. Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science. 2005 Jan 14;307(5707):262-5. PubMed.


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  1. This paper highlights an extremely important (but, unfortunately, often neglected) topic in the Alzheimer research community. Despite the plethora of research efforts and breakthroughs in areas such as genetics and immunology, details about the molecular mechanisms of Aβ assembly into amyloid fibrils are still lacking. Most scientists agree that unraveling the chemical mechanisms is absolutely essential for the development of specific inhibitors to prevent amyloidosis in humans. The most obvious missing details are high-resolution structural data, particularly regarding the soluble β-sheet aggregates and the amyloid fibril structures. Given that amyloid fibrils are not amenable to standard x-ray crystallography (i.e., they do not form crystals), lower-resolution analytical techniques, such as x-ray fibril diffraction, negative stain electron microscopy, and atomic force microscopy, have been employed. Although these methods have provided valuable data about the fibril morphology, including the discovery of “toxic” intermediates such as the “protofibrils,” they cannot provide critical atomic level structural information, such as what amino acids are interacting during the aggregation processes and whether or not the fibrils adopt unique, folded structures.

    Solid-state NMR is a technique that can address these issues, and has only recently been applied to amyloid fibril structure determination (Tycko, 2003). For biomolecules, solid-state NMR has unique capabilities in that it can be used with samples of limited solubility that often precipitate as non-crystalline solids. Solid-state NMR can provide accurate distances and torsion angles between site-specific 15N and/or 13C-labeled atoms and, under certain conditions, the NMR constraints can generate structural models on par with those obtained by solution NMR and x-ray.

    Based on negative stain electron microscopy and atomic force microscopy, Aβ amyloid fibrils have highly variable morphologies that are primarily twisted (also called parallel) assemblies of protein aggregates and filaments. It is thought that they arise from lateral association or from changes in the molecular structure at the protofilament level. With the combined use of solid-state NMR and electron microscopy, Petkova et al. clearly demonstrate that the latter possibility is the source of the different fibril morphologies, and that the accompanying neurotoxicity may result from variations in the intermolecular interactions that hold the fibrils together (such as salt bridges between residues containing oppositely charged side-chains).

    Petkova et al. also established that the fibril morphology and underlying molecular structures are highly dependent on the de novo solution conditions. Although it is well-known that the Aβ peptide aggregation state and secondary structure content (random, α-helix, or β-sheet) are extremely sensitive to the environmental variables (i.e., pH, temperature, solvent, ionic strength, peptide concentration) (Zagorski et al., 1999), and that the neurotoxicity to cortical cell cultures increases when the Aβ becomes aggregated as amyloid-like β-sheet structures (Hartley et al., 1999; Klein et al., 2001; Simmons et al., 1994; Walsh et al., 2002), the association of the neurotoxicity with the fibril structure has remained elusive. These issues were explored with the Aβ(1-40) peptide by performing careful parallel experiments, in which fibrils were grown by three methods: (1) quiescently (incubation only), (2) with agitation, or (3) by the addition of pre-aggregated seed peptide material. These alterations produced different fibril morphologies and structures, as revealed by transmission electron microscope images and solid-state NMR, as well as different neurotoxicity levels. This divergence could be due to different orientations of the amino acid side-chains on the fibril surfaces that lead to different biological activities. The solid-state NMR data suggest that weaker Asp23-Lys28 salt bridging is present in quiescently grown fibrils, while Lys16-Glu22 salt bridging is missing in fibrils obtained by agitation.

    The work of Petkova et al. has several important implications, most notably, that specific orientations of the Aβ peptide side-chains are critical parameters for controlling the β-sheet organization of amyloid fibrils and the accompanying neurotoxicity. One possibility is that other amyloid-forming proteins (besides the Aβ) may adopt similar molecular/fibril structures that promote comparable neurotoxic effects. These unique fibril structures, which are only visible by high-resolution techniques such as solid-state NMR, suggest that specific compounds could be targeted toward inhibiting amyloidosis of a group of proteins, possibly by altering the brain microenvironment by binding in a manner to prevent formation of certain salt bridge interactions that elicit the neurotoxicity. Such compounds could be designed using solid-state NMR-based molecular models.

    This article reminds the AD community that high-resolution structure determination is urgently needed in the amyloid research area, and that solid-state NMR has emerged as an indispensable tool in this endeavor.


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