title: Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.
abstract: Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsumura
authors.forename: Keiko
authors.initials: K
authors.lastname: Shinoda
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yamada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yokojima
authors.forename: Masafumi
authors.initials: M
authors.lastname: Inoue
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Shimada
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kikuchi
authors.forename: Dai
authors.initials: D
authors.lastname: Masui
authors.forename: Shigeki
authors.initials: S
authors.lastname: Hashimoto
authors.forename: Michio
authors.initials: M
authors.lastname: Sato
authors.forename: Akane
authors.initials: A
authors.lastname: Ito
authors.forename: Manami
authors.initials: M
authors.lastname: Akioka
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Takagi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Kiyokazu
authors.initials: K
authors.lastname: Nemoto
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Takamoto
authors.forename: Haruo
authors.initials: H
authors.lastname: Inoue
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Nakamura
authors.forename: Yo-ichi
authors.initials: Y
authors.lastname: Nabeshima
authors.forename: David B
authors.initials: DB
authors.lastname: Teplow
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.forename: Minako
authors.initials: M
authors.lastname: Hoshi
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D055503:Protein Multimerization; D051381:Rats
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D010455:Peptides