FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1980745617> ?p ?o ?g. }

• W1980745617 endingPage "33227" @default.
• W1980745617 startingPage "33220" @default.
• W1980745617 abstract "Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-β peptide (Aβ) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Aβ. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of β-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Aβ by increasing the β-cleavage of amyloid precursor protein, generating more substrate for an unmodified γ-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of β-catenin and slightly decreased Aβ production. It is concluded that the LiCl-mediated increase in Aβ production is not related to GSK3 inhibition. Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-β peptide (Aβ) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Aβ. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of β-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Aβ by increasing the β-cleavage of amyloid precursor protein, generating more substrate for an unmodified γ-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of β-catenin and slightly decreased Aβ production. It is concluded that the LiCl-mediated increase in Aβ production is not related to GSK3 inhibition. Alzheimer disease, the most frequent cause of dementia, is characterized by the presence of typical microscopic lesions in the brain of affected patients. The coexistence of intraneuronal neurofibrillary tangles and extracellular senile plaques allows confirmation of the clinical diagnosis of the disease (1Braak H. Braak E. Acta Neuropathol. (Berl.). 1991; 82: 239-259Crossref PubMed Scopus (11761) Google Scholar). Neurofibrillary tangles are made of paired helical filaments (PHFs) 2The abbreviations used are: PHF, paired helical filament; GSK3, glycogen synthase kinase 3; Aβ, amyloid-β peptide; APP, amyloid precursor protein; sαAPP, soluble α-amyloid precursor protein; BACE1, β-site APP-cleaving enzyme 1; βCTF, C-terminal fragment of APP produced by β-cleavage; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine tert-butyl ester; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; FRET, fluorescence resonance energy transfer. containing the microtubule-associated protein, tau (2Brion J.P. Couck A.M. Passareiro E. Flament-Durand J. J. Submicrosc. Cytol. 1985; 17: 89-96PubMed Google Scholar, 3Delacourte A. Defossez A. J. Neurol. Sci. 1986; 76: 173-186Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 4Grundke-Iqbal I. Iqbal K. Quinlan M. Tung Y.C. Zaidi M.S. Wisniewski H.M. J. Biol. Chem. 1986; 261: 6084-6089Abstract Full Text PDF PubMed Google Scholar). In Alzheimer disease, tau is hyperphosphorylated, and many serine and threonine residues (5Brion J.P. Hanger D.P. Couck A.M. Anderton B.H. Biochem. J. 1991; 279: 831-836Crossref PubMed Scopus (101) Google Scholar, 6Reynolds C.H. Betts J.C. Blackstock W.P. Nebreda A.R. Anderton B.H. J. Neurochem. 2000; 74: 1587-1595Crossref PubMed Scopus (315) Google Scholar) that are found to be phosphorylated in PHF tau can be phosphorylated by GSK3 in both in vitro and transfected cells (7Sperber B.R. Leight S. Goedert M. Lee V.M. Neurosci. Lett. 1995; 197: 149-153Crossref PubMed Scopus (198) Google Scholar, 8Hanger D.P. Hughes K. Woodgett J.R. Brion J.P. Anderton B.H. Neurosci. Lett. 1992; 147: 58-62Crossref PubMed Scopus (657) Google Scholar, 9Lovestone S. Reynolds C.H. Latimer D. Davis D.R. Anderton B.H. Gallo J.M. Hanger D. Mulot S. Marquardt B. Stabel S. Woodgett J.R. Miller C.J. Curr. Biol. 1994; 4: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Senile plaques contain an amyloid core that is mainly constituted of amyloid-β peptide (Aβ) (10Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3683) Google Scholar), which is derived from the amyloid precursor protein (APP) (11Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar, 12Octave J.N. Acta Neurol. Belg. 1995; 95: 197-209PubMed Google Scholar). The APP gene encodes 10 different APP isoforms (13Octave J.N. Rev. Neurosci. 1995; 6: 287-316Crossref PubMed Scopus (37) Google Scholar) with an amino acid content varying from 365 to 770 amino acids. The neuronal APP is a single pass type I transmembrane protein containing 695 amino acids (11Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar) that is processed by amyloidogenic and nonamyloidogenic catabolic pathways. The β-cleavage of APP, catalyzed by the well characterized aspartyl protease β-site APP-cleaving enzyme 1 (BACE1) (14Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3327) Google Scholar), produces a C-terminal fragment of APP (βCTF), which is further cleaved by γ-secretase to generate Aβ. The γ-secretase activity is found as a multiprotein complex containing at least four different proteins: Aph-1, nicastrin, presenilin, and Pen-2 (15De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 16Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar). APP can also be cleaved within the Aβ sequence by an α-secretase. The α-cleavage of APP generates a soluble N-terminal fragment (sαAPP) and a 83-membrane-anchored C-terminal fragment (C83). Experimental evidence indicates that the α-cleavage of APP695 could be performed by members of the desintegrin and metalloprotease family, ADAM10 and ADAM17 (17Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (986) Google Scholar). The short intracellular C-terminal domain of APP can be phosphorylated in vitro and in vivo by several protein kinases such as cdk5, c-Jun N-terminal kinase, and GSK3 (18Aplin A.E. Gibb G.M. Jacobsen J.S. Gallo J.M. Anderton B.H. J. Neurochem. 1996; 67: 699-707Crossref PubMed Scopus (164) Google Scholar, 19Iijima K. Ando K. Takeda S. Satoh Y. Seki T. Itohara S. Greengard P. Kirino Y. Nairn A.C. Suzuki T. J. Neurochem. 2000; 75: 1085-1091Crossref PubMed Scopus (208) Google Scholar, 20Standen C.L. Brownlees J. Grierson A.J. Kesavapany S. Lau K.F. McLoughlin D.M. Miller C.C. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (112) Google Scholar). Another substrate of GSK3 is β-catenin, an essential protein of the Wnt signaling pathway. In the absence of a Wnt ligand, GSK3 activity is not inhibited, resulting in the phosphorylation of soluble β-catenin for ubiquitin-proteasome-mediated degradation (21van Noort M. Meeldijk J. van der Zee R. Destree O. Clevers H. J. Biol. Chem. 2002; 277: 17901-17905Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Alternatively, as a result of GSK3 inactivation by Wnt signaling, intracellular levels of β-catenin increase, allowing its binding to components of the high mobility group family of transcription factors and its translocation into the nucleus. LiCl, an inhibitor of GSK3 (22Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar), reduces the phosphorylation of tau in rat cultured neurons (23Munoz-Montano J.R. Moreno F.J. Avila J. Diaz-Nido J. FEBS Lett. 1997; 411: 183-188Crossref PubMed Scopus (312) Google Scholar, 24Hong M. Chen D.C. Klein P.S. Lee V.M. J. Biol. Chem. 1997; 272: 25326-25332Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 25Lovestone S. Davis D.R. Webster M.T. Kaech S. Brion J.P. Matus A. Anderton B.H. Biol. Psychiatry. 1999; 45: 995-1003Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Interestingly, LiCl was recently reported to decrease Aβ production in both transfected cells and transgenic mice (26Sun X. Sato S. Murayama O. Murayama M. Park J.M. Yamaguchi H. Takashima A. Neurosci. Lett. 2002; 321: 61-64Crossref PubMed Scopus (161) Google Scholar, 27Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1101) Google Scholar, 28Su Y. Ryder J. Li B. Wu X. Fox N. Solenberg P. Brune K. Paul S. Zhou Y. Liu F. Ni B. Biochemistry. 2004; 43: 6899-6908Crossref PubMed Scopus (290) Google Scholar, 29Ryder J. Su Y. Liu F. Li B. Zhou Y. Ni B. Biochem. Biophys. Res. Commun. 2003; 312: 922-929Crossref PubMed Scopus (113) Google Scholar). Therefore, lithium could be a combined therapeutic agent, inhibiting both the phosphorylation of tau and the production of Aβ. In this study, transfected CHO cells and rat cultured neurons expressing human APP695 and producing human Aβ were treated with LiCl. This GSK3 inhibitor decreased the phosphorylation of tau in neurons and induced the nuclear translocation of β-catenin in both CHO cells and neurons. It was found that LiCl increased the β-secretase activity and consequently increased the amount of βCTF generated from human APP695. The cleavage of βCTF by an unchanged γ-secretase activity led to an overproduction of Aβ. SB415286, another GSK3 inhibitor, induced the nuclear translocation of β-catenin and slightly decreased neuronal Aβ production. Taken together, these results clearly demonstrate that LiCl stimulates the amyloidogenic pathway of human APP independently of its inhibition of GSK3. Materials and Antibodies—Fetal calf serum, cell culture media, and NuPage™ 4–12% bis-Tris gels were purchased from Invitrogen. Protein A-Sepharose CL-4B was from Amersham Biosciences. Protease inhibitors were from Roche Applied Science. MTT reagent was obtained from Sigma. DAPT, a functional γ-secretase inhibitor (30Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint A.P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Ni B. Nissen J.S. Porter W.J. Potts B.D. Reel J.K. Stephenson D. Su Y. Shipley L.A. Whitesitt C.A. Yin T. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (803) Google Scholar) was a kind gift from L. Mercken (Aventis, Vitry-sur-seine, France). LiCl was obtained from Merck (Darmstadt, Germany). SB415286, an ATP-competitive inhibitor of GSK3, was kindly provided by A. Goffinet (Université catholique de Louvain, Brussels, Belgium). The mouse WO-2 monoclonal antibody raised against amino acids 5–8 of human Aβ was purchased from the Genetics Company. Rabbit polyclonal anti-actin serum was purchased from Sigma. B19 rabbit polyclonal serum recognized tau in a phosphorylation independent-manner (31Brion J.P. Hanger D.P. Bruce M.T. Couck A.M. Flament-Durand J. Anderton B.H. Biochem. J. 1991; 273: 127-133Crossref PubMed Scopus (143) Google Scholar). Mouse monoclonal PHF1 antibody specifically recognizing tau phosphorylated at Ser396/Ser404 was kindly provided by P. Davies and S. Greenberg. Mouse monoclonal β-catenin antibody was purchased from Transduction Laboratories (Lexington, KY). The polyclonal antibody anti-p58 was a kind gift from J. Saraste (University of Bergen, Norway). The rabbit polyclonal antibodies directed against BACE1 were from Oncogene Research (La Jolla, CA). Secondary antibodies were from Amersham Biosciences. Cell Cultures—Transfected CHO cell lines expressing human APP695 were cultured as described previously (32Essalmani R. Macq A.F. Mercken L. Octave J.N. Biochem. Biophys. Res. Commun. 1996; 218: 89-96Crossref PubMed Scopus (23) Google Scholar). Cells were seeded at a density of 3.6 × 104 cells/cm2 in 6-, 12-, or 96-well culture dishes 24 h prior to treatment. Primary cultures of cortical neurons were prepared from 17-day-old Wistar rat embryos as described previously (33Macq A.F. Czech C. Essalmani R. Brion J.P. Maron A. Mercken L. Pradier L. Octave J.N. J. Biol. Chem. 1998; 273: 28931-28936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Cells were plated in 6-, 12-, or 96-well culture dishes (4 × 105 cells/cm2) on glass coverslips (1.2 × 105 cells/cm2) pretreated with poly-l-lysine (10 μg/ml in phosphate-buffered saline) and cultured for 6 days in vitro in Neurobasal™ medium supplemented with 2% B-27 and 0.5 mm l-glutamine prior to infection with recombinant adenoviruses. Under these conditions, neuronal cultures (up to 98% of neurons) display high differentiation and survival rates (34Brewer G.J. J. Neurosci. Res. 1995; 42: 674-683Crossref PubMed Scopus (481) Google Scholar). Recombinant Adenoviruses, Neuronal Infection, and Treatment— The construction and purification of adenoviruses encoding human APP695 were performed as described previously (35Lemarchand P. Jaffe H.A. Danel C. Cid M.C. Kleinman H.K. Stratford-Perricaudet L.D. Perricaudet M. Pavirani A. Lecocq J.P. Crystal R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6482-6486Crossref PubMed Scopus (181) Google Scholar, 36Stratford-Perricaudet L.D. Makeh I. Perricaudet M. Briand P. J. Clin. Investig. 1992; 90: 626-630Crossref PubMed Scopus (574) Google Scholar). After 6 days in vitro, neuronal cultures were infected at a multiplicity of infection of 100 for 4 h in a minimal volume of culture medium. Infection medium was then replaced by fresh culture medium for 4 days before treatment. Under these conditions, at least 75% of neurons stably express the proteins encoded by recombinant adenoviruses (33Macq A.F. Czech C. Essalmani R. Brion J.P. Maron A. Mercken L. Pradier L. Octave J.N. J. Biol. Chem. 1998; 273: 28931-28936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Neuronal and CHO cells were treated for 24 h with 5 mm LiCl and/or 25 μm SB415286 (two GSK3 inhibitors) (37Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee C.L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar) or for 8 h with 250 nm DAPT (a γ-secretase inhibitor) (30Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint A.P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Ni B. Nissen J.S. Porter W.J. Potts B.D. Reel J.K. Stephenson D. Su Y. Shipley L.A. Whitesitt C.A. Yin T. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (803) Google Scholar). Survival Assays—Cells survival was measured by the colorimetric MTT assay. Cells grown in 96-well dishes were incubated for 2 h at 37 °C in fresh culture medium containing 0.5 mg/ml MTT. The medium was removed, and dark blue crystals that had formed were dissolved by adding 100 μl/well lysis solution (isopropyl alcohol/0.04 n HCl). Absorbance was measured at 492 nm using a microplate reader (PerkinElmer Life Sciences). Protein Analysis by Western Blotting and Densitometric Quantification—Culture media and cell lysates were analyzed by Western blotting as described previously (33Macq A.F. Czech C. Essalmani R. Brion J.P. Maron A. Mercken L. Pradier L. Octave J.N. J. Biol. Chem. 1998; 273: 28931-28936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Cell lysates (10 μg of protein) and culture medium (15 μl) were separated in NuPage™ 4–12% bis-Tris gels (Invitrogen) and blotted onto nitrocellulose membranes. Membranes were incubated overnight at 4 °C with primary antibody followed by secondary antibody coupled to horseradish peroxidase. Immunoreactive bands were detected by ECL (Amersham Biosciences). For quantification of cellular APP, membranes were stripped and reincubated with an anti-actin antibody. The ratio between immunoreactive proteins and actin was quantified with an electrophoresis Gel Doc 2000 imaging system coupled to a Quantity One™ software (Bio-Rad). Immunoprecipitation of βCTF Fragment—βCTF production was monitored by immunoprecipitation of cell lysates. Four million neurons or 0.5 million CHO cells were plated in each well of a 6-well plate. Following treatment, cells were washed three times with phosphate-buffered saline and then scraped and pelleted in cold phosphate-buffered saline. Cells were solubilized in radioimmune precipitation assay buffer containing protease inhibitors (1 μg/ml pepstatin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride; purchased from Roche Applied Science). The samples were precleared overnight at 4 °C by incubation with 5 mg of protein A-Sepharose. After removal of protein A-Sepharose by centrifugation (15,800 × g for 3 min), the samples were immunoprecipitated with 1.5 μg of WO-2 antibody for 12 h at 4 °C. Protein A-Sepharose (5 mg) was then added overnight. The samples were centrifuged at 15,800 × g for 3 min, and the pellets were washed three times with 1× radioimmune precipitation assay buffer (25 mm Tris, pH 7.4, 0.5% Triton X-100, 0.5% Nonidet P-40) and once with TBS buffer (10 mm Tris, pH 7.5). Pellets were incubated for 10 min at 96 °C in sample buffer (125 mm Tris, pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, 1% bromphenol blue). The samples were then centrifuged at 15,800 × g for 3 min, and the supernatants were loaded onto NuPage™ 4–12% bis-Tris gels (Invitrogen) and analyzed by Western blotting (see above). Quantification of Aβ Production—Aβ production was monitored by immunoprecipitation of cell culture medium. The quantification of extracellular Aβ-(1–40) was performed by ELISA (BIOSOURCE, Camarillo, CA). For both approaches, culture medium was collected, treated with protease inhibitors (1 μg/ml pepstatin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride), and cleared by centrifugation (15,800 × g for 5 min, 4 °C). Supernatants (100 μl) were recovered for Aβ quantification by fluorescent sandwich ELISA assay according to the manufacturer's instructions. Previous experiments showed that there was no cross-reaction between Aβ-(1–40) and Aβ-(1–42) recognition (38Kienlen-Campard P. Octave J.N. Peptides (N. Y.). 2002; 23: 1199-1204Crossref PubMed Scopus (17) Google Scholar). Fluorescence emission was measured on an HTS 7000 Plus plate reader (PerkinElmer Life Sciences) at excitation/emission wavelengths of 485 nm/535 nm, respectively. Immunoprecipitation was performed in 1 ml of the culture medium as described previously (33Macq A.F. Czech C. Essalmani R. Brion J.P. Maron A. Mercken L. Pradier L. Octave J.N. J. Biol. Chem. 1998; 273: 28931-28936Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Immunocytochemistry—Cells were fixed with 4% (w/v) paraformaldehyde in a phosphate buffer (0.1 m, pH 7.4) The immunohistochemical labeling was performed using the ABC method. Briefly, fixed cells were treated with H2O2 to inhibit endogenous peroxidase and incubated with blocking solution (10% (v/v) normal horse serum in TBS (0.01 m Tris, 0.15 m NaCl, pH 7.4)). After overnight incubation with the diluted primary antibody, the fixed cells were sequentially incubated with horse anti-mouse antibodies conjugated to biotin (Vector, Burlingame, CA) followed by the ABC complex (Vector). Peroxidase activity was revealed using diaminobenzidine as chromogen. β- and γ-Secretase Activity Assay—The β- and γ-secretase activities were measured by FRET-based in vitro assays. CHO cells were washed, scraped in cold phosphate-buffered saline, and pelleted by centrifugation (1,000 × g for 5 min, 4 °C). One hundred μl of extraction buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 0.01% Triton X-100) was added to the cell pellet (∼1.5 × 106 cells). After brief sonication (5 s), cellular extracts were cleared by centrifugation (10,000 × g for 10 min, 4 °C), and enzymatic activities were measured in the supernatant. The β-secretase assay was carried out on ∼ 20 μg of proteins in reaction buffer containing the fluorogenic NH2RE(EDANS)EVNLDAEFK(DABCYL)R-OH β-secretase specific substrate (39Ermolieff J. Loy J.A. Koelsch G. Tang J. Biochemistry. 2000; 39: 12450-12456Crossref PubMed Scopus (135) Google Scholar) according to the manufacturer's instructions (BioVision, Mountain View, CA). After 1.5 h of incubation at 37 °C in the dark, samples were analyzed in a HTS 7000 Plus fluorescence microplate reader with excitation/emission wavelengths of 360/535 nm, respectively. Positive controls were performed with purified human β-secretase (10 μg/ml, final concentration). The β-secretase inhibitor used in the assays was Z-VLL-CHO (40Abbenante G. Kovacs D.M. Leung D.L. Craik D.J. Tanzi R.E. Fairlie D.P. Biochem. Biophys. Res. Commun. 2000; 268: 133-135Crossref PubMed Scopus (39) Google Scholar). The γ-secretase activity was measured by the QTL Lightspeed™ γ-secretase assay (QTL Biosystems, Santa Fe, NM). Approximately 10 μg of cellular extract were mixed to γ-secretase substrate (GVVIATVK, flanked by biotin and a fluorescence quencher, 20 μm) in 20 μl (final volume) of assay buffer (50 mm Tris-HCl, pH 7, 2 mm EDTA, 0.05% bovine serum albumin, 2 mm reduced glutathione). After 2 h of incubation in the dark, 40 μl of QTL Sensor™ (41Kumaraswamy S. Bergstedt T. Shi X. Rininsland F. Kushon S. Xia W. Ley K. Achyuthan K. McBranch D. Whitten D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7511-7515Crossref PubMed Scopus (205) Google Scholar) was added, and the fluorescence was measured in a microplate reader with excitation/emission wavelengths of 430/595 nm, respectively. Results are expressed as β- and γ-secretase activities/mg of cellular protein. Analytical Subcellular Fractionation—Cells were incubated with 4 μg/ml [125I]transferrin for 5 min, 37 °C. Under these conditions, transferrin is an early endosomal marker. Cells were recovered in 0.25 m sucrose containing 1 mm EDTA, 3 mm imidazole buffered at pH 7.4, and complete protease inhibitors (Roche Applied Science). Cellular suspension was homogenized in a tight Dounce homogenizer. A low-speed nuclear fraction was pelleted at 1,000 × g for 10 min and washed three times by resuspension and sedimentation. Pooled postnuclear supernatants were further sedimented at 100,000 × g for 60 min in a Ti50 rotor (Beckman). The high-speed pellet was resuspended in 0.3 ml of homogenization buffer, mixed with 2.3 m sucrose to reach 1.28 g/ml in density, and layered at the bottom of a linear sucrose gradient (from 1.10 to 1.24 g/ml in density). After floatation by centrifugation at 200,000 g for 22 h in a Sw40 rotor (Beckman), 12 fractions were collected and analyzed for protein content. Western blotting and quantifications were performed as described above. Results are represented as normalized histograms (42Leighton F. Poole B. Beaufay H. Baudhuin P. Coffey J.W. Fowler S. De Duve C. J. Cell Biol. 1968; 37: 482-513Crossref PubMed Scopus (825) Google Scholar). Statistical Analysis—The number of samples (n) in each experimental condition is indicated in the legends to Figs. 1, 2, 4, 5, 6, and 7. Unpaired t test were performed to compare two experimental conditions. Otherwise, statistical analysis was performed by one-way analysis of variance followed by Bonferroni's multiple comparison post-hoc test. Each Western blot presented in Figs. 1, 2, 5, and 6 are representative of at least three independent experiments.FIGURE 2LiCl increases the level of βCTF produced by CHO cells expressing human APP695. CHO cells expressing human APP695 were incubated in the presence or absence of 5 mm LiCl for 24 h or 250 nm DAPT for 8 h. A, expression of human APP695 was analyzed by Western blotting using the WO-2 antibody (upper panel). Cellular levels of βCTF were analyzed after immunoprecipitation with the WO-2 antibody (lower panel). B, the βCTF/APP signal ratio was quantified and expressed (mean ± S.E.) as percentage of nontreated (NT) controls; n = 3; ***, p < 0.001 when compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4LiCl increases β-secretase activity in CHO cells. CHO cells were incubated in the presence or in the absence of 5 mm LiCl for 24 h or 250 nm DAPT for 8 h. The β-(A) and γ-secretase (B) activity were measured in cell extracts by FRET-based in vitro assays. Results are expressed as percentage of untreated controls (NT); n = 6; ***, p < 0.001 when compared with control. **, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5LiCl inhibits GSK3 in rat cultured neurons. Rat cultured neurons (APP–) or neurons infected by a recombinant adenovirus encoding human APP695 (APP+) were analyzed by Western blotting using the WO-2 antibody. A, expression of APP in cellular extracts (upper left). Protein loading was controlled by incubating membranes with an anti-actin antibody (lower left). Expression of sαAPP (upper right) and detection of extracellular Aβ after immunoprecipitation (lower right) are shown. B, neuronal survival was measured using the MTT assay. Results (mean ± S.E.) are given as the percentage of survival of untreated neurons (NT); n = 20. C, tau or phospho-tau (P-tau) were analyzed by Western blotting using the B19 (upper panel) or the PHF1 (lower panel) antibody. D, tau phosphorylation was quantified by measuring the PHF1/B19 signal ratio and expressed (mean ± S.E.) as percentage of untreated neurons; n = 5; ***, p < 0.001 when compared with control. E, immunolabeling of untreated neurons or neurons incubated in the presence of LiCl with an anti-β-catenin antibody, indicating the LiCl-mediated concentration of β-catenin in nuclei. Scale bars,10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6LiCl increases the amount of βCTF, Aβ, and BACE1 protein in rat cultured neurons. Rat cortical neurons expressing human APP695 (APP+) were incubated in the presence (+) or in the absence (–) of 250 nm DAPT for 8 h (A and C) or 5 mm LiCl for 24 h (B–D). Cell extracts were analyzed by Western blotting. A and B, detection of βCTF after immunoprecipitation with the WO-2 antibody (left). The βCTF/APP signal ratio was quantified and expressed (mean ± S.E.) as percentage of untreated controls; n = 4; ***, p < 0.001 when compared with control (right). C, the extracellular Aβ-(1–40) was quantified by ELISA. Results are given as mean ± S.E. (left) of extracellular Aβ-(1–40) (pg/ml) or expressed as percentage (right) of nontreated controls; n = 7; ***, p < 0.0001 when compared with the control. D, expression of BACE1 protein in cellular extracts (upper left). Protein loading was controlled by incubating membranes with an anti-actin antibody (lower left). The BACE1/actin signal ratio was quantified and expressed (mean ± S.E.) as percentage of untreated controls (NT); n = 4; ***, p < 0.001 when compared with control (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7GSK3 inhibitor (SB415286) does not modify neuronal production of Aβ. Rat cortical neurons expressing human APP695 were incubated for 24 h in the presen" @default.
• W1980745617 created "2016-06-24" @default.
• W1980745617 creator A5014952360 @default.
• W1980745617 creator A5018505718 @default.
• W1980745617 creator A5018865223 @default.
• W1980745617 creator A5019999293 @default.
• W1980745617 creator A5066959404 @default.
• W1980745617 creator A5077055662 @default.
• W1980745617 creator A5079849514 @default.
• W1980745617 date "2005-09-01" @default.
• W1980745617 modified "2023-10-16" @default.
• W1980745617 title "Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3" @default.
• W1980745617 cites W1483703465 @default.
• W1980745617 cites W1501075116 @default.
• W1980745617 cites W1502707849 @default.
• W1980745617 cites W1527604508 @default.
• W1980745617 cites W1567624342 @default.
• W1980745617 cites W1611407165 @default.
• W1980745617 cites W1754645121 @default.
• W1980745617 cites W1806182908 @default.
• W1980745617 cites W1963728714 @default.
• W1980745617 cites W1970293963 @default.
• W1980745617 cites W1971541898 @default.
• W1980745617 cites W1972158411 @default.
• W1980745617 cites W1979993283 @default.
• W1980745617 cites W1982958012 @default.
• W1980745617 cites W1986200442 @default.
• W1980745617 cites W1987341255 @default.
• W1980745617 cites W1987693973 @default.
• W1980745617 cites W1997177821 @default.
• W1980745617 cites W2019576037 @default.
• W1980745617 cites W2024098928 @default.
• W1980745617 cites W2030182984 @default.
• W1980745617 cites W2034201162 @default.
• W1980745617 cites W2039550162 @default.
• W1980745617 cites W2046847750 @default.
• W1980745617 cites W2052742260 @default.
• W1980745617 cites W2054183594 @default.
• W1980745617 cites W2056181922 @default.
• W1980745617 cites W2064644219 @default.
• W1980745617 cites W2069584842 @default.
• W1980745617 cites W2070045579 @default.
• W1980745617 cites W2071633123 @default.
• W1980745617 cites W2073128231 @default.
• W1980745617 cites W2073292164 @default.
• W1980745617 cites W2076305899 @default.
• W1980745617 cites W2077845288 @default.
• W1980745617 cites W2078680897 @default.
• W1980745617 cites W2081881251 @default.
• W1980745617 cites W2090705035 @default.
• W1980745617 cites W2104962877 @default.
• W1980745617 cites W2108149965 @default.
• W1980745617 cites W2127778297 @default.
• W1980745617 cites W2133019124 @default.
• W1980745617 cites W2153450253 @default.
• W1980745617 cites W2153471866 @default.
• W1980745617 cites W2156989115 @default.
• W1980745617 doi "https://doi.org/10.1074/jbc.m501610200" @default.
• W1980745617 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16014628" @default.
• W1980745617 hasPublicationYear "2005" @default.
• W1980745617 type Work @default.
• W1980745617 sameAs 1980745617 @default.
• W1980745617 citedByCount "41" @default.
• W1980745617 countsByYear W19807456172012 @default.
• W1980745617 countsByYear W19807456172013 @default.
• W1980745617 countsByYear W19807456172014 @default.
• W1980745617 countsByYear W19807456172016 @default.
• W1980745617 countsByYear W19807456172019 @default.
• W1980745617 countsByYear W19807456172020 @default.
• W1980745617 crossrefType "journal-article" @default.
• W1980745617 hasAuthorship W1980745617A5014952360 @default.
• W1980745617 hasAuthorship W1980745617A5018505718 @default.
• W1980745617 hasAuthorship W1980745617A5018865223 @default.
• W1980745617 hasAuthorship W1980745617A5019999293 @default.
• W1980745617 hasAuthorship W1980745617A5066959404 @default.
• W1980745617 hasAuthorship W1980745617A5077055662 @default.
• W1980745617 hasAuthorship W1980745617A5079849514 @default.
• W1980745617 hasBestOaLocation W19807456171 @default.
• W1980745617 hasConcept C104629339 @default.
• W1980745617 hasConcept C179104552 @default.
• W1980745617 hasConcept C181199279 @default.
• W1980745617 hasConcept C182996813 @default.
• W1980745617 hasConcept C184235292 @default.
• W1980745617 hasConcept C185592680 @default.
• W1980745617 hasConcept C192118531 @default.
• W1980745617 hasConcept C2780990938 @default.
• W1980745617 hasConcept C55493867 @default.
• W1980745617 hasConceptScore W1980745617C104629339 @default.
• W1980745617 hasConceptScore W1980745617C179104552 @default.
• W1980745617 hasConceptScore W1980745617C181199279 @default.
• W1980745617 hasConceptScore W1980745617C182996813 @default.
• W1980745617 hasConceptScore W1980745617C184235292 @default.
• W1980745617 hasConceptScore W1980745617C185592680 @default.
• W1980745617 hasConceptScore W1980745617C192118531 @default.
• W1980745617 hasConceptScore W1980745617C2780990938 @default.
• W1980745617 hasConceptScore W1980745617C55493867 @default.
• W1980745617 hasIssue "39" @default.
• W1980745617 hasLocation W19807456171 @default.

• next