An unexpected functional link between lysosomal thiol reductase and mitochondrial manganese superoxide dismutase

Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

Mitochondrial superoxide dismutase SOD2, but not cytosolic SOD1, plays a critical role in protection against glutamate-induced oxidative stress and cell death in HT22 neuronal cells

Oxidative cell death is an important contributing factor in neurodegenerative diseases. Using HT22 mouse hippocampal neuronal cells as a model, we sought to demonstrate that mitochondria are crucial early targets of glutamate-induced oxidative cell death. We show that when HT22 cells were transfected with shRNA for knockdown of the mitochondrial superoxide dismutase (SOD2), these cells became more susceptible to glutamate-induced oxidative cell death. The increased susceptibility was accompanied by increased accumulation of mitochondrial superoxide and loss of normal mitochondrial morphology and function at early time points after glutamate exposure. However, overexpression of SOD2 in these cells reduced the mitochondrial superoxide level, protected mitochondrial morphology and functions, and provided resistance against glutamate-induced oxidative cytotoxicity. The change in the sensitivity of these SOD2-altered HT22 cells was neurotoxicant-specific, because the cytotoxicity of hydrogen peroxide was not altered in these cells. In addition, selective knockdown of the cytosolic SOD1 in cultured HT22 cells did not appreciably alter their susceptibility to either glutamate or hydrogen peroxide. These findings show that the mitochondrial SOD2 plays a critical role in protecting neuronal cells from glutamate-induced oxidative stress and cytotoxicity. These data also indicate that mitochondria are important early targets of glutamate-induced oxidative neurotoxicity.     

Targeting IFN-γ-inducible lysosomal thiol reductase overcomes chemoresistance in AML through regulating the ROS-mediated mitochondrial damage

The persistence of leukemia stem cells (LSCs) is one of the leading causes of chemoresistance in acute myeloid leukemia (AML). To explore the factors important in LSC-mediated resistance, we use mass spectrometry to screen the factors related to LSC chemoresistance and defined IFN-γ-inducible lysosomal thiol reductase (GILT) as a candidate. We found that the GILT expression was upregulated in chemoresistant CD34+ AML cells. Loss of function studies demonstrated that silencing of GILT in AML cells sensitized them to Ara-C treatment both in vitro and in vivo. Further mechanistic findings revealed that the ROS-mediated mitochondrial damage plays a pivotal role in inducing apoptosis of GILT-inhibited AML cells after Ara-C treatment. The inactivation of PI3K/Akt/ nuclear factor erythroid 2-related factor 2 (NRF2) pathway, causing reduced generation of antioxidants such as SOD2 and leading to a shifted ratio of GSH/GSSG to the oxidized form, contributed to the over-physiological oxidative status in the absence of GILT. The prognostic value of GILT was also validated in AML patients. Taken together, our work demonstrated that the inhibition of GILT increases AML chemo-sensitivity through elevating ROS level and induce oxidative mitochondrial damage-mediated apoptosis, and inhibition of the PI3K/Akt/NRF2 pathway enhances the intracellular oxidative state by disrupting redox homeostasis, providing a potentially effective way to overcome chemoresistance of AML.     

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.     

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.     

Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice

Presynaptic nerve terminals require high levels of ATP for the maintenance of synaptic function. Failure of synaptic mitochondria to generate adequate ATP has been implicated as a causative event preceding the loss of synaptic networks in neurodegenerative disease. Endogenous oxidative stress has often been postulated as an etiological basis for this pathology, but has been difficult to test in vivo. Inactivation of the superoxide dismutase gene (Sod2) encoding the chief defense enzyme against mitochondrial superoxide radicals results in neonatal lethality. However, intervention with an SOD mimetic extends the life span of this model and uncovers a neurodegenerative phenotype providing a unique model for the examination of in vivo oxidative stress. We present here studies on synaptic termini isolated from the frontal cortex of Sod2 null mice demonstrating impaired bioenergetic function as a result of mitochondrial oxidative stress. Cortical synaptosomes from Sod2 null mice demonstrate a severe decline in mitochondrial spare respiratory capacity in response to physiological demand induced by mitochondrial respiratory chain uncoupling with FCCP or by plasma membrane depolarization induced by 4-aminopyridine treatment. However, Sod2 null animals compensate for impaired oxidative metabolism in part by the Pasteur effect allowing for normal neurotransmitter release at the synapse, setting up a potentially detrimental energetic paradigm. The results of this study demonstrate that high-throughput respirometry is a facile method for analyzing specific regions of the brain in transgenic models and can uncover bioenergetic deficits in subcellular regions due to endogenous oxidative stress.     

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.     

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.     

Mitochondrial oxidative stress causes chromosomal instability of mouse embryonic fibroblasts

Reactive oxygen species are an inevitable by-product of mitochondrial respiration. It has been estimated that between 0.4 and 4% of molecular oxygen is converted to the radical superoxide (O2*-) and this level is significantly influenced by the functional status of the mitochondria. It is well established that exogenous oxidative stress and high doses of mitochondrial poisons such as paraquat and carbonyl cyanide 4 (trifluoromethoxy) phenylhydrazone (FCCP) can lead to genomic instability. In this report we show for the first time that endogenous mitochondrial oxidative stress in standard cell culture conditions results in nuclear genomic instability in primary mouse embryonic fibroblasts (MEFs). We show that lack of mitochondrial superoxide dismutase in MEFs leads to a severe increase of double strand breaks, end-to-end fusions, chromosomal translocations, and loss of cell viability and proliferative capacity. Our results predict that endogenous mitochondrial oxidative stress can induce genomic instability, and therefore may have a profound effect in cancer and aging.     

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.     

Pharmacogenomic profiling of an oxidative stress-mediated spongiform encephalopathy

The majority of cellular superoxide is generated in the mitochondria as a by-product of normal oxidative metabolism. In the mitochondria, superoxide is detoxified by manganese superoxide dismutase (SOD2). Mice lacking SOD2 demonstrate a multifaceted neonatal lethal phenotype, including a spongiform encephalopathy that is preventable through antioxidant treatment. The molecular events behind the observed pathology in the cortex of these mice are unknown. We hypothesized that the lack of SOD2 would result in significant changes in cortical gene expression and that therapeutically beneficial antioxidant treatment would normalize the expression of some genes, providing insight into the mechanism by which mitochondrial oxidative stress results in neurodegeneration. We report the identification of gene expression profiles associated with this paradigm, which characterize the degree of response to the pharmacologic intervention. We have identified specific pathways targeted by endogenous oxidative stress, including glutathione metabolism, iron metabolism, and cell-survival pathways centering on the kinase AKT. The normalization of expression of some of these pathways by antioxidant treatment suggests approaches to treating disease in which endogenous oxidative stress plays a role.     

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth’s surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O₂^•− generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.     

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.     

Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.     

Role of mitochondria in toxic oxidative stress

Oxidative stress and mitochondrial oxidative damage have been implicated in the etiology of numerous common diseases. The critical mitochondrial events responsible for oxidative stress-mediated cell death (toxic oxidative stress), however, have yet to be defined. Several oxidative events implicated in toxic oxidative stress include alterations in mitochondrial lipids (e.g., cardiolipin), mitochondrial DNA, and mitochondrial proteins (eg. aconitase and uncoupling protein 2). Furthermore, recent findings indicate the enrichment of mitochondrial membranes with vitamin E protects cells against the toxic effects of oxidative stress. This review briefly summarizes the role of these mitochondrial events in toxic oxidative stress, including: 1) the protective role of mitochondrial vitamin E in toxic oxidative stress, 2) the role of mitochondrial DNA in toxic oxidative stress, 3) the interaction between cardiolipin and cytochrome c in mitochondrial regulation of apoptosis, 4) the role of mitochondrial aconitase in oxidative neurodegeneration, and 5) the role of mitochondrial uncoupling protein 2 in the pathogenesis of type 2 diabetes.     

Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.     

神经退化性疾病生物能量代谢和氧化应激研究进展

衰老是导致几种常见的神经系统退化性疾病的主要危险因素,包括帕金森氏病（Ｐａｒｋｉｎｓｏｎ’ｓ ｄｉｓｅａｓｅ ＰＤ）,肌萎缩性侧索硬化（Ａｍｙｏｔｒｏｐｈｉｃ ｌａｔｅｒａｌ ｓｃｌｅｒｏｓｉｓ,ＡＬＳ）,早老性痴呆（Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ ＡＤ）和亨廷顿氏病（Ｈｕｎｔｉｎｇｔｏｎ’ｓ ｄｉｓｅａｓｅ ＨＤ）。最近研究表明,神经退化性疾病涉及到线粒体缺陷,氧化应激等因素。在脑和其它组织中,老化可导致线粒体功能的损伤和氧化损伤的增强。ＰＤ病人中,已发现线粒体复合酶体Ⅰ活性降低,氧化损伤增加和抗氧化系统活性的改变。在几例家族性ＡＬＳ病人中,也发现Ｃｕ、Ｚｎ超氧化物歧化酶（Ｃｕ,Ｚｎ ＳＯＤ）基因的突变,导致Ｃｕ、Ｚｎ超氧化物歧化酶活性减低;散发的ＡＬＳ病人氧化损伤增高。在ＨＤ病人中已发现能量代谢异常