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 beta (Abeta) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Abeta-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Abeta-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 Abeta 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 Abeta 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 Abeta-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Abeta exposure.
Highlights
Common form of dementia in the elderly, is characterized by extracellular neuritic plaques containing the amyloid beta (A) peptide1–42 and intracellular neurofibrillary tangles composed mainly of hyperphosphorylated tau protein
nicotinamide adenine dinucleotide phosphate (NADPH) is essential for maintaining glutathione and thioredoxin (Trx) in a reduced state through reactions catalyzed by glutathione reductase and thioredoxin reductase (TrxR), respectively [8]
A-Resistant PC12 Cells Display Increased Prx Levels—Previous studies have shown that A-resistant clonal populations of PC12 nerve cells are resistant to multiple forms of oxidative stress [2, 5, 7, 25]
Summary
Culture Conditions and Experimental Treatment—The PC12 clonal cell line, their A-resistant derivatives, and their culture conditions have been previously described [2, 5]. PC12 cells and primary cultures were seeded in 35-mm dishes at 5 ϫ 105 and 2 ϫ 106 cells/ dish, respectively, and co-transfected with either pcDNA or FLAGtagged Prx expression constructs along with an enhanced green fluorescent protein expression vector (Clontech, Palo Alto, CA) at a 3:1 molar ratio for a total of 4 g of DNA per dish using Lipofectamine 2000 (Invitrogen) in serum-free media. Immunoblotting—After exposure to various oxidative stimuli, ϳ1 ϫ 106 cells were washed twice with phosphate-buffered saline (PBS) and incubated in ice-cold PBS with 40 mM iodoacetamide for 5 min to prevent thiol-disulfide exchange and inhibit post-lysis oxidation of free cysteines These cells were harvested in 0.1 ml of digitonin extraction buffer (10 mM PIPES, pH 6.8, 0.015% (w/v) digitonin, 300 mM sucrose, 100 mM
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