26 July 2010. Amyloid plaques and neurofibrillary tangles of tau proteins are the two classic hallmarks of Alzheimer disease, but the connection between their two respective proteins—Aβ and tau—has remained mysterious. Now for the first time, a paper appearing July 22 in Cell details a molecular mechanism that links tau to Aβ toxicity at the synapse. Researchers led by Jürgen Götz and Lars Ittner at the University of Sydney, Australia, show that tau has a previously unknown role in the dendrite. Tau targets the Src kinase Fyn to the N-methyl-D-aspartic acid (NMDA) receptor, these authors report. This allows tau to mediate Aβ-induced excitotoxicity at the synapse. When tau is deleted or mistargeted in an AD model mouse, survival and memory improve to those of wild-type levels, although plaque burden and Aβ levels do not change. The same group shows, in a July 19 PNAS paper, that hyperphosphorylation of tau in a tau mouse model can be successfully treated with sodium selenate, leading to rescue of memory, motor performance, and neurogeneration. Both findings suggest promising new tau-based strategies for the treatment of dementias. Lars Ittner presented these data on July 15 in the very last session of the International Conference on Alzheimer’s Disease in Honolulu, Hawaii, where a diminished crowd of diehards gave it a favorable reception.
“I am very enthusiastic about th[is] paper for several reasons,” Lennart Mucke of the University of California San Francisco, wrote to ARF (see full comment below).
During the last decade, researchers led by Mike Hutton, then at the Mayo Clinic in Jacksonville, Florida, and Jürgen Götz, who at the time worked with Roger Nitsch at the University of Zurich, have shown that Aβ can worsen tau pathology, and therefore Aβ must act upstream of tau (see ARF related news story on Götz et al., 2001 and Lewis et al., 2001). Aβ is known to have excitotoxic effects in people and in animal models (see Amatniek et al., 2006; Palop et al., 2007 and ARF related news story; and Minkeviciene et al., 2009 and ARF related news story). The story leapt a step forward again when researchers led by Erik Roberson and Lennart Mucke at the University of California in San Francisco tied tau to excitotoxicity by showing that the removal of tau protein in an AD mouse model protected neurons from Aβ and other excitotoxic insults (see ARF related news story on Roberson et al., 2007). Nonetheless, it was not clear how tau mediated excitotoxicity.
One clue came from the fact that tau protein contains a binding site for Fyn kinase. Fyn mills around at the post-synaptic density in wild-type mice, where it phosphorylates the 2b subunit of the NMDA receptor (NR2b). This strengthens the interaction of the NMDA receptor with the post-synaptic density protein 95 (PSD-95), and leads to excitotoxic downstream signaling. Overexpression of Fyn increases Aβ toxicity (see Chin et al., 2004 and Chin et al., 2005).
First authors Ittner and Yazi Ke looked for Fyn in a tau knockout (KO) mouse, and found it to be reduced by two-thirds at the synapse. This indicates that tau plays an important role in targeting Fyn to the synapse, although some Fyn arrives at the synapse independently of tau. Ittner and colleagues then generated a transgenic mouse that expresses a truncated version of the tau protein (Δtau74) under a neuronal promoter. The truncated version lacks microtubule-binding domains and cannot form aggregates, but includes the amino-terminal projection domain with its binding site for Fyn kinase. Truncated tau localizes to the membrane of the cell body, but is not present in dendrites, and so is incapable of targeting Fyn to the synapses themselves.
Ittner and colleagues found that in the Δtau74 transgenic mouse, Fyn was down by three-quarters at the synapse, despite the presence of endogenous tau. It turns out that truncated tau acts as a dominant-negative mutation by competing with endogenous tau to bind Fyn and mistarget it. As evidence of this, in the Δtau74 mouse, co-immunoprecipitation with Fyn mostly pulls down truncated tau, not endogenous tau. As might be expected with less Fyn at the synapse, in both Δtau74 and tau-null mice there was less phosphorylation of NR2b, and fewer NR subunits co-immunoprecipitated with PSD-95, indicating a weaker interaction. Importantly, Δtau74 and tau-null mice were less susceptible to seizures, which result from overstimulation. Despite these changes, synaptic currents were normal in both tau mutant strains.
The authors then looked at what effect these changes in tau might have on AD by crossing the two tau strains with an AD mouse model (APP23), both independently and in combination. Both tau deletion and transgenic tau independently improved the memory of APP23 mice to wild-type levels. Both tau double-crosses also survived longer than the APP23 mice, which have a premature mortality phenotype, and in fact, the combination of transgenic tau with endogenous tau deletion fully rescued survival. The tau crosses also reduced excitotoxicity in the APP23 mice, decreasing the severity of seizures. Significantly, Aβ levels and plaque load were unchanged in these double-crosses, indicating that tau acts downstream of Aβ. Importantly, Ittner and colleagues used a different tau KO strain and different AD mouse strain than the 2007 study by Roberson et al., and yet they saw the exact same effect, demonstrating that this finding is robust and not dependent on a particular mouse strain.
These results implied that the NMDA receptor-PSD-95 interaction is a crucial feature of Aβ-induced excitotoxicity. To test this, the authors made use of a small peptide, Tat-NR2B9c, which has been shown to interfere with the NMDA receptor PSD-95 interaction and is already known to reduce excitotoxicity in a mouse model of ischemia (see Aarts et al., 2002). When primary neuronal cultures from wild-type mice were treated with this peptide, the neurons became more resistant to cell death induced by Aβ treatment. The authors then used osmotic mini-pumps to infuse the peptide into APP23 mice for eight weeks. Treated mice had fewer seizures, their memory improved, and their survival returned to near wild-type levels, even several months after treatment.
The results suggest several therapeutic possibilities, Götz said. Reducing tau levels can improve symptoms in AD mouse models, and therefore might be beneficial in people. Another exciting avenue might be to treat with a peptide or, better yet, small molecule that disrupts the NR2b-PSD-95 interaction, or with the tau projection domain, since these interventions weaken excitotoxicity without interfering with normal synaptic transmission. It is especially intriguing that a narrow therapeutic window of peptide treatment led to long-term protection, Götz said, and one of the more fascinating questions he intends to pursue is what might be the biological basis of that window. Other questions include discovering how Aβ acts to exert toxicity. Does it act extracellularly or intracellularly? The authors would also like to investigate whether the interaction between tau and PSD-95 is direct or indirect, Götz said.
Previous research had shown that Aβ can accelerate an existing tau pathology, but “these new findings show that Aβ toxicity is dependent on the presence of tau, and provide a molecular mechanism for that,” Götz said. The authors have also demonstrated a critical role for tau in dendrites, in contrast to the traditional conception of tau as an axonal protein. The findings are not in disagreement with previous work on tau, Götz said. “I believe there are different cellular compartments of tau, because tau most likely is not free; it exists always bound to something.” The majority of tau is bound to microtubules, and when it becomes hyperphosphorylated, it detaches from microtubules and forms tangles in cell cytoplasm (see Geschwind, 2003). By contrast, the dendritic pool of tau is small, Götz said.
In the second paper, the authors focused on hyperphosphorylated tau, rather than dendritic tau. First author Janet van Eersel used two tau mutant mouse models: pR5 mice that develop neurofibrillary tangles (NFTs) at six months, and K3 mice that develop parkinsonism and memory impairment. The authors showed that treatment with the small compound sodium selenate reduced tau phosphorylation and eliminated NFTs in both tau mouse models, in vitro and in vivo. Selenium is a crucial trace element in brain. Some forms, such as sodium selenite, are associated with toxicity; however, the authors found no toxic effects from sodium selenate, a more oxidized form of selenium, after four months of treatment.
Protein phosphatase 2A (PP2A) is a major phosphatase responsible for tau dephosphorylation, and both the level and activity of PP2A are down in the AD brain. The authors found that selenate treatment greatly increased the amount of PP2A that co-immunoprecipitated with tau, implying that selenate stabilizes the tau-PP2A complexes, allowing the phosphatase to more readily dephosphorylate tau. To test this idea, van Eersel and colleagues crossed the pR5 mouse with the Dom5 transgenic mouse, which expresses a dominant-negative form of PP2A, and demonstrated that selenate treatment was no longer able to reduce tau phosphorylation and NFTs.
Since sodium selenate mitigates tau pathologies in several tau model mice strains, it is a promising compound for drug development, Götz said. “It amazes me how this compound works, and you see it works on several levels,” Götz said, explaining that it not only reduces tau phosphorylation and tangle formation, but it also ameliorates motor deficits in the K3 mice and memory impairment in the pR5 mice, and prevents neurodegeneration of cerebellar basket cells.—Madolyn Bowman Rogers.
Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A, van Eersel J, Wölfing H, Chieng BC, Christie MJ, Napier IA, Eckert A, Staufenbiel M, Hardeman E, Götz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s disease mouse models. Cell 2010 Jul 22. Abstract
Van Eersel J, Ke YD, Liu X, Delerue F, Kril JJ, Götz J, Ittner LM. Sodium selenate mitigates tau pathology, neurodegeneration, and functional deficits in Alzheimer’s disease models. Proc Natl Acad Sci USA. 2010 Jul 19. Abstract