Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Manami Akioka,Masataka Kinjo,Keiko Shinoda,Akane Ito,Kazuya Kikuchi,Michio Sato,Hisayoshi Takamoto,Shinsuke Takagi,Yo‐Ichi Nabeshima ,Takayuki Ohnishi,Satoko Matsumura,Dai Masui,Masafumi Inoue,David B. Teplow,Shigeki Hashimoto,Shinichiro Nakamura,Yu Nakamura ,Kiyokazu Nemoto,Mayumi Yamada,Takahiro Shimada ,Yutaka Hasegawa,Haruo Inoue,Minako Hoshi,Satoshi Yokojima

doi:10.1074/jbc.m110.181313

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.

Highlights

Nonfibrillar assemblies of amyloid ␤-protein (A␤) are considered to play primary roles in Alzheimer disease (AD)
These findings suggest that native ASPDs might be a candidate for A␤ assemblies that directly cause neuronal loss in human AD brains
Excess A␤1–5 (DAEFR) or A␤16–20 (KLVFF) had no effect on ASPD formation, suggesting that amino acid residues around A␤1–5 or A␤16–20 are not involved in ASPD formation

Summary

EXPERIMENTAL PROCEDURES

Materials—ASPD-specific antibodies, a rabbit polyclonal rpASD1 (Kd Ϸ 5 pM) and a hamster monoclonal haASD1 (Kd Ϸ 0.5 pM), have been produced in our laboratory and recognize epitopes distinct from those present on dimers, A11 antibodyreactive 12-mers, or fibrils [21]. ASPDs were prepared in vitro from 50 ␮M solutions of A␤1–42 (with or without 0.1 ␮M NTR or K16TR; a probe ratio of 1/500) in F12 buffer without riboflavine, L-glutamine, and phenol red by slowly rotating the solutions at 4 °C for 16.5 h [10] Their quality was confirmed by dot blotting, TEM, and toxicity assays. If all components have equal brightness, the relative abundance of each assembly can be obtained directly from the distribution of assembly diffusion time. This is true for ASPDs and early fibril intermediates, but not for fibrils containing more than one fluorophore. Other Methods—Immunoprecipitation (IP), TEM, fluidphase imaging of ASPDs by atomic force microscopy, toxicity assays, and statistics are described in the supplemental “Experimental Procedures.”

RESULTS

IP eluate

DISCUSSION