title: Intracellular site of gamma-secretase cleavage for Abeta42 generation in neuro 2a cells harbouring a presenilin 1 mutation.
abstract: Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway.
authors.affiliation: Department of Dementia Research, National Institute for Longevity Sciences, Obu Aichi, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Sudoh
authors.forename: G
authors.initials: G
authors.lastname: Hua
authors.forename: Y
authors.initials: Y
authors.lastname: Kawamura
authors.forename: K
authors.initials: K
authors.lastname: Maruyama
authors.forename: H
authors.initials: H
authors.lastname: Komano
authors.forename: K
authors.initials: K
authors.lastname: Yanagisawa
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D020126:Brefeldin A / Q000494:pharmacology; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009154:Mutation; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis*; D053764:Presenilin-1; D021382:Protein Sorting Signals / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D016229:Amyloid beta-Peptides; D017931:DNA Primers; D008565:Membrane Proteins; D010446:Peptide Fragments; D053764:Presenilin-1; D021382:Protein Sorting Signals; C075222:amyloid beta-protein (1-42); D020126:Brefeldin A; D008985:Monensin; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse