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.
Aging; Epigenesis; Epigenetics; DNA hydroxymethylation; 5-hydroxymethylcytosine; Caloric restriction; Antioxidants; superoxide dismutase (SOD); Hippocampus
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.
reactive oxygen species; antioxidant; learning and memory; synaptic plasticity; aging
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.
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.
Radiation; brain; neurogenesis; oxidative stress; inflammation; SOD
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.
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.
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.
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.
superoxide dismutase 2; pancreatic neoplasms; proteomics; genomics; epigenetic process
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.
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.
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).
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).
human SOD1 enzyme; endotoxic shock; transgenic mice; protective effect
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.
Oxidative stress; Longevity
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.
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.
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.
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.
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.
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.
aging; diet; hippocampus; transcription; proteosome; ubiquitin
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.
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.
DNA methyltransferases (DNMTs) play an important role in establishing and maintaining DNA methylation. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer, and their contribution to aberrant DNA methylation has been proposed. Here, we generated HEK 293T cells stably transfected with each of 13 different DNMTs (DNMT1, two DNMT3A isoforms, nine DNMT3B isoforms and DNMT3L) and assessed the DNA methylation changes induced by each DNMT. We obtained DNA methylation profiles of DNA repetitive elements and 1505 CpG sites from 808 cancer-related genes. We found that DNMTs have specific and overlapping target sites and their DNA methylation target profiles are a reflection of the DNMT domains. By examining H3K4me3 and H3K27me3 modifications in the 808 gene promoter regions using promoter ChIP-on-chip analysis, we found that specific de novo DNA methylation target sites of DNMT3A1 are associated with H3K4me3 modification that are transcriptionally active, whereas the specific target sites of DNMT3B1 are associated with H3K27me3 modification that are transcriptionally inactive. Our data suggest that different DNMT domains are responsible for targeting DNA methylation to specific regions of the genome, and this targeting might be associated with histone modifications.
Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the C-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic are bred with Eμ-Myc transgenic mice, which model aggressive B cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared to Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the re-distribution of DNA methylation characterizing virtually every human tumor.
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.
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).
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.
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.
Oxidative stress; SOD1; MMP-9; brain hemorrhage
Radiation therapy of the CNS, even at low doses, can lead to deficits in neurocognitive functions. Reduction in hippocampal neurogenesis is usually, but not always, associated with cognitive deficits resulting from radiation therapy. Generation of reactive oxygen species is considered the main cause of radiation-induced tissue injuries, and elevated levels of oxidative stress persist long after the initial cranial irradiation. Consequently, mutant mice with reduced levels of the mitochondrial antioxidant enzyme, Mn superoxide dismutase (MnSOD or Sod2), are expected to be more sensitive to radiation-induced changes in hippocampal neurogenesis and the related functions. In this study, we showed that MnSOD deficiency led to reduced generation of immature neurons in Sod2−/+ mice even though progenitor cell proliferation was not affected. Compared to irradiated Sod2+/+ mice, which showed cognitive defects and reduced differentiation of newborn cells towards the neuronal lineage, irradiated Sod2−/+ mice showed normal hippocampal-dependent cognitive functions and normal differentiation pattern for newborn neurons and astroglia. However, we also observed a disproportional decrease in newborn neurons in irradiated Sod2−/+ following behavioral studies, suggesting that MnSOD deficiency may render newborn neurons more sensitive to stress from behavioral trainings following cranial irradiation. A positive correlation between normal cognitive functions and normal dendritic spine densities in dentate granule cells was observed. The data suggest that maintenance of synaptic connections, via maintenance of dendritic spines, may be important for normal cognitive functions following cranial irradiation.
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.
Dietary Restriction; Electron Microscopy; Serial Reconstruction; Synapses; Hippocampus; Rat
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.
breast cancer; SNPs; DNMT-1; DNMT-3B; risk
DNA methylation plays an important role in carcinogenesis through epigenetic silencing of tumor suppressor genes. Aberrant methylation usually results from changes in the activity of DNA methyltransferases (DNMTs). Some studies show that the overexpression of the DNMTs may lead to aberrant methylation of tumor suppressor genes. Also the overexpression of DNMTs may be related to methylation status of their genes. Due to limited number of studies on DNMT3B promoter methylation, this study was performed to quantitatively measure the methylation level of DNMT3B gene in archival formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Using differential high resolution melting analysis (D-HRMA) technology, the methylation level of DNMT3B gene promoter was quantified in 98 breast cancer FFPE tissues and also 10 fresh frozen normal tissue samples. Statistical analyses used for analyzing the correlation between the methylation and clinical variables. All the normal samples were found to be methylated at the DNMT3B promoter (the average methylation level 3.34%). Patients were identified as hypo-methylated (mean methylation level 0.8%), methylated (mean methylation level 2.48%) and hyper-methylated (mean methylation level 10.5%). Statistical analysis showed a significant correlation between the methylation status and the sample type, cancer type and tumor size. Also the methylation level was significantly associated with histologic grade. It is concluded that quantification of DNMT3B promoter methylation might be used as a reliable and sensitive diagnostic and prognostic tool in breast cancer. Also D-HRMA is demonstrated as a rapid and cost effective method for quantitative evaluation of promoter methylation.
DNA methylation; DNMT3B; Breast cancer; High resolution melting analysis; FFPE
Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady state levels of key antioxidant enzymes with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing pro-apoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD +/− mice. The effect of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development was determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in pre-malignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.
MnSOD; ROS; Apoptosis; Bcl-2; Oncogenesis; Chromosomal Instability
Mice deficient in superoxide dismutase 1 (Sod1−/− mice) develop many features seen in patients with age-related macular degeneration (AMD) including choroidal neovascularization (NV). We sought to determine if the absence of SOD1 contributes to the pro-angiogenic environment in the subretinal space or whether it is completely secondary to other changes in Bruch’s membrane and the retinal pigmented epithelium (RPE) that precede the development of choroidal NV. Expression of vascular endothelial growth factor (VEGF) in photoreceptors or ischemia resulted in significantly more NV in Sod1−/− compared to Sod1+/+ mice. The compromised antioxidant defense system in Sod1−/− mice contributes to the pro-angiogenic environment, because treatment of Sod1−/− mice with a mixture of antioxidants caused a significant reduction in ischemia-induced retinal NV. Wild type mice treated with the same antioxidants also showed reduced ischemia-induced retinal NV, reduced VEGF-induced subretinal NV, and reduced choroidal NV at Bruch’s membrane rupture sites. These data suggest that reactive oxygen species contribute to several types of ocular NV. This could explain why in the Age-Related Eye Disease Trial, antioxidant treatment reduced conversion from non-neovascular to neovascular AMD and severe vision loss, and suggest that potent antioxidants should be considered for other diseases complicated by ocular NV.
DNA methyltransferases (DNMTs) are key regulators of DNA methylation and have crucial roles in carcinogenesis, embryogenesis and epigenetic modification. In general, DNMT1 has enzymatic activity affecting maintenance of DNA methylation, whereas DNMT3A and DNMT3B are involved in de novo methylation events. Although DNMT genes are well known in mammals including humans and mice, they are not well studied in avian species, especially the laying hen which is recognized as an excellent animal model for research on human ovarian carcinogenesis. Results of the present study demonstrated that expression of DNMT1, DNMT3A and DNMT3B genes was significantly increased, particularly in the glandular epithelia (GE) of cancerous ovaries, but not normal ovaries. Consistent with this result, immunoreactive 5-methylcytosine protein was predominantly abundant in nuclei of stromal and GE cells of cancerous ovaries, but it was also found that, to a lesser extent, in nuclei of stromal cells of normal ovaries. Methylation-specific PCR analysis detected hypermethylation of the promoter regions of the tumor suppressor genes in the initiation and development of chicken ovarian cancer. Further, several microRNAs, specifically miR-1741, miR-16c, and miR-222, and miR-1632 were discovered to influence expression of DNMT3A and DNMT3B, respectively, via their 3′-UTR which suggests post-transcriptional regulation of their expression in laying hens. Collectively, results of the present study demonstrated increased expression of DNMT genes in cancerous ovaries of laying hens and post-transcriptional regulation of those genes by specific microRNAs, as well as control of hypermethylation of the promoters of tumor suppressor genes.
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.
Oxidative damage; Mn superoxide dismutase; Pathology; Aging