Akio Fukumori

Presenilins mediate a dual intramembranous γ-secretase cleavage of Notch-1

Following ectodomain shedding, Notch‐1 undergoes presenilin (PS)‐dependent constitutive intramembranous endoproteolysis at site‐3. This cleavage is similar to the PS‐dependent γ‐secretase cleavage of the β‐amyloid precursor protein (βAPP). However, topological differences in cleavage resulting in amyloid β‐peptide (Aβ) or the Notch‐1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N‐terminal Notch‐1 Aβ‐like fragment (Nβ). Analysis of Nβ by MALDI‐TOF MS revealed that Nβ is cleaved at a novel site (site‐4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of βAPP at position 40 and 42 of the Aβ domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimers disease‐associated PS1 mutations similar to the pathological endoproteolysis of βAPP. Considering these similarities between intramembranous processing of Notch and βAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/γ‐secretase.

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid β-peptide (Aβ) is generated via proteolytic processing of the β-amyloid precursor protein by β- and γ-secretase. γ-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Aβ42 and concomitantly increase the levels of the rather benign Aβ38. Here we show that Aβ42 and Aβ38 generation occur independently from each other. The amount of Aβ42 produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of γ-secretase, appeared to correlate with the respective age of onset in patients. However, Aβ38 levels did not show a negative correlation with the age of onset. Modulation of γ-secretase activity by sulindac sulfide reduced Aβ42 in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Aβ42 or only rather subtle effects. Strikingly, even the mutations that showed no effect on Aβ42 levels allowed a robust increase of Aβ38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Aβ42 and to decrease Aβ38. For mutants that predominantly produce Aβ42, the ability of fenofibrate to further increase Aβ42 levels became diminished, whereas Aβ38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Aβ38 and Aβ42 production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Aβ42-lowering capacity of γ-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to γ-secretase modulators.

Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1†

ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val→Gly and the Lys→Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

Secretion of the Notch-1 Aβ-like peptide during Notch signaling

The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 Aβ-like peptide (Nβ)) should be produced. Here we showed that Nβ species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel Nβ, Nβ21 and C-terminally elongated Nβ25, which were produced in an ∼5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated Aβ. PS pathogenic mutants increased the production of the longer species of Aβ (Aβ42) from β-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of Nβ. Strikingly, chemicals that modify the Aβ42 level caused parallel changes in the Nβ25 level. These results demonstrated that the characteristics of C-terminal elongation of Nβ and Aβ are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of Aβ- or Nβ-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for Aβ42 production.

Three-Amino Acid Spacing of Presenilin Endoproteolysis Suggests a General Stepwise Cleavage of γ-Secretase-Mediated Intramembrane Proteolysis

Presenilin (PS1 or PS2) is the catalytic component of the γ-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimers disease (AD)-characterizing amyloid β-peptide. PS is cleaved during complex assembly into its characteristic N- and C-terminal fragments. Both fragments are integral components of physiologically active γ-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of γ-secretase has been defined and numerous substrates were identified, the cellular mechanism of the endoproteolytic cleavage of PS is still unclear. We addressed this pivotal question by investigating whether familial AD (FAD)-associated PS1 mutations affect the precision of PS endoproteolysis in a manner similar to the way that such mutations shift the intramembrane cleavage of γ-secretase substrates. We demonstrate that all FAD mutations investigated still allow endoproteolysis to occur. However, the precision of PS1 endoproteolysis is affected by PS1 mutations. Comparing the cleavage products generated by a variety of PS1 mutants revealed that specifically cleavages at positions 293 and 296 of PS1 are selectively affected. Systematic mutagenesis around the cleavage sites revealed a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis reminiscent to the ε-, ζ-, and γ-cleavages described for typical γ-secretase substrates, such as the β-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by γ-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

Novel γ-Secretase Enzyme Modulators Directly Target Presenilin Protein

Background: γ-Secretase modulators (GSMs) hold great potential as anti-Alzheimer disease drugs, but their molecular target(s) are not established. Results: The catalytic subunit of γ-secretase, presenilin, was identified as a direct target of novel GSMs. Conclusion: Enzyme-targeting GSMs establish allosteric modulation as a mechanism of GSM action. Significance: The identification of presenilin as GSM target may contribute to the development of therapeutically active GSMs. γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid β-peptide (Aβ42). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the β-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aβ42 is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aβ42 without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The β-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.

Purification, Pharmacological Modulation, and Biochemical Characterization of Interactors of Endogenous Human γ-Secretase†

Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimers disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimers disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.

Generation of Alzheimer Disease-associated Amyloid β42/43 Peptide by γ-Secretase Can Be Inhibited Directly by Modulation of Membrane Thickness

Background: γ-Secretase-mediated intramembrane proteolysis generates Aβ42/43, pathogenic peptides implicated in causing Alzheimer disease (AD). Results: Reconstitution of γ-secretase in model membranes reveals that Aβ42/43 generation can be lowered in thick membranes. Conclusion: Membrane thickness is a crucial factor influencing the activity of γ-secretase to generate Aβ42/43. Significance: Targeting the lipid environment of γ-secretase by increasing membrane thickness may provide a therapeutic strategy for AD. Pathogenic generation of amyloid β-peptide (Aβ) by sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). β-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37–43-amino acid long Aβ species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aβ species, in particular that of the pathogenic longer variants Aβ42 and Aβ43, is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aβ42/43 species was markedly attenuated in thick membranes, that of the major and rather benign Aβ40 species was enhanced. Moreover, the increased production of Aβ42/43 by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aβ42/43 species.

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

Pathogenic generation of the 42-amino acid variant of the amyloid β-peptide (Aβ) by β- and γ-secretase cleavage of the β-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Aβ42 production by γ-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Aβ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of γ-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Aβ domain regarding their response to GSMs. We found that Aβ42-increasing familial AD mutations of the γ-secretase cleavage site domain responded robustly to Aβ42-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Aβ42 produced. We thus expect that familial AD patients carrying mutations at the γ-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Aβ species (Aβ41, Aβ39) could also be lowered besides Aβ42. Moreover, certain mutations simultaneously increased Aβ42 and the shorter peptide Aβ38, arguing that the proposed precursor-product relationship of these Aβ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Aβ42 generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.

APP mutants respond to γ-secretase modulators

Pathogenic generation of the 42-amino acid variant of the amyloid β-peptide (Aβ) by β- and γ-secretase cleavage of the β-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Aβ42 production by γ-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Aβ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of γ-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Aβ domain regarding their response to GSMs. We found that Aβ42-increasing familial AD mutations of the γ-secretase cleavage site domain responded robustly to Aβ42-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Aβ42 produced. We thus expect that familial AD patients carrying mutations at the γ-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Aβ species (Aβ41, Aβ39) could also be lowered besides Aβ42. Moreover, certain mutations simultaneously increased Aβ42 and the shorter peptide Aβ38, arguing that the proposed precursor-product relationship of these Aβ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Aβ42 generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.