Maria Rosa Ciriolo

Mitochondrial dysfunction in neurodegenerative diseases associated with copper imbalance.

Copper is an essential transition metal ion for the function of key metabolic enzymes, but its uncontrolled redox reactivity is source of reactive oxygen species. Therefore a network of transporters strictly controls the trafficking of copper in living systems. Deficit, excess, or aberrant coordination of copper are conditions that may be detrimental, especially for neuronal cells, which are particularly sensitive to oxidative stress. Indeed, the genetic disturbances of copper homeostasis, Menkes and Wilsons diseases, are associated with neurodegeneration. Furthermore, copper interacts with the proteins that are the hallmarks of neurodegenerative disorders, such as Alzheimers disease, Parkinsons disease, prion diseases, and familial amyotrophic lateral sclerosis. In all cases, copper-mediated oxidative stress is linked to mitochondrial dysfunction, which is a common feature of neurodegeneration. In particular we recently demonstrated that in copper deficiency, mitochondrial function is impaired due to decreased activity of cytochrome c oxidase, leading to production of reactive oxygen species, which in turn triggers mitochondria-mediated apoptotic neurodegeneration.

Decrease of superoxide dismutase and glutathione peroxidase in liver of rats treated with hypolipidemic drugs

1. INTRODUCTION Clofibrate and other hypolipidemic drugs in- crease the number of hepatic peroxisomes and cause a corresponding increase of several peroxi- somal enzyme activities [ 1,2]. This effect regards HlOz-generating reactions (most typically, palmi- toyl-CoA oxidase activity) and catalase, which is able to destroy most of the Hz02 formed within the organelle. It is reasonable to expect that the net flux of Hz02 outside the organelle should be enhanced under hypolipidemic treatment in view of the fact that significant Hz02 diffusion is observed in per- oxisomes of untreated animals [3,4] and the increase of palmitoyl-CoA oxidase activity induced by clo- tibrate feeding is much higher than that of catalase [ 11. Moreover, hypolipidemic agents cause prolif- eration of hepatic smooth endoplasmic reticulum as well, and this produces a marked increase of cyto- chrome P450 [5], which is likely to be a major source of Ozand H202 in the cell as suggested by results obtained with subcellular preparations [6,7]. It seemed therefore interesting to investigate whether activities of cytoplasmic enzymes acting as a defense against ‘oxygen radicals’ (i.e., superoxide dis- mutase and glutathione peroxidase) are affected by feeding hypolipidemic drugs. The results reported here show that both enzymes are significantly de- creased in rat liver during treatment with clofibrate and procetofene, another hypolipidemic agent [S]. This effect is associated with an increased suscepti- bility of the tissue to enhanced peroxidative risk, as *

Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS.

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus‐induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4‐derived ROS in activating redox‐regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4‐mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.

Superoxide dismutase, glutathione peroxidase and catalase in oxidative hemolysis. A study of Fanconi's anemia erythrocytes

Abstract Superoxide dismutase, glutathione peroxidase and catalase were assayed in the erythrocytes of three patients of Fanconis anemia. Superoxide dismutase was found to be significantly decreased, as previously reported. The enzymes metabolizing H 2 O 2 are normal (glutathione peroxidase in the higher limits of the normal value). The abnormal erythrocytes were found to be as resistant (perhaps more resistant) as normal red blood cells to oxidative hemolysis induced by drugs. Malonyl dialdehyde production was found to be comparable to that of normal erythrocytes. It is concluded that a significant (30–40%) deficiency of superoxide dismutase, when associated to normal values of H 2 O 2 -removing enzymes, does not affect the antioxidative defense capability of erythrocytes, even in conditions of augmented oxidative injury.

Multiple electrophoretic variants of Cu, Zn superoxide dismutase as expression of the enzyme aging. Effects of H2O2, ascorbate and metal ions

Multiple electrophoretic bands, with RF identical to the natural molecular variants, are produced by treatment of purified Cu, Zn Superoxide dismutase with either H2O2 or ascorbate plus Fe(III) EDTA. The ascorbate reaction is also due to H2O2 since it is inhibited by catalase. However while H2O2 inactivates the enzyme, the electromorphs produced by ascorbate-Fe(III) EDTA have only slightly less activity than the native enzyme and this property parallels the natural situation. It is concluded that oxidative aging can be responsible for the multiple molecular variants of the natural enzyme, under conditions where the oxidant attack is preferentially directed to amino acid side chains outside the active site. Such conditions may occur when a metal ion coordinated to the protein surface undergoes a redox cycle with biological reductants, like ascorbate.

Prolonged copper depletion induces expression of antioxidants and triggers apoptosis in SH-SY5Y neuroblastoma cells.

SH-SY5Y neuroblastoma cells were cultured for up to three serial passages in the presence of the copper chelator triethylene tetramine (Trien). The copper-depleted neuroblastoma cell line obtained showed decreased activities of the copper enzymes Cu, Zn superoxide dismutase and cytochrome c oxidase with concomitant increases in reactive oxygen species. Mitochondrial antioxidants (Mn superoxide dismutase and Bcl-2) were up-regulated. Overexpression and activation of p53 were early responses, leading to an increase in p21. Eventually, copper-depleted cells detached from the monolayer and underwent apoptosis. Activation of up-stream caspase-9, but not caspase-8, suggested that apoptosis proceeds via a mitochondrial pathway, followed by caspase-3 activation. The addition of copper sulfate to the copper-depleted cells restored copper enzymes, normalized antioxidant levels and improved cell viability. We conclude that prolonged copper starvation in these replicating cells leads to mitochondrial damage and oxidative stress and ultimately, apoptosis.

An X-ray absorption study of the reconstitution process of bovine Cu, Zn superoxide dismutase by Cu(I)-glutathione complex

The CU(I)GSH complex has recently been shown to be a good candidate for delivering copper to the active site of Cu‐free Cu,Zn superoxide dismutase both in vivo and in vitro. In this work X‐ray absorption spectroscopy has been used to characterize the CU(I)GSH complex and to follow in vitro the reconstitution of Cu,Zn superoxide dismutase from the copper‐free protein and this complex. The results obtained indicate that the copper is directly transferred as Cu(I) from the GSH complex into the empty copper binding site. No evidence has been obtained for a ternary complex in which the metal is bound to both GSH and the protein.

Correlation between superoxide dismutase, glutathione peroxidase and catalase in isolated rat hepatocytes during fetal development

Abstract Superoxide dismutase, glutathione peroxidase and catalase activities were determined in isolated fetal rat hepatocytes of various ages and compared with the values of neonatal and adult cells. The developmental pattern of superoxide dismutase and glutathione peroxidase were very similar with a low constant activity in the fetal cells and a postnatal burst. On the contrary catalase begins to increase already since the 18th day of the fetal life. The results suggest a functional correlation of superoxide dismutase and glutathione peroxidase in the antioxidative enzyme defense of liver cells.

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism.

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37 degrees C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H2O2 and HbO2, may be related to the pathogenesis of hemolysis in favism.

Disturbances of Copper Homeostasis and Brain Function

Copper is involved in electron-transporting chains of chloroplasts and mitochondria and is essential for the activity of enzymes involved in the oxygen metabolism, including cytochrome-c oxidase, superoxide dismutase, dopamine-β-hydroxylase, tyrosinase, lysyl oxidase, and peptidil—glycine α-amidating monooxygenase (1). These copper-dependent enzymes are crucial to several processes of oxidative metabolism, namely respiration, detoxification, neurotransmitter synthesis, pigmentation, and maturation of connective tissue.