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 Alzheimer's 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 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 -peptide (A), the main constituent of senile plaques in Alzheimer's 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 A occurs in conjunction with free radical oxidative stress associated with the peptide. A(1–40) and several other fragments of the A 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 A-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 A(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 A toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

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.     

Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor

Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis.     

Interacting quantitative trait loci control loss of peripheral tolerance and susceptibility to autoimmune ovarian dysgenesis after day 3 thymectomy in mice

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by CD4(+)CD25(+) T cells and the development of autoimmune ovarian dysgenesis (AOD) in A/J and (C57BL/6J x A/J)F(1) (B6AF(1)) hybrids but not in C57BL/6J mice. Quantitative trait loci (QTL) linkage analysis using a B6AF(1) x C57BL/6J backcross population verified Aod1 and Aod2 that were previously mapped as qualitative traits. Additionally, three new QTL intervals, Aod3, Aod4, and Aod5, on chromosomes 1, 2, and 7, respectively, influencing specific subphenotypes of AOD were identified. QTL linkage analysis using the A x B and B x A recombinant inbred lines verified Aod3 and confirmed linkage to H2. Aod5 colocalized with Mater, an ovarian-specific autoantigen recognized by anti-ovarian autoantibodies in the sera of D3Tx mice. Sequence analysis of Mater identified allelic, strain-specific splice variants between A/J and C57BL/6J mice making it an attractive candidate gene for Aod5. Interaction analysis revealed significant epistatic effects between Aod1-5 and Gasa2, a locus associated with susceptibility to D3Tx-induced autoimmune gastritis, as well as with H2. These results indicate that the QTL controlling D3Tx-induced autoimmune phenomenon are both organ specific and more generalized in their effects with respect to the genesis and activity of the immunoregulatory mechanisms maintaining peripheral tolerance.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

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

Amyloid beta-peptide 1-42 [Abeta(1-42)] is central to the pathogenesis of Alzheimer's 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.