Journal of Alzheimers Disease & Parkinsonism
Open Access

Abstract

Small intermediate aggregates (oligomers and protofibrils) of amyloidogenic peptides and proteins have been shown to be neurotoxic in vitro and are believed to be the principal toxic species for brain neurons causing Alzheimer�s and other neurodegenerative diseases.1 However, cell toxicity and structural studies of oligomers and protofibrils of Aβ using solid-state NMR and microscopy (TEM, STEM and AFM) are challenging, because of the transient nature and structural diversity of oligomers and a high level of polymorphism of protofibrils and amyloid fibrils, usually all coexisting in the same macroscopic sample. Recently, β-sheet rich oligomers and protofibrils of a model Alzheimer�s Aβ(1-42) peptide, Aβ(1-42)CC, were stabilized by the specific double mutation (A21C and A30C) followed by intramolecular cysteine-cysteine cross-linking.2 Aβ(1-42)CC forms only oligomers and protofibrils, which exhibit ca 100-fold higher apoptotic caspase-3/7 activity (neurotoxicity to SH-SY5Y human neuroblastoma cells) compared with Aβ(1-42) amyloid fibrils.2 We report on a variety of important structural constraints in oligomers of uniformly (13C,15N) and selectively (13C,15N and 17O) labelled Aβ(1-42)CC using multi-dimensional correlation 13C-13C and 13C-15N solid-state NMR experiments. Structural constraints obtained from 2D 13C-13C and 13C-15N NMR on hydrated oligomers of recombinant U-13C,15N-Aβ(1-42)C21C30 reveal β-sheet secondary structure features and intermolecular packing of the C-terminal regions in oligomers. Using 2D 13C-13C DARR NMR, V24Cγ-K28Cε and V36Cγ-K16Cε cross-peaks were detected in different selectively (13C,15N) labelled Aβ(1-42)CC samples of oligomers suggesting spatial proximities between side-chains of these amino acid residues. These constraints together with the Cα and Cβ 13C having chemical shifts characteristic of β-sheet structures in proteins are consistent with a β-hairpin structure in the V24-K28 structural fragment and with a specific packing of the central (K16) and the C-terminal (V36) regions in Aβ(1-42) CC oligomers, respectively. In addition, we also explore implementation of 15N{17O} REAPDOR NMR on selectively (13C,15N and 17O) labelled Aβ(1-42)CC for the direct probing of hydrogen bonding in oligomers: The method has successfully been validated on Aβ amyloid fibrils.

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