Gel formation in protein amyloid aggregation: a physical mechanism for cytotoxicity

Abstract

Amyloid fibers are associated with disease but have little chemical reactivity. We investigated the formation and structure of amyloids to identify potential mechanisms for their pathogenic effects. We incubated lysozyme 20 mg/ml at 55C and pH 2.5 in a glycine-HCl buffer and prepared slides on mica substrates for examination by atomic force microscopy. Structures observed early in the aggregation process included monomers, small colloidal aggregates, and amyloid fibers. Amyloid fibers were observed to further self-assemble by two mechanisms. Two or more fibers may merge together laterally to form a single fiber bundle, usually in the form of a helix. Alternatively, fibers may become bound at points where they cross, ultimately forming an apparently irreversible macromolecular network. As the fibers assemble into a continuous network, the colloidal suspension undergoes a transition from a Newtonian fluid into a viscoelastic gel. Addition of salt did not affect fiber formation but inhibits transition of fibers from linear to helical conformation, and accelerates gel formation. Based on our observations, we considered the effects of gel formation on biological transport. Analysis of network geometry indicates that amyloid gels will have negligible effects on diffusion of small molecules, but they prevent movement of colloidal-sized structures. Consequently gel formation within neurons could completely block movement of transport vesicles in neuronal processes. Forced convection of extracellular fluid is essential for the transport of nutrients and metabolic wastes in the brain. Amyloid gel in the extracellular space can essentially halt this convection because of its low permeability. These effects may provide a physical mechanism for the cytotoxicity of chemically inactive amyloid fibers in neurodegenerative disease.

Figure 1. Components of aggregating lysozyme.

Left: AFM image showing various structures seen in aggregating lysozyme on mica substrate: (a) colloidal spheres, (b) primary fibers, (c) compound fibers, (d) amorphous aggregates, (e) a continuous layer of protein monomers bound to substrate. Right: After 1∶1000 dilution the continuous protein layer is no longer present. Colloidal spheres are still seen (a), as are numerous discrete particles (b) with a height of nm (n = 20), probably representing individual lysozyme monomers.

Figure 2. Transition to helical structure is inhibited by high salt concentration.

Lysozyme aggregating in buffer with no salt (left) or 30 mM NaCl (center) demonstrate transformation of virtually all fibers to helical structure after incubation for 11 days. The helical curve of the lysozyme fiber bound to the substrate typically appears from above as a sinusoid (left, inset) although other patterns such as twisted ribbons are occasionally seen. The triangular symbols show the length of one turn of the helix is approximately.4m. Fibers forming in buffer with 150 mM NaCl (right) demonstrate virtually no helical structure even after 31 days.

Figure 3. Effect of time and salt concentration on fiber helicity.

Newly formed fibers are linear at all salt concentrations; at zero and 30 mM NaCl the fraction of fibers displaying helical morphology increases consistently with time until all fibers are helical. This change occurs more rapidly at lower salt concentrations. At 150 nM NaCl essentially all fibers remain linear.

Figure 4. Fiber bundle formation.

Lysozyme fibers aggregate by lateral association to form bundles. Left, center: Arrows identify points at which two fibers join to form a bundle. Appearance of the bundle suggests the fibers wrap around each other in a spiral fashion, a process described by Terech as helix formation. This requires the fibers be free to rotate. Right: Lysozyme fibers in a bundle may remain separated over a short distance and then rejoin, suggesting fiber helices must be aligned for bundling to occur.

Figure 5. Gel formation in lysozyme.

AFM images of lysozyme amyloid fiber network after 30 days incubation. The mixture is a firm gel at this point. (A) Lysozyme gel, undiluted, showing dense network structure. (B) When diluted with water 1∶100 and gently mixed, the gel swells and can be applied to the substrate in a monolayer, showing a range of fiber sizes, bound together at points of contact. (C) Detail of B, showing occasional merging of fibers at points of contact. (D) Gel was diluted in water (1∶10,000) and vortexed ×30 sec. Gel fibers are broken into short fragments but remain joined at points of contact. This indicates development of strong covalent linkages between fibers, typical of an irreversible macromolecular network or IRMAN .

Figure 6. Macromolecular networks formed from tau and A-beta.

Left: TEM image from Ruben of intracellular neurofibrillary tangle composed of tau protein, extracted from human brain tissue. The sample was replicated by coating with 2 nm of platinum-carbon and 12 nm of carbon. The organic material was then dissolved in sulfuric acid and the metallic replica imaged by TEM. The NFT is composed of helical fibers which form a three-dimensional network with the fibers merging at points of contact, indicating that they are bound together. The spaces between the fibers form pores of consistent size, generally less than 100 nm. These features are characteristic of an irreversible macromolecular network or IRMAN, a structure identified by Terech as one of the four types of gel-forming networks . Right: AFM image from Moores of human amyloid beta peptide (A-beta), the primary constituent of extracellular plaques in Alzheimer's disease, induced to aggregate on a substrate. The image demonstrates that A-beta can also spontaneously assemble to form an amyloid fiber network with the structural characteristics of a gel.