Jaroslaw Kanski

Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins.

Protein oxidation, one of a number of brain biomarkers of oxidative stress, is increased in several age-related neurodegenerative disorders or animal models thereof, including Alzheimers disease, Huntingtons disease, prion disorders, such as Creutzfeld-Jakob disease, and alpha-synuclein disorders, such as Parkinsons disease and frontotemporal dementia. Each of these neurodegenerative disorders is associated with aggregated proteins in brain. However, the relationship among protein oxidation, protein aggregation, and neurodegeneration remain unclear. The current rapid progress in elucidation of mechanisms of protein oxidation in neuronal loss should provide further insight into the importance of free radical oxidative stress in these neurodegenerative disorders.

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 Alzheimers disease in which oxidative stress is implicated.

Methionine residue 35 is important in amyloid β-peptide-associated free radical oxidative stress

Amyloid beta-peptide (Abeta), the central constituent of senile plaques in Alzheimers disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In the current study Abeta(1-40), found in AD brain, and the amyloid fragment Abeta(25-35) were used in conjunction with electron paramagnetic resonance spin trapping techniques to demonstrate that these peptides mediate free radical production. The methionine residue in these peptides is believed to play an important role in their neurotoxicity. Substitution of methionine by structurally similar norleucine in both Abeta(1-40) and Abeta(25-35), and the substitution of methionine by valine, or the removal of the methionine in Abeta(25-35), abrogates free radical production and protein oxidation of and toxicity to hippocampal neurons. These results are discussed with relevance to the hypothesis that neurodegeneration in Alzheimers disease may be due in part to Abeta-associated free radical oxidative stress that involves methionine, and to the use of spin trapping methods to infer mechanistic information about Abeta.

Methionine residue 35 is critical for the oxidative stress and neurotoxic properties of Alzheimer’s amyloid β-peptide 1–42

Amyloid beta-peptide 1-42 [Abeta(1-42)] is central to the pathogenesis of Alzheimers disease (AD), and the AD brain is under intense oxidative stress. Our laboratory combined these two aspects of AD into the Abeta-associated free radical oxidative stress model for neurodegeneration in AD brain. Abeta(1-42) caused protein oxidation, lipid peroxidation, reactive oxygen species formation, and cell death in neuronal and synaptosomal systems, all of which could be inhibited by free radical antioxidants. Recent studies have been directed at discerning molecular mechanisms by which Abeta(1-42)-associated free radical oxidative stress and neurotoxicity arise. The single methionine located in residue 35 of Abeta(1-42) is critical for these properties. This review presents the evidence supporting the role of methionine in Abeta(1-42)-associated free radical oxidative stress and neurotoxicity. This work is of obvious relevance to AD and provides a coupling between the centrality of Abeta(1-42) in the pathogenesis of AD and the oxidative stress under which the AD brain exists.

Apolipoprotein E modulates Alzheimer’s Aβ(1–42)-induced oxidative damage to synaptosomes in an allele-specific manner

Several functional differences have been reported among the three human e2, e3, and e4 alleles of apolipoprotein E (apoE). One functional difference lies in the antioxidant potential of these alleles; e4 has the poorest potential. Interestingly, e4 also correlates with increased oxidative damage in the Alzheimers disease (AD) brain, which may explain why the inheritance of the e4 allele is a risk factor for the onset of AD. Beta-amyloid (Abeta) is also intimately involved in AD and promotes oxidative damage in vitro; therefore, we have examined the role of the different apoE alleles in modulating Abeta(1-42)-induced oxidation to synaptosomes. Measurement of specific markers of oxidation in synaptosomes isolated from mice that express one of the human apoE alleles indicates that Abeta-induced increases of these markers can be modulated by apoE in an allele-dependent manner (e2>e3>e4). Increases in reactive oxygen species formation and protein and lipid oxidation were always greatest in e4 synaptosomes as compared to e2 and e3 synaptosomes. Our data support the role of apoE as a modulator of Abeta toxicity and, consistent with the antioxidant potentials of the three alleles, suggest that the e4 allele may not be as effective in this role as the e2 or e3 alleles of apoE. These results are discussed with reference to mechanistic implications for neurodegeneration in the AD brain.

Elevation of brain glutathione by γ-glutamylcysteine ethyl ester protects against peroxynitrite-induced oxidative stress

Elevation of glutathione (GSH) has been recognized as an important method for modulating levels of reactive oxygen species (ROS) in the brain. We investigated the antioxidant properties of γ‐glu‐cys‐ethyl ester (GCEE) in vitro and its ability to increase GSH levels upon in vivo i.p. injection. GCEE displays antioxidant activity similar to GSH as assessed by various in vitro indices such as hydroxyl radical scavenging, dichlorofluorescein fluorescence (DCF), protein specific spin labeling, glutamine synthetase (GS) activity, and protein carbonyls. Intraperitoneal injection of GCEE to gerbils resulted in a 41% increase in brain total GSH levels in vivo as determined by the DTNB‐GSH reductase recycling method. Gerbils injected with buthionine sulfoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase, had 40% less total brain glutathione. Gerbils injected with BSO followed by a GCEE injection had GSH levels similar to vehicle‐injected controls, suggesting that GCEE upregulates GSH biosynthesis by providing γ‐glutamylcysteine and not cysteine. Cortical synaptosomes from GCEE‐injected animals were less susceptible to peroxynitrite‐induced oxidative damage as assessed by DCF fluorescence, protein‐specific spin labeling, and GS activity. These experiments suggest that GCEE is effective in increasing brain GSH levels and may potentially play an important therapeutic role in attenuating oxidative stress in neurodegenerative diseases associated with oxidative stress such as Alzheimer disease.

Substitution of isoleucine-31 by helical-breaking proline abolishes oxidative stress and neurotoxic properties of Alzheimer's amyloid β-peptide (1-42)

Alzheimer’s disease (AD) brain is characterized by excess deposition of the 42-amino acid amyloid β-peptide [Aβ(1–42)]. AD brain is under intense oxidative stress, and we have previously suggested that Aβ(1–42) was associated with this increased oxidative stress. In addition, we previously demonstrated that the single methionine residue of Aβ(1–42), residue 35, was critical for the oxidative stress and neurotoxic properties of this peptide. Others have shown that the C-terminal region of Aβ(1–42) is helical in aqueous micellar solutions, including that part of the protein containing Met35. Importantly, Cu(II)-binding induces α-helicity in Aβ in aqueous solution. Invoking the i + 4 rule of helices, we hypothesized that the carbonyl oxygen of Ile31 would interact with the S atom of Met35 to change the electronic environment of the sulfur such that molecular oxygen could lead to the production of a sulfuramyl free radical on Met35. If this hypothesis is correct, a prediction would be that breaking the helical interaction of Ile31 and Met35 would abrogate the oxidative stress and neurotoxic properties of Aβ(1–42). Accordingly, we investigated Aβ(1–42) in which the Ile31 residue was replaced with the helix-breaking amino acid, proline. The α-helical environment around Met35 was completely abolished as indicated by circular dichroism (CD)-spectroscopy. As a consequence, the aggregation, oxidative stress, Cu(II) reduction, and neurotoxic properties of Aβ(1–42)I31P were completely altered compared to native Aβ(1–42). The results presented here are consistent with the notion that interaction of Ile31 with Met35 may play an important role in the oxidative processes of Met35 contributing to the toxicity of the peptide.

Role of glycine-33 and methionine-35 in Alzheimer’s amyloid β-peptide 1–42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptides toxicity in Alzheimers disease brain.

The Hydrophobic Environment of Met35 of Alzheimer's Aβ(1-42) is Important for the Neurotoxic and Oxidative Properties of the Peptide

In Alzheimers disease (AD) brain increased lipid peroxidation is found. Amyloid β-peptide [Aβ(1–42)] induces oxidative stress (including lipid peroxidation) and neurotoxicity, and the single methionine residue (Met35), is important for these properties. In the current study, we tested the hypothesis that removal of Met35 from lipid bilayer would abrogate the oxidative stress and neurotoxic properties of Aβ(1–42), i.e. we tested the hypothesis and found that lipid peroxidation initiated by oxidation of the Met35 is an early event in Aβ(1–42) neurotoxicity. Substitution of negatively charged aspartic acid for glycine residue 37 is not predicted to bring the Met35 residue out of the hydrophobic lipid bilayer. In this study, we showed that G37D substitution in Aβ(1–42) completely abolishes neurotoxic and oxidative processes associated with the parent peptide. This is demonstrated by the lack of cell toxicity and protein oxidation in contrast to the treatment with native Aβ(1–42). Additionally, the G37D peptide does not display the aggregation properties that are associated with native Aβ as seen in the thioflavin T (ThT) assay and fibril morphology. The results presented in this work are thus consistent with the notion of the importance of methionine 35 of Aβ(1–42) in the lipid-initiated oxidative cascade and subsequent neurotoxicity in AD brain.

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 Alzheimers disease.