How do Aβ oligomers cause synapses to wither in Alzheimer’s disease? In the April 10 Neuron, researchers led by Franck Polleux at the Scripps Research Institute, La Jolla, California, propose a pathway that leads from Aβ oligomers via two kinases, including AMP-activated kinase (AMPK), to tau phosphorylation and eventually to synapse loss. In a mouse model of AD, blocking either kinase or preventing tau phosphorylation at a single site prior to development of plaques preserved dendritic spines. The data suggest that this pathway plays a critical role in the synaptotoxic effects of Aβ, Polleux told Alzforum. In addition, “It is one of the first really clear kinase pathways to directly link Aβ oligomers to tau phosphorylation,” he said. It remains to be seen whether blocking this pathway would stop other aspects of AD pathology, such as amyloid accumulation and neuron loss, he noted. The findings, all from mouse studies, could have implications for proposed AD trials of metformin, an AMPK activator.
Other researchers found the data intriguing. “The paper makes a compelling case linking AMPK activation to spine degeneration in vivo,” Sung Yoon at Ohio State University, Columbus, told Alzforum.
AMPK is a key metabolic sensor in many cell types. It drew Polleux’s attention because it has been shown to be highly activated in neurons of AD brains, as well as in several other tauopathies, and other neurodegenerative diseases, including amyotrophic lateral sclerosis, and Huntington’s (see Vingtdeux et al., 2011). A previous study by Yoon found that Aβ oligomers switch on AMPK, which in turn ramps up Aβ production in an apparent feed-forward loop (see ARF related news story). Meanwhile, researchers led by David Carling at Imperial College London, U.K., reported that after Aβ activates AMPK, the kinase phosphorylates tau at several sites (see Thornton et al., 2011). While these findings implicate AMPK in AD pathology, other work contradicts this, showing that the kinase stimulates autophagy and helps degrade Aβ in APP/PS1 mice (see Vingtdeux et al., 2010; Vingtdeux et al., 2011).
To study the role of AMPK, first author Georges Mairet-Coello prepared synthetic Aβ oligomers using the method developed at William Klein's lab at Northwestern University, Evanston, Illinois (see Lambert et al., 1998). They confirmed that this preparation activated AMPK in mouse hippocampal and cortical neuronal cultures. As expected, after 24 hours of exposure to 1 μM Aβ, the neurons lost dendritic spines. The authors prevented this loss by inhibiting calcium/calmodulin-dependent kinase kinase 2 (CAMKK2), which normally turns on AMPK. Likewise, neurons from CAMKK2 or AMPK knockout mice kept their synapses when exposed to Aβ. This suggested that both kinases are necessary for Aβ to poison synapses. The authors then demonstrated that activation of this pathway even in the absence of Aβ sufficed to wither spines. Overexpression of CAMKK2 or AMPK, or activation of AMPK by several pharmacological means, mimicked the toxic effects of Aβ, they report.
Polleux and colleagues wondered if they could prevent synapse loss in vivo by blocking this pathway. They used in utero electroporation to express a dominant-negative, kinase-dead version of either CAMKK2 or AMPK in hippocampal neurons of embryos of the APP transgenic J20 line. After the embryos were born, the scientists counted spines in young mice at three months of age. While untreated transgenics had lost a significant fraction of their spines compared to wildtype controls, the mice that expressed the “dead” kinases, which block the endogenous active ones, had normal numbers of spines. At this age, the animals do not yet have amyloid plaques but they do overexpress mutant human APP and generate oligomeric Abeta.
How might AMPK be damaging spines? The authors linked AMPK activation to tau pathology. First they confirmed previous findings that AMPK phosphorylates tau at serine residue S262, in tau’s microtubule-binding region. Phosphorylation at this location makes the protein detach from microtubules, and has been hypothesized to prime pathological hyperphosphorylation (see Biernat et al., 1993; ARF related news story on Nishimura et al., 2004). When the authors expressed a mutated form of tau, S262A, which cannot be phosphorylated at this site, spines stayed healthy after exposure to Aβ both in vitro and in vivo. Tau phosphorylation at S262 therefore seems to mediate the synaptotoxic effects of Aβ, the authors conclude. Previous studies reported that early in AD, free tau wanders into dendrites, where it overexcites and poisons synapses through Fyn kinase (see ARF related news story on Ittner et al., 2010; ARF related news story on Zempel et al., 2010; Hoover et al., 2010).
In future work, Polleux will cross AMPK knockout mice with several different AD mouse models to examine the effect of chronically suppressing the kinase. Besides protecting synapses, would this approach prevent other features of AD, such as amyloid accumulation, neuron loss, and cognitive decline? Polleux will also test whether AMPK acts through other downstream mechanisms besides tau phosphorylation. If blocking AMPK can preserve synapses, does this have therapeutic potential? Polleux does not believe AMPK itself would make a good therapeutic target because it does so many things in different cell types. CAMKK2 might make a better drug target, as it is highly enriched in neurons, he suggested.
While these findings paint activated AMPK as a cause of AD pathology in J20 mice, previous studies found that turning on this kinase clears amyloid in APP/PS1 mice, suggesting the kinase can have divergent effects on synapses and amyloid. Sylvain Lesné at the University of Minnesota, Minneapolis, told Alzforum it will be interesting to test whether AMPK activation also curbs amyloid plaques in the J20 mice. For her part, Yoon suggested that the effects of AMPK may depend on environmental triggers. She pointed to a recent paper that found that the AMPK activator metformin affects worms differently depending on what bacteria the animals eat (see Cabreiro et al., 2013).
The question of what AMPK does has important clinical implications, as metformin is a widely-used treatment for Type II diabetes that is being considered for AD. Led by Jose Luchsinger at Columbia University, at least one phase 2 trial in amnestic MCI has already been completed, although no results have been reported (see also Luchsinger, 2010). “Before metformin goes into large clinical trials in the U.S., I think we need to test whether AMPK activation is beneficial or detrimental,” Yoon said. Polleux agreed, noting that, “In our hands, any drug, including metformin, that activates AMPK is as synaptotoxic as Aβ.” The use of metformin in people should be re-evaluated to make sure it is not harming cognition, Polleux suggested.
Lesné told Alzforum that the current results need repeating to find out how central the proposed mechanism is to AD pathogenesis. “There is pretty good evidence that there’s probably not just one pathway connecting Aβ oligomers to tau dysfunction and phosphorylation, but multiple pathways,” he said. He also would have liked to see the authors more extensively characterize the Aβ oligomers they used in vitro. Researchers in the field recommend using multiple rigorous techniques and oligomer-specific antibodies to determine the exact species of Aβ used in experiments (see Alzforum webinar). This allows other labs to reproduce findings, which has been lacking in Aβ oligomer research. Likewise, Lesné said, it would be interesting to know precisely what mixture of Aβ species is present in the three-month-old J20 mice, and what effect each type has on tau. “The challenge ahead lies in determining which Aβ oligomer is acting in which pathway, and what consequences each has on tau phosphorylation, oligomerization and aggregation,” he told Alzforum.—Madolyn Bowman Rogers
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