Alavez started with an interest in amyloid-binding dyes and related molecules, he said, because “these chemical compounds have a particular chemical structure that allows them to physically interact with proteins prone to aggregate.” Perhaps, he hypothesized, they might bind to small oligomeric aggregates and interfere with amyloid formation. Others have proposed that polyphenols (Porat et al., 2006) and the dye Congo red (Frid et al., 2006) can block amyloid fibrillization.

The first experiment was a flop; Alavez treated worms with the dyes and the worms died at the normal time. A year later, he decided to try again with higher drug concentrations. This time, the worms kept wriggling for four, even five weeks; five of the 10 compounds he tried created Methuselah nematodes. Thioflavin T extended lifespan by up to 78 percent; curcumin and another molecule, rifamicin, added up to 45 percent to a worm’s days. And they were active days, too; age-matched worms on thioflavin were much more motile than their untreated counterparts (see videos).

Control: Untreated worms that survive to 20 days barely move.

Treated: 20-day-old worms treated with thioflavin T still slither across the plate.

Image credits: Silvestre Alavez, Buck Institute for Research on Aging, Novato, California

The researchers wondered if thioflavin T might also battle diseases of aging, such as Alzheimer’s disease. They used two worm models to test this idea: one expressing an amyloid-β peptide and the other producing a polyglutamine peptide. Normally, these worms become paralyzed. Under thioflavin or curcumin treatment, half or fewer of the amyloid-β worms froze in their tracks. The effect on the polyglutamine strain was not as dramatic, but was statistically significant.

Finally, the researchers wondered whether thioflavin T directly represses aggregation, or if it interacts indirectly with worm genes and proteins to extend lifespan. RNAi interference of several genes blocked thioflavin T’s ability to reduce paralysis, indicating those genes were part of a thioflavin T-induced pathway. Chaperones and proteins involved in ubiquitin-mediated proteolysis and lysosomal protein degradation were essential to the thioflavin T’s effects on paralysis. Conversely, RNAi for some genes increased the drug’s effects, indicating that those proteins normally stand in the way of the drug’s activity. According to these experiments, Daf-16, a FOXO-like transcription factor involved in aging (see ARF related news story on Wolff et al., 2006 and Berman et al., 2006), represses this life-prolonging pathway. Skn-1, a transcription factor that promotes stress response and longevity was necessary for thioflavin T’s efficacy. Skn-1 is the homologue of mammalian Nrf-2; it promotes stress resistance and longevity.

In another set of experiments designed to measure lifespan, the researchers focused on Daf-16, Hsf-1, and Skn-1—transcription factors needed for normal lifespan. Hsf-1 is a chaperone that promotes disaggregation (see ARF related news story). A mutation in any of these proteins abolished thioflavin T’s life-extending effects.

Lithgow proposes that aging itself results from a loss of protein homeostasis, and neurodegeneration is an extreme version of this process (see also ARF related news story on David et al., 2010). He and Alavez found that an amyloid-specific antibody, A11, binds to unknown protein structures in elderly worms even if they do not express amyloid-β, suggesting there are many kinds of aggregates in old animals.

Lithgow imagines that in aging neurons, the production of misfolded protein “garbage” simply outpaces the trash removal system. Large aggregates like amyloid-β are the most noticeable fallout from poor homeostasis, but many other proteins may also be affected. “Who knows how many other oligomers we have forming in our bodies that never fibrillize into visible lesions…yet still cause problems?” agreed Rudy Tanzi of Massachusetts General Hospital, who was not involved in the study.

The mechanism by which thioflavin improved these worms’ lives, however, remains unknown. Tanzi, who is co-founder of a company developing small molecules that transport metal ions, speculated that the dye dissolved amyloids, freeing up metal ions that the cell needs (see ARF related news story on Adlard et al., 2008). Lithgow suspects that the dyes, as a first step, bind to amyloid and other misfolded proteins. “By some means, this may trigger a genomic response,” he said. However, Alavez was unable to show conclusively that thioflavin bound to amyloids. In immuno-localization studies of amyloid-β, he observed that the dye was located near the aggregates, but also all over the cell. He fears that fixing and staining the tissue may have rinsed away some of the thioflavin, clouding the results.

“I am not aware that thioflavin T slows Aβ aggregation…most people use this environment-sensitive fluorophore because it doesn’t alter the kinetics of aggregation directly,” wrote Jeff Kelly of The Scripps Research Institute in La Jolla, California, in an e-mail to ARF. “It is not clear to me that this compound quantitatively alters aggregate load…the ability of thioflavin T to interact with and promote the proper folding of misfolding-prone proteins seems unlikely,” added Kelly, who was not part of the study.

However it does it, thioflavin appears to activate the cell’s stress response machinery, which supports protein homeostasis and keeps the worms alive. Several investigators not involved in the study questioned what that mechanism might be. “It is not clear exactly how these compounds turn on these pathways,” said Chris Link of the University of Colorado in Boulder. He noted that the effect could be direct or indirect. The mechanism might, but does not necessarily have to, involve amyloid binding, said Greg Cole of the University of California in Los Angeles. Alternatively, he posited, thioflavin T and the like might simply be toxins, to which the cell responds by ramping up protective mechanisms. Indeed, high doses of thioflavin T were lethal, and worms on lower doses had reduced fertility.

Basic Yellow 1 joins curcumin (see ARF related news story on Yang et al., 2004), Congo red, and methylene blue (see ARF related news story and Necula et al., 2007)—not to mention red wine (see ARF related news story on Ladiwala et al., 2010), coffee (Dostal et al., 2010), and blueberries (see ARF related news story on Joseph et al., 2003)—in a veritable rainbow of potentially protective compounds. Another colorful stain for amyloid, and an analogue of thioflavin T, is Pittsburgh compound B, or PIB. Radioactive PIB lights up plaques on a PET scan (see ARF related news story on Bacskai et al., 2007). “That is encouraging; at least someone has actually put that into a person,” Lithgow said. Neither thioflavin T nor curcumin cross the blood-brain barrier, but other related life-prolonging compounds do, Alavez said.

“It does not want to make me treat people with thioflavin T derivatives just yet,” cautioned Bill Klunk of the University of Pittsburgh, who was not involved with the study. “It is a first step of many more needed steps.”—Amber Dance.

Reference:
Alavez S, Vantipalli MC, Zucker DJS, Klang IM, Lithgow GJ. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature, 2011 March 30. Abstract