D.A. Butterfield

Proteomic identification of proteins specifically oxidized by intracerebral injection of amyloid β-peptide (1–42) into rat brain: Implications for Alzheimer’s disease

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimers disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: A nutritional approach

Summary. Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimer’s disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis.Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response.Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimer’s disease, in fact, involves a chronic inflammatory response associated with both brain injury and β-amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.

Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain

The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and alpha-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, alpha-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

A neuronal model of Alzheimer's disease: an insight into the mechanisms of oxidative stress-mediated mitochondrial injury.

Alzheimers disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

The Antioxidant Vitamin E Modulates Amyloid β-Peptide-Induced Creatine Kinase Activity Inhibition and Increased Protein Oxidation: Implications for the Free Radical Hypothesis of Alzheimer's Disease

Amyloid β-peptide (Aβ), the main constituent of senile plaques in Alzheimers disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of Aβ occurs in conjunction with free radical oxidative stress associated with the peptide. Aβ(1–40) and several other fragments of the Aβ sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca2+ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that Aβ-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate Aβ(25–35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated Aβ toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD.

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.

Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure

Evidence is presented that free radical stress can directly induce physico-chemical alterations in rodent neocortical synaptosomal membrane proteins. Synaptosomes were prepared from gerbil cortical brain tissue and incubated with 3 mM ascorbate and various concentrations of exogenous Fe2+ for 30-240 min at 37 degrees C. Synaptosomes were then lysed and covalently labeled with the protein thiol-selective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperdin-1-oxyl) and subjected to electron paramagnetic resonance (EPR) spectrometry. In separate experiments, synaptosomal membranes were labeled with the thiol-specific spin label MTS ((1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate), or the lipid-specific spin probe 5-NS (5-nitroxide stearate). Free radical stress induced by iron/ascorbate treatment has a rigidizing effect on the protein infrastructure of these membranes, as appraised by EPR analysis of membrane protein-bound spin label, but no change was detected in the lipid component of the membrane. These results are discussed with reference to potential oxidative mechanisms in aging and neurological disorders.

Brain Distribution and Toxicological Evaluation of a Systemically Delivered Engineered Nanoscale Ceria

Engineered nanoscale ceria is used as a diesel fuel catalyst. Little is known about its mammalian central nervous system effects. The objective of this paper is to characterize the biodistribution of a 5-nm citrate-stabilized ceria dispersion from blood into brain and its pro- or antioxidant effects. An approximately 4% aqueous ceria dispersion was iv infused into rats (0, 100, and up to 250 mg/kg), which were terminated after 1 or 20 h. Ceria concentration, localization, and chemical speciation in the brain were assessed by inductively coupled plasma mass spectrometry, light and electron microscopy (EM), and electron energy loss spectroscopy (EELS). Pro- or antioxidative stress effects were assessed as protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-trans-nonenal in hippocampus, cortex, and cerebellum. Glutathione reductase, glutathione peroxidase, manganese superoxide dismutase, and catalase levels and activities were measured in hippocampus. Catalase levels and activities were also measured in cortex and cerebellum. Na fluorescein and horseradish peroxidase (HRP) were given iv as blood-brain barrier (BBB) integrity markers. Mortality was seen after administration of 175-250 mg ceria/kg. Twenty hours after infusion of 100 mg ceria/kg, brain HRP was marginally elevated. EM and EELS revealed mixed Ce(III) and Ce(IV) valence in the freshly synthesized ceria in vitro and in ceria agglomerates in the brain vascular compartment. Ceria was not seen in microvascular endothelial or brain cells. Ceria elevated catalase levels at 1 h and increased catalase activity at 20 h in hippocampus and decreased catalase activity at 1 h in cerebellum. Compared with a previously studied approximately 30-nm ceria, this ceria was more toxic, was not seen in the brain, and produced little oxidative stress effect to the hippocampus and cerebellum. The results are contrary to the hypothesis that a smaller engineered nanomaterial would more readily permeate the BBB.

Disruption of Thiol Homeostasis and Nitrosative Stress in the Cerebrospinal Fluid of Patients with Active Multiple Sclerosis: Evidence for a Protective Role of Acetylcarnitine

Recent studies suggest that NO and its reactive derivative peroxynitrite are implicated in the pathogenesis of multiple sclerosis (MS). Patients dying with MS demonstrate increased astrocytic inducible nitric oxide synthase activity, as well as increased levels of iNOS mRNA. Peroxynitrite is a strong oxidant capable of damaging target tissues, particularly the brain, which is known to be endowed with poor antioxidant buffering capacity. Inducible nitric oxide synthase is upregulated in the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. We have recently demonstrated in patients with active MS a significant increase of NOS activity associated with increased nitration of proteins in the cerebrospinal fluid (CSF). Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, in the present study, MS patients were treated for 6 months with acetylcarnitine and compared with untreated MS subjects or with patients noninflammatory neurological conditions, taken as controls. Western blot analysis showed in MS patients increased nitrosative stress associated with a significant decrease of reduced glutathione (GSH). Increased levels of oxidized glutathione (GSSG) and nitrosothiols were also observed. Interestingly, treatment of MS patients with acetylcarnitine resulted in decreased CSF levels of NO reactive metabolites and protein nitration, as well as increased content of GSH and GSH/GSSG ratio. Our data sustain the hypothesis that nitrosative stress is a major consequence of NO produced in MS-affected CNS and implicate a possible important role for acetylcarnitine in protecting brain against nitrosative stress, which may underlie the pathogenesis of MS.

Ischemia/reperfusion-induced changes in membrane proteins and lipids of gerbil cortical synaptosomes

The effects of transient bilateral carotid occlusion on the physical state of synaptosomal membrane proteins and lipids were studied in adult and aged gerbils employing electron paramagnetic resonance. Transient ischemia was produced in adult and aged gerbils by bilateral occlusion of the common carotid arteries with reperfusion times ranging from 0 to 24 h. Synaptosomes of the cerebral cortices were isolated and labeled with a protein-specific spin probe (2,2,6,6-tetramethyl-4-maleimido-piperidine-1-oxyl) and a lipid-specific spin probe (5-doxylstearic acid). Changes in the physical state of the protein peaked at 60 min reperfusion for both adult and aged gerbil models, with a more intense change in aged, but did not return to control values by 24 h. A biphasic change occurred with the lipid-specific label in both the aged and adult models. The onset of the first phase of change occurred at an earlier time (30 min reperfusion) for aged gerbil tissue than for adult tissue (between 3 and 6 h reperfusion), while the second phase of change occurred at 12 h reperfusion for both adult and aged. These results are consistent with the hypothesis that protein oxidation and lipid peroxidation are direct results of free radicals produced during the reperfusion following ischemia and that protein oxidation may be intensified by peroxidation of the surrounding lipids. Phospholipase A2 activation is implicated to cause changes in membrane phospholipid organization as seen in these studies.