(760e) Hydroxylated Flavones Reduce Amyloid-β Induced Calcium Influx

Alzheimer's disease (AD) is
the most common form of dementia, characterized by brains that have developed extracellular
plaques and intracellular tangles. 
The formation of plaques, which are composed of aggregated amyloid-β protein (Aβ), is largely
believed to be the underlying cause of the disease.  Mounting evidence suggests that the
plaques themselves do not induce neurodegeneration; it is instead induced by Aβ oligomers, which are formed in an intermediate step of
plaque formation.  Healthy individuals
have low levels of Aβ in blood stream and
cerebrospinal fluid that may be neurotrophic;
therefore, complete elimination of the protein is not likely a viable method of
AD prevention.  Rather, the current
therapeutic goal is to prevent nascent Aβ monomers
from aggregating into toxic oligomers.  Unfortunately, the current diagnostic
criteria for AD often do not present in early stages of the disease when it is
believed intervention can be successful.  As a result, naturally occurring compounds
that are capable of inhibiting Aβ aggregation
would be the ideal therapeutic as they can be safely incorporated into a
population's diet in a strategy similar to iodized salt.

Although old age is generally considered
the biggest risk factor for AD, the disease is markedly more prevalent in
developed countries.  By taking
advantage of differences in diets between cultures, epidemiological studies
have demonstrated a correlation between the increased intake of polyphenols and
reduced incidence of AD.  The
failure of the Western diet to incorporate foods containing polyphenols may be
a contributing factor to the AD epidemic. 
Many fruits, vegetables, and cereals contain flavonoids, which are the
most common polyphenolic compounds found in the human
diet.  The most basic flavonoid is
the three aromatic ring structure, flavone.  While many flavone derivatives exist,
this study focuses on the base structure and two derivatives with hydroxyl
functionalized aromatic rings: 3',4'‑dihydroxyflavone
(DHF) and 5,7,3',4'5'‑pentahydroxyflavone (PHF).  We explored the therapeutic potential of
these three compounds by examining their ability to inhibit Aβ aggregation using both solution and in vitro assays.

Aβ_1‑42 oligomers were prepared in solution prior to cellular
exposure.  Synthetic monomer was solubilized
in dimethyl sulfoxide before the addition of phosphate
buffered saline (PBS), which initiates oligomerization at room temperature.  Flavone, DHF, or PHF was added prior to
PBS in order to allow any inhibition to occur.  The resulting products formed in solution
were stabilized in sodium dodecyl
sulfate (SDS).  These oligomers
were then resolved by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) and electroblotted to
analyze size distribution.  Western
blot analysis demonstrates the presence of DHF and PHF in the oligomerization reactions reduces the maximum size of
detectable oligomers.  In parallel
experiments, cultures of the neuroblastoma cell line
SH‑SY5Y were challenged with the reaction products immediately after
formation.  Intracellular Ca²⁺
ion levels were measured with confocal microscopy via Fluo-3 fluorescence.  Cells exposed to Aβ_1‑42 oligomers exhibit Ca²⁺ influx, which is an
early indicator of cell death commonly associated with deleterious proteins.  Oligomers formed with DHF induce little Ca²⁺
influx similar to untreated controls whereas oligomers formed with PHF induce
the same level of Ca²⁺ influx as oligomers alone.

Polyphenol structure influences
the effect upon both Aβ oligomerization and
physiological activity.  PHF greatly
reduces the size of oligomers but does not prevent Ca²⁺ influx,
suggesting it keeps the oligomers in their most toxic state.  Alternatively, the ability of DHF to
reduce both the formation of larger Aβ_1‑42 oligomers as well as the toxic effects of Ca²⁺
influx suggests bioavailability of DHF could reduce AD symptoms.