Mitochondrial Mn-superoxide Dismutase Research Articles

PKCε activator protects hippocampal microvascular disruption and memory defect in 3×Tg-Alzheimer's disease mice with cerebral microinfarcts.

Current evidence suggests that microvessel disease is involved in Alzheimer's disease (AD). Cerebrovascular disease correlates with cardiovascular disease and is complicated in ≈40% of AD patients. The protein kinase C (PKC) ε activator DCPLA can stimulate human antigen (Hu) R that prevents degradation and promotes the translation of mitochondrial Mn-superoxide dismutase (MnSOD) and vascular endothelial growth factor-A (VEGF) mRNAs. To induce brain microinfarcts, we injected triple transgenic (3×Tg) and wild-type (WT) control mice with microbeads (20 μm caliber) into common carotid arteries, with or without the DCPLA-ME (methyl-ester) for 2 weeks. After water maze training, mice at 16 months old were examined for confocal immunohistochemistry at a single cell or microvessel level in the hippocampal CA1 area, important for spatial memory storage, and in the dorsal hippocampus by western blots. In 3×Tg mice without cerebral microinfarcts, an accelerating age-related increase in (mild) oxidative stress and hypoxia inducible factor (HIF)-1α, but a reduction in VEGF, mitochondrial transcription factor A (TFAM), and MnSOD were associated with capillary loss. The change was less pronounced in arterioles. However, in 3×Tg mice with cerebral microinfarcts, increasing arteriolar diameter and their wall cells were related with the strong oxidative DNA damage 8-hydroxy-2'-deoxyguanosine (8-OHdG), apoptosis (cleaved caspase 3), and sustained hypoxia (increased HIF-1α and VEGF/PKCε/extracellular signal regulated kinase or ERK pathway). Microocclusion enhanced the loss of the synaptic marker spinophilin, astrocytic number, and astrocyte-vascular coupling areas and demyelination of axons. DCPLA-ME prevented spatial memory defect; strong oxidative stress-related apoptosis; sustained hypoxia (by reducing HIF-1α and VEGF); and exaggerated cell repair in arteriolar walls, pericapillary space dilation, neuro-glial-vascular disruption, and demyelination. In conclusion, in 3×Tg mice with cerebral microinfarcts, sustained hypoxia (increased HIF-1α and VEGF signals) is dominant with arteriolar wall thickening, and DCPLA has a protective effect on sustained hypoxia.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

Hippocampus mitochondrial MnSOD activation by a SIRT3 activator, honokiol, correlates with its deacetylation and upregulation of FoxO3a and PGC1α in a rat model of ammonia neurotoxicity.

We have recently reported that honokiol (HKL), by activating mitochondrial SIRT3, normalizes reactive oxygen species level and mitochondrial integrity in hippocampus of the moderate grade hepatic encephalopathy (MoHE) rat model of ammonia neurotoxicity. To delineate the mechanism by which HKL does so, the present study describes activity versus level of the deacetylated mitochondrial Mn-superoxide dismutase (MnSOD) and expression of MnSOD versus levels of its main transcription regulators, FoxO3a and PGC1α, in the hippocampus of the MoHE rats. MoHE in rat was developed by administration of 100 mg/kg bw thioacetamide i.p. for 10 days. The study parameters were compared between the control, the MoHE rats and the MoHE rats treated with HKL (10 mg/Kg b.w.) for 7 days. As compared to control, the hippocampus mitochondria from MoHE rats showed a significantly declined activity of MnSOD vs enhanced lipid peroxidation coinciding with the increased level of its acetylated form. The HKL treatment could, however, normalize all these parameters in those MoHE rats. Also, a significantly reduced expression of MnSOD in the hippocampus of the MoHE rats coincided with a similar decline in transcript level of Foxo3a and Pgc1α. This was consistent with the reduced level of immuno-stained Foxo3a and Pgc1α proteins in hippocampus DG, CA1 and CA3 regions of those MoHE rats. However, all these factors were observed to be restored back to their normal levels due to the treatment with HKL. As HKL is a specific activator of mitochondrial SIRT3, these findings suggest involvement of Sirt3 activation led deacetylation of MnSOD and upregulation of its transcription activators, FoxO3a and PGC1α, in restoring mitochondrial MnSOD level in the hippocampus of the MoHE rat model of ammonia neurotoxicity.

Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis.

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.

Iron robbery by intracellular pathogen via bacterial effector–induced ferritinophagy

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3-induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants.

Effect of UV-B radiation on morphology, phenolic compound production, gene expression, and subsequent drought stress responses in chili pepper (Capsicum annuum L.)

It has been suggested that accumulation of flavonoids could be a key step in development of plant tolerance to different environmental stresses. Moreover, it has been recognized that abiotic stresses such as drought and UV-B radiation (280–315 nm) induce phenolic compound accumulation, suggesting a role for these compounds in drought tolerance. The aim of the present study was to evaluate the effect of UV-B exposure on chili pepper (Capsicum annuum, cv. ‘Coronel’) plant performance, phenolic compound production, and gene expression associated with response to subsequent drought stress. Additionally, the phenotypic response to drought stress of these plants was studied. UV-B induced a reduction both in stem length, stem dry weight and number of floral primordia. The largest reduction in these variables was observed when combining UV-B and drought. UV-B-treated well-watered plants displayed fructification approximately 1 week earlier than non-UV-B-treated controls. Flavonoids measured epidermally in leaves significantly increased during UV-B treatment. Specifically, UV-B radiation significantly increased chlorogenic acid and apigenin 8-C-hexoside levels in leaves and a synergistic increase of luteolin 6-C-pentoside-8-C-hexoside was obtained by UV-B and subsequent drought stress. Gene expression of phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes also increased during UV-B treatments. On the other hand, expression of genes related to an oxidative response, such as mitochondrial Mn-superoxide dismutase (Mn-SOD) and peroxidase (POD) was not induced by UV-B. Drought stress in UV-B-treated plants induced mitochondrial Mn-SOD gene expression. Taken together, the UV-B treatment did not induce significant tolerance in plants towards drought stress under the conditions used.

Analysis of the acute response of Galleria mellonella larvae to potassium nitrate

Potassium nitrate (E252) is widely used as a food preservative and has applications in the treatment of high blood pressure however high doses are carcinogenic. Larvae of Galleria mellonella were administered potassium nitrate to establish whether the acute effects in larvae correlated with those evident in mammals. Intra-haemocoel injection of potassium nitrate resulted in a significant increase in the density of circulating haemocytes and a small change in the relative proportions of haemocytes but haemocytes showed a reduced fungicidal ability. Potassium nitrate administration resulted in increased superoxide dismutase activity and in the abundance of a range of proteins associated with mitochondrial function (e.g. mitochondrial aldehyde dehydrogenase, putative mitochondrial Mn superoxide dismutase), metabolism (e.g. triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase) and nitrate metabolism (e.g. aliphatic nitrilase, glutathione S-transferase). A strong correlation exists between the toxicity of a range of food preservatives when tested in G. mellonella larvae and rats. In this work a correlation between the effect of potassium nitrate in larvae and mammals is shown and opens the way to the utilization of insects for studying the in vivo acute and chronic toxicity of xenobiotics.

The stilbenes resveratrol, pterostilbene and piceid affect growth and stress resistance in mammalian cells via a mechanism requiring estrogen receptor beta and the induction of Mn-superoxide dismutase

The mitochondrial antioxidant enzyme, Mn superoxide dismutase (MnSOD), has been shown to confer cytoprotection and to regulate cell cycle progression. Resveratrol, a phytoestrogen found in red wines and other foods, has been previously reported to increase MnSOD protein levels and activity both in vitro and in vivo. Numerous structural analogues of resveratrol produced via the same stilbene synthesis pathway (e.g. pterostilbene and piceid) and also present in foods and red wine may be capable of eliciting the same effects. Furthermore, in humans resveratrol is rapidly metabolized to resveratrol-4′-sulfate, resveratrol-3-glucuronide and other metabolites in vivo. Although these metabolites may accumulate to relatively high levels in plasma and tissues, little is known about their biological activities. Here the activities were compared of these stilbenes and stilbene metabolites in mammalian cells. Two key cellular activities associated with resveratrol were examined: inhibition of proliferative growth and increased stress resistance (important anti-cancer and cell protective activities, respectively). While resveratrol-4′-sulfate and resveratrol-3-glucuronide had no effect on either cell growth or stress resistance, both pterostilbene and piceid were at least as effective as resveratrol. Using pharmacological and genetic approaches, it was found that the effects of pterostilbene and piceid required an induction of the mitochondrial enzyme MnSOD and intact mitochondrial respiration. In addition, using estrogen receptor beta (ERbeta) knockout mouse myoblasts, it was demonstrated that the effects of stilbene compounds on cell growth and stress resistance all require ERbeta. Taken together, these results indicate that resveratrol, pterostilbene and piceid all activate the same mitochondrial response in mammalian cells, and therefore these latter two molecules might be as effective as resveratrol in eliciting positive health outcomes in vivo.

Oiwa, a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase, Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Val-9Ala and Ile + 58Thr polymorphism of MnSOD in Parkinson's disease

Objectives To investigate the polymorphism distribution of Val-9Ala and Ile + 58Thr of the Mn-superoxide dismutase (Mn-SOD) gene among subjects with Parkinson's disease (PD) by analyses of genders and clinical severity. Design and Methods We examined the DNA genotypes of Val-9Ala and Ile + 58Thr from 295 PD subjects and 111 controls by nucleotide sequencing and BsaWI restriction. Results Ala/Ala homozygosity was found in four PD subjects but not in the controls. All of the genotypes at codon + 58 among the examined samples were Ile/Ile homozygotes. Although higher carrier rate of Ala allele among PD subjects than the controls, there were no differences by analyses of the genders and clinical severity. Conclusion The higher Ala-allele carrier rate among PD subjects may suggest a possible higher amount of mitochondrial Mn-SOD rendering higher intracellular stress in PD. In this study the polymorphisms at codons -9 and + 58 did not give informative association evidences with PD.

Evaluation of abiotic stress tolerance of genetically modified potatoes (Solanum tuberosum cv. Desiree)

Abiotic stresses such as drought and extremes of temperature commonly reduce both yield and quality of potato. This study investigated the potential to use gene transfer technology to enhance the tolerance of potato to commonly encountered abiotic stresses. Agrobacterium mediated transformation was used to create lines of potato (cv. Desiree) that over-expressed either a wheat mitochondrial Mn superoxide dismutase (SOD3:1), dehydrin 4 (DHN 4) isolated from barley, a cold-inducible transcriptional factor DREB/CBF1 isolated from canola or ROB5, a stress inducible gene isolated from bromegrass that encodes for a heat stable LEA group 3-like protein. The transgenes were under the control of either a constitutive 35S promoter or a stress-induced Arabidopsis COR78 promoter. Yield potential of the transformed lines was evaluated under drought stress conditions in a greenhouse trial and under non-irrigated conditions in field trials conducted over 4 years in Saskatoon, Saskatchewan. In the years when the field trials experienced significant drought stress (2001, 2003 and 2006) many of the transformed lines produced higher yields than the control. However, under relatively cooler, wetter conditions (2005 cropping season) yields of most transformed lines were equivalent or inferior to the non-transformed parental line. Under non-stressed conditions, transformations utilizing the stress-induced COR78 promoter were higher yielding than transformations based on the constitutive 35S promoter. Combining the ROB5, DHN or SOD3.1 transgenes with the COR78 promoter all showed significant potential to enhance yields under moisture stress. All of the transgenes appeared to enhance the heat stress tolerance (44°C) of whole plants or excised leaves, with lines transformed with SOD3.1 showing the greatest effect. In low temperature stress trials conducted under controlled environment conditions and in the field, lines over-expressing SOD3:1 showed an enhanced capacity to grow at sub-optimal temperatures (10°C), while lines transformed with SOD3.1 or ROB5 had greater tolerance of freezing temperatures than the parental line. These results are encouraging as even a small degree of enhancement of stress tolerance has the potential to produce significant economic benefits in high value/stress sensitive crops such as potato.

Superoxide dismutase: an all-purpose gene for agri-biotechnology

The objective of this study was to evaluate the performance of transgenic canola (Brassica napus) plants over-expressing a wheat mitochondrial Mn superoxide dismutase (Mn SOD3.1) subjected to environmental stresses in the field and in controlled environments. Mn SOD3.1 was regulated by either CaMV 35S or Arabidopsis COR78 promoters. RT-PCR and a SOD enzyme activity assays demonstrated Mn SOD3.1 was expressed at both the mRNA and protein levels. Enzyme activity assays exhibited total SOD activity was up to 41.8% and up to 26.7% higher in the 35S:SOD3.1 and in the COR78:SOD3.1 transgenic plants than in the control, respectively. Germination studies, conducted at suboptimal (8°C) and optimal (23°C) temperatures, identified transgenic lines that germinated earlier than the control. In the field under drought conditions, several transgenic lines emerged earlier than the control. In both greenhouse and field environments, several transgenic lines were significantly taller than the control and over 50% of the transgenic canola lines flowered 7–14 days earlier than the control. Over expression of Mn SOD3.1 enhanced heat, drought and cold tolerance both in the field and under artificial stress conditions. This is one of the first tests conducted on transgenic canola plants subjected to field conditions.

Antioxidant enzymatic activities after experimental subarachnoid hemorrhage in rats.

Lipid peroxidation has been hypotesized as one of possible factors involved in the pathogenesis of neuronal damage and delayed vasospasm after subarachnoid hemorrhage. In the brain there are anti-oxidant enzymatic systems which act as scavengers of superoxides and free radicals. In the present study the pattern of enzymatic anti-oxidant activities (Cu-Zn and Mn superoxide dismutase, and glutathione peroxidase) was investigated in an experimental model of subarachnoid hemorrhage in the rat in order to verify whether the hemorrhagic insult may be responsible for an impairment of such anti-oxidant systems. Enzymatic activities were assayed in three different rat brain areas (cerebral cortex, hippocampus and brain stem) of sham-operated and at 30 min, 1, 6 and 48 h after subarachnoid hemorrhage induction. After the hemorrhage induction the Cu-Zn superoxide dismutase activity in cerebral cortex was significantly reduced at all the set times (p < .05), while Mn-superoxide dismutase activity was significantly decreased since 1 h (p < .05) until 48 h (p < .05). Glutathione peroxidase activity was significantly reduced only in the late phase (48 h) of subarachnoid hemorrhage (p < .01). In the hippocampus, all enzymatic activities were significantly reduced in the late phase. In the brain stem Cu-Zn superoxide dismutase was significantly impaired at 1 and 6 h (p < .05) after subarachnoid hemorrhage induction, while in the late phase (48 h) reached the control value. The mitochondrial Mn-superoxide dismutase was significantly reduced since 1 h (p < .05) until 48 h (p < .02) after subarachnoid hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal dexamethasone exposure in the common marmoset monkey enhances gene expression of antioxidant enzymes in the aorta of adult offspring

Human epidemiological studies have indicated that low birth weight associated with an adverse intrauterine environment is related to a greater incidence of cardiovascular disorders in later life. In the foetus, endogenous glucocorticoids generally increase if there is intrauterine nutrient deficiency. The consequent glucocorticoid hyperexposure has been hypothesised to cause in utero programming of atherogenic genes. We investigated the effect of oral treatment with the synthetic glucocorticoid dexamethasone during early or late pregnancy in marmoset monkeys on oxidative and antioxidant status in the offspring. Urinary concentrations of F2-isoprostanes were quantified as markers for in vivo oxidative stress. Expression of the mRNAs for the antioxidant enzymes cytosolic glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (GPx-4), cytosolic Cu,Zn-superoxide dismutase (SOD1), mitochondrial Mn-superoxide dismutase (SOD2), glutathione reductase (GSR), modifier subunit of glutamate-cysteine ligase (GCLM) and catalase were determined in the aorta. Three groups of pregnant marmosets (10 animals per group) were treated orally for one week with vehicle, or with dexamethasone (5 mg/kg daily) during two gestation windows: early dexamethasone group, pregnancy day 42–48, and late dexamethasone group, pregnancy day 90–96. In one male sibling of each litter (10 males per group), aortas were taken at 2 years of age. In the late dexamethasone group a higher aortic mRNA expression for GPx-1 (p < 0.023), MnSOD (p < 0.016), GCLM (p < 0.019) and GSR (p < 0.014) in comparison to the controls was observed. Aortic expression in the early dexamethasone group was statistically significantly higher only for GSR mRNA (p < 0.038). No significant changes in urinary F2-isoprostane concentrations between controls, early and late dexamethasone groups at 2 years of age were observed. Hence, prenatal exposure to dexamethasone in the third trimester leads to increased mRNA expression of several aortic antioxidant enzymes in the offspring. This expression pattern was not temporally related to oxidative stress, and it may reflect in utero re-programming of aortic antioxidant gene expression during prenatal glucocorticoid exposure.

Kainate-induced mitochondrial oxidative stress contributes to hippocampal degeneration in senescence-accelerated mice

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.

Role of glutathione peroxidase in the ontogeny of hippocampal oxidative stress and kainate seizure sensitivity in the genetically epilepsy-prone rats

Oxidative stress may contribute to epileptogenicity in genetic models of epilepsy. To address this, we examined the enzymatic activity of cytosolic Cu/Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), and glutathione peroxidase (GPx) in the developing hippocampus of genetically epilepsy-prone rats (GEPR-9s). We also measured changes in the GSH/GSSG ratio, lipid peroxidation, and protein oxidation at post-natal days (PD) 7, 30, and 90, respectively. Compared with control Sprague-Dawley (SD) rats, GEPR-9s showed similar SOD-1 and SOD-2 activity but lower GPx activity. Epilepsy-prone rats also showed lower GSH/GSSG ratios than controls, and more lipid peroxidation (as measured by malondialdehyde levels) and protein oxidation (as measured by carbonyl levels). Treatment with kainic acid (KA) resulted in more pronounced seizures, less GPx activity, and lower GSH/GSSG ratios in GEPR-9s than in controls, but KA did not significantly affect SOD-1 or SOD-2 activity, suggesting that GEPR-9s do not compensate for reduced GPx activity by increasing SOD. Moreover, KA treatment resulted in significantly a lower GSH/GSSG ratio and GPx-like immunoreactivity and higher malondialdehyde and carbonyl levels in GEPR-9s than in controls. These findings were more evident in GEPR-9s at PD 90 than at PD 30, indicating that oxidative stress is age-dependent. Double-labeling immunocytochemical analysis demonstrated co-localization of GPx-immunoreactive glia-like cells and reactive astrocytes, as labeled by glial fibrillary acidic protein (GFAP). This suggests that mobilization of astroglial cells for synthesis of GPx protein is a response to KA insult, intended to decrease the neurotoxicity induced by peroxides. These responses were more pronounced in control SD rats than in GEPR-9s. Our results suggest that impairment of the GPx (including glutathione)-mediated antioxidant system contributed to epileptogenesis in GEPR-9s.

NO‐induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn‐SOD activity and expression via RAS/ERK1/2 pathway

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn-superoxide dismutase (Mn-SOD) expression and activity. In addition, the possible involvement of Ras/extracellular-regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30-min sublethal OGD (95% N(2) and 5% CO(2)). Then, after a 24-h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn-SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L-NAME, a well known NOS inhibitor, the increase in Mn-SOD expression occurring during IPC was reduced and, as a result, IPC-induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn-SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn-SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn-SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post-transcriptionally activates Mn-SOD expression and activity, thus promoting neuroprotection during preconditioning.

Inadequate Cytoplasmic Antioxidant Enzymes Response Contributes to the Oxidative Stress in Human Hypertension

Untreated hypertensive patients show increased oxidative stress and decreased antioxidant enzyme activity in mononuclear cells. Therefore, the objective of this study was to determine whether or not the low antioxidant enzyme activity observed in mononuclear cells of hypertensive subjects is in part dependent on a defective activity of antioxidant mechanisms. Activity and mRNA level of antioxidant enzymes, CuZn- and Mn-superoxide dismutases, catalase, glutathione peroxidase type 1, and glutathione reductase were simultaneously measured in mononuclear cells of controls (n = 38) and hypertensive subjects (n = 35), in the absence of and during antihypertensive treatment. An increase in oxidative stress and a decrease in the activity of cytoplasmic enzymes were observed in untreated hypertensive patients. Concurrently, CuZn-superoxide dismutase and glutathione reductase mRNA levels were significantly reduced, and glutathione peroxidase type 1 mRNA was slightly reduced. In contrast, increased activity and mRNA levels of the mitochondrial Mn-superoxide dismutase were observed. Antihypertensive treatment, nonpharmacologic with or without a drug regimen of beta-blocker or angiotensin AT1 receptor blocker was administered for a 3-month period. Afterward, after the improvement in oxidative stress during treatment, a recovery of the cytoplasmic antioxidant enzymatic activity and a more profound decrease in mRNA levels were observed for CuZn-superoxide dismutase, glutathione peroxidase type 1, and glutathione reductase. Meanwhile mitochondrial enzymatic activity decreased, as did the mRNA level. The inadequate response of the main cytoplasmatic antioxidant systems, as well as of the enzymes participating in the maintenance of glutathione levels, may contribute to the vulnerability of hypertensives to oxidative stress.