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

2007; Elsevier BV; Volume: 282; Issue: 42 Linguagem: Inglês

10.1074/jbc.m700869200

1083-351X

Robert C. Cumming, Richard Dargusch, Wolfgang Fischer, David Schubert,

Cholinesterase and Neurodegenerative Diseases

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 β (Aβ) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Aβ-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Aβ-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 Aβ 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 Aβ 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 Aβ-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Aβ exposure. 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 β (Aβ) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Aβ-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Aβ-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 Aβ 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 Aβ 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 Aβ-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Aβ exposure. A wide body of evidence has implicated oxidative damage in the pathogenesis of Alzheimer disease (AD) 3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β peptide; MS, mass spectrometry; ROS, reactive oxygen species; Prx, peroxiredoxin; Prx-SO2, sulfinylated Prx; PrxSO3, sulfonylated Prx; Trx, thioredoxin; TrxR, Trx reductase; PPP, pentose phosphate pathway; LC-MS, liquid chromatography electrospray ionization mass spectrometry; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ANOVA, analysis of variance; tg, transgenic.3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β peptide; MS, mass spectrometry; ROS, reactive oxygen species; Prx, peroxiredoxin; Prx-SO2, sulfinylated Prx; PrxSO3, sulfonylated Prx; Trx, thioredoxin; TrxR, Trx reductase; PPP, pentose phosphate pathway; LC-MS, liquid chromatography electrospray ionization mass spectrometry; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ANOVA, analysis of variance; tg, transgenic. (1Butterfield D.A. Griffin S. Munch G. Pasinetti G.M. J. Alzheimers Dis. 2002; 4: 193-201Crossref PubMed Scopus (137) Google Scholar). AD, the most common form of dementia in the elderly, is characterized by extracellular neuritic plaques containing the amyloid beta (Aβ) peptide1–42 and intracellular neurofibrillary tangles composed mainly of hyperphosphorylated tau protein. Several studies have shown that Aβ exposure increases levels of hydrogen peroxide (H2O2), lipid peroxidation, and protein oxidation (carbonylation) in cultured neurons (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 3Aksenov M.Y. Aksenova M.V. Markesbery W.R. Butterfield D.A. J. Mol. Neurosci. 1998; 10: 181-192Crossref PubMed Scopus (40) Google Scholar, 4Butterfield D.A. Lauderback C.M. Free Radic. Biol. Med. 2002; 32: 1050-1060Crossref PubMed Scopus (863) Google Scholar). Increased oxidative damage to lipids, DNA, and proteins is also found in AD brains (1Butterfield D.A. Griffin S. Munch G. Pasinetti G.M. J. Alzheimers Dis. 2002; 4: 193-201Crossref PubMed Scopus (137) Google Scholar). Because the exogenous addition of antioxidants or catalase protects cultured nerve cells from Aβ toxicity (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar), it is likely that free radicals play a critical role in Aβ 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 Aβ 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 Aβ can actually rescue neurons from stressful conditions (6Yankner B.A. Duffy L.K. Kirschner D.A. Science. 1990; 250: 279-282Crossref PubMed Scopus (1909) Google Scholar). However, the mechanism of Aβ resistance is only poorly understood. Previously, a series of Aβ-resistant clones was derived from the rat pheochromocytoma cell line PC12 by growth for 4 months in the presence of high concentrations Aβ and subsequent cloning (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar). Aβ-resistant nerve cell clones not only survive exposure to toxic concentrations of Aβ but are also less sensitive to hydrogen peroxide (H2O2) and t-butyl-H2O2, in part due to increased expression and activities of the antioxidant enzymes glutathione peroxidase and catalase (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar). Further analysis revealed that Aβ-resistant nerve cells also have an enhanced flux of glucose through both the glycolytic and pentose phosphate pathway (PPP) (7Soucek T. Cumming R. Dargusch R. Maher P. Schubert D. Neuron. 2003; 39: 43-56Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). As a result of enhanced glycolysis and PPP activity, Aβ-resistant cells produce higher levels of reducing equivalents such as reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is essential for maintaining glutathione and thioredoxin (Trx) in a reduced state through reactions catalyzed by glutathione reductase and thioredoxin reductase (TrxR), respectively (8Arner E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1995) Google Scholar). Both the glutathione and TrxR systems are essential for maintaining intracellular protein sulfhydryls in a reduced state. Peroxiredoxins (Prxs) are a widely expressed group of peroxidases that reduce H2O2, peroxynitrite, and a range of organic hydroperoxides using reducing equivalents provided by the Trx/TrxR system (9Wood Z.A. Schroder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2118) Google Scholar). Recent studies have indicated that Prxs can also mediate signal transduction processes elicited by various growth factors and cytokines (10Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (615) Google Scholar). Prxs, which exist as homodimers (with the exception of Prx6), use redox-active cysteines to reduce peroxides and are divided into two classes, the 1-Cys and 2-Cys Prxs, based upon the number of conserved cysteines involved in catalysis (9Wood Z.A. Schroder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2118) Google Scholar). Typical 2-Cys Prxs, the largest class of Prxs, undergo a catalytic cycle in which the N-terminal cysteine (generally near residue 50) is oxidized by H2O2 to a cysteine-sulfenic acid (Cys-SOH) that then reacts with the C-terminal cysteine (near residue 170) of another subunit to produce an intermolecular disulfide. This disulfide is then reduced by the Trx/TrxR system, completing the catalytic cycle. Formation of the disulfide bond is a slow process, and the sulfenic intermediate is occasionally hyperoxidized to a sulfinic acid (Cys-SO2H) or even a sulfonic acid (Cys-SO3H), resulting in inactivation of Prx peroxidase activity (11Woo H.A. Chae H.Z. Hwang S.C. Yang K.S. Kang S.W. Kim K. Rhee S.G. Science. 2003; 300: 653-656Crossref PubMed Scopus (469) Google Scholar). Oxidation of the catalytic cysteine to a Cys-SO2H in Prxs 1–4 is reversed by a reaction catalyzed by sulfiredoxin (12Woo H.A. Jeong W. Chang T.S. Park K.J. Park S.J. Yang J.S. Rhee S.G. J. Biol. Chem. 2005; 280: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). No mechanism of sulfinic acid reduction in Prx5 and Prx6 isoforms has yet been identified. Moreover, over-oxidation of all Prx isoforms to a Cys-SO3H state is irreversible. Of the six known mammalian Prx isoforms, Prx1–4 belong to the typical 2-Cys class, and Prx5 is an atypical 2-Cys that forms an intramolecular disulfide as part of its catalytic cycle (9Wood Z.A. Schroder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2118) Google Scholar). Prx6 belongs to the 1-Cys class which possesses only one catalytic cysteine. Oxidized Prx6 (Cys-SOH) is either directly reduced by ascorbate (13Monteiro G. Horta B.B. Pimenta D.C. Augusto O. Netto L.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4886-4891Crossref PubMed Scopus (167) Google Scholar) or via a reaction catalyzed by glutathione S-transferase π (14Ralat L.A. Manevich Y. Fisher A.B. Colman R.F. Biochemistry. 2006; 45: 360-372Crossref PubMed Scopus (157) Google Scholar). Prx1, Prx2, and Prx6 are found mainly in the cytosol, Prx3 is found in mitochondria, and Prx4 is a secreted protein (9Wood Z.A. Schroder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2118) Google Scholar). Prx5 is localized to the cytosol, mitochondria, and peroxisomes (15Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Initial studies examining Prx1 and Prx2 expression in human brain revealed that Prx1 was expressed primarily in astrocytes, whereas Prx2 was expressed exclusively in neurons in the cerebral cortex, hippocampus, cerebellum, basal ganglia, substantia nigra, and spinal cord. A more recent immunohistochemical study in mouse brain revealed cytoplasmic expression of Prx2–5 in neurons throughout multiple regions of the brain (16Jin M.H. Lee Y.H. Kim J.M. Sun H.N. Moon E.Y. Shong M.H. Kim S.U. Lee S.H. Lee T.H. Yu D.Y. Lee D.S. Neurosci. Lett. 2005; 381: 252-257Crossref PubMed Scopus (90) Google Scholar). Prx1 and Prx6 expression was detected in microglia and astrocytes. respectively. These findings suggest that the varied distribution of Prx isoforms in the brain may serve distinct roles that may reflect the different functions and biochemical activities of their host cells. Several studies have shown that Prx expression is elevated in neurodegenerative diseases such as AD, Parkinson disease, Creutzfeldt-Jakob disease, and amyotrophic lateral sclerosis (17Krapfenbauer K. Engidawork E. Cairns N. Fountoulakis M. Lubec G. Brain Res. 2003; 967: 152-160Crossref PubMed Scopus (238) Google Scholar, 18Basso M. Giraudo S. Corpillo D. Bergamasco B. Lopiano L. Fasano M. Proteomics. 2004; 4: 3943-3952Crossref PubMed Scopus (222) Google Scholar, 19Krapfenbauer K. Yoo B.C. Fountoulakis M. Mitrova E. Lubec G. Electrophoresis. 2002; 23: 2541-2547Crossref PubMed Scopus (44) Google Scholar, 20Kato S. Kato M. Abe Y. Matsumura T. Nishino T. Aoki M. Itoyama Y. Asayama K. Awaya A. Hirano A. Ohama E. Acta Neuropathol. (Berl). 2005; 110: 101-112Crossref PubMed Scopus (50) Google Scholar). However, it is not known if increased Prx expression in these diseases occurs as a general response to nerve cell death or is a protective mechanism elicited by disease-specific stimuli. Here we show that nerve cells selected for Aβ-resistance exhibit increased expression of specific Prx isoforms that are less susceptible to oxidative inactivation. Transfection of Prx1 in either PC12 cells or primary hippocampal neurons confers increases resistance to Aβ. Exposure of rat cortical primary neurons to Aβ causes a time-dependent increase in Prx oxidation that is countered by a thiol-specific antioxidant. In addition, cortical tissue from post-mortem AD patients exhibits increased expression of Prx1, whereas Prx2 exists in a more oxidized (acidic) form in AD brains. These findings suggest that increased Prx expression and the ability to maintain Prxs in a reduced state is part of a specific neuroprotective mechanism that occurs in response to Aβ accumulation. Understanding these mechanisms of Aβ resistance may be important for designing therapies to increase both the antioxidant and reductive capacity of nerve cells in AD patients. Culture Conditions and Experimental Treatment—The PC12 clonal cell line, their Aβ-resistant derivatives, and their culture conditions have been previously described (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar). Rat primary cortical and hippocampal cultures were prepared and cultured under standard conditions (21Li Y. Maher P. Schubert D. Neuron. 1997; 19: 453-463Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). For Aβ treatment, a stock solution (0.5 mm) of the Aβ1–42 peptide (Bachem) was freshly prepared in distilled water and allowed to form fibrils for 4 h at room temperature before the addition to cell culture media. Primary cultures were treated with 10 μm Aβ1–42 peptide for various time periods as indicated. In some cases primary cultures were co-treated with 1 mm N-acetylcysteine (Sigma). Expression constructs containing FLAG-tagged versions of both wild type and cysteine mutant (C52S) Prx cDNAs were generously supplied by Dr. Hyunjung Ha (Chungbuk National University, Cheonju Korea). PC12 cells and primary cultures were seeded in 35-mm dishes at 5 × 105 and 2 × 106 cells/dish, respectively, and co-transfected with either pcDNA or FLAG-tagged 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 μg of DNA per dish using Lipofectamine 2000 (Invitrogen) in serum-free media. Plasmid DNA was mixed with 4 μl of Lipofectamine 2000 in Opti-MEM media (Invitrogen) added to PC12 cell cultures, and 6 h later the transfection media was replaced with regular media supplemented with 10% fetal bovine serum. Primary hippocampal neurons were transfected based on a protocol optimized for transfecting this cell type (22Ohki E.C. Tilkins M.L. Ciccarone V.C. Price P.J. J. Neurosci. Methods. 2001; 112: 95-99Crossref PubMed Scopus (103) Google Scholar). In brief, plasmid DNA was mixed with 8 μl of Lipofectamine 2000 in Opti-MEM media (500 μl total volume) and allowed to form a complex for 20 min at room temperature. The DNA-Lipofectamine complex was then added to the primary hippocampal culture, and after 6 h the transfection media was replaced with Neurobasal media with B27 supplement (Invitrogen) and 0.5 mm l-glutamine. Transfection efficiencies were ∼70 and 20% for PC12 and primary hippocampal neurons, respectively. One day after transfection, 20 μm Aβ1–42 peptide was added to the experimental dishes, and cell viability was determined at 24-h intervals. Twenty random fields of cells transfected with the Prx constructs (∼500 cells total) were scored for green fluorescence at 400× magnification using a Leica DMIRB microscope equipped with a mercury lamp and appropriate filters. Cell viability was assessed by comparing the average number of green positive cells after Aβ treatment versus no treatment. Cell viability data were based on the average of three separate experiments. Immunoblotting—After exposure to various oxidative stimuli, ∼1 × 106 cells were washed twice with phosphate-buffered saline (PBS) and then 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 NaCl, 3 mm MgCl2, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 40 mm iodoacetamide) followed by rocking on ice for 10 min. After centrifugation at 2500 × g, extracts were collected, and protein concentrations were determined using the Lowry assay (Bio-Rad). After the addition of an equal volume of 2× loading buffer (80 mm Tris, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromphenol blue, 4% β-mercaptoethanol) protein extracts (20 μg) were resolved using 12% SDS-PAGE precast gels (Bio-Rad) electroblotted onto Immobilon P membrane (Millipore, Bedford, MA) and blocked with 1% milk, 3% bovine serum albumin in Tris-buffered saline. Blots were hybridized with antibodies against Prx-specific isoforms, Trx-1 (Lab-Frontier, Seoul, Korea), thioredoxin reductase 1 (Upstate Biotechnology, Inc., Lake Placid, NY), FLAG, or actin (Sigma) overnight and, after washing, were further hybridized with appropriate horseradish peroxidate-conjugated secondary antibodies (Bio-Rad). Detection was performed using ECL Western blotting detection reagents (Amersham Biosciences). Densitometric Analysis—Western blots were scanned using a GS-800 calibrated densitometer equipped with Quantity One software (Bio-Rad). Gel bands were scanned three times and normalized to actin. In studies using cell lines, the densitometric values from three separate experiments were averaged. For protein expression studies using human post-mortem brain samples, densitometric values were normalized to actin, and then the total values for both the control and AD sample sets were averaged. Brain Tissue—Autopsied brain samples were obtained from Dr. Carol Miller at the Alzheimer Disease Research Center, Los Angeles, CA. All tissue samples were removed from the same area of the mid-frontal cortex and immediately quick-frozen. AD cases and controls were matched pairwise for age, sex, and in most cases post-mortem time. All AD patients had a clinical history of dementia, and histological analysis of mid-frontal cortex samples revealed plaque density scores in the low to frequent range according to The Consortium to Establish a Registry for Alzheimer's Disease criterion (1 = sparse, 1–5; 3 = moderate, 6–20; 5 = frequent, 21–30 or above). Patient details are summarized in Table 1.TABLE 1Control and AD patient detailsPatientDiagnosisAgeSexPMTPDSahC1Normal94Female4.50A1AD95Female4.53C2Normal87Female60A2AD86Female61C3Normal88Male20A3AD85Male4.51C4Normal69Male80A4AD75Male25C5Normal91FemaleN/A0A5AD90Female4.55C6Control92Female70A6AD90Female7.53C7Control80Male120A7AD77Male35C8Control71Female110A8AD72Female55 Open table in a new tab Frozen tissue samples were partially thawed, and ∼100-mg pieces were removed and minced in 5× weight/volume extraction buffer containing 50 mm Tris, pH 7.5, 2% SDS, and a protease inhibitor mixture. After sonication and centrifugation, supernatants were collected, and protein extract concentrations were determined using the Lowry assay. Tissue protein extracts (20 μg) were analyzed by immunoblotting as described above. Two-dimensional Gel Electrophoresis and Mass Spectrometry Analysis—Because the type and amount of proteins vary dramatically with the growth state of cultured cells (23Garrels J.I. J. Biol. Chem. 1979; 254: 7961-7977Abstract Full Text PDF PubMed Google Scholar), care was taken to grow and plate cells under identical conditions where comparisons of protein content were to be made. Exponentially dividing cultures were dissociated and replated at 5 × 105 cells/100-mm tissue culture plate. The next day 10 μm Aβ1–42 was added, and 24 h later the cells were harvested for two-dimensional gel electrophoresis. 0.5 ml of 8 m urea, 4% (w/v) CHAPS, 40 mm Tris, 0.2% Bio-Lyte 3–10 ampholytes (Bio-Rad), and 50 mm dithiothreitol were added to the plate, and the cells were scraped into a microcentrifuge tube. DNase1/RNase (100/50 μg/ml) was added to the lysate and incubated for 30 min at room temperature. After centrifugation at 14,000 × g, the protein amount was determined, and 300 μg of supernatant was loaded onto pH 4–7 isoelectric focusing strips (Bio-Rad) and electrophoresed to 60,000 V-h on a Bio-Rad Protean isoelectric focusing machine. The strip was then applied to the top of an 18-cm Bio-Rad precast 12% acrylamide gel and electrophoresed at 25 milliamps per gel until the dye front was at the bottom of the gel. Gels were fixed in 50% methanol overnight and silver stained according to the method of Shevchenko et al. (24Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7814) Google Scholar). Gel spots from two-dimensional gels were excised, in-gel-digested with trypsin (24Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7814) Google Scholar), and analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-MS). Briefly, samples were loaded onto a fused silica capillary column (PicoFrit Column, New Objective, Woburn, MA) packed with reversed phase material (Zorbax C-18, 5-μm particle size, Agilent, Santa Clara, CA). The mobile phase consisted of aqueous 0.1% formic acid (A buffer) and 0.1% formic acid in 80% acetonitrile/water (B buffer). Elution was achieved by a gradient of 5 to 70% B buffer in 65 min at a flow rate of 150 nl/min. The eluant was electrosprayed into a Bruker Esquire 3000 Plus quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Spectra were measured for the three most intense species in each time window. For each mass, MS and MS/MS spectra were recorded. After two sets of spectra, the parent mass was excluded for 1 min. The complete data set for each gel spot was searched using the Mascot algorithm (Matrix Science, London, UK) against a recent release of the non-redundant NCBI data base. Only results that gave significant Mascot scores were reported. Statistical Analysis—Changes in protein expression levels or cell viability were analyzed by ANOVA followed by Tukey post hoc testing using GraphPad InStat software. A p value <0.05 was considered significantly significant. Aβ-Resistant PC12 Cells Display Increased Prx Levels—Previous studies have shown that Aβ-resistant clonal populations of PC12 nerve cells are resistant to multiple forms of oxidative stress (2Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2047) Google Scholar, 5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar, 7Soucek T. Cumming R. Dargusch R. Maher P. Schubert D. Neuron. 2003; 39: 43-56Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25Dargusch R. Schubert D. J. Neurochem. 2002; 81: 1394-1400Crossref PubMed Scopus (41) Google Scholar). Although part of this resistance is attributable to an up-regulation of antioxidant and glycolytic enzymes, the full repertoire of differentially expressed proteins in Aβ-resistant cells has not been examined. We, therefore, compared protein profiles between an Aβ-resistant clone (PC12r7) and its parental cell line (PC12) using two-dimensional gel electrophoresis and liquid chromatography mass spectrometry (LC-MS) analysis to identify other proteins that may contribute to Aβ resistance. The range of proteins that displayed the most dramatic differences in abundance and pI focused between pH 5.3 and 6.5 with a molecular mass between 15 and 50 kDa. Among this subset of proteins ∼26 spots were identified that varied between the sensitive and resistant cells (Fig. 1). In agreement with earlier work (7Soucek T. Cumming R. Dargusch R. Maher P. Schubert D. Neuron. 2003; 39: 43-56Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), a number of glycolytic enzymes (Table 2) were up-regulated in the PC12r7 clone. Essentially identical results were obtained with another resistant clone, PC12r1. When PC12 cells were transiently exposed to 10 μm Aβ1–42, the same group of enzymes was up-regulated including triosephosphate isomerase, phosphoglycerate mutase, α-enolase, and aldolase A (Table 2). In addition, several antioxidant proteins were also elevated in the resistant and Aβ-treated cells including biliverdin reductase and Prx6.TABLE 2MS identification of proteins in PC12 control, PC12r7, and PC12 cells treated with AβSpot no.PC12PC12r7AβProtein nameAccession no.Molecular massMascot scorePeptideskDa1↓+-Heat shock protein 2720466522,8791313↓+-Ribosomal protein S121392899214,2591052↓+-NDPK25592614517,2726622↓+-Histone H2B47767613,8511785↓+-Ub-conj. enzyme E2N1675881017,1136413↓+↓Histone 13006139113,9842119↓+↓Phosphoglycerate mutase type b824881928,8281684↓+↓Ribosomal protein S205609027113,3641334↓+↓Ub-conj. enzyme E2N1675881017,11310824++↓Ub-conj. enzyme E2N1675881017,1131114++↓Ribosomal protein S196266137115,782662++↓Histone H2B47767613,8516425-+-Ran/TC4-binding protein73924123,568952-+-Ribosomal protein S196266137115,782923-+-Histone 13006139113,984602-+-Ribosomal protein S121392899214,259582-+-Ub-conj. enzyme E2N1675881017,1135126+↓↓Cu,Zn SOD20701215,7002737+↓↓Ub-conj. enzyme E2N1675881017,11310347+↓+Stmn13832824217,27834588-++Ca2+-regulated heat-stable protein2275814215,8968959-+-Transgelin 26155702822,3792064-+-αB-Crystallin5758019,945190510↓++Peroxiredoxin 61675834824,803392911++↓Peroxiredoxin 61675834824,8032806++↓Peroxiredoxin 31196813228,3031062++↓Heat shock protein 2720466522,879169412+↓↓Peroxiredoxin 31196813228,3032832+↓↓Lysophospholipase 2724215624,77866113+-↓Heat shock protein 2720466522,8793158+-↓Ubiquitin thiolesterase9293424,754228614+↓+Heat shock protein 2720466522,87949614+↓+Peroxiredoxin 61675834824,80385215+↑+Bilverdin reductase1387928622,1833086+↑+Peroxiredoxin 61675834824,8031135+↑+Transketolase1201825267,64483216+-+Stmn13832824217,2781974+-+Lysophospholipase 2724215624,77854217-+-Triosephosphate isomerase 11262107426,904332918+↓+Triosephosphate isomerase 11262107426,9041795+↓+Peroxiredoxin 61675834824,803110419-+-α-Enolase5092683347,0984911120+-↓Proteasome 28 subunit a6109821428,6172385+-↓Heat shock protein 2720466522,879122321+--Heat shock protein 2720466522,8793387+--Ubiquitin thiolesterase9293424,754166522+↑↓Aldolase A697848739,327294523+-+Heat shock protein 2720466522,87933124+↓+Aldolase A697848739,32755613+↓+α-Enolase5092683347,098106425+↓↓Glyceraldehyde-3-phosphate dehydrogenase5618835,813148626+-↓Glyceraldehyde-3-phosphate dehydrogenase5618835,813602+-↓Acat25403534141,10847127+--Heat shock protein 2720466522,879742 Open table in a new tab Curiously, Prx6 was identified in five different locations in the gels (Fig. 1). Although increased catalase and glutathione peroxidase activity have been detected in Aβ-resistant cells (5Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar), the observation that Prx6 expression was elevated in Aβ-resistant cells was new. Because multiple proteins can reside in the same spot and mask the relative protein abundance of Prx isoforms on silver-stained two-dimensional gels, we examined expression levels of all six Prx isoforms in lysates from both parental and Aβ-resistant PC12 cell lines by immunoblotting with isoform-specific antibodies. Although the same amount of protein per lane was loaded, all Prx levels were also standardized against an actin loading control. As expected, PC12-resistant clones displayed elevated