(683d) Ability of Polyphenols to Attenuate Alzheimer's Disease By Reducing Nuclear Factor- ?B Activation Through Inhibition of Both Amyloid- ? 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.