Tanuja Koppal

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

Abstract : 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 Alzheimers disease (AD) and amyotrophic lateral sclerosis, among others. Continuing our laboratorys 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 β‐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‐α‐phenylnitrone and Vitamin Ea

ABSTRACT: 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 prevously 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‐sensitve 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‐α‐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.

Elevated oxidative stress in models of normal brain aging and Alzheimer's disease

Age-associated neurodegenerative disorders are becoming more prevalent as the mean age of the population increases in the United States over the next few decades. Both normal brain aging and Alzheimers disease (AD) are associated with oxidative stress. Our laboratory has used a wide variety of physical and biochemical methods to investigate free radical oxidative stress in several models of aging and AD. Beta-amyloid (A beta), the peptide that constitutes the central core of senile plaques in AD brain, is associated with free radical oxidative stress and is toxic to neurons. This review summarizes some of our studies in aging and A beta-associated free radical oxidative stress and on the modulating effects of free radical scavengers on neocortical synaptosomal membrane damage found in aging and A beta-treated systems.

The Free Radical Antioxidant Vitamin E Protects Cortical Synaptosomal Membranes from Amyloid β-Peptide(25-35) Toxicity But Not from Hydroxynonenal Toxicity: Relevance to the Free Radical Hypothesis of Alzheimer's Disease

Amyloid β-peptide (Aβ) is a key factor in the neurotoxicity of Alzheimers disease (AD). Recent research has shown that Aβ-mediated neurotoxicity involves free radicals and that Aβ peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of Aβ peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with Aβ or HNE. The free radical dependence of β-amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of Aβ. In contrast, HNE was still toxic in the presence of vitamin E. These results support our Aβ-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD.

Vitamin E as an antioxidant/free radical scavenger against amyloid beta-peptide-induced oxidative stress in neocortical synaptosomal membranes and hippocampal neurons in culture: insights into Alzheimer's disease.

Amyloid beta-peptide (Abeta), the major constituent in senile plaques in Alzheimers disease (AD) brain, is thought by many researchers to be central to neurotoxicity in AD brain. Increasing evidence from many laboratories indicates that AD brain is under oxidative stress, with strong evidence of protein oxidation, lipid peroxidation, and peroxynitrite damage. A link between the central role of Abeta and oxidative stress in AD brain may be Abeta-associated free radical oxidative stress. If so, antioxidants such as vitamin E should modulate Abeta-induced oxidative damage and neurotoxicity in brain cells. This review summarizes studies of Abeta-associated free radical oxidative stress and its inhibition by vitamin E in cortical synaptosomal membranes and hippocampal neuronal cells in culture. Taken together with the recent report that vitamin E slows the progression of AD, this review strongly supports a central role of Abeta-associated free radical oxidative stress in neurotoxicity in AD brain.

[48] Amyloid β-peptide-associated free radical oxidative stress, neurotoxicity, and Alzheimer's disease

Given the increasing evidence of oxidative stress in AD brain and studies from different perspectives that appear to show a converging, central role for A beta in the pathogenesis and etiology of AD, insight into A beta-associated free radical oxidative stress will likely lead to a greater understanding of AD and, potentially, to better therapeutic strategies in this disorder. This article outlined methods to investigate markers of oxidative stress induced by A beta in brain membrane systems. Especially important are markers for protein oxidation, lipid peroxidation, and ROS generation by A beta. Oxidative stress and its sequelae are likely related to both necrotic and apoptotic mechanisms of neurotoxicity, and A beta-associated free radical oxidative stress may be of fundamental importance in Alzheimers disease etiology and pathogenesis. The methods described here provide some means for investigating this possibility.

Vitamin E protects against Alzheimer's amyloid peptide (25-35)-induced changes in neocortical synaptosomal membrane lipid structure and composition

magnetic resonance EPR spin trapping and spin labeling, measuring A b-induced protein and lipid oxidation, and investigating effects of A b on oxidation of and dysfunc- tions in proteins of brain membranes and neuronal cultures wx and their prevention by free radical antioxidants 2,6,7 . We proposed that A b-associated free radical lipid per- oxidation and the peroxidation product 4-hydroxynonenal .HNE may be related to the primary mechanism for brain wx

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.

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 degreesC 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.

Investigation of Peroxynitrite Induced Oxidative Stress in Red Blood Cells Monitored by Luminol-Dependent Chemiluminescence.

Peroxynitrite-induced oxidative stress was investigated in red blood cells by luminol-dependent chemiluminescence. Red blood cells are susceptible to free radical damage due to the abundance of oxygen, presence of iron and other agents such as polyunsaturated fatty acids. Peroxynitrite (ONOO-), formed by the reaction of superoxide radical with nitric oxide radical, has been shown to cause protein and lipid oxidation. Addition of ONOO- showed a dramatic increase in chemiluminescence, but treatment with glutathione ethyl ester, an antioxidant, partially reduced the increase in chemiluminescence. The results show that red blood cells are a good model to mimic oxidative stress conditions and are discussed with relevance to neurodegenerate disorders.