Alzheimer’s disease (AD), the most common form of dementia, is characterized by extracellular plaques in the brain created when monomeric amyloid-β (Aβ) protein aggregates into fibrillar structures. Soluble Aβ aggregates, including oligomers that form along the reaction pathway, are believed to be the primary toxic species and have been shown to increase the production of reactive oxygen species (ROS). Polyphenols have been suggested as a complimentary AD therapeutic based on epidemiological evidence that polyphenol-rich diets correlate with a reduced incidence of AD. In particular, many flavonoids, a subclass of polyphenols, have demonstrated the ability to inhibit Aβ aggregation thereby neutralizing the protein’s damaging effects. Alternatively, polyphenols may counteract Aβ-induced cellular responses by neutralizing ROS through their antioxidant properties. This study sought to identify polyphenols that can reduce Aβ-induced apoptosis by inhibiting Aβ oligomer formation and/or reducing ROS.
Polyphenols investigated include quercetin (QUE) and some of its most common metabolites, rhamnetin (RHA), isorhamnetin (IRHA), and tamarixetin (TAM). To identify polyphenols capable of inhibiting the formation of oligomers, polyphenols were added in 10-fold excess to monomeric Aβ1-42prior to induction of oligomerization. The resulting quantity of oligomers formed was analyzed via SDS-PAGE and Western blot. To identify polyphenols capable of neutralizing ROS, antioxidant capacity was evaluated using the OxiSelect™ Oxygen Radical Antioxidant Capacity Activity (ORAC) Assay. To verify that high antioxidant capability led to a reduction in Aβ-induced intracellular ROS, SH-SY5Y human neuroblastoma cells were treated with either oligomers alone or simultaneously with oligomers and polyphenol. Addition of DCFH-DA, which is converted into fluorescent DCF in the presence of intracellular ROS, was used to determine the resulting ROS. To assess the effect of oligomer inhibition and antioxidant capacity on Aβ-induced apoptosis, SH-SY5Y cells were treated with either oligomers formed in the presence of polyphenols or with oligomers formed in the absence of polyphenols followed by addition of polyphenols at concentrations capable of reducing ROS. Cells were stained using TUNEL, which identifies breaks in the DNA strand characteristic of apoptosis. Apoptosis was visualized using a fluorescence microscope and quantified using MATLAB.
A volumetric analysis was used to quantify the amount of oligomers formed in the absence or presence of polyphenols. Only IRHA was unable to reduce Aβ oligomers 250-100 kDa in size, while QUE reduced these oligomers by 88.3 ± 2.4%. QUE, RHA, and TAM all reduced Aβ oligomers <100 kDa in size by approximately 50%, while IRHA remained the weakest inhibitor, only reducing oligomers by 26.2 ± 3.8%. Antioxidant capabilities were quantified relative to Trolox, a vitamin E analog. All compounds exhibited antioxidant capability similar to Trolox, although IRHA and TAM had decreased antioxidant capacity compared to QUE and RHA. This trend was also observed in the reduction of intracellular ROS, with only QUE and RHA significantly reducing Aβ-induced intracellular ROS. When present during oligomerization, all compounds were able to reduce Aβ-induced apoptosis by at least 30%. Polyphenol reduction of ROS also protected against apoptosis.
Characterization of polyphenols for oligomer inhibition and antioxidant capabilities led to the identification of polyphenols with varying degrees of both activities. This panel of compounds demonstrated that both inhibiting Aβ aggregation and reducing ROS can attenuate Aβ-induced apoptosis associated with AD. Future studies will examine the capabilities of these polyphenols to synergistically attenuate apoptosis to determine their potential as dual action drugs for AD.