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• W1987261678 abstract "Nerve growth factor (NGF) can induce apoptosis in neural cells via activation of the low affinity neurotrophin receptor p75NTR. NADE (p75NTR-associated celldeath executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the death domain of p75NTR in 293T, PC12, and nnr5 cells (Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M. T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L. A., and Sato, T. A. (2000) J. Biol. Chem. 275, 17566–17570). We performed extensive mutational analysis on NADE, to better characterize its structural and functional features. Truncation of a minimal region, including amino acid residues 41–71 of NADE, was found to be sufficient to induce apoptosis. The designated regulatory region includes the C-terminal amino acid residues (72–112) and is essential for NGF-dependent regulation of NADE-induced apoptosis. Furthermore, the mutants with amino acid substitutions in the leucine-rich nuclear export signal (NES) sequence (residues 90–100) abolished the export of NADE from the nucleus to the cytoplasm. Mutation of the NES also abolished self-association of NADE, its interaction with p75NTR, and NGF-dependent apoptosis. Expression of a fragment of NADE (amino acid residues 81–124) blocked NGF-induced apoptosis in oligodendrocytes, suggesting that this region has a dominant negative effect on NGF/p75NTR-induced apoptosis. These studies identify distinct regions of NADE that are involved in regulating specific functions involved in p75NTR signal transduction. Nerve growth factor (NGF) can induce apoptosis in neural cells via activation of the low affinity neurotrophin receptor p75NTR. NADE (p75NTR-associated celldeath executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the death domain of p75NTR in 293T, PC12, and nnr5 cells (Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M. T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L. A., and Sato, T. A. (2000) J. Biol. Chem. 275, 17566–17570). We performed extensive mutational analysis on NADE, to better characterize its structural and functional features. Truncation of a minimal region, including amino acid residues 41–71 of NADE, was found to be sufficient to induce apoptosis. The designated regulatory region includes the C-terminal amino acid residues (72–112) and is essential for NGF-dependent regulation of NADE-induced apoptosis. Furthermore, the mutants with amino acid substitutions in the leucine-rich nuclear export signal (NES) sequence (residues 90–100) abolished the export of NADE from the nucleus to the cytoplasm. Mutation of the NES also abolished self-association of NADE, its interaction with p75NTR, and NGF-dependent apoptosis. Expression of a fragment of NADE (amino acid residues 81–124) blocked NGF-induced apoptosis in oligodendrocytes, suggesting that this region has a dominant negative effect on NGF/p75NTR-induced apoptosis. These studies identify distinct regions of NADE that are involved in regulating specific functions involved in p75NTR signal transduction. Many mammalian cells undergo apoptosis in response to various stimuli, including DNA damage, growth factor deprivation, and abnormal expression of oncogenes or tumor suppressor genes (1.Raff M.C. Barres B.A. Burne J.F. Coles H.S. Ishizuka Y. Jacobson M.D. Science. 1993; 262: 695-700Crossref PubMed Scopus (1344) Google Scholar, 2.Thompson C.B. Science. 1995; 262: 1456-1462Crossref Scopus (6162) Google Scholar, 3.Vaux D.L. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2239-2244Crossref PubMed Scopus (907) Google Scholar). Apoptosis induced by these various signals appears to be mediated by a common set of downstream elements that act as regulators and effectors of programmed cell death. Apoptosis also occurs during the course of normal development. In neural cells, neurotrophins have been shown to promote apoptosis during development (4.Bibel M. Barde Y.A. Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (863) Google Scholar). Nerve growth factor (NGF) 1The abbreviations used are: NGFnerve growth factorICDintracellular domainp75NTRp75 neurotrophin receptorGFPgreen fluorescence proteinPBSphosphate-buffered salineGSTglutathione S-transferaseFITCfluorescein isothiocyanateDMEMDulbecco's modified Eagle's mediumDAPI4,6-diamidino-2-phenylindoleTrkAtyrosine kinase receptorMAPmitogen-activated proteinMAPKKMAP kinase kinaseTNFtumor necrosis factorTRAFTNF receptor-associated factorICDintracellular domainNADEp75NTR-associated cell death executorNESnuclear export signalWTwild-typeHEKhuman embryonic kidneyBMEBasal Medium EagleALLNN-acetyl-Leu-Leu-norleucinalLMBleptomycin BPMSFphenylmethylsulfonyl fluorideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingMORT1mediator of receptor-induced toxicityFADDFas-associating death domain protein1The abbreviations used are: NGFnerve growth factorICDintracellular domainp75NTRp75 neurotrophin receptorGFPgreen fluorescence proteinPBSphosphate-buffered salineGSTglutathione S-transferaseFITCfluorescein isothiocyanateDMEMDulbecco's modified Eagle's mediumDAPI4,6-diamidino-2-phenylindoleTrkAtyrosine kinase receptorMAPmitogen-activated proteinMAPKKMAP kinase kinaseTNFtumor necrosis factorTRAFTNF receptor-associated factorICDintracellular domainNADEp75NTR-associated cell death executorNESnuclear export signalWTwild-typeHEKhuman embryonic kidneyBMEBasal Medium EagleALLNN-acetyl-Leu-Leu-norleucinalLMBleptomycin BPMSFphenylmethylsulfonyl fluorideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingMORT1mediator of receptor-induced toxicityFADDFas-associating death domain proteinwas first identified as a growth factor that is required for the survival of specific neuronal cells during normal development (5.Levi-Montalcini R. EMBO J. 1987; 6: 1145-1154Crossref PubMed Scopus (476) Google Scholar). However, more recently, it has been shown that NGF has other diverse effects on the nervous system, including differentiation and apoptosis (4.Bibel M. Barde Y.A. Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (863) Google Scholar, 5.Levi-Montalcini R. EMBO J. 1987; 6: 1145-1154Crossref PubMed Scopus (476) Google Scholar, 6.Rabizadeh S. Bredesen D.E. Dev. Neurosci. 1994; 16: 207-211Crossref PubMed Scopus (64) Google Scholar). nerve growth factor intracellular domain p75 neurotrophin receptor green fluorescence protein phosphate-buffered saline glutathione S-transferase fluorescein isothiocyanate Dulbecco's modified Eagle's medium 4,6-diamidino-2-phenylindole tyrosine kinase receptor mitogen-activated protein MAP kinase kinase tumor necrosis factor TNF receptor-associated factor intracellular domain p75NTR-associated cell death executor nuclear export signal wild-type human embryonic kidney Basal Medium Eagle N-acetyl-Leu-Leu-norleucinal leptomycin B phenylmethylsulfonyl fluoride terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling mediator of receptor-induced toxicity Fas-associating death domain protein nerve growth factor intracellular domain p75 neurotrophin receptor green fluorescence protein phosphate-buffered saline glutathione S-transferase fluorescein isothiocyanate Dulbecco's modified Eagle's medium 4,6-diamidino-2-phenylindole tyrosine kinase receptor mitogen-activated protein MAP kinase kinase tumor necrosis factor TNF receptor-associated factor intracellular domain p75NTR-associated cell death executor nuclear export signal wild-type human embryonic kidney Basal Medium Eagle N-acetyl-Leu-Leu-norleucinal leptomycin B phenylmethylsulfonyl fluoride terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling mediator of receptor-induced toxicity Fas-associating death domain protein NGF recognizes at least two cell surface receptors, the high affinity tyrosine kinase receptor (TrkA) and the low affinity non-tyrosine kinase type receptor p75 neurotrophin receptor (p75NTR) (7.Klein R. Jing S.Q. Nanduri V. O'Rourke E. Barbacid M. Cell. 1991; 65: 189-197Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 8.Chao M.V. Bothwell M.A. Ross A.H. Loprpwski H. Lanahanm A.A. Buck C.R. Sehgal A. Science. 1986; 232: 518-521Crossref PubMed Scopus (350) Google Scholar, 9.Johnson D. Lanahan A. Buck C.R. Sehgal A. Morgan C. Mercer E. Bothwell M. Chao M.V. Cell. 1986; 47: 545-554Abstract Full Text PDF PubMed Scopus (736) Google Scholar). TrkA contains a tyrosine kinase motif within its intracellular region. Binding of NGF to TrkA activates the kinase and subsequently induces phosphorylation of multiple substrates that lead to the activation of mitogen-activated protein (MAP) kinase, phosphatidylinositol 3-kinase, and other intracellular signaling cascades (10.Peng X. Greene L.A. Kaplan D.R. Stephens R.M. Neuron. 1995; 15: 395-406Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 11.Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1284) Google Scholar). The TrkA receptor promotes cell survival and initiates differentiation signals in neuronal cells (12.Kaplan D.R. Stephens R.M. J. Neurobiol. 1994; 25: 1404-1417Crossref PubMed Scopus (471) Google Scholar, 13.Lachyankar M.B. Condon P.J. Quesenberry P.J. Litofsky N.S. Recht L.D. Ross A.H. Exp. Neurol. 1997; 144: 350-360Crossref PubMed Scopus (119) Google Scholar). In contrast, p75NTR, which is a member of the tumor necrosis factor (TNF) receptor superfamily, mediates neurotrophin-induced apoptosis (14.Dechant G. Barde Y.A. Curr. Opin. Neurobiol. 1997; 7: 413-418Crossref PubMed Scopus (203) Google Scholar). Several lines of evidence from various systems, including cultured cells as well as knockout and transgenic mice, suggest that p75NTR has a pro-apoptotic role (15.Bredesen D.E. Rabizadeh S. Trends Neurosci. 1997; 18: 321-326Google Scholar, 16.Maidan M. Lachance C. J. Neurosci. 1997; 17: 6988-6998Crossref PubMed Google Scholar, 17.Miller F.D. Kaplan D.R. Cell Death Differ. 1998; 5: 343-345Crossref PubMed Scopus (24) Google Scholar, 18.Frade J.M. Rodriguez-Tebar A. Barde Y.A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (662) Google Scholar, 19.von Schack D. Casademunt E. Schweigreiter R. Meyer M. Bibel M. Dechant G. Nature Neurosci. 2001; 4: 977-978Crossref PubMed Scopus (214) Google Scholar). Furthermore, NGF induces apoptosis in terminally differentiated primary oligodendrocytes expressing p75NTR (20.Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (712) Google Scholar). These studies suggested that p75NTR is involved in NGF-induced apoptosis. The only known consensus motif within the intracellular domain of p75NTR is a death domain, similar to that found in the p55 tumor necrosis factor receptor (p55TNFR) and in Fas. However, the molecular mechanisms by which p75NTR mediates pro-apoptotic signaling have not been well characterized. Recently, members of the TNF receptor-associated factor (TRAF) family, FAP-1, SC-1, NRIF, NRAGE, and RhoA, have been reported to interact with the intracellular domain (ICD) of p75NTR (21.Khursigera G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 22.Ye X. Mehlen P. Rabizadeh S. VanArsdale T. Zhang H. Shin H. Wang J.J.L. Leo E. Zapata J. Hauser C.A. Reed J.C. Bredesen D.E. J. Biol. Chem. 1999; 274: 30202-30208Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 23.Irie S. Hachiya T. Rabizadeh S. Maruyama W. Mukai J. Li Y. Reed J.C. Bredesen D.E. Sato T.A. FEBS Lett. 1999; 460: 191-198Crossref PubMed Scopus (74) Google Scholar, 24.Chittka A. Chao M.V. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10705-10710Crossref PubMed Scopus (111) Google Scholar, 25.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (153) Google Scholar, 26.Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 27.Yamashita T. Tucker K.L. Barde Y.A. Neuron. 1999; 24: 585-593Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Of these, TRAF2, NRIF, and NRAGE have been reported to function in the p75NTR-mediated apoptotic pathway. Co-expression of TRAF2 with p75NTR enhanced cell death (22.Ye X. Mehlen P. Rabizadeh S. VanArsdale T. Zhang H. Shin H. Wang J.J.L. Leo E. Zapata J. Hauser C.A. Reed J.C. Bredesen D.E. J. Biol. Chem. 1999; 274: 30202-30208Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), whereas co-expression of TRAF6 was cytoprotective (21.Khursigera G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). NRIF is known to block cell division. The retinae ofnrif −/− mice show reduced cell death, and this reduction is quantitatively similar to that seen inp75−/− and ngf−/−mice (25.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (153) Google Scholar). NRAGE binds with p75NTR both in vitro andin vivo and blocks the physical association of p75NTR with TrkA. NRAGE overexpression facilitates cell cycle arrest and permits NGF-dependent apoptosis within sympathetic neuron precursor cells (26.Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). However, the mechanisms of p75NTR-mediated signal transduction are still not fully understood. Recently, we identified a novel protein that we termed p75NTR-associated cell death executor (NADE), which associated with the p75NTR(ICD) in a yeast two-hybrid screen (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). A data base search using the putative NADE amino acid sequence identified a homologous, partially characterized human cDNA clone termed HGR74/Bex3 (29.Brown A.L. Kay G.F. Hum. Mol. Genet. 1999; 8: 611-619Crossref PubMed Scopus (54) Google Scholar). Co-expression of NADE and p75NTR induced cell death in 293T cells. NGF induced a dose-dependent association of NADE with the death domain of p75NTR, and p75NTR·NADE-induced cell death required NGF but not BDNF, NT-3, or NT-4/5. Similar results were also obtained in PC12 and PC12 nnr5 cells (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). After forebrain ischemia, p75NTR and NADE gene expression were induced in degenerating rat hippocampal CA1 neurons. NADE contributes to p75NTR-induced cortical neuronal death (30.Park J.A. Lee J.-Y. Sato T.A. Koh J.-Y. J. Neurosci. 2000; 20: 9096-9103Crossref PubMed Google Scholar). Furthermore, 14-3-3 proteins associate with NADE and play a role in p75NTR·NADE-mediated apoptosis in HEK293 cells, PC12 nnr5 cells, and oligodendrocytes (31.Kimura M.T. Irie S. Shoji-Hoshino S. Mukai J. Nadano D. Oshimura M. Sato T.A. J. Biol. Chem. 2001; 276: 17291-17300Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). NADE has a nuclear export signal (NES) consensus motif, which is both necessary and sufficient to mediate nuclear export of large carrier proteins (32.Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1061) Google Scholar). Recent reports have demonstrated that the cellular localization of many proteins is tightly regulated by this motif, including that of HIV-Rev (33.Fischer U. Huber J. Boelens W.C. Mattaj I.W. Lhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar), PKI (34.Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (991) Google Scholar), and MAPKK (35.Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Thus, the NES-mediated intracellular transport system is a widely conserved mechanism that controls the subcellular localization of proteins in cells. In this study, we have analyzed three issues relating to the functional and structural properties of mouse NADE. First, by mutational analysis, we have defined the subregions of NADE that are required for NGF-induced cell death. Second, we have shown that NADE contains a functional NES domain and that this sequence is responsible for self-association, interaction with p75NTR and induction of cell death. Finally, we have identified a dominant negative form of NADE, comprised of amino acids 81–124, which inhibited NGF-induced apoptosis in oligodendrocytes. NADE (WT) (pcDNA3.1/Myc-His(−)A/mNADE WT) was constructed as described previously (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Expression vectors for mouse NADE deletion mutants were constructed by PCR amplification of NADE coding sequences using oligonucleotide pairs as shown, digesting the resulting fragments with XhoI/BamHI, and ligating the resulting fragments into XhoI/BamHI-digested pcDNA3.1/Myc-His(−) A: for N-(1–120), FX29 (5′-ATCCTCGAGCGATCATGGCCAATGTCCAC-3′) and RB360 (5′-ATCGGATCCGAATTCATCATGGTGATC-3′); for N-(1–112), FX29 and RB336 (5′-ATCGGATCCGTTAGACAGCTCCCCCAT-3′); for N-(1–100), FX29 and RB300 (5′-ATCGGATCCTCTCAGCTGTAGCTCCCT-3′); for N-(1–71), FX29 and RB213 (5′-ATCGGATCCGTCATTCATCTGCCTGTT-3′); for N-(1–20), FX29 and RB60 (5′-ATCGGATCCTTCCTGTCCATTCTGCAG-3′); for N-(41–124), FX121 (5′-ATCCTCGAGACCATGCACAACCATAACCACAAC-3′) and RB27 (5′-ATCGGATCCAGGCATAAGGCAGAATTC-3′); for N-(81–124), FX241 (5′-ATCCTCGAGACCATGGAAATGTTCATGGAGGAG-3′) and RB27; for N-(101–124), FX301 (5′-ATCCTCGAGACCATGAATTGTCTACGCATCCTT-3′) and RB27; for N-(41–71), FX121 and RB213; for FLAG-tagged N(WT), FX29, and RBFLAG (5′-CGCGGATCCTCACTTATCGTCGTCATCCTTGTAATCAGGCATAAGGCAGAATTC-3′). Point mutants for NADE were constructed by PCR amplification ofNADE coding sequences using oligonucleotide pairs as described below, followed by digesting the resulting PCR product withDpnI: for N(L99A) and GFP-N(L99A), in which Leu-99 is replaced with Ala, F-L99A (5′-AGGGAGCTACAGGCGAGAAATTGTCTA-3′), and R-L99A (5′-TAGACAATTTCTCGCCTGTAGCTCCCT-3′); for N(L94A/L97A/L99A) and GFP-N(L94A/L97A/L99A), in which Leu-94, Leu-97, and Leu-99 are replaced with Ala, F-L97A (5′-AAAGCTTAGGGAGGCACAGCTGAGAAA-3′), R-L97A (5′-TTTCTCAGCTGTGCCTCCCTAAGCTTT-3′) and F-L97A/L99A (5′-AGGGAGGCACAGGCGAGAAATTGTCTA-3′), R-L97A/L99A (5′-TAGACAATTTCTCGCCTGTGCCTCCCT-3′) and F-L94A/L97A (5′-ATCCGGAGAAAGGCTAGGGAGGCACA-3′), R-L94A/L97A (5′-TGTGCCTCCCTAGCCTTTCTCCGGAT-3′). Expression plasmids encoding fusion proteins of green fluorescence protein (GFP) and NADE proteins were constructed as follows: GFP cDNA was PCR-amplified from pEGFP-N2 (CLONTECH, Palo Alto, CA) by using the primer pair 5′-CTAGCTAGCATCATGGTGAGCAAGGGCGAG-3′ and 5′-CCGCTCGAGTCTTGTACAGCTCGTCCAT-3′. The product was cloned into theNheI and XhoI sites of pcDNA3.1/Myc-His(−)A/mNADE. Expression plasmids for glutathioneS-transferase (GST)-fused p75NTR proteins have been described previously (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). pVP16/N(WT) and pVP16/N(L94A/L97A/L99A) were constructed by PCR amplification of N(WT) and N(L94A/L97A/L99A), respectively, using oligonucleotide pairs as shown, digesting the resulting fragments withBamHI/NotI, and ligating the fragments intoBamHI/NotI-digested pVP16: forward primer (5′-GGGATCCTAATGGCCAATGTCCACCAGGAA-3′) and reverse primer (5′-ATAGTTTAGCGGCCGCAATCAAGGCATAAGGCA-3′). pBTM116/N(WT) was constructed by PCR amplification of N(WT) using oligonucleotide pairs as shown, digesting the resulting fragments withBamHI/SalI, and ligating the fragments intoBamHI/SalI-digested BTM116: forward primer (5′-GGGATCCTAATGGCCAATGTCCACCAGGAA-3′) and reverse primer (5′-TGCGGTCGACGCTAAGGCATAAGGCAGAA-3′). pBTM116/p75DD was constructed by PCR amplification of pcDNA3/p75NTR using oligonucleotide pairs as shown, digesting the resulting fragments withEcoRI/SalI and ligating the fragments intoEcoRI/SalI-digested BTM116: forward primer (5′-CGGAATTCAAGGGTGATGGCAACCTC-3′) and reverse primer (5′-GCGTCGACTCAGTAACCCAGCTCGCCTGC-3′). Adenoviral vectors were constructed using the Adeno-X Expression system (CLONTECH) according to the protocol recommended by the manufacturer. pShuttle/Myc-GFP, pShuttle/Myc-N(WT), and pShuttle/Myc-N-(81–124) were constructed by digesting the pcDNA3.1/Myc-His(−)A/GFP, pcDNA3.1/Myc-His(−)A/N(WT), and pcDNA3.1/Myc-His(−)A/N-(81–124) withNheI/AflII, and ligating the resulting fragments into the NheI/AflII sites of the pShuttle vector. pAdeno-X/Myc-GFP, pAdeno-X/Myc-N(WT), and pAdeno-X/Myc-N-(81–124) were constructed by digesting pShuttle constructs with PI-SceI/I-CeuI and ligating the resulting fragments into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK) 293 cells. The adenoviral particles were propagated in HEK293 cells and purified on cesium chloride gradients. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy and/or Western blot analysis. Adenoviral infection was performed on oligodendrocytes that were cultured for 7 days in Basal Medium Eagle (BME), which is described under “Cell Culture and Transfection.” Cells were infected with 4–10 plaque-forming units/cell of adenovirus in BME. After incubation for 6 h at 37 °C, cultures were replaced with fresh BME for 18 h. For the apoptosis assay, infected oligodendrocytes were treated with 100 ng/ml NGF. N-Acetyl-Leu-Leu-norleucinal (ALLN), NGF, leptomycin B (LMB), and the anti-FLAG monoclonal antibody were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-rabbit IgG-Alexa Fluor 568, and anti-mouse IgG-Alexa Fluor 350 were obtained from Molecular Probes (Eugene, OR). The anti-α-NADE polyclonal antibody was prepared as described previously (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The anti-O1 mouse monoclonal antibody was a kind gift from Dr. S. Pfeiffer. The anti-myc polyclonal antibody was obtained from MBL International Corp. (Watertown, MA). The anti-myc monoclonal antibody was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). 293T cells were obtained from the American Type Culture Collection; 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum. For transfection, 293T cells (1.5 × 106 per 100-mm dish) were transiently transfected with 25 μg of DNA using the calcium phosphate method in DMEM supplemented with 10% fetal bovine serum and cultured for 10 h. After withdrawing the serum, the cells were treated with 100 ng/ml NGF for 36 h. Oligodendrocytes were prepared as described previously (36.Gu C. Casaccia-Bonnefil P. Srinivasan A. Chao M.V. J. Neurosci. 1999; 19: 3043-3049Crossref PubMed Google Scholar). Mixed glial cultures were prepared from P2-rat cortex. After plating on poly-d-lysine-coated 75-cm2flasks, cultures were grown at 37 °C in a humidified incubator with 5% CO2 for 7 days. Oligodendrocytic cultures were typically preplated and grown for 24 h in NM15 media (minimal essential medium supplemented with 15% FCS, 6 mg/ml glucose, 10 units/ml penicillin, and 10 mg/ml streptomycin). Cultures were then switched to oligodendrocyte differentiation media, containing BME:Ham's F-12 (1:1 v/v) supplemented with 6 mg/mld-glucose, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml transferrin, 25 μg/ml insulin, 20 nm progesterone, 60 μm putrescine, 30 nm selenium, 6.6 mm glutamine, and 0.5 μm thyroxine. 293T cells were plated onto glass coverslips and transfected with GFP-containing constructs. At 24 h after transfection, cells were fixed with 3.7% paraformaldehyde, washed with phosphate-buffered saline (PBS), and stained with Hoechst 33342 to visualize the nucleus. The images of representative fields were captured on a fluorescence microscopy EFD-3 (Nikon) and recorded with a SPOT-RT digital charge-coupled device camera (Diagnostic Instruments). Subcellular fractionation was performed as described previously (37.Stegh A.H. Schickling O. Ehret A. Scaffidi C. Petehansel C. Hofmann T.G. Grummt I. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 5974-5986Crossref PubMed Scopus (114) Google Scholar). Transfected 293T cells were washed in PBS. Half of the cells were used for preparation of cytoplasmic and nuclear extracts, respectively. For preparation of cytoplasmic extracts, the cells were resuspended in 300 μl of buffer A (50 mm HEPES, pH 7.4, 50 mm KCl, 5 mm EDTA, 2 mm MgCl2, 0.1 mm dithiothreitol, 20 μm leupeptin, 10 μg/ml pepstatin A, 1 mm PMSF, and 1 mmbenzamidine), and allowed to swell by incubation for 10 min on ice. The cells were gently homogenized with a Dounce homogenizer. Small aliquots of the lysate were taken and stained with trypan blue to determine the progression of cell lysis. Homogenization was continued until more than 95% of the cells were broken. After centrifugation at 1500 ×g, the resultant supernatant was carefully removed and supplemented with 60 μl of 6-fold concentrated standard reducing sample buffer. For preparation of nuclei, cells were incubated on ice for 10 min in buffer B (10 mm HEPES, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.1 mm dithiothreitol, 20 μm leupeptin, 10 μg/ml pepstatin A, 1 mm PMSF, and 1 mmbenzamidine) and homogenized as described for the preparation of cytoplasmic extracts. When more than 95% of the cells were lysed, the salt concentration was adjusted to 150 mm, and the suspension was layered over 20% (w/v) sucrose in buffer B and centrifuged at 800 × g. The nuclei were washed, resuspended in 300 μl of buffer B, and supplemented with 60 μl of 6-fold concentrated standard reducing sample buffer. In vitro translated [35S]methionine-labeled proteins were generated with the TnT-coupled reticulocyte lysate system (Promega, Madison, WI). Binding assays were performed as described previously (28.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The transfected 293T cells were lysed in a buffer containing 20 mmTris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.2% Nonidet P-40, 1 mm PMSF, 1 mm benzamidine, 50 μg/ml leupeptin, 7 μg/ml pepstatin A, and 25 μmALLN). The lysates were then centrifuged at 15,000 × gfor 15 min at 4 °C. Myc was then immunoprecipitated from the resulting supernatants with 0.8 μg of an anti-Myc monoclonal antibody coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Inc., Uppsala, Sweden). The immune complexes were then subjected to SDS-PAGE on 12.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membrane was blocked for 5 h at room temperature in blocking buffer containing 10% skim milk and 0.1% NaN3 in PBS. FLAG-tagged constructs were detected by Western blotting with an anti-FLAG monoclonal antibody and visualized with the ENHANCE chemiluminescence system (Amersham Biosciences, Inc.). The O1 mouse monoclonal antibody was used to identify oligodendrocytes. Cells were gently washed with PBS, and viable cells were incubated with the O1 antibody for 30 min at room temperature (1:100 dilution in Hanks' balanced salt solution buffer containing 3% bovine serum albumin and 3% fetal calf serum). For double-staining, cells were then fixed with 3.7% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min on ice, and processed for TUNEL as described below. Cells were incubated with the anti-Myc polyclonal antibody in PBS containing 1% bovine serum albumin and 1% normal goat serum (Sigma) for 1 h at room temperature. After several rinses with PBS, cells were incubated with the anti-rabbit IgG-Alexa Fluor 568 and anti-mouse IgG-Alexa Fluor 350 at room temperature for 30 min. These antibodies were used at 1:100 dilutions in PBS supplemented with 0.5 m NaCl and 1% normal goat serum. In situ detection of apoptotic oligodendrocytes was performed by using a TUNEL method (38.McGahon A.J. Martin S.J. Bissonnette R.P. Mahboubi A. Shi Y. Mogil R.J. Nishioka W.K. Green D.R. Methods Cell Biol. 1995; 46: 153-185Crossref PubMed Scopus (512) Google Scholar). After incubation with the O1 antibody, oligodendrocytes were fixed and permeabilized, as described above. After several rinses, samples were processed for TUNEL using the in situ cell death detection assay according to the protocol suggested by the manufacturer (MBL International Corp.). In some experiments, Alexa 568-dUTP (Molecular Probes) was used instead of FITC·dUTP as a substrate for the TUNEL reaction. Double-stained cells were visualized by fluorescence microscopy EFD-3 (Nikon) and recorded with a SPOT-RT digital charge-coupled device camera (Diagnostic Instruments). Apoptotic cells were determined by counting the percentage of TUNEL-positive cells among O1-positive cells in four fields across the" @default.
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• W1987261678 title "Structure-Function Analysis of NADE" @default.
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