Analysis of Notch function in presomitic mesoderm suggests a gamma-secretase-independent role for presenilins in somite differentiation

The role of Notch signaling in general and presenilin in particular was analyzed during mouse somitogenesis. We visualize cyclical production of activated Notch (NICD) and establish that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, which have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without gamma-secretase, presenilin may have a gamma-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1(V-->G)) experience fortuitous cleavage of N1(V-->G) by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth is via the differential activation of Notch signaling

The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the γ-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-γ-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfection of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the γ-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.     

Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Activation of Notch signaling requires intramembranous cleavage by γ-secretase to release the intracellular domain. We previously demonstrated that presenilin and nicastrin, components of the γ-secretase complex, are required for neuronal survival in the adult cerebral cortex. Here we investigate whether Notch1 and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2, and Notch1/Notch2 conditional knock-out (cKO) mice. Unexpectedly, we did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to ～2 years of age, whereas conditional inactivation of presenilin or nicastrin using the same αCaMKII-Cre transgenic mouse caused progressive, striking neuronal loss beginning at 4 months of age. More surprisingly, we failed to detect any reduction of Notch1 and Notch2 mRNAs and proteins in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively, even though Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notch1/Notch2 pups, where Notch1 and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. Together, these results demonstrate that Notch1 and Notch2 are not involved in the age-related neurodegeneration caused by loss of presenilin or γ-secretase and suggest that there is no detectable expression of Notch1 and Notch2 in pyramidal neurons of the adult cerebral cortex.     

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.     

A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.     

Apoptotic activities of wild-type and Alzheimer's disease-related mutant presenilins in Drosophila melanogaster.

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. We show that loss of presenilin function in Drosophila melanogaster increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact approximately 300-kD precursor and its approximately 120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. We describe a genetic model in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and we find that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin.     

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.     

aph-1 and pen-2 are required for Notch pathway signaling,gamma-secretase cleavage of betaAPP,and presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.     

Functional implications of the presenilin dimerization: reconstitution of gamma-secretase activity by assembly of a catalytic site at the dimer interface of two catalytically inactive presenilins

Presenilins are the catalytic components of gamma-secretase, an intramembrane-cleaving protease whose substrates include beta-amyloid precursor protein (betaAPP) and the Notch receptors. These type I transmembrane proteins undergo two distinct presenilin-dependent cleavages within the transmembrane region, which result in the production of Abeta and APP intracellular domain (from betaAPP) and the Notch intracellular domain signaling peptide. Most cases of familial Alzheimer's disease are caused by presenilin mutations, which are scattered throughout the coding sequence. Although the underlying molecular mechanism is not yet known, the familial Alzheimer's disease mutations produce a shift in the ratio of the long and short forms of the Abeta peptide generated by the gamma-secretase. We and others have previously shown that presenilin homodimerizes and suggested that a presenilin dimer is at the catalytic core of gamma-secretase. Here, we demonstrate that presenilin transmembrane domains contribute to the formation of the dimer. In-frame substitution of the hydrophilic loop 1, located between transmembranes I and II, which modulates the interactions within the N-terminal fragment/N-terminal fragment dimer, abolishes both presenilinase and gamma-secretase activities. In addition, by reconstituting gamma-secretase activity from two catalytically inactive presenilin aspartic mutants, we provide evidence of an active diaspartyl group assembled at the interface between two presenilin monomers. Under our conditions, this catalytic group mediates the generation of APP intracellular domain and Abeta but not Notch intracellular domain, therefore suggesting that specific diaspartyl groups within the presenilin catalytic core of gamma-secretase mediate the cleavage of different substrates.     

The gamma-secretase complex: machinery for intramembrane proteolysis

Gamma-secretase is a membrane protease complex that possesses presenilin as a catalytic subunit. Presenilin generates amyloid beta peptides in the brains of Alzheimer's patients and is indispensable to Notch signaling in tissue development and renewal. Recent studies have revealed how presenilin is assembled with its cofactor proteins and acquires the gamma-secretase activity: Aph-1 and nicastrin initially form a subcomplex to bind and stabilize presenilin, and then Pen-2 confers the gamma-secretase activity and facilitates endoproteolysis of presenilin. Understanding the mechanism of gamma-secretase cleavage will help to clarify how intercellular cell signaling through transmembrane proteins is regulated by intramembrane proteolysis, and will hopefully eventually lead to a cure for Alzheimer's disease.     

Overexpression of human CRB1 or related isoforms, CRB2 and CRB3, does not regulate the human presenilin complex in culture cells

The presenilin proteins (PS1 and PS2) with their partners (NCT, Aph1, and Pen2) are the major components of the high molecular weight gamma-secretase complex which facilitates the intramembraneous cleavage of various type 1 transmembrane proteins, including the amyloid-beta precursor protein (APP) and the Notch receptor. Additional gamma-secretase complex components may be involved in regulation of its activity and specificity. A recent investigation indicated that the Crumbs protein is a negative regulator of Notch signaling and may act by repressing gamma/epsilon-secretase activity in Drosophila [Herranz, H., Stamataki, E., Feiguin, F., and Milan, M. (2006) EMBO Rep. 7, 297-302]. To address this question, we investigated potential functional interactions between the human Crumbs homologues (CRB1, CRB2, and CRB3) and presenilin complexes which mediate gamma/epsilon-secretase cleavage of APP and Notch. We found no evidence for direct interaction between CRB1, CRB2, or CRB3 and presenilin complex components. Furthermore, overexpression of human CRB1 and related isoforms, CRB2 and CRB3, had no effect on the levels of presenilin complex components, on NCT maturation or on PS endoproteolysis, and did not alter Abeta AICD or NICD production. These results suggest that, in mammalian cells at least, Crumbs is unlikely to be a significant direct modulator of presenilin-dependent gamma/epsilon-secretase activity.     

APH-2/Nicastrin Functions in LIN-12/Notch Signaling in the Caenorhabditis elegans Somatic Gonad

Nicastrin is a recently identified member of high-molecular weight complexes containing presenilin. The Caenorhabditis elegans homolog of nicastrin, aph-2, was shown to be required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. We show that this latter defect is related to the SEL-12/presenilin egg-laying defect. We also show that aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. We illustrate a new role for aph-2 in facilitating lin-12 signaling in the somatic gonad, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Finally, we demonstrate that nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins.     

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Here we present evidence for a new role: stimulating growth in response to Hedgehog. We show that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and we describe strong genetic interactions between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic.     

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.     

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.     

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.     

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.     

Antioxidant proteins TSA and PAG interact synergistically with Presenilin to modulate Notch signaling in Drosophila

Alzheimer’s disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer’s disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study we expressed a presenilin transgene in Drosophila wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. We found that while expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer’s pathogenesis.     

The gamma-secretase complex: membrane-embedded proteolytic ensemble

Gamma-secretase is responsible for the proteolytic processing of a variety of membrane-associated fragments derived from type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor. This enzyme is composed of four different integral membrane proteins: presenilin, nicastrin, Aph-1, and Pen-2. During assembly and maturation of the protease complex, presenilin is endoproteolyzed into two subunits, each of which contributes one aspartate to the active site of an aspartyl protease. Substrate apparently interacts with an initial docking site before passing in whole or in part between the two presenilin subunits to the internal water-containing active site. The ectodomain of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Sites for allosteric regulation on the protease complex allow selective inhibition or modulation of APP processing without interfering with Notch signaling, and such selective agents may represent promising leads for the development of Alzheimer's disease therapeutics. Elucidation of detailed structural features of gamma-secretase and other membrane-embedded proteases is the next frontier in understanding how these enzymes carry out hydrolysis within the lipid bilayer.