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ELISA Analysis of Β-Secretase Cleavage of the Swedish Amyloid

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ELISA Analysis of Β-Secretase Cleavage of the Swedish Amyloid Journal of Neurochemistry, 2002, 80, 1019–1028 ELISA analysis of b-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways Michelle L. Steinhilb,* R. Scott Turner and James R. Gautà *Institute of Gerontology and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA Department of Neurology, University of Michigan Medical Center, Ann Arbor, Michigan, USA àVeterans Affairs Medical Center Geriatric Research, Education, and Clinical Center, Ann Arbor, Michigan, USA Abstract endocytic pathways. Using a dilysine retrieval motif to retain Limiting beta amyloid (Ab) production could become an APPsw in the ER, we discovered that less than 15% of the important therapeutic target in Alzheimer’s disease (AD). Ab is b-secretase cleavage was still detected. Experiments utilizing derived by the sequential cleavage of amyloid precursor pro- endocytosis-impaired mutants of APPsw revealed that little or tein (APP) by b- and c-secretases. A double missense no b-secretase cleavage of APPsw appears to take place mutation in APP found in a Swedish pedigree (APPsw) ele- through endocytosis. Surprisingly, deletion of the entire cyto- vates Ab40 and Ab42 production. Ab production and, there- plasmic tail increased both APPswb and Ab secretion, sug- fore, b-secretase cleavage of APPsw reportedly occur in the gesting that protein interactions with this region normally endoplasmic reticulum (ER), Golgi and endocytic compart- impede b-secretase cleavage. These results suggest that ments. However, the relative contribution of b-secretase APPsw is cleaved by b-secretase late in the secretory path- cleavage occurring in each compartment has not been way. determined. Experiments described here use a novel ELISA to Keywords: Alzheimer’s disease, BACE, b-secretase, endo- measure the b-cleaved product, APPswb. Using this ELISA, plasmic reticulum. we provide new information regarding the relative amount of J. Neurochem. (2002) 80, 1019–1028. b-secretase cleavage of APPsw that occurs in secretory and Amyloid precursor protein (APP) is processed into Ab40 and treatment is also capable of blocking alternate secondary Ab42, by b- and c-secretases, which, respectively, cleave pathways that may become major sites of Ab generation in within the extracellular and the transmembrane regions of neurons if only the primary site is inhibited. Such mech- this type I integral membrane protein (for review see Selkoe anisms include determining the cellular location(s) of b- and 1998). Alzheimer’s disease (AD) is hypothesized to result from the aggregation and deposition of these peptides into amyloid plaques in brain. A double missense mutation in Received 5 September, 2001; revised manuscript received 7 December, APP identified in a Swedish pedigree with early onset 2001; accepted 10 December, 2001. familial Alzheimer’s disease (FAD) (APPsw; K651N/ Address correspondence and reprint requests to James R. Gaut, Pre- M652L) enhances cleavage by b-secretase. This, in turn, ventive Medicine, St Jude Children’s Research Hospital, 332 N. Lau- increases both Ab40 and Ab42 secretion (Citron et al. 1994; derdale, Memphis, TN 38105, USA. E-mail: [email protected] Haass et al. 1995). Abbreviations used:Ab peptide, a 40 or 42 amino acid peptide derived from APP; p3, peptide derived from a- and c-secretase cleavage of APP; To develop strategies for therapeutic treatment of AD, a CTF, carboxyl terminal fragment; APP, amyloid precursor protein; thorough understanding of all the molecular mechanisms APPsw, APP bearing the Swedish mutation (KM/NL); BACE, beta-site capable of generating amyloid peptides is required. APP APP cleaving enzyme; APPswb, soluble b-secretase cleaved APPsw bearing the Swedish mutation is an important biochemical fragment; APPswa, soluble a-secretase cleaved APPsw fragment; APPa, tool since it has been established that expression of APPsw soluble a-secretase cleaved APP fragment; APPb, soluble b-secretase cleaved APP fragment; CHO, Chinese hamster ovary cells; HEK293, in non-neuronal cells more closely mimics processing of human embryonic kidney cells; SDS–PAGE, sodium dodecyl sulfate APPwt by neurons (Forman et al. 1997). A complete polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; TMB, understanding of amyloid generation will ensure that a 3,3¢, 5,5¢-tetramethylbenzidine. Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 1019–1028 1019 1020 M. L. Steinhilb et al. c-secretase cleavage of APPsw. However, determining the that deletion of the cytoplasmic tail of APPsw significantly cellular site(s) of Ab peptide derivation is more complex than increased cleavage by b-secretase. This suggests that originally thought. Production of Ab in Chinese hamster cytoplasmic proteins that interact with the carboxyl terminal ovary (CHO) cells involves internalization of APP from the tail of APPsw affect its vulnerability to cleavage by cell surface (Koo and Squazzo 1994). Furthermore, Ab b-secretase. secretion is severely decreased in non-neuronal cells expres- sing APP constructs possessing cytoplasmic tail mutations that impair endocytosis (Perez et al. 1999). APPsw process- Materials and methods ing is different from APP in that blocking endocytosis of the Swedish mutant by deleting the cytoplasmic internalization Cell lines, expression systems, and antibodies signal still permits production of Ab peptide (Citron et al. Human endothelial kidney 293 (HEK293), Chinese hamster ovary 1995). This suggests that amyloid peptides could be K1 (CHOK1), and mouse neuroblastoma (N2a) cells (American Type Culture Collection, Rockville, MD, USA) were used for generated from APPsw within the secretory pathway. How- transient transfections. All cell lines were grown in Dulbecco’s ever, other experiments revealed that Ab peptide could also modified Eagle medium (DMEM) supplemented with 10% fetal calf be derived through processing of APPsw endocytosed from serum, glutamine, non-essential amino acids (Life Technologies, the cell surface in addition to the secretory pathway (Perez Inc., Rockville, D, USA) and penicillin/streptomycin/fungizone et al. 1996). Prior to the studies presented here, it was (BioWhittaker, Inc., Walkersville, MD, USA) as described elsewhere unclear how much the secretory and endocytic pathways (Yang et al. 1998). For transfection experiments, cells were seeded contribute to Ab peptide production from APPsw. 1 day prior to use at 1 · 106 cells/6-cm dish and transfected with The specific locations of intracellular amyloid peptide LipofectAMINE (Life Technologies, Inc.) reagent as described by generation within the secretory pathway may be distinctly the manufacturer. APPsw containing a carboxyl-terminal double different for Ab40 and Ab42. Treating HEK 293 cells with lysine motif (KK) was engineered with two single-base changes brefeldin A (BFA) to accumulate APP within the endoplas- using PCR site-directed mutagenesis resulting in a QM to KK modification at amino acids 747 and 748, respectively (Q747K/ mic reticulum (ER) results in increased generation of M748K; APP-751 isoform numbering) similar to Chyung et al. intracellular Ab peptides that terminated at amino acid 42, (1997). The Swedish mutation (K651M/N652L) was introduced into but not at 40 (Wild-Bode et al. 1997). Similarly, treating the APP YENP mutants (kindly provided by Dr R.G. Perez, human NT2 neurons with BFA eliminated production of University of Pittsburgh) and all mutants were cloned into the intracellular Ab40 but not Ab42 (Cook et al. 1997). Indeed, pCDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA, evidence of b-secretase cleavage in the ER has been USA). All resulting cDNAs were sequenced to verify that only the described in NT2 neurons (Chyung et al. 1997; Skovronsky correct mutational changes were made. The monoclonal antibody et al. 1998), hippocampal neurons (Hartmann et al. 1997; 22C11 was purchased from Roche Molecular Biochemicals (Indi- Annaert et al. 1999) and fibroblasts isolated from PS-1 anapolis, IN, USA; Anti-Alzheimer Precursor Protein A4) and knockout mice (Xia et al. 1998). Recently, an enzyme with recognizes amino acids 60–100 of the amino terminus. 8E5 is a all the characteristics of b-secretase has been identified and mouse monoclonal antibody raised to amino acids 444–592 of human APP that does not react with mouse APP and was a generous appears to localize to the late Golgi and endosomes (Hussain gift from Dr D. Schenk (Elan Pharmaceuticals). et al. 1999; Vassar et al. 1999; Yan et al. 1999; Haniu et al. 2000). Thus, evidence of b-secretase activity has been Pulse-chase analysis, immunoprecipitation, electrophoresis, reported within the endocytic pathway as well as within the and western blotting ER, the Golgi and trans Golgi network of the secretory Forty-four hours after transient transfection, cells were washed in pathway in neuronal and non-neuronal cells. Determining the Dulbecco’s phosphate-buffered saline (PBS; Life Technologies, relative amount of b-secretase cleavage of APPsw occurring Inc.) and incubated in cysteine-free/methionine-free DMEM (Life within each of these subcellular compartments would Technologies, Inc.) for 30 min. One 6-cm plate containing 2 · 106 35 enhance our understanding of the mechanisms of Ab peptide cells was used per sample. Cells were labeled with [ S]methionine/ 35 generation. [ S]cysteine (50 lCi/mL; ICN Pharmaceuticals, East Hill, NY, We developed an ELISA capable of measuring APPswb,a USA) for 60 min. Labeling was terminated
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