One Small Molecule Detoxifies Different Forms of Aβ

Aggregates of Aβ peptide have long been known to be a major player in Alzheimer disease (AD) neurodegeneration, but in recent years, mounting evidence has pointed to soluble oligomers of Aβ, rather than amyloid fibrils, as the most toxic species. This has potentially complicated the development of AD treatments, since an effective therapeutic molecule might need to have activity for several harmful species—but there’s hope. In the May 28 Journal of Biological Chemistry, researchers led by Peter Tessier at Rensselaer Polytechnic Institute, Troy, New York, report that a small molecule can rather specifically remodel several kinds of toxic Aβ aggregates, including soluble oligomers and fibrils, but not non-toxic forms. The authors tested resveratrol, a small polyphenolic compound present in red wine. They discovered that resveratrol selectively converted three toxic Aβ conformers into high-molecular-weight, “off-pathway” aggregates that were no longer toxic in a cellular assay. Their results suggest that toxic Aβ species, despite their differences in structure, may have some underlying biochemical similarity that could make them susceptible to a single therapeutic agent. It also adds to the growing body of evidence that Aβ toxicity can be reduced by forming larger Aβ aggregates, rather than smaller ones.

Resveratrol is only one of a suite of small molecules that have garnered interest for their ability to break up or prevent harmful Aβ aggregates. Researchers led by Giulio Pasinetti at the Mount Sinai School of Medicine in New York have shown that polyphenolic compounds derived from grape seeds can inhibit Aβ aggregation in vitro and in vivo (see ARF related news story on Wang et al., 2008). Curcumin, a spice found in curry, has been shown by Greg Cole, Sally Frautschy, and colleagues at the University of California, Los Angeles, to have the ability to disassemble amyloid plaques and inhibit Aβ oligomerization (see ARF related news story on Yang et al., 2004). Another compound of great interest is epigallocatechin gallate (EGCG), a polyphenol found in green tea, which has been demonstrated by Erich Wanker and others in Germany to steer Aβ peptides into high-molecular-weight, unstructured, non-toxic forms (see ARF related news story on Ehrnhoefer et al., 2008). Resveratrol has been previously shown to inhibit amyloid fibril formation and reduce Aβ toxicity (see Feng et al., 2009 and Rivière et al., 2007), but has been disappointing as a therapeutic, in part because it does not cross the blood-brain barrier.

Despite increasing data demonstrating the Aβ remodeling powers of small polyphenolic compounds, little is known about how these molecules exert their effects, and why they act on some types of aggregates and not others. Researchers led by Charlie Glabe at the University of California, Irvine, discovered that small molecules can selectively act on specific Aβ forms (see Necula et al., 2007), but how they do this remains unclear. Likewise, it’s not yet known why some forms of aggregated Aβ are toxic, while others are not. To try to tease apart these structural questions, Tessier and colleagues chose to use resveratrol as a chemical probe.

“We still don’t understand how differences in protein conformation confer differences in toxicity,” Tessier said. “Molecules like resveratrol can be helpful in identifying what are the conformational or structural features of toxic species relative to non-toxic conformers.”

Building on work by the Glabe lab, first author Ali Reza Ladiwala began by developing methods for preparing apparently homogenous populations of specific Aβ conformers. By varying the incubation conditions, the authors were able to induce a solution of monomeric Aβ to form either soluble toxic Aβ oligomers, toxic fibrillar intermediates, or toxic amyloid fibrils. The authors confirmed the identity of these species using the A11 antibody developed in Glabe’s lab, which specifically recognizes soluble toxic Aβ oligomers (Kayed et al., 2003). They also used the Glabe lab’s “officer” OC antibody and thioflavin T, which are specific for both fibrillar intermediates and mature fibrils.

Intriguingly, after two days of incubation, soluble toxic oligomers spontaneously transformed into non-toxic “off-pathway” oligomers. These were insoluble and no longer bound A11, yet they were structurally indistinguishable from toxic oligomers. Using circular dichroism spectrometry, the researchers determined that both toxic and non-toxic Aβ oligomers possessed random coil secondary structures, while fibrillar intermediates and amyloid fibrils were both rich in β-sheets. The authors also used atomic force microscopy to directly visualize the size and structure of the aggregates.

The authors then added resveratrol to each solution. They discovered that resveratrol converted soluble toxic oligomers, fibrillar intermediates, and amyloid fibrils into a high-molecular-weight, unstructured species that was not toxic in a PC12 cell culture. Resveratrol had no effect on non-toxic Aβ oligomers, even though these possess the same secondary structure as the toxic ones.

Toxic and non-toxic Aβ oligomers “clearly have some difference in structure that must encode their difference in toxicity,” Tessier said. Another structural clue to toxicity is that the β-sheet rich fibril and the random coil soluble oligomer are both recognized by resveratrol and converted into the same non-toxic end product, Tessier said. “Our work starts to suggest there must be structural or conformational similarities between [these forms] that have not been realized previously.”

This finding adds to the body of evidence suggesting that high-molecular-weight Aβ conformers are often less toxic than smaller species. For example, research led by Andrew Dillin at The Salk Institute, La Jolla, California, on the effects of the insulin signaling pathway has shown that bundled Aβ is less toxic than soluble Aβ, and that Aβ toxicity can be reduced by aggregation into higher-molecular-weight species (see ARF related news story on Cohen et al., 2009 and ARF related news story on Cohen et al., 2006). The new resveratrol results “are in line with the protective effects [of suppressing insulin signaling] that we found in worms and mice that forced toxic oligomers into less toxic fibrils,” Dillin said in an e-mail. “In the future, it will be very interesting to determine if resveratrol can also have this effect in vivo. The most interesting part of the paper is that resveratrol showed selectivity to toxic Aβ species, but not non-toxic species.”

David Teplow, University of California, Los Angeles, found the suggestion that resveratrol can have similar effects with different toxic oligomers especially interesting. “In the future, it would be helpful to develop a much more explicit structural definition and characterization of the five different Aβ assembly states, because that might help you tease out exactly what resveratrol is or isn’t doing.” Teplow said it would also be useful to know what epitopes the A11 antibody is recognizing in the soluble oligomers, which are not present in the non-toxic oligomers.

Despite resveratrol’s ability to remodel Aβ in vitro, Pasinetti cautioned that resveratrol itself should not be considered a therapeutic agent, due to its limited bioavailability. He also stressed that the concentration of resveratrol found in red wine is too small for dietary consumption, such as moderate enjoyment of red wine, to produce the desired biological effect.

Tessier does not expect resveratrol itself to become a therapeutic candidate, either. He believes it has continued value for investigating structural questions. He finds it interesting that such a simple molecule—just two aromatic groups with hydroxyls on it—is nonetheless able to selectively recognize certain Aβ conformers. His lab is now studying how the structure of resveratrol affects its remodeling activity, specifically probing the possibility that resveratrol may be interacting with aromatic side chains in Aβ. “One hypothesis we’re pursuing is that differences in packing or stacking of aromatic residues within these different Aβ conformers could be a key signature of differences in their toxicity,” Tessier said. His lab would also like to examine how other classes of small molecules with anti-aggregation activity exert their effects on toxic Aβ conformers. He believes this information could eventually guide the development of new therapeutic candidates.—Madolyn Bowman Rogers

Comments

  1. Resveratrol was reported in 8,000 publications and 7,332 biological studies so far. Most cases reported an activity in the range of 1-50 mMol for the inhibition of enzymes and protein-protein interaction. What is the underlying principle for this extremely promiscuous activity? As a 3,5-diphenol, resveratrol is less prone to be oxidized to quinoid structures. Quinones may derive from curcumin, which is teratogenic in zebrafish larvae at 2 uMol/L, or EGCG, which was classified as weakly embryotoxic.

    Thus, there seems to be a good reason for the very low oral bioavailability of most polyphenols: their brain permeation is close to zero. The Seliwanov reaction of carbohydrates and polyphenols occurs readily at 37°C in 0.1 N HCl; this will impair the oral availability of such dirty compounds already upon digestion both in rats or humans. The pharmaceutical industry is very reluctant to pursue polyphenolic lead structures. The investigation of cleaner compounds may get us to the relevant issues faster.

    References:

    Wang CC, Chu KO, Chong WS, Li WY, Pang CP, Shum AS, Lau TK, Rogers MS. Tea epigallocatechin-3-gallate increases 8-isoprostane level and induces caudal regression in developing rat embryos. Free Radic Biol Med. 2007 Aug 15;43(4):519-27. PubMed.

    View all comments by Boris Schmidt

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References

News Citations

  1. Grape-derived Polyphenols Fight Amyloid, Head to Clinic
  2. Curry Ingredient Spices Things Up by Blocking Aβ Aggregation
  3. A Fortune in Tea Leaves—Extract Blocks Amyloid Formation
  4. Long Life With Tight Plaques—Repressing IGF-1 Protects AD Mice
  5. Aggregation/Disaggregation: Longevity Genes Protect Worms Against Aβ Toxicity

Paper Citations

  1. Wang J, Ho L, Zhao W, Ono K, Rosensweig C, Chen L, Humala N, Teplow DB, Pasinetti GM. Grape-derived polyphenolics prevent Abeta oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer's disease. J Neurosci. 2008 Jun 18;28(25):6388-92. PubMed.
  2. Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, Chen PP, Kayed R, Glabe CG, Frautschy SA, Cole GM. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem. 2005 Feb 18;280(7):5892-901. PubMed.
  3. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker EE. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat Struct Mol Biol. 2008 Jun;15(6):558-66. PubMed.
  4. Feng Y, Wang XP, Yang SG, Wang YJ, Zhang X, Du XT, Sun XX, Zhao M, Huang L, Liu RT. Resveratrol inhibits beta-amyloid oligomeric cytotoxicity but does not prevent oligomer formation. Neurotoxicology. 2009 Nov;30(6):986-95. PubMed.
  5. Rivière C, Richard T, Quentin L, Krisa S, Mérillon JM, Monti JP. Inhibitory activity of stilbenes on Alzheimer's beta-amyloid fibrils in vitro. Bioorg Med Chem. 2007 Jan 15;15(2):1160-7. PubMed.
  6. Necula M, Kayed R, Milton S, Glabe CG. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007 Apr 6;282(14):10311-24. PubMed.
  7. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 2003 Apr 18;300(5618):486-9. PubMed.
  8. Cohen E, Paulsson JF, Blinder P, Burstyn-Cohen T, Du D, Estepa G, Adame A, Pham HM, Holzenberger M, Kelly JW, Masliah E, Dillin A. Reduced IGF-1 signaling delays age-associated proteotoxicity in mice. Cell. 2009 Dec 11;139(6):1157-69. PubMed.
  9. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science. 2006 Sep 15;313(5793):1604-10. PubMed.

Further Reading

Papers

  1. Urbanc B, Betnel M, Cruz L, Bitan G, Teplow DB. Elucidation of amyloid beta-protein oligomerization mechanisms: discrete molecular dynamics study. J Am Chem Soc. 2010 Mar 31;132(12):4266-80. PubMed.
  2. Yamin G, Ruchala P, Teplow DB. A peptide hairpin inhibitor of amyloid beta-protein oligomerization and fibrillogenesis. Biochemistry. 2009 Dec 8;48(48):11329-31. PubMed.
  3. Ono K, Condron MM, Teplow DB. Structure-neurotoxicity relationships of amyloid beta-protein oligomers. Proc Natl Acad Sci U S A. 2009 Sep 1;106(35):14745-50. PubMed.

Primary Papers

  1. Ladiwala AR, Lin JC, Bale SS, Marcelino-Cruz AM, Bhattacharya M, Dordick JS, Tessier PM. Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers. J Biol Chem. 2010 Jul 30;285(31):24228-37. PubMed.

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