title: The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel.
abstract: BACKGROUND
  During intramembrane proteolysis of β-amyloid protein precursor (βAPP) by presenilin (PS)/γ-secretase, ε-cleavages at the membrane-cytoplasmic border precede γ-cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε-cleavages, rather than the γ-cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε-cleavage-derived), instead of that of Aβ42.
  
  
  METHODS
  Cell-free γ-secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI-TOF MS analysis was carried out to detect the N-termini of AICD species. Aβ and AICD species were measured by ELISA and immunoblotting techniques.
  
  
  RESULTS
  Effects on the ε-cleavage by AD-associated pathological mutations around the ε-cleavage sites (i.e., βAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε-cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aβ42 production. Only AICDε51, though, and not Aβ42 production, dramatically changed with modifications to the cell-free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes.
  
  
  CONCLUSIONS
  Our current data show that the generation ratio of AICDε51 and Aβ42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aβ42 generation, measurement of the relative level of Aβ42-related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε-cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε-cleavage as a means for decreasing Aβ42 production.
authors.affiliation: Department of Integrated Medicine, Division of Internal Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Mori
authors.forename: Masayasu
authors.initials: M
authors.lastname: Okochi
authors.forename: Shinji
authors.initials: S
authors.lastname: Tagami
authors.forename: Taisuke
authors.initials: T
authors.lastname: Nakayama
authors.forename: Kanta
authors.initials: K
authors.lastname: Yanagida
authors.forename: Takashi S
authors.initials: TS
authors.lastname: Kodama
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tatsumi
authors.forename: Kana
authors.initials: K
authors.lastname: Fujii
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tanimukai
authors.forename: Ryota
authors.initials: R
authors.lastname: Hashimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Morihara
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Tanaka
authors.forename: Takashi
authors.initials: T
authors.lastname: Kudo
authors.forename: Satoru
authors.initials: S
authors.lastname: Funamoto
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ihara
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Takeda
mesh_terms: D000368:Aged; D000544:Alzheimer Disease / Q000235:genetics* / Q000503:physiopathology*; D053829:Amyloid Precursor Protein Secretases / Q000502:physiology*; D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D053763:Presenilins / Q000502:physiology*
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D053763:Presenilins; C075222:amyloid beta-protein (1-42); C538897:amyloid beta-protein (40-42); D053829:Amyloid Precursor Protein Secretases