272560 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.
Aβ1‑42 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 Aβ1‑42 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 Aβ1‑42 oligomers as well as the toxic effects of Ca2+ influx suggests bioavailability of DHF could reduce AD symptoms.
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