Copper-zinc Superoxide Dismutase Research Articles

Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases.

Greater than 160 missense mutations in copper-zinc superoxide dismutase-1 (SOD1) can cause amyotrophic lateral sclerosis (ALS). These mutations produce conformational changes that reveal novel antibody binding epitopes. A monoclonal antibody, clone C4F6 - raised against the ALS variant G93A of SOD1, has been identified as specifically recognizing a conformation shared by many ALS mutants of SOD1. Attempts to determine whether non-mutant SOD1 adopts a C4F6-reactive conformation in spinal tissues of sporadic ALS (sALS) patients has produced inconsistent results. To define the epitope recognized by C4F6, we tested its binding to a panel of recombinant ALS-SOD1 proteins expressed in cultured cells, producing data to suggest that the C4F6 epitope minimally contains amino acids 90–93, which are normally folded into a tight hairpin loop. Multiple van der Waals interactions between the 90–93 loop and a loop formed by amino acids 37–42, particularly a leucine at position 38, form a stable structure termed the β-plug. Based on published modeling predictions, we suggest that the binding of C4F6 to multiple ALS mutants of SOD1 occurs when the local structure within the β-plug, including the loop at 90–93, is destabilized. In using the antibody to stain tissues from transgenic mice or humans, the specificity of the antibody for ALS mutant SOD1 was influenced by antigen retrieval protocols. Using conditions that showed the best discrimination between normal and misfolded mutant SOD1 in cell and mouse models, we could find no obvious difference in C4F6 reactivity to spinal motor neurons between sALS and controls tissues.Electronic supplementary materialThe online version of this article (doi: 10.1186/2051-5960-2-55) contains supplementary material, which is available to authorized users.

Solid-state NMR studies of metal-free SOD1 fibrillar structures

Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90%) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central β-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

Blood Volatile Organic Compounds as Potential Biomarkers for Amyotrophic Lateral Sclerosis: an Animal Study in the SOD1 G93A Mouse

Amyotrophic lateral sclerosis (ALS) is a rapid progressive motor neuron disease. Currently, there are no specific or reliable biomarkers for the diagnosis of this disease, and there are no effective medical treatments. The early diagnosis and treatment of this disease has the potential to prolong the survival of ALS patients, but typically, approximately 1 year passes between the onset of symptoms and the diagnosis of this disease. Therefore, there is an urgent need to find specific biomarkers to enable early diagnosis and therapeutic intervention in this disease. Analyzing the volatile organic compounds (VOCs) present in the blood and exhaled breath is a useful and convenient approach for investigating potential biomarkers. In this study, we examined the VOCs present in blood samples from copper zinc superoxide dismutase 1 (SOD1) glycine to alanine mutation at position 93 (G93A) mice to determine whether a specific biomarker pattern exists in these transgenic mice. Blood samples from ALS mice and their age-matched littermates were analyzed using gas chromatography-mass spectrometry. A total of 12 independent compounds associated with oxidative stress were identified at the early stage of disease. The data show that there is a specific pattern of blood VOCs in ALS mice that could potentially be used as biomarkers that could improve the diagnosis of this disease. Furthermore, these compounds could also potentially be used to monitor the response to neuroprotective agents and to help us better understand the underlying mechanisms of ALS.

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni(2+)- and other metal-ion-substituted wild-type copper-zinc superoxide dismutases.

The dimeric Cu-Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu2Co2SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co(2+), Ni(2+) is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu2Ni2SOD1 and acting as a very good mimic of the native Cu ion in Ni2Zn2SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini's paramagnetic NMR pursuits of metalloproteins. We report Ni(2+) binding to apo wild-type SOD1 and a time-dependent Ni(2+) migration from the Zn site to the Cu site, and the preparation and characterization of Ni2Ni2SOD1, which shows coordination properties similar to those of Cu2Cu2SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.

The crystal structure of SOD5: an unusual copper‐only superoxide dismutase (774.2)

Superoxide dismutases (SODs) are antioxidant enzymes that catalyze the dismutation of superoxide into molecular oxygen and hydrogen peroxide. Candida albicans is a polymorphic fungus that causes the majority of fungal infections in humans. In the immunocompetent population, colonization of C. albicans can result in acute infections of the mucosal surfaces both in the oral and genital cavities, however, immunocompromised individuals encounter life‐threatening systemic infections. Several factors contribute to the pathogenesis of C. albicans including the expression of SOD5; a postulated copper‐zinc SOD. SOD5 enables C. albicans to evade the immune response of the host, namely the oxidative burst. In the absence of SOD5, reactive oxygen species (ROS) accumulate and C. albicans exhibit a severe loss in viability. Our studies sought to examine the crystal structure of SOD5. The protein was overexpressed in E. coli and due to its insolubility; it was denatured, refolded, and further purified. Following protein purification, a sedimentation velocity experiment was performed. The data reveal that SOD5 is a monomeric protein in solution. The protein was then crystallized and the structure of SOD5 was determined confirming that SOD5 is a novel monomeric SOD possessing a copper‐only active site and lacking a zinc binding site. In addition, this protein was devoid of the electrostatic loop that typically characterizes all SOD1s. The SOD5 copper site is solvent accessible, cupped in an electrostatic architecture that could serve in superoxide guidance.

Guanabenz delays the onset of disease symptoms, extends lifespan, improves motor performance and attenuates motor neuron loss in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disease characterized by the loss of motor neurons in the motor cortex, brain stem and spinal cord. Currently, there is no cure for this lethal disease. Although the mechanism underlying neuronal cell death in ALS remains elusive, growing evidence supports a crucial role of endoplasmic reticulum (ER) stress in the pathogenesis of ALS. Recent reports show that guanabenz, a novel inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, possesses anti-prion properties, attenuates ER stress and reduces paralysis and neurodegeneration in mTDP-43 Caenorhabditis elegans and Danio rerio models of ALS. However, the therapeutic potential of guanabenz for the treatment of ALS has not yet been assessed in a mouse model of ALS. In the present study, guanabenz was administered to a widely used mouse model of ALS expressing copper zinc superoxide dismutase-1 (SOD1) with a glycine to alanine mutation at position 93 (G93A). The results showed that the administration of guanabenz significantly extended the lifespan, delayed the onset of disease symptoms, improved motor performance and attenuated motor neuron loss in female SOD1 G93A mice. Moreover, western blotting results revealed that guanabenz dramatically increased the levels of phosphorylated-eIF2α (P-eIF2α) protein, without affecting total eIF2α protein levels. The results also revealed a significant decrease in the levels of the ER chaperone glucose-regulated protein 78 (BiP/Grp78) and markers of another two ER stress pathways, activating transcription factor 6α (ATF6α) and inositol-requiring enzyme 1 (IRE1). In addition, guanabenz increased the protein levels of anti-apoptotic B cell lymphoma/lewkmia-2 (Bcl-2), and down-regulated the pro-apoptotic protein levels of C/EBP homologous protein (CHOP), Bcl-2-associated X protein (BAX) and cytochrome C in SOD1 G93A mice. Our findings indicate that guanabenz may represent a novel therapeutic candidate for the treatment of ALS, a lethal human disease with an underlying mechanism involving the attenuation of ER stress and mitochondrial stress via prolonging eIF2α phosphorylation.

Effect of exenatide on the cardiac expression of adiponectin receptor 1 and NADPH oxidase subunits and heart function in streptozotocin-induced diabetic rats

BackgroundThis study investigated the effect of exenatide on the cardiac expression of adiponectin receptor 1 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and heart function in streptozotocin-induced diabetic rats.MethodsMale Sprague–Dawley rats were randomly divided into four groups, i.e. control group, diabetic group, diabetic treated with low doses of exenatide (2 μg · kg−1.d−1) and diabetic treated with high doses of exenatide (10 μg · kg−1.d−1). Diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg body weight). At the termination after exenatide treatment for eight weeks, following anesthesia of the rats, a catheter was inserted into the left ventricle through the right common carotid artery for measurement of left ventricular pressure, which included left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the maximal rate of rise and decline of ventricular pressure (±dp/dt[max]). Plasma and myocardial adiponectin levels, and the expressions of myocardial adiponectin receptor 1, p22phox, NADPH oxidase 4 (NOX4), glucose transporter type 4 (Glut4), AMPK-α, phosphorylated-AMPK-α, connective tissue growth factor (CTGF) and copper zinc superoxide dismutase (Cu-Zn-SOD) were assayed.ResultsHeart function, plasma adiponectin levels, the protein expression of myocardial phosphorylated-AMPK-α, the mRNA expression of myocardial Glut4, and the positive expression of myocardial Cu-Zn-SOD were significantly decreased in diabetic. The protein expression of myocardial adiponectin receptor 1, the mRNA expression of myocardial p22phox and NOX4, and the positive expression of myocardial CTGF were significantly increased in diabetic. Low and high doses of exenatide treatment significantly attenuated these changes in diabetic rats.ConclusionsThese results suggest that exenatide may contribute to the improvement of the heart function in diabetic rats by down-regulating the expression of myocardial adiponectin receptor 1, p22phox and NOX4, and up-regulating plasma adiponectin level and the expression of myocardial AMPK-α, Glut4 and Cu-Zn-SOD.

Effects of dietary inorganic copper on growth performance and immune responses of juvenile beluga,Huso huso

A 12-week feeding trial was conducted to evaluate the optimum dietary inorganic copper (copper sulphate) in juvenile beluga, Huso huso. Eight semi-purified diets containing 1.1 (Cu1.0), 3.5 (Cu4.0), 7.1 (Cu7.0), 9.7 (Cu10), 13.1 (Cu13), 25.1 (Cu25), 49.9 (Cu50) and 195 (Cu195) mg Cu kg−1 diet in the form of CuSO4.5H2O were fed to fish of initial body weight 8.49 ± 0.32 g and length 11.85 ± 0.66 cm (mean ± SD) in triplicate groups in a flow-through system. Weight gain (WG) of fish fed Cu10 and Cu13 diets was significantly higher than that of fish fed Cu1.0, Cu4.0, Cu25, Cu50 and Cu195 diets (P < 0.05). Whole-body and muscle crude protein increased with dietary Cu up to the supplementation level of 13.1 mg kg−1 diet and then decreased. Whole-body lipid content was negatively correlated, while whole-body ash was positively correlated with dietary copper concentration. Hepatic copper–zinc superoxide dismutase activity of fish fed Cu10 and Cu13 diets was significantly higher than that of fish fed Cu1.0, Cu4.0 and Cu195 diets. Hepatic thiobarbituric acid-reactive substances of fish fed Cu13 diet was significantly lower than those of fish fed the other diets except for that of fish fed Cu10 diet. Aspartate aminotransferase, alanine aminotransferase and copper accumulation in tissues increased with dietary copper. Broken-line analysis of WG suggested that the optimum dietary Cu level was 10.3 mg Cu kg−1 diet. Therefore, these results may indicate that the optimum dietary Cu levels could be greater than 10.3 mg Cu kg−1 diet but less than 13.1 mg Cu kg−1 diet in juvenile beluga, when copper sulphate is used as the dietary source of inorganic copper.

Comparative analysis of the neuroprotective effects of ginsenosides Rg1 and Rb1 extracted fromPanax notoginsengagainst cerebral ischemia

Panax notoginseng, a traditional Chinese medicine, has been used for thousands of years to treat ischemic patients. More than 20 saponin components have been isolated from P. notoginseng root and identified chemically. However, these different chemical components have different roles. In this study we compared the neuroprotective mechanisms of ginsenosides Rg1, Rb1, Rg1/Rb1, and panax notoginsenoside (PNS) against injuries caused by cerebral ischemia-reperfusion (I/R). Our results show that all of these treatments significantly reduced infarction volume and alleviated neurological deficits caused by cerebral I/R. The increase in malondialdehyde (MDA) concentration was inhibited by these treatments in the hippocampus. The decreased expressions of thioredoxin-1 (Trx-1), copper-zinc superoxide dismutase (SOD-1), protein kinase B (PKB/Akt), and nuclear factor-kappa B (NF-κB) caused by cerebral I/R were restored by these treatments. The expression of heat shock protein 70 (HSP70) was enhanced in the middle cerebral artery occlusion (MCAO) group, as well as in all of the treatment groups. These results suggest that Rg1 and Rb1 have similar roles in protecting the brain from ischemic damage; however, neither Rg1/Rb1 nor PNS have synergistic effects, thus either Rg1 or the Rb1 monomer should be considered as a pharmacological neuroprotective strategy for use in the case of ischemic stroke.

Activation of the cnidarian oxidative stress response by ultraviolet light, polycyclic aromatic hydrocarbons and crude oil

Organisms are continuously exposed to reactive chemicals capable of causing oxidative stress and cellular damage. Antioxidant enzymes, such as superoxide dismutases (SODs) and catalases, are present in both prokaryotes and eukaryotes and provide an important means of neutralizing such oxidants. Studies in cnidarians have previously documented the occurrence of antioxidant enzymes (transcript expression, protein expression and/or enzymatic activity), but most of these studies have not been conducted in species with sequenced genomes or included phylogenetic analyses, making it difficult to compare results across species due to uncertainties in the relationships between genes. Through searches of the genome of the sea anemone Nematostella vectensis Stephenson, one catalase gene and six SOD family members were identified, including three copper/zinc-containing SODs (CuZnSODs), two manganese-containing SODs (MnSODs) and one copper chaperone of SOD (CCS). In 24 h acute toxicity tests, juvenile N. vectensis showed enhanced sensitivity to combinations of ultraviolet radiation (UV) and polycyclic aromatic hydrocarbons (PAHs, specifically pyrene, benzo[a]pyrene and fluoranthene) relative to either stressor alone. Adult N. vectensis exhibited little or no mortality following UV, benzo[a]pyrene or crude oil exposure but exhibited changes in gene expression. Antioxidant enzyme transcripts were both upregulated and downregulated following UV and/or chemical exposure. Expression patterns were most strongly affected by UV exposure but varied between experiments, suggesting that responses vary according to the intensity and duration of exposure. These experiments provide a basis for comparison with other cnidarian taxa and for further studies of the oxidative stress response in N. vectensis.

The Morphological Features and Mitochondrial Oxidative Stress Mechanism of the Retinal Neurons Apoptosis in Early Diabetic Rats

This paper aims to explore the relationship of retinal neuron apoptosis and manganese superoxidase dismutase (MnSOD) at early phase of diabetic retinopathy. Sprague-Dawley rats were grouped into normal controls and diabetics. Data were collected after 4, 8, and 12 weeks (n = 12). The pathological changes and ultrastructure of the retina, the apoptosis rate of retinal neurons by TdT-mediated dUTP nick end label (TUNEL), mRNA expressions of MnSOD and copper-zinc superoxide dismutase (Cu–Zn SOD), and the activities of total SOD (T-SOD) and subtypes of SOD were tested. For the controls, there was no abnormal structure or apoptosis of retinal neurons at any time. There was no change of structure for rats with diabetes at 4 or 8 weeks, but there was a decrease of retinal ganglion cells (RGCs) number and thinner inner nuclear layer (INL) at 12 weeks. The apoptosis ratio of RGCs was higher than that of the controls at 8 and 12 weeks (P < 0.001). The activity and mRNA levels of MnSOD were lower in diabetics at 4, 8, and 12 weeks (P < 0.05). In summary, the apoptosis of the retinal neurons occurred at 8 weeks after the onset of diabetes. Retinal neuron apoptosis in early diabetic rats may be associated with the decreased activity and mRNA of MnSOD.

The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach.

Protein glycation is initiated by a nucleophilic addition reaction between the free amino group from a protein, lipid or nucleic acid and the carbonyl group of a reducing sugar. This reaction forms a reversible Schiff base, which rearranges over a period of days to produce ketoamine or Amadori products. The Amadori products undergo dehydration and rearrangements and develop a cross-link between adjacent proteins, giving rise to protein aggregation or advanced glycation end products (AGEs). A number of studies have shown that glycation induces the formation of the β-sheet structure in β-amyloid protein, α-synuclein, transthyretin (TTR), copper-zinc superoxide dismutase 1 (Cu, Zn-SOD-1), and prion protein. Aggregation of the β-sheet structure in each case creates fibrillar structures, respectively causing Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and prion disease. It has been suggested that oligomeric species of glycated α-synuclein and prion are more toxic than fibrils. This review focuses on the pathway of AGE formation, the synthesis of different types of AGE, and the molecular mechanisms by which glycation causes various types of neurodegenerative disease. It discusses several new therapeutic approaches that have been applied to treat these devastating disorders, including the use of various synthetic and naturally occurring inhibitors. Modulation of the AGE-RAGE axis is now considered promising in the prevention of neurodegenerative diseases. Additionally, the review covers several defense enzymes and proteins in the human body that are important anti-glycating systems acting to prevent the development of neurodegenerative diseases.

Analysis of oxidative stress enzymes and structural and functional proteins on human aortic tissue from different aortopathies.

The role of oxidative stress in different aortopathies is evaluated. Thirty-two tissue samples from 18 men and 14 women were divided into: 4 control (C) subjects, 11 patients with systemic arterial hypertension (SAH), 4 with variants of Marfan's syndrome (MV), 9 with Marfan's syndrome (M), 2 with Turner's syndrome, and 2 with Takayasu's arteritis (TA). Aorta fragments were homogenized. Lipoperoxidation (LPO), copper-zinc and manganese superoxide dismutase (Mn and Cu-Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), endothelial nitric oxide synthase (eNOS), nitrates and nitrites (NO3 −/NO2 −), and type IV collagen, and laminin were evaluated. There was an increase in Mn- and Cu-Zn-SOD activity in SAH, MV, M, and Turner's syndrome. There was also an increase in CAT activity in M and Turner' syndrome. GPx and GST activity decreased and LPO increased in all groups. eNOS was decreased in SAH, MV, and M and NO3 −/NO2 − were increased in SAH and TA. Type IV collagen was decreased in Turner's syndrome and TA. Laminin γ-1 was decreased in MV and increased in M. In conclusion, similarities and differences in oxidative stress in the different aortopathies studied including pathologies with aneurysms were found with alterations in SOD, CAT, GPx, GST, and eNOS activity that modify subendothelial basement membrane proteins.

A genetic reduction in antioxidant function causes elevated aggression in mice.

Male-male aggression can have a large influence on access to mates, particularly in highly territorial animals such as mice. It has been suggested that males with impaired antioxidant defence and a consequential increased susceptibility to oxidative stress may have a reduced ability to invest in aggressive behaviours, which could limit their mating opportunities and reproductive success. Oxidative stress occurs as a result of an uncontrolled over-production of reactive oxygen species (ROS) in relation to defence mechanisms (such as antioxidants), and can cause damage to a variety of different cellular components. Impairments in specific aspects of antioxidant defence, leading to oxidative stress, can limit investment in some reproductive traits in males, such as sperm quality and the production of sexual signals to attract mates. However, a direct effect of impaired antioxidant defence on aggressive behaviour has not, to our knowledge, been reported. In this study, we demonstrate that mice with experimentally elevated sensitivity to oxidative stress (through inhibition of copper-zinc superoxide dismutase, Sod1) actually show the opposite response to previous predictions. Males completely deficient in SOD1 are more aggressive than both wild-type males and males that express 50% of this antioxidant enzyme. They are also faster to attack another male. The cause of this increased aggression is unknown, but this result highlights that aggressive behaviour in mice is not highly constrained by inhibited Sod1 expression, in contrast to other reproductive traits known to be impaired in this mouse model.

SOD1 oxidation and formation of soluble aggregates in yeast: Relevance to sporadic ALS development

Misfolding and aggregation of copper–zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS.

Neuron‐specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD‐knockout mice

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

Comparative structural and conformational studies on H43R and W32F mutants of copper–zinc superoxide dismutase by molecular dynamics simulation

Recently, mutations in copper–zinc superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (fALS), a progressive neurodegenerative disease involving motor neuron loss, paralysis and death. It is mainly due to protein misfolding and aggregation resulting from the enhanced peroxidase activity of SOD1 mutants. In this study, we have carried out a 20ns molecular dynamics simulation for wild type (WT), H43R and W32F mutated SOD1's dimer and compared their structure and conformational properties by extracting several quantitative properties from the trajectory to understand the pathology of fALS disease. Our results show considerable differences in H43R compared to WT and W32F mutated SOD1, such as increasing distances between the critical residues results in open conformation at the active site, strong fluctuations in the important loops (Zinc and electrostatic loops) and weakening of important hydrogen bonds especially between N (His 43/Arg 43) and carbonyl oxygen (His 120) in agreement with the experimental report. The calculated buried surface area of dimer interface for WT, H43R and W32F are 682, 726 and 657Å2 respectively, representing the loss of dimerization in H43R. Essential dynamics reveal that overall motions of WT and W32F are mainly involved in three to four eigenvectors, but in H43R the overall motions are mainly in the first eigenvector. These data thus provide a unifying description for the structural destabilization, enhanced peroxidase activity, loss of dismutation activity and increase in aggregation propensity in the pathology of fALS diseases.

Ambient temperature enhanced freezing tolerance of Chrysanthemum dichrum CdICE1Arabidopsis via miR398

BackgroundICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance.ResultsUsing EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid assays, we confirmed that CdICE1 binds specifically to the MYC element in the CdDREBa promoter and activates transcription. In addition, overexpression of CdICE1 enhanced Arabidopsis freezing tolerance after transition from 23°C to 4°C or 16°C. We found that after acclimation to 4°C, CdICE1, like Arabidopsis AtICE1, promoted expression of CBFs (CRT/DRE Binding Factor) and their genes downstream involved in freezing tolerance, including COR15a (Cold-Regulated 15a), COR6.6, and RD29a (Responsive to Dessication 29a). Interestingly, we observed that CdICE1-overexpressing plants experienced significant reduction in miR398. In addition, its target genes CSD1 (Copper/zinc Superoxide Dismutase 1) and CSD2 showed inducible expression under acclimation at 16°C, indicating that the miR398-CSD pathway was involved in the induction of freezing tolerance.ConclusionsOur data indicate that CdICE1-mediated freezing tolerance occurs via different pathways, involving either CBF or miR398, under acclimation at two different temperatures.

Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E2Zn2SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E2Zn2SOD = Cu-free derivative of bovine liver copper-zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu(2+) level with clear red-to-yellow color change based on specific biomolecular recognition of E2Zn2SOD for Cu(2+) ion. The inorganic-organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu(2+) ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu(2+) with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10(-7)-10(-3) M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu(2+) changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu(2+) in live cells.

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization, production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.