Alzheimer’s Disease (AD) is the most common neurodegenerative disease and the 6thleading cause of death in the US. One neurological marker of AD is the deposition of extracellular plaques composed of aggregates of the amyloid-β (Aβ) protein. As described by the amyloid cascade hypothesis, Aβ aggregation follows a nucleation-dependent pathway, beginning with protein monomer forming nuclei that grow into soluble aggregates and proceed to form the insoluble fibrils deposited in AD brain. In turn, these aggregate species are associated with increased intracellular reactive oxygen species (ROS) as well as cellular apoptosis. While such understanding of AD pathogenesis has increased significantly, AD remains the one disease in the top 10 that has no therapy, prevention, or cure. Among the most effective pharmaceuticals, 15% have been developed based on natural compounds or their derivatives. Thus, novel AD therapies can be developed and improved if natural compounds can be identified that effectively inhibit Aβ aggregation. Soy isoflavones (SIF), a subclass of naturally occurring polyphenols, are known for their beneficial effects in a myriad of diseases and cancers. While other subclasses of polyphenols have been studied for their ability to modulate Aβ growth and toxicity, SIFs remain unexplored. Here, four SIFS are studied for their ability to alter key steps in the Aβ aggregation pathway: genistein (GEN), genistin (O-GEN), daidzein (DEN), and daidzin (O-DEN). These compounds vary in hydroxylation as well as glycosylation on the fused center ring structure.
To examine the earliest stages of aggregation, Aβ oligomerization was induced by combining DMSO-solubilized Aβ1-42 with a 10-fold excess of inhibitor followed by dilution into PBS. Oligomer formation was monitored via SDS-PAGE and Western blotting to quantify oligomer size. Additionally, oligomer samples were loaded onto APTES treated mica for AFM imaging. To study the overall process of aggregation, SEC-purified Aβ1-40 was agitated with physiological salt in the presence of a 10-fold excess of compound and monitored using thioflavin T to detect aggregate β-sheet structure. Results were quantified as fold extension in lag time and reduction in equilibrium plateau. At terminal time points, samples were loaded onto copper grids for TEM imaging of fibril morphology. The antioxidant capacity of SIFs was evaluated using the OxiSelectTM Oxygen Radical Antioxidant Capacity (ORAC) assay. To confirm the antioxidant action of SIFs in cell culture, SH-SY5Y human neuroblastoma cells were treated simultaneously with SIFs and oligomers formed in the absence of SIFs. Cells were incubated with DCFH-DA, a probe that decomposes into fluorescent DCF in the presence of intracellular ROS, allowing for quantification of reduction in intracellular ROS.
When present during oligomerization, all SIFs reduced the amount of 25-100 kDa oligomers in size, while only GEN and O-DEN significantly reduced the amount larger 100-250 kDa oligomers. Additionally, GEN, O-GEN, DEN, and O-DEN increased the amount of monomer, trimer, and tetramer species. Together, these results suggest that SIFs keep small Aβ aggregates from progressing to larges sizes. In monomer aggregation, a corresponding effect was observed in lag extension. Through AFM and TEM, visual morphology changes were also apparent in oligomer and burblier aggregates, respectively. SIFs were also good antioxidants that were able to modulate Aβ-induced ROS.
In conclusion, all SIFs tested exhibit an effect on Aβ oligomerization and aggregation, as well as intracellular ROS, suggesting their potential as dual action therapies. Future work will determine the effect of SIFs on Aβ-induced cellular apoptosis to confirm that these drug-like compound properties have therapeutic potential.