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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

Knockdown efficacy of GPx4 in retinal precursor R28 cells.(A) The knockdown of GPx4 mRNA was confirmed by real-time RT-PCR. Data are mean ± SEM. (n = 3–4). **p < 0.01. (B) The knockdown of GPx4 protein was also confirmed by Western blot in triplicate.
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pone.0130467.g002: Knockdown efficacy of GPx4 in retinal precursor R28 cells.(A) The knockdown of GPx4 mRNA was confirmed by real-time RT-PCR. Data are mean ± SEM. (n = 3–4). **p < 0.01. (B) The knockdown of GPx4 protein was also confirmed by Western blot in triplicate.

Mentions: First, we confirmed a ubiquitous expression of GPx4 in mouse retinas (Fig 1). The effects of GPx4 silencing in retinal cells were then evaluated using R28 cells. R28 cells were transfected with GPx4 siRNA to specifically knockdown GPx4. Two days after transfection, a favorable efficiency was confirmed at both mRNA (Fig 2A) and protein levels (Fig 2B) measured by real-time RT-PCR and Western blot, respectively.


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)

Knockdown efficacy of GPx4 in retinal precursor R28 cells.(A) The knockdown of GPx4 mRNA was confirmed by real-time RT-PCR. Data are mean ± SEM. (n = 3–4). **p < 0.01. (B) The knockdown of GPx4 protein was also confirmed by Western blot in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470664&req=5

pone.0130467.g002: Knockdown efficacy of GPx4 in retinal precursor R28 cells.(A) The knockdown of GPx4 mRNA was confirmed by real-time RT-PCR. Data are mean ± SEM. (n = 3–4). **p < 0.01. (B) The knockdown of GPx4 protein was also confirmed by Western blot in triplicate.
Mentions: First, we confirmed a ubiquitous expression of GPx4 in mouse retinas (Fig 1). The effects of GPx4 silencing in retinal cells were then evaluated using R28 cells. R28 cells were transfected with GPx4 siRNA to specifically knockdown GPx4. Two days after transfection, a favorable efficiency was confirmed at both mRNA (Fig 2A) and protein levels (Fig 2B) measured by real-time RT-PCR and Western blot, respectively.

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