Drosophila nicastrin is essential for the intramembranous cleavage of notch.

The catalytic subunit of gamma-secretase is thought to be Presenilin, which is required for both the cleavage of APP and in the processing of Notch. Presenilin is found in a multisubunit complex that also contains Nicastrin. Nicastrin has been implicated in APP processing, but its role in Notch signaling remains unclear. Here we show that Drosophila Nicastrin is required for Notch signaling, and acts specifically at the S3 cleavage step. Partially processed Notch accumulates apically in nicastrin and presenilin mutant follicle cells. nicastrin and presenilin mutations also disrupt the spectrin cytoskeleton, suggesting that the gamma-secretase complex has another function in Drosophila in addition to its role in processing Notch and APP.     

Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.     

Cell-type-specific processing of the amyloid precursor protein by Presenilin during Drosophila development

The cleavage of proteins within their transmembrane domain by Presenilin (PS) has an important role in different signalling pathways and in Alzheimer's disease. Nevertheless, not much is known about the regulation of PS activity. It has been suggested that substrate recognition by the PS complex depends only on the size of the extracellular domain independent of the amino-acid sequence and that PS activity is constitutive in all cells that express the minimal components of the complex. We report here the development of an in vivo reporter system that allowed us to analyse the processing of human amyloid precursor protein (APP) and the Notch receptor tissue specifically during Drosophila development in the living organism. Using this system, we demonstrate differences between APP and Notch processing and show that PS-mediated cleavage of APP can be regulated in different cell types independent of the size of the extracellular domain.     

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.     

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53

The gamma-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. gamma-Secretase cleavage of APP releases the Abeta peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active gamma-secretase complex.     

Notch and presenilin: a proteolytic mechanism emerges.

Presenilins are needed for proteolytic processing of transmembrane proteins of the Notch/Lin-12 family and for cleavage of the amyloid precursor protein. Accumulating evidence now strongly implicates Presenilin as the catalytic core of a multiprotein complex that executes an unusual intramembranous cleavage of its substrates. In the case of amyloid precursor protein, this cleavage contributes to the generation of small, toxic amyloid peptides that trigger the pathological development of Alzheimer's disease. In the Notch/Lin-12 pathway, Presenilin-mediated cleavage of the receptor is a crucial feature of ligand-induced receptor activation and signal transduction. In this pathway, the Presenilins perform a regulated cleavage event that follows additional processing steps during receptor maturation and ligand-induced ectodomain removal.     

Identifying genes that interact with Drosophila presenilin and amyloid precursor protein

The γ‐secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of γ‐secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin‐interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin‐dependent Notch‐related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease. genesis 47:246–260, 2009. © 2009 Wiley‐Liss, Inc.     

Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-beta precursor protein and Notch

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.     

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.     

Notch cleavage: Nicastrin helps Presenilin make the final cut

Presenilin is thought to be the proteolytic component of gamma-secretase, responsible for the intramembranous cleavage of substrates that include the activated Notch receptor. Recent studies have identified the novel protein Nicastrin as another essential component of the Presenilin/gamma-secretase complex.     

Hibris, a Drosophila nephrin homolog, is required for presenilin-mediated Notch and APP-like cleavages

Drosophila Hibris (Hbs), a member of the Nephrin Immunoglobulin Super Family, has been implicated in myogenesis and eye patterning. Here, we uncover a role of Hbs in Notch (N) signaling and γ-secretase processing. Loss of hbs results in classical N-signaling-associated phenotypes in Drosophila, including eye patterning, wing margin, and sensory organ specification defects. In particular, hbs mutant larvae display altered γ-secretase-dependent Notch proteolytic processing. Hbs also interacts molecularly and genetically with Presenilin (Psn) and other components of the γ-secretase complex. This Hbs function appears conserved, as mammalian Nephrin also promotes N signaling in mammalian cells. Our data suggest that Hbs is required for Psn maturation. Consistent with its role in Psn processing, Hbs genetically interacts with the Drosophila β-amyloid protein precursor-like (Appl) protein, the homolog of mammalian APP, the cleavage of which is associated with Alzheimer's disease. Thus, Hbs/Nephrin appear to share a general requirement in Psn/γ-secretase regulation and associated processes.     

Low density lipoprotein receptor-related protein (LRP) interacts with presenilin 1 and is a competitive substrate of the amyloid precursor protein (APP) for gamma-secretase

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a gamma-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for gamma-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that gamma-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a gamma-secretase docking site located in close proximity to PS1. This is analogous to APP-gamma-secretase interactions. Finally, we show that LRP competes with APP for gamma-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the Abeta40, Abeta42, and p3 peptides without changing the total level of APP expression. In addition, transfection with the beta-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.     

Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.

The amyloid-beta protein (Abeta) is strongly implicated in the pathogenesis of Alzheimer's disease. The final step in the production of Abeta from the amyloid precursor protein (APP) is proteolysis by the unidentified gamma-secretases. This cleavage event is unusual in that it apparently occurs within the transmembrane region of the substrate. Studies with substrate-based inhibitors together with molecular modeling and mutagenesis of the gamma-secretase cleavage site of APP suggest that gamma-secretases are aspartyl proteases that catalyze a novel intramembranous proteolysis. This proteolysis requires the presenilins, proteins with eight transmembrane domains that are mutated in most cases of autosomal dominant familial Alzheimer's disease. Two conserved transmembrane aspartates in presenilins are essential for gamma-secretase activity, suggesting that presenilins themselves are gamma-secretases. Moreover, presenilins also mediate the apparently intramembranous cleavage of the Notch receptor, an event critical for Notch signaling and embryonic development. Thus, if presenilins are gamma-secretases, then they are also likely the proteases that cleave Notch within its transmembrane domain. Another protease, S2P, involved in the processing of the sterol regulatory element binding protein, is also a multipass integral membrane protein which cleaves within or very close to the transmembrane region of its substrate. Thus, presenilins and S2P appear to be members of a new type of polytopic protease with an intramembranous active site.     

A sensitive and quantitative assay for measuring cleavage of presenilin substrates.

The presenilin (PS) proteins are components of the gamma-secretase activity, which is central in the pathogenesis of Alzheimer's disease. Here we present a novel cell-based reporter gene assay for the quantification of PS-controlled gamma-secretase cleavage of the Alzheimer amyloid precursor protein (APP). We show that this assay offers several advantages, including increased sensitivity and specificity, improved quantification of cleavage, and simultaneous detection of all gamma-secretase cleavages in APP. Furthermore, the APP assay can be used in parallel with a similar assay that records gamma-secretase cleavage of a Notch receptor. The use of these assays to analyze the effects of two known gamma-secretase inhibitors and postulated PS active site mutants on APP and Notch processing demonstrated that inhibitors and mutants that differently affect Notch and APP cleavage can be identified rapidly. The possibility in using these assays for high throughput screening of candidate gamma-secretase inhibitors for APP and Notch in parallel opens up new vistas to systematically search for novel inhibitors that selectively block APP cleavage while not affecting Notch signaling.     

Presenilin affects arm/beta-catenin localization and function in Drosophila

Presenilin is an essential gene for development that when disrupted leads to a neurogenic phenotype that closely resembles Notch loss of function in Drosophila. In humans, many naturally occurring mutations in Presenilin 1 or 2 cause early onset Alzheimer's disease. Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions were not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in Armadillo and exhibited reduced Armadillo at the plasma membrane that is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein which may be delta-catenin. Our results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome.     

Alzheimer’s disease and Notch signaling

Cleavage of the amyloid precursor protein (APP) by γ-secretase generates a neurotoxic amyloid β-peptide (Aβ) that is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients. Presenilin is the catalytic member of the γ-secretase proteolytic complex and mutations in presenilins are the major cause of early-onset familial Alzheimer’s disease. In addition to APP, γ-secretase substrates include Notch1 homologues, Notch ligands Delta and Jagged, and additional type I membrane proteins, raising concerns about mechanism-based toxicities that might arise as a consequence of inhibiting γ-secretase. Notch signaling is involved in tumorigenesis as well as in determining the fates of neural and nonneural cells during development and in adults. Alterations in proteolysis of the Notch by γ-secretase could be involved in the pathogenesis of AD. Inconsistently, several recent observations have indicated that enhanced Notch signaling and expression could be instrumental in neurodegeneration in AD. Therefore, detailed and precise study of Notch signaling in AD is important for elucidating diverse mechanisms of pathogenesis and potentially for treating and preventing Alzheimer’s disease.     

Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage

Research on Alzheimer's disease led to the identification of a novel proteolytic mechanism in all metazoans, the presenilin/gamma-secretase complex. This unique intramembrane-cleaving aspartyl protease is required for the normal processing of Notch, Jagged, beta-amyloid precursor protein (APP), E-cadherin, and many other receptor-like proteins. We recently provided indirect evidence of gamma-secretase activity at the cell surface in HeLa cells following inhibition of receptor-mediated endocytosis. Here, we directly identify and isolate gamma-secretase as an intact complex (Presenilin, Nicastrin, Aph-1, and Pen-2) from the plasma membrane, both in overexpressing cell lines and endogenously. Inhibition of its proteolytic activity allowed cell surface gamma-secretase to be captured in association with its plasma membrane-localized APP substrates (C83 and C99). Moreover, non-denaturing isolation of the intact enzyme complex revealed that cell surface gamma-secretase can specifically generate amyloid beta-protein from an APP substrate and similarly cleave a Notch substrate. These data directly establish the proteolytic function of gamma-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role. We conclude that presenilin/gamma-secretase exists as a mature complex at the cell surface, where it interacts with and can cleave its substrates, consistent with an essential function in processing many adhesion molecules and receptors required for cell-cell interaction or intercellular signaling.     

Nicastrin functions as a gamma-secretase-substrate receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.     

Notch and the amyloid precursor protein are cleaved by similar gamma-secretase(s)

Gamma-secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the gamma-secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP gamma-like proteolysis. In an effort to resolve this issue directly, we established an in vitro gamma-secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and N100Flag. Analysis with specific gamma-secretase inhibitors, dominant-negative gamma-secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same gamma-secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).     

Characterization of Drosophila Presenilin and its colocalization with Notch during development.

Mutant Presenilin proteins cause early-onset familial Alzheimer's disease in humans and Caenorhabditis elegans Presenilins may facilitate Notch receptor signaling. We have isolated a Drosophila Presenilin homologue and determined the spatial and temporal distribution of the encoded protein as well as its localization relative to the fly Notch protein. In contrast to previous mRNA in situ studies, we find that Presenilin is widely expressed throughout oogenesis, embryogenesis, and imaginal development, and generally accumulates at comparable levels in neuronal and nonneuronal tissues. Double immunolabeling with Notch antibodies revealed that Presenilin and Notch are coexpressed in many tissues throughout Drosophila development and display partially overlapping subcellular localizations, supporting a possible functional link between Presenilin and Notch.