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Brain-Specific Superoxide Dismutase 2 Deficiency Causes Perinatal Death with Spongiform Encephalopathy in Mice.

Izuo N, Nojiri H, Uchiyama S, Noda Y, Kawakami S, Kojima S, Sasaki T, Shirasawa T, Shimizu T - Oxid Med Cell Longev (2015)

Bottom Line: Manganese superoxide dismutase (Mn-SOD, SOD2) is a mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide.B-Sod2(-/-) showed perinatal death, along with severe growth retardation.Furthermore, brain lipid peroxidation was significantly increased in the B-Sod2(-/-), without any compensatory alterations of the activities of other antioxidative enzymes, such as catalase or glutathione peroxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Aging Medicine, Chiba University Graduate School of Medicine, Inohana, Chuo-ku, Chiba 260-8670, Japan.

ABSTRACT
Oxidative stress is believed to greatly contribute to the pathogenesis of various diseases, including neurodegeneration. Impairment of mitochondrial energy production and increased mitochondrial oxidative damage are considered early pathological events that lead to neurodegeneration. Manganese superoxide dismutase (Mn-SOD, SOD2) is a mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide. To investigate the pathological role of mitochondrial oxidative stress in the central nervous system, we generated brain-specific SOD2-deficient mice (B-Sod2(-/-)) using nestin-Cre-loxp system. B-Sod2(-/-) showed perinatal death, along with severe growth retardation. Interestingly, these mice exhibited spongiform neurodegeneration in motor cortex, hippocampus, and brainstem, accompanied by gliosis. In addition, the mutant mice had markedly decreased mitochondrial complex II activity, but not complex I or IV, in the brain based on enzyme histochemistry. Furthermore, brain lipid peroxidation was significantly increased in the B-Sod2(-/-), without any compensatory alterations of the activities of other antioxidative enzymes, such as catalase or glutathione peroxidase. These results suggest that SOD2 protects the neural system from oxidative stress in the perinatal stage and is essential for infant survival and central neural function in mice.

No MeSH data available.


Related in: MedlinePlus

Generation of B-Sod2−/−. (a) DNA fragments amplified from the wild (500-bp) or lox allele (358-bp) were detected in tail DNA. The Cre transgene was confirmed by PCR with Cre primers. A 208 bp DNA fragment was detected in knockout and heterozygous mice using Cre PCR primers. A 401 bp DNA fragment amplified from the deletion allele was detected in the brains of knockout and heterozygous mice. (b) The results of Western blot analysis of SOD2 protein in two-week-old B-Sod2−/− and control. Protein extracts from the brain, liver, kidney, and heart of B-Sod2−/− or control were immunoblotted with anti-SOD2 or anti-actin antibodies. (c) The growth rate of body weight increase in B-Sod2−/− (n = 24), indicated by closed circle, progressively decreased compared with control (n = 29, ∗P < 0.05), indicated by open circle. (d) The brain weight of B-Sod2−/− at 16–19 days of age (n = 24) was significantly lower than that of brains from age-matched control (n = 22, ∗∗∗P < 0.005). (e) The cumulative survival of B-Sod2−/− (n = 29), indicated by closed circle, and control (n = 29), indicated by open circle.
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fig1: Generation of B-Sod2−/−. (a) DNA fragments amplified from the wild (500-bp) or lox allele (358-bp) were detected in tail DNA. The Cre transgene was confirmed by PCR with Cre primers. A 208 bp DNA fragment was detected in knockout and heterozygous mice using Cre PCR primers. A 401 bp DNA fragment amplified from the deletion allele was detected in the brains of knockout and heterozygous mice. (b) The results of Western blot analysis of SOD2 protein in two-week-old B-Sod2−/− and control. Protein extracts from the brain, liver, kidney, and heart of B-Sod2−/− or control were immunoblotted with anti-SOD2 or anti-actin antibodies. (c) The growth rate of body weight increase in B-Sod2−/− (n = 24), indicated by closed circle, progressively decreased compared with control (n = 29, ∗P < 0.05), indicated by open circle. (d) The brain weight of B-Sod2−/− at 16–19 days of age (n = 24) was significantly lower than that of brains from age-matched control (n = 22, ∗∗∗P < 0.005). (e) The cumulative survival of B-Sod2−/− (n = 29), indicated by closed circle, and control (n = 29), indicated by open circle.

Mentions: In order to investigate the physiological and pathological role of SOD2 in the brain, we generated B-Sod2−/− using the Cre-loxP system. We used nestin-Cre transgenic mice for the selective expression of Cre protein in the neurogenic lineage, such as in neurons, astrocytes, and oligodendrocytes, which are mainly in brain [16], in early prenatal stage in coordination with nestin expression from E7 [17]. As shown in Figure 1, crossbreeding homozygous SOD2-flox mice, which were set as control, with nestin-Cre transgenic mice gave rise to B-Sod2−/−. Genomic DNA extracted from brain tissues was analyzed by PCR to detect the deleted fragment from the genomic SOD2 gene. Corresponding to the deletion allele, a 401 bp DNA fragment was specifically amplified by PCR from the brains of B-Sod2−/− and heterozygous mice, while no fragment was amplified in control mice (Figure 1(a)). Western blot analyses further showed a specific loss of SOD2 expression in the brain, but not in the liver, kidney, or heart of B-Sod2−/− compared with control (Figure 1(b)). The slight expression of SOD2 protein in the brains of B-Sod2−/− could be derived from nonneurogenic lineage cells, such as microglia or endothelial cells. B-Sod2−/− were born in normal Mendelian ratio and in the neonatal stage, and we were unable to find any differences in the visible appearance or body size between B-Sod2−/− and control (data not shown). However, at 10 days of age, B-Sod2−/− began to exhibit a delayed increase in body weight (Figure 1(c)), and the brain weight of B-Sod2−/− was significantly lower than that of control at the age of 16–19 days (Figure 1(d)), probably caused by growth retardation. Finally, the B-Sod2−/− began to die from 12 days of age (Figure 1(e)), and all of B-Sod2−/− died by 25 days of age, with a median survival rate of 19.6 ± 3.6 days (Figure 1(e)). These results suggest that SOD2 expression in brain is required for the postnatal survival of mice.


Brain-Specific Superoxide Dismutase 2 Deficiency Causes Perinatal Death with Spongiform Encephalopathy in Mice.

Izuo N, Nojiri H, Uchiyama S, Noda Y, Kawakami S, Kojima S, Sasaki T, Shirasawa T, Shimizu T - Oxid Med Cell Longev (2015)

Generation of B-Sod2−/−. (a) DNA fragments amplified from the wild (500-bp) or lox allele (358-bp) were detected in tail DNA. The Cre transgene was confirmed by PCR with Cre primers. A 208 bp DNA fragment was detected in knockout and heterozygous mice using Cre PCR primers. A 401 bp DNA fragment amplified from the deletion allele was detected in the brains of knockout and heterozygous mice. (b) The results of Western blot analysis of SOD2 protein in two-week-old B-Sod2−/− and control. Protein extracts from the brain, liver, kidney, and heart of B-Sod2−/− or control were immunoblotted with anti-SOD2 or anti-actin antibodies. (c) The growth rate of body weight increase in B-Sod2−/− (n = 24), indicated by closed circle, progressively decreased compared with control (n = 29, ∗P < 0.05), indicated by open circle. (d) The brain weight of B-Sod2−/− at 16–19 days of age (n = 24) was significantly lower than that of brains from age-matched control (n = 22, ∗∗∗P < 0.005). (e) The cumulative survival of B-Sod2−/− (n = 29), indicated by closed circle, and control (n = 29), indicated by open circle.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4537744&req=5

fig1: Generation of B-Sod2−/−. (a) DNA fragments amplified from the wild (500-bp) or lox allele (358-bp) were detected in tail DNA. The Cre transgene was confirmed by PCR with Cre primers. A 208 bp DNA fragment was detected in knockout and heterozygous mice using Cre PCR primers. A 401 bp DNA fragment amplified from the deletion allele was detected in the brains of knockout and heterozygous mice. (b) The results of Western blot analysis of SOD2 protein in two-week-old B-Sod2−/− and control. Protein extracts from the brain, liver, kidney, and heart of B-Sod2−/− or control were immunoblotted with anti-SOD2 or anti-actin antibodies. (c) The growth rate of body weight increase in B-Sod2−/− (n = 24), indicated by closed circle, progressively decreased compared with control (n = 29, ∗P < 0.05), indicated by open circle. (d) The brain weight of B-Sod2−/− at 16–19 days of age (n = 24) was significantly lower than that of brains from age-matched control (n = 22, ∗∗∗P < 0.005). (e) The cumulative survival of B-Sod2−/− (n = 29), indicated by closed circle, and control (n = 29), indicated by open circle.
Mentions: In order to investigate the physiological and pathological role of SOD2 in the brain, we generated B-Sod2−/− using the Cre-loxP system. We used nestin-Cre transgenic mice for the selective expression of Cre protein in the neurogenic lineage, such as in neurons, astrocytes, and oligodendrocytes, which are mainly in brain [16], in early prenatal stage in coordination with nestin expression from E7 [17]. As shown in Figure 1, crossbreeding homozygous SOD2-flox mice, which were set as control, with nestin-Cre transgenic mice gave rise to B-Sod2−/−. Genomic DNA extracted from brain tissues was analyzed by PCR to detect the deleted fragment from the genomic SOD2 gene. Corresponding to the deletion allele, a 401 bp DNA fragment was specifically amplified by PCR from the brains of B-Sod2−/− and heterozygous mice, while no fragment was amplified in control mice (Figure 1(a)). Western blot analyses further showed a specific loss of SOD2 expression in the brain, but not in the liver, kidney, or heart of B-Sod2−/− compared with control (Figure 1(b)). The slight expression of SOD2 protein in the brains of B-Sod2−/− could be derived from nonneurogenic lineage cells, such as microglia or endothelial cells. B-Sod2−/− were born in normal Mendelian ratio and in the neonatal stage, and we were unable to find any differences in the visible appearance or body size between B-Sod2−/− and control (data not shown). However, at 10 days of age, B-Sod2−/− began to exhibit a delayed increase in body weight (Figure 1(c)), and the brain weight of B-Sod2−/− was significantly lower than that of control at the age of 16–19 days (Figure 1(d)), probably caused by growth retardation. Finally, the B-Sod2−/− began to die from 12 days of age (Figure 1(e)), and all of B-Sod2−/− died by 25 days of age, with a median survival rate of 19.6 ± 3.6 days (Figure 1(e)). These results suggest that SOD2 expression in brain is required for the postnatal survival of mice.

Bottom Line: Manganese superoxide dismutase (Mn-SOD, SOD2) is a mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide.B-Sod2(-/-) showed perinatal death, along with severe growth retardation.Furthermore, brain lipid peroxidation was significantly increased in the B-Sod2(-/-), without any compensatory alterations of the activities of other antioxidative enzymes, such as catalase or glutathione peroxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Aging Medicine, Chiba University Graduate School of Medicine, Inohana, Chuo-ku, Chiba 260-8670, Japan.

ABSTRACT
Oxidative stress is believed to greatly contribute to the pathogenesis of various diseases, including neurodegeneration. Impairment of mitochondrial energy production and increased mitochondrial oxidative damage are considered early pathological events that lead to neurodegeneration. Manganese superoxide dismutase (Mn-SOD, SOD2) is a mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide. To investigate the pathological role of mitochondrial oxidative stress in the central nervous system, we generated brain-specific SOD2-deficient mice (B-Sod2(-/-)) using nestin-Cre-loxp system. B-Sod2(-/-) showed perinatal death, along with severe growth retardation. Interestingly, these mice exhibited spongiform neurodegeneration in motor cortex, hippocampus, and brainstem, accompanied by gliosis. In addition, the mutant mice had markedly decreased mitochondrial complex II activity, but not complex I or IV, in the brain based on enzyme histochemistry. Furthermore, brain lipid peroxidation was significantly increased in the B-Sod2(-/-), without any compensatory alterations of the activities of other antioxidative enzymes, such as catalase or glutathione peroxidase. These results suggest that SOD2 protects the neural system from oxidative stress in the perinatal stage and is essential for infant survival and central neural function in mice.

No MeSH data available.


Related in: MedlinePlus