The conversion of the β-amyloid (Aβ) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer’s disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aβ or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem, we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in the self-assembly of Aβ42, the most aggregation-prone variant of Aβ, are analyzed quantitatively. By applying this approach, we report that bexarotene, an anticancer drug approved by the U.S. Food and Drug Administration, selectively targets the primary nucleation step in Aβ42 aggregation, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aβ42 deposition and its consequences in a Caenorhabditis elegans model of Aβ42-mediated toxicity. These results suggest that the prevention of the primary nucleation of Aβ42 by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer’s disease and, more generally, that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

Keywords

These results can now be taken further as they offer the possibility of investigating the mechanism of inhibition of the Aβ42 aggregation process by therapeutic molecules ( 28 , 29 , 32 ). Approaches using chemical kinetics, which do not require prior knowledge of the elusive structures of the toxic species and are not limited by the need for very tight binding of small molecules to the aggregation-prone proteins, provide highly sensitive methods for the quantitative detection of the effects of potential therapeutic molecules on the aggregation process. By adopting this strategy, we show here that bexarotene, which is an anticancer drug approved by the U.S. Food and Drug Administration (FDA), selectively targets the primary nucleation step in the self-assembly of Aβ, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aβ aggregation and its consequence in a Caenorhabditis elegans model of Aβ-mediated toxicity.

A therapeutic strategy of this type can now be proposed by exploiting recent major advances in our understanding of the molecular processes underlying amyloid formation. These advances are the result of the innovative application of chemical kinetics to the study of protein aggregation ( 29 , 30 ). The availability of highly reproducible data obtained from kinetic measurements based on thioflavin T (ThT) fluorescence ( 31 ) has recently allowed us to define the Aβ42 aggregation mechanism in terms of its underlying molecular events ( 30 ). We observed that once a small but critical concentration of Aβ42 aggregates has been generated through primary nucleation of monomers, surface-catalyzed secondary nucleation becomes the dominant process, whereby the surfaces of the fibrils once formed serve as catalytic sites for the generation of toxic oligomeric species ( 30 ). These oligomers can then grow and convert into additional fibrils, thus further promoting the formation of additional toxic species in a highly effective catalytic cycle ( 30 ).

A key molecular pathway that underlies AD involves the aggregation of Aβ42, the 42-residue form of the β-amyloid (Aβ) peptide, which is a fragment produced by the proteolytic cleavage of the amyloid precursor protein ( 3 – 5 , 8 , 9 ). Aβ42 is an intrinsically disordered peptide ( 10 ) that self-assembles into fibrillar aggregates observed in the brains of AD patients ( 5 , 9 ). Inhibiting the self-assembly of Aβ42 has, therefore, emerged as a major potential therapeutic strategy against AD ( 11 – 20 ), although no small molecule designed to achieve this effect has yet shown clinical efficacy ( 21 ). Such clinical failures are caused at least in part by the incomplete knowledge of the molecular mechanisms underlying the generation of toxic species and of the processes by which small molecules are able to interfere with the aggregation pathway of Aβ42. In addition, it is increasingly evident that prefibrillar oligomeric species, rather than mature amyloid fibrils and plaques, represent the main pathogenic agents in AD and other neurodegenerative conditions ( 3 , 22 – 27 ). Accordingly, effective therapeutic strategies are unlikely to consist of a nonspecific suppression of Aβ42 fibril formation but involve the targeting of specific species in a controlled intervention at precise microscopic steps during the complex aggregation process of Aβ42 ( 28 ).

The incidence of Alzheimer’s disease (AD) is increasing rapidly as the global population ages. It is estimated that 44 million people currently suffer from AD and that this number will exceed 135 million by 2050 ( 1 ). AD is one of more than 40 related disorders ( 2 ) characterized by the misfolding of soluble proteins and their subsequent conversion into amyloid fibrils ( 3 – 9 ).

RESULTS

Chemical kinetics-based therapeutic strategies allow to combat Aβ aggregation The drug discovery strategy that we describe in this work to target the aggregation of Aβ42 consists of four main steps (Fig. 1). First, a fragment-based approach is applied to identify small molecules that could interfere with Aβ aggregation (Fig. 1, step 1). Fragment-based drug design approaches are based on the screening of a limited number of small molecules to identify fragments with low binding affinities, typically with K D values in the high micromolar to millimolar range (33, 34). When combined together, some of these fragments result in molecules containing multiple favorable interactions that bind more tightly than the initial fragments to the target of interest. To implement this strategy, we generated a set of 164 fragments derived from 88 compounds reported in the literature to interact with Aβ. These fragments were then screened for chemically related compounds in four small-molecule databases (ChEMBL, PubChem, ZINC, and DrugBank), resulting in the identification of 16,850 molecules. This fragment-based library contains 386 FDA-approved drugs, which have the potential to be effective candidates for preclinical lead development. Indeed, such an approach offers exciting opportunities to repurpose existing licensed therapeutic compounds for use in AD with the benefit of providing a more rapid route to the clinic than through novel drug discovery approaches (35, 36). 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). To test this strategy, we selected two compounds from this library with different chemical scaffolds, bexarotene and tramiprosate (Fig. 1, step 1). Tramiprosate, which has been unsuccessful in phase 3 AD clinical trials (identifier: NCT0088673), was reported in preclinical development to bind soluble Aβ species, to maintain them in a nonfibrillar form, to decrease Aβ-induced neurotoxicity, and to reduce amyloid plaques and cerebral levels of Aβ in mice (37). By contrast, bexarotene is still in phase 2 AD clinical trials (identifier: NCT01782742). This small molecule is a retinoid X receptor agonist approved by the FDA for the treatment of cutaneous T cell lymphoma and has shown an ability to restore cognitive function to some degree in AD (38–44). Although this activity has been reported to be associated with an enhancement of apolipoprotein E levels, and thus of Aβ42 clearance, conflicting reports have been published and the detailed mechanism of action remains uncertain (38–44). Indeed, this molecule has also been reported to compete with cholesterol for binding to Aβ42, thus inhibiting its cholesterol-induced oligomerization (45). The next step in our general strategy is to apply chemical kinetics to elucidate the mechanism of aggregation of the protein of interest (Fig. 1, step 2). In the specific case of Aβ42, as mentioned in the Introduction, we have recently provided a comprehensive description of the mechanism (32). The kinetic analysis is then repeated in the presence of selected compounds to identify the microscopic events that are specifically inhibited and hence to define the species with which the compound interacts (Fig. 1, step 3). Indeed, a potential drug candidate could bind to monomers, oligomers, or fibril surface or ends, and therefore different microscopic steps could be affected. Sequestration of monomers could result in a decrease in the rates of all microscopic events shown in Fig. 1 (that is, k n for primary nucleation, k 2 for secondary nucleation, and k + for elongation), whereas sequestering oligomers is expected to affect k n and k 2 . On the other hand, targeting fibrils could decrease either k + or k 2 depending on whether inhibitors bind to fibril ends or surfaces, respectively (28). Finally, we evaluate the observed effect of the molecules on the formation of toxic species in vivo (Fig. 1, step 4). The inhibition of specific microscopic steps in Aβ42 aggregation is expected to have different effects on the generation of toxic oligomers (28). More specifically, inhibiting primary nucleation will strongly delay the aggregation reaction and should not affect the total load of toxic oligomers generated during the reaction. However, an increase or a decrease in the number of toxic oligomers is anticipated to be the result of the suppression of either elongation or secondary nucleation, respectively (28). These two latter processes represent interchangeable pathways as inhibiting elongation is expected to redirect the aggregation reaction toward secondary nucleation, which will consequently increase the number of toxic oligomers, and vice versa (28).

Bexarotene, but not tramiprosate, delays Aβ42 fibril formation To investigate the effects of the two small molecules selected for this study on the individual microscopic steps underlying the aggregation process of Aβ42, we carried out a global analysis of the aggregation profiles acquired at different concentrations of both Aβ42 and each of the two compounds in turn (Fig. 2). We monitored Aβ42 fibril formation in vitro in the absence and in the presence of tramiprosate and of bexarotene using a highly reproducible ThT-based protocol described previously (31). Unlike earlier reports (37), we observed no effects of tramiprosate on Aβ42 aggregation even when present in 20-fold excess (Fig. 2A and fig. S1A). By contrast, we observed a progressive delay in Aβ42 aggregation with increasing concentrations of bexarotene (Fig. 2, B and C, and fig. S1B). The scaling of the half-times in such reaction profiles as a function of the total protein concentration follows a power law whose exponent contains important information about the microscopic events underlying the macroscopic aggregation behavior (32). In the presence of bexarotene, although the lag times of the aggregation reaction increase as the concentration is increased, the scaling exponent remains unaffected. This result suggests that, under these conditions, bexarotene delays the aggregation reaction by inhibiting primary nucleation but does not affect the relative contributions of primary and secondary nucleation to the overall aggregation reaction. 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. These experiments show that the effect of bexarotene on Aβ42 aggregation is substantial. The addition of a fourfold excess of bexarotene to a 2 μM sample of Aβ42 increased the time to half-completion of the aggregation reaction compared to that of Aβ42 alone by a factor of 2 (that is, 2.30 ± 0.02 hours for Aβ42 alone and 4.50 ± 0.06 hours for Aβ42 in the presence of bexarotene) (Fig. 3A). To further probe this effect, and to exclude any possible interference of bexarotene on ThT binding to Aβ42 fibrils and hence on the resulting fluorescence, we monitored fibril formation under conditions where ThT was not present by removing aliquots of solution at a series of different time points and measuring the extent of fibril formation using atomic force microscopy (AFM) and immunochemistry (Fig. 3, B to D). Fractions were collected throughout the entire reaction to monitor the events during the three phases of Aβ42 aggregation (that is, lag, growth, and saturation) as completely as possible. AFM images show that after 2.1 hours, fibrillar structures are visible in the absence of bexarotene but not in its presence, thus providing independent evidence for a bexarotene-induced delay of fibril formation (Fig. 3B). These results were extended (Fig. 3C) by probing the quantities of Aβ42 at nine different time points during the aggregation using either sequence-sensitive W0-2 or fibril-sensitive OC primary antibodies (see Materials and Methods). W0-2 antibodies recognize the sequence spanning residues 4 to 10 (that is, the N terminus) that is not likely to be involved in the hydrophobic core of any aggregated species and thus bind to all types of Aβ species. W0-2 antibodies indicated the presence of similar quantities of Aβ42 at the different time points during its aggregation reaction (Fig. 3C, upper panel), whereas fibril-specific OC antibodies that recognize only fibrillar species (Fig. 3C, lower panel) showed, in agreement with the kinetic analysis and the AFM images, a delay of 2 hours in the time required for half-completion of the aggregation process. 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. We further probed the quantity of Aβ42 converted into fibrils by comparing the intensities of the dots to a dot-blot assay that was performed on a range of concentrations between 4 and 0.1 μM Aβ42 fibrils using the OC fibril antibodies (Fig. 3D). Fibrils were collected after incubation for 3 hours of a freshly prepared 4 μM solution of monomeric Aβ42 (Fig. 3D, lower panel), and then the solution was diluted to yield 12 samples with concentrations ranging from 4 to 0.1 μM (Fig. 3D, lower panel). Analysis of the dot-blot data indicated that the quantities of fibrils formed at the reaction half-times of the 2 μM Aβ42 sample in the absence and in the presence of bexarotene (Fig. 3C, lower panel, time points 6 and 7) were indeed closely similar to those formed during the aggregation reaction of an Aβ42 sample of 1 μM concentration, consistent with no interference from ThT on the aggregation reaction (Fig. 3D, lower panel).

Bexarotene specifically inhibits the primary nucleation of Aβ42 aggregation We then carried out a quantitative analysis of the effects of bexarotene by matching the aggregation profiles on the basis of the rate laws derived from a master equation that relates the macroscopic time evolution of the quantity of fibrils to the rate constants of the different microscopic events (28, 29). In this approach, the aggregation profiles in the presence of the inhibitor are described by introducing into the rate laws suitable perturbations to each of the microscopic rate constants evaluated in the absence of the inhibitor. The modifications of the rate constants required to describe the aggregation profiles in the presence of different inhibitor concentrations are then indicative of the specific process affected by the presence of the compound. The aggregation profiles of 5 μM Aβ42 in the presence of concentrations of bexarotene in the range of 5 to 25 μM show that the experimental data are extremely well described when the primary nucleation rate constant, k n , is specifically decreased. By contrast, the experimental data are not consistent with predictions made by altering the rate constants of secondary nucleation or of elongation, k 2 and k + , respectively (Fig. 4, A to C). The data therefore reveal that, under these conditions, bexarotene specifically modifies the primary nucleation pathway (that is, k n k + ) with no (or very little) detectable effect on secondary pathways in the aggregation reaction (that is, k 2 k + ) (Fig. 4D). Similar results were obtained at different Aβ42 concentrations (fig. S2). 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 (k n is the rate of primary nucleation, k + is the rate of elongation, and k 2 is the rate of secondary nucleation; K represents in each case either k n k + or k 2 k + ). Note the significant decrease in primary pathways, k n k + , when compared to secondary pathways, k 2 k + , 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 (k 2 ) as obtained from the aggregation kinetics in (G). To further strengthen these conclusions, we carried out an additional series of measurements of the aggregation kinetics of Aβ42 under conditions where the primary nucleation step was bypassed by the introduction of preformed fibrils (that is, seeds) to the reaction mixture. In such a situation, the contribution of primary nucleation to the reaction kinetics is negligible, as the conversion of soluble peptide into mature fibrils is greatly accelerated by secondary nucleation and elongation reactions promoted by the seeds (22). In the presence of 10% fibril seeds, where elongation of the preformed fibril seeds is the dominant mechanism, no effect was observed on the aggregation kinetics of 2 μM Aβ42 even at a 10-fold excess of bexarotene (Fig. 4E), whereas the corresponding aggregation process under unseeded conditions was completely inhibited for 15 hours (fig. S2A). Furthermore, we also measured the aggregation kinetics of a 2 μM sample of Aβ42 in the presence of 5% of preformed fibril seeds. Under these conditions, primary nucleation is completely bypassed, whereas both surface-catalyzed secondary nucleation and elongation significantly contribute to the overall kinetics (Fig. 4F). These experiments show a concentration-dependent bexarotene-induced delay that corresponds to a decrease in the rate of surface-catalyzed secondary nucleation (Fig. 4, G and H). These findings show that, although the elongation of fibrils is essentially unaffected by bexarotene, this compound has a large and highly selective effect on the nucleation of Aβ42, with the effect being more selective toward primary rather than secondary nucleation. The observation that bexarotene inhibits Aβ42 aggregation by specifically perturbing both primary and secondary nucleation could, in principle, result from the interaction of bexarotene with Aβ42 monomers, with primary and secondary oligomers, or indeed with both monomers and oligomers (Fig. 4F). However, binding to monomers would affect the rates of all the microscopic steps in the overall reaction under the conditions studied here (28), enabling us to conclude that bexarotene specifically interacts primarily with Aβ42 oligomers. This conclusion is further supported by nuclear magnetic resonance (NMR) spectroscopy measurements, where no significant perturbations of the chemical shifts could be observed in the heteronuclear single-quantum correlation (HSQC) spectra of 25 μM 15N-labeled monomeric Aβ42 before and after the addition of a fivefold excess of bexarotene, indicating that binding to the monomeric form of Aβ42 is likely to be negligible (figs. S3 and S4). Furthermore, these findings provide novel insights into the structural features of primary and secondary oligomers of Aβ42, suggesting that primary and secondary nuclei may have similar structural features, and hence can interact with bexarotene in a similar manner, and that the same residues of Aβ42 may be involved in both primary and secondary nucleation events.

Targeting primary nucleation delays the formation of toxic species of Aβ42 We next explored whether the delay in Aβ42 fibril formation resulting from inhibiting primary nucleation by bexarotene could be associated with a delay in the formation of neurotoxic species, as the perturbation of different microscopic steps in Aβ42 aggregation has different effects on the rate of generation of toxic oligomers (28). Thus, for example, decreasing the primary nucleation rate is expected to delay the overall aggregation reaction but not to affect the total number of toxic oligomers generated during the reaction (28). An increase or decrease in the number of toxic oligomers is, however, likely to result from the suppression of elongation or secondary nucleation, respectively (28). To examine this issue, we performed numerical simulations of the total rate of formation of oligomers, from both primary and secondary processes in the aggregation reaction of a 0.5 μM solution of Aβ42 in the absence and presence of a 20-fold excess of bexarotene (see Materials and Methods). Because bexarotene preferentially targets primary nucleation, it is expected to delay the entire aggregation process, that is, to delay the generation of toxic species without necessarily decreasing the amount of the toxic species. On the basis of our previous findings, primary nucleation is a very rapid process that is directly bypassed by secondary nucleation once a small but critical concentration of seeds has been formed (22). We have therefore used the lowest concentration of Aβ42 at which aggregation can be observed (31) and monitored its aggregation in the presence of high concentrations of bexarotene to inhibit the primary nucleation step as strongly as possible. The simulations show that by delaying the primary nucleation of Aβ42 in the presence of bexarotene, a delay in the formation of Aβ42 oligomers occurs but without decreasing the total amount of oligomers formed over the time course of the aggregation process (Fig. 5, A and B). Indeed, the formation of oligomers follows a parabolic evolution in that the number of oligomers formed during the aggregation of Aβ42 alone starts to decrease at the point where the number in the presence of bexarotene started to increase. 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 k 2 = 1 × 106 M−2 s−1, k + = 3 × 106 M−1 s−1, and k n = 1 × 104 M−1 s−1. Dotted red lines show the behavior in the presence of bexarotene, where the nucleation rate constant, k n , 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. To verify these predictions, we measured the cytotoxicity in human neuroblastoma cells (SH-SY5Y) resulting from the aggregation of 0.5 μM Aβ42 in the absence and presence of a 20-fold excess (that is, 10 μM) of bexarotene by evaluating the levels of caspase-3, an early marker of apoptosis (see Materials and Methods). Indeed, caspase-9 is an initiator enzyme that mediates apoptotic pathways after mitochondrial damage and activates effector caspases, such as caspase-3, by cleaving their inactive proforms and initiating the apoptotic cascade. Staurosporine, which is a broad-spectrum kinase inhibitor known to activate the apoptosis pathway, was used as a positive control (see Materials and Methods). Aliquots of Aβ42 solutions were removed at different time points during the lag phase of the aggregation reaction in the absence and presence of bexarotene and incubated with SH-SY5Y neuroblastoma cells. In its monomeric form (before incubation, Fig. 5C), Aβ42 showed no detectable toxicity irrespective of the presence of bexarotene. In the absence of bexarotene, the species formed after incubation for 0.3 hours (Fig. 5D) and 4 hours (Fig. 5E) exhibited a level of toxicity 65 ± 5% higher than that in the presence of bexarotene. However, for the samples incubated for 7 hours, the level of toxicity generated in the presence of bexarotene increased to a value similar to that observed in its absence whereas the toxicity of Aβ alone decreased. This further supports a bexarotene-induced delay of the formation of Aβ42 toxic species in cell (Fig. 5F).