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Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways.

The PI3K/mTOR signaling cascade is fundamental in T-cell activation and fate decisions. We showed the distinct regulation of PI3K/mTOR in regulatory and effector T-cells and proposed the potential therapeutic benefit of targeting this pathway to control the balance between effector and regulatory T-cell activities. Substantial adverse effects in long-term clinical usage of Rapamycin suggest the use of alternative treatments in restraining effector T-cell function in transplant patients. We hypothesize that dual PI3K/mTOR inhibitors may represent an immunosuppressant alternative. Here we show that dual PI3K/mTOR PI-103 and PKI-587 inhibitors interfered IL-2-dependent responses in T-cells. However, in contrast to the inhibitory effects in non-Treg T-cell proliferation and effector functions, dual inhibitors increased the differentiation, preferential expansion and suppressor activity of iTregs. Rapamycin, PI-103 and PKI-587 targeted different signaling events and induced different metabolic patterns in primary T-cells. Similar to Rapamycin, in vivo administration of PI-103 and PKI-587 controlled effectively the immunological response against allogenic skin graft. These results characterize specific regulatory mechanisms of dual PI3K/mTOR inhibitors in T-cells and support their potential as a novel therapeutic option in transplantation. This article is protected by copyright. All rights reserved.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria following DOX treatment. A redox proteomics method involving 2D electrophoresis followed by mass spectrometry and investigation of protein data bases identified several HNE-modified mitochondria proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle (TCA) and electron transport chain (ETC). The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate (OCR) was also significantly reduced after DOX treatment. The treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnP, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

α-Tocopherol (vitamin E) is an essential dietary antioxidant with important neuroprotective functions. α-Tocopherol deficiency manifests primarily in neurological pathologies, notably cerebellar dysfunctions such as spinocerebellar ataxia. To study the roles of α-tocopherol in the cerebellum, we used the α-tocopherol transfer protein for the murine version (Ttpa(-/)(-)) mice which lack the α-tocopherol transfer protein (TTP) and are a faithful model of vitamin E deficiency and oxidative stress. When fed vitamin E-deficient diet, Ttpa(-/)(-) mice had un-detectable levels of α-tocopherol in plasma and several brain regions. Dietary supplementation with α-tocopherol normalized plasma levels of the vitamin, but only modestly increased its levels in the cerebellum and prefrontal cortex, indicating a critical function of brain TTP. Vitamin E deficiency caused an increase in cerebellar oxidative stress evidenced by increased protein nitrosylation, which was prevented by dietary supplementation with the vitamin. Concomitantly, vitamin E deficiency precipitated cellular atrophy and diminished dendritic branching of Purkinje neurons, the predominant output regulator of the cerebellar cortex. The anatomic decline induced by vitamin E deficiency was paralleled by behavioral deficits in motor coordination and cognitive functions that were normalized upon vitamin E supplementation. These observations underscore the essential role of vitamin E and TTP in maintaining CNS function, and support the notion that α-tocopherol supplementation may comprise an effective intervention in oxidative stress-related neurological disorders.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment, inability to perform activities of daily living and mood changes. Acetylcholinesterase inhibitors or NMDA glutamate receptor antagonists are currently used for the treatment of AD, but only the former have weak beneficial effects on cognitive function.The aim of this review is to provide an overview of the main pharmacological features of both current drugs and new compounds which are still under clinical development for the treatment of AD.The discovery of new drugs acting at the early stage of AD could be considered as a 'medical need' and inhibitors of γ-secretase or monoclonal antibodies against Aβ seemed good options. However, inhibitors of γ-secretase, that is, tarenflurbil or semagacestat, were discontinued due to their lack of cognitive improvement or unacceptable side effects. A careful evaluation of the risk:benefit ratio should be considered for monoclonal antibodies since, by increasing the disaggregation of fibrillar amyloid-β-peptide (Aβ), they could increase the neurotoxicity of soluble Aβ oligomers. In conclusion, the discovery of new drugs efficacious in AD subjects is an ambitious goal, however, and one that will require close, active collaboration by pharmacologists, chemists and clinicians.

Aging is characterized by a gradual and continuous loss of physiological functions and responses particularly marked in the central nervous system. Reactive oxygen species (ROS) can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since proteins are the major components of biological systems and regulate multiple cellular pathways, oxidative damage of key proteins are considered to be the principal molecular mechanisms leading to age-related deficits. Recent evidences support the notion that a decrease of energy metabolism in the brain contribute to neuronal loss and cognitive decline associated with aging. In the present study we identified selective protein targets which are oxidized in aged rats compared with adult rats. Most of the oxidatively modified proteins we found in the present study are key proteins involved in energy metabolism and ATP production. Oxidative modification of these proteins was associated with decreased enzyme activities. In addition, we also found decreased levels of thiol reducing system. Our study demonstrated that oxidative damage to specific proteins impairs energy metabolism and ATP production thus contributing to shift neuronal cells towards a more oxidized environment which ultimately might compromise multiple neuronal functions. These results further confirm that increased protein oxidation coupled with decreased reducing systems are characteristic hallmarks of aging and aging-related degenerative processes.

Alzheimer's disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

There is significant evidence to show that aging is characterized by a stochastic accumulation of molecular damage and by a progressive failure of maintenance and repair processes. Protective mechanisms exist in the brain which are controlled by vitagenes and include members of the heat shock system, heme oxygenase-I, and Hsp70 as critical determinants of brain stress tolerance. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and the present study reports that treatment for 4 months of senescent rats with acetyl-L-carnitine induces heme oxygenase-1 as well as Hsp70 and SOD-2. This effect was associated with upregulation of GSH levels, prevention of age-related changes in mitochondrial respiratory chain complex expression, and decrease in protein carbonyls and HNE formation. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Particularly, modulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimer's disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis. Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimer's disease, in fact, involves a chronic inflammatory response associated with both brain injury and beta-amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.

Recent studies suggest that NO and its reactive derivative peroxynitrite are implicated in the pathogenesis of multiple sclerosis (MS). Patients dying with MS demonstrate increased astrocytic inducible nitric oxide synthase activity, as well as increased levels of iNOS mRNA. Peroxynitrite is a strong oxidant capable of damaging target tissues, particularly the brain, which is known to be endowed with poor antioxidant buffering capacity. Inducible nitric oxide synthase is upregulated in the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. We have recently demonstrated in patients with active MS a significant increase of NOS activity associated with increased nitration of proteins in the cerebrospinal fluid (CSF). Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, in the present study, MS patients were treated for 6 months with acetylcarnitine and compared with untreated MS subjects or with patients noninflammatory neurological conditions, taken as controls. Western blot analysis showed in MS patients increased nitrosative stress associated with a significant decrease of reduced glutathione (GSH). Increased levels of oxidized glutathione (GSSG) and nitrosothiols were also observed. Interestingly, treatment of MS patients with acetylcarnitine resulted in decreased CSF levels of NO reactive metabolites and protein nitration, as well as increased content of GSH and GSH/GSSG ratio. Our data sustain the hypothesis that nitrosative stress is a major consequence of NO produced in MS-affected CNS and implicate a possible important role for acetylcarnitine in protecting brain against nitrosative stress, which may underlie the pathogenesis of MS.

Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta-peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.

Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3e, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70 kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2.

Recent evidence indicates that alterations in brain polyamine metabolism may be critical for nerve cell survival after a free radical initiated neurodegenerative process. It has been shown previously that A beta(1-42) and A beta(25-35) are toxic to neurons through a free radical dependent oxidative mechanism. Treatment of rat embryonic hippocampal neuronal cultures with A beta-peptides increased ornithine decarboxylase (ODC) activity and spermidine uptake, suggesting that oxidative stress upregulates the polyamine mechanism for the repair of free radical damage. Pretreatment of the cells with vitamin E prior to A beta exposure decreased ODC activity and spermidine uptake to control level. This study is the first to demonstrate that A beta treated cells show an increased polyamine metabolism in response to free radical mediated oxidative stress and that the free radical scavenger vitamin E prevents these attenuations. These results are discussed with reference to Alzheimer's disease.

Amyloid beta-peptide (Abeta), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of Abeta occurs in conjunction with free radical oxidative stress associated with the peptide. Abeta(1-40) and several other fragments of the Abeta sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca2+ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that Abeta-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate Abeta(25-35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated Abeta toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD.

Peroxynitrite (ONOO ) is a highly reactive, oxidizing anion with a half-life of

Amyloid beta-peptide (Abeta) is a key factor in the neurotoxicity of Alzheimer's disease (AD). Recent research has shown that Abeta-mediated neurotoxicity involves free radicals and that Abeta peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of Abeta peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with Abeta or HNE. The free radical dependence of beta-amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of Abeta. In contrast, HNE was still toxic in the presence of vitamin E. These results support our Abeta-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD.

Amyloid beta-peptide (Abeta), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown that the neurotoxicity of Abeta occurs in conjunction with free-radical oxidative stress associated with the peptide. In the present study, we investigated the temporal relations among the formation of Abeta-associated free radicals, the oxidative damage to, and the activation of antioxidant defense mechanisms in rat embryonic hippocampal neuronal culture subjected to toxic Abeta(25-35). Temporal electron paramagnetic resonance (EPR) spectroscopy results show that synthetic Abeta(25-35) forms free radicals rapidly after solubilization with a high signal intensity at initial time points. At those time points, neuronal toxicity and oxidative stress gradually increase as assessed by reduction of 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide, trypan blue exclusion, formation of reactive oxygen species, and detection of protein carbonyl levels. The latter occurs before neurotoxicity. When the EPR signal intensity of Abeta solution decreases at later time points, neuronal toxicity levels off and remains the same until the end of the experiment. The oxidative-sensitive enzyme creatine kinase (CK) (brain isoform) (CK-BB) content increases at initial points of the Abeta treatment in correlation with the EPR signal to keep the CK activity constant, presumably to overcome the Abeta-induced oxidative insult. CK-BB content returns to normal levels by the end of the experiment. CK activity normalized to CK content implies the presence of inactivated CK molecules during the treatment. Both Mn SOD and Cu/Zn superoxide dismutase (SOD) mRNA levels show robust increases initially, which later return to control level with decreasing oxidative insult. These results are consistent with the notion that Abeta(25-35) promotes a rapid free-radical oxidative stress to neurons, which respond by modulating various oxidative stress-handling genes.

The effect of aging and diet restriction (DR) on the activity of creatine kinase (CK), glutamine synthetase (GS) and protein carbonyl formation in the cerebellum, hippocampus and cortex of male and female brown Norway (BN) rats has been investigated. It was demonstrated that CK activity in three different regions of the rat brain declines with age by 30%. Age-related decrease of GS activity was only 10-13% and did not reach statistical significance. Consistent with previously published studies, age-related increase of protein carbonyl content in each brain area studied has been observed. Preventive effects of a caloric restricted diet on the age-associated protein oxidation and changes of the activity of CK and GS in the brain was observed for both aging male and female BN rats. DR delayed the accumulation of protein carbonyls. Age-related changes of CK activity in rat brain were abrogated by DR. The activity of GS in the brain of old rats subjected to the caloric restricted diet was higher than that in the brain of young animals fed ad libitum. The results are consistent with the notion that DR may relieve age-associated level of oxidative stress and lessen protein damage.

Alzheimer disease (AD) is one of the most disabling disorders of the elderly and the number of people worldwide facing dementia is expected to dramatically increase in the near future. Thus, one of the major concerns of modern society is to identify putative biomarkers that serve as a valuable early diagnostic tool to identify a subset of patients with increased risk to develop AD. An ideal biomarker should be present in blood before dementia is clinically confirmed, have high sensitivity and specificity, and be reproducible. Proteomics platforms offer a powerful strategy to reach these goals and recently have been demonstrated to be promising approaches. However, the high variability of technologies and studied populations has led to contrasting results. To increase specificity, we analyzed both protein expression profiles and oxidative modifications (carbonylation) of plasma proteins in mild cognitive impairment (MCI) and AD subjects compared with age-matched controls. Most of the proteins found to have differential levels in MCI and AD confirmed results already obtained in other cohort studies. Interestingly, we applied for the first time in MCI a redox proteomics approach to specifically identify oxidized proteins. Among them, haptoglobin, one of the most abundantly secreted glycoproteins with chaperone function, was found to be either increasingly downregulated or increasingly oxidized in AD and MCI compared with controls. We also demonstrated that in vitro oxidation of haptoglobin affects the formation of amyloid-β fibrils, thus suggesting that oxidized haptoglobin is not able to act as an extracellular chaperone to prevent or slow formation of amyloid-β aggregates. Another chaperone protein, α2-macroglobulin, was found to be selectively oxidized in AD patients compared with controls. Our findings suggest that alterations in proteins acting as extracellular chaperones may contribute to exacerbating amyloid-β toxicity in the peripheral system and may be considered a putative marker of disease progression.

In order to study oxidative stress in peripheral cells of Alzheimer's disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named "unfolded p53", was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients.

Evidence is presented that free radical stress can directly induce physico-chemical alterations in rodent neocortical synaptosomal membrane proteins. Synaptosomes were prepared from gerbil cortical brain tissue and incubated with 3 mM ascorbate and various concentrations of exogenous Fe2+ for 30-240 min at 37 degrees C. Synaptosomes were then lysed and covalently labeled with the protein thiol-selective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperdin-1-oxyl) and subjected to electron paramagnetic resonance (EPR) spectrometry. In separate experiments, synaptosomal membranes were labeled with the thiol-specific spin label MTS ((1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate), or the lipid-specific spin probe 5-NS (5-nitroxide stearate). Free radical stress induced by iron/ascorbate treatment has a rigidizing effect on the protein infrastructure of these membranes, as appraised by EPR analysis of membrane protein-bound spin label, but no change was detected in the lipid component of the membrane. These results are discussed with reference to potential oxidative mechanisms in aging and neurological disorders.

The acridine-based, potential Alzheimer's disease therapeutic agents, tacrine and velnacrine, were incubated with rat or gerbil neocortical synaptosomal membranes. Electron paramagnetic resonance employing a protein-specific spin label was used to monitor this interaction. Analogous to their effects in erythrocyte membranes [Butterfield and Rangachari (1992) Biochem. Biophys. Res. Commun. 185: 596–603], in the present studies both agents decreased segmental motion of spin labeled synaptosomal membrane proteins, consistent with increased cytoskeletal protein-protein interactions (0.001

Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid-β peptide (Aβ) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) [PDAPP mice, line J20], as evidenced by increased levels of protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-nonenal. This oxidative damage was dependent on the methionine 35 residue within the Aβ peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discovery-based proteomics studies; therefore, two-dimensional gel-based expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with non-transgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, ρ GDP-dissociation inhibitor 1, T-complex protein 1 subunit α A, α-enolase, peptidyl-prolyl cis-trans isomerase (Pin-1), and ATP synthase subunit α mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidatively-modified proteins: phosphatidylethanolamine-binding protein 1 and Pin-1 with decreased levels of protein 3-nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of Aβ deposition and clues with which to further direct studies on elucidating AD pathogenesis.

Engineered nanoscale ceria is used as a diesel fuel catalyst. Little is known about its mammalian central nervous system effects. The objective of this paper is to characterize the biodistribution of a 5-nm citrate-stabilized ceria dispersion from blood into brain and its pro- or antioxidant effects. An approximately 4% aqueous ceria dispersion was iv infused into rats (0, 100, and up to 250 mg/kg), which were terminated after 1 or 20 h. Ceria concentration, localization, and chemical speciation in the brain were assessed by inductively coupled plasma mass spectrometry, light and electron microscopy (EM), and electron energy loss spectroscopy (EELS). Pro- or antioxidative stress effects were assessed as protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-trans-nonenal in hippocampus, cortex, and cerebellum. Glutathione reductase, glutathione peroxidase, manganese superoxide dismutase, and catalase levels and activities were measured in hippocampus. Catalase levels and activities were also measured in cortex and cerebellum. Na fluorescein and horseradish peroxidase (HRP) were given iv as blood-brain barrier (BBB) integrity markers. Mortality was seen after administration of 175-250 mg ceria/kg. Twenty hours after infusion of 100 mg ceria/kg, brain HRP was marginally elevated. EM and EELS revealed mixed Ce(III) and Ce(IV) valence in the freshly synthesized ceria in vitro and in ceria agglomerates in the brain vascular compartment. Ceria was not seen in microvascular endothelial or brain cells. Ceria elevated catalase levels at 1 h and increased catalase activity at 20 h in hippocampus and decreased catalase activity at 1 h in cerebellum. Compared with a previously studied approximately 30-nm ceria, this ceria was more toxic, was not seen in the brain, and produced little oxidative stress effect to the hippocampus and cerebellum. The results are contrary to the hypothesis that a smaller engineered nanomaterial would more readily permeate the BBB.

Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as "chemobrain." While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor alpha (TNF-alpha), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive dysfunction.

Background The UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS), which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes. Results The expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized. Conclusions Despite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional) proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.

This study points out different behaviour between HEK cells overexpressing wild-type or mutant APP when exposed to oxidative insult. Although apparently both APPwt and APPmut overexpression conferred resistance to oxidative insult, some differences in terms of degree of protection was observed in the two clones. We found that the two clones differed, especially, in terms of redox profile. HEK-APPmut cells were characterized by higher levels of oxidative markers in comparison with HEK-APPwt. In addition, SOD activity appeared more efficient in HEK-APPwt than in HEK-APPmut, thus justifying the differences in terms of cell survival in the two clones. We suggest that, according to “hormesis theory”, in HEK-APPwt cells low amount of oxidative stress can exert a beneficial effect that at a higher intensity results harmful. In contrast, HEK-APPmut cells lost this stress resistance probably because the degree of oxidative stress is too high and the antioxidant enzymes are themselves compromised.

Tumor necrosis factor-alpha (TNF-alpha), a ubiquitous pro-inflammatory cytokine, is an important mediator in the immune-neuroendocrine system that affects the CNS. The present study demonstrates that treatment with TNF-alpha activates microglia to increase TNF-alpha production in primary cultures of glial cells isolated from wild-type (WT) mice and mice deficient in the inducible form of nitric oxide synthase (iNOSKO). However, mitochondrial dysfunction in WT neurons occurs at lower concentrations of TNF-alpha when neurons are directly treated with TNF-alpha or co-cultured with TNF-alpha-treated microglia than iNOSKO neurons similarly treated. Immunofluorescent staining of primary neurons co-cultured with TNF-alpha-treated microglia reveals that the antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), is co-localized with nitrotyrosine in WT but not in iNOSKO primary neuronal cells. Importantly, the percentage of surviving neurons is significantly reduced in WT neurons compared with iNOSKO neurons under identical treatment conditions. Together, the results suggest that TNF-alpha activates microglia to produce high levels of TNF-alpha and that production of nitric oxide (NO) in neurons is an important factor affecting MnSOD nitration and subsequent mitochondrial dysfunction.

Acrolein, the most reactive of the alpha,beta-unsaturated aldehydes, is endogenously produced by lipid peroxidation, and has been found increased in the brain of patients with Alzheimer's disease. Although it is known that acrolein increases total protein carbonylation and impairs the function of selected proteins, no study has addressed which proteins are selectively carbonylated by this aldehyde. In this study we investigated the effect of increasing concentrations of acrolein (0, 0.005, 0.05, 0.5, 5, 50 microM) on protein carbonylation in gerbil synaptosomes. In addition, we applied proteomics to identify synaptosomal proteins that were selectively carbonylated by 0.5 microM acrolein. Acrolein increased total protein carbonylation in a dose-dependent manner. Proteomic analysis (two-dimensional electrophoresis followed by mass spectrometry) revealed that tropomyosin-3-gamma isoform 2, tropomyosin-5, beta-actin, mitochondrial Tu translation elongation factor (EF-Tu(mt)) and voltage-dependent anion channel (VDAC) were significantly carbonylated by acrolein. Consistent with the proteomics studies that have identified specifically oxidized proteins in Alzheimer's disease (AD) brain, the proteins identified in this study are involved in a wide variety of cellular functions including energy metabolism, neurotransmission, protein synthesis, and cytoskeletal integrity. Our results suggest that acrolein may significantly contribute to oxidative damage in AD brain.

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease. One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide, which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Amyloid beta-peptide is the main component of senile plaques and generates free radicals ultimately leading to neuronal damage of membrane lipids, proteins and nucleic acids. Therefore, interest in the protective role of different antioxidant compounds has been growing for treatment of Alzheimer's disease and other oxidative stress-related disorders. Among different antioxidant drugs, much interest has been devoted to "thiol-delivering" compounds. Tricyclodecan-9-yl-xanthogenate is an inhibitor of phosphatidylcholine specific phospholipase C, and recent studies reported its ability to act as a glutathione-mimetic compound. In the present study, we investigate the in vivo ability of tricyclodecan-9-yl-xanthogenate to protect synaptosomes against amyloid beta-peptide-induced oxidative stress. Gerbils were injected i.p. with tricyclodecan-9-yl-xanthogenate or with saline solution, and synaptosomes were isolated from the brain. Synaptosomal preparations isolated from tricyclodecan-9-yl-xanthogenate injected gerbils and treated ex vivo with amyloid beta-peptide (1-42) showed a significant decrease of oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3-nitrotyrosine levels) and lipid peroxidation (4-hydroxy-2-nonenal levels). Our results are consistent with the hypothesis that modulation of free radicals generated by amyloid beta-peptide might represent an efficient therapeutic strategy for treatment of Alzheimer's disease and other oxidative-stress related disorders. Based on the above data, we suggest that tricyclodecan-9-yl-xanthogenate is a potent antioxidant and could be of importance for the treatment of Alzheimer's disease and other oxidative stress-related disorders.

Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFgamma-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.

The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and alpha-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, alpha-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

Summary. Proteomics offers the opportunity elucidate the complex protein interactions of cellular systems by studying the products of genes, i.e., proteins, and their structure, function and localization. The purpose of proteomics is to explain the information contained in the genome sequences in order to provide clues on cellular events, especially related to disease. Our proteomic approach has made possible the identification of specifically oxidized proteins in Alzheimer’s disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in ADrelated neurodegeneration. This represents an example of the use of proteomics to solve biological problems related to disease. The field, which is still in its infancy, represents a very promising way to elucidate mechanism of disease at a protein level. However, the techniques that support its development present several limitations and require introduction of new tools and innovation in order to achieve a fast, reliable and sensitive method to understand normal biological processes and their regulation as well as these cellular properties in disease.

Oxidative stress induced by amyloid beta-peptide (A beta) has been implicated in the neurodegeneration observed in Alzheimer's disease (AD) brain. However, the mechanism by which the predominant form of A beta found in AD brains, A beta(1--42), causes oxidative stress and neurotoxicity remains unknown. Numerous laboratories have used the smaller 11-amino acid fragment of the full-length peptide, A beta(25--35), as a convenient alternative in AD investigations since the smaller peptide mimics several of the toxicological and oxidative stress properties of the native full-length peptide. Our observation that the truncated peptide is more rapidly toxic and causes more oxidative damage than the parent A beta(1--42) led us to investigate the cause for this enhanced toxicity of A beta(25--35) in order to gain insight into the mechanism of action of these peptides. These studies reveal that two different mechanisms may be operative in the two peptides; however, the single methionine residue in the peptides appears to play a crucial role in both mechanisms. That methionine is C-terminal in A beta(25--35) seems to be the cause for its exaggerated effects. When the next amino acid in the sequence of A beta(1--42) (valine) is appended to A beta(25--35), the resultant peptide, A beta(25--36), in which methionine is no longer C-terminal, is neither toxic to cultured neurons nor does it cause oxidative damage. Additionally, oxidizing the sulfur of methionine to a sulfoxide abrogates the damaging effects of both A beta(25--35) and A beta(1--42). The putative mechanistic role of methionine in the observed properties of A beta peptides is discussed in the context of the obtained results as is the role of A beta(1--42)-induced oxidative stress in the neurodegeneration found in AD brain.

Creatine kinase (CK) BB, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells. CK BB levels are reduced in brain regions affected by neurodegeneration in Alzheimer's disease (AD), Pick's disease, and Lewy body dementia, and this reduction is not a result of decreased mRNA levels. This study demonstrates that posttranslational modification of CK BB plays a role in the decrease of CK activity in AD brain. The specific CK BB activity and protein carbonyl content were determined in brain extracts of six AD and six age-matched control subjects. CK BB activity per microgram of immunoreactive CK BB protein was lower in AD than in control brain extracts, indicating the presence of inactive CK BB molecules. The analysis of specific protein carbonyl levels in CK BB, performed by two-dimensional fingerprinting of oxidatively modified proteins, identified CK BB as one of the targets of protein oxidation in the AD brain. The increase of protein carbonyl content in CK BB provides evidence that oxidative posttranslational modification of CK BB plays a role in the loss of CK BB activity in AD.

Amyloid beta-peptide (Abeta), the major constituent in senile plaques in Alzheimer's disease (AD) brain, is thought by many researchers to be central to neurotoxicity in AD brain. Increasing evidence from many laboratories indicates that AD brain is under oxidative stress, with strong evidence of protein oxidation, lipid peroxidation, and peroxynitrite damage. A link between the central role of Abeta and oxidative stress in AD brain may be Abeta-associated free radical oxidative stress. If so, antioxidants such as vitamin E should modulate Abeta-induced oxidative damage and neurotoxicity in brain cells. This review summarizes studies of Abeta-associated free radical oxidative stress and its inhibition by vitamin E in cortical synaptosomal membranes and hippocampal neuronal cells in culture. Taken together with the recent report that vitamin E slows the progression of AD, this review strongly supports a central role of Abeta-associated free radical oxidative stress in neurotoxicity in AD brain.

beta-Amyloid peptide (A beta), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of A beta form free radical species in aqueous solution. It was proposed that A beta-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic A beta can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic A beta(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by A beta, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of A beta is not iron or peroxide-dependent. Our present observations consistently show that toxic A beta does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that A beta(25-35) induces carbonyl formation in BSA. Our results demonstrate that beta-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.

The amyloid beta-peptide (A beta) that accumulates as insoluble plaques in the brain in Alzheimer's disease can be directly neurotoxic and can increase neuronal vulnerability to excitotoxic insults. The mechanism of A beta toxicity is unclear but is believed to involve generation of reactive oxygen species (ROS) and loss of calcium homeostasis. We now report that exposure of cultured rat hippocampal neurons to A beta 1–40 or A beta 25–35 causes a selective reduction in Na+/K(+)-ATPase activity which precedes loss of calcium homeostasis and cell degeneration. Na+/K(+)-ATPase activity was reduced within 30 min of exposure to A beta 25–35 and declined to less than 40% of basal level by 3 hr. A beta did not impair other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. Experiments with ouabain, a specific inhibitor of the Na+/K(+)-ATPase, demonstrated that impairment of this enzyme was sufficient to induce an elevation of [Ca2+]i and neuronal injury. Impairment of Na+/K(+)-ATPase activity appeared to be causally involved in the elevation of [Ca2+]i and neurotoxicity since suppression of Na+ influx significantly reduced A beta- and ouabain-induced [Ca2+]i elevation and neuronal death. Neuronal degeneration induced by ouabain appeared to be of an apoptotic form as indicated by nuclear condensation and DNA fragmentation. The antioxidant free radical scavengers vitamin E and propylgallate significantly attenuated A beta- induced impairment of Na+/K(+)-ATPase activity, elevation of [Ca2+]i and neurotoxicity, suggesting a role for ROS. Finally, exposure of synaptosomes from postmortem human hippocampus to A beta resulted in a significant and specific reduction in Na+/K(+)-ATPase and Ca(2+)-ATPase activities, without affecting other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. These data suggest that impairment of ion-motive ATPases may play a role in the pathogenesis of neuronal injury in Alzheimer's disease.

We have previously reported (Hensley et al., Proc. Natl. Acad. Sci. USA (1994) in press) that beta-amyloid peptide fragments in aqueous media, in a metal-independent reaction, produce reactive peptide free radicals and reactive oxygen species. In contrast to the hours or days necessary to produce neurotoxicity and a detectable free radical for beta-amyloid, the extremely neurotoxic A beta(25-35) fragment of beta-amyloid peptide produces a detectable radical in minutes. We now report that A beta(25-35) is a potent lipoperoxidation initiator, as inferred from peptide-mediated reduction of nitroxyl stearate spin labels bound to rodent neocortical synaptosomal membranes. A beta(25-35) rapidly quenches the paramagnetism of membrane-bound 12-nitroxyl stearate spin probe deep within the lipid bilayer, but reacts poorly with the 5-nitroxyl isomer whose paramagnetic center is near the lipid/water interface. A beta(35-25), the non-neurotoxic reverse sequence of A beta(25-35), shows little proclivity to reduce either spin label. These findings are formulated into a "molecular shrapnel" model of neuronal membrane damage in Alzheimer's disease.

Alzheimer's disease (AD), the major dementing disorder of the elderly, is associated with cholinergic neuronal loss and decreased activity of choline acetyltransferase (CAT). Previous biophysical studies had suggested an altered conformation of membrane proteins in AD erythrocyte ghosts. Since erythrocytes have a choline transport system and cholinergic neurons are implicated in AD, the present experiments were undertaken to determine if the efflux rate of [14C]choline was altered in AD erythrocytes. The mean efflux rate constant was highly significantly increased (P less than 0.01) by greater than 25% in 9 drug-free AD patients compared to 9 sex-matched, drug-free controls of similar age. These results are discussed in terms of potential molecular mechanisms to account for cholinergic neuronal loss in AD.