Biomedical subjects

D A Butterfield

Publications and source records attributed to D A Butterfield.

At least 37 records · Page 2Linked to original sources

PubMed2001

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.

Alzheimer Disease↗

PubMed2001

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.

Acetylcysteine↗

PubMed2001

Protein oxidation in the brain in Alzheimer's disease.

In this study we used immunohistochemistry and two-dimensional fingerprinting of oxidatively modified proteins (two-dimensional Oxyblot) together to investigate protein carbonyl formation in the Alzheimer's disease brain. Increased protein oxidation was detected in sections from the hippocampus and parahippocampal gyrus, superior and middle temporal gyri of six Alzheimer's disease and six age-matched control human subjects, but not in the cerebellum. In two brain regions severely affected by Alzheimer's disease pathology, prominent protein carbonyl immunoreactivity was localized in the cytoplasm of neurons without visual pathomorphological changes and degenerating neurons, suggesting that intracellular proteins might be significantly affected by oxidative modifications. Following two-dimensional electrophoresis the positions of some individual proteins were identified using specific antibodies, and immunoblot analysis for protein carbonyls was performed. These studies demonstrated the presence of protein carbonyl immunoreactivity in beta-tubulin, beta-actin and creatine kinase BB in Alzheimer's disease and control brain extracts. Protein carbonyls were undetectable in spots matching glial fibrillary acidic protein and tau isoforms. Specific protein carbonyl levels in beta-actin and creatine kinase BB were significantly higher in Alzheimer's disease than in control brain extract. beta-Tubulin did not demonstrate a significant increase in specific protein carbonyl content in Alzheimer's disease brains. We suggest that oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the Alzheimer's disease brain.

Actins↗

PubMed2001

Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide.

Amyloid beta-peptide (Abeta) is heavily deposited in the brains of Alzheimer's disease (AD) patients. Free-radical oxidative stress, particularly of neuronal lipids, proteins and DNA, is extensive in those AD brain areas in which Abeta is abundant. Recent research suggests that these observations might be linked, and it is postulated that Abeta-induced oxidative stress leads to neurodegeneration in AD brain. Consonant with this postulate, Abeta leads to neuronal lipid peroxidation, protein oxidation and DNA oxidation by means that are inhibited by free-radical antioxidants. Here, we summarize current research on phospholipid peroxidation, as well as protein and DNA oxidation, in AD brain, and discuss the potential role of Abeta in this oxidative stress.

Alzheimer Disease↗

PubMed2001

5-Aminosalicylic acid protection against oxidative damage to synaptosomal membranes by alkoxyl radicals in vitro.

The antioxidant properties of 5-aminosalicylic acid in vitro were evaluated in a synaptosomal membrane system prepared from gerbil cortical synaptosomes using EPR spin labeling and spectroscopic techniques. MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl) and 5-NS (5-nitroxide stearate) spin labels were used to assess changes in protein oxidation and membrane lipid fluidity, respectively. Synaptosomal membranes were subjected to oxidative stress by incubation with 1 mM azo-bis(isobutyronitrile) (AIBN) or 1 mM 2,2'-azobis(amidino propane) dihydrochloride (AAPH) at 37 degrees C for 30 minutes. The EPR analyses of the samples showed significant oxidation of synaptosomal proteins and a decrease in membrane fluidity. 5-Aminosalicylic acid also was evaluated by means of FRAP (the ferric reducing ability of plasma) test as a potential antioxidant. 5-Aminosalicylic acid also showed protection against the oxidation in gerbil cortical synaptosomes system caused by AIBN and AAPH. These results are consistent with the notion of antioxidant protection against free radical induced oxidative stress in synaptosomal membrane system by this agent.

Alcohols↗

PubMed2001

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.

ATP-Binding Cassette Transporters↗

PubMed2001

Brain oxidative stress in animal models of accelerated aging and the age-related neurodegenerative disorders, Alzheimer's disease and Huntington's disease.

Oxidative stress in brain is emerging as a potential causal factor in aging and age-related neurodegenerative disorders. Brain tissue from living patients is difficult to acquire; hence, animal models of aging and age-related neurodegenerative disorders, though not perfect models, have provided tissue to study the role of oxidative stress in these disorders. In this review, the central role of oxidative damage in brain in models of accelerated aging (progeria and Werner's syndrome) and the age-related neurodegenerative disorders, Alzheimer's disease and Huntington's disease, will be presented and evaluated. To the extent that the animal models faithfully mirror their respective disorders, and based on the totality of the studies, it is apparent that oxidative stress, the excess of free radicals over the means of scavenging these harmful agents, may play critical roles in the molecular basis of accelerated aging, Alzheimer's disease, and Huntington's disease.

Aging, Premature↗

PubMed2001

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.

Animals↗

PubMed2000

Methamphetamine toxicity is attenuated in mice that overexpress human manganese superoxide dismutase.

We have investigated methamphetamine (MA) toxicity in transgenic mice that overexpress the human form of mitochondrial manganese superoxide dismutase (MnSOD). Our results reveal a significant reduction in the long-term depletion of striatal dopamine and protein oxidation following repeated administration of MA in transgenic vs. non-transgenic littermates. These findings support the notion that ROS contribute to MA-induced brain damage and suggest that mitochondria may play an important role in this form of neurodegeneration.

Animals↗

PubMed2000

Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1.

Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, aN = 8.01 [2N], aH = 5.26 [4H], aH = 2.72 [4H]), or ADP-ribose. Reaction of glycoaldehyde with poly-L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo.

Acetaldehyde↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Review: Alzheimer's amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity.

Alzheimer's disease, the major dementing disorder of the elderly that affects over 4 million Americans, is related to amyloid beta-peptide, the principal component of senile plaques in Alzheimer's disease brain. Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of Alzheimer's disease brain. Our laboratory united these two observations in a model to account for neurodegeneration in Alzheimer's disease brain, the amyloid beta-peptide-associated oxidative stress model for neurotoxicity in Alzheimer's disease. Under this model, the aggregated peptide, perhaps in concert with bound redox metal ions, initiates free radical processes resulting in protein oxidation, lipid peroxidation, reactive oxygen species formation, cellular dysfunction leading to calcium ion accumulation, and subsequent neuronal death. Free radical antioxidants abrogate these findings. This review outlines the substantial evidence from multiidisciplinary approaches for amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity and protection against these oxidative processes and cell death by free radical scavengers. In addition, we review the strong evidence supporting the notion that the single methionine residue of amyloid beta-peptide is vital to the oxidative stress and neurotoxicological properties of this peptide. Further, we discuss studies that support the hypothesis that aggregated soluble amyloid beta-peptide and not fibrils per se are necessary for oxidative stress and neurotoxicity associated with amyloid beta-peptide.

Alzheimer Disease↗

PubMed2000

In-vivo glutathione elevation protects against hydroxyl free radical-induced protein oxidation in rat brain.

Glutathione deficiency has been associated with a number of neurodegenerative diseases including Lou Gehrig's disease, Parkinson's disease, and HIV. A crucial role for glutathione is as a free radical scavenger. Alzheimer's disease (AD) brain is characterized by oxidative stress, manifested by protein oxidation, lipid oxidation, oxidized glutathione, and decreased activity of glutathione S-transferase, among others. Reasoning that elevated levels of endogenous glutathione would offer protection against free radical-induced oxidative stress, rodents were given in vivo injections of N-acetylcysteine (NAC), a known precursor of glutathione, to study the vulnerability of isolated synaptosomal membranes treated with Fe2+/H2O2, a known hydroxyl free radical producer. Protein carbonyls, a marker of protein oxidation, were measured. NAC significantly increased endogenous glutathione levels in cortical synaptosome cytosol (P < 0.01). As reported previously, protein carbonyl levels of the Fe2+/H2O2-treated synaptosomes were significantly higher compared to that of non-treated controls (P < 0.01), consistent with increased oxidative stress. In contrast, protein carbonyl levels in Fe2+/H2O2-treated synaptosomes isolated from NAC-injected animals were not significantly different from saline-injected non-treated controls, demonstrating protection against hydroxyl radical induced oxidative stress. These results are consistent with the notion that methods to increase endogenous glutathione levels in neurodegenerative diseases associated with oxidative stress, including AD, may be promising.

Acetylcysteine↗

PubMed2000

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-Peptides↗

PubMed2000

Oxidative modification of creatine kinase BB in Alzheimer's disease brain.

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

Aged↗

PubMed2000

Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid.

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.

Amyloid beta-Peptides↗

PubMed2000

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.

Acetylcysteine↗