Amyloid-β (Aβ) comes in many forms, including monomers, oligomers, protofibrils, and fibrils. Protofibrils have emerged as perhaps the most toxic form of the peptide (see ARF related news story and ARF news story), but because these species form slowly from, and are in equilibrium with, other forms of Aβ, experimental data can be difficult to interpret. In this week’s online edition of PNAS, researchers report that a small molecule, calmidazolium chloride (CLC), can accelerate formation of, and stabilize, protofibrils. The findings could help the study of protofibril toxicity and formation.

Ronald Wetzel and colleagues from the University of Tennessee and Louisiana State University found CLC when searching a chemical library for compounds that would inhibit fibril growth. Instead, they found several compounds that stimulated Aβ aggregation—one of them, CLC, accelerated aggregation by around sixfold.

CLC appears to act directly on free Aβ because when first author Angela Williams and colleagues added the compound to a solution of Aβ monomers, the normal lag seen in fibril formation, which can be up to several days, was eliminated. In fact, after a few hours, almost all of the Aβ monomers had aggregated.

The CLC aggregates resembled protofibrils, in that they did not react well with thioflavin T, a fibril-binding flavin with much lower affinity for protofibrils. A protofibril-like structure was also suggested by direct physical measurements. When Williams and colleagues analyzed the CLC aggregates using electron microscopy, they found curvilinear, rough-edged structures, just like protofibrils. In addition, hydrogen-deuterium exchange experiments, which are used to measure accessibility of amino acids to solvent, showed that about 28 of the amino acids in the CLC aggregates are free to swap their hydrogens for deuteriums. This is typical of protofibrils, but in fibrils, only about 12 amino acids are free to exchange protons, reflecting a much more tightly bound structure. CLS also seems to have a stabilizing effect because the authors found that it took 14 days for protofibrils to convert to thioflavin T-binding species, and then the conversion was only about 20 percent of normal—conversion in the absence of CLC is almost complete by 6-8 days.

The CLC-induced protofibrils do differ in some respects from spontaneously formed protofibrils, however. Williams and colleagues found that residues 21-30 of Aβ40 are involved in aggregation in the presence of CLC, while these residues are less important for formation of natural protofibrils. The authors suggest that “the CLC protofibril may involve Aβ in more of a simple extended hairpin structure with a long, relatively flexible loop, involving residues 22-29, spanning the two β-structure segments.”

The authors also suggest that “although counterintuitive, it remains conceivable that compounds that stabilize protofibril structure may also have therapeutic value,” adding that “…a drug that facilitates sequestration of monomeric Aβ into stabilized protofibrils may provide therapeutic advantage, protecting cells while giving the body time to remove or inactivate these peptides.” The burning question, of course, is whether CLC-protofibrils are less or more toxic than spontaneously formed species. —Tom Fagan

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  1. Indeed, since some time I have been thinking about the need to search for protofibril stabilizers, i.e., substances, possibly but not necessarily amphipathic, able to interact with early oligomers of misfolded proteins or peptides by masking their exposed hydrophobic patches. Such an approach might make prefibrillar aggregates more stable, allowing them not only to arrest their growth into more stable and less toxic mature fibrils (which might appear undesirable), but also to make them much less toxic. This view is conceivable if we assume that toxicity is, at least in most cases, a consequence of aggregate instability and surface activity following exposure of hydrophobic clusters, allowing them to interact with other cellular components, notably membranes.

    The revised paper apparently proceeds in this direction and it appears very important to check whether the CLC-induced protofibrils display the same toxicity to cells as those obtained in the absence of CLC. I expect they are much less toxic, but I am keen to read these data.

References

News Citations

  1. Earliest Amyloid Aggregates Fingered As Culprits, Disrupt Synapse Function in Rats
  2. Oligomers in AD: Too Much of a Bad Thing?

Further Reading

No Available Further Reading

Primary Papers

  1. . Structural properties of Abeta protofibrils stabilized by a small molecule. Proc Natl Acad Sci U S A. 2005 May 17;102(20):7115-20. PubMed.