Researchers have unearthed a new victim of presenilin 1’s voracious appetite. According to a study published September 6 in Science Signaling, the enzyme cleaves STIM1—a calcium sensor in the endoplasmic reticulum that promotes the replenishment of the cation when stores run low. The researchers, led by King-Ho Cheung of the University of Hong Kong, reported that familial Alzheimer’s disease (FAD) mutations in presenilin enhanced cleavage of STIM1, causing calcium influx to flag. The calcium dearth crumbled mature dendritic spines on the surface of neurons. The researchers proposed that in addition to its role in churning out Aβ peptides by processing APP, presenilin 1 also contributes to neuronal dysfunction via its destruction of STIM1.
“We have known for some time that presenilin mutations associated with FAD alter calcium signaling in neurons, but how this occurred was a mystery,” commented Stanley Thayer of the University of Minnesota Medical School in Minneapolis, who was not involved in the study. “The report by Tong et al. provides a mechanism linking the altered calcium signaling to the enzymatic activity of γ-secretase.”
Normally, neuronal stimulation triggers a release of calcium from the ER, which is then rapidly replaced by an influx of calcium through channels on the cell surface. This replenishment occurs when STIM1 and STIM2 calcium receptors in the ER sense low calcium levels and communicate with receptors on the cell surface to promote calcium influx. This so-called capacitative calcium entry (CCE), also known as store-operated calcium entry (SOCE), reportedly weakens in capacitative calcium entry cells expressing mutant presenilin (see Leissring et al., 2000; Yoo et al., 2000).
Researchers have proffered multiple explanations for this, including the idea, proposed by Ilya Bezprozvanny’s group at the University of Texas Southwestern Medical Center in Dallas, that PS1 is itself an ER calcium release channel (see Sep 2006 news; Jun 2010 news). As a postdoc in Kevin Foskett’s lab at the University of Pennsylvania in Philadelphia, Cheung published several studies supporting yet another mode of PS1 influence on calcium signaling, through an interaction with inositol triphosphate (IP3) receptors in the ER membrane that promotes ER calcium release (see Jun 2008 news on Cheung et al., 2008; Cheung et al., 2010; May 2014 news). Bezprozvanny subsequently reported that PS1 somehow lowers the expression of STIM2, thus preventing influx of calcium through that route as well (see Apr 2014 news).
First author Benjamin Chun-Kit Tong and colleagues wondered if calcium influx related to PS1’s protease activity. PS1 only gains catalytic activity as part of the larger γ-secretase complex, and despite its purported solo role as a calcium leak channel, earlier studies indicated PS1 effects on calcium homeostasis partially require catalytic activity (see Akbari et al., 2004; Bojarski et al., 2009).
The researchers started by comparing CCE in neuroblastoma cells expressing either wild-type PS1 or PS1 harboring the M146L, A246E, V97L, or A136G mutations. They depleted ER calcium by stimulating the cells with an acetylcholine receptor agonist in the absence of calcium, then monitored calcium influx after adding the cation back to the medium. Using single-cell calcium imaging, they found that cells expressing any of the mutant forms of PS1 took up calcium more slowly—and took up less of it—than cells expressing normal PS1. The same was true when the researchers performed the experiments using skin fibroblasts from AD patients with PS1 mutations. Tellingly, CCE occurred at normal levels in all the cells expressing mutant forms of PS1 when the researchers pretreated the cells with DAPT, a γ-secretase inhibitor. Together, these findings indicated that FAD-linked PS1 mutations attenuated calcium replenishment, and that PS1’s catalytic activity was required for this effect.
Further experiments indicated that STIM1 and PS1 physically interacted in the ER, and that mutated forms of PS1 prevented the oligomerization of STIM1 in response to waning ER calcium. This oligomerization allows ER-bound STIM1 to move to plasma membrane junctions, where it interacts with the ORAI-1 receptor, which ultimately triggers calcium influx through the plasma membrane. Using a combination of fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF), the researchers reported that the M146L mutation in PS1 prevented STIM1 oligomerization and bungled its hook-up with ORAI-1, thus preventing calcium influx. Again, this effect was abolished when the researchers treated the cells with a γ-secretase inhibitor.
How might mutated PS1 thwart STIM1 oligomerization? The researchers hypothesized that STIM1 was a γ-secretase substrate. Like APP, STIM1 is a type I transmembrane protein, and the amino acid sequence of its transmembrane region is similar to that of APP. In support of their hypothesis, the researchers found a very faint band, representing a potential STIM1 cleavage product, in western blots prepared from cells expressing wild-type or mutant PS1. In an in vitro γ-secretase assay, the M146L-PS1 cleaved STIM1 more efficiently than did the wild-type enzyme. This mutant also cleaved the transmembrane region of APP more efficiently than did wild-type PS1.
To determine whether PS1’s cleavage of STIM1 would exact a physiological toll, the researchers next measured the stability of mature, mushroom-shaped dendritic spines on the surface of hippocampal neurons. The influx of calcium triggered by STIM1 is necessary for the maintenance of these spines, which are crucial for memory. The researchers transfected primary rat hippocampal neurons with wild-type or M146L-PS1, and found that both the density of mature spines and the number of neurites decorated with those spines was lower in cells expressing the mutated version of PS1. When the researchers either overexpressed STIM1 or inhibited γ-secretase, dendritic spine numbers were restored to normal. These results indicated that enhanced cleavage of STIM1 by mutated PS1 reduced calcium influx, leading to the collapse of mature dendritic spines.
Incapacitated. In cells with normal PS1, STIM1 oligomerization triggers CCE and maintains mature spines. In mutant PS1 neurons, subpar calcium replenishment caused by cleavage of STIM1 destabilizes spines. [Image courtesy of Tong et al., Science Signaling, 2016.]
The findings strengthen the “calcium hypothesis of AD,” first proposed more than two decades ago, commented Jacek Kuznicki of the International Institute of Molecular and Cell Biology in Warsaw (see Khachaturian, 1994). He drew connections with the current paper and the previous observations from Bezprozvanny’s lab that FAD mutations in PS1 decrease expression of STIM2, albeit in a γ-secretase independent fashion. “The common pathophysiological outcome is the reduced number of mushroom spines in FAD PS1 neurons,” he wrote. He added that targeting such calcium entry pathways, rather than Aβ, may be an attractive therapeutic strategy.
Bezprozvanny commented that while the results jibe with those from his lab in some ways, the researchers will need to account for the variable effects of different presenilin mutations. “Many PS1-FAD mutants reduce γ-secretase activity, and it remains to be determined how the proposed model works for these mutants, as they would be expected to have reduced levels of STIM cleavage when compared to the wild type,” he wrote to Alzforum (see full comment below).
Cheung agreed it is unclear how FAD-linked mutations in PS1 would enhance STIM1 cleavage, but speculated that the unique environment of the ER might influence the substrate specificity and activity of the enzyme. He plans to test more PS1 mutations for their effects on STIM1 processing in the future, and also to test the hypothesis that PS1 cleaves STIM2 as well. He added that it was possible the PS1/STIM1 pathway was only relevant in people with certain mutations, although problems with calcium homeostasis are a common feature of both familial and sporadic forms of the disease. Just as researchers are busy designing γ-secretase modulators that preferentially block the enzyme’s cleavage of APP, Cheung proposed a similar strategy to block γ-secretase destruction of STIM1.
Thayer added that alterations in the STIM1 pathway could explain some of the adverse effects of more general γ-secretase inhibitors. “Clearly, effects on STIM will be an important consideration for drug development targeted to the γ-secretase complex,” he wrote.
Mark Mattson of the National Institute on Aging in Bethesda, Maryland, noted, as did the authors, that calcium homeostasis requires a delicate balance. Before laying out therapeutic strategies, researchers will need to further investigate the normal physiological role of PS1 in limiting calcium entry, he suggested. He added that studying the pathway in the context of age-related changes, such as oxidative stress, mitochondrial dysfunction, and neuronal hyperactivity, will be crucial. Creating an AD model mouse with a version of STIM1 that cannot be cleaved by γ-secretase would allow researchers to test the role of the STIM1/PS1 pathway in AD pathogenesis, he said.—Jessica Shugart
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