3 September 2010. Inhibition of the γ-secretase enzyme that snips amyloid precursor protein (APP) to form Aβ has long been seen as a therapeutic option for Alzheimer disease, but finding a safe, effective inhibitor has proved frustrating. The risk of side effects from γ-secretase inhibition is high, in part because this secretase also cuts Notch, an essential protein for numerous biological functions. The recent cancellation of a high-profile γ-secretase inhibitor clinical trial (see ARF related news story) is the latest disappointment for approaches targeting this secretase. A paper in yesterday’s Nature online offers a new tack for this field by reporting the discovery of a γ-secretase activating protein (GSAP) that acts specifically to promote the binding of γ-secretase to APP, but not to Notch. Researchers led by Paul Greengard at the Rockefeller University in New York show that inhibition of GSAP reduces the production of Aβ by 40 to 50 percent both in vitro and in AD model mice, while having no effect on Notch processing. This suggests that GSAP inhibition could be a promising therapeutic approach to lowering amyloid levels while avoiding toxic side effects. Although the results still need to be replicated by other labs, the news has generated excitement in the field.
“This is a tour de force work that goes from identifying a protein, to making a mouse model where [the authors] actually show that this is a potential target for therapeutics,” said Gopal Thinakaran, of the University of Chicago in Illinois. “This is a very elegant set of studies, and the data are compelling.”
Several other proteins that modulate γ-secretase activity have been identified in recent years, including transmembrane protein TMP21 (see ARF related news story on Chen et al., 2006), G-coupled protein receptor 3 (see ARF related news story on Thathiah et al., 2009), and the γ-secretase complex component Aph1 (see ARF related news story on Serneels et al., 2009). What distinguishes GSAP, said Thinakaran, is that the authors have described a detailed mechanism of action for it, making it an attractive drug target.
Previous work by Greengard and colleagues had shown that the anti-cancer drug imatinib, also known as Gleevec, acted as a γ-secretase modulator, lowering production of Aβ38, 40, and 42 without affecting Notch processing (see ARF related news story on Netzer et al., 2003). Imatinib itself is not a good AD drug candidate because it does not enter the brain. Therefore Greengard and colleagues set out to understand how it blocked the secretase complex so they could develop a better drug. First author Gen He added a radiolabeled, photoactivatable nitrogen group to imatinib, then incubated this derivative with a membrane preparation containing the γ-secretase complex. When He and colleagues illuminated this preparation with ultraviolet light, the nitrogen bonds broke, creating reactive free radicals that bound to any proteins in the vicinity and radiolabeled them. Surprisingly, none of the known γ-secretase components bound the radiolabel, but a novel 16 kilodalton protein did. After purification, mass spectrometry identified this small protein as the C-terminal region of an uncharacterized protein known as pigeon homologue protein, which the authors renamed γ-secretase activating protein (GSAP).
He and colleagues characterized GSAP by using co-immunoprecipitations, showing that it binds both the γ-secretase complex and the membrane-bound, β-secretase-cleaved C-terminal fragment of APP. An experiment using truncated forms of APP showed that GSAP binds a region of about 10 amino acids that lies just next to the membrane on the cytoplasmic side of the precursor. Other experiments demonstrated that imatinib inhibits γ-secretase by interfering with the binding of GSAP to APP. In contrast, He and colleagues showed that GSAP does not bind Notch or affect Notch cleavage, explaining how imatinib spares Notch processing.
The crucial question, however, is what effect GSAP inhibition has on Aβ production. The authors addressed this first in cell cultures, where they used short interfering RNA to knock down GSAP expression by about three-quarters. This lowered production of Aβ38, 40, and 42 by about 50 percent each. Imatinib had no additional effect on Aβ production, confirming that the drug acts through GSAP. The authors then looked at the effects of blocking the activating protein in vivo by making a transgenic mouse that expressed short hairpin RNA for GSAP under a tetracycline inducible promoter. He and colleagues crossed these mice with double-transgenic animals carrying both APP with the Swedish mutation and a presenilin mutation (APPswe/PS1ΔE9). Induction of the interfering RNA for six months reduced GSAP RNA levels by 85 percent. Levels of Aβ40 and 42 dropped some 40 percent, as did the number of amyloid plaques (see figure below). These effects on amyloid were similar to those seen in AD mice treated with a global γ-secretase inhibitor, dibenzazepine, but without the toxic side effects created by faulty Notch processing.
GSAP Promotes Aβ Production
In double-transgenic APP/PS1 mice, GSAP knockdown (right) attenuates Aβ production and plaque deposition in comparison to controls (left). Image credit: Nature Publishing Group
This demonstration of in vivo relevance is significant, said Michael Wolfe of Harvard Medical School. “Assuming that this is validated, it’s quite an important advance. You always increase your chances of finding better [therapeutic] agents when you know what the target is.”
Nonetheless, numerous questions remain about GSAP’s mode of action. Thinakaran said it would be important to know whether GSAP is bound to γ-secretase throughout the neuron, or whether there are specific subcellular compartments that favor the interaction, particularly in light of data showing that presynaptically as well as post-synaptically released Aβ may harm synapses. Another question is whether GSAP binds γ-secretase and APP in a one-to-one fashion. Greengard said his team believes the three proteins act as a trimer. To prove this, however, would require more detailed molecular modeling, Thinakaran said.
One notoriously controversial question in the field concerns the γ-secretase cleavage sites on APP. The transmembrane cleavage at the γ site releases toxic Aβ species, while a few amino acids farther toward the C-terminus, the ε site cleavage is the classic way to produce the APP intracellular domain (AICD), which may have important signaling roles. Some work suggests that these cleavages happen sequentially, first at the ε site and then at γ (see Gu et al., 2001; Lefranc-Jullien et al., 2006; Takami et al., 2009). This scenario was supported by a recent study showing that autoproteolysis of presenilin, the catalytic subunit of γ-secretase, occurs in a stepwise fashion (see ARF related news story). Other papers suggest, however, that the two types of cleavage can occur independently of each other (see Wiley et al., 2005; Kume et al., 2006; Bentahir et al., 2006). He and colleagues uncovered evidence in favor of independent cleavage. The Rockefeller scientists showed, in an in vitro assay, that adding recombinant GSAP increased the levels of Aβ produced by γ cleavage, while decreasing the amount of AICD produced by ε cleavage. Greengard said the cleavage sites were distinguished by the use of antibodies specific for each cleavage fragment.
If independent cleavage is occurring, as the paper suggests, some scientists wonder what the downstream consequences of this would be, and what becomes of the unusual fragments produced. Wolfe points out that, “Whenever you produce an Aβ [fragment], you produce AICD. It’s a one-to-one correspondence.” Wolfe would like to see what happened to the corresponding Aβ and AICD fragments that were not detected in this assay, i.e., long Aβ49 produced by ε cleavage, and long AICD produced by γ cleavage. Bart De Strooper, of the University of Leuven, Belgium, also finds this issue puzzling. He noted, “The question of what happens with Aβ49 has surprisingly not been addressed in the paper” (see full comment below). In an accompanying News & Views article, Peter St George-Hyslop and Gerold Schmitt-Ulms at the University of Toronto ask, “Are these amino-terminal fragments left in the membrane as Aβ49, or are they degraded by some other mechanism that does not generate disease-associated amyloid-β peptides?” Thinakaran speculates that “maybe the γ-secretase complex is capable of both sequential and independent cleavage, and this difference between the two modes of cleavage is dictated by the presence or absence of GSAP.”
For the moment, Greengard and colleagues are focused on the therapeutic potential of GSAP. Greengard said they are looking for the protein that cleaves GSAP into its mature form, with the hope that this protein itself might make an appealing therapeutic target. They are also looking for more proteins that bind to GSAP, Greengard explained, to see if GSAP has other functions in the cell that might be affected by knockdown of the modulator. Finally, he stated that they are also collaborating with molecular geneticists to see if GSAP mutations are associated with AD. It is not clear if the researchers have immediate plans to screen for small-molecule GSAP inhibitors.
Paul Fraser of the University of Toronto, Canada, who led the discovery of TMP21, commented that this new paper “validates the notion that regulators [of γ-secretase] are going to be important. There has to be some way of keeping a hold on this protease so it doesn’t randomly traipse around and chew up things it shouldn’t.” He said the fact that GSAP “appears to be a positive regulator of γ-secretase opens a door that nobody knew was there before. It’s exciting stuff.”—Madolyn Bowman Rogers.
He G, Luo W, Li P, Remmers C, Netzer WJ, Hendrick J, Bettayeb K, Flajolet M, Gorelick F, Wennogle LP, Greengard P. Gamma-secretase activating protein is a therapeutic target for Alzheimer’s disease. Nature 2010 Sep 2. Abstract
George-Hyslop PS, Schmitt-Ulms G. Selectively tuning gamma-secretase. Nature 2010 Sep 2. Abstract