title: The A673T mutation in the amyloid precursor protein reduces the production of β-amyloid protein from its β-carboxyl terminal fragment in cells.
abstract: BACKGROUND
  The A673T mutation in the amyloid precursor protein (APP) protects against Alzheimer's disease by reducing β-amyloid protein (Aβ) production. This mutation reduced the release of the soluble APP fragment (sAPPβ), which is processed by β-secretase, suggesting a concomitant decrease in the β-carboxyl fragment of APP (C99), which is a direct substrate of γ-secretase for Aβ production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A673T as the cause of reduced Aβ production. Here, we investigated the effect of the A673T mutation in C99 on γ-cleavage in cells.
  
  
  RESULTS
  We found that the level of C99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild-type APP, although the release of sAPPβ was significantly reduced in the APP A673T cells. In addition, our reconstituted β-secretase assay demonstrated no significant difference in β-cleavage on an APP fragment carrying the A673T mutation compared with the wild-type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the Aβ numbering) produced roughly half the level of Aβ compared with the wild-type C99, suggesting that the C99 A2T is an insufficient substrate of γ-secretase in cells. A cell-free γ-secretase assay revealed that Aβ production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with γ-secretase components in the raft fractions were reduced significantly.
  
  
  CONCLUSIONS
  Our data indicate that the A673T mutation in APP alters the release of sAPPβ, but not the C99 level, and that the C99 A2T is an inefficient substrate for γ-secretase in cell-based assay.
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Kokawa
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan.
authors.forename: Seiko
authors.initials: S
authors.lastname: Ishihara
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Fujiwara
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Nobuhara
authors.affiliation: Department of Neuropathology, Graduate School of Brain Sciences, Doshisha University, Kizugawa, Kyoto, 619-0225, Japan.
authors.forename: Minori
authors.initials: M
authors.lastname: Iwata
authors.affiliation: Department of Neuropathology, Graduate School of Brain Sciences, Doshisha University, Kizugawa, Kyoto, 619-0225, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ihara
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan. sfunamot@mail.doshisha.ac.jp.
authors.forename: Satoru
authors.initials: S
authors.lastname: Funamoto
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D002793:Cholic Acids / Q000494:pharmacology; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments; D013997:Time Factors; D014162:Transfection
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D002793:Cholic Acids; D010446:Peptide Fragments; D049452:Green Fluorescent Proteins; C048531:chapso; D053829:Amyloid Precursor Protein Secretases