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Mitochondrial dysfunction caused by oxidative stress appears at early stages of aging and age-related diseases. Plasma membrane redox enzymes act in a compensatory manner to decrease oxidative stress and supply reductive capacity to ensure cell survival. Plasma membrane redox enzymes transfer electrons from NAD(P)H to oxidized ubiquinone and α-tocopherol, resulting in inhibition of further oxidative damage. Plasma membrane redox enzymes and their partners are affected by aging, leading to progression of neurodegenerative disease pathogenesis. Up-regulating plasma membrane redox enzymes via calorie restriction and phytochemicals make cells more resistant to oxidative damage under stress conditions by maintaining redox homeostasis and improving mitochondrial function. Investigation into plasma membrane redox enzymes can provide mechanistic details underlying the relationships between plasma membrane redox enzymes and mitochondrial complexes and provide a good therapeutic target for prevention and delay of neurodegenerative disorders.

Modest levels of reactive oxygen species (ROS) are necessary for intracellular signaling, cell division, and enzyme activation. These ROS are later eliminated by the body’s antioxidant defense system. High amounts of ROS cause carcinogenesis by altering the signaling pathways associated with metabolism, proliferation, metastasis, and cell survival. Cancer cells exhibit enhanced ATP production and high ROS levels, which allow them to maintain elevated proliferation through metabolic reprograming. In order to prevent further ROS generation, cancer cells rely on more glycolysis to produce ATP and on the pentose phosphate pathway to provide NADPH. Pro-oxidant therapy can induce more ROS generation beyond the physiologic thresholds in cancer cells. Alternatively, antioxidant therapy can protect normal cells by activating cell survival signaling cascades, such as the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway, in response to radio- and chemotherapeutic drugs. Nrf2 is a key regulator that protects cells from oxidative stress. Under normal conditions, Nrf2 is tightly bound to Keap1 and is ubiquitinated and degraded by the proteasome. However, under oxidative stress, or when treated with Nrf2 activators, Nrf2 is liberated from the Nrf2-Keap1 complex, translocated into the nucleus, and bound to the antioxidant response element in association with other factors. This cascade results in the expression of detoxifying enzymes, including NADH-quinone oxidoreductase 1 (NQO1) and heme oxygenase 1. NQO1 and cytochrome b5 reductase can neutralize ROS in the plasma membrane and induce a high NAD+/NADH ratio, which then activates SIRT1 and mitochondrial bioenergetics. NQO1 can also stabilize the tumor suppressor p53. Given their roles in cancer pathogenesis, redox homeostasis and the metabolic shift from glycolysis to oxidative phosphorylation (through activation of Nrf2 and NQO1) seem to be good targets for cancer therapy. Therefore, Nrf2 modulation and NQO1 stimulation could be important therapeutic targets for cancer prevention and treatment.

In this study, we demonstrate that activation of AMP-activated protein kinase (AMPK) with glabridin alleviates adiposity and hyperlipidemia in obesity. In several obese rodent models, glabridin decreased body weight and adiposity with a concomitant reduction in fat cell size. Further, glabridin ameliorated fatty liver and plasma levels of triglyceride and cholesterol. In accordance with these findings, glabridin suppressed the expression of lipogenic genes such as sterol regulatory element binding transcription factor (SREBP)-1c, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD)-1 in white adipose tissues and liver, whereas it elevated the expression of fatty acid oxidation genes such as carnitine palmitoyl transferase (CPT)1, acyl-CoA oxidase (ACO), and peroxisome proliferator-activated receptor (PPAR)α in muscle. Moreover, glabridin enhanced phosphorylation of AMPK in muscle and liver and promoted fatty acid oxidation by modulating mitochondrial activity. Together, these data suggest that glabridin is a novel AMPK activator that would exert therapeutic effects in obesity-related metabolic disorders.

The cause of elevated level of amyloid β-peptide (Aβ42) in common late-onset sporadic (AD) has not been established. Here we show that the membrane lipid peroxidation product 4-hydroxynonenal (HNE) is associated with amyloid and neurodegenerative pathologies in AD, and that it enhances γ-secretase activity and Aβ42 production in neurons. The γ-secretase substrate receptor, nicastrin was found to be modified by HNE in cultured neurons and in brain specimens from AD patients, in which HNE-nicastrin levels were found to be correlated with increased γ-secretase activity and Aβ plaque burden. Furthermore, HNE modification of nicastrin enhanced its binding to the γ-secretase substrate, amyloid precursor protein (APP) C99. In addition, the stimulation of γ-secretase activity and Aβ42 production by HNE were blocked by an HNE-scavenging histidine analog in a 3xTgAD mouse model of AD. These findings suggest a specific molecular mechanism by which oxidative stress increases Aβ42 production in AD, and identify HNE as a novel therapeutic target upstream of the γ-secretase cleavage of APP.

A number of epidemiological studies have demonstrated a strong association between the incidence of neurodegenerative disease and pesticide exposure. Fluazinam (FZN) is a preventative fungicide from the pyridinamine group that was introduced in the 1990s and that quickly established itself as a new standard for the control of blight caused by Phytophthora infestans in potatoes. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of neuronal cell death in response to FZN and showed that FZN was cytotoxic to SH-SY5Y cells in a concentration- and time-dependent manner. Additionally, we showed that FZN treatment significantly decreased the neuron numbers including dopaminergic neurons and mitochondrial complex I activity. The cytotoxic effects of FZN were associated with an increase in reactive oxygen species (ROS) generation because pretreatment with N-acetyl cysteine, an anti-oxidant, reduced cell death. We showed that neuronal cell death in response to FZN was due to apoptosis because FZN increased cytochrome C release into the cytosol and activated caspase-3 through the accumulation of p53. FZN also reduced the levels of Bcl-2 protein but increased the levels of Bax. Our results provide insight into the molecular mechanisms of FZN-induced apoptosis in neuronal cells.

The nature of mitochondrial dysfunction in dopaminergic neurons in familial Parkinson's disease (PD) is unknown. We characterized the pathophenotypes of dopaminergic neuronal cells that were deficient in PINK1 or DJ-1, genes with mutations linked to familial PD. Both PINK1- and DJ-1-deficient dopaminergic neurons had the increased production of ROS, severe mitochondrial structural damages and complex I deficits. A striking decrease in complex IV activity was also prominent by the PINK1-deficiency. The complex I deficits were relatively PD-specific and were significantly improved by an antioxidant Trolox. These data suggest that mitochondrial deficits are severe in dopaminergic neurons in familial PD and antioxidant-mediated functional recovery is feasible.

Increased cell mass is one of the characteristics of senescent cells, but this event has not been clearly defined. When subcellular organellar mass was estimated with organelle-specific fluorescence dyes, we observed that most membranous organelles progressively increase in mass during cell senescence. This increase was accompanied by an increase in membrane lipids and augmented expression of lipogenic enzymes, such as fatty acid synthase (FAS), ATP citrate lyase, and acetyl-CoA carboxylase. The mature form of sterol regulatory element-binding protein (SREBP)-1 was also elevated. Increased expression of these lipogenic effectors was further observed in the liver tissues of aging Fischer 344 rats. Ectopic expression of mature form of SREBP-1 in both Chang cells and primary young human diploid fibroblasts was enough to induce senescence. Blocking lipogenesis with FAS inhibitors (cerulenin and C75) and via siRNA-mediated silencing of SREBP-1 and ATP citrate lyase significantly attenuated H(2)O(2)-induced senescence. Finally, old human diploid fibroblasts were effectively reversed to young-like cells by challenging with FAS inhibitors. Our results suggest that enhanced lipogenesis is not only a common event, but also critically involved in senescence via SREBP-1 induction, thereby contributing to the increase in organelle mass (as a part of cell mass), a novel indicator of senescence.

The objective of the present study was to examine the role of selenium in the metabolism of Aβ and in Aβ-induced neuronal death. Selenium treatment significantly reduced Aβ40, Aβ42, and sAPPβ production by reducing Aβ producing β-secretase and γ-secretase activities. The lipid peroxidation product 4-Hydroxynonenal (HNE)-induced transcription of β-secretase (BACE1) was blocked by selenium. Finally, our data show that selenium protects against HNE and Aβ-mediated toxicity in primary cultured neurons. The present study suggests that selenium may be able to salvage the neuronal degeneration of Alzheimer's disease, thereby limiting β-amyloid production and neuronal death.

The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD(+)/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H2O2 (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H2O2 and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD(+)/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases.

The plasma membrane (PM) contains redox enzymes that provide electrons for energy metabolism and recycling of antioxidants such as coenzyme Q and α-tocopherol. Brain aging and neurodegenerative disorders involve impaired energy metabolism and oxidative damage, but the involvement of the PM redox system (PMRS) in these processes is unknown. Caloric restriction (CR), a manipulation that protects the brain against aging and disease, increased activities of PMRS enzymes (NADH-ascorbate free radical reductase, NADH-quinone oxidoreductase 1, NADH-ferrocyanide reductase, NADH-coenzyme Q10 reductase, and NADH-cytochrome c reductase) and antioxidant levels (α-tocopherol and coenzyme Q10) in brain PM during aging. Age-related increases in PM lipid peroxidation, protein carbonyls, and nitrotyrosine were attenuated by CR, levels of PMRS enzyme activities were higher, and markers of oxidative stress were lower in cultured neuronal cells treated with CR serum compared with those treated with ad libitum serum. These findings suggest important roles for the PMRS in protecting brain cells against age-related increases in oxidative and metabolic stress.

Oxidative stress over time leads to the accumulation of damaged macromolecules and to profound physiological changes that are associated with several age-related diseases. The plasma membrane redox system (PMRS) appears to attenuate oxidative stress acting as a compensatory mechanism during the aging process. The PMRS appears to play a protective role during mitochondrial dysfunction to provide cells with a survival mechanism by lowering oxidative stress. The PMRS accomplishes this by producing more NAD(+) for glycolytic ATP production via transfer of electrons from intracellular reducing equivalents to extracelluar acceptors. Ubiquinone and alpha-tocopherol are key antioxidant molecules in the plasma membrane that are affected by aging and can be up-regulated by dietary interventions such as calorie restriction (CR). Up-regulation of PMRS activity leads to cell survival and membrane homeostasis under stress conditions and during calorie restriction. Further studies of the PMRS may provide not only additional information on the mechanisms involved in aging and CR, but may provide therapeutic targets for the prevention and treatment of age-related diseases.

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.

4-Hydroxy-2,3-trans-nonenal (HNE) is a neurotoxic unsaturated aldehyde end-product of lipid peroxidation. The addition of HNE to NT-2 and SK-N-MC cell lines induces apoptosis and we now investigated the time-course of events occurring prior to apoptosis. Treatment of both NT-2 and SK-N-MC cell lines with HNE led to HNE association with the proteasome, increased levels of protein carbonyls and ubiquitinated proteins, and decreased proteasomal function. There was also decreased metabolic activity, cytochrome c release and activation of caspase 3, followed by apoptotic changes including chromatin condensation, cell shrinkage and DNA fragmentation and laddering. Overexpression of mutant superoxide dismutase 1 proteins associated with amyotrophic lateral sclerosis decreased proteasomal activities in the absence of HNE and accelerated the apoptosis induced by HNE. By contrast, overexpression of wild-type superoxide dismutase 1 did not affect basal levels of proteasomal activity. The data suggest that accumulation of ubiquitinated proteins and impairment of proteasomal function are important events in HNE toxicity. We propose that the proteasomal system is a significant target of HNE neurotoxicity in a wide range of neurodegenerative diseases, especially if abnormal proteins are being expressed.

Mutations in Parkin (a ubiquitin protein ligase) are involved in autosomal recessive juvenile parkinsonism, but it is not known how they cause nigral cell death. We examined the effect of Parkin overexpression on cellular levels of oxidative damage, antioxidant defenses, nitric oxide production, and proteasomal enzyme activity. Increasing expression of Parkin by gene transfection in NT-2 and SK-N-MC cells led to increased proteasomal activity, decreased levels of protein carbonyls, 3-nitrotyrosine-containing proteins, and a trend to a reduction in ubiquitinated protein levels. Transfection of these cells with DNA encoding three mutant Parkins associated with autosomal recessive juvenile parkinsonism (Del 3–5, T240R, and Q311X) gave smaller increases in proteasomal activity and led to elevated levels of protein carbonyls and lipid peroxidation. Turnover of the mutant proteins was slower than that of the wild-type protein, and both could be blocked by the proteasome inhibitor, lactacystin. A rise in levels of nitrated proteins and increased levels of NO /NO was also observed in cells transfected with mutant Parkins, apparently because of increased levels of neuronal nitric-oxide synthase. The presence of mutant Parkin in substantia nigra in juvenile parkinsonism may increase oxidative stress and nitric oxide production, sensitizing cells to death induced by other insults.

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 microM) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO(-)) upon proteins. Higher levels (25 microM) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 microM) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO.) (measured as levels of NO2- plus NO3-) by the cells; this was inhibited by N-nitro-L-arginine methyl ester (L-NAME), which also decreased cell death induced by 25 microM lactacystin and decreased levels of 3-nitrotyrosine. The NO. production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.

Gastric cancer is one of the most common malignant tumors and the second cause of cancer-related deaths worldwide. Naphthoquinones such as juglone and plumbagin are compounds used extensively to overcome resistance to chemotherapeutic agents in cancers due to their cytotoxic role. This study is the first to investigate the anti-cancer effect of naphthazarin (Naph), one of the naphthaquinones, in human gastric cancer AGS cells. We showed that Naph exhibited effective preferential cell growth inhibition via G2/M phase arrest and apoptosis, which was associated with reduced levels of Cdc2 and Cdc25C expression. Naph also increased cleaved caspase-3 and Poly ADR(adenosine diphosphate ribose) Polymerase expression, γ-H2AX expression (an indicator of DNA double strand breaks) and DNA fragmentation. We also found the generation of reactive oxygen species is a critical mediator in Naph-induced cell growth inhibition and apoptosis. The non-protein antioxidant, glutathione significantly abolished Naph-mediated inhibition of cell growth and apoptosis. Taken together, our findings showed that Naph not only inhibited cell growth, but also induced apoptosis of AGS cells, suggesting that Naph may be a potential candidate for cancer therapy against gastric cancers.

The plasma membrane redox system (PMRS) of nicotinamide adenine dinucleotide (NADH)-related enzymes plays a key role in the maintenance of cellular energetics. During the aging process, neural cells are particularly sensitive to impaired energy metabolism and oxidative damage, but the involvement of the PMRS in these processes is unknown. Here, we used human neuroblastoma cells with either elevated or reduced levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS regulates neuronal stress responses. Cells with elevated NQO1 levels were more resistant to death induced by 2-deoxyglucose, potassium cyanide (energetic stress), and lactacystin (proteotoxic stress), but were not protected from being killed by H(2)O(2) and serum withdrawal. The NAD(+)(an oxidized form of NADH)/NADH ratio was maintained at a significantly higher level in cells overexpressing NQO1, consistent with enhanced levels of NQO1 activity. Levels of the neuroprotective transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and nuclear factor (erythroid-derived 2)-like 2, and the protein chaperone HSP70 were elevated in cells overexpressing NQO1. Cells in which NQO1 levels were decreased by RNA interference exhibited increased vulnerability to death induced by 2-deoxyglucose and lactacystin. Thus, a higher NAD(+)/NADH ratio and activation of adaptive stress response pathways are enhanced by the PMRS in neuroblastoma cells, enabling them to maintain redox homeostasis under conditions of energetic and proteotoxic stress. These findings have implications for the development of therapeutic interventions for neural tumors and neurodegenerative conditions.

Impairment of the ubiquitin–proteasome system (UPS) for degrading abnormal proteins leads to protein aggregates and increased protein oxidation/nitration. This study was performed to show that interference with polyubiquitination in the presence of 4-hydroxy-2,3- trans -nonenal (HNE) has similar consequences. Levels of polyubiquitin chains were not increased in NT-2 and SK-N-MC cells overexpressing a dominant-negative mutant form of ubiquitin (K48R) in response to HNE compared to wild-type transfectants. Increased oxidative (GSH, protein carbonyls and lipid peroxidation) and nitrative damage (nitric oxide production and elevated protein nitration) were aggravated in the mutant transfectants. These data show that initial oxidative/nitrative damage (due to HNE) and interference with ubiquitination (induced by mutant ubiquitin or HNE) can cause common characteristics of neurodegenerative diseases. These data suggest that impairment of the UPS at different levels may be a common mechanism in neurodegeneration and that more such defects remain to be identified.

Membrane-associated oxidative stress has been implicated in the synaptic dysfunction and neuronal degeneration that occurs in Alzheimer's disease (AD), but the underlying mechanisms are unknown. Enzymes of the plasma membrane redox system (PMRS) provide electrons for energy metabolism and recycling of antioxidants. Here, we show that activities of several PMRS enzymes are selectively decreased in plasma membranes from the hippocampus and cerebral cortex of 3xTgAD mice, an animal model of AD. Our results that indicate the decreased PMRS enzyme activities are associated with decreased levels of coenzyme Q(10) and increased levels of oxidative stress markers. Neurons overexpressing the PMRS enzymes (NQO1 or cytochrome b5 reductase) exhibit increased resistance to amyloid β-peptide (Aβ). If and to what extent Aβ is the cause of the impaired PMRS enzymes in the 3xTgAD mice is unknown. Because these mice also express mutant tau and presenilin-1, it is possible that one or more of the PMRS could be adversely affected by these mutations. Nevertheless, the results of our cell culture studies clearly show that exposure of neurons to Aβ1-42 is sufficient to impair PMRS enzymes. The impairment of the PMRS in an animal model of AD, and the ability of PMRS enzyme activities to protect neurons against Aβ-toxicity, suggest enhancement PMRS function as a novel approach for protecting neurons against oxidative damage in AD and related disorders.

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H(2)O(2)), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.

Regular exercise can counteract the adverse effects of aging on the musculoskeletal and cardiovascular systems. In males the normal aging process is associated with reductions in testosterone production’(Matsumoto 2002); (synder 2001) and impaired spermatogenesis, but the underlying mechanisms and their potential modification by exercise are unknown. Here we report that lifelong regular exercise (running) protects the testes against maturation as well as age related changes, and that this effect of running is associated with decreased amounts of oxidative damage to proteins, lipids and DNA in spermatogenic and Leydig cells. Six month-old male mice were divided into a sedentary group and a group which ran an average of 1.75 km/day, until the mice reached the age of 20 months. Seminiferous tubules of runners exhibited a full complement of cells at different stages of the spermatogenic process and a clear central lumen with large numbers of spermatozoa, in contrast to sedentary mice which exhibited disorganized spermatogenic cells and lacked spermatocytes in a central lumen. Levels of protein carbonyls, nitrotyrosine, lipid peroxidation products and oxidatively modified DNA were significantly greater in spermatogenic and Leydig cells of sedentary mice compared to runners. These findings suggest that lifelong regular exercise suppresses aging of the testes by a mechanism that involves reduced oxidative damage to spermatogenic and Leydig cells.

Uric acid is a major antioxidant in the blood of humans that can protect cultured neurons against oxidative and metabolic insults. However, uric acid has a very low solubility which compromises its potential clinical use for neurodegenerative disorders. Here we describe the synthesis, characterization and preclinical development of neuroprotective methyl- and sulfur-containing analogs of uric acid with increased solubility. In vitro and cell culture screening identified 1,7-dimethyluric acid (mUA2) and 6,8-dithiouric acid (sUA2) as two analogs with high antioxidant and neuroprotective activities. When administered intravenously in mice, uric acid analogs mUA2 and sUA2 lessened damage to the brain and improved functional outcome in an ischemia-reperfusion mouse model of stroke. Analogs sUA2 and mUA2 were also effective in reducing damage to the cerebral cortex when administered up to 4 h after stroke onset in a permanent middle cerebral artery occlusion mouse model. These findings suggest a therapeutic potential for soluble analogs of uric acid in the treatment of stroke and related neurodegenerative conditions.

Mutations in parkin are involved in some cases of autosomal recessive juvenile parkinsonism (AR-JP), but it is not known how they result in nigral cell death. We examined the effect of parkin overexpression on the response of cells to various insults. Wild-type and AR-JP-associated mutant parkins (Del3-5, T240R, and Q311X) were overexpressed in NT-2 and SK-N-MC cells. Overexpressed wild-type parkin delayed cell death induced by serum withdrawal, H2O2, 1-methyl-4-phenylpyridinium (MPP+), or 4-hydroxy-2-trans-nonenal (HNE) but did not delay cell death caused by the proteasome inhibitor lactacystin. Increases in damage to proteins (protein carbonyls and 3-nitrotyrosine) were attenuated by wild-type parkin after serum withdrawal or exposure to H2O2, MPP+, or HNE but not after exposure to lactacystin. The mutant parkins (of all types) markedly accelerated cell death in response to all the insults, accompanied by increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation, and 3-nitrotyrosine and decreased levels of GSH. The viability loss induced by all the insults showed apoptotic features. The presence of parkin mutations in substantia nigra in Parkinson's disease may increase neuronal vulnerability to a range of toxic insults. © 2005 Wiley-Liss, Inc.

Inhibition of the proteasomal pathway for degrading abnormal proteins leads to protein aggregation, increased oxidative damage and increased protein nitration. We now show that interference with polyubiquitination has similar consequences. Expression of a dominant-negative mutant form of ubiquitin (K48R) in NT-2 and SK-N-MC cells caused decreased cell growth rates and increased oxidative damage (protein carbonyls and lipid peroxidation), nitric oxide production and elevated protein nitration. It also rendered cells highly sensitive to 4-hydroxy-2,3-trans-nonenal, a neurotoxic end-product of lipid peroxidation, hydrogen peroxide and deprivation of growth factors. Overexpression of wild-type ubiquitin did not produce these effects. Our data show that interference with the ubiquitin-proteasome pathway at a different point and by a different mechanism can produce many of the common features of human neurodegenerative diseases, such as increased lipid peroxidation, protein oxidation and protein nitration. We suggest that defects in this pathway at multiple points could produce the common features of neurodegenerative diseases, and that more such defects remain to be discovered.

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls. Bcl-2 transfectants of both cell lines were more resistant to H(2)O(2) and showed increases in GSH level and Cu/Zn-superoxide dismutase (SOD1) activity, but not in Mn-superoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SK-N-MC cells. Overexpression of Bcl-2 did not significantly decrease levels of oxidative DNA damage (measured as 8-hydroxyguanine) or lipid peroxidation, but it decreased levels of 3-nitrotyrosine in both cell lines and protein carbonyls in SK-N-MC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl-2 overexpression to decrease 3-nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species; this was confirmed as decreased production of NO(2)(-)/NO(3)(-) in the transfected cells and a fall in the level of nNOS protein.

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.