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. 2012 May 1;52(9):1820-7.
doi: 10.1016/j.freeradbiomed.2012.02.043. Epub 2012 Mar 6.

Gpx4 ablation in adult mice results in a lethal phenotype accompanied by neuronal loss in brain

Si-Eun Yoo  1 , Liuji Chen, Ren Na, Yuhong Liu, Carmen Rios, Holly Van Remmen, Arlan Richardson, Qitao Ran
Affiliations

V体育安卓版 - Gpx4 ablation in adult mice results in a lethal phenotype accompanied by neuronal loss in brain

Si-Eun Yoo et al. Free Radic Biol Med. .

Abstract (VSports在线直播)

Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme important in reducing hydroperoxides in membrane lipids and lipoproteins. Gpx4 is essential for survival of embryos and neonatal mice; however, whether Gpx4 is required for adult animals remains unclear. In this study, we generated a floxed Gpx4 mouse (Gpx4(f/f)), in which exons 2-4 of Gpx4 gene are flanked by loxP sites. We then cross-bred the Gpx4(f/f) mice with a tamoxifen (tam)-inducible Cre transgenic mouse (R26CreER mice) to obtain mice in which the Gpx4 gene could be ablated by tam administration (Gpx4(f/f)/Cre mice). After treatment with tam, adult Gpx4(f/f)/Cre mice (6-9 months of age) showed a significant reduction of Gpx4 levels (a 75-85% decrease) in tissues such as brain, liver, lung, and kidney. Tam-treated Gpx4(f/f)/Cre mice lost body weight and died within 2 weeks, indicating that Gpx4 is essential for survival of adult animals. Tam-treated Gpx4(f/f)/Cre mice exhibited increased mitochondrial damage, as evidenced by the elevated 4-hydroxylnonenal (4-HNE) level, decreased activities of electron transport chain complexes I and IV, and reduced ATP production in liver. Tam treatment also significantly elevated apoptosis in Gpx4(f/f)/Cre mice VSports手机版. Moreover, tam-treated Gpx4(f/f)/Cre mice showed neuronal loss in the hippocampus region and had increased astrogliosis. These data indicate that Gpx4 is essential for mitochondria integrity and survival of neurons in adult animals. .

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Figures

Figure 1
Figure 1
Generation of a floxed Gpx4 mouse. A. Illustrations of the wild-type Gpx4 (WT) gene allele, the targeting vector, the targeted allele, and the floxed allele of Gpx4 gene. The black boxes indicate exons: E1, E1a, and E2–E6 (left to right). The targeting vector was electroporated into ES cells. After standard selection and screening procedures, ES cells were used to produce mice with the targeted allele of Gpx4 gene. Mice with the targeted allele of Gpx4 gene were then cross-bred with Flpe mice to generate mice with a floxed allele of Gpx4 gene, in which E2, E3 and E4 are flanked by two loxP sites. B&C. Southern blot results showing the presence of the targeted allele in ES cells (the left lane). B. Genomic DNA from two ES cell clones was digested with HindIII and hybridized with Probe 2. The 15.5 kb fragment and the 11.0 kb fragment were indicative of the WT allele and the targeted allele, respectively. C. Genomic DNA from two ES cell clones was digested with EcoRI and hybridized with Probe 1. The 5.3 kb fragment and the 7.6 kb fragment were indicative of the WT allele and the targeted allele, respectively. The graph was rearranged. D. Identification of the floxed allele of Gpx4 gene by PCR. Using primers described in the Materials and Methods, a 200bp fragment and a 600bp fragment were amplified from the WT allele and the floxed allele of tail DNA, respectively. The genotypes are shown above.
Figure 2
Figure 2
Lethal phenotype of adult Gpx4(f/f)/Cre mice treated with tam. A. Body weights of Gpx4(f/f)/Cre mice and Gpx4(f/f) mice (6–9 months of age) during and after tam treatment (4 injections of tam) are shown (n=19 mice per group). *: p <0.05 B. Survival of tam-treated Gpx4(f/f)/Cre mice (n = 21) and tam-treated Gpx4(f/f) mice (n = 21). The curves were found to be significantly different by Log-Rank test.
Figure 3
Figure 3
Reduced Gpx4 levels in tissues from tam-treated adult Gpx4(f/f)/Cre mice. Gpx4 levels were measured by Western blots (Representative blots are shown. Some graphs were rearranged.) in liver, lung, kidney, and brain (cortex and hippocampus) from tam-treated Gpx4(f/f)/Cre mice and tam-treated Gpx4(f/f) mice. The mean levels of Gpx4 protein (the ratio of Gpx4 to β-actin determined by densitometry) in Gpx4(f/f) mice were assigned as 1 arbitrarily, and the relative data representing mean ± SEM are shown. *: p <0.05. n=3
Figure 4
Figure 4
Effect of Gpx4 ablation on mitochondrial function. A. The levels of 4-HNE protein adducts in liver whole homogenates and mitochondrial fractions of tam-treated Gpx4(f/f)/Cre mice and tam-treated Gpx4(f/f) mice. The activities of electron transport chain complex I (B), II (C), III (D), IV (E), and ATP generation (F) of isolated mitochondria from liver were measured in tam-treated Gpx4(f/f)/Cre mice and tam-treated Gpx4(f/f) mice. The data representing mean ± SEM are shown. (*: p <0.05, n=3). G/M: glutamate plus malate; Suc/Rot: succinate plus rotenone.
Figure 5
Figure 5
Elevated apoptosis in livers from tam-treated Gpx4(f/f)/Cre mice. A&B. Graphs of liver sections stained for the presence of double-strand DNA breaks from tam-treated Gpx4(f/f) mice (A) and tam-treated Gpx4(f/f)/Cre mice (B). Arrows indicate cells positive for double-strand DNA breaks. C. The levels (expressed as percentages) of apoptotic cells in livers of tam-treated Gpx4(f/f) mice and tam-treated Gpx4(f/f)/Cre mice. The values are expressed as mean ± SEM. *: p<0.05. n=3. D. A graph of Western blots showing levels of caspase-3 and cleaved caspase-3 in tam-treated Gpx4(f/f)/Cre mice and tam-treated Gpx4(f/f) mice. E. The levels of cleaved caspase-3 in tam-treated Gpx4(f/f) mice and tam-treated Gpx4(f/f)/Cre mice. F. The levels of cytosolic cyt. c in tam-treated Gpx4(f/f) mice and tam-treated Gpx4(f/f)/Cre mice. The mean level of cleaved caspase-3 or cytosolic cyt. c in Gpx4(f/f) mice was assigned as 1 arbitrarily, and the relative data representing mean ± SEM are shown. *: p <0.05, n=3.
Figure 6
Figure 6
Neurodegeneration in tam-treated Gpx4(f/f)/Cre mice. A&B. Images of brain sections of hippocampus regions stained with an anti-NeuN antibody from tam-treated Gpx4(f/f)mice (A) and tam-treated Gpx4(f/f)/Cre mice (B). Arrows indicate areas of neuronal loss. C. A graph of Western blots showing NeuN and Synaptophysin (syn) levels in hippocampus of tam-treated Gpx4(f/f) mice and tam-treated Gpx4(f/f) /Cre mice. D&E. Quantified results of NeuN levels (D) and Syn levels (E). The mean level of NeuN or Syn in Gpx4(f/f) mice was assigned as 1 arbitrarily, and the relative data representing mean ± SEM are shown. *: p <0.05, n=3.
Figure 7
Figure 7
Astrocyte activation in hippocampus of tam-treated Gpx4(f/f)/Cre mice. A&B. Images of brain sections of hippocampus regions stained with an anti-GFAP antibody from tam-treated Gpx4(f/f) mice (A) and tam-treated Gpx4(f/f)/Cre mice (B). C. A graph of Western blots showing GFAP protein levels in hippocampus of tam-treated Gpx4(f/f) mice and tam-treated Gpx4(f/f) /Cre mice. D. Quantified results of GFAP protein levels. The mean level of GFAP in Gpx4(f/f) mice was assigned as 1 arbitrarily, and the relative data representing mean ± SEM are shown. *: p <0.05, n=3.
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