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1.  Histone Deacetylase 2 in the Mouse Hippocampus: Attenuation of Age-Related Increase by Caloric Restriction 
Current Alzheimer research  2013;10(8):868-876.
The aging process in the hippocampus is associated with aberrant epigenetic marks, such as DNA methylation and histone tail alterations. Recent evidence suggests that caloric restriction (CR) can potentially delay the aging process, while upregulation of antioxidants may also have a beneficial effect in this respect. We have recently observed that CR attenuates age-related changes in the levels of the epigenetic molecules DNA methyltransferase 3a, 5-methylcytidine (5-mC) and 5-hydroxymethylcytosine in the mouse hippocampus while overexpression of the antioxidant Cu/Zn superoxide dismutase 1 (SOD1) does not. However, the impact of aging on the levels of histone-modifying enzymes such as histone deacetylase 2 (HDAC2) in the hippocampus has not been studied in much detail. Here, we investigated immunoreactivity (IR) of HDAC2 in three subregions of the hippocampus (dentate gyrus, CA3 and CA1-2) of mice taken from large cohorts of aging wild-type and transgenic mice overexpressing normal human SOD1, which were kept under normal diet or CR from weaning onwards. Independent from the genotype, aging (between 12 and 24 months) increased levels of HDAC2 IR in the hippocampus. Moreover, CR prevented this age-related increase, particularly in the CA3 and CA1-2 subregions, while SOD1 overexpression did not. Quantitative image analyses showed that HDAC2 IR correlated positively with 5-mC IR while these markers were shown to colocalize in the nucleus of hippocampal cells. Together with recent literature reports, these findings suggest that altered levels of epigenetic regulatory proteins including HDAC2 regulate age-related changes in the mouse hippocampus and that CR may prevent these age-related changes.
PMCID: PMC3966721  PMID: 24093534
Aging; epigenesis; histone deacetylase 2 (HDAC2); caloric restriction; hippocampus
2.  Age-related increase in levels of 5-hydroxymethylcytosine in mouse hippocampus is prevented by caloric restriction 
Current Alzheimer research  2012;9(5):536-544.
Aberrations in epigenetic marks have been associated with aging of the brain while caloric restriction (CR) and upregulation of endogenous antioxidants have been suggested as tools to attenuate the aging process. We have recently observed age-related increases in levels of 5-methylcytidine (5-mC) and DNA methyltransferase 3a (Dnmt3a) in the mouse hippocampus. Most of those age-related changes in these epigenetic relevant markers were prevented by CR but not by transgenic overexpression of the endogenous antioxidant superoxide dismutase 1 (SOD1). As recent work has suggested a distinct role for hydroxymethylation in epigenetic regulation of gene expression in the brain, the current study investigated age-related changes of 5-hydroxymethylcytosine (5-hmC) in the mouse hippocampus, and furthermore tested whether CR and transgenic upregulation of SOD1 affected any age-related changes in 5-hmC. Immunohistochemical analyses of 5-hmC in 12- and 24-month-old wild-type and transgenic mice overexpressing SOD1, which were kept under either a control or a calorie restricted diet, revealed an increase of 5-hmC immunoreactivity occurring with aging in the hippocampal dentate gyrus, CA3 and CA1–2 regions. Moreover, CR, but not overexpression of SOD1, prevented the age-related increase in the CA3 region. These region-specific findings indicate that the aging process in mice is connected with epigenetic changes and suggest that the beneficial actions of CR may be mediated via epigenetic mechanisms such as methylation and hydroxymethylation of DNA.
PMCID: PMC3561726  PMID: 22272625
Aging; Epigenesis; Epigenetics; DNA hydroxymethylation; 5-hydroxymethylcytosine; Caloric restriction; Antioxidants; superoxide dismutase (SOD); Hippocampus
3.  Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer’s disease patients 
Neurobiology of aging  2013;34(9):2091-2099.
Epigenetic dysregulation of gene expression is thought to be critically involved in the pathophysiology of Alzheimer’s disease (AD). Recent studies indicate that DNA methylation and DNA hydroxymethylation are 2 important epigenetic mechanisms that regulate gene expression in the aging brain. However, very little is known about the levels of markers of DNA methylation and hydroxymethylation in the brains of patients with AD, the cell-type specificity of putative AD-related alterations in these markers, as well as the link between epigenetic alterations and the gross pathology of AD. The present quantitative immunohistochemical study investigated the levels of the 2 most important markers of DNA methylation and hydroxymethylation, that is, 5-methylcytidine (5-mC) and 5-hydroxymethylcytidine (5-hmC), in the hippocampus of AD patients (n = 10) and compared these to non demented, age-matched controls (n = 10). In addition, the levels of 5-hmC in the hippocampus of a pair of monozygotic twins discordant for AD were assessed. The levels of 5-mC and 5-hmC were furthermore analyzed in a cell-type and hippocampal subregion–specific manner, and were correlated with amyloid plaque load and neurofibrillary tangle load. The results showed robust decreases in the hippocampal levels of 5-mC and 5-hmC in AD patients (19.6% and 20.2%, respectively). Similar results were obtained for the twin with AD when compared to the non-demented co-twin. Moreover, levels of 5-mC as well as the levels of 5-hmC showed a significant negative correlation with amyloid plaque load in the hippocampus (rp = −0.539, p = 0.021 for 5-mC and rp = −0.558, p = 0.016 for 5-hmC). These human postmortem results thus strengthen the notion that AD is associated with alterations in DNA methylation and hydroxymethylation, and provide a basis for further epigenetic studies identifying the exact genetic loci with aberrant epigenetic signatures.
doi:10.1016/j.neurobiolaging.2013.02.021
PMCID: PMC3955118  PMID: 23582657
Alzheimer’s disease; Epigenetics; DNA methylation; DNA hydroxymethylation; Amyloid
4.  Molecular consequences of SOD2 expression in epigenetically silenced pancreatic carcinoma cell lines 
British Journal of Cancer  2007;97(8):1116-1123.
Manganese superoxide dismutase (SOD2) is an enzyme that catalyses the dismutation of superoxide in the mitochondria, leading to reduced levels of reactive oxygen species. Reduced expression levels of SOD2 have been shown to result in increased DNA damage and sod2 heterozygous mice have increased incidences of cancer. It has also been shown that SOD2 expression is lost in pancreatic cell lines, with reintroduction of SOD2 resulting in decreased rate of proliferation. The mechanism of decreased SOD2 expression in pancreatic carcinoma has not been previously determined. We demonstrate, through sodium bisulphite sequencing, that the sod2 locus is methylated in some pancreatic cell lines leading to a corresponding decrease in SOD2 expression. Methylation can be reversed by treatment with zebularine, a methyltransferase inhibitor, resulting in restored SOD2 expression. Furthermore, we demonstrate that sensitivity of pancreatic carcinoma cell lines to 2-methoxyestradiol correlates with SOD2 expression and SOD2 modulation can alter the sensitivity of these cells. Using both genomics and proteomics, we also identify molecular consequences of SOD2 expression in MIA-PaCa2 cells, including dephosphorylation of VEGFR2 and the identification of both SOD2-regulated genes and transcription factors with altered binding activity in response to SOD2 expression.
doi:10.1038/sj.bjc.6604000
PMCID: PMC2360443  PMID: 17895890
superoxide dismutase 2; pancreatic neoplasms; proteomics; genomics; epigenetic process
5.  Hippocampal Long-term Potentiation, Memory, and Longevity in Mice that Overexpress Mitochondrial Superoxide Dismutase 
Superoxide has been shown to be critically involved in several pathological manifestations of aging animals. In contrast, superoxide can act as a signaling molecule to modulate signal transduction cascades required for hippocampal synaptic plasticity. Mitochondrial superoxide dismutase (SOD-2 or Mn-SOD) is a key antioxidant enzyme that scavenges superoxide. Thus, SOD-2 may not only prevent aging-related oxidative stress, but may also regulate redox signaling in young animals. We used transgenic mice overexpressing SOD-2 to study the role of mitochondrial superoxide in aging, synaptic plasticity and memory-associated behavior. We found that overexpression of SOD-2 had no obvious effect on synaptic plasticity and memory formation in young mice, and could not rescue the age-related impairments in either synaptic plasticity or memory in old mice. However, SOD-2 overexpression did decrease mitochondrial superoxide in hippocampal neurons, and extended the lifespan of the mice. These findings increase our knowledge of the role of mitochondrial superoxide in physiological and pathological processes in the brain.
doi:10.1016/j.nlm.2006.10.003
PMCID: PMC1847321  PMID: 17129739
reactive oxygen species; antioxidant; learning and memory; synaptic plasticity; aging
6.  Mice Deficient in Both Mn Superoxide Dismutase and Glutathione Peroxidase-1 Have Increased Oxidative Damage and a Greater Incidence of Pathology but No Reduction in Longevity 
To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1−/−, Sod2+/−Gpx1+/−, Sod2+/−Gpx1−/−, and wild-type control mice. Oxidative damage was elevated in Sod2+/−Gpx1−/− mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2+/−Gpx1−/− mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.
doi:10.1093/gerona/glp132
PMCID: PMC2781787  PMID: 19776219
Oxidative stress; Longevity
7.  Nutrient-Dependent Requirement for SOD1 in Lifespan Extension by Protein Restriction in Drosophila melanogaster 
Aging cell  2012;11(5):783-793.
Summary
Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target-of-rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP and low calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high, and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.
doi:10.1111/j.1474-9726.2012.00842.x
PMCID: PMC3444681  PMID: 22672579
8.  Reduced Susceptibility of DNA Methyltransferase 1 Hypomorphic (Dnmt1N/+) Mice to Hepatic Steatosis upon Feeding Liquid Alcohol Diet 
PLoS ONE  2012;7(8):e41949.
Background
Methylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated.
Methodology/Principal Findings
Dnmt1 wild type (Dnmt1+/+) and hypomorphic (Dnmt1N/+) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in Dnmt1+/+ mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, Dnmt1N/+ mice were less susceptible to alcoholic steatosis compared to Dnmt1+/+ mice. Expression of several key genes involved in alcohol (Aldh3b1), lipid (Ppara, Lepr, Vldlr, Agpat9) and xenobiotic (Cyp39a1) metabolism, and oxidative stress (Mt-1, Fmo3) were significantly (P<0.05) altered in Dnmt1N/+ mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of Agpat9, Lepr, Mt1 and Ppara were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice.
Conclusions/Significance
Although feeding alcohol diet reduced Dnmtase activity, the loss of one copy of Dnmt1 protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.
doi:10.1371/journal.pone.0041949
PMCID: PMC3414497  PMID: 22905112
9.  Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector 
BMC Neuroscience  2006;7:35.
Background
Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated.
Results
Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced.
Conclusion
When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.
doi:10.1186/1471-2202-7-35
PMCID: PMC1462999  PMID: 16638118
10.  Radiation-Induced Reductions in Neurogenesis are Ameliorated in Mice Deficient in CuZnSOD or MnSOD 
Free radical biology & medicine  2009;47(10):1459-1467.
Ionizing irradiation significantly affects hippocampal neurogenesis and is associated with cognitive impairments; these effects may be influenced by an altered microenvironment. Oxidative stress is a factor that has been shown to affect neurogenesis, and one of the protective pathways to deal with such stress involves the antioxidant enzyme superoxide dismutase (SOD). This study addressed how the deficiency of cytoplasmic (SOD1) or mitochondrial (SOD2) SOD impacts radiation effects on hippocampal neurogenesis. Wild type (WT), SOD 1 and SOD2 knock out (KO) mice received a single x-ray dose of 5 Gy, and quantification of the survival and phenotypic fate of newly generated cells in the dentate subgranular zone was performed 2 months later. Radiation exposure reduced neurogenesis in WT mice but had no apparent effect in KO mice, although baseline levels of neurogenesis were reduced in both SOD KO strains prior to irradiation. Additionally, there were marked and significant differences between WT and both KO strains in how irradiation affected newly generated astrocytes and activated microglia. The mechanism(s) responsible for these effects are not yet known, but a pilot in vitro study suggests a ‘protective’ effect of elevated levels of superoxide. Overall, these data suggest that under conditions of SOD deficiency, there is a common pathway dictating how neurogenesis is affected by ionizing irradiation.
doi:10.1016/j.freeradbiomed.2009.08.016
PMCID: PMC2767469  PMID: 19703553
Radiation; brain; neurogenesis; oxidative stress; inflammation; SOD
11.  Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia 
Journal of Clinical Investigation  1999;103(7):1055-1066.
Extracellular superoxide dismutase (EC-SOD, or SOD3) is the major extracellular antioxidant enzyme in the lung. To study the biologic role of EC-SOD in hyperoxic-induced pulmonary disease, we created transgenic (Tg) mice that specifically target overexpression of human EC-SOD (hEC-SOD) to alveolar type II and nonciliated bronchial epithelial cells. Mice heterozygous for the hEC-SOD transgene showed threefold higher EC-SOD levels in the lung compared with wild-type (Wt) littermate controls. A significant amount of hEC-SOD was present in the epithelial lining fluid layer. Both Tg and Wt mice were exposed to normobaric hyperoxia (>99% oxygen) for 48, 72, and 84 hours. Mice overexpressing hEC-SOD in the airways attenuated the hyperoxic lung injury response, showed decreased morphologic evidence of lung damage, had reduced numbers of recruited inflammatory cells, and had a reduced lung wet/dry ratio. To evaluate whether reduced numbers of neutrophil infiltration were directly responsible for the tolerance to oxygen toxicity observed in the Tg mice, we made Wt and Tg mice neutropenic using anti-neutrophil antibodies and subsequently exposed them to 72 hours of hyperoxia. Both Wt and Tg neutrophil-depleted (ND) mice have less severe lung injury compared with non-ND animals, thus providing direct evidence that neutrophils recruited to the lung during hyperoxia play a distinct role in the resultant acute lung injury. We conclude that oxidative and inflammatory processes in the extracellular lung compartment contribute to hyperoxic-induced lung damage and that overexpression of hEC-SOD mediates a protective response to hyperoxia, at least in part, by attenuating the neutrophil inflammatory response.
PMCID: PMC408251  PMID: 10194479
12.  Amyloidβ-induced Impairments in Hippocampal Synaptic Plasticity are Rescued by Decreasing Mitochondrial Superoxide 
Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer’s disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid beta peptide (Aβ1–42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by DPI or by genetically deleting gp91phox, the key enzymatic component of NADPH oxidase, had no effect on Aβ-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aβ-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aβ-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aβ-induced impairments in hippocampal synaptic plasticity.
doi:10.1523/JNEUROSCI.6566-10.2011
PMCID: PMC3095121  PMID: 21490199
13.  Overexpression of antioxidant enzymes in diaphragm muscle does not alter contraction-induced fatigue or recovery 
Experimental physiology  2009;95(1):222-231.
Low levels of reactive oxygen species (ROS) production are necessary to optimize muscle force production in unfatigued muscle. In contrast, sustained high levels of ROS production have been linked to impaired muscle force production and contraction-induced skeletal muscle fatigue. Using genetically engineered mice, we tested the hypothesis that the independent transgenic overexpression of catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD; SOD1) or manganese superoxide dismutase (MnSOD; SOD2) antioxidant enzymes would negatively affect force production in unfatigued diaphragm muscle but would delay the development of muscle fatigue and enhance force recovery after fatiguing contractions. Diaphragm muscle from wild-type littermates (WT) and from CAT, SOD1 and SOD2 overexpressing mice were subjected to an in vitro contractile protocol to investigate the force–frequency characteristics, the fatigue properties and the time course of recovery from fatigue. The CAT, SOD1 and SOD2 overexpressors produced less specific force (in N cm−2) at stimulation frequencies of 20–300 Hz and produced lower maximal tetanic force than WT littermates. The relative development of muscle fatigue and recovery from fatigue were not influenced by transgenic overexpression of any antioxidant enzyme. Morphologically, the mean cross-sectional area (in μm2) of diaphragm myofibres expressing myosin heavy chain type IIA was decreased in both CAT and SOD2 transgenic animals, and the percentage of non-contractile tissue increased in diaphragms from all transgenic mice. In conclusion, our results do not support the hypothesis that overexpression of independent antioxidant enzymes protects diaphragm muscle from contraction-induced fatigue or improves recovery from fatigue. Moreover, our data are consistent with the concept that a basal level of ROS is important to optimize muscle force production, since transgenic overexpression of major cellular antioxidants is associated with contractile dysfunction. Finally, the transgenic overexpression of independent endogenous antioxidants alters diaphragm skeletal muscle morphology, and these changes may also contribute to the diminished specific force production observed in these animals.
doi:10.1113/expphysiol.2009.049650
PMCID: PMC2910396  PMID: 19783618
14.  Overexpression of Mn Superoxide Dismutase Does Not Increase Life Span in Mice 
Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4–6 months) and old (26–28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.
doi:10.1093/gerona/glp100
PMCID: PMC2759571  PMID: 19633237
Oxidative damage; Mn superoxide dismutase; Pathology; Aging
15.  Gene Expression in the Hippocampus: Regionally Specific Effects of Aging and Caloric Restriction 
We measured changes in gene expression, induced by aging and caloric restriction (CR), in three hippocampal subregions. When analysis included all regions, aging was associated with expression of genes linked to mitochondrial dysfunction, inflammation, and stress responses, and in some cases, expression was reversed by CR. An age-related increase in ubiquintination was observed, including increased expression of ubiquitin conjugating enzyme genes and cytosolic ubiquitin immunoreactivity. CR decreased cytosolic ubiquitin and upregulated deubiquitinating genes. Region specific analyses indicated that CA1 was more susceptible to aging stress, exhibiting a greater number of altered genes relative to CA3 and the dentate gyrus (DG), and an enrichment of genes related to the immune response and apoptosis. CA3 and the DG were more responsive to CR, exhibiting marked changes in the total number of genes across diet conditions, reversal of age-related changes in p53 signaling, glucocorticoid receptor signaling, and enrichment of genes related to cell survival and neurotrophic signaling. Finally, CR differentially influenced genes for synaptic plasticity in CA1 and CA3. It is concluded that regional disparity in response to aging and CR relates to differences in vulnerability to stressors, the availability of neurotrophic, and cell survival mechanisms, and differences in cell function.
doi:10.1016/j.mad.2010.10.006
PMCID: PMC3032012  PMID: 21055414
aging; diet; hippocampus; transcription; proteosome; ubiquitin
16.  Influence of Viral Vector–Mediated Delivery of Superoxide Dismutase and Catalase to the Hippocampus on Spatial Learning and Memory During Aging 
Antioxidants & Redox Signaling  2012;16(4):339-350.
Abstract
Aims: Studies employing transgenic mice indicate that overexpression of superoxide dismutase 1 (SOD1) improves memory during aging. It is unclear whether the improvement is due to a lifetime of overexpression, decreasing the accumulation of oxidized molecules, or if increasing antioxidant enzymes in older animals could reduce oxidative damage and improve cognitive function. We used adeno-associated virus to deliver antioxidant enzymes (SOD1, SOD2, catalase [CAT], and SOD1+CAT) to the hippocampus of young (4 months) and aged (19 months) F344/BN F1 male rats and examined memory-related behavioral performance 1 month and 4 months postinjection. Results: Overexpression of antioxidant enzymes reduced oxidative damage; however, memory function was not related to the level of oxidative damage. Increased expression of SOD1, initiated in advanced age, impaired learning. Increased expression of SOD1+CAT provided protection from impairments associated with overexpression of SOD1 alone and appears to guard against cognitive impairments in advanced age. Innovation: Viral vector gene delivery provides a novel approach to test the hypothesis that increased expression of antioxidant enzymes, specifically in hippocampal neurons, will provide protection from age-related cognitive decline. Further, expression of multiple vectors permits more detailed investigation of mechanistic pathways. Conclusion: Oxidative stress is a likely component of aging; however, it is unclear whether increased production of reactive oxygen species or the accumulation of oxidative damage is the primary cause of functional decline. The results provide support for the idea that altered redox-sensitive signaling rather than the accumulation of damage may be of greater significance in the emergence of age-related learning and memory deficits. Antioxid. Redox Signal. 16, 339–350.
doi:10.1089/ars.2011.4054
PMCID: PMC3246419  PMID: 21942371
17.  Superoxide dismutase 1 knockdown induces oxidative stress and DNA methylation loss in the prostate 
Increased oxidative stress and concordant DNA methylation changes are found during aging and in many malignant processes including prostate cancer. Increased oxidative stress has been shown to inhibit DNA methyltransferase in in vitro assays, but whether this occurs in vivo is unknown. To generate increased oxidative stress we utilized mice containing mutations in the CuZnSOD (Sod1) gene, a major superoxide dismutase in mammals. Increased 8-hydroxy-2′-deoxyguanosine, an adduct indicating oxidative damage, was found in liver and prostate tissues at 2 and 12 mo Sod1+/− mice compared to controls. prostate tissues from Sod1+/− mice demonstrated decreased weight at 2 mo compared to controls, but this difference was not significant at 12 mo. histologic changes were not seen. Global DNA methylation was significantly decreased at 2 mo in the prostate in Sod1+/− mice. 11p15 containing the epigenetically modulated insulin-like growth factor 2 (Igf2) and H19 genes, both which display oncogenic functions, may be particularly sensitive to oxidative stress. CpG island methylation at an intergenic CTCF binding site and the Igf2 P3 promoter was decreased in Sod1 mutants compared to controls
This is the first in vivo study to show that a deficiency of Sod1 leads to a decrease in DNA methylation. These studies indicate that increased oxidative stress, a factor implicated in neoplasia, can induce DNA hypomethylation in prostate tissues.
PMCID: PMC3825185  PMID: 20458166
prostate cancer; superoxide dismutase 1; DNA methylation; oxidative stress; Sod1; Igf2; H19
18.  Two-stage Case-control Study of DNMT-1 and DNMT-3B Gene Variants and Breast Cancer Risk 
Aberrant DNA methylation of CpG islands is a common epigenetic alteration found in cancers. DNA methylation is typically mediated by DNA methyltransferases (DNMTs). Only two studies have evaluated DNMT-3B and/or DNMT-1 gene polymorphisms in relation to breast cancer risk, and results have been inconsistent. We comprehensively evaluated genetic variations in the DNMT-1 and DNMT-3B genes with breast cancer risk among the participants of the Shanghai Breast Cancer Study (SBCS), a large-scale, two-stage, case-control study. Of the 25 SNPs in the DNMT-1 and DNMT-3B genes analyzed, only one (rs8101866) reached a normal significance level (p=0.042). This association, however, was no longer statistically significant after adjustment for multiple comparisons. Our data suggest that there is no apparent association of common DNMT-1 and DNMT-3B polymorphisms with the risk of breast cancer among Chinese women.
doi:10.1007/s10549-009-0569-9
PMCID: PMC3493111  PMID: 19798569
breast cancer; SNPs; DNMT-1; DNMT-3B; risk
19.  Manganese Superoxide Dismutase Deficiency Exacerbates Ischemic Brain Damage Under Hyperglycemic Conditions by Altering Autophagy 
Translational stroke research  2011;2(1):42-50.
Both preischemic hyperglycemia and suppression of SOD2 activity aggravate ischemic brain damage. This study was undertaken to assess the effect of SOD2 mutation on ischemic brain damage and its relation to the factors involved in autophagy regulation in hyperglycemic wild-type (WT) and heterozygous SOD2 knockout (SOD2−/+) mice subjected to 30-min transient focal ischemia. The brain samples were analyzed at 5 and 24 h after recirculation for ischemic lesion volume, superoxide production, and oxidative DNA damage and protein levels of Beclin 1, damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3). The results revealed a significant increase in infarct volume in hyperglycemic SOD2−/+ mice, and this was accompanied with an early (5 h) significant rise in superoxide production and reduced SOD2 activity in SOD2−/+ mice as compared to WT mice. The superoxide production is associated with oxidative DNA damage as indicated by colocalization of the dihydroethidium (DHE) signal with 8-OHdG fluorescence in SOD2−/+ mice. In addition, while ischemia in WT hyperglycemics increased the levels of autophagy markers Beclin 1, DRAM, and LC3, ischemia in hyperglycemic, SOD2-deficient mice suppressed the levels of autophagy stimulators. These results suggest that SOD2 knockdown exacerbates ischemic brain damage under hyperglycemic conditions via increased oxidative stress and DNA oxidation. Such effect is associated with suppression of autophagy regulators.
PMCID: PMC3124221  PMID: 21720543
Cerebral ischemia; SOD2; Hyperglycemia; Oxidative stress; Autophagy
20.  Caloric restriction experience reprograms stress and orexegenic pathways and promotes binge-eating 
Long-term weight management by dieting has a high failure rate. Pharmacological targets have focused on appetite reduction, while less is understood as to the potential contributions of the stress state during dieting in long-term behavioral modification. In a mouse model of moderate caloric restriction in which a 10–15% weight loss similar to human dieting is produced, we examined physiological and behavioral stress measures. Following three weeks of restriction, mice showed significant increases in immobile time in a tail suspension test and stress-induced corticosterone levels. Increased stress was associated with brain region specific alterations of corticotropin-releasing factor (CRF) expression and promoter methylation, changes that were not normalized with re-feeding. Similar outcomes were produced by high fat diet withdrawal, an additional component of human dieting. In examination of long-term behavioral consequences, previously restricted mice showed a significant increase in binge-eating of a palatable high fat food during stress exposure. Orexegenic hormones, melanin concentrating hormone (MCH) and orexin, were significantly elevated in response to the high fat diet only in previously restricted mice. Further, administration of the MCH receptor-1 antagonist GSK-856464 significantly reduced total caloric intake in these mice during high fat access. These results reveal reprogramming of key central pathways involved in regulating stress responsivity and orexegenic drives by moderate caloric restriction experience. In humans, such changes would be expected to reduce treatment success by promoting behaviors resulting in weight re-gain, and suggest that management of stress during dieting may be beneficial in long-term maintenance.
doi:10.1523/JNEUROSCI.1955-10.2010
PMCID: PMC3034235  PMID: 21123586
dieting; stress; reward; epigenetic
21.  Critical role for CuZn-superoxide dismutase in preventing spontaneous intracerebral hemorrhage during acute and chronic hypertension in mice 
Backgrounds and Purpose
Superoxide is associated with spontaneous intracerebral hemorrhage (ICH) during hypertension. The goal of this study was to test the hypothesis that changes in superoxide, in genetically altered mice with deletion and overexpression of CuZn-superoxide dismutase (SOD1), modulate susceptibility to ICH.
Methods
Chronic hypertension was produced by infusion of angiotensin II (AngII) and an inhibitor of nitric oxide synthase in drinking water in SOD1 transgenic (SOD1Tg) mice, SOD1 deficient (SOD1−/−) mice, and their respective wild-type (WT)-littermates. Acute hypertension was produced by daily injections of AngII in some mice with chronic hypertension to produce ICH. We evaluated susceptibility to ICH, oxidative stress [superoxide, NAD(P)H oxidase activity, SOD activity], gene expression, and activity of matrix metalloproteinases (MMPs).
Results
Incidence, size, and number of ICH were reduced in SOD1Tg mice, and were increased in SOD1−/− mice compared to their WT-littermates. Levels of superoxide increased in the brain even before developing ICH in WT-littermates, while levels of superoxide remained low in SOD1Tg mice. Changes in level of MMP-9 paralleled oxidative stress in SOD1Tg mice and WT-littermates. Moreover, levels of superoxide and MMP-9 were greater in SOD1−/− mice than WT-littermates after induction of ICH. Active MMPs colocalized on cerebral vessels that appeared to lead toward regions with ICH.
Conclusions
These results suggest that superoxide contributes to the pathogenesis of spontaneous ICH, possibly through activation of MMP-9, and that SOD1 protects against spontaneous ICH during hypertension.
doi:10.1161/STROKEAHA.109.569616
PMCID: PMC2847648  PMID: 20150548
Oxidative stress; SOD1; MMP-9; brain hemorrhage
22.  Irradiation Enhances Hippocampus-Dependent Cognition in Mice Deficient in Extracellular Superoxide Dismutase 
Hippocampus  2011;21(1):72-80.
The effects of ionizing irradiation on the brain are associated with oxidative stress. While oxidative stress following irradiation is generally viewed as detrimental for hippocampal function, it might have beneficial effects as part of an adaptive or preconditioning response to a subsequent challenge. Here we show that in contrast to what is seen in wild-type mice, irradiation enhances hippocampus-dependent cognitive measures in mice lacking extracellular superoxide dismutase. These outcomes were associated with genotype-dependent effects on measures of oxidative stress. When cortices and hippocampi were analyzed for nitrotyrosine formation as an index of oxidative stress, the levels were chronically elevated in mice lacking extracellular superoxide dismutase. However, irradiation caused a greater increase in nitrotyrosine levels in wild-type mice than mice lacking extracellular superoxide dismutase. These paradoxical genotype-dependent effects of irradiation on measures of oxidative stress and cognitive function underscore potential beneficial effects associated with chronic oxidative stress if it exists prior to a secondary insult such as irradiation.
doi:10.1002/hipo.20724
PMCID: PMC2891276  PMID: 20020436
23.  Age-Related Synapse Loss In Hippocampal CA3 Is Not Reversed By Caloric Restriction 
Neuroscience  2010;171(2):373-382.
Caloric restriction (CR) is a reduction of total caloric intake without a decrease in micronutrients or a disproportionate reduction of any one dietary component. While CR attenuates age-related cognitive deficits in tasks of hippocampal-dependent memory, the cellular mechanisms by which CR improves this cognitive decline are poorly understood. Previously, we have reported age-related decreases in key synaptic proteins in the CA3 region of the hippocampus that are stabilized by lifelong CR. In the present study, we examined possible age-related changes in the functional microcircuitry of the synapses in the stratum lacunosum-moleculare (SL-M) of the CA3 region of the hippocampus, and whether lifelong CR might prevent these age-related alterations. We used serial electron microscopy to reconstruct and classify SL-M synapses and their postsynaptic spines. We analyzed synapse number and size as well as spine surface area and volume in young (10 mos.) and old (29 mos) ad libitum fed rats and in old rats that were calorically restricted from 4 months of age. We limited our analysis to SL-M because previous work demonstrated age-related decreases in synaptophysin confined to this specific layer and region of the hippocampus. The results revealed an age-related decrease in macular axo-spinous synapses that was not reversed by CR that occurred in the absence of changes in the size of synapses or spines. Thus, the benefits of CR for CA3 function and synaptic plasticity may involve other biological effects including the stabilization of synaptic proteins levels in the face of age-related synapse loss.
doi:10.1016/j.neuroscience.2010.09.022
PMCID: PMC2995889  PMID: 20854882
Dietary Restriction; Electron Microscopy; Serial Reconstruction; Synapses; Hippocampus; Rat
24.  Dnmt1 Overexpression Causes Genomic Hypermethylation, Loss of Imprinting, and Embryonic Lethality 
Molecular and Cellular Biology  2002;22(7):2124-2135.
Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.
doi:10.1128/MCB.22.7.2124-2135.2002
PMCID: PMC133685  PMID: 11884600
25.  Overexpression of human SOD1 improves survival of mice susceptible to endotoxic shock 
Background
Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.
Methods
Transgenic (tg) mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1) tg mice with mixed genetic background (tg-JAX); (2) wild-type (WT) littermates of tg-JAX strain (WT-JAX); (3) tg mice with C57BL/6J background (tg-TX); (4) WT littermates of tg-TX strain (WT-TX). Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval) were analyzed using Kaplan–Meier survival curves.
Results
Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX) compared with their WT littermates (WT-JAX and WT-TX, respectively). Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX), but such aberrant changes were not observed in tg-TX mice. There was no difference in the survival time between tg-JAX and WT-JAX groups after endotoxic shock (P > 0.05). However, the survival time in the tg-TX group was more than twofold longer than that in the WT-TX group (P < 0.05). In addition, WT-JAX mice survived significantly longer than WT-TX mice (P < 0.05).
Conclusion
Aberrant decrease of endogenous SOD1 activities may have overshadowed the effect of overexpression of SOD1 in tg mice (tg-JAX). Mice with C57BL/6J background (tg-TX) are more susceptible to lipopolysaccharide-induced endotoxic shock than those with mixed genetic background (tg-JAX). Overexpression of SOD1 is protective only in mice with C57BL/6J background (tg-TX).
doi:10.2147/JIR.S32073
PMCID: PMC3413208  PMID: 22879777
human SOD1 enzyme; endotoxic shock; transgenic mice; protective effect

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