21 February 2013. In 2008, Rember™, aka methylene blue, created a stir when the drug’s sponsors reported that it slowed cognitive decline in a Phase 2 trial for Alzheimer’s disease, while other scientists questioned that interpretation (see ARF related news story). The dye had been used for decades in various industrial, research, and therapeutic settings. In Alzheimer’s patients the dye is claimed to break up aggregates of tau, the principal component of neurofibrillary tangles, but exactly how it interacts with tau is a gray area. This month, some clues appeared in a paper published online February 11 in the applied chemistry journal Angewandte Chemie. Researchers led by Markus Zweckstetter at the Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, report that methylene blue oxidizes tau, but not in the way one might expect. "The surprise was that methylene blue oxidizes cysteine sulfhydryl groups to sulfenic, sulfinic, and sulfonic acids," Zweckstetter told Alzforum. "That keeps tau in the monomeric state, blocking the misfolding that initializes aggregation."
The Phase 2 trial has focused interest on how the dye works. Researchers confirmed that methylene blue disrupts tau aggregates (see ARF related news story; Taniguchi et al., 2005), and Zweckstetter wanted to understand the mechanism behind that interaction.
To get at it, first author Elias Akoury and colleagues turned to biophysics. They incubated tau with the dye and tracked structural changes with NMR spectroscopy. Akoury saw that methylene blue broadened dramatically the spectral component attributable to two cysteine residues in tau. Methylene blue is a known oxidation/reduction agent, and cysteines can be oxidized to disulfides. However, the spectra were not consistent with a disulfide bond. They were consistent with the sequential addition of oxygen to the cysteines to generate the three different moieties that are sulfenic, sulfinic, and sulfonic acid. The finding makes sense, said Zweckstetter, because methylene blue blocks aggregation of all isoforms of tau, even those that have two cysteines. Those isoforms can make intermolecular disulfides, which speed up aggregation. If the dye oxidized those cysteines to disulfides, then that would accelerate, not delay, formation of larger tau complexes.
Does sequential oxidation of cysteine explain how methylene blue works as a therapeutic in people? Claude Wischik heads TauRx Therapeutics, the Singapore-based biotech company that develops the drug for tauopathies including Alzheimer’s. He thinks not. In a written comment to Alzforum (see below), Wischik noted that while a redox mechanism of action is attractive, "it could only potentially explain a preventative effect on tau aggregation, but not on disaggregation of already assembled native paired-helical fragments."
Zweckstetter and coauthor Eckhard Mandelkow from the German Center for Neurodegenerative Diseases, Bonn, disagree. "In principle, any aggregation inhibitor not only inhibits (or prevents) aggregation, but also disassembles preformed aggregates, as long as the aggregates are in dynamic equilibrium with their free subunits," Mandelkow replied via e-mail. In fact, Mandelkow's group previously showed that tau aggregates disassemble to a degree in vivo when tau monomers are removed from the picture by turning off a human tau transgene in mice (see ARF related news story on Sydow et al., 2011). Zweckstetter noted that many compounds that block tau aggregation also promote disaggregation with about the same potency, or IC50 (see both full comments below).
The strongest evidence that methylene blue prevents tau aggregation by oxidizing cysteines comes from replacing the cysteines with other amino acids. Cysteine-free tau does aggregate, and Zweckstetter was unable to prevent these mutant proteins from doing so by incubating them with the dye, even at high concentrations. "We unambiguously proved that methylene blue-driven modification of the tau cysteine is essential for inhibition of tau aggregation," he told Alzforum.
The story gets more complicated, however. Both in vivo and in vitro, methylene blue loses methyl groups to yield the derivatives azure A and azure B. Both of these also oxidize tau cysteines in the same way as the parent dye, but there's a twist. The azure compounds also bind to aromatic amino acids and thereby block β-sheet stacking. "These compounds strike a double hit to prevent tau aggregation," said Zweckstetter.
Do these new mechanisms instill confidence in current trials of a methylene blue formulation in people with AD or frontotemporal lobar degeneration (see ARF related news story)? The field has seen purported amyloid busters fail before (see ARF related news story), and skepticism of new attempts to bust protein aggregates is rife. "The field will have to remain suspicious for a while," Mandelkow told Alzforum. "However, contrary to other drugs, methylene blue has a long history of being well tolerated. It is efficacious in a number of disease states, it is FDA approved [for methemoglobinemia and other non-neurodegenerative disorders], and it is cheap. Our experiments with transgenic mice so far suggest that cognitive decline can be prevented in principle. Whether it can also be reversed remains to be seen," he added.
Researchers involved in the upcoming trials declined to comment, as did some others who see the field as a quagmire. John Trojanowski from the University of Pennsylvania, Philadelphia, cautioned that the rationale behind the drug remains murky. "It has a wonderful safety profile, since it has been around a long time, so aside from turning urine and sclera blue/green, there should be few adverse events. But clearly, it has an undefined mechanism of action and appears to have pleiotropic effects, from oxidizing tau to interfering with microtubule assembly," Trojanowski told Alzforum via e-mail.
Peter Davies, a tau expert at Albert Einstein College of Medicine, New York, had a slightly different take. Davies wondered how important it is to block tau aggregation versus soluble oligomers, an open question for both tau and amyloid-β (see ARF related news story). "The field seems to have drifted towards the idea that it is not the aggregates that are the problem," he wrote to Alzforum, "I have not been very impressed with the data from humans that I have seen so far, but the Phase 3 studies, if they were clearly positive, would be a very strong argument that aggregation was an important event in human disease." See full comment below.—Tom Fagan.
Akoury E, Pickhardt M, Gajda M, Biernat J, Mandelkow E, Zweckstetter M. Mechanistic Basis of Phenothiazine-Driven Inhibition of tau Aggregation. Angew Chem Int Ed Engl. 2013 Feb 11. Abstract