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, 93 (11), 1728-39

Basal Brain Oxidative and Nitrative Stress Levels Are Finely Regulated by the Interplay Between Superoxide Dismutase 2 and p53

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Basal Brain Oxidative and Nitrative Stress Levels Are Finely Regulated by the Interplay Between Superoxide Dismutase 2 and p53

Eugenio Barone et al. J Neurosci Res.

Abstract

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

Keywords: MnSOD; p53; RRID:AB_10618757; RRID:AB_10850321; RRID:AB_1840351; RRID:AB_2049199; RRID:AB_2256876; RRID:AB_476744; RRID:AB_881705; RRID:AB_958795; biliverdin reductase-A; heme oxygenase-1; oxidative stress.

Conflict of interest statement

Conflict of interest statement

The authors all state that there are no conflicts of interests associated with the research presented in this paper.

Figures

Figure 1
Figure 1. In vivo oxidative and nitrative modifications observed in mitochondria isolated from the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) protein carbonyls (PC) levels; (B) protein-bound 4-hydroxy-2-nonenal (HNE) levels and (C) 3-nitrotyrosine (3-NT) levels measured in the mitochondrial fraction. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of three replicates of each individual sample (n=6) per group. **p<0.01 versus wild type or the respective single mutant mice (ANOVA).
Figure 2
Figure 2. In vivo oxidative and nitrative modifications observed in nuclei and cytosol isolated from the brain of of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) protein carbonyls (PC) levels, (B) protein-bound 4-hydroxy-2-nonenal (HNE) levels and (C) 3-nitrotyrosine (3-NT) levels measured in the nuclear fraction; (D) PC, (E) HNE and (F) 3-NT levels measured in the cytosolic fraction. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of three replicates of each individual sample (n=6) per group. *p< 0.05, **p<0.01 and ***p<0.001 versus wild type or the respective single mutant mice (ANOVA).
Figure 3
Figure 3. In vivo oxidative and nitrative modifications observed in the whole homogenate obtained from the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) protein carbonyls (PC) levels; (B) protein-bound 4-hydroxy-2-nonenal (HNE) levels and (C) 3-nitrotyrosine (3-NT) levels measured in the mitochondrial fraction. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of three replicates of each individual sample (n=6) per group. *p<0.05 and ***p<0.001 versus wild type or the respective single mutant mice (ANOVA).
Figure 4
Figure 4. Expression levels of heme oxygenase-1 (HO-1) and thioredoxin-1 evaluated in the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) HO-1 and (B) thioredoxin-1 protein levels measured in the membrane fraction isolated from the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and those of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+)) as described under materials and methods. Representative gels are shown. Protein levels were normalized to the loading control β-actin. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of n=6 individual sample per group. *P <0.05 versus wild type or the respective single mutant mice (ANOVA).
Figure 5
Figure 5. Expression levels of Biliverdin Reductase-A (BVR-A) evaluated in the cytosol and in the nuclei isolated form the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) cytosolic and (B) nuclear BVR-A protein levels measured in the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and those of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+)) as described under materials and methods. Representative gels are shown. Protein levels were normalized to the loading control β-actin. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of n=6 individual sample per group. *P <0.05 versus wild type or the relatives single mutant mice (ANOVA).
Figure 6
Figure 6. Expression levels of the nuclear factor-erythroid 2 related factor 2 (Nrf-2) evaluated in the cytosol and in the nuclei isolated form the brain of of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+))
(A) cytosolic and (B) nuclear Nrf-2 protein levels measured in the brain of wild type (WT), SOD2 transgenic (TgSOD2), SOD2 heterozygous knock-out (SOD2(−/+)) and those of the mice carrying the double mutation (p53(−/−)xTgSOD2; p53(−/−)xSOD2(−/+)) as described under materials and methods. Representative gels are shown. Protein levels were normalized to the loading control β-actin. Densitometric values shown are given as percentage of the wild type (WT) group, set as 100%. Data are expressed as mean ± SE of n=6 individual sample per group. *P <0.05 versus wild type or the relatives single mutant mice (ANOVA)

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