Effect of Amyloid Fibril Oxidation on its Seeding Potency

Abstract

Extracellular plaques composed of β-sheet rich amyloid-beta (Aβ) fibrils are believed to cause neurodegeneration and cognitive decline in Alzheimer's disease. Fibrils can form through a primary nucleation-dependent aggregation pathway or secondary nucleation mechanism in which preformed fibrils cause Aβ monomers to misfold and aggregate into amyloid fibrils. As Aβ aggregation is believed to occur during the prolonged asymptomatic stage before neurodegeneration, it is important to gain insights into the aggregation process. Photodynamic therapy utilizes photosensitizing molecules to produce reactive oxygen species, which in turn can oxidize an array of biomolecules including proteins. We have recently shown that a novel Aβ fibril sensor, the anionic phenylene ethynylene oligomer OPE1-, exhibits controllable photosensitizing activity. When bound to Aβ fibrils, OPE1- becomes highly fluorescent, sensitizes the production of singlet oxygen, and induces fibril oxidation upon irradiation. As the singlet oxygen species is short-lived, the controlled photosensitization thus lowers off-target oxidation. In this study we evaluated the effects of OPE1- induced Aβ40 fibril oxidation on its seeding potency. Aβ40 aggregation seeded by non-oxidized and oxidized fibrils were characterized by Thioflavin-T fluorescence assay and compared. Morphologies of the fibrils produced by both seeding conditions were imaged by transmission electron microscopy and fibril secondary structures were analyzed by circular dichroism spectroscopy. Our results thus far show that oxidation of the fibril seeds does not alter Aβ40 aggregation rates or its seeding potency after 3 days of incubation. Furthermore, fibrils produced in the presence of oxidized and non-oxidized fibril seeds showed similar morphologies on TEM images. We plan to further investigate the effect of oxidation on fibril cell toxicity. Results from this study contribute to a better understanding of amyloid protein aggregation via secondary nucleation pathway, which is important in understanding Alzheimer's disease process.