Synaptosomal Membrane Proteins Research Articles

Proteomic Analysis of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor Complexes

The AMPA receptor (AMPA-R) is a major excitatory neurotransmitter receptor in the brain. Identifying and characterizing the neuronal proteins interacting with AMPA-Rs have provided important information about the molecular mechanisms underlying synaptic transmission and plasticity. In this study, to identify more AMPA-R interactors in vivo, we performed proteomic analyses of AMPA-R complexes from the brain. AMPA-R complexes were isolated from the brain through various combinations of biochemical techniques for solubilization, enrichment, and immunoprecipitation. Mass spectrometry analyses of these isolated complexes identified several novel components of the AMPA-R complexes as well as some previously identified components. The identification of these novel components helps to further define the complex mechanisms involved in the regulation of AMPA receptor function and synaptic plasticity.

Proteomic identification of binding partners for the brain metabolite lanthionine ketimine (LK) and documentation of LK effects on microglia and motoneuron cell cultures.

Lanthionine ketimine (LK) represents a poorly understood class of thioethers present in mammalian CNS. Previous work has indicated high-affinity interaction of LK with synaptosomal membrane protein(s), but neither LK binding partners nor specific bioactivities have been reported. In this study, LK was chemically synthesized and used as an affinity agent to capture binding partners from mammalian brain lysate. Liquid chromatography with electrospray ionization-mass spectrometry of electrophoretically separated, LK-bound proteins identified polypeptides implicated in axon remodeling or vesicle trafficking and diseases including Alzheimer's disease and schizophrenia: collapsin response mediator protein-2/dihydropyrimidinase-like protein-2 (CRMP2/DRP2/DPYSL2), myelin basic protein, and syntaxin-binding protein-1 (STXBP1/Munc-18). Also identified was the recently discovered glutathione-binding protein lanthionine synthetase-like protein-1. Functional consequences of LK:CRMP2 interactions were probed through immunoprecipitation studies using brain lysate wherein LK was found to increase CRMP2 coprecipitation with its partner neurofibromin-1 but decreased CRMP2 coprecipitation with beta-tubulin. Functional studies of NSC-34 motor neuron-like cells indicated that a cell-permeable LK-ester, LKE, was nontoxic and protective against oxidative challenge with H(2)O(2). LKE-treated NSC-34 cells significantly increased neurite number and length in a serum concentration-dependent manner, consistent with a CRMP2 interaction. Finally, LKE antagonized the activation of EOC-20 microglia by inflammogens. The results are discussed with reference to possible biochemical origins, paracrine functions, neurological significance, and pharmacological potential of lanthionyl compounds.

Pantothenate prevents disturbances in the synaptosomal glutathione system and functional state of synaptosomal membrane under oxidative stress conditions

Parameters of the redox status of the synaptosomal glutathione system and the functional state of the synaptosomal membrane of rat brain were investigated after activation of free radical processes in vitro. A decrease in the content of reduced glutathione along with a simultaneous increase in that of its oxidized form and inhibition of Na+,K+-ATPase and acetylcholine esterase were observed for the model of oxidative stress. These parameters correlated with changes in the total phospholipids: cholesterol ratio and in the balance of thiol-and disulphide groups of proteins of synaptosomal membranes. Preincubation of synaptosomes with 0.5 mM sodium pantothenate prevented the negative effects of the Fenton reaction, but had no effect in the presence of the buthionine-sulfoximine inhibitor of glutathione biosynthesis. Our results provide evidence for the regulatory effect of the glutathione redox status on the structural and functional state of synaptosomal membranes, and for its possible modulation by the CoA biosynthetic precursor.

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies

In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer’s disease in which oxidative stress is implicated.

Oxidative stress in synaptosomal proteins from mutant presenilin-1 knock-in mice: implications for familial Alzheimer's disease.

Presenilin-1 (PS-1) is a transmembrane protein that may be involved in the processing of amyloid precursor protein (APP). Mutations in PS-1 are the major cause of familial Alzheimer's disease (AD). AD brain is under significant oxidative stress, including protein oxidation. In the present study, protein oxidation was compared in synaptosomes from knock-in mice expressing mutant human PS-I (M146V mutation) and from wild-type mice expressing non-mutant human PS-1. Synaptosomal membrane protein conformational alterations associated with oxidative stress were measured using electron paramagnetic resonance (EPR) in conjunction with a protein-specific spin-label. Direct synaptosomal protein oxidation was assessed by a carbonyl detection assay. Synaptosomal proteins from PS-1 mutant mice displayed increased oxidative stress as measured by both techniques, compared with synaptosomal proteins from wild type mice. These data suggest that PS-1 mutations cause oxidative alterations in synaptosomal membrane protein structure and oxidative modification of synaptosomal proteins. Our findings suggest that familial AD may be associated with oxidative stress that may play a pivotal role in neuronal dysfunction and death.

Antioxidant activity of the organotellurium compound 3-[4-( N, N-dimethylamino)benzenetellurenyl]propanesulfonic acid against oxidative stress in synaptosomal membrane systems and neuronal cultures

Antioxidant activities of 3-[4-( N, N-dimethylamino) benzenetellurenyl]propanesulfonic acid sodium salt (NDBT) were evaluated in solution, red blood cells, synaptosomal membranes, and cultured hippocampal neuronal cells after exposure to peroxynitrite (ONOO −) and hydroxyl radicals. The organotellurium compound NDBT possesses significant activity towards hydrogen peroxide and/or the hydroxyl radical in solution, demonstrated by inhibition of hydroxylation of terephthalic acid. In addition, the compound displayed great antioxidant abilities as shown by: reduction of ONOO −-induced 2,7-dichlorofluorescein (DCF) fluorescence in synaptosomes; complete prevention of lipid peroxidation in synaptosomes caused by OH radicals (TBARS), and significant prevention of protein oxidation caused by ONOO − and OH, indexed by the levels of protein carbonyls in synaptosomes and neuronal cells. The presence of the compound abolished neuronal cell death caused by ONOO −. Further, the compound was effective in preventing the oxidative changes in synaptosomal membrane protein conformation and crosslinking (EPR spin labeling). Finally, the organotellurium molecule attenuated peroxynitrite-induced, luminol-dependent chemiluminescence in red blood cells — an index of cellular oxidation. These findings demonstrate the great potential of the antioxidant and are consistent with the notion that NDBT may have a role to play in modulating oxidative stress in neurodegenerative disorders, including Alzheimer’s disease.

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.

Effect of exogenous and endogenous antioxidants on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions: insights into Huntington's disease.

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of complex II in the mitochondria. 3-NP toxicity has gained acceptance as an animal model of Huntington's disease (HD). In the present study, we confirmed that rats injected with 3-NP (20 mg/kg, i.p., daily for 4 days) exhibit increased oxidative stress in both striatum and cortical synaptosomes as well as lesions in the striatum. Synaptosomal membrane proteins from rats injected with 3-NP exhibited a decrease in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation, and western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal protein oxidation. Treatment of rats with the brain-accessible free radical spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO; 30 mg/kg, i.p., daily 2 h before 3-NP injection) or with N-acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3-NP injection), a known glutathione precursor, before 3-NP treatments protects against oxidative damage induced by 3-NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3-NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3-NP neurotoxicity and perhaps against HD as well.

3-nitropropionic acid induced in vivo protein oxidation in striatal and cortical synaptosomes: insights into Huntington's disease.

3-nitropropionic acid (3-NP) administered systemically daily for 4 days to rats inhibits mitochondrial oxidative phosphorylation and induces selective lesions in the striatum in a manner reminiscent of Huntington's disease (HD). To investigate the potential oxidative nature of these lesions, rats were injected with 3-NP (20 mg/kg, i.p. daily for 4 days) and subsequently isolated brain synaptosomal membranes were examined for evidence of oxidative stress. Brain synaptosomal membrane proteins from rats injected with 3-NP exhibited a decreased in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation (76% of control), and Western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal membrane protein oxidation (248% of control). Similar results were obtained in synaptosomes isolated from striatum and from cerebral cortex, demonstrating that the oxidative changes are not restricted to the lesion site. Moreover, increased oxidative stress was evident prior to the appearance of morphological lesions. These data are consistent with the hypothesis that 3-NP-induced striatal lesions, and perhaps those in HD, are associated with oxidative processes.

ATP αS is a ligand for P2Y receptors in synaptosomal membranes: solubilization of [ 35S]ATP αS binding proteins associated with G-proteins

ATP αS was established as a P2Y receptor-specific ligand for assaying the solubilization of functional native P2Y receptors from synaptosomal membranes. These receptors are not yet amenable to biochemical studies. High-affinity [ 35S]ATP αS binding sites in synaptosomal membranes, solubilized with Brij58, retained the binding affinity and ligand specificity (ATP αS=ATP>2-MeSATP>ADP, ADP βS>AMP⪢ α, β-MeATP) corresponding to P2Y receptors. Mg 2+ but not Ca 2+, enhanced high-affinity [ 35S]ATP αS binding 30-fold, supporting specific recognition by P2Y receptors. ATP αS stimulated P2Y receptor-mediated [ 35S]GTP γS binding equipotently with ATP in synaptosomal membranes and in Brij58-solubilized proteins demonstrating the association with G-proteins. Anion-exchange chromatography of solubilized synaptosomal membrane proteins yielded two fractions in which [ 35S]ATP αS binding was regulated by GTP γS/Mg 2+, thus possibly by heterotrimeric G-proteins. After a second chromatographic step (hydroxyapatite) the regulation of high-affinity [ 35S]ATP αS binding by Mg 2+ was still present, whereas the regulation by GTP γS/Mg 2+ was lost indicating the dissociation from G-proteins. Thus, conditions were found to stabilize ligand binding activity of solubilized P2Y receptors and to solubilize P2Y receptors associated with G-proteins.

In vivo modulation of rodent glutathione and its role in peroxynitrite-induced neocortical synaptosomal membrane protein damage

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, leads to the oxidation of proteins, lipids, and DNA, and nitrates thiols such as cysteine and glutathione, and amino acids like tyrosine. Previous in vitro studies have shown glutathione to be an efficient scavenger of peroxynitrite, protecting synaptosomal membranes from protein oxidation, the enzyme glutamine synthetase from inactivation, and preventing the death of hippocampal neurons in culture. The current study was undertaken to see if in vivo modulation of glutathione levels would affect brain cortical synaptosomal membrane proteins and their subsequent reaction with peroxynitrite. Glutathione levels were depleted, in vivo, by injecting animals with 2-cyclohexen-1-one (CHX, 100 mg/kg body weight), and levels of glutathione were enhanced by injecting animals with N-acetylcysteine (NAC, 200 mg/kg body weight), which gets metabolized to cysteine, a precursor of glutathione. Changes in membrane protein conformation and structure in synaptosomes subsequently isolated from these animals were examined using electron paramagnetic resonance, before and after in vitro addition of peroxynitrite. The animals injected with the glutathione depletant CHX showed greater damage to the membrane proteins both before and after peroxynitrite treatment, compared to the non-injected controls. The membrane proteins from animals injected with NAC were comparable to controls before peroxynitrite treatment and were partially protected against peroxynitrite-induced damage. This study showed that modulation of endogenous glutathione levels can affect the degree of peroxynitrite-induced brain membrane damage and may have potential therapeutic significance for oxidative stress-associated neurodegenerative disorders.

Peroxynitrite-induced alterations in synaptosomal membrane proteins: insight into oxidative stress in Alzheimer's disease.

Peroxynitrite (ONOO ) is a highly reactive, oxidizing anion with a half-life of <1 s that is formed by reaction of superoxide radical anion with nitric oxide. Several reports of ONOO--induced oxidation of lipids, proteins, DNA, sulfhydryls, and inactivation of key enzymes have appeared. ONOO- has also been implicated as playing a role in the pathology of several neurodegenerative disorders, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis, among others. Continuing our laboratory's interest in free radical oxidative stress in brain cells in AD, the present study was designed to investigate the damage to brain neocortical synaptosomal membrane proteins and the oxidation-sensitive enzyme glutamine synthetase (GS) caused by exposure to ONOO-. These synaptosomal proteins and GS have previously been shown by us and others to have been oxidatively damaged in AD brain and also following treatment of synaptosomes with amyloid beta-peptide. The results of the current study showed that exposure to physiological levels of ONOO- induced significant protein conformational changes, demonstrated using electron paramagnetic resonance in conjunction with a protein-specific spin label, and caused oxidation of proteins, measured by the increase in protein carbonyls. ONOO- also caused inactivation of GS and led to neuronal cell death examined in a hippocampal cell culture system. All these detrimental effects of ONOO- were successfully attenuated by the thiol-containing antioxidant tripeptide glutathione. This research shows that ONOO- can oxidatively modify both membranous and cytosolic proteins, affecting both their physical and chemical nature. These findings are discussed with reference to the potential involvement of ONOO- in AD neurodegeneration.

Structural and functional changes in proteins induced by free radical-mediated oxidative stress and protective action of the antioxidants N-tert-butyl-alpha-phenylnitrone and vitamin E.

The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have previously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation-sensitive enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence-accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8-sensitive line were compared to the SAMR1-resistant mice following 14 days of daily PBN treatment at a dose of 30 mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These components may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging.

Cryopreservation of rat cortical synaptosomes and analysis of glucose and glutamate transporter activities, and mitochondrial function

Direct comparisons of synaptic functional parameters in brain tissues from different groups of experimental animals and different samples from post mortem human brain are often hindered by the inability to perform assays at the same time. To circumvent these difficulties we developed methods for cryopreservation and long-term storage of neocortical synaptosomes. The synaptosomes are suspended in a cryopreservation medium containing 10% dimethylsulfoxide and 10% fetal bovine serum, and are slowly cooled to −80°C and then stored in liquid nitrogen. The function of plasma membrane glucose and glutamate transporters, and mitochondrial electron transport activity and membrane potential were measured in fresh, cryopreserved (CP), and non-cryopreserved freeze–thawed (NC) synaptosomes. Glucose and glutamate transporter activities, and mitochondrial functional parameters in CP synaptosomes were essentially identical to those in fresh unfrozen synaptosomes. Glucose and glutamate transport were severely compromised in NC synaptosomes, whereas mitochondrial function and cellular esterase activity were largely maintained. Electron paramagnetic resonance studies in conjunction with a protein-specific spin label indicated that cryopreservation did not alter the physical state of synaptosomal membrane proteins. These methods provide the opportunity to generate stocks of functional synaptosomes from different experiments or post mortem samples collected over large time intervals. Themes: Excitable membranes and synaptic transmission Topics: Presynaptic mechanisms

The lipid peroxidation product, 4-hydroxy-2-trans-nonenal, alters the conformation of cortical synaptosomal membrane proteins.

Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta). A beta-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of A beta and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1-10 microM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and A beta-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 microM). The results obtained are discussed with relevance to the hypothesis of A beta-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.

Effect of Verapamil on Changes in Brain Protein Phosphorylation after Oral Administration of Tri-O-cresyl Phosphate to Hens

The endogenous phosphorylation of specific brain proteins in white Leghorn laying hens treated with tri-o-cresyl phosphate (TOCP) with and without verapamil, a calcium channel blocker, which has been shown to ameliorate clinical signs and nervous lesions associated with organophosphate-induced delayed neuropathy (OPIDN), was determined. Verapamil was given im at the dose of 7 mg/kg/day for 4 days beginning 1 day before administration of TOCP (750 mg/kg, po). Phosphorylation of crude synaptosomal (P2) membrane proteins was assayedin vitrousing [γ-32P]ATP as a phosphate donor. Following resolution of brain membrane proteins by SDS-PAGE, specific protein phosphorylation was detected by autoradiography and quantified by densitometry TOCP caused, 21 days after dosing, an enhancement of the phosphorylation of membrane proteins with apparent molecular weights of 40, 55, and 60 kDa by as much as 253, 322, and 609%, respectively. Verapamil administered to TOCP-treated hens abolished the enhancement and even caused some inhibition. This indicates that calcium channel blocker may contribute to the amelioration of OPIDN by preventing the enhancement of phosphorylation of brain membrane proteins.

Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl-alpha-phenylnitrone.

According to the free radical theory of aging, reactive oxygen species cause oxidative damage, proposed to be an underlying factor of the aging process. In the current study, we have used electron paramagnetic resonance spin labeling, measurements of protein carbonyl content, an index of protein oxidation, and determination of the activity of glutamine synthetase (an oxidatively sensitive enzyme) to report that cortical synaptosomal membranes from the senescence accelerated-prone (SAMP8) mouse showed structural characteristics of free radical oxidative stress relative to the senescence accelerated-resistant (SAMR1) mouse. The SAMP8 mouse exhibited a decrease in the relevant EPR parameter consistent with oxidative stress (P < 0.002), a decreased glutamine synthetase activity (P < 0.05), and an increased protein carbonyl content (P < 0.01) compared with these parameters in the SAMR1 mouse. Further, because free radical trapping compounds have been demonstrated to extend maximum life span and improve cognition in SAMP8 mice, we investigated the protective nature of the known free radical scavenger, N-tert-butyl-alpha-phenylnitrone (PBN), on the physical state of cortical synaptosomal membrane proteins. For 14 days, SAMR1 and SAMP8 mice were injected with 30 mg/kg PBN while the controls were injected with the corresponding volume of saline. Characteristic of less oxidized systems, cortical synaptosomal membranes from the PBN-injected SAMP8 mouse exhibited a return toward normal values of the relevant EPR parameter [the M1 = +1 low-field weakly immobilized line/M1 = +1 low-field strongly immobilized line (W/S) ratio of a protein-specific spin label] (P < 0.001) compared with that from saline-injected SAMP8 mice. In SAMR1 mice, in contrast to SAMP8, there was no significant change in the conformation of membrane proteins or protein carbonyl content of cortical synaptosomal membranes from the PBN-injected and saline-injected SAMR1 mice, showing that PBN itself did not induce conformational changes in cortical synaptosomal membrane proteins. The results are discussed with reference to the use of free radical scavengers as potential anti-aging agents.

Effect of 2-cyclohexene-1-one-induced glutathione diminution on ischemia/reperfusion-induced alterations in the physical state of brain synaptosomal membrane proteins and lipids

Glutathione is able to protect membrane proteins from oxidative stress. In ischemia/reperfusion injury, free radicals cause synaptosomal membrane protein and lipid oxidation that is prevented by the free radical scavenger N-tert-butyl- α-phenylnitrone [Hall N. C. et al. (1995) Neuroscience 64, 81–89; 69, 591–600]. We wondered if diminution of glutathione would lead to further membrane alterations. Accordingly, the effects of glutathione depletion, by intraperitoneal administration of 2-cyclohexene-1-one, on the physical state of cortical synaptosomal membrane proteins and lipids, with and without global ischemia/reperfusion, were studied in vivo and in vitro in adult and aged gerbils utilizing electron paramagnetic resonance spectrometry. 2-Cyclohexene-1-one (100 mg/kg, i.p.) was administered 30 min prior to 10-min ischemia followed by 1 or 14 h reperfusion. This glutathione reduction agent was also administered to gerbils under the same temporal schedule in the absence of ischemia and compared to untreated controls. Synaptosomal membranes were labeled with a protein-specific spin label, 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl, or a lipid-specific spin probe, 5-doxylstearic acid. There were no significant changes in the physical state of the lipid portion of synaptosomal membranes when comparing ischemia reperfusion and 2-cyclohexene-1-one-treated ischemia reperfusion in either the adult or aged gerbils. However, glutathione depletion without ischemia/reperfusion caused significant changes in the physical state of the protein portion of cortical synaptosomal membranes in both the adult and aged models. Glutathione depletion, without ischemia/reperfusion, in the adult model showed a maximum change at 3 h that returned to control values by 14 h. In contrast, the aged model showed significant changes at 1 h reperfusion, which did not return to control values by 14 h reperfusion. Glutathione depletion combined with ischemia/reperfusion caused initial protein change in both adult and aged models at 1 h reperfusion, which did not return toward control values by 14 h reperfusion. The results of this study suggest that glutathione depletion increases the severity of membrane protein damage associated with ischemia/reperfusion injury.

Prevention of hyperoxia-induced alterations in synaptosomal membrane-associated proteins by N-tert-butyl-alpha-phenylnitrone and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol).

Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age-related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to 24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N-tert-butyl-alpha-phenylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPR) protein-specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of either of the two free radical scavengers. After 30 min, the gerbils were exposed to 90-100% O2 for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol-specific label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl). As in our earlier study, the EPR spectral parameter of MAL-6-labeled membranes, the W/S ratio, decreased with hyperoxia (p < 0.00001). This effect was lessened significantly with administration of PBN (p < 0.0003) or Tempol (p < 0.00003). For the GS and protein carbonyl assays, cortical proteins were used. The activity of the GS decreased with hyperoxia (p < 0.000005), and this effect likewise was lessened with administration of PBN (p < 0.004) or Tempol (p < 0.002). The protein carbonyl content increased with hyperoxia (p < 0.0002), and there was a protective effect found with Tempol (p < 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as potential antiaging agents.