19 February 2010. Among theories that espouse something besides Aβ as the primary instigator of Alzheimer disease, the calcium dysregulation hypothesis has remained in vogue for some time (see ARF Live Discussion). New research offers more evidence for overlap between the amyloid and calcium theories. In this week’s Journal of Neuroscience, Brad Hyman, Massachusetts General Hospital, Charlestown, and colleagues report that Aβ leads to misshapen neurons and spine loss, in part by activating the calcium-dependent phosphatase calcineurin (CaN). Blocking CaN signaling in AD mice prevents these morphological abnormalities. Furthermore, Aβ may trigger calcium changes with potentially far-reaching effects beyond those seen in neurons. As described in an ASN Neuro paper published online January 25, Gabriel Silva and colleagues at the University of California, San Diego, have developed an astrocyte culture system whereby Aβ peptides induce short-lived calcium waves associated with later effects on proteins that mark gliosis. Together, the studies raise the possibility that calcium signaling carries out much of Aβ’s dirty work in neurodegenerative disease.
Several scientists commented on the thoroughness of the study, which moves from cultured neurons to in vivo models and offers a potential clinical connection to boot—a rarity in the basic science literature. “They tried to connect some major dots,” said Beth Stutzmann of Rosalind Franklin University in North Chicago, Illinois. “I was impressed at how dense the data was surrounding a very specific topic.”
Having shown in vivo that neurons near Aβ plaques lose their spines and become deformed (Spires et al., 2005; Meyer-Luehmann et al., 2008 and ARF related news story), Hyman’s team used these morphological changes as a readout for Aβ toxicity and designed the current study to probe how they come about. They had a hunch that calcineurin would be involved. Previous work has implicated the enzyme in synaptic plasticity (Winder and Sweatt, 2001) and learning and memory (Klee et al., 1979), and dialing calcineurin up or down in mice can impair or strengthen memory, respectively (Malleret et al., 2001; Mansuy, 2003). Moreover, the Hyman lab imaged neurons in AD mice and found elevated intracellular calcium in dendritic spines that fizzle when in close proximity to plaques (Kuchibhotla et al., 2008 and ARF related news story).
In the present study, first author Hai-Yan Wu and colleagues developed an in vitro model to study the mechanisms behind Aβ-induced morphological changes. Cortical neurons from Tg2576 embryos make high levels of Aβ40 and Aβ42, and the researchers drove up internal calcium levels in wild-type neurons by exposing them to conditioned media (CM) from Tg2576 cultures. Treated wild-type cells developed morphological abnormalities similar to those reported earlier in AD transgenic neurons (Spires et al., 2005; Meyer-Luehmann et al., 2008), and had increased calcineurin activation, as measured by enhanced nuclear localization of a key calcineurin substrate, nuclear factor of activated T cells, cytoplasmic 4. NFATc4 did not over-accumulate in the nuclei of wild-type neurons treated with Tg2576 conditioned media that was immunodepleted using Aβ-specific antibodies, confirming the NFATc4 effect was Aβ-induced.
Furthermore, Hyman’s team could prevent the neurodegenerative morphologies by blocking calcineurin activity—either by transfecting cells with an inhibitory peptide (AKAP79), or by treating them with a potent calcineurin inhibitor (FK506) or with a peptide that blocks calcineurin-NFAT interaction (VIVIT). They showed this in Tg2576 neurons and Tg2576 CM-treated wild-type neurons.
The researchers demonstrated that calcineurin/NFAT signaling is not only essential, but also sufficient, for mediating Aβ’s ill effects on neuronal morphology. Even in the absence of Aβ, they could induce the dendritic abnormalities and spine loss by expressing a 45 kD constitutively active calcineurin fragment in cultured wild-type neurons.
Moving to in vivo models, Wu and colleagues injected a fluorescence-labeled version of this calcineurin fragment into the brains of wild-type mice and saw the same morphological phenotypes using multiphoton microscopy. To show the converse, they injected AD transgenic mice (APP/PS1) with the calcineurin inhibitory peptide AKAP79 and found milder neuronal and dendritic abnormalities relative to AD mice injected with a vector control.
To top things off, the authors made a case that these findings could mean something clinically. Analyzing cortical brain samples from six AD patients, they found increased nuclear expression of NFATc4 and of the 45 kD constitutively active form of calcineurin, compared with the same number of control brain specimens.
Stutzmann’s lab has plasticity and physiology data that also suggest a role for calcineurin in Aβ-induced neurodegeneration. In studies with brain slices from wild-type and triple transgenic (3xTg) AD mice, manipulation of intracellular calcium stores creates vast differences in long-term depression, she told ARF. Stutzmann presented preliminary aspects of this data at last year’s Society for Neuroscience meeting.
Work by Chris Norris, University of Kentucky, Lexington, and colleagues offers further support for the importance of calcineurin/NFAT signaling in Aβ toxicity. At SfN, his team reported that hippocampal tissue from people with mild cognitive impairment (MCI) have elevated levels of NFAT1 as well as the 45 kD calcineurin fragment relative to healthy controls (Abdul et al., 2009 and ARF related news story). “It fits very nicely with the story of calcineurin being activated during the course of AD,” Norris said. “It seems to be happening early in the disease.” He and colleagues have rodent data suggesting that calcineurin activity is heightened even during normal aging (Foster et al., 2001; Norris et al., 2005).
Calcineurin/NFAT pathways may have connections with AD beyond direct activation by Aβ. Recent work suggests that NFAT1 increases Aβ levels by regulating transcription of BACE1, one of the enzymes needed to cleave Aβ peptide from its precursor (Cho et al., 2009). In ongoing studies, Norris and colleagues are selectively targeting NFAT isoforms in astrocytes of APP/PS1 mice and getting “really interesting results on synaptic function, plasticity and neuroinflammation,” he told ARF.
The ASN Neuro paper by UCSD researchers also suggests a key role for calcium signaling in astrocytes. Demonstrating in rat astrocyte cultures what Brian Bacskai, Hyman, and other Mass General colleagues reported for in vivo models last year (Kuchibhotla et al., 2009 and ARF related news story), Silva’s team, led by first author Siu-Kei Chow, showed that low (5 micromolar) concentrations of Aβ1-40 can induce transient intercellular calcium waves that associate with increases in several markers of reactive gliosis detectable half a day later. “What really struck me about this was the time course. You have Aβ setting up later events within minutes, essentially,” said Terrence Town, Cedars-Sinai Medical Center, Los Angeles, California, in an interview with ARF (see Town’s additional written comment below). The calcium waves appeared in 10 minutes and disappeared in about an hour, he said, “and yet at 12 hours you have these effects on GFAP and S100B expression. That, to me, is the most salient point.”
How far these later events extend remains an open question. “I wonder if there are even more protracted events that are set up by these low micromolar levels of Aβ,” Town said, noting past research suggesting that Aβ-induced increases in intracellular calcium can make neurons susceptible to excitotoxicity (Mattson et al., 1992). “It raises the question of whether these calcium waves set in motion a series of events culminating in neurotoxicity,” he said.
This possibility also draws support from a recent paper by Town and colleagues suggesting that the current findings may be relevant to AD. They show that forced expression of human S100B leads to exacerbated AD-like pathology in Tg2576 mice (see ARF related news story on Mori et al., 2010).—Esther Landhuis.
Wu HY, Hudry E, Hashimoto T, Kuchibhotla K, Rozkalne A, Fan Z, Spires-Jones T, Xie H, Arbel-Ornath M, Grosskreutz CL, Bacskai BJ, Hyman BT. Amyloid beta Induces the Morphological Neurodegenerative Triad of Spine Loss, Dendritic Simplification, and Neuritic Dystrophies through Calcineurin Activation. J Neurosci. 2010 Feb 17;30(7):2636-2649. Abstract
Chow SK, Yu D, MacDonald CL, Buibas M, Silva GA. Amyloid beta-peptide directly induces spontaneous calcium transients, delayed intercellular calcium waves and gliosis in rat cortical astrocytes. ASN Neuro. 2010 Jan 25;2(1): e00026. Abstract