NFκB-dependent Control of BACE1 Promoter Transactivation by Aβ42

Abstract

β-Amyloid (Aβ) peptides that accumulate in Alzheimer disease are generated from the β-amyloid precursor protein (βAPP) by cleavages by β-secretase BACE1 and by presenilin-dependent γ-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific γ-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive γ-secretase. The overexpression of APP intracellular domain (AICD), the γ/ϵ-secretase-derived C-terminal product of β-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPϵ (the N-terminal product of βAPP generated by ϵ-secretase cleavage harboring the Aβ domain but lacking the AICD sequence), suggesting that the production of Aβ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type βAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Aβ-potentiating Swedish mutation. This effect was mimicked by exogenous application of Aβ42 but not Aβ40 or by transient transfection of cDNA encoding Aβ42 sequence. The IκB kinase inhibitor BMS345541 prevents Aβ-induced BACE1 promoter transactivation suggesting that NFκB could mediate this Aβ-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated βAPP does not modify the transactivation of BACE1 promoter constructs lacking NFκB-responsive element. Furthermore, APP/β-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Aβ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Aβ production linked to wild-type or Swedish mutated βAPP overexpression modulates BACE1 promoter transactivation and activity via an NFκB-dependent pathway. β-Amyloid (Aβ) peptides that accumulate in Alzheimer disease are generated from the β-amyloid precursor protein (βAPP) by cleavages by β-secretase BACE1 and by presenilin-dependent γ-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific γ-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive γ-secretase. The overexpression of APP intracellular domain (AICD), the γ/ϵ-secretase-derived C-terminal product of β-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPϵ (the N-terminal product of βAPP generated by ϵ-secretase cleavage harboring the Aβ domain but lacking the AICD sequence), suggesting that the production of Aβ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type βAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Aβ-potentiating Swedish mutation. This effect was mimicked by exogenous application of Aβ42 but not Aβ40 or by transient transfection of cDNA encoding Aβ42 sequence. The IκB kinase inhibitor BMS345541 prevents Aβ-induced BACE1 promoter transactivation suggesting that NFκB could mediate this Aβ-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated βAPP does not modify the transactivation of BACE1 promoter constructs lacking NFκB-responsive element. Furthermore, APP/β-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Aβ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Aβ production linked to wild-type or Swedish mutated βAPP overexpression modulates BACE1 promoter transactivation and activity via an NFκB-dependent pathway. Alzheimer disease (AD) 3The abbreviations used are:ADAlzheimer diseasePSpresenilinsβAPPβ-amyloid precursor proteinAββ-amyloid peptideAICDAPP intracellular domainAPLP2β-amyloid precursor protein-like protein 2CREBcAMP-response element-binding proteinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineNEPneprilysin. is characterized by abnormal deposition of a set of hydrophobic peptides called amyloid β (Aβ) peptides. The increase of cerebral Aβ levels is one of the common denominators characterizing both sporadic and familial forms of AD and therefore, if not demonstrated as the etiological cause of AD pathology, is often considered as a key factor contributing to the degenerative process (1Hardy J.A. Higgins G.A. Science. 1992; 256: 184-185Crossref PubMed Scopus (5110) Google Scholar). 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PS belong to the high molecular weight γ-secretase complex, which also includes Aph-1, Pen-2, and nicastrin (21De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 22Haass C. EMBO J. 2004; 23: 483-488Crossref PubMed Scopus (486) Google Scholar). Besides their contribution to the intramembranous cleavage of βAPP, PS participate in the proteolytic activation/degradation of a series of other substrates involved in many vital functions (23Thinakaran G. Parent A.T. Pharmacol. Res. 2004; 50: 411-418Crossref PubMed Scopus (60) Google Scholar), thereby explaining the apparent remarkable pleiotropy of these proteins (24Checler F. IUBMB Life. 1999; 48: 33-39Crossref PubMed Google Scholar). Among other functions, PS contribute to cellular adhesion, cell death control, and cell signaling (23Thinakaran G. Parent A.T. Pharmacol. Res. 2004; 50: 411-418Crossref PubMed Scopus (60) Google Scholar, 25Popescu B.O. Ankarcrona M. J. 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Here we show that PS control the promoter transactivation of BACE1, and we establish that this PS-dependent effect is related to its associated γ-secretase activity. Furthermore, we demonstrate that BACE1 activity and promoter transactivation were directly linked to exacerbated production of Aβ and unrelated to AICD generation. Finally, we establish that Aβ-mediated transactivation of BACE1 promoter involves the NFκB pathway. Cell Cultures and Transfections—Fibroblasts devoid of PS1 and PS2 and βAPP/APLP2 double knock-out fibroblasts have been described previously (33Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 34Heber S. Herms J. Gajic V. Hainfellner J.A. Aguzzi A. Rülicke T. Kretzschmar H. von Koch C. Sisodia S.S. Tremml P. Lipp H.-P. Wolfer D.P. Müller U. J. Neurosci. 2000; 20: 7951-7963Crossref PubMed Google Scholar). HEK293 overexpressing wild-type βAPP, Swedish mutated βAPP, APPϵ, wild-type PS1, and mutated AA-PS1 were obtained and cultured as described previously (35Chevallier 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, 36Lefranc-Jullien S. Sunyach C. Checler F. J. Neurochem. 2006; 97: 807-817Crossref PubMed Scopus (21) Google Scholar). Transient transfections of 2 μg of total cDNA were carried out with Lipofectamine 2000 reagent (Invitrogen) according to previously reported procedures (37Alves da Costa C. Sunyach C. Pardossi-Piquard R. Sevalle J. Vincent B. Boyer N. Kawarai T. Girardot N. St George Hyslop P. Checler F. J. Neurosci. 2006; 26: 6377-6385Crossref PubMed Scopus (154) Google Scholar). Transgenic Mouse Brain Tissue Preparations—Brains from Fe65 or Fe65/AICD transgenic mice (38Ryan K.A. Pimplikar S.W. J. Cell Biol. 2005; 171: 327-335Crossref PubMed Scopus (156) Google Scholar) were homogenized with 10 mm Tris-HCl, pH 7.5, and lysed with a Dounce homogenizer (50 strokes). Samples were immediately assayed for β-secretase activity and analyzed for their endogenous BACE1 expression as described below (39Lefranc-Jullien S. Lisowski V. Hernandez J.F. Martinez J. Checler F. Br. J. Pharmacol. 2005; 145: 228-235Crossref PubMed Scopus (29) Google Scholar). Western Blot Analysis and Antibodies—BACE1, βAPP, APLP2, Tip60, ADAM10, and Fe65 were separated on 8% Tris/glycine gel acrylamide; PS were analyzed on 12% Tris/glycine gels, and AICDs and Aβ were separated on 16.5% Tris/Tricine gels as described previously (40Alves da Costa C. Paitel E. Mattson M.P. Amson R. Telerman A. Ancolio K. Checler F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4043-4048Crossref PubMed Scopus (114) Google Scholar). Proteins were transferred onto Hybond-C nitrocellulose membranes (Amersham Biosciences) and then probed overnight with the following appropriate antibodies: anti-BACE1 (Zymed Laboratories Inc.); anti-N terminus of PS1 (kindly provided by Dr. G. Thinakaran); 9E10 anti-Myc antibody to label AICDs (Aventis); anti-Fe65, anti-hemagglutinin (Tip60), 6E10 anti-Aβ, BR188 antibody that recognizes the C terminus of APP (provided by Dr. M. Goedert, Cambridge, UK); WO2 antibody (The Genetics Co., Zurich, Switzerland) recognizes 5-8 sequence of Aβ; 2H3 antibody (provided by Dr. D. Schenk, San Francisco) raised against 1-12 sequence of Aβ, 22C11 antibody (Roche Applied Science); anti-actin and anti-tubulin (Sigma); and anti-ADAM10 antibodies (Euromedex, Souffelmeyersheim, France). Immunological complexes were revealed with appropriate secondary antibody and detected using an electrochemiluminescence method with the Lumi-light Western blotting substrate (Roche Applied Science) as described previously (41Pardossi-Piquard R. Petit A. Kawarai T. Sunyach C. Alves da Costa C. Vincent B. Ring S. D'Adamio L. Shen J. Müller U. St George-Hyslop P. Checler F. Neuron. 2005; 46: 541-554Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). BACE1 and ADAM10 Fluorimetric Assay—Cells were lysed with 10 mm Tris-HCl, pH 7.5, and then homogenates were monitored for their BACE1 activity as described previously (39Lefranc-Jullien S. Lisowski V. Hernandez J.F. Martinez J. Checler F. Br. J. Pharmacol. 2005; 145: 228-235Crossref PubMed Scopus (29) Google Scholar, 42Andrau 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). Briefly, samples (30 μg of proteins diluted in 10 mm acetate buffer, pH 4.5) were incubated in a final volume of 100 μl of the above acetate buffer containing BACE1 substrate (10 μm, (7-methoxycoumarin-4-yl)acetyl-SEVNLDAEFRK(2,4-dinitrophenyl)-RRNH2; R & D Systems) in the absence or presence of the previously described BACE1 inhibitor JMV2764 (50 μm) (39Lefranc-Jullien S. Lisowski V. Hernandez J.F. Martinez J. Checler F. Br. J. Pharmacol. 2005; 145: 228-235Crossref PubMed Scopus (29) Google Scholar). BACE1 activity corresponds to the JMV2764-sensitive fluorescence recorded at 320 and 420 nm as excitation and emission wavelengths, respectively as described (39Lefranc-Jullien S. Lisowski V. Hernandez J.F. Martinez J. Checler F. Br. J. Pharmacol. 2005; 145: 228-235Crossref PubMed Scopus (29) Google Scholar). ADAM10 activity was monitored as described previously (43Alfa Cissé M. Gandreuil C. Hernandez J. Martinez J. Checler F. Vincent B. Biochem. Biophys. Res. Commun. 2006; 347: 254-260Crossref PubMed Scopus (22) Google Scholar). Transactivation of BACE1 Promoter—Empty vector or cDNA encoding rat BACE1 promoter or deletion constructs in-frame with luciferase (44Lange-Dohna C. Zeitschel U. Gaunitz F. Perez-Polo J.R. Bigl V. Rossner S. J. Neurosci. Res. 2003; 73: 73-80Crossref PubMed Scopus (50) Google Scholar) were co-transfected with β-galactosidase (to normalize transfection efficiencies). HEK293 cells and fibroblasts devoid of presenilins were cultured in 12-well dishes or 60-mm-diameter dishes and transfected with 2 or 8 μg of the total amount of cDNA, respectively. For Aβ transfection experiments, cells were transfected with a total amount of 3 μg of cDNA encoding rat BACE1 promoter, β-galactosidase, and either empty vector or constructs harboring Aβ-(1-42), Aβ-(42-1), or Aβ-(1-40) (45Ohyagi Y. Asahara H. Chui D.H. Tsuruta Y. Sakae N. Miyoshi K. Yamada T. Kikuchi H. Taniwaki T. Murai H. Ikezoe K. Furuya H. Kawarabayashi T. Shoji M. Checler F. Iwaki T. Makifuchi T. Takeda K. Kira J-I. Tabira T. FASEB J. 2005; 19: 255-257Crossref PubMed Scopus (240) Google Scholar). Thirty hours after transfection, cells were harvested with phosphate-buffered saline/EDTA (5 mm), pelleted by centrifugation (1000 × g, 5 min), lysed with 50 μl of lysis buffer (luciferase kit Promega), centrifuged for 5 min at 2000 rpm, and then luciferase activity was measured with 10 μl of supernatant and 50 μl of luciferase assay (Promega). Effect of γ-Secretase Inhibitor Treatment on BACE1 Promoter Transactivation and Activity—Cells were treated for 16 h with various concentrations of the γ-secretase inhibitor DFK167 (46Wolfe M.S. Citron M. Diehl T.S. Xia W. Donkor I.O. Selkoe D.J. J. Med. Chem. 1998; 41: 6-9Crossref PubMed Scopus (218) Google Scholar) or corresponding amounts of Me2SO (all DFK167 stocks were made at 10 mm in 100% Me2SO). Cells were harvested with phosphate-buffered saline/EDTA (5 mm), pelleted by centrifugation (1000 × g, 5 min), and lysed with Tris-HCl (10 mm, pH 7.5) or with 50 μl of lysis buffer (luciferase kit Promega) to measure BACE1 activity and rat BACE1 promoter activity, respectively. Effect of Exogenous Αβ on BACE1 Promoter Transactivation—Twenty four hours after transfection of rat BACE1 promoter, medium was replaced with Opti-MEM medium (Sigma) containing fetal bovine serum (2%) and complemented with phosphoramidon (10 μm) to prevent Aβ degradation. The cells were then treated for 48 h with various concentrations of synthetic Aβ42 or Aβ40 (Bachem). Effect of Treatment with the IκB Kinase Inhibitor BMS345541—HEK293 overexpressing wild-type βAPP, Swedish mutated βAPP, or Aβ-treated cells were incubated for 18 h with the IκB kinase inhibitor BMS345541 ((4(2′-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline), 30 μm) or a corresponding amount of Me2SO in Opti-MEM medium containing 2% of fetal bovine serum. Analysis of Aβ40 Production—Cells were allowed to secrete Aβ in Opti-MEM medium (Sigma) for 8 or 16 h in the presence of phosphoramidon (10 μm) (to prevent the degradation of secreted Aβ), in the absence or in the presence of DFK167 (50 μm), collected, supplemented with RIPA buffer (10 mm Tris-HCl, pH 8, EDTA 5 mm, NaCl 150 mm), and then incubated overnight with a 100-fold dilution of FCA18 or FCA3340 (47Barelli H. Lebeau A. Vizzavona J. Delaere P. Chevallier N. Drouot C. Marambaud P. Ancolio K. Buxbaum J.D. Khorkova O. Heroux J. Sahasrabudhe S. Martinez J. Warter J-M. Mohr M. Checler F. Mol. Med. 1997; 3: 695-707Crossref PubMed Google Scholar). Aβ40 was immunoprecipitated with protein A-Sepharose, analyzed onto a 16.5% Tris/Tricine gel, Western blotted, and revealed with 6E10 as described previously (36Lefranc-Jullien S. Sunyach C. Checler F. J. Neurochem. 2006; 97: 807-817Crossref PubMed Scopus (21) Google Scholar). Stastistical Analysis—Statistical analysis was performed with Prism software (Graphpad, San Diego) using the Student-Newman-Keul's multiple comparison test for one-way analysis of variance or unpaired t test for pairwise comparison. Presenilin 1 and Presenilin 2 Deficiency Affects BACE1 Maturation, Activity, and Promoter Transactivation—We examined the influence of presenilins on the expression of BACE1. In wild-type fibroblasts, pro-BACE1 and mature BACE1 were detected at their previously reported molecular weights (Fig. 1A) (48Vassar R. J. Mol. Neurosci. 2001; 17: 157-170Crossref PubMed Scopus (170) Google Scholar). Interestingly, the depletion of both PS1 and PS2 lowers the level of mature BACE1 (44.4 ± 10.1% compared with control, n = 6, p < 0.01) without affecting pro-BACE1 (Fig. 1, B and C). In PS-deficient fibroblasts, an additional band harboring BACE-1-like immunoreactivity was detected above the mature form of the enzyme (Fig. 1A). The identity of this protein is still unknown, but we can rule out the possibility that this protein derives from abnormal prohormone convertase processing that would have occurred in the absence of PS because this protein remains totally insensitive to the convertase inhibitor α1-antitrypsin Portland variant (49Anderson E.D. Thomas L. Hayflick J.S. Thomas G. J. Biol. Chem. 1993; 268: 24887-24891Abstract Full Text PDF PubMed Google Scholar, 50Benjannet S. Savaria D. Laslop A. Munzer J.S. Chrétien M. Marcinkiewicz M. Seidah N. J. Biol. Chem. 1997; 272: 26210-26218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar and data not shown). We examined whether PS deficiency-associated reduction of mature BACE1 expression was associated with a decrease of BACE1 enzymatic activity. Thus, the depletion of endogenous PS triggers a significant reduction of BACE1 activity (27.4 ± 4.8% compared with control, n = 6, p < 0.0001; see Fig. 1D). The similar decrease in both mature BACE1 expression and activity suggests that the above-described unidentified protein apparently does not harbor catalytic properties. Of most interest, we establish that fibroblasts devoid of PS display drastically lower BACE1 promoter transactivation (77.4 ± 7.4% compared with control, n = 9, p < 0.0001; see Fig. 1E). Overall, the above data suggested that PS modulate BACE1 activity by apparently controlling this protease at both transcriptional and post-transcriptional levels. Presenilin-dependent γ-Secretase Activity Is Involved in the Control of BACE1—We examined whether the modulation of BACE1 activity could be linked to the enzymatic activity displayed by the PS-dependent γ-secretase complex. Two distinct sets of experiments suggest that it is indeed the case. First, we assessed the influence of a double mutation thought to yield a catalytically inactive PS1 (D257A/D385A-PS1, AA-PS1 (51Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar)). Unlike wild-type PS1, which slightly increases BACE1 activity (Fig. 2B), AA-PS1 overexpression (Fig. 2A) reduces BACE1 activity (53.4 ± 8.2% compared with WT-PS1, n = 3, p < 0.01; Fig. 2B). Second, we assessed the influence of DFK167, a γ-secretase inhibitor that physically interacts with PS (52Esler W.P. Kimberly W.T. Ostaszewski B. Diehl T.S. Moore C.L. Tsai J-Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (506) Google Scholar), on BACE1 activity. As a matter of controls, we first show that DFK167 does not directly interfere with BACE1 activity (Fig. 3A) but indeed abolishes Aβ production in HEK293 cells expressing Swedish mutated βAPP (Fig. 3B). Clearly, DFK167 reduces cellular BACE1 activity in wild-type fibroblasts (40.9 ± 3.6% for 10 μm DFK167 and 46.9 ± 4% for 50 μm DFK167 compared with control untreated cells, n = 16, p < 0.001; Fig. 3C) as well as BACE1 expression (Fig. 3C). However, the γ-secretase inhibitor neither alters BACE1 expression nor activity in PS-deficient cells (Fig. 3D). Altogether, both mutational and pharmacological data indicate that PS-induced modulation of BACE1 activity was apparently linked to PS-associated γ-secretase activity.FIGURE 3Effect of the γ-secretase inhibitor DFK167 on BACE1 expression and activity in wild-type and PS-/- fibroblasts. A and B, DFK167 inhibits Aβ production but does not directly affect BACE1 activity in vitro. A, BACE1 activity was fluorimetrically monitored in PS+/+ homogenates in the absence (Ct) or in the presence of DFK167 (50 μm). B, HEK293 overexpressing Swedish mutated βAPP were incubated for 16 h with phosphoramidon (10 μm), in the absence (Ct) or in the presence of either DFK167 (50 μm) or corresponding amount of Me2SO (DMSO). Secreted Aβ was monitored after immunoprecipitation with FCA18 antibody and analyzed after 16.5% Tris/Tricine electrophoresis and Western blot with 6E10 antibody as described under “Materials and Methods.” PS+/+ (C) and PS-/- (D) fibroblasts were treated for 16 h with the indicated concentration of DFK167 or adequate amount of Me2SO (DMSO), and then endogenous BACE1 expression and activity were measured as described under “Materials and Methods.” Bars correspond to BACE1 activity expressed as percent of that observed in untreated cells (Me2SO, taken as 100) and are the means ± S.E. of 16 independent determinations. *, p < 0.001; ns, not statistically significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) AICDs Do Not Modulate BACE1 Activity, in Vitro and in Vivo—Because the modulation of BACE1 was apparently linked to PS-dependent catalytic events, we postulated that the control of BACE1 should involve catabolites generated by PS-dependent γ-secretase-associated processes and harboring transcription factor properties. In this context, we examined whether the C-terminal products of βAPP hydrolysis could contribute to the modulation of BACE1 activity. Thus, these fragments called AICD (APP intracellular domain) have been shown to be derived from DFK167-sensitive γ- and ϵ-secretase proteolytic cleavages (53Passer B. Pellegrini L. Russo C. Siegel R.M. Lenardio M.J. Schettini G. Bachmann M. Tabaton M. D'Adamio L. J. Alzheimer Dis. 2000; 2: 289-301Crossref PubMed Scopus (202) Google Scholar, 54Kume H. Maruyama K. Kametani F. Int. J. Mol. Med.