Increase in Expression Levels and Resistance to Sulfhydryl Oxidation of Peroxiredoxin Isoforms in Amyloid β-Resistant Nerve Cells

Abstract

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid ␤ (A␤) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that A␤-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. A␤-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased A␤ resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to A␤ display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in A␤-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant A␤ exposure.A wide body of evidence has implicated oxidative damage in the pathogenesis of Alzheimer disease (AD) 3 (1). AD, the most common form of dementia in the elderly, is characterized by extracellular neuritic plaques containing the amyloid beta (A␤) peptide 1-42 and intracellular neurofibrillary tangles composed mainly of hyperphosphorylated tau protein. Several studies have shown that A␤ exposure increases levels of hydrogen peroxide (H 2 O 2 ), lipid peroxidation, and protein oxidation (carbonylation) in cultured neurons (2-4). Increased oxidative damage to lipids, DNA, and proteins is also found in AD brains (1). Because the exogenous addition of antioxidants or catalase protects cultured nerve cells from A␤ toxicity (2, 5), it is likely that free radicals play a critical role in A␤ cytotoxicity. Therefore, cellular mechanisms that either prevent or remove reactive oxygen species (ROS) or reverse damage to cellular components after ROS exposure may play a key role in blocking A␤ toxicity. Although widespread nerve cell death occurs in the brains of AD patients, some neurons are spared, indicating that certain populations of cells survive the same conditions that kill neighboring cells. Early studies had shown that low concentrations of A␤ can actually rescue neurons from stressful conditions (6). However, the mechanism of A␤ resistance is only poorly understood. Previously, a series of A␤-resistant clones w...

Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid β and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production

“…Although difficult to study in patients, models of A␤ resistance have been generated in vitro following the continual exposure of cultured nerve cells to concentrations of A␤ that would otherwise be toxic and the eventual emergence of surviving clonal nerve cell populations. Analysis of these A␤-resistant nerve cells revealed up-regulation of anti-oxidant enzymes compared with the sensitive parental cells (12,13). Additionally these cells displayed an increased resistance to a wide array of neurotoxins, suggesting that acquisition of A␤ resistance also confers resistance to a variety of environmental stresses (14).…”

“…Twelve-month-old tg-AD mice and age matched controls were perfused with saline and protease inhibitor mixture, and brain regions were snap-frozen and stored at Ϫ80°C. Frozen tissue samples were partially thawed, and ϳ100-mg pieces were removed and minced in a 5ϫ weight/volume extraction buffer containing 50 mM Tris, pH 7.5, 2% SDS, and protease inhibitor mixture (12,15). Following sonication and centrifugation, supernatants were collected, and protein extracts were quantified using the Lowry assay.…”

“…At this time cells were treated with 10 M A␤ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Cells were harvested in 2% SDS buffer as described above at 4,8,12,16,24,36, and 48 h post-A␤ treatment. PDK1 and LDHA were visualized by Western blot analysis as described above, and protein quantification was performed using ImageJ software.…”

“…Protein extracts were quantified by a Lowry assay, resolved by 12% SDS-PAGE, and electroblotted onto PVDF membrane (Bio-Rad) (12). Membranes were probed with the following antibodies: polyclonal anti-LDHA (1:1000; Cell Signaling, Danvers, MA), polyclonal anti-PDK1 (1:1000; StressGen, San Diego), and a monoclonal anti-␤-actin (1:2000; Cell Signaling, Danvers, MA) followed by incubation with an appropriate horseradish peroxidase-conjugated secondary antibody (BioRad).…”

Interactome mapping suggests new mechanistic details underlying Alzheimer's disease

“…Hypothetical role of ECSIT as molecular link between oxidative stress, inflammation, and mitochondrial dysfunction in AD Chronic Ab exposure increases protein oxidation in cultured neurons and in AD brains, indicating that mitochondria play a critical role in Ab cytotoxicity and thereby in the pathogenesis of AD (Cumming et al 2007). In the AD-PIN network we detected several modules linking redox signaling and immune responses.…”