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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 ?-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 ?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 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.

SUBMITTER: Matsumura S

PROVIDER: S-EPMC3064209 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

Matsumura Satoko S Shinoda Keiko K Yamada Mayumi M Yokojima Satoshi S Inoue Masafumi M Ohnishi Takayuki T Shimada Tetsuya T Kikuchi Kazuya K Masui Dai D Hashimoto Shigeki S Sato Michio M Ito Akane A Akioka Manami M Takagi Shinsuke S Nakamura Yoshihiro Y Nemoto Kiyokazu K Hasegawa Yutaka Y Takamoto Hisayoshi H Inoue Haruo H Nakamura Shinichiro S Nabeshima Yo-ichi Y Teplow David B DB Kinjo Masataka M Hoshi Minako M

The Journal of biological chemistry 20110203 13

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 cor ...[more]

PMID: 21292768

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Project description:Amyloid fibrils are highly ordered protein aggregates that are associated with several pathological processes, including prion propagation and Alzheimer's disease. A key issue in amyloid science is the need to understand the mechanical properties of amyloid fibrils and fibers to quantify biomechanical interactions with surrounding tissues, and to identify mechanobiological mechanisms associated with changes of material properties as amyloid fibrils grow from nanoscale to microscale structures. Here we report a series of computational studies in which atomistic simulation, elastic network modeling, and finite element simulation are utilized to elucidate the mechanical properties of Alzheimer's Abeta(1-40) amyloid fibrils as a function of the length of the protein filament for both twofold and threefold symmetric amyloid fibrils. We calculate the elastic constants associated with torsional, bending, and tensile deformation as a function of the size of the amyloid fibril, covering fibril lengths ranging from nanometers to micrometers. The resulting Young's moduli are found to be consistent with available experimental measurements obtained from long amyloid fibrils, and predicted to be in the range of 20-31 GPa. Our results show that Abeta(1-40) amyloid fibrils feature a remarkable structural stability and mechanical rigidity for fibrils longer than approximately 100 nm. However, local instabilities that emerge at the ends of short fibrils (on the order of tens of nanometers) reduce their stability and contribute to their disassociation under extreme mechanical or chemical conditions, suggesting that longer amyloid fibrils are more stable. Moreover, we find that amyloids with lengths shorter than the periodicity of their helical pitch, typically between 90 and 130 nm, feature significant size effects of their bending stiffness due the anisotropy in the fibril's cross section. At even smaller lengths (50 nm), shear effects dominate lateral deformation of amyloid fibrils, suggesting that simple Euler-Bernoulli beam models fail to describe the mechanics of amyloid fibrils appropriately. Our studies reveal the importance of size effects in elucidating the mechanical properties of amyloid fibrils. This issue is of great importance for comparing experimental and simulation results, and gaining a general understanding of the biological mechanisms underlying the growth of ectopic amyloid materials.

Dataset Information

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

Publications

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

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