Research ArticleNEUROSCIENCE

An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease

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Science Advances  12 Feb 2016:
Vol. 2, no. 2, e1501244
DOI: 10.1126/sciadv.1501244
  • Fig. 1 Schematic illustration of the drug discovery strategy described in this work.

    The strategy consists of four steps: (1) A fragment-based approach that allows the identification of small molecules that interact with the aggregation-prone system, here Aβ42, including FDA-approved molecules for drug repurposing. (2) An in vitro kinetic analysis that identifies the specific molecular steps in the Aβ42 aggregation mechanism responsible for the generation of toxic species. (3) A further kinetic analysis to determine the mechanism of inhibition associated with the molecules identified in step 1. (4) An evaluation of the effects of these molecules on the formation of toxic species in vivo. In particular, the inhibition of primary nucleation is predicted to delay the aggregation without affecting the total number of oligomers generated by the aggregation process, whereas inhibiting elongation or secondary nucleation is predicted either to increase or to decrease the number of toxic oligomers, respectively (see text).

  • Fig. 2 Bexarotene, but not tramiprosate, delays the formation of Aβ42 fibril formation.

    (A) Kinetic profiles of Aβ42 aggregation under quiescent conditions at a concentration of 3 μM (open circles) and 4 μM (open squares) in the absence or in the presence of a fourfold excess of tramiprosate. (B) Kinetic profiles of Aβ42 aggregation under quiescent conditions at a concentration of 3 μM (open circles) and 4 μM (open squares) in the absence or in the presence of a fourfold excess of bexarotene. (C) Average half-time of the aggregation reaction as a function of the initial monomer concentration in the absence or in the presence of fourfold excess of bexarotene.

  • Fig. 3 Bexarotene delays Aβ42 fibril formation in a label-free environment.

    (A) Kinetic profiles of the aggregation of 2 μM Aβ42 under quiescent conditions in the absence and in the presence of a fourfold excess of bexarotene; the table below the graph shows the equivalent of the different time points in hours (represented in black solid lines in the graph) at which aliquots of Aβ42 were removed from a solution of 2 μM peptide undergoing aggregation. (B) AFM images of Aβ42 species in the absence and in the presence of a fourfold excess of bexarotene. Images were acquired with tapping mode in air on aliquots of the Aβ42 solutions that were removed from the aggregation reaction at the 1.2- and 2.1-hour time points. Fibrillar structures can be observed after 2.1 hours only in the absence of bexarotene. (C) Time course of the formation of 2 μM Aβ42 fibrils as assessed by antibody binding. The quantity of Aβ42 that was detected by the sequence-specific W0-2 antibody (upper panel) remained unchanged during the complete time course of the reaction on the total quantity of Aβ42 (in solution or as aggregates). The fibril-specific OC antibody (lower panel), however, probes only fibrillar structures that can be seen to have formed earlier in the absence of bexarotene than in its presence. The extent of the observed delay (highlighted in red) is in complete accord with the aggregation profiles shown in (A). (D) Calibration of the dependence of Aβ42 fibril mass concentration to the dot-blot intensity of the fibril-specific OC antibody. (Top) Kinetic profile of 4 μM Aβ42 by means of ThT fluorescence. AFM image of typical mature Aβ42 fibrils acquired with tapping mode in air, formed at pH 8. (Bottom) Dot-blot intensities obtained from binding of the fibril-specific OC antibody to a serial dilution of Aβ42 fibrils that were collected after 3 hours of incubation of a fresh 4 μM Aβ42 monomer. Fibril concentrations were in the range of 4 to 0.1 μM.

  • Fig. 4 Bexarotene selectively targets the primary nucleation of Aβ42.

    (A to C) Kinetic profiles of the aggregation reaction of 5 μM Aβ42 in the absence or in the presence of a 1:1 or 5:1 concentration ratio of bexarotene to Aβ42 (represented by different colors). The solid lines show predictions for the resulting reaction profiles when secondary nucleation (A), fibril elongation (B), or primary nucleation (C) is inhibited by bexarotene. Only the prediction for the case where primary nucleation alone is inhibited closely fits the experimental data. (D) Evolution of the apparent reaction rate constants with increasing concentration ratios of bexarotene (kn is the rate of primary nucleation, k+ is the rate of elongation, and k2 is the rate of secondary nucleation; K represents in each case either knk+ or k2k+). Note the significant decrease in primary pathways, knk+, when compared to secondary pathways, k2k+, as the concentration of bexarotene is increased. (E) Kinetic profiles of 2 μM Aβ42 without (blue) and with the addition of 10% of preformed seed fibrils in the absence or in the presence of a 2-, 5-, 7-, and 10-fold excess of bexarotene (represented by different colors). Note the rapid increase in theslope of the aggregation reaction in the presence of preformed seed fibrils compared to that of the reaction without the addition of preformed fibrils. (F) Simulations showing identical curves for the aggregation profile of a 2 μM Aβ42 sample in the presence of 5% of preformed fibril seeds where primary nucleation events either contribute (gray) or are negligible (brown). (G) Effect of 0.5- and 5-fold excess of bexarotene on the aggregation kinetics of a 2 μM Aβ42 sample in the presence of 5% of preformed fibril seeds. (H) Effect of 0.5- and 5-fold excess of bexarotene on the rates of surface-catalyzed secondary nucleation (k2) as obtained from the aggregation kinetics in (G).

  • Fig. 5 Bexarotene delays the formation of Aβ42 toxic species in neuroblastoma cells.

    (A and B) Numerical simulations of the reaction profiles (A) and nucleation rates (B) for a solution of 0.5 μM Aβ42 in the absence and presence of a 20-fold excess of bexarotene. Blue lines correspond to a control aggregation reaction in the absence of bexarotene, with reaction rate constants k2 = 1 × 106 M−2 s−1, k+ = 3 × 106 M−1 s−1, and kn = 1 × 104 M−1 s−1. Dotted red lines show the behavior in the presence of bexarotene, where the nucleation rate constant, kn, has been decreased to 1 × 102 1 M−1 s−1. A delay in the evolution of the total nucleation rate (that is, of both primary and secondary nucleation) is observed. (C to F) Levels of activated caspase-3 as an indicator of the cytotoxic effects of Aβ42 species on a human neuroblastoma cell line (SH-SY5Y); the fluorescence values have been normalized (see Materials and Methods). Aliquots of 0.5 μM Aβ42, in the absence and in the presence of 10 μM bexarotene, were removed from the aggregation reaction at 0 hour (C), 0.3 hour (D), 4 hours (E), and 7 hours (F). A.U., arbitrary units. Percentage differences between the fluorescence values of Aβ42 in the absence or in the presence of bexarotene (gray circles). These results show that detectable quantities of toxic Aβ42 species are formed in the presence of bexarotene only after 7 hours of incubation at 37°C, in agreement with a bexarotene delay of the formation of Aβ42 toxic species.

  • Fig. 6 Bexarotene restores the motility of C. elegans models of Aβ42-mediated toxicity by preventing Aβ42 aggregation.

    (A) Experimental procedure for the measurement of the effects of bexarotene on the frequency of body bends and on the quantity of aggregates in C. elegans GMC101 (that is, the Aβ worm model) and CL2122 (that is, the control worm model) models. Bexarotene was given to the worms at larval stages L1 and L4. (B) Measurements of the effect of increasing concentrations of bexarotene ranging from 5 to 10 μM on the frequency of body bends in the Aβ worm model. Normalized values with respect to day 0 are shown. The experimental data are shown for a single experiment but are representative in each case of three independent experiments. Complete recovery of the motility of the Aβ worm model can be observed at 10 μM bexarotene; the inset shows the dose dependence of the effects of bexarotene on Aβ worms at day 3 of adulthood. (C) In vivo imaging of aggregates stained using the amyloid-specific dye NIAD-4 in the absence and in the presence of 10 μM bexarotene; images from days 6 and 9 only are shown for clarity. (D) Time course of the reaction of amyloid aggregates formed in the Aβ worms in the absence and in the presence of 1 μM bexarotene. Quantification of fluorescence intensity was performed using ImageJ software (see Materials and Methods). In all panels, error bars represent the SEM. (E) Insoluble fraction of the protein extracts from C. elegans in the presence and in the absence of bexarotene with immunodetection of Aβ and α-tubulin (see Materials and Methods).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/2/e1501244/DC1

    Fig. S1. Comparison of the effects of bexarotene and tramiprosate on Aβ42 aggregation.

    Fig. S2. Quantitative evaluation of the effect of bexarotene on the rate constants of Aβ42 aggregation.

    Fig. S3. HSQC spectra of 15N-Aβ42 in the absence and in the presence of fivefold excess of bexarotene.

    Fig. S4. Interaction between 15N-Aβ42 and bexarotene.

    Fig. S5. Effect of bexarotene on Aβ42 aggregation in C. elegans.

    Fig. S6. Effect of DMSO on Aβ42 aggregation.

    Fig. S7. Toxicity induced by bexarotene in SH-SY5Y human neuroblastoma cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Comparison of the effects of bexarotene and tramiprosate on Aβ42 aggregation.
    • Fig. S2. Quantitative evaluation of the effect of bexarotene on the rate constants of Aβ42 aggregation.
    • Fig. S3. HSQC spectra of 15N-Aβ42 in the absence and in the presence of fivefold excess of bexarotene.
    • Fig. S4. Interaction between 15N-Aβ42 and bexarotene.
    • Fig. S5. Effect of bexarotene on Aβ42 aggregation in C. elegans.
    • Fig. S6. Effect of DMSO on Aβ42 aggregation.
    • Fig. S7. Toxicity induced by bexarotene in SH-SY5Y human neuroblastoma cells.

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