Showing papers on "Oxidative stress published in 2002"

Free Radicals in the Physiological Control of Cell Function

Abstract: At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

Cellular response to oxidative stress: signaling for suicide and survival.

Abstract: Reactive oxygen species (ROS), whether produced endogenously as a consequence of normal cell functions or derived from external sources, pose a constant threat to cells living in an aerobic environment as they can result in severe damage to DNA, protein, and lipids. The importance of oxidative damage to the pathogenesis of many diseases as well as to degenerative processes of aging has becoming increasingly apparent over the past few years. Cells contain a number of antioxidant defenses to minimize fluctuations in ROS, but ROS generation often exceeds the cell's antioxidant capacity, resulting in a condition termed oxidative stress. Host survival depends upon the ability of cells and tissues to adapt to or resist the stress, and repair or remove damaged molecules or cells. Numerous stress response mechanisms have evolved for these purposes, and they are rapidly activated in response to oxidative insults. Some of the pathways are preferentially linked to enhanced survival, while others are more frequently associated with cell death. Still others have been implicated in both extremes depending on the particular circumstances. In this review, we discuss the various signaling pathways known to be activated in response to oxidative stress in mammalian cells, the mechanisms leading to their activation, and their roles in influencing cell survival. These pathways constitute important avenues for therapeutic interventions aimed at limiting oxidative damage or attenuating its sequelae.

Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification.

Abstract: Reactive oxygen species (ROS) and other radicals are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degenerative and other diseases, inflammation, aging, and development. ROS are well recognized for playing a dual role as deleterious and beneficial species. The objectives of this review are to describe oxidative stress phenomena, terminology, definitions, and basic chemical characteristics of the species involved; examine the biological targets susceptible to oxidation and the defense mechanisms of the organism against these reactive metabolites; and analyze methodologies, including immunohistochemical markers, used in toxicological pathology in the visualization of oxidative stress phenomena. Direct detection of ROS and other free radicals is difficult, because these molecules are short-lived and highly reactive in a nonspecific manner. Ongoing oxidative damage is, thus, generally analyzed by measurement of secondary products including derivatives of amino acids, nuclei acids, and lipid peroxidation. Attention has been focused on electrochemical methods based on voltammetry measurements for evaluating the total reducing power of biological fluids and tissues. This approach can function as a tool to assess the antioxidant-reducing profile of a biological site and follow changes in pathological situations. This review thus includes different topics essential for understanding oxidative stress phenomena and provides tools for those intending to conduct study and research in this field.

Plant responses to abiotic stresses: heavy metal‐induced oxidative stress and protection by mycorrhization

Abstract: The aim of this review is to assess the mode of action and role of antioxidants as protection from heavy metal stress in roots, mycorrhizal fungi and mycorrhizae. Based on their chemical and physical properties three different molecular mechanisms of heavy metal toxicity can be distinguished: (a) production of reactive oxygen species by autoxidation and Fenton reaction; this reaction is typical for transition metals such as iron or copper, (b) blocking of essential functional groups in biomolecules, this reaction has mainly been reported for non-redox-reactive heavy metals such as cadmium and mercury, (c) displacement of essential metal ions from biomolecules; the latter reaction occurs with different kinds of heavy metals. Transition metals cause oxidative injury in plant tissue, but a literature survey did not provide evidence that this stress could be alleviated by increased levels of antioxidative systems. The reason may be that transition metals initiate hydroxyl radical production, which can not be controlled by antioxidants. Exposure of plants to non-redox reactive metals also resulted in oxidative stress as indicated by lipid peroxidation, H(2)O(2) accumulation, and an oxidative burst. Cadmium and some other metals caused a transient depletion of GSH and an inhibition of antioxidative enzymes, especially of glutathione reductase. Assessment of antioxidative capacities by metabolic modelling suggested that the reported diminution of antioxidants was sufficient to cause H(2)O(2) accumulation. The depletion of GSH is apparently a critical step in cadmium sensitivity since plants with improved capacities for GSH synthesis displayed higher Cd tolerance. Available data suggest that cadmium, when not detoxified rapidly enough, may trigger, via the disturbance of the redox control of the cell, a sequence of reactions leading to growth inhibition, stimulation of secondary metabolism, lignification, and finally cell death. This view is in contrast to the idea that cadmium results in unspecific necrosis. Plants in certain mycorrhizal associations are less sensitive to cadmium stress than non-mycorrhizal plants. Data about antioxidative systems in mycorrhizal fungi in pure culture and in symbiosis are scarce. The present results indicate that mycorrhization stimulated the phenolic defence system in the Paxillus-Pinus mycorrhizal symbiosis. Cadmium-induced changes in mycorrhizal roots were absent or smaller than those in non-mycorrhizal roots. These observations suggest that although changes in rhizospheric conditions were perceived by the root part of the symbiosis, the typical Cd-induced stress responses of phenolics were buffered. It is not known whether mycorrhization protected roots from Cd-induced injury by preventing access of cadmium to sensitive extra- or intracellular sites, or by excreted or intrinsic metal-chelators, or by other defence systems. It is possible that mycorrhizal fungi provide protection via GSH since higher concentrations of this thiol were found in pure cultures of the fungi than in bare roots. The development of stress-tolerant plant-mycorrhizal associations may be a promising new strategy for phytoremediation and soil amelioration measures.

Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage.

Abstract: The objectives of this study were to determine whether differences in the size and composition of coarse (2.5-10 micro m), fine (< 2.5 microm), and ultrafine (< 0.1 microm) particulate matter (PM) are related to their uptake in macrophages and epithelial cells and their ability to induce oxidative stress. The premise for this study is the increasing awareness that various PM components induce pulmonary inflammation through the generation of oxidative stress. Coarse, fine, and ultrafine particles (UFPs) were collected by ambient particle concentrators in the Los Angeles basin in California and used to study their chemical composition in parallel with assays for generation of reactive oxygen species (ROS) and ability to induce oxidative stress in macrophages and epithelial cells. UFPs were most potent toward inducing cellular heme oxygenase-1 (HO-1) expression and depleting intracellular glutathione. HO-1 expression, a sensitive marker for oxidative stress, is directly correlated with the high organic carbon and polycyclic aromatic hydrocarbon (PAH) content of UFPs. The dithiothreitol (DTT) assay, a quantitative measure of in vitro ROS formation, was correlated with PAH content and HO-1 expression. UFPs also had the highest ROS activity in the DTT assay. Because the small size of UFPs allows better tissue penetration, we used electron microscopy to study subcellular localization. UFPs and, to a lesser extent, fine particles, localize in mitochondria, where they induce major structural damage. This may contribute to oxidative stress. Our studies demonstrate that the increased biological potency of UFPs is related to the content of redox cycling organic chemicals and their ability to damage mitochondria.

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress

Abstract: Reactive oxygen species are required for cell proliferation but can also induce apoptosis1. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis2. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans3,4,5,6, and we conclude that this model also extends to mammalian systems.

Regulation and function of ascorbate peroxidase isoenzymes

Abstract: Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.

Signal transduction during oxidative stress

Abstract: As an unfortunate consequence of aerobic life, active oxygen species (AOS) are formed by partial reduction of molecular oxygen. Plants possess a complex battery of enzymatic and non-enzymatic antioxidants that can protect cells from oxidative damage by scavenging AOS. It is becoming evident that AOS, which are generated during pathogen attack and abiotic stress situations, are recognized by plants as a signal for triggering defence responses. An overview of the literature is presented on the signalling role of AOS in plant defence responses, cell death, and development. Special attention is given to AOS and redox-regulated gene expression and the role of kinases and phosphatases in redox signal transduction.

Water stress‐induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up‐regulates the activities of antioxidant enzymes in maize leaves

Abstract: The interrelationship among water-stress-induced abscisic acid (ABA) accumulation, the generation of reactive oxygen species (ROS), and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) was investigated in leaves of detached maize (Zea mays L.) plants exposed to -0.7 MPa water stress induced by polyethylene glycol (PEG 6000). Time-course analyses of ABA content, the production of ROS, and the activities of antioxidant enzymes in water-stressed leaves showed that a significant increase in the content of ABA preceded that of ROS, which was followed by a marked increase in the activities of these antioxidant enzymes. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA, and also reduced the increased generation of ROS and the up-regulation of these antioxidant enzymes in water-stressed leaves. A mild oxidative stress induced by paraquat, which generates O - 2 and then H 2 O 2 , resulted In a significant enhancement in the activities of antioxidant enzymes in non-water-stressed leaves. Pretreatment with some ROS scavengers, such as Tiron and dimethylthiourea (DMTU), and an inhibitor of NAD(P)H oxidase, diphenyleneiodonium (DPI), almost completely arrested the increase In ROS and the activities of these antioxidant enzymes induced by water stress or ABA treatment. These data suggest that water stress-Induced ABA accumulation triggers the increased generation of ROS, which, In turn, leads to the up-regulation of the antioxidant defence system.

Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra.

Abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra (p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO-) or hydroxyl radicals (OH.), or to be a prooxidant effect of treatment with L-Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.

Oxidative stress and antioxidative system in plants

Abstract: Free radicals and other active derivatives of oxygen are inevitable by-products of biological redox reactions. Reactive oxygen species inactivate enzymes and damage important cellular components. The increased production of toxic oxygen derivatives is considered to be a universal or common feature of stress conditions. Plant and other organisms have evolved a wide range of mechanisms to contend with this problem. The antioxidant defence system of the plant comprises a variety of antioxidant molecules and enzymes. The effects of the action of free radicals on membranes include the induction of lipid peroxidation and fatty acid de-esterification. Both ethylene biosynthesis and membrane breakdown, which appear to be closely linked, seem to involve free radicals, although the sequence of events generating these free radicals is still poorly understood. It is clear that the capacity and activity of the antioxidative defence system are important in limiting oxidative damage and in destroying active oxygen species that are produced in excess of those normally required for metabolism. Transgenic plants offered us a means by which to achieve complete understanding of the roles of the enzymes involved in protection against stress of many types, environmental and induced. Studies on transformed plants expressing increased activities of single enzymes of the antioxidative defence system indicate that it is possible to confer a degree of tolerance to stress by these means. The advent of plant transformation has placed within our grasp the possibility of engineering greater stress tolerance in plants by enhancements of the antioxidative defence system.

Amyloid β-peptide (1-42)-induced Oxidative Stress and Neurotoxicity: Implications for Neurodegeneration in Alzheimer's Disease Brain. A Review

Abstract: Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid beta-peptide (1-42) [Abeta(1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated Abeta(1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of Abeta(1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of Abeta(1-42) is helical, and invoking the i + 4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of Abeta(1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of Abeta(1-42). The free radical scavenger vitamin E prevented A(beta (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for Abeta-associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to Abeta(1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). Abeta inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production (alpha-enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of Abeta(1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of Abeta(1-42)-induced oxidative stress and neurodegeneration in AD.

Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients.

Abstract: We examined the pancreatic islet lesions in Japanese patients with Type II diabetes mellitus to determine if the damage was related to oxidative stress. Morphometric analyses were performed on immunostained sections of the tail portion of the pancreas from 14 diabetic and 15 non-diabetic patients. Amyloid deposition and oxidative stress-induced tissue damage were evaluated by Congo-red staining and immunostaining. Resistance to oxidative stress was assessed from immunostaining results for Cu, Zn-superoxide dismutase (SOD). Expression of (pro)insulin mRNA was assessed by in situ hybridisation. The pancreas from diabetic patients had amyloid deposition in about 15 % of the islets, intensified reactions of 8-OHdG and HNE, as well as reduced expression of SOD. Islet volume density of beta cells and total beta-cell mass in the pancreas from diabetic patients were reduced by 22 % (p < 0.001) and 30 % (p < 0.05). Islet volume density and total mass of (pro)insulin mRNA-positive cells were similarly reduced in diabetic patients by 22 % (p < 0.001) and 39 % (p < 0.05), respectively. Islet volume density of A cells was increased by 20 % (p < 0.001) but total mass did not change. There were no changes in volume densities of islet, D and PP cells. Reduced beta-cell volume density correlated with increased positive staining of 8-OHdG. Japanese Type II diabetic patients show a reduction of beta-cell mass and evidence of increased oxidative stress-related tissue damage that is correlated with the extent of the beta-cell lesions. [Diabetologia (2002) 45: 85–96]

Role of NRF2 in protection against hyperoxic lung injury in mice

Abstract: NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95-98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2-/-) and wild-type mice (Nrf2+/+). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2-/- mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2+/+ mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2+/+ mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.

Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease.

Abstract: Recent epidemiological and clinical data suggest that persons with low folic acid levels and elevated homocysteine levels are at increased risk of Alzheimer's disease (AD), but the underlying mechanism is unknown. We tested the hypothesis that impaired one-carbon metabolism resulting from folic acid deficiency and high homocysteine levels promotes accumulation of DNA damage and sensitizes neurons to amyloid β-peptide (Aβ) toxicity. Incubation of hippocampal cultures in folic acid-deficient medium or in the presence of methotrexate (an inhibitor of folic acid metabolism) or homocysteine induced cell death and rendered neurons vulnerable to death induced by Aβ. Methyl donor deficiency caused uracil misincorporation and DNA damage and greatly potentiated Aβ toxicity as the result of reduced repair of Aβ-induced oxidative modification of DNA bases. When maintained on a folic acid-deficient diet, amyloid precursor protein (APP) mutant transgenic mice, but not wild-type mice, exhibited increased cellular DNA damage and hippocampal neurodegeneration. Levels of Aβ were unchanged in the brains of folate-deficient APP mutant mice. Our data suggest that folic acid deficiency and homocysteine impair DNA repair in neurons, which sensitizes them to oxidative damage induced by Aβ.

Oxidative stress and male infertility: from research bench to clinical practice.

Abstract: Extensive research in our center at the Cleveland Clinic indicates that the seminal oxidative stress test has diagnostic and prognostic capabilities beyond those of conventional tests of sperm quality or functions. An oxidative stress test can accurately discriminate between fertile and infertile men and identify patients with a clinical diagnosis of male-factor infertility who are likely to initiate a pregnancy if they are followed over a period of time. In addition, the test can help select subgroups of patients with infertility in which oxidative stress is a significant factor, and who may benefit from antioxidant supplementation. Incorporation of such a test into routine andrology laboratory practice may be of particular importance to the future management of male infertility. In recent years, the generation of reactive oxygen species (ROS) in the male reproductive tract has become a real concern because of their potential toxic effects at high levels on sperm quality and function. ROS are highly reactive oxidizing agents belonging to the class of free radicals (Aitken, 1994). A free radical is defined as ‘‘any atom or molecule that possesses one or more unpaired electrons’’ (Warren et al, 1987). Recent reports have indicated that high levels of ROS are detected in semen samples of 25% to 40% of infertile men (de Lamirande et al, 1995; Padron et al, 1997). However, a strong body of evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities (Aitken, 1999). Spermatozoa, like all cells living in aerobic conditions, constantly face the oxygen (O2) paradox: O2 is required to support life, but its metabolites such as ROS can modify cell functions, endanger cell survival, or both (de Lamirande and Gagnon, 1995). Hence, ROS must be continuously inactivated to keep only a small

An In Vitro Model of Parkinson's Disease: Linking Mitochondrial Impairment to Altered α-Synuclein Metabolism and Oxidative Damage

Abstract: Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson's disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitin- and alpha-synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nm rotenone for 1-4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-synuclein and ubiquitin. After a latency of >2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.

The harlequin mouse mutation downregulates apoptosis-inducing factor

Abstract: Harlequin (Hq) mutant mice have progressive degeneration of terminally differentiated cerebellar and retinal neurons. We have identified the Hq mutation as a proviral insertion in the apoptosis-inducing factor (Aif) gene, causing about an 80% reduction in AIF expression. Mutant cerebellar granule cells are susceptible to exogenous and endogenous peroxide-mediated apoptosis, but can be rescued by AIF expression. Overexpression of AIF in wild-type granule cells further decreases peroxide-mediated cell death, suggesting that AIF serves as a free radical scavenger. In agreement, dying neurons in aged Hq mutant mice show oxidative stress. In addition, neurons damaged by oxidative stress in both the cerebellum and retina of Hq mutant mice re-enter the cell cycle before undergoing apoptosis. Our results provide a genetic model of oxidative stress-mediated neurodegeneration and demonstrate a direct connection between cell cycle re-entry and oxidative stress in the ageing central nervous system.