NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305204200
ISSN1083-351X
AutoresJong‐Min Lee, Andy Y. Shih, Timothy H. Murphy, Jeffrey A. Johnson,
Tópico(s)GDF15 and Related Biomarkers
ResumoNF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes (∼10%) and neurons (∼90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult. NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes (∼10%) and neurons (∼90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult. The antioxidant-responsive element (ARE) 1The abbreviations used are: ARE, antioxidant-responsive element; NQO1, NAD(P)H:quinone oxidoreductase-1; GST, glutathione S-transferase; Nrf2, NF-E2-related factor-2; PD, Parkinson's disease; MPP+, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine; DIV, days in vitro; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein-2; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GCLC, glutamatecysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; RT, reverse transcription; Ad, adenovirus; GFP, green fluorescent protein; m.o.i., multiplicity of infection; dtBHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester. plays an important role in the expression of genes encoding phase II detoxification enzymes and antioxidant proteins such as NAD(P)H: quinone oxidoreductase-1 (NQO1), glutathione S-transferases (GSTs), glutamate-cysteine ligase, and heme oxygenase-1 (1Rushmore T.H. 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A. 1994; 91: 9926-9930Crossref PubMed Scopus (1248) Google Scholar), is the principal component leading to ARE-driven gene expression. Recently, Nrf2 target genes were identified by oligonucleotide microarray analysis, and the gene lists suggest that Nrf2 is important in combating electrophiles and reactive oxygen species (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 13Thimmulappa R.K. Mai K.H. Srisuma S. Kensler T.W. Yamamoto M. Biswal S. Cancer Res. 2002; 62: 5196-5203PubMed Google Scholar, 14Shih A.Y. Johnson D.A. Wong G. Kraft A.D. Jiang L. Erb H. Johnson J.A. Murphy T.H. J. Neurosci. 2003; 23: 3394-3406Crossref PubMed Google Scholar). Many studies have shown that Nrf2 plays a critical role in protecting cells from oxidative stress. Chan et al. reported that Nrf2 protects liver from acetaminophen-induced injury (15Chan K. Han X.D. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4611-4616Crossref PubMed Scopus (654) Google Scholar) and lung from butylated hydroxytoluene-induced toxicity (16Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (526) Google Scholar). Cho et al. (17Cho H.Y. Jedlicka A.E. Reddy S.P. Kensler T.W. Yamamoto M. Zhang L.Y. Kleeberger S.R. Am. J. Respir. Cell Mol. Biol. 2002; 26: 175-182Crossref PubMed Scopus (590) Google Scholar) demonstrated that Nrf2 knockout mice are more sensitive to hyperoxia-induced lung injury. Recently, our laboratory reported that Nrf2-/- primary astrocytes are more susceptible to oxidative stress and inflammation compared with Nrf2+/+ astrocytes (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Pretreatment of these Nrf2+/+ (but not Nrf2-/-) astrocytes with t-butylhydroquinone, which induces Nrf2 nuclear translocation resulting in coordinate up-regulation of ARE-driven genes, attenuates H2O2- and platelet-activating factor-induced cell death (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Similarly, t-butylhydroquinone-mediated protective effects have been shown in rodent and human neuroblastoma cells (18Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar, 19Li J. Lee J.-M. Johnson J.A. J. Biol. Chem. 2002; 277: 388-394Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). These observations suggest that the coordinate up-regulation of antioxidant genes is the key to protecting cells from oxidative stress and that Nrf2 is a master regulator of ARE-driven antioxidant gene expression, a process we refer to as programmed cell life (19Li J. Lee J.-M. Johnson J.A. J. Biol. Chem. 2002; 277: 388-394Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Parkinson's disease (PD) is a progressive neurodegenerative disease caused by degeneration of dopaminergic neurons in the substantia nigra. Although the underlying mechanism by which dopaminergic neurons degenerate is not clear, oxidative stress has been implicated to play a role in the neuronal cell death associated with PD (20Adams Jr., J.D. Chang M.L. Klaidman L. Curr. Med. Chem. 2001; 8: 809-814Crossref PubMed Scopus (136) Google Scholar). In support of this, many studies have shown decreased antioxidant levels in PD patients as well as a protective effect of antioxidants in animal models of PD. For example, GSH and coenzyme Q10 have been reported to play an important role in protecting dopaminergic neurons not only in human PD patients, but also in animal PD models (21Nakamura K. Wang W. Kang U.J. J. Neurochem. 1997; 69: 1850-1858Crossref PubMed Scopus (78) Google Scholar, 22Beal M.F. Matthews R.T. Tieleman A. Shults C.W. Brain Res. 1998; 783: 109-114Crossref PubMed Scopus (228) Google Scholar, 23Shults C.W. Oakes D. Kieburtz K. Beal M.F. Haas R. Plumb S. Juncos J.L. Nutt J. Shoulson I. Carter J. Kompoliti K. Perlmutter J.S. Reich S. Stern M. Watts R.L. Kurlan R. Molho E. Harrison M. Lew M. the Parkinson Study GroupArch. Neurol. 2002; 59: 1541-1550Crossref PubMed Scopus (937) Google Scholar, 24Bharath S. Hsu M. Kaur D. Rajagopalan S. Andersen J.K. Biochem. 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Neuron. 2003; 37: 899-909Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Furthermore, superoxide dismutase and glutathione peroxidase have been reported to play a protective role in animal models of PD (28Przedborski S. Kostic V. Jackson-Lewis V. Naini A.B. Simonetti S. Fahn S. Carlson E. Epstein C.J. Cadet J.L. J. Neurosci. 1992; 12: 1658-1667Crossref PubMed Google Scholar, 29Zhang J. Graham D.G. Montine T.J. Ho Y.S. J. Neuropathol. Exp. Neurol. 2000; 59: 53-61Crossref PubMed Scopus (101) Google Scholar, 30Klivenyi P. Andreassen O.A. Ferrante R.J. Dedeoglu A. Mueller G. Lancelot E. Bogdanov M. Andersen J.K. Jiang D. Beal M.F. J. Neurosci. 2000; 20: 1-7Crossref PubMed Google Scholar). Interestingly, our recent oligonucleotide microarray study with mouse primary astrocytes showed that the expression of many of these protective antioxidant genes such as glutamate-cysteine ligase, superoxide dismutase, glutathione peroxidase, and ferritin is dependent on Nrf2. Together, these observations suggest that coordinate upregulation of these programmed cell life genes through an Nrf2-ARE pathway may protect neurons from reactive oxygen species-induced neuronal cell death. In this study, we raised and tested the hypothesis that decreased levels of detoxification enzymes and antioxidant proteins in Nrf2-/- neuronal cultures result in increased susceptibility to oxidative stress. To investigate this hypothesis, we used Nrf2-/- and Nrf2+/+ neuronal cultures treated with 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPP+) or rotenone. Both of these compounds inhibit mitochondrial respiration (complex I) and are widely used in animal and/or cell culture models of PD (31Offen D. Beart P.M. Cheung N.S. Pascoe C.J. Hochman A. Gorodin S. Melamed E. Bernard R. Bernard O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5789-5794Crossref PubMed Scopus (212) Google Scholar, 32Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2979) Google Scholar, 33Sherer T.B. Betarbet R. Stout A.K. Lund S. Baptista M. Panov A.V. Cookson M.R. Greenamyre J.T. J. Neurosci. 2002; 22: 7006-7015Crossref PubMed Google Scholar). Furthermore, oligonucleotide microarray analysis was used to identify genes associated with this Nrf2-dependent differential sensitivity to inhibition of mitochondrial respiration. Primary Neuronal Culture—Nrf2+/- mice were bred with Nrf2+/- mice, and primary neuronal cultures were prepared individually. Cerebral cortices from littermate embryos (gestation day 16) were removed, placed in ice-cold Hanks' balanced salt solution (3 ml/embryo; Invitrogen), centrifuged (300 × g, 2 min), and digested individually (0.5 mg/ml trypsin (Invitrogen) in Hanks' balanced salt solution, 37 °C, 10 min). Tissues were washed twice with Hanks' balanced salt solution and resuspended in minimal essential medium with Earle's salt (Invitrogen) containing heat-inactivated (55 °C, 30 min) fetal bovine serum (10%; Atlanta Biologicals, Inc.) and horse serum (10%; Atlanta Biologicals, Inc.). Cell suspensions were sieved through cell strainers (70 μm; Falcon) and plated at a density of 1 × 106 cells/ml. After 45 min of initial plating, the medium was changed to neurobasal medium ([Ca2+] = 1.8 mm) supplemented with B27 Minus AO (antioxidant) (Invitrogen). Cultures were maintained at 37 °C in a humidified tri-gas incubator (5% O2, 90% N2, and 5% CO2; Forma Scientific, Inc.). The Nrf2 genotype of each culture was determined by a PCR-based method as described previously (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Cultures were used for experiments between 2 and 7 days in vitro (DIV). Over 90% of the cells in the cultures (both Nrf2-/- and Nrf2+/+) were neurons as determined by immunostaining of the astrocyte-specific marker glial fibrillary acidic protein (GFAP; Dako Corp.) and the neuron-specific markers microtubule-associated protein-2 (MAP2; Chemicon International, Inc.) and βIII-tubulin (Promega) (data not shown). In addition, there was no difference in the ratio of cell type (neurons versus astrocytes) between Nrf2+/+ and Nrf2-/- cultures (data not shown). NQO1 Activity—NQO1 enzymatic activity was determined by a colorimetric method for whole cell extracts (34Prochaska H.J. Santamaria A.B. Anal. Biochem. 1988; 169: 328-336Crossref PubMed Scopus (460) Google Scholar) and by histochemistry for fixed cultures as described previously (35Johnson D.A. Andrews G.K. Xu W. Johnson J.A. J. Neurochem. 2002; 81: 1233-1241Crossref PubMed Scopus (142) Google Scholar). Cytotoxicity Assay—3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS; Promega) cytotoxicity assay and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL; Roche Applied Science) staining were performed according to the manufacturers' protocols. Immunohistochemistry—Cells on chamber slides were rinsed with phosphate-buffered saline; fixed in 4% paraformaldehyde for 20 min; and blocked with phosphate-buffered saline containing 1% goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 for 1 h. Cells were incubated with primary antibodies (rabbit anti-MAP2 (1:200), mouse anti-βIII-tubulin (1:200), rabbit anti-GFAP (1:500), and rabbit anticleaved caspase-3 (1:200) (Cell Signaling); rabbit anti-Nrf2 (1:2000) and rabbit anti-GST Mu1 (1:1000) (Biotrin); and rabbit anti-GCLC (1:1000)) overnight at 4 °C, followed by incubation for 1 h with Texas Red- or fluorescein-conjugated secondary antibodies (1:200; Vector Laboratories, Inc.). Hoechst 33258 was used for nuclear counterstaining. Fluorescence microscope pictures were taken under the same conditions. Western Blot Analysis and Total GSH Measurement—Western blot analysis for GST Mu1, GCLM, and GCLC were performed, and total GSH levels were measured as described previously (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Reverse Transcription (RT)-PCR—Total RNA was isolated by TRIzol reagent (Invitrogen), and cDNA was synthesized using a reverse transcription system (Promega) according to the manufacturers' protocols. Aliquots of cDNA were used for PCR amplification with Taq DNA polymerase (Promega). PCR primers specific to each gene are as follows: Nrf2 exons 1–3, 5′-AGTTCTCGCTGCTCGGACTA-3′ and 5′-AGGCATCTTGTTTGGGAATG-3′; Nrf2 exon 5, 5′-TCTCCTCGCTGGAAAAAGAA-3′ and 5′-AATGTGCTGGCTGTGCTTTA-3′; NQO1, 5′-CATTCTGAAAGGCTGGTTTGA-3′ and 5′-CTAGCTTTGATCTGGTTGTCAG-3′; GST Alpha4, 5′-GCCAAGTACCCTTGGTTGAA-3′ and 5′-CAATCCTGACCACCTCAACA-3′; GST Mu1, 5′-CTCCCGACTTTGACAGAAGC-3′ and 5′-CAGGAAGTCCCTCAGGTTTG-3′; GST Mu3, 5′-AATCTTGGCCTGGATTTTCC-3′ and 5′-GAGGAAGCGGCTACTCTTCA-3′; GCLM, 5′-ACCTGGCCTCCTGCTGTGTG-3′ and 5′-GGTCGGTGAGCTGTGGGTGT-3′; GCLC, 5′-ACAAGCACCCCCGCTTCGGT-3′ and 5′-CTCCAGGCCTCTCTCCTCCC-3′; visinin-like 1, 5′-AAGTCATGGAGGACCTGGTG-3′ and 5′-CAGGCCATCCTCATTCATTT-3′; calbindin-28K, 5′-TGTGGCACATTCTTTTCTGC-3′ and 5′-TGGCTACCTTCCCTTACCAA-3′; synaptotagmin, 5′-GACAAAAGTCCACCGGAAAA-3′ and 5′-CTCGAACGGAACTTCAAAGC-3′; hippocalcin, 5′-AGGAAGGGGCTAAGCAAGAC-3′ and 5′-CGTGAGTGCACGAGAGAGAG-3′; and β-actin, 5′-AGAGCATAGCCCTCGTAGAT-3′ and 5′-CCCAGAGCAAGAGAGGTATC-3′. Oligonucleotide Microarray Analysis—Total RNA was obtained from Nrf2-/- and Nrf2+/+ primary neuronal cultures. Biotinylated cRNA was prepared, and fragmented cRNA was hybridized to MG U74Av2 arrays (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 19Li J. Lee J.-M. Johnson J.A. J. Biol. Chem. 2002; 277: 388-394Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Affymetrix Microarray Suite Version 5.0 was used to scan and analyze the relative abundance of each gene (analysis parameters: scaling target signal, 2500; Alpha1, 0.04; Alpha2, 0.06; Tau, 0.015; Gamma1L and Gamma1H, 0.025; Gamma2L and Gamma2H, 0.05; and pertubation, 1.1). Data were analyzed by rank analysis as previously described (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 19Li J. Lee J.-M. Johnson J.A. J. Biol. Chem. 2002; 277: 388-394Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The reproducibility of comparisons was based on the coefficient of variation (cv; S.D./mean) for the -fold change of ranked genes. A distribution curve of the cv was used to determine a cv cutoff value. The cutoff values were cv < 1.0- and ≥1.3-fold for increased genes and cv > -1.0- and ≤ -1.3-fold for decreased genes. Gene categorization was based on the NetAffex Database. 2Available at www.affymetrix.com. Adenoviral Infection—Recombinant adenoviral vectors (Ad-GFP, expressing GFP alone; and Ad-Nrf2, expressing GFP and Nrf2) were constructed using the Cre-lox system (Canadian Stroke Network Core Facility, University of Ottawa) (36Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). The Nrf2 cDNA was excised from pEF/Nrf2 (4Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar) by NotI and HindIII restriction. Titers of all viruses were determined on HEK293 cells. Nrf2-/- primary neuronal cultures were infected with Ad-GFP or Ad-Nrf2 at a multiplicity of infection (m.o.i.) of 50 at 2 DIV for 48 h. Typically, 10–20% of the neurons and virtually 100% of the astrocytes were infected with viral vectors as determined by GFP fluorescence and MAP2 and GFAP staining. Nrf2-dependent NQO1 Gene Expression in Primary Neuronal Cultures—Initially, we compared the level of expression and enzymatic activity of a known Nrf2-dependent ARE-driven gene, NQO1, in Nrf2+/+ and Nrf2-/- primary neuronal cultures. The expression level of NQO1 was dramatically greater in Nrf2+/+ neuronal cultures (Fig. 1A). Similarly, the NQO1 activity of Nrf2+/+ neuronal cultures was significantly higher than that of Nrf2-/- neuronal cultures (Fig. 1B). This correlated with a complete lack of NQO1 histochemical staining in the Nrf2-/- neuronal cultures (Fig. 1C) and implies that the lack of Nrf2 significantly reduced the Nrf2-ARE signaling pathway in primary neuronal cultures. It was also noted, as published previously by our laboratory (35Johnson D.A. Andrews G.K. Xu W. Johnson J.A. J. Neurochem. 2002; 81: 1233-1241Crossref PubMed Scopus (142) Google Scholar), that NQO1 staining was isolated to the astrocytes in this mixed culture system. Differential Sensitivity to Mitochondrial Toxins—To investigate whether this loss of Nrf2-dependent gene expression confers increased sensitivity of neurons to oxidative stress, we treated primary neuronal cultures with the well known mitochondrial toxins MPP+ (31Offen D. Beart P.M. Cheung N.S. Pascoe C.J. Hochman A. Gorodin S. Melamed E. Bernard R. Bernard O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5789-5794Crossref PubMed Scopus (212) Google Scholar) and rotenone (32Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2979) Google Scholar, 33Sherer T.B. Betarbet R. Stout A.K. Lund S. Baptista M. Panov A.V. Cookson M.R. Greenamyre J.T. J. Neurosci. 2002; 22: 7006-7015Crossref PubMed Google Scholar). These compounds induce neuronal cell death by inhibiting mitochondrial complex I, resulting in decreased ATP levels, loss of membrane potential, increased reactive oxygen species generation, and increased [Ca2+]i. As shown in Fig. 2A, Nrf2-/- neuronal cultures were much more sensitive to MPP+- or rotenone-induced cytotoxicity. Rotenone (0.1 μm) and MPP+ (50 μm) induced 85% cell death (equivalent to 100% neuronal cell death) in Nrf2-/- neuronal cultures (Fig. 2A). The remaining MTS activity is presumably due to the surviving astrocytes. Strikingly, the same concentrations of MPP+ and rotenone induced little or no cell death in Nrf2+/+ neuronal cultures, respectively (Fig. 2A). This is reflected in the numbers of TUNEL-positive cells in MPP+- or rotenone-treated Nrf2-/- neuronal cultures (Fig. 2B). There were far greater numbers of TUNEL-positive cells in the Nrf2-/- neuronal cultures than in the Nrf2+/+ neuronal cultures (Fig. 2B). Double labeling with selective astrocyte or neuronal markers indicated that these apoptotic cells were neurons, not astrocytes (data not shown). MPP+ and rotenone also preferentially activated the caspase-3 pathway in Nrf2-/- cultured neurons as shown by immunostaining for the cleaved caspase-3 (Fig. 3).Fig. 3Preferential activation of caspase-3 in Nrf2-/- neuronal cultures by mitochondrial toxins. Primary neuronal cultures were treated with vehicle (0.01% Me2SO), MPP+ (50 μm), or rotenone (0.1 μm). After 24 h, cells were stained for cleaved caspase-3 as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of Nrf2 Target Genes—To identify the Nrf2 target genes conferring this observed neuroprotection, we performed oligonucleotide microarray analysis on Nrf2+/+ and Nrf2-/- primary neuronal cultures. A total of 142 increased and 175 decreased genes were identified using rank analysis (R ≥ 4, R ≤ -4), followed by cutoff values for cv (cv < 1.0, cv > -1.0) and -fold change (-fold ≥ 1.3, -fold ≤ -1.3). The major functional categories of Nrf2 target genes in primary neuronal cultures are 1) detoxification/antioxidant/reducing potential, 2) calcium homeostasis, 3) growth factors, 4) signaling, 5) receptor/channel/carrier protein, 6) neuron-specific proteins, and 7) defense/immune/inflammation (Table I). 3A complete list of all increased and decreased genes, including genes of unknown function and expressed sequence tags, is available upon request. Although many of the classical Nrf2-dependent ARE-driven genes such as NQO1, GSTs, and GCLC were increased by Nrf2 in primary neuronal cultures, the final list of Nrf2 target genes in these neuronal cultures was quite distinct from those of other cell types (11Lee J.-M. Calkins M.J. Chan K. Kan Y.W. Johnson J.A. J. Biol. Chem. 2003; 278: 12029-12038Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 13Thimmulappa R.K. Mai K.H. Srisuma S. Kensler T.W. Yamamoto M. Biswal S. Cancer Res. 2002; 62: 5196-5203PubMed Google Scholar). The gene classifications of calcium homeostasis, receptor/channel/carrier protein, and neuron-specific proteins, for instance, were unique to the primary neuronal cultures.Table IIdentification of Nrf2 target genes in primary neuronal culturesGeneAccession no.R.Signal of Nrf2-/--Fold ± S.E.CVDetoxification/antioxidant/reducing potentialNQO1U129614120.84.9 ± 2.240.91GST Alpha4L060478440.04.7 ± 2.060.88GST Mu1J03952822,336.82.2 ± 0.110.10GST Mu3J0395385050.81.7 ± 0.030.03Malic enzyme, supernatantJ0265264830.81.6 ± 0.190.25GCLCU8541441256.11.3 ± 0.130.19Catalase-1M2939471486.01.3 ± 0.040.06Calcium HomeostasisVisinin-like 1D21165610,455.31.8 ± 0.440.48Calbindin-28KD2635251265.11.8 ± 0.380.42Synaptotagmin-1D3779285245.51.6 ± 0.160.20HippocalcinAB01520062892.91.5 ± 0.240.32Synaptotagmin-5AB02680648830.21.4 ± 0.190.27Nucleobindin-2AJ2225864453.01.4 ± 0.090.13Calmodulin IIIAI842328627,383.91.4 ± 0.160.24Ryanodine receptor-3X8393441485.91.3 ± 0.060.09Growth factorsNerve growth factor-γAV043739438.12.7 ± 0.460.34Fibroblast growth factor-13AF02073741481.61.3 ± 0.160.24Brain-derived neurotrophic factorX5557341610.61.3 ± 0.150.23SignalingRegulator of G-protein signaling 11AF06193441677.32.4 ± 0.750.63Rho GEF-3A18537064453.81.6 ± 0.270.34Protein-tyrosine phosphatase receptor type DD1390342446.41.4 ± 0.200.29Neuronal GEFAW05034664796.31.4 ± 0.140.19Protein kinase inhibitor-αAW125442421,774.31.4 ± 0.170.25GABA-A receptor-α2M6237483896.41.4 ± 0.110.16Megakaryocyte-associated tyrosine kinaseD4524362644.91.4 ± 0.090.13Phosphatidylinositol-4-phosphate 5-kinase type 1-γAB0069164543.11.3 ± 0.150.22Mitogen-activated protein kinase-10L3523647146.51.3 ± 0.130.20Protein-tyrosine phosphatase receptor type OU3746556840.51.3 ± 0.130.20Protein kinase C-βX5353241782.61.3 ± 0.040.07G-protein-γ3AF069953435,274.81.3 ± 0.130.20GABA-A receptor-1U1441846611.11.3 ± 0.160.23GABA-B receptor-1AL078630613,361.31.3 ± 0.130.21Adenylate cyclase-activating polypeptide-1AB0101494955.81.3 ± 0.160.26Inosine 5′-phosphate dehydrogenase-1U0097852082.81.3 ± 0.110.17Receptor/channel/carrier proteinPotassium voltage-gated channel shaker-related β1AF0330034444.92.5 ± 0.750.60Potassium large conductance Ca2+-activated channel Mα1U0938363434.31.4 ± 0.140.21Solute carrier family 7 (y+ system), member 8AW1227066563.71.4 ± 0.100.15Solute carrier family 3, member 1AW12115469570.41.3 ± 0.080.11Chloride channel 4-2Z4991644720.21.3 ± 0.160.24Potassium voltage-gated channel shaker-related β2U6559241033.01.3 ± 0.110.18Solute carrier family 1, member 1U7352141276.61.3 ± 0.170.26Solute carrier family 30 (zinc transporter), member 4AF00410062080.81.3 ± 0.020.04Neuron-specific proteinsKinesin family member C2U92949478.04.1 ± 1.240.60TubbyU546434184.13.1 ± 0.560.37Nasal embryonic LH-RH factorAI84956541258.82.1 ± 0.290.28Myocyte enhancer factor-2CL1317184204.22.0 ± 0.310.31Neurogenic differentiation-1U280684543.71.6 ± 0.290.35L1 cell adhesion moleculeX1287562279.11.6 ± 0.280.34ReelinU2470388198.41.6 ± 0.260.31Secretogranin IIX6883762516.51.5 ± 0.200.27Chromogranin BX5142967396.81.4 ± 0.160.22Synaptosome-associated protein, 25 kDaM22012611,907.01.4 ± 0.180.26Vesicular membrane protein p24D8320661555.51.4 ± 0.080.12SynaptophysinX9581842938.41.4 ± 0.110.17Synapsin IIAF09686744310.71.4 ± 0.110.17Prolyl endopeptidaseAB0220534799.21.4 ± 0.110.16Amyloid-β (A4) precursor protein binding A2L3467642672.21.3 ± 0.140.20Dynactin-3AF098508612,963.51.3 ± 0.070.10Defense/immune/inflammationSmall inducible cytokine B subfamily, member 5U272676350.72.3 ± 0.630.56Platelet-activating factor acerylhydrolaseU34
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