Takuma Okada

Formation of Toxic Aβ(1–40) Fibrils on GM1 Ganglioside-Containing Membranes Mimicking Lipid Rafts: Polymorphisms in Aβ(1–40) Fibrils

The abnormal aggregation and deposition of amyloid beta protein (Abeta) on neuronal cells are critical to the onset of Alzheimers disease. The entity (oligomers or fibrils) of toxic Abeta species responsible for the pathogenesis of the disease has been controversial. We have reported that the Abeta aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Abeta(1-40) aggregates formed with GM1-ganglioside-containing membranes. Abeta(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 degrees C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Abeta fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Abeta(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Abeta(1-40) fibrils exhibit polymorphisms.

Inhibition of the formation of amyloid β-protein fibrils using biocompatible nanogels as artificial chaperones

The formation of fibrils by amyloid β‐protein (Aβ) is considered as a key step in the pathology of Alzheimers disease (AD). Inhibiting the aggregation of Aβ is a promising approach for AD therapy. In this study, we used biocompatible nanogels composed of a polysaccharide pullulan backbone with hydrophobic cholesterol moieties (cholesterol‐bearing pullulan, CHP) as artificial chaperones to inhibit the formation of Aβ‐(1–42) fibrils with marked amyloidgenic activity and cytotoxicity. The CHP‐nanogels incorporated up to 6–8 Aβ‐(1–42) molecules per particle and induced a change in the conformation of Aβ from a random coil to α‐helix‐ or β‐sheet‐rich structure. This structure was stable even after a 24‐h incubation at 37 °C and the aggregation of Aβ‐(1–42) was suppressed. Furthermore, the dissociation of the nanogels caused by the addition of methyl‐β‐cyclodextrin released monomeric Aβ molecules. Nanogels composed of amino‐group‐modified CHP (CHPNH2) with positive charges under physiological conditions had a greater inhibitory effect than CHP‐nanogels, suggesting the importance of electrostatic interactions between CHPNH2 and Aβ for inhibiting the formation of fibrils. In addition, CHPNH2 nanogels protected PC12 cells from Aβ toxicity.

Controlled Production of Amyloid β Peptide from a Photo-Triggered, Water-Soluble Precursor “Click Peptide“

In biological experiments, poor solubility and uncontrolled assembly of amyloid β peptide (Aβ) 1–42 pose significant obstacles to establish an experiment system that clarifies the function of Aβ1–42 in Alzheimers disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water‐soluble photo‐“click peptide” with a coumarin‐derived photocleavable protective group that is based on an O‐acyl isopeptide method. The click peptide had nearly 100‐fold higher water solubility than Aβ1–42 and did not self‐assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Aβ1–42. Moreover, the click peptide afforded Aβ1–42 quickly under physiological conditions (pH 7.4, 37 °C) by photoirradiation followed by an O–N intramolecular acyl migration. Because the in situ production of intact Aβ1–42 from the click peptide could improve the difficulties in handling Aβ1–42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Aβ1–42 in AD.

Ganglioside-mediated aggregation of amyloid β-proteins (Aβ): comparison between Aβ-(1―42) and Aβ-(1―40)

J. Neurochem. (2011) 116, 851–857.

Design, synthesis, and biophysical properties of a helical Aβ1–42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid beta-peptide (Abeta) into beta-sheet-rich aggregates is a crucial step in the etiology of Alzheimers disease. Helical forms of Abeta have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Abeta analog would be useful to investigate the role of helical intermediates in fibrillization by Abeta. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Abeta1-42 with 1,6-bismaleimidohexane. The analog assumed a weak alpha-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Abeta1-42 formed a beta-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Abeta analog significantly reduced the amyloid formation and cytotoxicity by Abeta1-42. Thus, our helical Abeta1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Abeta, but also an inhibitor of Abeta-induced cytotoxicity.