27 June 2008. It’s not all about amyloid and tau. Amid a torrent of discoveries touting toxic clusters of each as pathological hallmarks of Alzheimer disease, other scientists have trod a less beaten path. They consider AD pathogenesis from a different, but not necessarily mutually exclusive, perspective—one that pins the blame on intraneuronal calcium dysregulation. Two papers published this week in Neuron and Cell give “Calcinists” reason for cheer, and may even begin to forge a more holistic AD hypothesis incorporating their doctrine along with those of Baptists and Tauists.
In the Neuron paper, scientists report how sophisticated electrophysiology techniques, along with biochemical and functional assays, suggest a new mechanism by which mutant presenilins (PS1 and PS2)—two of three known genes linked to early-onset AD—can cause intraneuronal calcium signaling to run amok. Led by J. Kevin Foskett of the University of Pennsylvania, Philadelphia, the researchers propose that mutant PS1 and PS2 boost calcium outflow from the ER through interactions with the inositol 1,4,5-trisphosphate receptor (InsP3R) calcium release channel, and that these calcium perturbations are linked with enhanced Aβ production.
Writing in Cell, researchers led by Philippe Marambaud at the Feinstein Institute for Medical Research in Manhasset, New York, identified and characterized a novel gene, CALHM1 (calcium homeostasis modulator 1), encoding a transmembrane glycoprotein with calcium channel-like properties. The researchers also found an AD-linked CALHM1 polymorphism that increased Aβ production by disturbing CALHM1-mediated calcium permeability in transfected cells. Based on these findings, they propose that CALHM1 controls Aβ levels and that variants of this gene may increase susceptibility to late-onset AD.
“These two calcium channel studies each provide a detailed, mechanistic approach for translating how gene mutations linked to AD can directly alter calcium channel function,” wrote Grace Stutzmann of Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, in an e-mail to ARF. In a recent review, Stutzmann discusses evidence for why AD pathogenesis can be seen as a lifelong “Calciumopathy” (see Stutzmann, 2007).
A large number of studies using various experimental systems—including cells from AD patients, cultured neuronal and non-neuronal cells expressing mutant PS proteins, and primary brain neurons from mutant PS transgenic mice—have established the connection between mutant presenilins and aberrant intracellular calcium regulation. To get a handle on the mechanism behind these long-standing observations, Foskett and collaborators applied a unique patch-clamp technique he developed years ago for recording ion channel activity at the single-molecule level.
First author King-Ho Cheung and colleagues did one set of recordings in Sf9 cells (an insect line expressing the InsP3R isoform most closely related and functionally similar to the type 1 channel in mammalian brain) infected with wild-type or mutant PS1 (M146L) baculovirus. The team also rigged up chicken DT40 cells (which express native InsP3R) that stably express the same PS1 proteins. In both systems, expression of mutant PS1 exerted powerful stimulatory effects on InsP3R gating activity (i.e., higher open probability and mean open time, lower mean closed time) at saturating and subsaturating IP3 levels. The researchers saw a similar trend in Sf9 cells expressing mutant PS2 (N141I) and, importantly, in mutant PS1-transfected cortical neurons from mouse brain. That these effects were seen in ER membrane patches from isolated nuclei suggested a biochemical association between PS and InsP3R—which indeed was shown, for wild-type and mutant forms of PS1 and PS2, in immunoprecipitates of Sf9 lysates. The wild-type PS proteins had some effects on InsP3R activity as well. PS1 enhanced the channel’s mean open time, though to a lesser extent than mutant PS1, and it increased the mean closed time. PS2 also increased both mean closed and open times.
To test whether the InsP3R-PS interaction had AD-relevant functional consequences, coauthors Diana Shineman and Virginia Lee, also at the University of Pennsylvania, evaluated Aβ production in APP-transfected DT40 cells that also expressed wild-type or mutant PS1. Mutant PS1 increased Aβ40 and Aβ42 levels by about two- and threefold, respectively, relative to control cells. But when the researchers made similar measurements in InsP3R-deficient cells, they saw no such Aβ enhancement by mutant PS1. These findings suggest that the changes in APP processing by mutant PS1 depend on its interactions with InsP3R.
In a phone conversation with this reporter, Foskett noted that his team had initially tested whether the presenilin proteins themselves behaved as ion channels. This idea was proposed in an earlier study by Ilya Bezprozvanny and colleagues at the University of Texas Southwestern Medical Center, Dallas (see ARF related news story). “Our preliminary experiments couldn’t find any evidence for that,” Foskett said.
For the CALHM1 channel connection, lead author Ute Dreses-Werringloer and colleagues used the Alzgene database and a bioinformatics-based tool developed by coauthor Fabien Campagne (Skrabanek and Campagne, 2001) to look for potential late-onset AD (LOAD) risk factors among genes expressed specifically in the hippocampus. Their search pulled out CALHM1, an as yet uncharacterized gene encoding a glycoprotein expressed primarily in adult brain and localized to the ER and plasma membrane. The gene shares sequence similarities with the ion selectivity filter of the N-methyl-D-aspartate (NMDA) receptor, the researchers found. In experiments with CALHM1-transfected neuronal cell lines, the scientists showed that CALHM1 forms multimers and controls cytosolic calcium concentration. Foskett was also a coauthor in this work, as his group did electrophysiology studies that helped finalize CALHM1’s identification as a calcium channel component.
In genetic screens of more than 3,400 subjects in five independent European cohorts, coauthor Jean-Charles Lambert of the Institut Pasteur de Lille, France, and colleagues determined that the frequency of a CALHM1 polymorphism was significantly higher (allele-specific odds ratio = 1.44) in AD cases.
The researchers showed that in APP-transfected cells this polymorphism (P86L) reduced cytosolic calcium levels, presumably by interfering with CALHM1-mediated calcium permeability, and increased Aβ production about twofold.
“Both papers have gone out of their way to pay homage to the amyloid hypothesis in an effort to gain legitimacy for their findings,” wrote Zaven Khachaturian in an e-mail to ARF. Khachaturian is president and CEO of the Lou Ruvo Brain Institute in Las Vegas, Nevada. As a former director of Alzheimer’s research at the National Institutes of Health, he first proposed the calcium hypothesis for AD and brain aging in the early 1980s (for the most up-to-date version, see Khachaturian, 1994). “It is interesting that both CALHM1 and PS1/PS2 mutations affect amyloid production, which in fact might be a crucial step in neurodegeneration,” Khachaturian wrote. “However, it is equally likely that the disruption in cytosolic calcium concentration in and of itself might be the culprit—without the amyloid.”
In the Calcinists’ view, intraneuronal calcium dysregulation could arise in a variety of ways, many through the normal aging process. The calcium buildup could give rise to numerous possible scenarios, Khachaturian suggested—microtubule disassembly, axoplasmic flow disruptions, activation of proteases (perhaps those that cleave transmembrane proteins such as APP), to name a few. “If the excessive amyloid is playing a toxic role, it’s doing that in addition to the underlying problems with calcium,” Khachaturian said in a phone interview.
Foskett told ARF his group has looked past amyloid and begun to examine their mutant PS-expressing cells for more general effects of calcium dysregulation—for example, generation of reactive oxygen species and activation of calcium-regulated transcription factors. “This is turning out to be a remarkable story I can’t talk about right now,” he said.
Foskett and colleagues are also starting to look for the PS/InsP3R-induced phenotypes in AD transgenic mice. One approach is to look at genes upregulated as a result of exaggerated calcium signaling in their cell culture models, and then ask whether the gene expression profile looks similar in AD mouse brains. “It’s a little bit indirect,” he said, “but it’s a way of asking whether what we’ve been doing in vitro is corroborated in vivo.”
For their part, Marambaud and colleagues are generating a CALHM1 knockout mouse. They plan to cross these animals with APP transgenic mice to see if CALHM1 deficiency boosts Aβ deposition and promotes cognitive decline, Marambaud told ARF.
As Bezprozvanny sees it, the CALHM1 and PS/InsP3R findings are just the tip of the AD-calcium iceberg. “There is no doubt future studies will uncover additional connections between calcium signaling and amyloid processing,” he wrote in an e-mail to ARF. “It may well be that the ‘calcium hypothesis of AD’ and the ‘amyloid hypothesis of AD’ are much more closely related than it initially appeared.”—Esther Landhuis.
Cheung K-H, Shineman D, Mueller M, Cárdenas C, Mei L, Yang J, Tomita T, Iwatsubo T, Lee V M-Y, Foskett JK. Mechanism of Ca2+ Disruption in Alzheimer’s Disease by Presenilin Regulation of InsP3 Receptor Channel Gating. Neuron. 26 June 2008;58:871-883. Abstract
Dreses-Werringloer U, Lambert J-C, Vingtdeux V, Zhao H, Vais H, Siebert A, Jain A, Koppel J, Rovelet-Lecrux A, Hannequin D, Pasquier F, Galimberti D, Scarpini E, Mann D, Lendon C, Campion D, Amouyel P, Davies P, Foskett JK, Campagne F, Marambaud P. A Polymorphism in CAHLM1 Influences Ca2+ Homeostasis, Abeta Levels, and Alzheimer’s Disease Risk. Cell. 27 June 2008;133:1149-1161. Abstract