Antagonistic Effects of β-Site Amyloid Precursor Protein-cleaving Enzymes 1 and 2 on β-Amyloid Peptide Production in Cells
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m300169200
ISSN1083-351X
AutoresGuriqbal S. Basi, Normand L. Frigon, Robin Barbour, Tam Doan, Grace Gordon, Lisa McConlogue, Sukanto Sinha, Michelle Zeller,
Tópico(s)RNA Interference and Gene Delivery
ResumoThe deposition of extracellular β-amyloid peptide (Aβ) in the brain is a pathologic feature of Alzheimer's disease. The β-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the β-site, promotes Aβ production. A second integral membrane aspartyl protease related to BACE1, referred to as β-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the β-cleavage site in transfected cells. The role of endogenous BACE2 in Aβ production remains unresolved. We investigated the role of endogenous BACE2 in Aβ production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88–94% in cells. Selective inactivation of BACE1 by RNA interference results in decreased β-cleaved secreted APP and Aβ peptide secretion from cells, as expected. Selective inactivation of BACE2 by RNAi results in increased β-cleaved secreted APP and Aβ peptide secretion from cells. Simultaneous targeting of both enzymes by RNA interference does not have any net effect on Aβ released from cells. Our observations of changes in APP metabolism and Aβ are consistent with a role of BACE2 in suppressing Aβ production in cells that co-express both enzymes. The deposition of extracellular β-amyloid peptide (Aβ) in the brain is a pathologic feature of Alzheimer's disease. The β-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the β-site, promotes Aβ production. A second integral membrane aspartyl protease related to BACE1, referred to as β-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the β-cleavage site in transfected cells. The role of endogenous BACE2 in Aβ production remains unresolved. We investigated the role of endogenous BACE2 in Aβ production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88–94% in cells. Selective inactivation of BACE1 by RNA interference results in decreased β-cleaved secreted APP and Aβ peptide secretion from cells, as expected. Selective inactivation of BACE2 by RNAi results in increased β-cleaved secreted APP and Aβ peptide secretion from cells. Simultaneous targeting of both enzymes by RNA interference does not have any net effect on Aβ released from cells. Our observations of changes in APP metabolism and Aβ are consistent with a role of BACE2 in suppressing Aβ production in cells that co-express both enzymes. The production and deposition of insoluble Aβ 1The abbreviations used are: Aβ, β-amyloid peptide; APP, amyloid precursor protein; APPsw, Swedish mutant allele of APP; BACE, β-site APP cleaving enzyme; CTFs, carboxyl-terminal fragments; GFP, green fluorescent protein; RNAi, RNA interference; sAPP, secreted APP; sAPPβ, β-cleaved sAPP; sAPPα, α-cleaved sAPP; siRNA, small interfering RNA; wt, wild type; 293sw, HEK293 cells overexpressing APPsw; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; sAPPtot, total sAPP.1The abbreviations used are: Aβ, β-amyloid peptide; APP, amyloid precursor protein; APPsw, Swedish mutant allele of APP; BACE, β-site APP cleaving enzyme; CTFs, carboxyl-terminal fragments; GFP, green fluorescent protein; RNAi, RNA interference; sAPP, secreted APP; sAPPβ, β-cleaved sAPP; sAPPα, α-cleaved sAPP; siRNA, small interfering RNA; wt, wild type; 293sw, HEK293 cells overexpressing APPsw; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; sAPPtot, total sAPP. peptide in the brain results in the hallmark pathological feature of Alzheimer's disease (1Selkoe D.J. 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The decrease in Aβ production from BACE2 transfected cells has been attributed to the second cleavage site for BACE2 on APP (14Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar, 16Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (345) Google Scholar, 17Ehehalt R. Michel B. De Pietri Tonelli D. Zacchetti D. Simons K. Keller P. Biochem. Biophys. Res. Commun. 2002; 293: 30-37Crossref PubMed Scopus (53) Google Scholar, 18Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Crossref PubMed Scopus (97) Google Scholar, 19Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. 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RNAi is triggered by 21–23-nucleotide synthetic double-stranded RNAs termed small interfering RNA (siRNAs, we use “oligonucleotide” synonymously with siRNA in this report for convenience) in mammalian cells (23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8105) Google Scholar, 24Caplen N.J. Parrish S. Imani F. Fire A. Morgan R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9742-9747Crossref PubMed Scopus (924) Google Scholar, 25Paddison P.J. Caudy A.A. Hannon G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1443-1448Crossref PubMed Scopus (497) Google Scholar). We report here the selective knock-down of endogenous BACE1 or BACE2 message as well as protein by RNAi in HEK293 cell lines stably overexpressing APP695wt (wild type allele) or APPsw, the familial Alzheimer's disease allele of APP (the K595M,N596L Swedish double mutant). The selective knock-down of either enzyme leads to complementary alterations in APP metabolites and Aβ peptide secreted from cells. Our studies suggest that BACE2 inhibition elevates secretion of Aβ in cells co-expressing BACE1 and BACE2 and are of significance for amyloid-based therapeutics targeting these enzymes. Quantitative PCR—For RNA analysis, total RNA was prepared from cells at various time points post-transfection using Qiagen RNAeasy total RNA extraction kits. Total RNA was treated with DNase and quantitated by absorbance before quantitative PCR assay. Absence of contaminating DNA in the final RNA preparations was confirmed by a PCR assay with input RNA as template, omitting the reverse transcriptase. Quantitative RT-PCR was performed using an ABI 7700 Sequence Detector. All of the samples were assayed in triplicate. BACE1 primers, 5′-CCCGAAAACGAATTGGCTTT and 3′-GCTGCCGTCCTGAACTCATC, and probe, CTGTCAGCGCTTGCCATGTGCA; and BACE2 primers, 5′-CGTTTTCTCCATGCAGATGTGT and 3′-CCTCCGTTGGTCCCAGATC, and probe, AGCCGGCTTGCCCGTTGCT. Standard curves for BACE1 and 2 in HEK293 cells treated with siRNAs, as well as in primary brain cultures, were obtained using serial dilution of total RNA from untreated HEK293 cells. BACE message values per ng of input RNA were normalized to GAPDH message per ng of input RNA for each sample. BACE1 and 2 mRNA levels in cell lines were determined with standard curves using an in vitro synthesized RNA transcript as the standard. RNAi-mediated Knock-down of BACE1 and BACE2 in HEK293 Cells—The synthetic siRNAs to BACE1 used in this study are: B1 sense, CAGGAUCUGAAAAUGGACUGtt; B1 anti-sense, CAGUCCAUUUUCAGAUCCUGtt; B2 sense, UCUACGUUGUCUUUGAUCGGtt; B2 antisense, CCGAUCAAAGACAACGUAGAtt; B3 sense, CAGACGCUCAACAUCCUGGUtt; and B3 antisense, ACCAGGAUGUUGAGCGUCUGtt. The siRNAs to BACE2 used in this study are: C1 sense, GUGGGCAUGGGCGCACUGGtt; C1 antisense, CCAGUGCGCCCAUGCCCACtt; C3 sense, ACAGAGAGGUCUAGCACAUtt; C3 antisense, AUGUGCUAGACCUCUCUGUtt; C4 sense, UUGAAUCAGAGAAUUUCUUtt; and C4 antisense, AAGAAAUUCUCUGAUUCAAtt. These siRNA were synthesized and used essentially as described (23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8105) Google Scholar). HEK293 cells stably transfected with wild type APP695 (Amy5) (26Selkoe D.J. Podlisny M.B. Joachim C.L. Vickers E.A. Lee G. Fritz L.C. Oltersdorf T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7341-7345Crossref PubMed Scopus (539) Google Scholar) or APP695sw (293sw) were plated at a density of 200,000 cells/well in 24-well plates the morning of transfection and allowed to settle for 5 h prior to transfection. The Cells were transfected overnight with 200 ng of double-stranded RNA/well using Lipofectamine 2000® (Invitrogen) per the manufacturer's instructions. For quantitative analysis of APP metabolites and Aβ production in cells, all of the transfections were performed in triplicate, and the samples were assayed independently. For the experiment shown in Fig. 5, transfections with a combination of BACE1 and BACE2 oligonucleotides were conducted with 100 ng of each oligonucleotide, for a total of 200 ng of oligonucleotide/transfection (standard conditions) or 20 ng of total oligonucleotide (0.1× condition). No additional carrier nucleic acid was added for the 0.1× transfections. BACE1 and 2 protein knock-down was confirmed by co-transfecting siRNAs with 0.8-μg plasmid expression vectors encoding BACE1 or BACE2-Fc. Western Blot Analysis and Aβ Measurement in Transfected Cells— For protein knock-down studies, the cell lysates and conditioned media were collected at 48 h post-transfection. The cells were lysed in phosphate-buffered saline with 0.5% Nonidet P-40 supplemented with complete protease inhibitor mixture (Roche Applied Science) and analyzed by Western blot with the antibodies as noted below. Protein concentration in lysates was determined by BCA assay (Pierce). Fresh conditioned medium was collected from cells for 4 h at 2 days post-transfection by replacing transfection medium, and 10–20 μl/lane (normalized for protein concentration of the cognate lysate) were loaded onto gels for Western blot analysis. APP metabolites were detected with the antibodies previously described. Briefly, we used 8E5 for total sAPP (27Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Guido T. Hagopian S. Johnson-Wood K. Khan K. Lee M. Leibowitz P. Lieberburg I. Little S. Masliah E. McConlogue L. Montoya-Zavala M. Mucke L. Paganini L. Penniman E. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2227) Google Scholar), anti-6 for intracellular mature APP, as well as intracellular CTFs (28Oltersdorf T. Ward P.J. Henriksson T. Beattie E.C. Neve R.L. Lieberburg I. Fritz L.C. J. Biol. Chem. 1990; 265: 4492-4497Abstract Full Text PDF PubMed Google Scholar), and 192wt and 192sw for sAPPβ species produced from APPwt and APPsw, respectively (29Seubert P. Oltersdorf T. Lee M.G. Barbour R. Blomquist C. Davis D.L. Bryant K. Fritz L.C. Galasko D. Thal L.J. Lieberburg I. Schenk D.B. Nature. 1993; 361: 260-263Crossref PubMed Scopus (499) Google Scholar). The mouse monoclonal 7A6, preferentially recognizing the sAPPα neoepitope, was produced using peptide CEVHHQK (residues 607–612 from APP695/Aβ residues 11–16), coupled via the amino-terminal cysteine residue to sheep anti-mouse IgG as immunogen. Hybridomas from mice immunized with this peptide were screened for reactivity to peptide sequence CGGYEVHHQK (Aβ residues 10–16). Hybridoma clone 7A6 showed stronger reactivity toward the Aβ10–16 peptide than to a peptide spanning the α-secretase cleavage site (Aβ residues 1–28). A quantitation of the difference in reactivity of 7A6 toward the two peptides, presented in the supplemental figure, shows that 7A6 reactivity toward sAPP in conditioned medium is competed 100% by the immunizing peptide at 100 μg/ml but only partially by the overlapping peptide at 100 μg/ml. Competition by 10 μg/ml Aβ 11–16 is approximately equal to the level of competition seen with 100 μg/ml Aβ 1–28. The result presented in the supplemental figure indicates that 7A6 binds with ∼10 times greater preference to the sAPPα neo-epitope (ending at Aβ residue 16) than it does to a peptide which spans this site. Hence, although 7A6 is not expected to detect the sAPP species from BACE2 cleavage at Aβ residues 19 and 20 by virtue of the immunogen against which it was raised, it can potentially detect sAPP species extending beyond Aβ residue 16 because of a 10-fold weaker reactivity for residues between Aβ11 and Aβ16. Further characterization of monoclonal 7A6 included the ability to capture iodinated Aβ10–16 peptide. Total Aβ was quantitated by sandwich ELISA using monoclonal antibody 266-coated microtiter plates followed by biotinylated 3D6 as secondary reporter (30Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Lieberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. Usa. 1997; 94: 1550-1555Crossref PubMed Scopus (582) Google Scholar). This assay measures all Aβ species starting at position 1 and extending beyond position 28 (i.e. Aβ 1–x). Statistical analysis of Aβ values was performed using StatView software package (SAS, Cary, NC). All of the samples were analyzed by analysis of variance, followed by post-hoc analysis using Fischer's protected least significant difference test to determine p values, comparing the means of the normalized Aβ values of each treatment group to the mean of the normalized Aβ value from the no treatment control group and also to the mean of the normalized Aβ value from the lamin and GFP control groups. BACE1 protein expression was detected by rabbit polyclonal antisera 264 (immunized with a peptide corresponding to BACE1 residues 48–66 of the nascent polypeptide). BACE2 protein was detected as a chimeric Fc construct (residues 1–465 of BACE2 extracellular domain fused to H-CH2-CH3 domains of human Cγ1) followed by an horseradish peroxidase-conjugated goat anti-human IgG (Jackson Immunoresearch) to detect and quantify suppression of transfected BACE2. Parallel blots from equivalently loaded gels were probed with antibody to β-tubulin (Sigma) to confirm specificity of siRNAs for the targeted protein, as well as equal sample loading. BACE1 and BACE2 mRNA Are Co-expressed in Cells—A quantitative RT-PCR survey of different cell types reveals the co-expression of mRNA for BACE1 and BACE2 enzymes in a variety of cell lines (Fig. 1A). Of the brain cell types surveyed, we observed co-expression of the mRNA for both enzymes in astrocytes, whereas BACE1 was exclusively expressed in neurons and microglia (Fig. 1B). Oligodendrocyte precursor cells (purified from rat optic nerve) do not express detectable levels of either message (not shown). Although a discrepancy has been noted with regard to message and activity distribution of BACE1 (5Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1473) Google Scholar, 6Vassar 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 (3268) Google Scholar), we note that mRNA degradation is directly linked with protein expression, as well as with APP metabolite production, in our experiments using RNAi (see below) in HEK293 cells. siRNAs to BACE1 and BACE2 Reduce Expression of the Cognate Protein and mRNA in a Sequence-specific Manner—A detailed comparison of residue specificity between 8 substrate subsites spanning P4–P4′ revealed a very high degree of similarity between BACE1 and 2 (31Turner 3rd, R.T. Loy J.A. Nguyen C. Devasamudram T. Ghosh A.K. Koelsch G. Tang J. Biochemistry. 2002; 41: 8742-8746Crossref PubMed Scopus (56) Google Scholar, 32Turner III, R.T. Koelsch G. Hong L. Castanheira P. Ermolieff J. Ghosh A.K. Tang J. Castenheira P. Ghosh A. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (196) Google Scholar), underscoring the challenge in obtaining selective small molecule inhibitors. Hence, we addressed the role of BACE1 and BACE2 in Aβ production in HEK293 cells (a cell type that expresses message for both enzymes, labeled as A293 in Fig. 1A) by selective degradation of mRNA for each enzyme using RNAi. Synthetic siRNAs to BACE1 and BACE2 (“Experimental Procedures”) were tested for activity in an overexpression paradigm by co-transfecting HEK293 cells with the double-stranded RNAs along with BACE1 or BACE2-Fc expression vectors. Expression of BACE1 and BACE2-Fc in lysates from transfected cells was assessed by Western blot analysis. The results, shown in Fig. 2, indicate that BACE1 expression is reduced specifically by oligonucleotide B3, and BACE2-Fc expression is reduced specifically by oligonucleotide C3. Expression of BACE1 is not affected by an irrelevant lamin A/C oligonucleotide (23Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8105) Google Scholar) nor any of the BACE2 oligonucleotides tested. Likewise, BACE2 expression is not affected by any of the BACE1 oligonucleotides tested nor by irrelevant oligonucleotides targeting lamin A/C or GFP. We further characterized the specificity as well as the kinetics of the active siRNAs B3 (anti-BACE1) and C3 (anti-BACE2) for degradation of the cognate mRNA in 293sw cells. Total RNA was isolated from 293sw cells (expressing endogenous levels of BACE1 and BACE2) at varying time points following transfection with oligonucleotides B3, C3, or B3+C3, and mRNA levels were measured by quantitative RT-PCR. Lamin siRNA was used as a specificity control. The results from this experiment, shown in Fig. 3, confirm the specificity and reveal the kinetics of message degradation mediated by the siRNAs. The lamin A/C oligonucleotide does not affect BACE1 message over the 72-h time course surveyed (Fig. 3A). BACE2 message is likewise unaffected by the lamin oligonucleotide up to 48 h (Fig. 3B). Although BACE2 mRNA is reduced 50% at 72 h post-transfection by the lamin oligonucleotide, we note that our experiments assayed the effect of RNAi against BACE2 at 48 h post-transfection. BACE1 message is not affected by the active C3 oligonucleotide targeting BACE2 (Fig. 3A), nor is BACE2 message affected by the active BACE1 oligonucleotide B3 (Fig. 3D). Degradation of both transcripts by the respective oligonucleotides is evident within 6 h post-transfection (Fig. 3, B and C). BACE2 mRNA is suppressed 90% by 12 h post-transfection and remains suppressed for at least 72 h (3B, 3D). BACE1 message suppression appears less robust (∼60% suppression at 12 h) and more transient in comparison (return to base line by 72 h; Fig. 3C). In cells transfected simultaneously with both oligonucleotides, suppression of BACE1 mRNA at time points earlier than 48 h is attenuated when compared with BACE2 mRNA suppression (Fig. 3, C and D). Furthermore, the overall potency of the RNAi response to each target appeared to be attenuated when cells were treated with both oligonucleotides (see legend to Fig. 3). Extracts from cells transiently transfected with B3 siRNA plus BACE1 expression plasmid or C3 siRNA plus BACE2-Fc expression plasmid were analyzed by quantitative Western blots to determine the magnitude of protein suppression in an overexpression paradigm. Our results, shown in Fig. 4, indicate that BACE1 expression is reduced at least 8–16-fold (Fig. 4, A and B), whereas BACE2 is suppressed at least 16-fold (Fig. 4C). In summary, RNAi mediated suppression of BACE1 by oligonucleotide B3 and that of BACE2 by oligonucleotide C3 are highly sequence-specific. In addition, mRNA degradation correlates with a suppression of the cognate overexpressed protein. BACE1 and BACE2 Knock-down Alters APP Metabolites Released from Cells—The consequence of knocking down endogenous BACE2 or BACE1 on APP metabolism and Aβ production was studied in stably transfected cells overexpressing APPsw (293sw cells; Fig. 5) or APP695wt (Amy5 cells; Fig. 6). The high level of APP expression in these cells facilitates detection of metabolites produced by the two enzym
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