (B) Comparison of the number of TUNEL positive cells in the retina. Data are mean ± SEM (n = 9–10). **p < 0.05.
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pone.0130467.g009: TUNEL staining in the retina of GPx4+/− and GPx4+/+ mice treated with NMDA or vehicle.(A) TUNEL staining of the retina after NMDA or vehicle treatment. Scale bar, 30 μm. (B) Comparison of the number of TUNEL positive cells in the retina. Data are mean ± SEM (n = 9–10). **p < 0.05.

Mentions: Next, we evaluated the extension of TUNEL-positive cell death in GCL 24 h after injections with NMDA or the vehicle. TUNEL-positive cells were hardly observed in the vehicle-treated retinas of both GPx4+/+ and GPx4+/− mice (Fig 9). Intravitreal injection of NMDA induced TUNEL-positive cells in GCL of both GPx4+/+ and GPx4+/− mice; however, the number of TUNEL-positive cells in GCL was significantly higher in GPx4+/− mice than in GPx4+/+ mice (Fig 9A and 9B).


Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina.

Sakai O, Uchida T, Roggia MF, Imai H, Ueta T, Amano S - PLoS ONE (2015)

TUNEL staining in the retina of GPx4+/− and GPx4+/+ mice treated with NMDA or vehicle.(A) TUNEL staining of the retina after NMDA or vehicle treatment. Scale bar, 30 μm. (B) Comparison of the number of TUNEL positive cells in the retina. Data are mean ± SEM (n = 9–10). **p < 0.05.
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pone.0130467.g009: TUNEL staining in the retina of GPx4+/− and GPx4+/+ mice treated with NMDA or vehicle.(A) TUNEL staining of the retina after NMDA or vehicle treatment. Scale bar, 30 μm. (B) Comparison of the number of TUNEL positive cells in the retina. Data are mean ± SEM (n = 9–10). **p < 0.05.
Mentions: Next, we evaluated the extension of TUNEL-positive cell death in GCL 24 h after injections with NMDA or the vehicle. TUNEL-positive cells were hardly observed in the vehicle-treated retinas of both GPx4+/+ and GPx4+/− mice (Fig 9). Intravitreal injection of NMDA induced TUNEL-positive cells in GCL of both GPx4+/+ and GPx4+/− mice; however, the number of TUNEL-positive cells in GCL was significantly higher in GPx4+/− mice than in GPx4+/+ mice (Fig 9A and 9B).

Bottom Line: 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).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).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo of Medicine, Tokyo, Japan; Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.

ABSTRACT

Purpose: 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).

Conclusion: These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

No MeSH data available.


Related in: MedlinePlus