RAC1 Inhibition Targets Amyloid Precursor Protein Processing by γ-Secretase and Decreases Aβ Production in Vitro and in Vivo
2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês
10.1074/jbc.m507913200
ISSN1083-351X
AutoresLaurent Désiré, Jérôme Bourdin, Nadia Loiseau, Hélène Peillon, Virginie Picard, Catherine De Oliveira, Florence Bachelot, Bertrand Leblond, Thierry Taverne, Eric Beausoleil, S. Lacombe, Dominique Drouin, Fabien Schweighoffer,
Tópico(s)Drug Transport and Resistance Mechanisms
Resumoβ-Amyloid peptides (Aβ) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Aβ 40 and Aβ 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Aβ involves β-secretase and γ-secretase activities and is regulated by membrane trafficking of the proteins involved in Aβ production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Aβ 40 and Aβ 42 production but does not impact sAPPα levels and does not inhibit β-secretase. Rather, EHT 1864 modulates APP processing at the level of γ-secretase to prevent Aβ 40 and Aβ 42 generation. This effect does not result from a direct inhibition of the γ-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Aβ 40 and Aβ 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Aβ formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease. β-Amyloid peptides (Aβ) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Aβ 40 and Aβ 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Aβ involves β-secretase and γ-secretase activities and is regulated by membrane trafficking of the proteins involved in Aβ production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Aβ 40 and Aβ 42 production but does not impact sAPPα levels and does not inhibit β-secretase. Rather, EHT 1864 modulates APP processing at the level of γ-secretase to prevent Aβ 40 and Aβ 42 generation. This effect does not result from a direct inhibition of the γ-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Aβ 40 and Aβ 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Aβ formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease. Alzheimer disease (AD) 2The abbreviations used are: ADAlzheimer diseaseAββamyloidAPPamyloid precursor proteinsAPPαsoluble APP ectodomainBACEβsite APP-cleaving enzymeCTFcarboxyl-terminal fragmentDAPTN-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl esterELISAenzyme-linked immunosorbent assayFBSfetal bovine serumHEKhuman embryonic kidneyNICDNotch intracellular cytoplasmic domainswAPP-HEK293HEK cells expressing human APP695 containing the Swedish mutationPak1p21-activated kinase 1RBDRho-GTP binding domainILinterleukinEHT 18645-(5-(7-(trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholinomethyl)-4H-pyran-4-one dihydrochlorideEHT 40635-(5-(quinazolin-4-yloxy)pentyloxy)-2-((4-methylpiperazin-1-yl)methyl)-4H-pyran-4-oneMES4-morpholineethanesulfonic acidMCA7-methoxycoumarin-4-yl)acetylDNP2,4-dinitrophenylNMA2-(N-methylamino)benzoyl is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterized by the presence of amyloid plaques (1Hardy J.A. Higgins G.A. Science. 1992; 256: 184-185Crossref PubMed Scopus (5200) Google Scholar), intracellular neurofibrillary tangles, and pronounced cell death. The β-amyloid peptide (Aβ) (2Buxbaum J.D. Oishi M. Chen H.I. Pinkas-Kramarski R. Jaffe E.A. Gandy S.E. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10075-10078Crossref PubMed Scopus (504) Google Scholar) is the main constituent of senile plaques found in AD brains. Furthermore, extracellular Aβ 42 appears toxic to neurons in vitro and in vivo (reviewed in Ref. 3Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). Aβ is generated by proteolysis of an integral membrane protein, the amyloid precursor protein (APP), via at least two post-translational pathways. The amyloidogenic cleavage of APP is a sequential processing of APP initiated by β-secretase (BACE), which cleaves APP within the luminal domain or at the cell surface, generating the N terminus of Aβ (4Vassar R. J. Mol. Neurosci. 2004; 23: 105-114Crossref PubMed Scopus (306) Google Scholar). This cleavage generates several membrane-bound proteolytic C-terminal fragments (CTFs), such as the 99-residue β-CTF (also called C99), as well as the secreted APP ectodomain sAPPβ. The C terminus of Aβ is subsequently generated by intramembranous cleavage of CTFs by γ-secretase, producing either Aβ 40 or Aβ 42. The cleavages at residues 40–42 are referred to as γ-cleavage, and the cleavages at residues 49–52 are referred to as ϵ-cleavage (5Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Crossref PubMed Scopus (322) Google Scholar). The nonamyloidogenic cleavage of APP, which precludes Aβ generation, is mediated by α-secretase, a disintegrin and metalloproteinase 10, and a disintegrin and metalloproteinase 17, in a reaction believed to occur primarily on the plasma membrane. This proteolytic cleavage by α-secretase occurs within the Aβ region and produces soluble APP (sAPPα), the dominant processing product, and the residual membrane-bound 10-kDa CTF (CTFα, also called C83). Like C99, C83 is a substrate for γ-secretase, which cleaves C83 to generate the nonamyloidogenic p3 fragment. APP is also a substrate of caspase activities that cleave its cytosolic domain (6Weidemann A. Paliga K. Durrwang U. Reinhard F.B. Schuckert O. Evin G. Masters C.L. J. Biol. Chem. 1999; 274: 5823-5829Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Alzheimer disease βamyloid amyloid precursor protein soluble APP ectodomain βsite APP-cleaving enzyme carboxyl-terminal fragment N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester enzyme-linked immunosorbent assay fetal bovine serum human embryonic kidney Notch intracellular cytoplasmic domain HEK cells expressing human APP695 containing the Swedish mutation p21-activated kinase 1 Rho-GTP binding domain interleukin 5-(5-(7-(trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholinomethyl)-4H-pyran-4-one dihydrochloride 5-(5-(quinazolin-4-yloxy)pentyloxy)-2-((4-methylpiperazin-1-yl)methyl)-4H-pyran-4-one 4-morpholineethanesulfonic acid 7-methoxycoumarin-4-yl)acetyl 2,4-dinitrophenyl 2-(N-methylamino)benzoyl Multiple lines of evidence suggest that APP processing and Aβ generation are determined by dynamic interactions of APP with membrane microdomains, known as lipid rafts, which facilitate the production of Aβ (7Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (929) Google Scholar, 8Wolozin B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5371-5373Crossref PubMed Scopus (178) Google Scholar). Lipid rafts are rich in cholesterol and sphingolipids and are also the principal compartment in which Aβ is found (9Lee S.J. Liyanage U. Bickel P.E. Xia W. Lansbury Jr., P.T. Kosik K.S. Nat. Med. 1998; 4: 730-734Crossref PubMed Scopus (375) Google Scholar, 10Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (365) Google Scholar). BACE and γ-secretase also localize to these lipid raft microdomains, in endosomes and post-Golgi compartments, enabling them to cleave APP (11Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar, 12Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). More generally, lipid rafts contribute to trafficking of proteins and lipids in the secretory and endocytic pathways by regulating vesicle formation and sorting. They also act as signaling platforms for various pathways including GTPase-dependent actin rearrangements (13Jaksits S. Bauer W. Kriehuber E. Zeyda M. Stulnig T.M. Stingl G. Fiebiger E. Maurer D. J. Immunol. 2004; 173: 1628-1639Crossref PubMed Scopus (35) Google Scholar) induced by small GTP-binding proteins from the Rho family such as Rac, Rho, and Cdc42. These small G proteins are activated by GTP/GDP exchange and regulate a wide variety of cellular functions such as gene expression, cytoskeletal reorganization, and vesicle/secretory trafficking. The activated Cdc42 or Rac then activates the PAK Ser/Thr kinase family. Recent studies showed the participation of Rho in the formation of stress fibers, whereas activated Cdc42 induces the formation of filopodia, thin finger-like extensions containing actin bundles. Rac regulates the formation of lamellipodia or ruffles, curtain-like extensions often formed along the edge of the cell (see Ref. 14Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar for a review). In the brain, these small G proteins participate in the morphological changes of neurons, localized in growth cones, axons, dendritic trunks, and spines (15van Leeuwen F.N. van Delft S. Kain H.E. van der Kammen R.A. Collard J.G. Nat. Cell Biol. 1999; 1: 242-248Crossref PubMed Scopus (184) Google Scholar). In the mature brain, it has been shown that Rac1, but not Rho nor Cdc42, is present in the raft domain of neuronal membranes (16Kumanogoh H. Miyata S. Sokawa Y. Maekawa S. Neurosci. Res. 2001; 39: 189-196Crossref PubMed Scopus (40) Google Scholar). This was recently confirmed by an unbiased quantitative proteomics study revealing Rac1 as a raft-associated protein (17Foster L.J. De Hoog C.L. Mann M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5813-5818Crossref PubMed Scopus (730) Google Scholar). Other studies showed that activation of Rac1 is associated with its rapid recruitment into the lipid rafts, whereas Cdc42 is not, and that Rac1, but not Rho or Cdc42, regulates the assembly and export to the cell membrane of Golgi-derived lipid rafts (18Field K.A. Apgar J.R. Hong-Geller E. Siraganian R.P. Baird B. Holowka D. Mol. Biol. Cell. 2000; 11: 3661-3673Crossref PubMed Scopus (40) Google Scholar, 19Rozelle A.L. Machesky L.M. Yamamoto M. Driessens M.H. Insall R.H. Roth M.G. Luby-Phelps K. Marriott G. Hall A. Yin H.L. Curr. Biol. 2000; 10: 311-320Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). A number of recent studies have implicated the Rho family of small G proteins, including Rac1 itself, in the modulation of APP processing. Interestingly, two different aspects of APP processing appear to be controlled by Rac1 and other small G proteins: ectodomain shedding (20Maillet M. Robert S.J. Cacquevel M. Gastineau M. Vivien D. Bertoglio J. Zugaza J.L. Fischmeister R. Lezoualc'h F. Nat. Cell Biol. 2003; 5: 633-639Crossref PubMed Scopus (166) Google Scholar), which is a prerequisite for β-γ-secretase cleavage, and the β-γ-secretase cleavage itself (21Gianni D. Zambrano N. Bimonte M. Minopoli G. Mercken L. Talamo F. Scaloni A. Russo T. J. Biol. Chem. 2003; 278: 9290-9297Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Zambrano N. Gianni D. Bruni P. Passaro F. Telese F. Russo T. J. Biol. Chem. 2004; 279: 16161-16169Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 23Zhou Y. Su Y. Li B. Liu F. Ryder J.W. Wu X. Gonzales-DeWhitt P.A. Gelfanova V. Hale J.E. May P.C. Paul S.M. Ni B. Science. 2003; 302: 1215-1217Crossref PubMed Scopus (308) Google Scholar). In particular, overexpression of dominant negative (RacN17) or constitutively active (RacQL) mutants of Rac1 was demonstrated to inhibit or stimulate γ-secretase-mediated APP processing (21Gianni D. Zambrano N. Bimonte M. Minopoli G. Mercken L. Talamo F. Scaloni A. Russo T. J. Biol. Chem. 2003; 278: 9290-9297Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), respectively, which suggests that Rac1 is crucial for the homeostasis of endogenous Aβ production. It is now clear that APP processing is controlled by multiple pathways to provide a fine tuned processing of APP in physiological conditions. In the AD condition, consequent progress in the identification of dysregulated mechanisms controlling APP processing has been made. For example, Rac1, as well as other small G proteins, has been implicated in the pathways triggered by inflammatory mediators, such as interleukin (IL)-1β, IL-6, or tumor necrosis factor-α, and by growth factors, such as transforming growth factor-β or platelet-derived growth factor, that have been found to be up-regulated in AD brains (24Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (529) Google Scholar, 25Hawkins P.T. Eguinoa A. Qiu R.G. Stokoe D. Cooke F.T. Walters R. Wennstrom S. Claesson-Welsh L. Evans T. Symons M. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). These signaling pathways have been shown to stimulate the generation of Aβ (26Liao Y.F. Wang B.J. Cheng H.T. Kuo L.H. Wolfe M.S. J. Biol. Chem. 2004; 279: 49523-49532Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The involvement of Rac1 in AD is further stressed by the observation that an up-regulation of neuronal Cdc42/Rac1 occurs in selected neuronal populations of the AD brain in comparison with age-matched controls (27Zhu X. Raina A.K. Boux H. Simmons Z.L. Takeda A. Smith M.A. Int. J. Dev. Neurosci. 2000; 18: 433-437Crossref PubMed Scopus (83) Google Scholar). Therefore, an attractive hypothesis proposes a role for small G proteins, such as Rac1, in the control of APP processing and Aβ accumulation that occur in AD. We describe here a new molecule, EHT 1864, that inhibits Rac1 signaling and APP processing, lowering Aβ production in vitro and leading to a decrease in Aβ in the brain of guinea pigs. Since this molecule does not affect Notch processing and the neurotrophic α-secretase pathway, EHT 1864 represents a prototype of a new chemical series of interest for developing new treatments for AD. Materials and Compounds—EHT 1864 and EHT 4063 (Fig. 1) were synthesized as described in Ref. 28Leblond B. Petit S. Picard V. Taverne T. Schweighoffer F. World Patent WO2004076445. 2004; Google Scholar. All cell culture reagents were from Invitrogen (Cergy Pontoise, France) unless otherwise noted. NSC23766, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), BACE inhibitors, BACE, and γ-secretase fluorogenic substrates were obtained from Calbiochem (CliniSciences, Montrouge, France). Cell Culture and Treatments—Stably transfected HEK293 cells overexpressing human swAPP harboring the "Swedish" mutation (29Chevallier N. Jiracek J. Vincent B. Baur C.P. Spillantini M.G. Goedert M. Dive V. Checler F. Br. J. Pharmacol. 1997; 121: 556-562Crossref PubMed Scopus (38) Google Scholar) (swAPP-HEK293 cells) were maintained in modified Eagle's medium plus Earle's salt supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine (Sigma), 1× nonessential amino acids, and antibiotics. GC cars NIH3T3 cells (LGC PromoChem) were grown in high glucose Dulbecco's modified Eagle's medium plus Glutamax supplemented with 10% newborn serum and antibiotics. Human astrocytomas U87MG (ATCC number HTB-14) were grown at 37 °C in Dulbecco's modified Eagle's medium containing 1 mm glutamine, 10% FBS, and antibiotics. SH-SY5Y cells (ATCC number CRL-2266) were maintained in modified Eagle's medium/F-12K (1:1, v/v) supplemented with 10% FBS, 2 mm l-glutamine, 1× nonessential amino acids, 1× sodium pyruvate, and antibiotics. HeLa cells (ATCC number CCL 2) were grown in modified Eagle's medium supplemented with 10% FBS, 2 mm l-glutamine, and antibiotics. Cells were treated 48 h after plating in 10-cm plates with various concentrations of the indicated molecules or Me2SO as the vehicle for 16 or 24 h. To do so, medium was replaced with 5 ml of new medium in which treatments were performed. Total Me2SO dilution was 1:1000 in all cases. Cells were allowed to secrete in 5 ml of medium for 7 h in the presence of 1 μm phosphoramidon. Endogenous Rac GTPase Activation Assay—U87-MG cells were grown in a 150-mm diameter dish until they reached 80% confluence. The cells were then treated with the test compounds or the solvent only. Cells were then lysed in a buffer containing 0.5% Triton X-100, 10 mm Tris, pH 7.5, 25 mm KCl, 120 mm NaCl, and 1.8 mm CaCl2. Lysates were clarified, the protein concentrations were normalized, and the GTP-bound Rac1 in the lysates were measured using the Rac activation assay Biochem kit (Cytoskeleton) as per the manufacturer's recommendations. Endogenous Rho GTPase Activation Assay—GST-Rhotekin Rho-GTP binding domain (RBD) fusion protein beads were prepared as follows, in laboratory of A. Hall (University College, London, UK). BL21 DE3 pLysS strain transformed with pGEX2T Rhotekin RBD grown overnight in LB containing 50 μg/ml ampicillin and 25 μg/ml chloramphenicol was pelleted and then resuspended in GTLB I buffer (50 mm Tris, pH 8, 40 mm EDTA, 25% (w/v) sucrose, and 1 mm phenylmethylsulfonyl fluoride). Suspension was rotated on a wheel at 4 °C for 10–20 min. GTLB II buffer (50 mm Tris pH 8, 100 mm MgCl2, 0.2% (w/v) Triton X-100) was added, and suspension was rotated again for a further 10 min. Bacteria were sonicated on ice at 15 μm in 10-s bursts and centrifuged. Supernatant was carefully removed and transferred to 50-ml Falcon tubes. 1 ml of 50% glutathione-agarose beads was added, and the suspension was rotated on a wheel at 4 °C for 1 h. Beads were spun down for 20 s at no more than 2500 rpm. Supernatant was discarded, and beads were washed with cold wash buffer (50 mm Tris, pH 7.6, 50 mm NaCl, 5 mm MgCl2). Beads were transferred to Eppendorf tubes and spun down again, and the last traces of buffer were removed. Beads were then resuspended in wash buffer containing 50% glycerol (final glycerol concentration 25%), aliquoted, and stored at –80 °C. Pull-down experiments were performed as described for the Rac-GTPase activation assay except that the detection antibody was an anti-RhoA antibody (Tebu, Le Perray en Yvelines, France) used at a 1:750 dilution. Transient Expression Reporter Assays—Transcriptional activation of luciferase gene expression constructs was performed as described previously (30Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar). Briefly, 250,000 NIH3T3 cells/well were seeded in 6-well plates and were co-transfected 24 h later with plasmids prK5-RacV12 and reporter constructs using Lipofectamine Plus (Invitrogen). The compound of interest was added after the incubation with Lipofectamine. 24 h after transfection, cells were starved for an additional 24 h with Dulbecco's modified Eagle's medium supplemented with 0.5% FBS together with the appropriate doses of test compounds or the solvent only. Analyses of the cell lysates of the transiently transfected NIH3T3 cells were performed using the luciferase assay system (Promega) and Fluoroscan Ascent FL plate reader (Thermo LabSystems). All assays were performed in duplicate, and results shown represent the mean ± S.E. of four independent experiments for each reporter gene. We did not use an internal standard in the transfections, since all three promoters tested responded to active Rac overexpression to varying extents. However, consistent and reproducible data were obtained in different assays performed using several plasmid preparations, and we monitored protein concentration for yield in the cell extracts as well as expression of the tagged, exogenous protein by Western blotting. The reporter constructs 5× Gal4-Luc plus Gal4-c-Jun, HIV-Luc bearing NF-κB binding sites (30Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar), and cyclin D1-Luc (31Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar) were described previously and are a kind gift of Dr. Channing J. Der (University of North Carolina, Chapel Hill, NC). The expression plasmid pRK5-RacV12 was described previously (32Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-404Abstract Full Text PDF PubMed Scopus (3084) Google Scholar) and is a kind gift of Dr. Alan Hall (University College, London, UK). Western Blot Analyses—swAPP-HEK293 cells were scraped and lysed in CelLytic-M (Sigma). Protein concentrations were determined by the Bradford procedure. Equal protein quantities were separated on a 10% SDS-polyacrylamide gel and transferred to Hybond-C (Amersham Biosciences) membranes. After transfer, membranes were blocked with 5% nonfat milk and incubated overnight with the primary antibody anti-APP antibody at 1:1000 (AHP538; Serotec), allowing the detection of both APP (resolved as doublets in some experiments) and C99 CTF. For sAPPα detection, cells were allowed to secrete for 7 h. Media were collected and cleared by centrifugation, and then equal amounts were loaded on 10% SDS-PAGE and subjected to Western blot with 6E10 monoclonal antibody (1:1000). Immunological complexes were revealed with an anti-mouse peroxidase (1:5000; Jackson Laboratories) antibody followed by ECL (Amersham Biosciences). NotchΔE Transfection and Notch-1 Cleavage Assays in HeLa Cells— HeLa cells in 10-cm plates were transiently transfected with the expression vector pSC2+ΔE3MV-6MT, which overexpresses truncated Notch-1, lacking most of the Notch extracellular domain, and has a C-terminal Myc tag, (NotchΔE). This truncated form of Notch is the substrate of γ-secretase (33Kopan R. Schroeter E.H. Weintraub H. Nye J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1683-1688Crossref PubMed Scopus (425) Google Scholar). 1 day post-transfection, cultures were preincubated with EHT 1864 or the γ-secretase inhibitor DAPT for 18 h at the indicated concentrations, and then CelLytic-M lysates were processed for detection of the Notch intracellular domain (NICD) by Western blotting using anti-Myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1000. In Vivo Delivery of Inhibitors—EHT 1864 or vehicle (physiological saline) were injected in male Hartley albino guinea pigs, weighing 250–270 g at delivery and obtained from Charles River Laboratories (L'Arbresle, France), once a day for 15 consecutive days by the intraperitoneal route. 1 h after the final administration, the guinea pigs were killed; brains were immediately extracted and immersed in an oxygenated (95% O2, 5% CO2) physiological saline bath placed on ice (1–2 °C); and superficial vessels were removed. The whole brains were dissected to provide left and right cortices, which were weighed, snap-frozen in liquid nitrogen, and stored at –80 °C, separately. The maximum time between sacrifice and snap freezing was less than 15 min. Measurements of Aβ 40 and Aβ 42—Stably transfected swAPP-HEK293 cells or confluent SH-SY5Y cells were incubated for 7 h in the presence of phosphoramidon (1 μm) (Sigma). Media and cell lysates were collected as above, centrifuged, normalized to total protein, and assayed for Aβ 40 and Aβ 42 by sandwich ELISA according to the manufacturer's instructions (BIOSOURCE International). For Aβ 42 detection, samples were concentrated on YM3 Microcon columns (Millipore Corp.). For in vivo samples, the protocol ensured a final concentration of guanidine of <0.1 m, as recommended by the manufacturer, and ELISA standards included guanidine. Right cortices were homogenized for 3 h at room temperature in 5 m guanidine HCl, 50 mm Tris-HCl, and pH 8 with a protease inhibitor mixture (Roche Applied Science). Tissue homogenates were diluted 1:1 (v/v) in BSAT-DPBS buffer (Dulbecco's phosphate-buffered saline with 5% bovine serum albumin and 0.03% Tween 20), pH 7.4, and were centrifuged at 20,000 × g for 20 min at 4 °C. Supernatants were diluted 1:1 (v/v) in ELISA kit sample buffer, normalized to total protein, and assayed for Aβ 40 and Aβ 42 by sandwich ELISA according to the manufacturer's instructions. For Aβ 42 detection, samples were concentrated on YM3 Microcon columns (Millipore). BACE Assay—Human BACE1 cDNA was generated by reverse transcription-PCR from human brain mRNA samples (Biocat) and cloned into the pcDNA3 expression vector. Subsequently, a HEK293 cell line stably expressing BACE1 was generated and used as a source of BACE1. An in vitro assay was developed based on previous studies (34Ermolieff J. Loy J.A. Koelsch G. Tang J. Biochemistry. 2000; 39: 12450-12456Crossref PubMed Scopus (135) Google Scholar, 35Andrau D. Dumanchin-Njock C. Ayral E. Vizzavona J. Farzan M. Boisbrun M. Fulcrand P. Hernandez J.F. Martinez J. Lefranc-Jullien S. Checler F. J. Biol. Chem. 2003; 278: 25859-25866Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) using a quenched fluorogenic substrate containing the Swedish mutation MCA-SEVNLDAEFK(DNP)-NH2 (Substrate V; Calbiochem). Proteins were extracted in 20 mm MES, 1% Triton X-100 plus protease inhibitor mixture by incubation on ice for 30 min. The assay was carried out in black 96-well plates (ATGC) in a volume of 200 μl of reaction buffer (25 mm MES, 25 mm sodium acetate, 25 mm Tris, pH 4.4), containing 25 μl of the preparation plus 15 μm peptide Substrate V. Excitation was performed at 320 nm, and the reaction kinetics were monitored by measuring the fluorescence emission at 420 nm on a Fluoroscan Ascent FL plate reader (Thermo LabSystems). Controls included purified recombinant human BACE501 protein (R&D Systems) diluted at 1 μg/well in 200 μl of 0.1 m sodium acetate buffer (pH 4.4), the BACE substrate analog inhibitor III (Glu-Val-Asn-statine-Val-Ala-Glu-Phe-NH2; Calbiochem), or substrate alone, and background fluorescence was subtracted from recorded BACE activity. Final Me2SO concentration was 1% (v/v) and did not affect the fluorescence or BACE activity. γ-Secretase Assay—Here, we implemented a γ-secretase assay allowing de novo Aβ generation in vitro, using cell membranes as the source of γ-secretase and endogenous C99 generated from swAPP as the substrate. Preparation of solubilized γ-secretase fractions was performed essentially as described previously (36Pinnix I. Musunuru U. Tun H. Sridharan A. Golde T. Eckman C Ziani-Cherif C Onstead L. Sambamurti K. J. Biol. Chem. 2001; 276: 481-487Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 37Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (501) Google Scholar) with the above modifications. All incubations were performed in the presence of Complete protease inhibitor mixture. Confluent plates of swAPP-HEK293 cells were lysed in 1 ml of ice-cold CelLytic-M (Sigma) and incubated for 15 min at 4 °C on a shaker. Cell debris and nuclei were removed by centrifugation at 1000 × g for 15 min at 4 °C. For membrane isolation, the supernatant solutions were centrifuged at 20,000 × g for 1 h at 4°C. After centrifugation, the ensuing pellets were resuspended in 100 μl of activity buffer (150 mm sodium citrate, pH 6.4) per cell plate and were defined as solubilized γ-secretase, as previously shown in Ref. 33Kopan R. Schroeter E.H. Weintraub H. Nye J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1683-1688Crossref PubMed Scopus (425) Google Scholar. Solubilized γ-secretase activity was induced at 37 °C for 2 h with or without the indicated treatments, and Aβ 40 generated de novo was quantified by ELISA. Control experiments used the internally quenched fluorogenic γ-secretase substrate NMA-GGVVIATVK(DNP)-DRDRDR-NH2 (λex = 355 nm; λem = 440 nm) from Calbiochem, which contains the C-terminal β-APP amino acid sequence that is cleaved by γ-secretase and the γ-secretase inhibitor DAPT (Calbiochem). Cytotoxicity Assays—Cell viability and cytotoxicity of the tested compounds were routinely assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or a released lactate dehydrogenase assay using the CytoTox 96 assay according to the manufacturer's instructions (Promega). Statistical Analysis—Mann-Whitney U test and Wilcoxon test were used to determine the significance between the data means. Significance values are as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus corresponding control. EHT 1864 Inhibits Rac1/Pak1 Interaction—To test whether EHT 1864 might affect Rac1 activity, U87-MG cells were treated with different concentrations of EHT 1864. We used a GST fusion protein containing the p21-binding domain of human p21-activated kinase 1 (Pak1) to affinity-precipitate endogenous active Rac1 (GTP-Rac1) from cell lysates in order to monitor the activation of the small GTPase Rac1. The GST-Pak-p21-binding domain fusion protein was incubated with cell lysate, and the effector pulled down active or GTP-Rac1 was detected by Western blot analysis using a specific Rac1 antibody. The Rhotekin protein specifically recognizes and binds to the active, GTP-bound, form of RhoA, R
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