382604 Quercetin and Derivatives Reduce Nuclear Factor-κB Activation Associated with Alzheimer's Disease through Inhibition of Both Amyloid-β Oligomer Formation and Reactive Oxygen Species
Alzheimer’s disease (AD) is the most common form of dementia and is characterized pathologically by extracellular plaques in the brain. These plaques are created when monomeric amyloid-β (Aβ) protein aggregates into fibrillar structures. Soluble Aβ aggregates, including oligomers, which form along the aggregation pathway, can induce the production of reactive oxygen species (ROS) that serve as second messengers for NF-κB activation. NF-κB activation has been shown previously in our lab to mediate cellular responses that are related to the elevated inflammation observed in AD brain. Polyphenols, compounds found in fruits, vegetables, and herbs, have been suggested as a complimentary therapeutic for AD based on epidemiological studies demonstrating that diets rich in polyphenols are associated 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 inhibit Aβ-induced cellular responses by neutralizing ROS through their antioxidant properties. This study sought to identify polyphenols that can inhibit Aβ aggregation and/or reduce ROS in order to determine the role of these characteristics in attenuating NF-κB activation.
Polyphenols investigated were from the flavonols subclass and included quercetin (QUE), kaempferol (KAE), fisetin (FIS), 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 via dilution from DMSO into PBS. The size of oligomers formed was analyzed via SDS-PAGE and Western blot. To identify polyphenols capable of neutralizing ROS stimulated by aggregated Aβ, antioxidant capacity was first evaluated using an antioxidant assay. To verify that high antioxidant capability led to a reduction in intracellular ROS, compounds were also tested for their ability to decrease ROS production in a neuronal cell culture model. To determine which mechanism, oligomer formation inhibition or ROS reduction, is most effective at reducing NF-κB activation, SH-SY5Y human neuroblastoma cells were treated with oligomers formed in the presence of polyphenols or treated with oligomers formed in the absence of polyphenols followed by addition of polyphenols capable of reducing ROS. Cells were fixed and stained with an anti-NF-κB antibody that selectively binds to the activated form of NF-κB. NF-κB activation was visualized using confocal microscopy and quantified.
A volumetric analysis was used to quantify the amount of oligomers formed in the absence (control) or presence of polyphenols. All compounds tested were found to reduce the population of Aβ oligomers formed in the 25 – 100 kD size range. In addition, all compounds except IRHA reduced the amount of larger oligomers formed (100 – 250 kD). Antioxidant capabilities were compared to the positive control Trolox, a vitamin E analog. KAE, FIS, IRHA, and TAM exhibited antioxidant capability similar to that of Trolox, while QUE and RHA were observed to have antioxidant capability significantly greater than Trolox. Furthermore, compounds with the highest antioxidant properties also produced the greatest reduction in intracellular ROS. Finally, neuronal models revealed that polyphenols are capable of reducing Aβ-induced NF-κB activation through both Aβ aggregation inhibition and ROS reduction.
Characterization of polyphenols for anti-aggregation and antioxidant activities 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 NF-κB activation associated with AD. Future studies will examine the capabilities of these polyphenols to synergistically attenuate NF-κB activation to further elucidate their potential as dual action drugs for AD.