Ability of Polyphenols to Attenuate Alzheimer's Disease By Reducing Nuclear Factor- ΚB Activation 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 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 that form along the aggregation pathway, have the ability to induce production of reactive oxygen species (ROS) resulting in various cellular responses, including increased cerebrovascular inflammatory responses associated with AD pathology.  Specifically, ROS serve as messengers for increased NF-κB activation, which has been shown previously by our laboratory to regulate endothelial monolayer permeability as well as endothelial adhesion of monocytes and subsequent transendothelial monocyte migration.  Epidemiological studies have shown that diets rich in polyphenols, compounds found in fruits, vegetables, and herbs, result in a reduced incidence of AD.  Many polyphenols of the flavonoid subclass can inhibit Aβ aggregation thereby neutralizing the protein's damaging effects.  Alternatively, polyphenols have been shown to inhibit Aβ induced cellular responses by neutralizing ROS through their antioxidant properties.  This study sought to identify polyphenols that can either inhibit Aβ aggregation or reduce ROS in order to determine the role of these two activities in reducing NF-κB activation.  Additionally, polyphenols that have both of these activities were identified to investigate possible synergy between the two mechanisms and the potential of polyphenols to serve as dual action drugs for AD.

Polyphenols investigated were from the subclass flavonoids and included various flavones, isoflavones, flavanols, and catechins in order to explore correlations between structure and inhibitory effects.  To identify polyphenols capable of inhibiting the formation of oligomers, polyphenols were added in 10-fold excess to monomeric Aβ_1-42 prior to induction of oligomerization.  The resulting 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 evaluated using the OxiSelect™ Oxygen Radical Antioxidant Capacity Activity Assay.  A Vitamin E analog, Trolox, was used as a standard.  To verify that high antioxidant capability led to a reduction in intracellular ROS, the 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, has the greatest effect on 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 for NF-kB (rabbit polyclonal anti-NF-κB–p65 antibody) and nuclear location (DAPI).  Localization of NF-κB-p65 subunits within the nucleus, indicating NF-kB activation, was quantified.

 

Flavones tested showed a correlation between the number of hydroxyl groups present and the ability to inhibit Aβ oligomer formation.  Results from the flavanols further suggest that not only the number of hydroxyl groups but also their placement is crucial for oligomer inhibition.  Isoflavones and catechins were shown to lack the ability to inhibit oligomer formation even with the presence of several hydroxyl groups.  The correlation between hydroxyl groups and antioxidant capacity was weaker, and at least one compound from each group exhibited antioxidant capability similar to that of Trolox.  Polyphenols with either anti-aggregation or antioxidant activity were capable of reducing the activation of NF-kB, and polyphenols possessing both activities were the most effective inhibitors of NF-kB activation.

Characterization of polyphenols for anti-aggregation and antioxidant activities led to the identification of polyphenols with each of these activities as well as polyphenols with both activities.  Distinct correlations were observed between the structure of the polyphenols and the ability to inhibit oligomer formation. This panel of compounds facilitated elucidation that both of these characteristics of polyphenols can protect against Ab-induced NF-kB activation associated with AD.  Furthermore, polyphenols that posses both activities can exert synergism to function as dual-action drugs.