31 May 2008. Fortune telling aside, green tea has been touted as a potential cure for a myriad of conditions, including cancer and neurodegenerative diseases such as Alzheimer and Parkinson diseases. Scientific evidence that the brew might work has just become stronger. In yesterday’s Nature Structural & Molecular Biology, researchers in Germany report that (-)-epigallocatechin gallate (EGCG), a polyphenol found in green tea, prevents both amyloid-β (Aβ) and α-synuclein from forming toxic oligomers. The work suggests that EGCG works as a generic inhibitor of amyloids, making it a potential lead for treatments of not only AD and PD but perhaps any amyloidosis.
This is not the first evidence that the polyphenol might in theory benefit AD patients. EGCG appears to block Aβ formation by stimulating α-secretase cleavage of the amyloid-β precursor protein (see ARF related news story), and it may even protect neurons against toxic forms of Aβ (see ARF related news story). How it achieves either is not clear, but researchers led by Erich Wanker at the Max Delbrueck Center for Molecular Medicine, Berlin, reported that EGCG influences the folding of mutant huntingtin and reduces its toxicity, suggesting that the polyphenol limits formation of dangerous oligomers (see Ehrnhoefer et al., 2006). Fibrillogenesis of α-synuclein, Aβ, and tau can also be prevented by polyphenols, suggesting that these compounds are particularly interesting as anti-amyloid agents.
To address the molecular basis for this anti-amyloid action, Wanker and colleagues have tested the effects of EGCG on both Aβ and α-synuclein aggregation using a variety of biochemical and biophysical methods. Their findings suggest that the polyphenol steers the peptides away from β-sheet-rich structures toward unstructured, non-toxic forms. There is growing evidence that these “off” pathways are often taken by amyloidogenic peptides, and finding ways to encourage them down that road could prove valuable therapeutically.
Joint first authors Dagmar Ehrnhoefer, Jan Bieschke, and colleagues first measured the effect of EGCG on aggregation of α-synuclein. NMR spectra showed that the polyphenol bound to the polypeptide backbone of the protein, while nitroblue tetrazolium (the dye turns blue in the presence of protein-bound EGCG) revealed that the polyphenol binds both monomers and SDS-stable oligomers. The consequences of binding were significant. The polyphenol prevented fibrillogenesis of α-synuclein as judged by thioflavin T fluorescence, and when the researchers looked at the α-synuclein aggregates in the electron microscope, they found that the usual, long (0.5-2.0 μm), 5-15 nm diameter fibrils did not form in the presence of EGCG; instead α-synuclein formed mostly spherical (~20 nm diameter), amorphous structures. Circular dichroism spectral changes that accompany aggregation of α-synuclein and are indicative of β-sheet formation were also absent when the protein was allowed to aggregate in the presence of EGCG, supporting the idea that the phenol prevents the “on” pathways that lead to formation of β-sheet-rich, toxic α-synuclein oligomers. Reactivity with the A11 antibody was also abolished when EGCG interferes with aggregation, another indication that the phenol steers the protein away from toxic pathways. A11, developed by Charlie Glabe and colleagues at University of California, Irvine, seems to recognize a common secondary structure shared by toxic amyloid of various origins (see ARF related news story).
The authors carried out similar experiments with Aβ. The nitroblue tetrazolium assay showed that EGCG binds to the peptide, and in the presence of the polyphenol the lag phase in formation of Aβ aggregates was prolonged. Electron microscopy showed that the aggregation products that did form were spherical amorphous structures rather than fibrils and, as with α-synuclein, the aggregates did not cross-react with A11.
In the case of both α-synuclein and Aβ, EGCG interfered with seeding reactions that normally accelerate the formation of fibrillar structures. For Aβ, a 5:1 ratio of EGCG to peptide was sufficient to completely suppress formation of amyloid from seeds. In both cases, too, the products formed in the presence of EGCG appeared to be non-toxic. When the researchers added the aggregates to PC12 cells they saw little effect on the reduction of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide), which is normally suppressed by toxic protofibrils and fibrils.
How EGCG works is not entirely clear. The primary sequence of the protein does not seem to be important, since the researchers found that it binds to unfolded bovine serum albumin just as readily as it binds to α-synuclein. “This suggests that EGCG targets the polypeptide main chain that is identical in all proteins and easily accessible under unfolded conditions,” write the authors. The fact that it binds to Aβ supports that idea. “On the basis of these results, we propose that EGCG should also influence the aggregation cascade of other natively unfolded polypeptides and proteins, such as islet amyloid polypeptide, tau or the prion protein,” write the authors.
Before you run off to boil the water, consider this. This work was carried out in vitro, and it is known that EGCG penetrates the brain poorly and is probably quickly metabolized by the body (see Zhu et al., 2000). Nonetheless, EGCG and perhaps other polyphenols could serve as a basis for developing more suitable compounds for therapeutic purposes.—Tom Fagan.
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 May 30; Abstract