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. 2010 Jul 30;285(31):24228-37.
doi: 10.1074/jbc.M110.133108. Epub 2010 May 28.

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers

Ali Reza A Ladiwala  1 , Jason C Lin, Shyam Sundhar Bale, Anna Marie Marcelino-Cruz, Moumita Bhattacharya, Jonathan S Dordick, Peter M Tessier
Affiliations

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers

Ali Reza A Ladiwala et al. J Biol Chem. .

Abstract

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Abeta1-42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Abeta monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their beta-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Abeta-mediated toxicity.

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Figures

FIGURE 1.
FIGURE 1.
Resveratrol selectively remodels soluble prefibrillar Aβ oligomers. Aβ conformers (25 μm) were assembled for 0–6 days without agitation (A–E) and incubated with resveratrol (Res., 20 μm), vanillin (Van, 20 μm) or the vehicle (1% DMSO) for 4 h (C–E). A, dot blot analysis of Aβ conformers probed with the conformation-specific antibodies A11 (specific for soluble oligomers) and OC (specific for fibrillar conformers) as well the 6E10 antibody (specific for the N terminus of Aβ). B, circular dichroism spectra of Aβ conformers relative to Aβ amyloid fibrils. C, image of SDS-PAGE gel after silver-staining. D, AFM images of Aβ conformers before and after incubation with resveratrol. The scale bar is 1 μm. E, cell toxicity analysis of Aβ conformers (2.5 μm Aβ, 0.8:1 resveratrol:Aβ molar ratio, n = 3). BSA, bovine serum albumin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
FIGURE 2.
FIGURE 2.
Dose dependence of resveratrol remodeling of soluble prefibrillar Aβ oligomers. Aβ-soluble oligomers (25 μm; formed after 1 day without agitation) were exposed to various concentrations of resveratrol (Res.) and control solutions for 4 h and monitored via SDS-PAGE (A), dot blots probed with conformation-specific (A11, soluble oligomers) and sequence-specific (6E10) antibodies (B), and cell toxicity analysis (2.5 μm, Aβ, n = 3) (C). Van, vanillin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
FIGURE 3.
FIGURE 3.
Resveratrol remodeling activity for Aβ fibrillar intermediates. Aβ (25 μm) conformers were assembled via mild agitation (250 rpm) for 5 days (A–E) and then incubated with resveratrol (Res., 20 μm), vanillin (Van., 20 μm), or the vehicle (1% DMSO) for 4 h (C–E). A, dot blot analysis of Aβ conformers is shown. B, circular dichroism spectra of Aβ conformers is shown. C, image of silver-stained, SDS-PAGE gel. D, AFM images of Aβ conformers before and after incubation with resveratrol. The scale bar is 1 μm. E, cell toxicity analysis of Aβ conformers (2.5 μm, 0.8:1 resveratrol:Aβ molar ratio, n = 3). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
FIGURE 4.
FIGURE 4.
Resveratrol remodeling activity for Aβ fibrils. Aβ (25 μm) conformers were assembled via mild agitation (lateral mixing rate of 250 rpm) in the absence of salt and monitored via dot blots probed with conformation-specific (OC, fibrillar conformer) and sequence-specific (6E10) antibodies (A) and AFM (scale bar is 1 μm) (B). In addition in B, Aβ fibrils were incubated with resveratrol (Res, 20 μm) or vanillin (Van, 20 μm) for 4 h.
FIGURE 5.
FIGURE 5.
Templating activity of Aβ conformers. Aβ fibrillar conformers (fibrillar intermediates (Int., A) and amyloid fibrils (B)) were incubated with resveratrol (res., 20 μm) for 0 or 4 h, and their templating activity was evaluated at 25 °C by incubating each insoluble conformer (5 wt % seed) with freshly dissolved Aβ monomers (25 μm) via ThT analysis (n = 5). RFU, relative fluorescence units.
FIGURE 6.
FIGURE 6.
Dose dependence of resveratrol remodeling of Aβ fibrillar intermediates. Aβ fibrillar intermediates (25 μm) were incubated with resveratrol (Res.) or control solutions for 4 h and monitored using SDS-PAGE (A), dot blot analysis using both conformation-specific (OC) and sequence-specific (6E10) antibodies (B), ThT (n = 5) (C), and cell toxicity analysis (2.5 μm Aβ; n = 3) (D). RFU, relative fluorescence units.
FIGURE 7.
FIGURE 7.
Dose dependence of resveratrol remodeling of Aβ amyloid fibrils. Aβ fibrils (25 μm) were incubated with resveratrol (Res.) or control solutions for 4 h and monitored using SDS-PAGE (A), cell toxicity analysis (2.5 μm Aβ; n = 3) (B), dot blot analysis using both conformation-specific (OC) and sequence-specific (6E10) antibodies (C), and ThT (n = 5) (D). In A, Aβ fibril samples (both before and after incubation with resveratrol) were also heated (100 °C, 10 min) as loading controls. Van., vanillin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RFU, relative fluorescence units.
FIGURE 8.
FIGURE 8.
Aβ assembly and resveratrol remodeling pathways. Aβ matures into A11-positive, soluble oligomers that are toxic, largely unstructured, ThT-negative, and SDS-soluble. For quiescent assembly conditions, soluble prefibrillar oligomers mature into non-toxic oligomers that are SDS-resistant, ThT- and A11-negative, and non-toxic. For agitated assembly conditions, soluble oligomers mature into OC-positive fibrillar intermediates that are SDS-resistant, ThT-positive, A11-negative, β-sheet rich, and mildly toxic. For agitated assembly conditions without salt, OC-positive fibrillar intermediates mature into amyloid fibrils that possess similar properties to fibrillar intermediates but are also seeding-competent and are more resistant to resveratrol (Res.) remodeling. Resveratrol remodels Aβ soluble oligomers, fibrillar intermediates, and amyloid fibrils into high molecular weight aggregates that are SDS-resistant, negative for multiple conformational probes (OC and A11 antibodies, and ThT), unstructured, seeding-incompetent, and non-toxic relative to Aβ monomers.
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