This is Part 3 of a seven-part series. See also Parts 1, 2, 4, 5, 6, 7.
31 December 2008. At the 8th Eibsee Meeting on Cellular Mechanisms of Alzheimer’s Disease, held October 29 to November 1 in Germany, Bart de Strooper of the University of Leuven and Flaams Instituut voor Biotechnologie in Leuven, Belgium, set the scene for a session on γ-secretase, the unusual intramembraneous aspartyl protease that releases the Aβ peptide once the BACE enzyme is done clipping APP. γ-secretase, a complex composed of presenilin (PS), nicastrin (NCT), Aph1, and Pen2, remains a central target of drug discovery.
Before he got into new data, de Strooper looked back for a minute to say that the field’s extensive discussion of whether familial AD mutations in presenilin cause a loss or a gain of function in this enzyme in his view has been constructive, because it led to an important change of how researchers think about amyloid (ARF Live Discussion, follow-up discussion). “In understanding the toxicity of amyloid, we must consider not just its quantity, but the ratio of Aβ species,” de Strooper said. γ-secretase spews out a whole range of different Aβ species. How it does that is not clear; however, a model first proposed by Yasuo Ihara of Japan’s Tokyo University, whereby it starts cutting at a particular cleavage site and then perhaps cuts processively every three amino acids, is drawing some support in the field.
The main biological function of γ-secretase is Notch signaling, de Strooper said, and that is why inhibitors cause immunological and gastrointestinal side effects. “But I do not think the story of γ-secretase inhibition is over,” de Strooper said. γ-secretase is a highly complex activity, and drilling deeper may reveal more specific drug targets. De Strooper pointed out years ago that the different presenilin and APH1 genes that exist in the human (and also mouse) genome make up four different γ-secretase complexes (de Strooper, 2003). At the Eibsee, de Strooper presented data that distinguish among some of these, and made a case that Aph1b/c-bearing complexes might be targeted for AD drug discovery, whereas Aph1a-bearing complexes should be left alone to handle Notch cleavage. The widespread expression pattern of Aph1a compared to a more specific, brain-only expression of Aph1b/c in mouse, as well as Aph1b expression in human brain, is consistent with Aph1b playing a role in AD, de Strooper reported.
In a previous study looking for a physiological function of Aph1b/c, the group had deleted the gene. The knockout mice displayed deficits in prepulse inhibition, working memory, and a drug response to antipsychotics that was reminiscent of schizophrenia and, together, suggested that Aph1b/c-containing γ-secretase functions in neuregulin1 cleavage during brain development (Dejaegere et al., 2008). More recently, the group asked whether targeting Aph1b/c might rescue an AD phenotype in mice. To address the question, the scientists crossed their Aph1b/c knockout strain with Mathias Jucker’s APP/PS1 mice that develop aggressive early amyloidosis, and checked for amyloid load, memory and behavior, and γ-secretase inhibitor side effect profile. The crosses had far fewer plaques than did the APP/PS1 strain alone, and differed on all other readouts, as well. “This means these various γ-secretase complexes have slightly different biochemical activities, and suggests that some complexes may be better drug targets than others,” de Strooper said. By implication, a compound that blocks only Aph1b, not Aph1a, might change the Aβ species distribution without hitting Notch. However, whether Aph1b directly affects AD is unknown (see also Aph1b on Alzgene). These issues are being hotly pursued in several γ-secretase laboratories around the world.
The possible neurodevelopmental role of Aph1b/c-containing γ-secretase raises parallels with BACE. This secretase also has a dual role in cleaving APP as well as neuregulin during developmental myelination (see ICAD news story; Savonenko et al., 2008; see also Part 5 of this series).
Tough Love in the γ Club
The Eibsee meeting convenes a “hard core” of γ-secretase aficionados. One thing these labs do, in a friendly spirit of scientific verification, is subject one another’s newest data to independent replication—or lack thereof, as the case may be. Sometimes, it’s the Eibsee presentations themselves that get their share of needling at the same venue in later years. For example, the finding that a GxGD motif in presenilin is important for the activity of the enzyme complex (Haass and Steiner, 2002; Xia and Wolfe, 2003) has held up in the hands of other scientists (e.g., Sato et al., 2006). This year at the conference, Harald Steiner, in Haass’ group at the University of Munich, added to it by describing how his group probed the motif by replacing its conserved glycines at positions 382 and 384. This demonstrated that these glycines are crucial active site elements without which the enzyme can’t function (see, e.g., Fluhrer et al., 2008). Two other findings have a harder time withstanding collegial scrutiny. For example, Steiner also presented data showing that his group has been unable, thus far, to replicate a finding reported previously at the conference, namely that the protein tmp21 is a regulatory subunit of γ-secretase (see Eibsee story and Chen et al., 2006). Steiner noted that in his laboratory, several different purification regimens all suggest that small amounts of tmp21, and also the other proposed complex component cd147 (Zhou et al., 2005), may indeed loosely associate with the complex, but are unlikely to be true components of it.
The corrective element of independent reproduction emerged a second time when several groups noted that their own data failed to support a published role for the complex component nicastrin. Well received at the time, that work had proposed that nicastrin serves as a kind of gatekeeper that recognizes the substrate and then passes it on to the active site for cleavage (see Eibsee story). The idea was that substrate recognition requires a particular glutamate residue on nicastrin; however, follow-up experiments by de Strooper’s and Phil Wong’s groups cast doubt on this notion when it turned out that γ-secretase recognized its substrate just fine, thank you, even when this glutamate on nicastrin was mutated (Chavez-Gutierrez et al., 2008). At this Eibsee conference, Regina Fluhrer in Haass’ group at the University of Munich corroborated de Strooper/Wong’s finding by studying signal peptide peptidases, the simpler cousins of γ-secretase. These enzymes require no complex, but Fluhrer found that they, too, perform a kind of length measurement of the substrate’s extramembraneous stub that had been ascribed exclusively to nicastrin, suggesting substrate recognition could occur more directly by way of structural features between the substrate and the enzyme’s transmembrane domain. In essence, scientists at the meeting said that nicastrin is no longer seen as indispensable for substrate recognition.—Gabrielle Strobel.
This is Part 3 of a seven-part series. See also Parts 1, 2, 4, 5, 6, 7.