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

Abstract

Nonfibrillar assemblies of amyloid ␤-protein (A␤) 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 A␤ labeled at the N terminus or Lys 16 , we uncovered two distinct assembly pathways. One leads to highly toxic 10 -15-nm spherical A␤ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ϳ330 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 ϳ128 kDa (ϳ32-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 A␤ 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 A␤ assembly steps at which inhibition may be beneficial.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

“…In other words, the NMR-visible amino acids, although derived from different Aβ regions, may exist in close proximity to form the ASPD surface (Fig. 7B), in accordance with our finding that specific binding of monoclonal mASD3 antibody to ASPD was blocked by pentapeptides covering Aβ 2-8 , Aβ [15][16][17][18][19] , or Aβ [19][20][21][22][23] (19). Because mASD3 antibody neutralizes ASPD toxicity (19), the NMR-identified ASPD surface is likely involved in binding to NAKα3.…”

“…2E), and appeared as ∼11.7 ± 1.6-nm spheres (n = 49) in TEM (Fig. 2C), consistent with previous data (18)(19)(20). We also used in vitro-reconstituted synthetic ASPD, which share essential characteristics with patient ASPD (19) (Table S1), as an analog.…”

“…As shown previously (19), mASD3 inhibited ASPD-induced neuronal death, but 6E10, targeting Aβ 3-8 , did not. ASPD concentration is expressed in terms of the average ASPD mass 128 kDa (20). (B) Cells were treated for 30 min with 140 nM synthetic ASPD as in A.…”

“…1C). ASPD concentration was determined using an average ASPD molecular weight of 128 kDa (20). See summary of the characteristics of patient and synthetic ASPD in Table S1.…”

“…We previously isolated neurotoxic Aβ oligomers, termed amylospheroids (ASPD), from the brains of AD patient (18)(19)(20). ASPD appear in transmission electron microscopic (TEM) images as spheres of diameter ∼11.9 ± 1.7 nm (19).…”