This is Part 3 of a three-part series. See also Part 1 and Part 2.
1 May 2008. A third area of γ-secretase research that drew attention at the Keystone conference held 24-29 March in Keystone, Colorado, is the hotly debated question of how presenilin mutations cause familial Alzheimer disease (FAD). Scientists wonder exactly how these mutations manage to shift the cleavage of APP in such a way that the ratio of Aβ40 and 42 changes enough for the more fibrillogenic Aβ42 to occur in excess over a period of years. The question has been framed in several ways. Some investigators asked whether FAD mutations cause a gain or a loss of presenilin function, some asked what that meant for drug development, and others questioned whether the distinction was real and important or merely semantic. The Alzforum has addressed the issue in two Live Discussions (see Davies/De Strooper discussion; see Shen/Kelleher discussion). At the Keystone conference, Christian Haass of Ludwig-Maximilians University in Munich, Germany, joined the debate with new data from his lab. In an opening plenary lecture, Haass first broadly summarized what’s known about amyloidogenic APP proteolysis and its players so far. Then he presented data suggesting that at least one FAD presenilin mutation his group studied in detail—the G384A mutation that sits smack in the crucial GxGD motif in the active site—slows down the processive cuts the enzyme usually performs on the substrate in such a way that relatively more Aβ42 gets made at the expense of the less harmful Aβ40, whose production plummets. In essence, then, the verdict is Solomonic: a relative loss of presenilin function (Aβ40 generation) amounts to a relative gain of toxic function (Aβ42 aggregation).
Haass arrived at this conclusion by first using SPP2b, a simpler member of the GxGD-type family of aspartyl proteases that previous research by his and other groups had defined. These enzymes make up the family of I-CLiPs. SPPs in particular come in handy for the study of the AD-relevant presenilin because they work alone, without requiring presenilin’s three protein partners Nicastrin, Aph-1, and Pen-2. Hence they are simpler to study but otherwise function almost identically to presenilin. They are also fascinating in their own right in that they operate as virtual mirror images of presenilin. The orientation by which their nine TMDs weave in and out of the lipid bilayer is flipped relative to that of presenilin; they cut substrates existing in type 2 orientation, while presenilin cuts substrates existing in the opposite type 1 orientation, and their cleavage products, too, are identical but opposite in orientation. “Their orientation across the membrane is opposite; everything else is in parallel,” Haass said.
SPP2b offered a handle to test the problem of the FAD mutations when Regina Fluhrer in Haass’ group found that this protease processes tumor necrosis factor α (TNFα, a type 2-oriented transmembrane substrate) in much the same way as presenilin cuts APP. The similarity even includes a few slightly different cleavage sites inside the membrane, much like the different cleavages known for presenilin. The major reason GxGD proteases perform multiple cuts starting from these different cleavage sites is to remove the hydrophobic sequence of the substrate as a part of its degradation, Haass speculated.
Fluhrer made the FAD presenilin G384A mutation in SPP2b (where it is a G420A mutation). To her surprise, she found that it produces an elongated TNFα intracellular domain (ICD), which, because of the reaction’s flipped orientation, corresponds to Aβ in presenilin cleavage. This means that an FAD mutation in SPP2b created a change corresponding to an Aβ40-to-42 shift in APP processing, Haass said. Further analysis of TNFα cleavage with this FAD mutation showed that the normal, stepwise cleavage that degrades the substrate and finally liberates the ICD slows down in the mutant and becomes much less efficient. It takes the mutant protease much longer to produce the smaller peptides. Indeed, the effect of the mutant can be mimicked by simply chilling the wild-type enzyme down to 20 degrees Celcius from the 37 degrees at which it usually operates, Haass showed.
The finding held true in the same way for G384A presenilin and Aβ generation: Aβ40 production slowed to a crawl; Aβ42 production stayed unchanged. The reasons for this are not proven yet, but Haass interprets it this way at present: prior research by Yasuo Ihara at the University of Tokyo and others has shown that γ-secretase has different production lines, whereby the secretase latches on at a slightly different starting point and then cleaves processively. The production line that latches on at the so-called ε site releases Aβ40, and that is the one most hampered by the mutation. This leaves more substrate for the production line that produces Aβ42. (The exact details of how processive cleavage works, and how the ε, ζ, and γ cleavage sites relate to how much of each final product is made remains unclear. Audience members asked those questions, but no γ-secretase speaker was willing to speculate, which is as good a hint as any that several γ-secretase labs are trying hard to get a grip on the problem.)
In essence, aggressive FAD mutations slow down the pace and efficiency of γ-secretase in a selective loss of function, Haass suggested. His stance adds new data and a new voice to a position articulated recently by Michael Wolfe and Bart de Strooper (see also Winklhofer et al., 2008). Furthermore, it fits with a recent study that found a relatively protective role of Aβ40 in amyloid deposition (Kim et al., 2007).
Haass closed his talk with a cautionary note about γ-secretase modulation. This drug development approach is being actively pursued at several different biotech and pharmaceutical companies, and Phase 3 clinical trial results about one older such compound, Flurizan, are expected at the ICAD conference to be held this July in Chicago. The premise of γ-secretase modulation grew out of the twin realizations that outright γ-secretase inhibition could be toxic and that certain NSAIDs are able to subtly tweak γ-secretase so as to produce less Aβ42 and more of the presumably innocuous Aβ38. New science is now raising fresh questions for scientists to address while developing this approach. For one, a Keystone speaker claimed that under certain conditions, Aβ38 can aggregate, too (see ARF related news story). For another, Haass presented data from his own lab that support a recent, separate paper by Sascha Weggen and colleagues at Heinrich-Heine-University in Duesseldorf, Germany. Weggen’s data implied not only that certain FAD-based models were unsuitable for finding effective γ-secretase modulators, but also that people carrying severe, early-onset FAD mutations were unlikely to respond to γ-secretase modulators even if those drugs do treat the majority of AD cases (Czirr et al., 2007).
For his part, Haass started out testing an underlying assumption of γ-secretase modulation. It is that Aβ38 and 42 generation are connected, i.e., that more of one means less of the other. This turned out to be wrong, Haass said. In data recently published (Page et al., 2008), the scientists found that the age of onset of FAD mutation indeed correlates with the amount of Aβ42, but not with the amount of Aβ38. Extending this observation to NSAIDs and to the γ-secretase inhibitors DAPT and Merck’s compound E, the scientists found that, unlike wild-type presenilin, certain FAD mutants fail to reduce their Aβ42 production in response to these drugs, yet they did respond with a robust increase in Aβ38, showing again how these two products are not connected. As do Weggen’s data on this issue (Czirr et al. 2008), Haass' results also imply that people with strong FAD presenilin mutations should not be treated with NSAIDs, Haass said. “I think the mutant γ-secretase is locked in a pathological conformation,” he added.
This emerging research reinforces a point of caution pharma researchers had raised in prior conversations with this reporter when pressed about why they resisted doing drug trials with families carrying eFAD mutations. Many obstacles, some of them surmountable, hold back such efforts. One scientific concern is that the genetic cause of AD may stand in the way of certain treatment mechanisms, and more research is needed to understand better which treatments stand the best chance of working in families with autosomal-dominant AD (for specific reference, see Drug Trials in EOAD essay; for general overview, see eFAD Research essay). The last word clearly is not spoken on this issue, but for now, it looks as if people with presenilin mutations are well advised to stay away from NSAIDs and look toward immunotherapies or small molecules such as scyllo-inositol instead.—Gabrielle Strobel.
Page RM, Baumann K, Tomioka M, Pérez-Revuelta BI, Fukumori A, Jacobsen H, Flohr A, Luebbers T, Ozmen L, Steiner H, Haass C. Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation. J Biol Chem. 2008 Jan 11;283(2):677-83. Abstract
Fluhrer R, Fukumori A, Martin L, Grammer G, Haug-Kröper M, Klier B, Winkler E, Kremmer E, Condron MM, Teplow DB, Steiner H, Haass C. Intramembrane proteolysis of GxGD-type aspartyl proteases is slowed by a familial Alzheimer disease-like mutation. J Biol Chem. 2008 Sep 3; Abstract
This is Part 3 of a three-part series. See also Part 1 and Part 2.