Iron accumulation in Alzheimer disease is a source of redox-generated free radicals

Damage from free radicals has been demonstrated in susceptible neuronal populations in cases of Alzheimer disease. In this study, we investigated whether iron, a potent source of the highly reactive hydroxyl radical that is generated by the Fenton reaction with H2O2, might contribute to the source of radicals in Alzheimer disease. We found, using a modified histochemical technique that relies on the formation of mixed valence iron complexes, that redox-active iron is associated with the senile plaques and neurofibrillary tangles—the pathological hallmark lesions of this disease. This lesion-associated iron is able to participate in in situ oxidation and readily catalyzes an H2O2-dependent oxidation. Furthermore, removal of iron was completely effected using deferoxamine, after which iron could be rebound to the lesions. Characterization of the iron-binding site suggests that binding is dependent on available histidine residues and on protein conformation. Taken together, these findings indicate that iron accumulation could be an important contributor toward the oxidative damage of Alzheimer disease.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger.

Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta-AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK-8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Mitochondrial adenine nucleotide translocase is modified oxidatively during aging

The purpose of this study was to test the hypothesis that elevation in protein oxidative damage during the aging process is a targeted rather than a stochastic phenomenon. Oxidative damage to proteins in mitochondrial membranes in the flight muscles of the housefly, manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl antibodies. Adenine nucleotide translocase (ANT) was found to be the only protein in the mitochondrial membranes exhibiting a detectable age-associated increase in carbonyls. The age-related elevation in ANT carbonyl content was correlated with a corresponding loss in its functional activity. Senescent flies that had lost the ability to fly exhibited a relatively higher degree of ANT oxidation and a greater loss of functional activity than their cohorts of the same age that were still able to fly. Exposure of flies to 100% oxygen resulted in an increase in the level of ANT carbonyl content and a loss in its activity. In vitro treatment of mitochondria with a system that generated hydroxyl free radicals caused an increase in ANT carbonyl level and a decrease in ANT exchange activity. ANT was also the only mitochondrial membrane protein exhibiting adducts of the lipid peroxidation product 4-hydroxynonenal. Results of this study indicate that proteins in mitochondrial membranes are modified selectively during aging.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis–linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant.

L-ascorbic acid (vitamin C) is a powerful reducing agent found in millimolar concentrations in plants, and is proposed to play an important role in scavenging free radicals in plants and animals. However, surprisingly little is known about the role of this antioxidant in plant environmental stress adaptation or ascorbate biosynthesis. We report the isolation of soz1, a semi-dominant ozone-sensitive mutant that accumulates only 30% of the normal ascorbate concentration. The results of genetic approaches and feeding studies show that the ascorbate concentration affects foliar resistance to the oxidizing gas ozone. Consistent with the proposed role for ascorbate in reactive oxygen species detoxification, lipid peroxides are elevated in soz1, but not in wild type following ozone fumigation. We show that the soz1 mutant is hypersensitive to both sulfur dioxide and ultraviolet B irradiation, thus implicating ascorbate in defense against varied environmental stresses. In addition to defining the first ascorbate deficient mutant in plants, these results indicate that screening for ozone-sensitive mutants is a powerful method for identifying physiologically important antioxidant mechanisms and signal transduction pathways. Analysis of soz1 should lead to more information about the physiological roles and metabolism of ascorbate.

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD.