The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer's disease brain: the role of Abeta1-42.

Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in several neurodegenerative disorders. In particular, glutamate uptake is inhibited in Alzheimer's disease (AD); however, the mechanism of decreased transporter activity is unknown. Oxidative damage in brain is implicated in models of neurodegeneration, as well as in AD. Glutamate transporters are inhibited by oxidative damage from reactive oxygen species and lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE). Therefore, we have investigated a possible connection between the oxidative damage and the decreased glutamate uptake known to occur in AD brain. Western blots of immunoprecipitated HNE-immunoreactive proteins from the inferior parietal lobule of AD and control brains suggest that HNE is conjugated to GLT-1 to a greater extent in the AD brain. A similar analysis of beta amyloid (Abeta)-treated synaptosomes shows for the first time that Abeta1-42 also increases HNE conjugation to the glutamate transporter. Together, our data provide a possible link between the oxidative damage and neurodegeneration in AD, and supports the role of excitotoxicity in the pathogenesis of this disorder. Furthermore, our data suggests that Abeta may be a possible causative agent in this cascade.

Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease.

Oxidative stress may be a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD) Huntington's, and Parkinson's diseases as well as amyotrophic lateral sclerosis. Acrolein is a highly reactive product of lipid peroxidation that is elevated in the brains of persons with AD. This alkenal potentially can react with proteins by Michael addition to alter their structure and function. In the present study, we used electron paramagnetic resonance in conjunction with a protein-specific spin label to monitor synaptosomal membrane protein conformational alterations induced by acrolein. A dose-dependent increased conformational alteration was observed. Consistent with this finding, protein carbonyl levels from protein-bound acrolein were significantly elevated. However, pretreatment of synaptosomes with glutathione ethyl ester (GEE) significantly ameliorated both the conformational alterations and protein carbonyls induced by acrolein. Based on this success, we tested the hypothesis that elevated levels of endogenous glutathione (GSH) would offer protection against acrolein-induced oxidative stress. In-vivo elevation of GSH (215% over control, P<0.04) was produced by i.p. injection of N-acetylcysteine (NAC), a known precursor of GSH. Synaptosomes were treated with vehicle or 2 nM acrolein, the level of this alkenal found in AD brain. In contrast to synaptosomes from control animals, which had significantly increased protein carbonyl levels following addition of 2 nM acrolein, synaptosomes that were isolated from NAC-treated rodents and treated with 2 nM acrolein showed no increased carbonyl levels compared to untreated controls. These results demonstrate protection by increased in-vivo GSH levels against acrolein-induced oxidative stress at levels found in AD brain and are consistent with the notion that methods to increase endogenous GSH levels in neurodegenerative diseases associated with oxidative stress may be promising.

D609 inhibits ionizing radiation-induced oxidative damage by acting as a potent antioxidant.

Tricyclodecan-9-yl-xanthogenate (D609) has been extensively studied in biological systems and exhibits a variety of biological functions, including antiviral, antitumor, and anti-inflammatory activities. Most of these activities have been largely attributed to the inhibitory effect of D609 on phosphatidylcholine-specific phospholipase C. However, as a xanthate derivative, D609 is a strong electrolyte and readily dissociates to xanthate anions and cations of alkali metals in solution. Xanthate anions and protonated xanthic acid contain a free thiol moiety and are highly reductive. This implies that D609 and other xanthate derivatives may function as potent antioxidants. Indeed, we found that D609 inhibited the Fenton reaction-induced oxidation of dihydrorhodamine 123 in a dose-dependent manner similar to that of pyrrolidinedithiocarbamate, a well known antioxidant. In addition, D609 inhibited the formation of the alpha-phenyl-tert-butylnitrone-free radical spin adducts and lipid peroxidation of synaptosomal membranes by the Fenton reagents. Furthermore, preincubation of lymphocytes with D609 resulted in a significant diminution of ionizing radiation (IR)-induced 1) production of reactive oxygen species; 2) decrease in intracellular reduced glutathione; 3) oxidative damage to proteins and lipids; and 4) activation of nuclear factor-kappaB. Moreover, when D609 (50 mg/kg i.v.) was administered to mice 10 min prior to total body IR (6.5 and 8.5 Gy), it protected the mice from IR-induced lethality. Thus, these results indicate that D609 is a potent antioxidant and has the ability to inhibit IR-induced cellular oxidative stress.

Vulnerability of synaptosomes from apoE knock-out mice to structural and oxidative modifications induced by A beta(1-40): implications for Alzheimer's disease.

Apolipoprotein E (apoE) plays an important role in the response to central nervous system injury. The e4 allele of apoE and amyloid beta-peptide (Abeta) are associated with Alzheimer's disease (AD) and may be central to the pathogenesis of this disorder. Recent studies demonstrate evidence for neurodegeneration and increased lipid peroxidation in transgenic mice lacking apoE (KO). In the current study, synaptosomes were prepared from apoE KO mice to determine the role of apoE in synaptic membrane structure and to determine susceptibility to oxidative damage by Abeta(1-40). ApoE KO mice exhibited structural modifications to lipid and protein components of synaptosomal membranes as determined by electron paramagnetic resonance in conjunction with lipid- and protein- specific spin labels. Incubation with 5 microM Abeta(1-40) resulted in more severe oxidative modifications to proteins and lipids in apoE KO synaptosomes as measured by protein carbonyls, an index of protein oxidation, and TBARs and protein-bound 4-hydroxynonenal (HNE), markers of lipid oxidation. Together, these data support a role for apoE in the modulation of oxidative injury and in the maintenance of synaptic integrity and are discussed with reference to alterations in AD brain.

The pyrrolopyrimidine U101033E is a potent free radical scavenger and prevents Fe(II)-induced lipid peroxidation in synaptosomal membranes.

The pyrrolopyrimidine U101033E is a therapeutic compound potentially useful in stroke, head injury and other oxidative stress conditions. Electron paramagnetic resonance (EPR) techniques of spin labeling and spin trapping in conjunction with measures of lipid and protein oxidation have been used to investigate the proposed antioxidant capacity of U101033E. We report potent antioxidant activity of this agent in aqueous cell-free solution as measured by spin trapping. U101033E significantly (P<0.005) reduces the formation of the EPR active spin trap N-t-butyl-alpha-phenylnitrone (PBN)-radical adduct by 17.1% at a concentration of 1 microM, four orders of magnitude less than the concentration of PBN. As measured by the decrease in signal intensity of lipid-resident nitroxide stearate spin probes, an EPR assay for lipid peroxidation, this pyrrolopyrimidine compound efficiently protected against hydroxyl radical-induced lipid peroxidation in cortical synaptosomal membranes deep within the membrane bilayer, but not closer to the membrane surface. In addition, U101033E partially prevents synaptosomal protein oxidation in the presence of Fe(II); however, U101033E demonstrates some protein oxidative effects itself. These results are supportive of the proposed role of U101033E as a lipid-specific antioxidant, especially for protection against lipid peroxidation that occurs deep within the membrane bilayer, but raise some potential concerns about the oxidative nature of this agent toward proteins.

Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice.

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.

Amyloid beta-peptide inhibits Na+-dependent glutamate uptake.

The purpose of this review is to summarize much of the work on the inhibition of the astroglial glutamate transporter in relation to excitotoxic neurodegeneration, in particular, inhibition of uptake by the beta-amyloid peptide (A beta) found in the Alzheimer's disease (AD) brain. There is evidence for oxidative stress in the AD brain, and A beta has been found to generate reactive oxygen species (ROS), thus adding to the stress or possibly initiating it. The oxidative inhibition of the glutamate transporter protein by A beta increases the vulnerability of glutamatergic neurons, and by rendering them susceptible to the excitotoxic insult that results from impaired glutamate uptake, A beta can be directly connected to the neurodegeneration that follows.