The natural product betulinic acid rapidly promotes amyloid-β fibril formation at the expense of soluble oligomers

Abstract

The biochemical hallmarks of Alzheimer’s disease (AD) are the aggregates of amyloid-β (Aβ) peptide that deposit in brains of AD patients as senile plaques. The monomeric Aβ undergoes aggregation in a nucleation-dependent manner to form insoluble fibrils. Emerging evidence suggests that the low-molecular-weight aggregates called “soluble oligomers” are the primary neurotoxic agents as opposed to the fibrils. Needless to say, developing Aβ aggregation inhibitors is imperative for a meaningful progress toward AD therapy. In this report, we have explored the in vitro interactions between a natural product called betulinic acid (BA) and Aβ42 peptide. BA has found its therapeutic use in several human pathologies including cancer, HIV-related AIDS, and nervous system disorders. The results from this study indicate that BA rapidly promotes the formation of Aβ42 fibrils and, in doing so, partly circumvents the formation of potentially neurotoxic soluble oligomers. Furthermore, the promotion of fibrils by BA seems to be specific for the fibril formation “on-pathway”, and it fails to interact with aggregates that are formed outside this obligatory pathway. The unique ability of BA to promote fibrils at the expense of oligomers along with its well-known pharmacological properties make BA a potential therapeutic agent for AD.

Figure 1

List of compounds used in this study.

Figure 2

Dose-dependent augmentation of Aβ42 aggregation by BA. Aggregation was measured via ThT fluorescence over time. (A) Incubations composed of 25 μM Aβ42 in 20 mM Tris, pH 8.0 at 37 °C with increasing amounts of BA dissolved in DMSO; 12.5 μM (■), 25 μM (○), 50 μM (▲), 100 μM (□). (B) Controls containing DMSO concentrations corresponding to 12.5, 25, 50, and 100 μM BA incubations, respectively, shown in (A): 0.625% (▲), 1.25% (○), 2.5% (□), and 5% (■). (C) Incubations comprising buffered 25 μM Aβ42 with increasing amounts of BA dissolved in EtOH: 12.5 μM (■), 25 μM (○), 50 μM (▲), and 100 μM (□). (D) Controls containing varing EtOH concentrations: 0.625% (■), 1.25% (○), 2.5% (▲), and 5% (□). (E) Difference plot of BA dissolved in DMSO (A) and DMSO controls (B). The control fluorescence at each time point was subtracted from the fluorescence of the BA samples to generate the plot, which shows the molar stoichiometric incubations, 1:0.5 (■), 1:1 (○), 1:2 (▲), and 1:4 (□). (F) Similar difference plot of BA dissolved in EtOH (C) and EtOH controls (D), which shows the molar stoichiometric incubations, 1:0.5 (■), 1:1 (○), 1:2 (▲), and 1:4 (□). The Aβ:BA stoichiometry is indicated by dotted arrows in (E) and (F).

Figure 3

Other assays also suggest rapid Aβ fibrillation induced by BA. (A) Turbidity assay was performed by measuring the absorbance at 400 nm for buffered 25 μM Aβ42 in 5% DMSO at 37 °C in the presence of 100 μM BA (○) or in the absence of BA (●). The difference plot is also shown (▲). (B) Kinetics of the incubations in (A) was monitored immediately after incubation by ThT fluorescence (gray line) or absorbance at 400 nm (black line). (C) Samples in (A) were subjected to fractionation via SEC after 24 h of incubation. The samples were subjected to centrifugation at 19,000g for 15 min, and the supernatants were loaded on to the Superdex-75 column. The sample containing BA is shown as a smooth line, whereas the control sample in the absence of BA is shown as a dotted line The monomers eluted at fractions 22–26, while the aggregates peaked near the void volume (V₀) between 15 and 17 (inset). (D) Sedimentation assay was performed by taking an aliquot of the coincubation of Aβ42 with 4-fold molar excess of BA and measuring ThT fluorescence after 24 h (T). In parallel, another aliquot of the same reaction was subjected to centrifugation at 19,000g for 15 min. The supernatant thus obtained was measured via ThT fluorescence (S). Three such data sets were averaged.

Figure 4

Aβ42 aggregation in the presence of BA monitored by immunoblots. Samples containing 25 μM Aβ42 were incubated alone (control) or with 100 μM BA (+BA) at 37 °C. Aliquots of the sample were taken at the indicated time points and were run on an SDS-PAGE gel followed by Western blotting and immunodetection. The lanes T and S represent total and supernatant (after spinning the sample at 19,000g for 15 min to remove fibrils) respectively, after 0, 24, and 48 h of incubation.

Figure 5

Secondary structure changes in Aβ42 upon incubation with BA. Coincubation reactions containing 4-fold molar excess of BA in EtOH similar to those shown in Figure 2C were monitored using far-UV CD (solid lines) along with a control in the absence of BA (dashed lines) at the indicated time points (A–D). (E) Difference plot obtained by subtracting the molar ellipticity values at 216 nm of the control sample from the sample containing BA (■), which was overlaid with the difference plot shown in Figure 2F for 1:4 stoichiometry (○).

Figure 6

Binding affinity between Aβ42 and BA measured by fluorescence anisotropy. The specific mutant F19W-Aβ42 was used as a probe to measure tryptophan anisotropy (r). Aliquots of mixtures containing 5 μM Aβ42 and 50 μM BA in 2.5% DMSO were titrated on a solution containing 5 μM Aβ42 in a similar solvent (●). The anisotropy was measured in quadruplets and averaged for each titration point. As a negative control, a similar experiment was performed using glycyl-glycine instead of BA (○). Three independent data sets were averaged and fit with a model for single site binding (eq 2).

Figure 7

Effect of BA on “off-pathway” oligomers. Lanes 1 and 2 show 25 μM Aβ42 containing 5% DMSO incubated with 5 mM lauric acid alone and in the presence of 100 μM BA, respectively. In parallel, the SEC isolated oligomers were also incubated alone and with 100 μM BA at 37 °C. Aliquots of the sample were taken at the indicated time points and were electrophoresed and immunoblotted. Lanes T and S represent total and supernatant (after spinning the sample at 19,000g for 15 min to remove fibrils), respectively, after 0 and 24 h. The double arrows indicate LFAOs of Aβ42.