Cắt đứt protein tiền chất amyloid của bệnh Alzheimer bởi protease aspartic xuyên màng BACE

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In situ hybridizations were performed using 10-μm sections through fresh frozen brain tissue obtained from adult Sprague-Dawley rats. Antisense riboprobes labeled with 35 S-UTP and 35 S-CTP were generated from rat BACE cDNA Bgl II–Kpn I fragment (nucleotides +815 to +1593). Hybridization and wash conditions were as described (36).

A synthetic peptide corresponding to the COOH-terminal 17 amino acids of BACE was synthesized conjugated to keyhole limpet hemocyanin (KLH) and used to raise a polyclonal antibody. The antibody and preimmune serum (negative control) were coupled to Protein A Sepharose for use in immunoprecipitation assays. Pieces of parietal cortex of human Alzheimer's disease and control brains obtained from Sun Health Research Institute (Arizona) were homogenized in lysis buffer (22) and the homogenate was spun at 20 000 g for 10 min at 4°C. For each immunoprecipitation about 25 mg wet weight equivalent were used. BACE immunoprecipitation from extracts of 293 cells transiently transfected with BACE were carried out as in (8) and the precipitates were subjected to SDS-PAGE followed by immunoblotting with the BACE antiserum as primary antibody.

For the generation of the BACE-HA cell line a BACE-HA fusion construct was made containing the nine–amino acid hemagglutinin epitope (37) fused in frame at the BACE COOH-terminus.

Immunocytochemistry of paraformaldehyde-fixed Triton X-100 permeabilized cells followed standard protocols (12). Primary antibodies and dilutions were the following: anti-HA monoclonal or polyclonal antibodies (Covance) 1:100; anti-58K (Sigma) 1:20; and anti-transferrin receptor (Dako) 1:20; anti-APPsβsw 1:500. Alexa 488 (green) and Alexa 594 (red) secondary antibodies (Molecular Probes) were used at 1:200 dilution.

Transfection metabolic labeling preparation of total cell lysates immunoprecipitation and protein radiosequencing were carried out as in (8). For immunoprecipitation of APP and its COOH-terminal fragments we used a polyclonal antibody that we raised to the last 20 amino acids of the cytoplasmic tail of APP. For immunopreciptation of Aβ we used the monoclonal antibody 4G8 raised to Aβ17-24 (Senetek). Quantitation of APP metabolites was done using time-resolved fluorescence sandwich ELISA assays with unmodified capture antibody and a biotinylated reporter antibody. Europium-labeled streptavidin was then reacted with the biotinylated antibody and the europium was quantitated by Delfia time-resolved fluorescence. For quantitation of total Aβ (Aβ 1−x ) we established an ELISA using monoclonal antibody 4G8 as capture and biotinylated monoclonal antibody 6E10 raised to Aβ1-17 (Senetek) as reporter. This assay detects all forms of Aβ with an intact NH 2 -terminus. For quantitation of all Aβ derivatives ending at amino acid 42 (Aβ x−42 ) we established an ELISA using anti-Aβ 42 a purified rabbit polyclonal antibody that specifically recognizes the 42 form of Aβ (Biosource International) as capture and biotinylated 4G8 as reporter. This assay detects all forms of Aβ and p3 that end at amino acid 42. A similar ELISA for detection of Aβ x−40 was established using anti-Aβ 40 a purified rabbit polyclonal antibody which specifically recognizes the 40 form of Aβ (Biosource International). For quantitation of APPsα we used a polyclonal antibody raised to the APP midregion (total APP) as capture and biotinylated 6E10 as reporter. For quantitation of APPsβsw we raised a polyclonal antibody specific for the β-secretase generated neoepitope of APPsw and used this antibody as capture followed by biotinylated monoclonal antibody 5A3 (total APP) (11) as reporter.

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Antisense oligonucleotides complementary to BACE mRNA were generated (Sequitur): AS#2 (5′-GUCCUGAACUCAUCGUGCACAUGGC-3′); AS#3 (5′-CAUCUGUGUCUCCUACUUGUGACCA-3′); and AS#4 (5′-CCAAGAGUAUUCCCGCCAGCAGAGU-3′). A reverse sequence control oligonucleotide for each antisense oligonucleotide was also synthesized. The following AS#2 mismatch control oligonucleotides were made: 2MIS (5′-GUCCUGAAGUCAUCGUCCACAUGGC-3′); 4MIS (5′-GUCCAGAAGUCAUCGUCCACUUGGC-3′); and 6MIS (5′-GGCCAGAAGUCAUCGUCCACUUGUC-3′). The APPsw cells were plated in 6-well dishes at 396 000 cells/well. After reaching ∼70 to 80% confluency cells were treated in triplicate with antisense or control oligonucleotide continuously for 48 hours in the presence of serum using a lipid delivery system (Sequitur). Cells were then washed and fresh medium was added for 6 to 8 hours of conditioning. Conditioned media were collected and assayed by APPsβsw APPsα Aβx-40 and Aβx-42 ELISAs. In addition cells from triplicate wells were harvested and pooled for isolation of polyadenylated [poly(A) + ] RNA (Micro-FastTrack; Invitrogen). Northern analysis was performed as described above. For normalization blots were stripped and reprobed with phospholipase A2 and glyceraldehyde-3 phosphate dehydrogenase cDNA probes (Clonetech; Palo Alto CA). Blots were visualized and quantitated using a Storm 860 phosphoImager (Molecular Dynamics).

For the soluble BACE-IgG cell line we generated a fusion construct encoding BACE amino acids 1–460 (ending at the start of the predicted transmembrane sequence) fused in frame with a three–amino acid linker (alanine-valine-threonine) and the Fc portion of human IgG1 (starting at amino acid 29; GenBank accession number ). BACE-IgG was purified from conditioned media of stably transfected 293T cells with Protein A columns. Substrate peptides were synthesized and labeled with dinitrophenol at P8' to allow easy ultraviolet detection of cleavage products after reversed phase high performance liquid chromatography. The APPwt substrate sequence was TTRPGSGLTNIKTEEISEV KM DAEFRHDK(dnp)G; in the Swedish mutant variant KM is substituted by NL and in the MV mutant M is substituted by V. All assays were performed with the same batch of BACE-IgG. Substrates were used at 30 μM in a 50-μl assay. All assays were buffered with 50 mM acetic acid (pH 4.5) unless otherwise indicated. Enzyme and substrate were incubated between 30 min and 18 hours at room temperature then the reaction mixtures were quenched and analyzed by reversed-phase HPLC. Both product and substrate were monitored by absorbance at 360 nm. Products were identified by retention time comparison with a reference peptide run under identical conditions.

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Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr. X indicates any residue.

We thank E. H. Koo for monoclonal antibody 5A3 T. Woolf for technical advice on antisense experiments G. Zajic for help with microscopy and N. Davidson T. Livelli and J. Ngai for helpful discussions. We also thank D. Paulin D. Olivares and G. Zajic for preparation of figures.