An unexpected observation from what was supposed to be a control experiment provides new insight into amyloid precursor protein (APP) processing. Researchers led by Lawrence Rajendran, University of Zurich, discovered that APP C-terminal fragments (CTFs) derived from α- or β-secretases, respectively, are handled differently by γ-secretase. The enzyme complex cleaves the longer βCTFs at the Aβ42 position more often than it cuts the shorter αCTFs there. The results may reflect differences in substrate positioning on the γ-secretase complex, and suggest a novel approach to tricking γ-secretase into making less of the toxic Aβ42 by targeting the substrate, rather than the enzyme, the researchers claim. The paper appeared June 6 in Cell Reports. 

Amyloid-β production starts with β-secretase (BACE) cleavage of APP to yield either an 89 or 99 amino acid CTF (C89 or C99). The CTFs contain a truncated extracellular domain, the transmembrane region, and the intracellular domain (AICD) of APP. They provide fodder for processing by γ-secretase. After first clipping C89/99 internally (ε cleavage) at either the Aβ48 or Aβ49 positions to release the AICD, γ-secretase continues in carboxypeptidase fashion to trim the remaining CTFs' three amino acids at a time. Ultimately, processing of C99 yields a mixture of Aβ42 and Aβ40 peptides. The C89 substrate gives rise to 42/40 mixtures of N-terminally truncated (called Aβ11-x, or Aβ') peptides. In the brain, Aβ' peptides appear to predominate, but Aβ peptides are amyloidogenic and abundant in plaques. Cleavage of APP by α-secretase yields a CTF (C83) that is cleaved by γ-secretase to generate non-amyloidogenic peptides, called p3s. 

Some pathogenic mutations in APP and in the γ-secretase catalytic subunit shift the 42/40 ratio toward Aβ42, and modulators of the enzyme can do the same. BACE and α-secretase had never been shown to alter the Aβ42:40 ratio. That’s why the first author Gabriele Siegel was puzzled when she treated primary mouse neurons in culture with a BACE inhibitor, and measured a two-thirds decrease in the 42/40 ratio of peptides secreted by the cells. “If you inhibit BACE, you expect to see a decrease in overall Aβ production, which we saw, but you don’t expect to decrease the 42/40 ratio,” she told Alzforum.

Soon Siegel hit on the explanation: The commercial antibody kit she used to measure Aβ peptides recognized not only Aβ and Aβ', but also p3. Unexpectedly, the p3 peptides were produced at a much lower 42/40 ratio than Aβ and Aβ'.

The results held up in cultured iPSC-derived human neurons, and in a cell-free system where Siegel directly measured the cleavage products of each CTF. Consistently, C83, the shortest substrate, produced the lowest 42/40 ratio. C99 (the parent of Aβ) produced a ratio 1.4-fold to twofold higher, and C89 gave the highest, elevating the Aβ'42/40 ratio by 1.6 and 2.4 times. “Very much to our surprise, there was no ranking according to length of the APP ectodomain. There is something very particular about C89 that it produces the highest ratio,” Siegel said. 

Variable Output: γ-secretase produces Aβ and Aβ' peptides with higher 42:40 ratios than p3. [Courtesy of Cell Reports, Siegel et al.]

To determine if the differential processing of the three CTFs occurred during the ε or γ cleavage, Siegel analyzed the AICDs products of the ε cleavage by mass spec. She found that all three CTFs gave rise to two AICDs, with relative ratios that matched what she saw for the 42/20 peptides (see scheme above). 

Her result suggested that the γ-secretase handled the CTFs differently from the get-go, but why? One possibility is that the substrates preferentially recognize different γ-secretases. Two of the four proteins in the γ-secretase complex (PSEN and Aph1) have genetic variants, which permits formation of at least six different complexes in mouse, and there are two different presenilins, the catalytic subunit of the enzyme. Siegel used siRNA in mouse neurons to systematically knock down each variant (PSEN1, PSEN2, APH1A, APH1B, and APH1C); this showed that changing the composition of the secretase complex had no effect on the basal 42/20 ratio, or the response to a BACE inhibitor. This left the researchers with the hypothesis that the lack of just six amino acids in the C83 ectodomain compared to C89 changed the interaction between substrate and γ-secretase complex enough to shift the site preference of the ε-cleavage. 

A new route to decreasing Aβ42 production could run through that ectodomain, the researchers posit. “Now that we know these N-terminal amino acids seem to modulate the 42/40 ratio, it may be possible to mask those residues in C99 to lower Aβ42 production without any need to interfere with the enzyme itself,” Rajendran said.

Harald Steiner of the Ludwig-Maximilians-University in Munich called the study interesting, and the results unexpected. The findings are in line with the hypothesis that interactions between APP extracellular domains and γ-secretase are critical for cleavage of the substrate, he wrote to Alzforum. But he said modulating cleavage by targeting CTFs may be a challenge. “One would need to have small compounds that interfere with the interaction of C99 or C89 with γ-secretase while leaving the interaction of C83 and that of other substrates intact. This is not easy and will probably require precise structural knowledge on the substrate-enzyme interactions,” Steiner added. 

Rajendran has hope for aptamers, small oligonucleotides that, much like antibodies, specifically bind to unique amino acid sequences. C99 aptamers would not affect other γ-secretase substrates, he said. He agrees with Steiner that molecular modeling of the γ-secretase-CTF complexes is needed to reveal fine details of substrate binding.

The study is “an initial and very interesting departure point for better understanding APP processing by γ-secretase,” said Lucia Chavez-Gutierrez, University of Leuven, Belgium. She said the idea that the CTF N-terminal exodomain helps determine the site of first cleavage needs more data to be proven.

The study also underscores that research on p3 and other γ-secretase products has been neglected, said Chavez-Gutierrez. “This paper highlights that if we inhibit BACE, we will increase p3, and invites us to study in more detail all aspects of APP processing,” she said. Other researchers echoed this sentiment, wanting to know the relevance of all the different Aβ, Aβ', and p3 peptides to plaques, perivascular amyloid, and to synaptic toxicity.—Pat McCaffrey

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  1. Siegel et al. identified the difference in the γ-secretase-mediated cleavage on APP substrate with distinct N-terminal length. Complex profiles of γ-cleavage on C83, C89, and C99 suggest that further systematic analysis for the enzymatic property of γ-secretase would be needed.

    This work highlights the pathological importance of not only Aβ' starting at Glu-11, but N-terminally truncated Aβ species. Because of limitations in biochemical detection, several labs, including ours, have utilized Swedish mutant APP, which shifts β-cleavage at +1 position, in our cell biological experiments. However, development of sensitive ELISA systems and mass spectrometry has revealed that naïve cells secrete relatively high amounts of N-terminally truncated Aβ (Seubert et al., 1992; Asami-Odaka et al., 1995; Wang et al., 1996).

    These Aβ levels/ratios might be influenced by cell type and culture conditions. Importantly, N-terminally truncated Aβ species deposited in senile plaques (Naslund et al., 1994) in addition to Aβ species modified after deposition such as pyroglutamylation. Thus, we must carefully interpret the effect of secretase modifiers in cells/mice expressing APP carrying the Swedish mutation.

    References:

    . Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids. Nature. 1992 Sep 24;359(6393):325-7. PubMed.

    . Long amyloid beta-protein secreted from wild-type human neuroblastoma IMR-32 cells. Biochemistry. 1995 Aug 15;34(32):10272-8. PubMed.

    . The profile of soluble amyloid beta protein in cultured cell media. Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry. J Biol Chem. 1996 Dec 13;271(50):31894-902. PubMed.

    . Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8378-82. PubMed.

  2. The paper is interesting. It shows that γ-secretase processes the different APP CTF substrates to different 42/40 peptide ratios, with those produced from C89 being the highest. This unexpected observation might suggest that C89 is presented to the γ-secretase active site differently than C99 or C83.

    In determining the interaction sites of C99 by photoaffinity mapping, we have previously shown that FAD mutations in presenilin-1 can cause altered substrate positioning of C99 (Fukumori and Steiner, 2016). Similar mispositioning may particularly occur for C89, giving rise to substantial alterations in the cleavage precision/processivity compared to C99 and C83. 

    The new data indicate that interactions of γ-secretase exosites with the extracellular APP domain (also see Fukumori and Steiner, 2016) are critical for substrate cleavage, a hypothesis that Akio Fukumori and I are currently exploring further.

  3. The most prominent feature of this paper is that C89 is a favored APP substrate cleaved at epsilon 48 by γ-secretase to produce AICD49-99, leading to Aβ42 product line (Takami et al., 2009). It suggests that ectodomain length of substrate influences not only cleavage efficiency but also substrate cleavage sites by this enzyme.

    We previously reported cleavage efficiency and cleavage sites on APP, APLP2, and Notch substrates containing different ectodomain length (namely C99 and C83 on APP; β-cleaved APLP2 and α-cleaved APLP2 on APLP2; ∆E Notch and S2 cleaved Notch on Notch1) (Funamoto et al., 2013). In either case, γ-secretase preferentially cleaved substrates containing short ectodomain (C83, α-cleaved APLP2, and S2 cleaved Notch).

    However, we found no difference in cleavage sites close to the membrane-cytoplasmic boundary (ε, ε-like, and S3 sites) between short and long ectodomain substrates on APP, APLP2, and Notch (Funamoto et al., 2013, see supplementary Fig. S3). Probably, C89-like substrates of APLP2 and Notch may exhibit modulation of the cleavage sites as shown in C89 of APP.

    The finding by Siegel and colleagues is quite interesting. However, it is reported that the Aβ42/Aβ40 ratio is modulated by concentrations of substrate and remnant detergent (Fraering et al., 2004Yin et al., 2007). We should be careful to assess the modulation of Aβ42/Aβ40 in vitro.

    References:

    . gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.

    . Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase. Nat Commun. 2013;4:2529. PubMed.

    . Purification and characterization of the human gamma-secretase complex. Biochemistry. 2004 Aug 3;43(30):9774-89. PubMed.

    . {gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE. J Biol Chem. 2007 Aug 10;282(32):23639-44. PubMed.

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  1. . The Alzheimer's Disease γ-Secretase Generates Higher 42:40 Ratios for β-Amyloid Than for p3 Peptides. Cell Rep. 2017 Jun 6;19(10):1967-1976. PubMed.