An Investigation on the Interaction of Various Natural Products with the Amyloid-β Peptide; Betulinic Acid Causes Rapid Amyloid-β Fibril Formation at the Expense of Soluble Oligomers
Date of Award
Honors College Thesis
In this report we investigate the interaction of the natural compounds asiatic acid (AA), asiaticoside (AS), umbelliferone (UM), and betulinic acid (BA) with the amyloid-β (Aβ) peptide, the aggregation of which is implicated to be the major pathogenic event in Alzheimer's Disease (AD). Screening of compounds for ability to affect Aβ aggregation was performed via ThT fluorescence. Only BA showed consistent deviation from controls, causing virtually instantaneous formation of large amounts of fibrils in a concentration-dependent fashion. Turbidity assays ensured the aggregation monitored via ThT was not experimental aberration. Kinetics utilizing ThT and turbidity indicated that BA causes immediate exponential aggregation before leveling out within 15 min. SEC demonstrated the decrease in monomer caused by BA within 24 h and indicated that BA led to a decrease in oligomeric species. Sedimentation assays confirmed that large amounts of fibrils were formed in 24 h. Circular dichroism (CD) showed that BA caused instantaneous conversion of Aβ monomer from random coil to β-sheet, and that the maximal difference in secondary structure from controls was seen at 24 h. Immunoblots confirmed 24 h fibril formation, and also indicated that BA prevented the formation of oligomeric species found in control samples. Fluorescence anisotropy confirmed that BA binds to Aβ with a KDapp of 11.02 ± 2.01 µM. We conclude that AA, AS, and UM do not directly affect Aβ aggregation, while BA promotes rapid Aβ fibrilization at the expense of soluble oligomeric species. Because oligomers are the most neurotoxic form of Aβ, this suggests that BA should be further investigated as a potential therapeutic for AD.
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Planchard, Matthew, "An Investigation on the Interaction of Various Natural Products with the Amyloid-β Peptide; Betulinic Acid Causes Rapid Amyloid-β Fibril Formation at the Expense of Soluble Oligomers" (2012). Honors Theses. 52.