InteractiveFly: GeneBrief

Nicastrin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Nicastrin is involved Notch signaling and along with Presenilin forms part of a large multiprotein complex. Drosophila nicastrin (nic) mutants display characteristic Notch-like phenotypes. Genetic and inhibitor studies indicate a function for Nicastrin in the gamma-secretase step of Notch processing, similar to Presenilin. Further, Nicastrin is genetically required for signaling from membrane-anchored activated Notch. In the absence of Nicastrin, Presenilin is destabilized and mature C-terminal subunits are absent. Partially processed Notch accumulates apically in nicastrin and presenilin mutant follicle cells. nicastrin and presenilin mutations disrupt the spectrin cytoskeleton, suggesting that the gamma-secretase complex has another function in Drosophila in addition to its role in processing Notch and the second target, ß amyloid protein precursor (APP). Nicastrin might recruit gamma-secretase substrates into the proteolytic complex as a prerequisite for Presenilin maturation and active complex assembly (Hu, 2002; López-Schier, 2002).

Recent studies have established that the crucial signal-generating cleavage of Notch depends upon a large protein complex containing Presenilin, with Presenilin itself likely performing the catalytic role in Notch proteolysis. The Presenilins are a family of polytopic membrane proteins having eight putative transmembrane domains and a large cytoplasmic loop, and they are localized predominantly in the ER/Golgi compartment. Mutations in the two known human Presenilin genes PS1 and PS2 account for most cases of early onset, autosomal dominant Alzheimer's disease. The PS mutations promote the generation of neurotoxic Aß peptide derivatives of amyloid precursor protein (APP) by altering the manner in which APP is cleaved at a site, termed the gamma-secretase site, within its transmembrane domain. These observations led to the hypothesis that Presenilin might itself be the long sought 'gamma-secretase' enzyme responsible for this cleavage. Several findings have now provided strong support for this idea. Elimination of PS1 activity in knockout mice leads to reduced gamma cleavage of APP, and mutagenesis of either of two conserved aspartate residues in PS1 interferes with APP cleavage. Moreover, PS1 and gamma-secretase activity can be biochemically copurified from solubilized HeLa cell membranes, and transition-state analog inhibitors of gamma-secretase label PS1 and PS2. Finally, database comparisons have led to the identification of a conserved motif in PS proteins exhibiting similarity to a short amino acid segment found in a family of bacterial aspartyl proteases (Hu, 2002 and references therein).

The assertion that Presenilin itself is a proteolytic enzyme has not been completely accepted, due to the difficulty in reconstituting in vitro the gamma-secretase proteolytic activity. Presenilin apparently functions as part of a large multiprotein complex (>2000 kD), which contains numerous other unidentified factors that might also be required for proteolytic activity of the intact complex. Another component of the complex, termed Nicastrin, has recently been identified, although the exact function of Nicastrin in the gamma-secretase cleavage of substrate proteins is unknown. Mammalian Nicastrin binds to C-terminal derivatives of APP that are substrates for gamma-secretase, and overexpression of engineered Nicastrin mutants influences cleavage of APP and subsequent Aß secretion (Yu, 2000). Nicastrin has also been implicated in the regulation of Notch proteolysis. Mutations in the C. elegans Nicastrin homolog aph-2 were isolated as mutants displaying Notch/Lin-12 pathway defects (Goutte, 2000), and overexpression of the engineered mammalian Nicastrin mutants has weak modulatory effects on Notch cleavage (Chen, 2001). Nicastrin also binds to membrane-tethered forms of Notch that are structurally analogous to the C-terminal derivatives of APP, suggesting that Nicastrin might generally bind to gamma-secretase proteolytic substrates (Chen, 2001). However, it has not been shown that Nicastrin is directly required for gamma-secretase-like proteolysis of the Notch receptor, or how the loss of Nicastrin might affect formation and/or function of the gamma-secretase complex (Hu, 2002).

Embryos that lack both maternal and zygotic nct display a strong neurogenic phenotype associated with a loss of Notch signaling. The cuticle fails to form except for a small patch on the dorsal side of the embryo, and there is a hyperplasia of the embryonic nervous system, as revealed by a large increase in the number of cells expressing the neuronal marker MAb22C10. nct^agro homozygous clones also produce the same phenotypes as Notch clones in other tissues. In the wing imaginal disc, Notch signaling establishes the dorsal-ventral compartment boundary, which is marked by the expression of Cut protein. In mosaic discs containing marked nct mutant clones, Cut is not expressed in mutant cells along the boundary. Mutant clones also produce other typical Notch phenotypes, such as large notches in the wing margin, thickening of the wing veins, and the loss or duplication of sensory bristles (López-Schier, 2002).

During stages 5-6 of oogenesis, Delta signals from the germline to activate Notch in the somatic follicle cells, inducing these cells to stop mitosis and differentiate, and this provides an excellent system for determining whether components of the Notch pathway are required in the signaling or responding cells. Whereas nct germline clones have no effect on follicle cell development, mutant follicle cell clones show an identical phenotype to clones of null mutations in Notch or psn. The cells continue dividing after stage 6 of oogenesis, as shown by the expression of the mitotic marker phospho-histone H3, and this causes an increase in cell number and decrease in cell size in mutant clones. Mutant cells also fail to differentiate, since they continue to express high levels of the immature follicle cell marker Fasiclin III (Fas III), and never express any markers of differentiated follicle cell types. For example, mutant clones at the posterior of the egg chamber cannot adopt a posterior fate in response to the Gurken signal from the oocyte, and these cells therefore fail to signal back to the germline to polarize the anterior-posterior axis of the oocyte. As a consequence, the oocyte does not repolarize at stage 7, and the germinal vesicle fails to migrate from the posterior to the anterior pole. This requirement for Nct is strictly cell autonomous, since mutant follicle cells always express Fas III, even in very small clones. Thus, nct is a novel neurogenic gene that is required cell autonomously for the transduction of the Notch signal in responding cells (López-Schier, 2002).

By isolating and characterizing nicastrin loss-of-function mutants in Drosophila, it has been demonstrated that Nicastrin is required both genetically and biochemically for proteolysis of membrane-tethered forms of Notch and associated Notch signaling activity. The Drosophila nicastrin mutants exhibit defects in Notch signaling and intramembranous cleavage that are indistinguishable from those seen in presenilin mutants. Notch-like phenotypes are observed in various tissues at all stages of development, and may be attributed to a specific failure in the gamma-secretase-like cleavage of Notch that normally generates an ~120 kDa intracellular signaling fragment from the activated receptor. Identical biochemical effects are observed for Drosophila Notch with the gamma-secretase inhibitor DFK-167, supporting the role of Nicastrin in this proteolytic event and suggesting that Drosophila Notch processing might be a useful model for investigating pharmacological inhibitors of gamma-secretase. Analysis of Notch immunoreactivity in genetic mosaics reveals that homozygous loss of nicastrin activity results in a subtle overaccumulation of Notch proteins at the apical intracellular surface. Similar observations have been reported for Drosophila Psn mutants, and are consistent with the idea that nic and Psn are necessary for cleavage and release of membrane-anchored Notch C-terminal fragments from the cell surface (Hu, 2002).

In mammalian cells, expression of engineered mutants of Nicastrin with amino acid substitutions or internal deletions in a conserved domain have been reported to have dramatic modulatory effects on APP processing and modest effects on Notch proteolysis. In an RNAi-based Drosophila S2 cell assay, expression of analogous engineered forms of Drosophila Nicastrin does not lead to any observable proteolytic activity toward Notch or any dominant-negative effects. These results indicate that some aspects of Nicastrin function might not be conserved among different species, or that differences in the assay systems might underlie the contradictory results (Hu, 2002).

The function of Nicastrin in the large gamma-secretase proteolytic complex is not well understood. Presenilins from different species show high levels of sequence conservation in their transmembrane domains, including conserved aspartyl residues thought to be important for catalysis, consistent with the proposed catalytic role in the intramembranous cleavage of membrane-bound substrates. In contrast, Nicastrins from various organisms display little conservation in their single transmembrane segment and short intracellular domain, suggesting that they are unlikely to participate directly in the proteolytic reaction within the lipid bilayer. The most highly conserved segments of Nicastrin are located in the large extracellular domain, while only a very small portion of Presenilin consisting of the short loops connecting the transmembrane domains is topologically exposed at the extracellular surface. Since Nicastrin binds to C-terminal membrane-anchored substrates of gamma-secretase (Yu, 2000; Chen, 2001), one speculative idea is that the extracellular domain of Nicastrin might act as a sensor that measures the size of the extracellular domain of bound substrates, a critical determinant of cleavage efficiency. Further functional characterization of the gamma-secretase complex will be necessary to evaluate this possibility (Hu, 2002).

Data showing that mature Presenilin protein is unstable in Drosophila cells lacking Nicastrin might simply be due to a general degradation of Presenilin in the absence of an essential interacting protein. However, other studies have suggested that steady-state levels of mature Presenilin are regulated by a specific 'stabilization/cleavage' mechanism in which most nascent full-length Presenilin is degraded before incorporation into the complex, while a small pool becomes stabilized by unknown factors and undergoes subsequent endoproteolytic maturation. Nicastrin might be a limiting cellular factor which influences the amount of Presenilin that is either stabilized or degraded. Some data indicate that binding of Nicastrin to Presenilin occurs prior to assembly of the large multiprotein gamma-secretase complex. For example, the aspartyl mutant PS1-D385A is unable to be incorporated into the complex, but is still able to bind human Nicastrin (Yu, 2000). Since Nicastrin is known to bind to Notch and APP C-terminal derivatives, one possibility is that the inactive PS holoprotein might only be stabilized and converted into an active protease following association with Nicastrin bound to an appropriate substrate (Hu, 2002).

In vivo reconstitution of γ-secretase in Drosophila results in substrate specificity

The intramembrane aspartyl protease γ-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimer's disease. This study describes a transgenic Drosophila model for in vivo-reconstituted γ-secretase, based on expression of epitope-tagged versions of the four core γ-secretase components, Presenilin, Nicastrin, Aph-1, and Pen-2. In agreement with previous cell culture and yeast studies, coexpression of these four components promotes the efficient assembly of mature, proteolytically active γ-secretase. In vivo-reconstituted γ-secretase has biochemical properties and a subcellular distribution resembling those of endogenous γ-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted γ-secretase toward different substrates, including markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor. These findings indicate that in vivo under physiological conditions, additional factors differentially modulate the activity of γ-secretase toward its substrates. Thus, this approach for the first time demonstrates the overall functionality of reconstituted γ-secretase in a multicellular organism and the requirement for substrate-specific factors for efficient in vivo cleavage of certain substrates (Stempfle, 2010).

The Presenilin (PS) protein family was first identified on the basis of dominant familial mutations inducing early-onset Alzheimer's disease. An important pathological feature of Alzheimer's disease is the formation of plaques caused by the deposition of amyloid peptides derived from the proteolytic cleavage of the amyloid precursor protein (APP). The pathogenic Aβ peptide is produced by the sequential cleavage of APP through β- and γ-secretases. The first cleavage by β-secretase occurs at an extracellular site near the transmembrane region of APP, leading to secretion of the extracellular domain (ECD). The remaining C-terminal fragment (CTF) serves as a substrate for γ-secretase, which mediates proteolysis inside the membrane region, releasing the APP intracellular domain (AICD) and the Aβ peptide (Stempfle, 2010).

PS contains two aspartate residues that are essential for the catalytic activity of the complex and that are thought to form the active center of the protease. Nct, Aph-1, and Pen-2 contribute to the maturation and stabilization of the complex. Furthermore, evidence has been obtained for a function of Nct in substrate recognition (Shah, 1995), a view challenged by a recent study suggesting that Nct is instead needed only for maturation of the complex (Chávez-Gutiérrez, 2008). Cell-based and cell-free assays have shown that only the coordinated overexpression of all four proteins leads to an increase in γ-secretase activity, arguing that they form the minimal active complex and that the assembly and maturation of the complex are highly regulated. In Drosophila, there is only one homolog of PS and Aph-1, whereas two homologs of each exist in mammalian cells (PS1/PS2 and Aph-1a/Aph-1b). Based on the fact that alternative aph-1a splice forms can be detected, it has been suggested, and subsequently demonstrated, that at least six distinct γ-secretase complexes exist in mammalian cells. However, to date, the precise compositions and architectures of these complexes are not known, and depending on the experimental conditions used, complexes with a molecular mass of 250, 500, or >2,000 kDa have been isolated in vitro. Recently, it was shown that a complex containing only one of each component displays in vitro activity, but it is unknown whether this activity is found in vivo as well. Furthermore, recently published interactome analyses of γ-secretase suggest that it interacts with a variety of other proteins, which could be important for maturation, localization, and/or enzymatic activity. Taken together, these findings illustrate a potential limitation with the analysis of purified γ-secretase complexes, namely, that their observed in vitro minimal activities might not fully reflect their full range of activities and cleavage efficiencies in vivo (Stempfle, 2010).

In one of the earliest models addressing PS activity and specificity, it was suggested that substrate recognition by the γ-secretase complex depends only on the size of the ECD and is sequence independent. To address this phenomenon in more detail, a reporter system was developed in Drosophila which monitors the cleavage of transmembrane proteins in vivo during Drosophila development, and it was shown that PS-mediated cleavage of APP is regulated in a cell-type-specific manner, independent of the size of the ECD. In the meantime, several studies have confirmed the existence of complex regulatory mechanisms that influence the cleavage efficiencies of different substrate CTFs (Stempfle, 2010).

To address the question of the activity of the core PS complex and the contribution of substrate-specific factors, the Drosophila PS complex was reconstituted in vivo by simultaneous overexpression of tagged versions of the four core components with the GAL4/upstream activation sequence (UAS) system, and its ability to cleave alternative model substrates was analyzed, including ones based on Notch and different APP/APLP family members. In contrast to mammalian γ-secretase, Drosophila γ-secretase is homogenous in composition due to the fact that single-copy genes encode each of its four core subunits. The Drosophila-based approach thus permits the analysis of substrate-specific cleavage efficiencies without the additional complication of multiple potential γ-secretase complexes having distinct enzymatic properties. It was found that in vivo-reconstituted Drosophila γ-secretase displayed biochemical and cell biological features similar to those of endogenous γ-secretase but exhibited substrate-specific effects with respect to its proteolytic activities toward Notch and APP/APLP proteins. These findings have potential implications for understanding the diverse functions of γ-secretase involving different cleaved substrates in different tissues, as well as the development of pharmacological inhibitors that could potentially target specific substrates (Stempfle, 2010).

REGULATION

Protein Interactions

aph-1 and pen-2 are required for Notch pathway signaling, gamma-Secretase cleavage of ßAPP, and Presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of ßAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 (Drosophila homolog: anterior pharynx defective 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 ßAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, which, like nicastrin, are required for the activity and accumulation of gamma-secretase (Francis, 2002).

To study the roles of aph-1 and pen-2 in APP and Notch cleavage, γ-secretase activity assays were developed in Drosophila tissue culture cells, similar to nuclear access assays that accurately report presenilin-dependent processing in Drosophila. To confirm that the Drosophila cell assays accurately measure γ-secretase-like activity, Dmel2 cells and developing flies were tested for sensitivity to previously described γ-secretase inhibitors. When applied to Drosophila embryos and larvae at intermediate concentrations, compound E (Seiffert, 2000) causes adult wing notching and rough eye phenotypes characteristic of Notch mutants and a presenilin hypomorphic mutant. Compound E also induces glp-1-like sterility when applied to developing hop-1 mutant C. elegans. These results demonstrate that this selective γ-secretase inhibitor interferes with Notch signaling in vivo in Drosophila and C. elegans. In Dmel2 cell assays, compound E inhibits membrane-tethered APP C99-GV reporter activity by 2- to 3-fold but has no effect on the non-membrane-bound APP C59-GV reporter activity. Similar reporter inhibition was obtained with two additional γ-secretase inhibitors. Membrane-tethered N^ECN-GV reporter activity is inhibited by compound E at very similar concentrations to C99-GV, while N^INTRA-GV reporter activity is unaffected (Francis, 2002).

To determine whether the γ-secretase activity observed in Dmel2 cells produces the same cleavage products as the human enzyme, secreted Aβ peptides released from the C99-GV substrate were measured using specific Aβ40 and Aβ42 ELISA assays. Aβ40 and Aβ42 are both detected, and, as in human cell supernatants, Aβ40 is approximately 10-fold more abundant than Aβ42. Production of both peptides is inhibited by compound E to levels below the limit of detection in ELISA assay conditions. The ability of compound E to completely inhibit Aβ production in this assay contrasted with the maximum 2- to 3-fold inhibition of N^ECN-GV and C99-GV reporter gene activity observed, suggesting that residual reporter gene activity may be a result of presenilin-independent nuclear access of the GV activator in Dmel2 cells. As described for mammalian presenilins, high concentrations of compound E cause an increase in ~50 kDa full-length presenilin levels, but the strong inhibition of Aβ production observed in this assay cannot be explained simply by reduction in presenilin CTF levels. These data confirm that Dmel2 cells have an endogenous γ-secretase activity with pharmacological and substrate cleavage properties similar to human γ-secretase (Francis, 2002).

RNAi was used to determine whether γ-secretase activity in Dmel2 cells is presenilin dependent. RNAi of Drosophila psn strongly reduces endogenous PSN C-terminal fragment (CTF) protein levels. psn RNAi also reduced secreted Aβ40 and Aβ42 peptides by 85% and decreased C99-GV and N^ECN-GV reporter gene activity by 50%–70% but had no effect on the intracellular domain reporters C59-GV or N^INTRA-GV. The APP cleavage reporter, Notch cleavage reporter, and Aβ ELISA assays thus each demonstrate presenilin dependence and provide independent measures of γ-secretase function (Francis, 2002).

RNAi of Drosophila aph-1, pen-2, or nicastrin (nct) results in reduction of secreted Aβ40 and Aβ42 and reduction in reporter gene activity elicited by both C99-GV and N^ECN-GV, but not by C59-GV or N^INTRA-GV. Inhibition in each case is approximately as strong as that observed for RNAi of presenilin itself. In addition, RNAi of aph-1 and pen-2, like that of nct, leads to strong reductions in PSN CTF protein levels, with no buildup of detectable full-length PSN. pen-2 RNAi is slightly less efficient and more variable in these assays than aph-1 or nct RNAi, in contrast to the identical behavior of pen-2 and aph-1 in all C. elegans in vivo genetic assays. The difference in efficiency of pen-2 versus aph-1 RNAi in Drosophila cells may thus reflect incomplete inactivation of pen-2 by RNAi rather than a functional difference between the two genes. These data reveal that aph-1 and pen-2, like nct, are necessary for γ-secretase activity and for the accumulation of processed presenilin protein in Drosophila cells (Francis, 2002).

Using a genetic approach in C. elegans, aph-1 and pen-2, two new, phylogenetically conserved members of the Notch pathway have been identified. aph-1 and pen-2 have very similar functions in C. elegans, since their individual mutant phenotypes and their genetic interaction phenotypes with aph-2, hop-1, and sel-12 are indistinguishable. aph-1 and pen-2 are required for all lin-12- and glp-1-mediated signaling events examined, and no phenotypes were observed in the aph-1 or pen-2 mutants that would suggest functions other than in lin-12/glp-1-dependent processes. It is concluded that aph-1 and pen-2 define new obligate members of the Notch signaling pathway (Francis, 2002).

The sel-12 enhancer screen identified only these two genes with strong effects on germline presenilin activity. Three other related genetic screens were performed for presenilin pathway components and additional alleles were isolated of aph-1, pen-2 and aph-2, but no additional genes were found with similar strong phenotypes. If other genes that are essential for presenilin function in C. elegans exist, they may be masked by genetic redundancy or may have additional functions and associated phenotypes that obscure their contributions to Notch signaling. These genetic studies suggest that aph-1, pen-2, and aph-2 define a set of genes that is unique in the strength and specificity of their interactions with the presenilin genes (Francis, 2002).

The lin-12(gf) suppression tests and Drosophila cell culture experiments on defined γ-secretase substrates presented in this study demonstrate that aph-1 and pen-2 are required for Notch and βAPP cleavages, specifically at the presenilin-mediated γ-secretase/S3 step. It was also found that aph-1 and pen-2, like nct, are required for accumulation of processed presenilin protein. Although this requirement could be the simple explanation for the loss of γ-secretase activity upon depletion of aph-1 and pen-2, recent studies have suggested that there may be separable requirements for nicastrin in Notch signaling and in accumulation or maintenance of presenilin protein. Similarly, cell culture data suggest that pen-2 RNAi can have stronger inhibitory effects on γ-secretase activity than on presenilin protein levels, suggesting that, as is the case for nicastrin, γ-secretase activity may be lost more rapidly than PSN-CTF stability or accumulation upon pen-2 depletion. Although the mechanism is not clear, accumulation of processed presenilin in Drosophila cells depends on aph-1, pen-2, and nct (Francis, 2002).

Despite their extensive functional similarities with aph-2, aph-1 and pen-2 behave distinctly from aph-2 in certain genetic interaction tests. (1) aph-2 mutants strongly interact with hop-1, but not with sel-12 mutants, to produce the glp-sterile phenotype, while aph-1 and pen-2 mutants exhibit sterility with sel-12, but not with hop-1 mutants. (2) aph-1 and pen-2 mutants or RNAi do not show additive phenotypes with each other, while aph-2 RNAi interacts with both aph-1 and pen-2 mutants to give partially penetrant sterility. (3) Rescue of either aph-1 or pen-2 is dependent on coexpression of human aph-1 and pen-2 genes together, but not on nicastrin, whereas aph-2 is rescued by human nicastrin alone. APH-2/nicastrin is a type 1 glycosylated transmembrane protein, while aph-1 and pen-2 are predicted to be polytopic integral membrane proteins, suggesting a basis for functional differences between aph-2 and the aph-1 and pen-2 genes. It is suggested that aph-1 and pen-2 interact closely with each other in the same process, perhaps at the same step, but in a role somewhat distinct from that of aph-2/nicastrin, to facilitate presenilin activity (Francis, 2002).

In rescue experiments it was found that human aph-1 and pen-2 are corequired for rescue. APH-1 and PEN-2 thus cooperate functionally, consistent with a model in which the two proteins associate directly. The APH-1 and PEN-2 proteins are highly conserved, but sequence divergence between the C. elegans and human APH-1 and PEN-2 proteins might interfere with functional cross-species APH-1/PEN-2 interactions and account for the corequirement observed for aph-1 and pen-2 mutant rescue. Cross-species interactions between a putative APH-1/PEN-2 unit and presenilins can occur because haph-1 and hpen-2 together are able to confer low-level rescue of pen-2 mutants in the absence of PS1 and because human PS1 or PS2 alone is able to rescue sel-12 mutants. However, coinjection of human PS1 together with haph-1 and hpen-2 improves the efficiency of pen-2 rescue, suggesting that the putative APH-1/PEN-2 protein complex also interacts with presenilin, and these interactions are more functional with intraspecific forms. APH-1 and PEN-2 are good candidates to be regulators and/or components of the high-molecular weight γ-secretase complex (Francis, 2002).

The role for γ-secretase in proteolysis of Notch, APP, and other cell surface transmembrane proteins suggests a site of action at the cell surface or in endocytic compartments. Some presenilin is detected in these locations, although most is found in ER and Golgi compartments. PEN-2::GFP expression in adult C. elegans, like SEL-12::GFP, is found primarily in internal membrane compartments, consistent with a model in which PEN-2 interacts with APH-1 and presenilin early in the secretory pathway. Endogenous APH-2 protein in early C. elegans embryos, in contrast, is localized primarily to the plasma membrane. In aph-1 and hop-1; sel-12 mutant embryos, APH-2 localizes instead in a perinuclear staining pattern consistent with ER/Golgi localization. Transport of APH-2/nicastrin to the cell surface is thus dependent on presenilins and APH-1. One model for APH-1 and PEN-2 function would be to facilitate trafficking of APH-2 to the cell surface, which in turn promotes trafficking of presenilin. However, genetic data favor a model in which APH-1 and PEN-2 together interact directly with presenilin or a presenilin/nicastrin complex to promote maturation and accumulation of the complex. It will be important to examine expression of endogenous proteins in the same cell types to determine to what extent APH-1 and PEN-2 colocalize with presenilins and nicastrin (Francis, 2002).

aph-1, pen-2, and aph-2/nicastrin define a core set of genes essential for the activity and accumulation of presenilin-dependent γ-secretase complexes. Several mechanisms could explain the failure to stably accumulate processed presenilin protein in the absence of APH-1, PEN-2, and APH-2/nicastrin proteins, including an inability to assemble, mature, transport, or stabilize the γ-secretase complex. If APH-1 and PEN-2 proteins interact directly with each other and with presenilin, these interactions could be transitory during early steps in γ-secretase complex assembly or may persist in the mature enzyme complex. Further studies of APH-1 and PEN-2 function should enhance the understanding of the γ-secretase enzyme and improve opportunities for the design of selective Alzheimer's disease therapeutics (Francis, 2002).

The role of presenilin cofactors in the gamma-secretase complex

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and ß-amyloid precursor protein (APP). Cleavage of APP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid 42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (also known as Pen-1 or Presenilin stabilization factor) and PEN-2. However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Drosophila APH-1 (Aph-1) is shown to increase the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex (Takasugi, 2003).

Presenilin is essential for gamma-secretase cleavage, which releases amyloid ß-peptide (Aß) and the intracellular domain of Notch by intramembranous proteolysis of ß-amyloid precursor protein (APP) and Notch, respectively. Presenilin mediates gamma-secretase function by forming a highly stable protein complex of high relative molecular mass (high-Mr) together with a set of cofactor proteins. In addition to nicastrin (NCT), a type I single-pass membrane glycoprotein, two additional putative presenilin cofactors have been identified: APH-1, a multi-transmembrane protein coded by a gene whose deletion leads to hypoplasia of the anterior pharynx in Caenorhabditis elegans, was found to be a Notch pathway member possibly involved in presenilin function; aph-1 was also identified as one of the presenilin enhancer genes (pen-1) together with pen-2, which codes for a double-membrane-spanning protein. NCT, APH-1 and PEN-2 are required for gamma-secretase function and accumulation of presenilin fragments, although an understanding of the differential roles of each cofactor in the formation of the high-Mr presenilin protein complex, and whether NCT, APH-1 and PEN-2 represent the principal presenilin cofactors to confer gamma-secretase activity, has remained elusive (Takasugi, 2003).

Drosophila S2 cells was stably transfected with Aph-1, and a significant increase in the levels of endogenous Psn holoprotein was found. This is markedly enhanced by expression of Aph-1 and Drosophila Nct, but is not observed in cells transfected with Nct alone. The levels of Psn fragments involved in the active form of gamma-secretase are not altered in either case. Stably co-transfected S2 cells overexpressing Aph-1 and Nct were treated with cycloheximide (CHX) to block total cellular protein synthesis, and the stability of Psn and other proteins was examined. In mock-transfected S2 cells (transfected with an empty vector alone) only small amounts of Psn holoprotein were detectable; these were rapidly degraded within about 4 h of CHX treatment, however, fragments of Psn were relatively more abundant and highly stable, in a manner similar to that of mammalian presenilin. In contrast, Psn holoprotein levels were significantly increased by overexpression of Aph-1 or of Aph-1 and Nct, and remained highly stabilized. Levels of Aph-1 and Nct in co-transfected S2 cells were also highly stable during the period of CHX treatment, suggesting that Psn (including holoprotein) forms a highly stabilized protein complex together with Aph-1 and Nct under these conditions. To gain support for this hypothesis, CHAPSO-solubilized membrane fractions of S2 cells stably transfected with Aph-1 and Nct were separated by glycerol velocity gradient centrifugation. Psn holoprotein in cells overexpressing Aph-1 and Nct was fractionated totally in high-Mr ranges of 232-440K together with Psn fragments. This was in contrast to the exclusive low-Mr distribution of short-lived Psn holoproteins in S2 cells in the absence of Aph-1 overexpression. Furthermore, most of the Aph-1 and Nct proteins were found in the high-Mr fractions. Taken together, these data support the hypothesis that Aph-1 represents the major 'stabilizing' cofactor of Psn that promotes the key step of stable high-Mr complex formation. However, the level of A secretion from cells with or without co-expression of Nct and Aph-1 was not significantly different, suggesting that some additional factors are needed for the upregulation of gamma-secretase activity (Takasugi, 2003).

To examine the role of PEN-2 (another putative cofactor of presenilin that harbours two membrane-spanning domains) in the formation and function of the gamma-secretase complex, double-stranded RNA-mediated interference (RNAi) experiments were performed. Unlike the results from Aph-1 or Nct RNAi, treatment of S2 cells with Pen-2 RNAi led to loss of fragment forms of Psn and accumulation of Psn holoprotein. Application of CHX to S2 cells treated with Pen-2 RNAi showed that the accumulated Psn holoprotein is highly stabilized and is recoverable in high-Mr fractions. In contrast to the result of the Pen-2 RNAi experiment, combinations of Pen-2 and Aph-1 or Pen-2 and Nct RNAi abolished the accumulation of either fragment or holoprotein forms of Psn. gamma-Secretase activities, as determined by an in vitro Aß generation assay using recombinant APP C100 (carboxy-terminal 99 amino acid residues of human APP) tagged with Flag, Myc and His as a substrate, were totally abolished by every combination of RNAi that diminished the Psn fragment. These data provide genetic evidence to support the hypothesis that Nct and Aph-1 act upstream of Pen-2 in the formation of the gamma-secretase complex. The accumulation of Psn holoprotein by Pen-2 RNAi was also observed in BG2 cells, a Drosophila neuronal cell line derived from primary cultures of larval central nervous system (Takasugi, 2003).

To examine further whether PEN-2 has a similar role in the formation of the gamma-secretase complex in mammalian cells, PEN-2 RNAi was performed by transfection of small interfering RNA (siRNA). Knockdown of PEN-2 expression in human HeLa cells results in the accumulation of highly stabilized presenilin 1 (PS1, also known as PSEN1) holoproteins accompanied by a decrease in the levels of PS1 fragments as well as of gamma-secretase activity, as evidenced by the accumulation of APP C83. It was confirmed that RNAi depletion of PEN-2 causes an accumulation of PS1 holoproteins in mammalian neuronal cells. Overexpression of human PEN-2 in Pen-2 RNAi-treated Drosophila S2 cells rescued the diminution in Psn fragments and attenuated the accumulation of Psn holoproteins (Takasugi, 2003).

Transient overexpression of Pen-2 was performed in S2 cells stably transfected with Aph-1 and Nct; an increase in the accumulation of Psn fragments was observed. To determine whether Pen-2, Aph-1, Nct and Psn function by directly binding to each other, co-immunoprecipitation studies were performed in CHAPSO-solubilized membrane fractions of S2 cells stably co-transfected with the three cofactors; any two of the components among Psn (holoprotein as well as fragments), Nct, Aph-1 or Pen-2 were found to be co-immunoprecipitated. In addition, Pen-2 was fractionated in the high-Mr ranges together with Psn, Nct or Aph-1 by glycerol velocity gradient centrifugation, suggesting that they function together and form a protein complex. Finally, it was found that secretion of Aß from S2 cells, as well as the total cellular gamma-secretase activity as determined by an in vitro Aß generation assay using recombinant APP C100 was significantly increased by the co-transfection of Pen-2, indicating that gamma-secretase activity is augmented by triple co-expression of Aph-1, Nct and Pen-2. Taken together, these data indicate that Pen-2 is a cofactor that is required for the final step of presenilin complex maturation after presenilin is stabilized in a holoprotein form and incorporated into a high-Mr protein complex, conferring gamma-secretase activities and leading to endoproteolysis of presenilin (Takasugi, 2003).

By combining RNAi inactivation and stable overexpression of cofactor proteins of Psn in Drosophila S2 cells, it was possible to dissect the process of high-Mr protein complex formation and the activation of gamma-secretase function of presenilin. Overexpression of Aph-1 is sufficient to elicit the stabilization and high-Mr complex formation of endogenous presenilin in a holoprotein form, although this high-Mr protein complex harbouring presenilin holoprotein is still inactive with respect to gamma-secretase. The final maturation step of the presenilin complex, which consists of presenilin fragments and demonstrates gamma-secretase activity, may then be mediated by the additional function of Pen-2 through directly interacting with the Psn-Aph-1-Nct complex1. This model is supported by the following observations: (1) Pen-2 RNAi facilitates the accumulation of stabilized Psn holoprotein, as is seen with Aph-1 overexpression; (2) Psn polypeptide accumulation is totally abolished by RNAi-mediated inactivation of Nct or Aph-1 expression; and (3) co-expression of Pen-2, Nct and Aph-1 renders the stabilized Psn holoprotein into the gamma-secretase-active fragment form. The latter result also suggests that APH-1, NCT and PEN-2 represent the set of major presenilin cofactors sufficient for the formation of the gamma-secretase complex; a large protease complex reminiscent of the proteasome that executes the cleavage of peptide bonds buried within the lipid bilayer. Further efforts to reconstitute gamma-secretase activity from recombinant proteins of the presenilin and the three cofactors in vitro will be needed to elucidate the configuration and function of presenilin complex and to develop therapeutic approaches to Alzheimer's disease based on inhibition of gamma-secretase (Takasugi, 2003).

In vivo reconstitution of γ-secretase in Drosophila results in substrate specificity

The intramembrane aspartyl protease γ-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimer's disease. This study describes a transgenic Drosophila model for in vivo-reconstituted γ-secretase, based on expression of epitope-tagged versions of the four core γ-secretase components, Presenilin, Nicastrin, Aph-1, and Pen-2. In agreement with previous cell culture and yeast studies, coexpression of these four components promotes the efficient assembly of mature, proteolytically active γ-secretase. In vivo-reconstituted γ-secretase has biochemical properties and a subcellular distribution resembling those of endogenous γ-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted γ-secretase toward different substrates, including markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor. These findings indicate that in vivo under physiological conditions, additional factors differentially modulate the activity of γ-secretase toward its substrates. Thus, this approach for the first time demonstrates the overall functionality of reconstituted γ-secretase in a multicellular organism and the requirement for substrate-specific factors for efficient in vivo cleavage of certain substrates (Stempfle, 2010).

The Presenilin (PS) protein family was first identified on the basis of dominant familial mutations inducing early-onset Alzheimer's disease. An important pathological feature of Alzheimer's disease is the formation of plaques caused by the deposition of amyloid peptides derived from the proteolytic cleavage of the amyloid precursor protein (APP). The pathogenic Aβ peptide is produced by the sequential cleavage of APP through β- and γ-secretases. The first cleavage by β-secretase occurs at an extracellular site near the transmembrane region of APP, leading to secretion of the extracellular domain (ECD). The remaining C-terminal fragment (CTF) serves as a substrate for γ-secretase, which mediates proteolysis inside the membrane region, releasing the APP intracellular domain (AICD) and the Aβ peptide (Stempfle, 2010).

PS contains two aspartate residues that are essential for the catalytic activity of the complex and that are thought to form the active center of the protease. Nct, Aph-1, and Pen-2 contribute to the maturation and stabilization of the complex. Furthermore, evidence has been obtained for a function of Nct in substrate recognition (Shah, 1995), a view challenged by a recent study suggesting that Nct is instead needed only for maturation of the complex (Chávez-Gutiérrez, 2008). Cell-based and cell-free assays have shown that only the coordinated overexpression of all four proteins leads to an increase in γ-secretase activity, arguing that they form the minimal active complex and that the assembly and maturation of the complex are highly regulated. In Drosophila, there is only one homolog of PS and Aph-1, whereas two homologs of each exist in mammalian cells (PS1/PS2 and Aph-1a/Aph-1b). Based on the fact that alternative aph-1a splice forms can be detected, it has been suggested, and subsequently demonstrated, that at least six distinct γ-secretase complexes exist in mammalian cells. However, to date, the precise compositions and architectures of these complexes are not known, and depending on the experimental conditions used, complexes with a molecular mass of 250, 500, or >2,000 kDa have been isolated in vitro. Recently, it was shown that a complex containing only one of each component displays in vitro activity, but it is unknown whether this activity is found in vivo as well. Furthermore, recently published interactome analyses of γ-secretase suggest that it interacts with a variety of other proteins, which could be important for maturation, localization, and/or enzymatic activity. Taken together, these findings illustrate a potential limitation with the analysis of purified γ-secretase complexes, namely, that their observed in vitro minimal activities might not fully reflect their full range of activities and cleavage efficiencies in vivo (Stempfle, 2010).

In one of the earliest models addressing PS activity and specificity, it was suggested that substrate recognition by the γ-secretase complex depends only on the size of the ECD and is sequence independent. To address this phenomenon in more detail, a reporter system was developed in Drosophila which monitors the cleavage of transmembrane proteins in vivo during Drosophila development, and it was shown that PS-mediated cleavage of APP is regulated in a cell-type-specific manner, independent of the size of the ECD. In the meantime, several studies have confirmed the existence of complex regulatory mechanisms that influence the cleavage efficiencies of different substrate CTFs (Stempfle, 2010).

To address the question of the activity of the core PS complex and the contribution of substrate-specific factors, the Drosophila PS complex was reconstituted in vivo by simultaneous overexpression of tagged versions of the four core components with the GAL4/upstream activation sequence (UAS) system, and its ability to cleave alternative model substrates was analyzed, including ones based on Notch and different APP/APLP family members. In contrast to mammalian γ-secretase, Drosophila γ-secretase is homogenous in composition due to the fact that single-copy genes encode each of its four core subunits. The Drosophila-based approach thus permits the analysis of substrate-specific cleavage efficiencies without the additional complication of multiple potential γ-secretase complexes having distinct enzymatic properties. It was found that in vivo-reconstituted Drosophila γ-secretase displayed biochemical and cell biological features similar to those of endogenous γ-secretase but exhibited substrate-specific effects with respect to its proteolytic activities toward Notch and APP/APLP proteins. These findings have potential implications for understanding the diverse functions of γ-secretase involving different cleaved substrates in different tissues, as well as the development of pharmacological inhibitors that could potentially target specific substrates (Stempfle, 2010).

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

The transmembrane glycoprotein Nicastrin was identified in a complex with the multipass membrane protein Presenilin. Presenilin mediates transmembrane cleavage of single-pass transmembrane proteins with short extracellular domains, including the ligand-activated form of the receptor Notch and beta-amyloid precursor protein (beta-APP). Transmembrane cleavage of Notch is essential for signal transduction, and transmembrane cleavage of beta-APP generates pathogenic amyloid peptides implicated in Alzheimer's disease. This study investigates the requirement for Nicastrin in Presenilin-mediated transmembrane cleavage. In Drosophila, loss of Nicastrin activity blocks the accumulation of Presenilin associated with the apical plasma membrane, abolishes Presenilin-dependent cleavage of the transmembrane domains of Notch and beta-APP, and abrogates Notch signal transduction (Chung, 2001).

Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor

Drosophila nicastrin mutations have been isolated by systematic lethal mutagenesis screening. nicastrin mutants exhibit defective cell fate specifications at all stages of development, similar to what has been observed in Notch and Presenilin mutants. Biochemical analysis of Notch proteolysis reveals that loss of either nicastrin or Presenilin activity affects the same step of Notch proteolysis that is blocked by a peptidomimetic gamma-secretase inhibitor compound. Consistent with these observations, Nicastrin is essential for signaling from a membrane-tethered form of constitutively activated Notch, but dispensable for signaling from a nuclearly localizing, nonmembrane-bound Notch intracellular domain. Using RNA interference (RNAi) in Drosophila S2 cells, it has been shown that absence of Nicastrin function is accompanied by a loss of mature Presenilin protein. Expression of human Presenilin in this assay is upregulated when endogenous Drosophila Presenilin is inactivated, similar to results seen with expression of heterologous Presenilins in transfected mammalian cells and transgenic mice. On the basis of these results and the known binding properties of Nicastrin, it is suggested that Nicastrin may bind gamma-secretase substrates and recruit them into the Presenilin-containing complex, which might be an obligatory step in stabilizing Presenilin during assembly of the active proteolytic complex (Hu, 2002).

To examine the role of Nicastrin in Notch receptor proteolysis and signaling, lesions were created in the Drosophila nicastrin (nic) gene by performing a lethal mutagenesis of the third chromosome region 96A21-C2, uncovered by the deficiency Df(3R)96B, an interval that harbors the nic gene. After screening ~4,000 mutagenized third chromosomes, four lethal alleles of a single complementation group corresponding to the nic gene were recovered. Three of the four alleles result in indistinguishable lethal phases and mutant phenotypes at the end of the larval period when assayed in trans to Df(3R)96B, and are thus likely to be complete or nearly complete loss-of-function alleles. Two of these alleles, Nic^A7 and Nic^J2, are predicted to encode truncated Nic proteins due to premature termination of translation. The fourth allele, Nic^E3, allows the flies to survive to a slightly later stage and is therefore likely to be a strong but not complete loss-of-function mutation. nic null mutant larvae secrete a pupal case and complete the last stages of larval development, but do not form any adult structures and instead collapse into an oily mass within the pupal case. This zygotic lethal null phenotype closely resembles those observed for certain Notch pathway genes in Drosophila, including Presenilin (Psn) and Suppressor of Hairless (Hu, 2002).

The postembryonic lethality of nic mutants suggested that an embryonic requirement for nic in Notch signaling might be masked by maternally deposited protein, as is the case for both Psn and Su(H) mutants. Adult female flies bearing nic germline clones were created, and it was found that embryos from these females that lack both maternal and zygotic Nicastrin activity exhibit a Notch-like lethal hyperplasia of the embryonic nervous system. A small fraction (<10%) of the maternally deficient nic mutant embryos can be zygotically rescued by wild-type male gametes to produce morphologically normal adult flies, as is also seen for Psn mutants. During larval development, one feature of wing imaginal disc morphogenesis that is mediated by Notch signaling is the emergence of individual sensory organ precursor (SOP) cells at defined positions. This process depends upon lateral inhibition among proneural cell clusters at each position to ensure that a single cell of the cluster adopts the proper SOP fate. Histochemical characterization of nic mutant wing discs using the SOP marker scabrous-lacZ reveals clusters of supernumerary SOP cells arising at certain normal SOP locations in addition to a developmental arrest phenotype, as seen in other neurogenic mutants. Lateral inhibition within the proneural cell clusters is severely impaired in nic mutant discs, as shown by the enlarged SOP territories and elevated scabrous-lacZ regulatory activity. Lastly, to circumvent the larval-pupal lethality of the nic mutations and examine potential Notch-like phenotypes in adult tissues, homozygous mutant nic somatic tissue clones were induced in heterozygous nic hosts. Several phenotypes characteristic of impaired Notch signaling were observed in these clones, including frequent wing notching and thoracic cuticle patches devoid of bristles. Taken together, these results indicate that Nicastrin is generally required at all developmental stages for some aspect of Notch function in Drosophila (Hu, 2002).

Next, the proteolysis of Notch was examined in nic mutant tissues, since Presenilin is required for proteolytic activation of the Notch receptor during signaling. Notch is synthesized as an ~300 kDa full-length protein, the majority of which is cleaved at a lumenal site by furin proteases within the trans-Golgi network. As a result, most mature Notch receptor at the cell surface is a heterodimer consisting of a large ~200 kDa N-terminal extracellular EGF-homologous region joined noncovalently to a smaller C-terminal Notch subunit containing a short extracellular stalk, the transmembrane domain, and the intracellular domain. Activation of this heterodimeric receptor is accompanied by removal of the extracellular domain by TNFalpha-converting enzyme (TACE)-mediated proteolysis at an extracellular site located just beyond the transmembrane domain or by subunit dissociation under some experimental conditions. In Drosophila, the TACE-related metalloprotease Kuzbanian is thought to execute this postfurin second cleavage. This event generates a membrane-anchored C-terminal segment of Notch, containing an intact intracellular domain and a short extracellular stub. This product is then efficiently cleaved within the membrane by a Presenilin-associated gamma-secretase-like activity, thereby liberating a soluble intracellular Notch fragment that translocates to the nucleus and activates transcription of Notch target genes (Hu, 2002 and references therein).

Notch proteins from nic mutants, Psn mutants, and wild-type animals were subjected to Western immunoblot analysis following extraction under gentle hypotonic shock conditions, which allows the selective resolution of the mature processed forms of Notch that comigrate as an ~120 kDa smear under harsher SDS-urea extraction conditions. This protocol also results in efficient ligand-independent activation of Notch, presumably due to dissociation of the extracellular Notch subunit at low ionic concentrations. As is the case with numerous studies involving ligand-independent truncated forms of Notch, the increased Notch activation achieved in this manner facilitates the analysis of Notch processing by generating relatively high levels of gamma-secretase-derived Notch polypeptides. All four nic loss-of-function mutations result in the complete absence of a faster migrating ~120 kDa major C-terminal fragment of Notch (DNIC-2) and overaccumulation of a more slowly migrating C-terminal ~120 kDa fragment (DNIC-1). A third ~120 kDa C-terminal fragment (DNIC-3) that migrates slightly faster than DNIC-2 is unaltered in the nic mutants, relative to wild-type. These effects are identical to those observed with homozygous loss-of-function mutations in the Drosophila Psn gene. Equivalent results were obtained using a cultured Drosophila Schneider-2 (S2) cell assay in which Psn and nic function were selectively eliminated by RNA-mediated interference (Hu, 2002).

To determine which of the three ~120 kDa C-terminal fragments corresponds to the gamma-secretase-generated signaling product of Notch, the inhibition profile of each band was characterized in S2 cells using a variety of pharmacological compounds and expression constructs. Mutagenesis of the putative furin cleavage site in the Notch expression construct, coexpression of a dominant-negative Kuzbanian construct, or treatment of the cells with Brefeldin A all blocked production of DNIC-1 and -2 with no observable effects on DNIC-3. Notch immunoreactivity was undetectable at the extracellular surface of the Brefeldin A-treated cells. Conversely, DNIC-3 alone is blocked by treatment of the cells with a broad spectrum of protease inhibitors that have no discernible effect on DNIC-1 and -2. These results suggest that DNIC-1 most likely corresponds to the C-terminal fragment of Notch produced by furin-mediated cleavage and/or the slightly smaller fragment thought to be generated following subsequent Kuzbanian cleavage. The data do not allow for the unambiguous identification of DNIC-1, because the dominant-negative Kuzbanian protein might interact with the extracellular region of Notch containing the nearby furin and Kuzbanian cleavage sites, interfering with both processing events. Since DNIC-2 shows a similar inhibition profile as DNIC-1, but is specifically blocked by loss of Psn or nic function with a corresponding overaccumulation of DNIC-1, DNIC-2 appears to be derived from DNIC-1. The inference that DNIC-2 is generated by gamma-secretase-like cleavage of DNIC-1 was confirmed by the finding that the appearance of DNIC-2 is specifically blocked by treatment of S2 cells with the pharmacological gamma-secretase inhibitor DFK-167. Unlike DNIC-1 and -2, DNIC-3 appears to be derived from a different Notch processing pathway that is independent of Brefeldin A-sensitive anterograde protein transport through the ER and Golgi compartments (Hu, 2002).

To confirm these results using a genetic assay, the ability of constitutively activated forms of Notch to signal in the absence of nic gene function was examined. A soluble intracellular C-terminal fragment of Notch lacking all extracellular and transmembrane sequences [N(intra)] possesses intrinsic signaling activity and suppresses neural precursor cell specification independent of both Notch ligand and Presenilin. In contrast, a membrane-anchored C-terminal fragment of Notch lacking only the extracellular domain (DeltaECN) also signals constitutively in the absence of ligand, but requires Presenilin activity for intramembranous cleavage and liberation of the signal-transducing intracellular Notch domain. The effects of N(intra) and DeltaECN on SOP formation in larval imaginal wing discs of wild-type flies and nicastrin mutant flies was assessed using immunostaining for the proneural antigen Scabrous to monitor SOP differentiation. N(intra) displays nearly complete SOP inhibition in both wild-type and nic mutant flies, whereas DeltaECN blocks SOP formation only in wild-type animals and has no detectable inhibitory effect on SOP formation in the nic mutants. Identical results were confirmed for DeltaECN and N(intra) in Psn mutants. The similar genetic requirements for Presenilin and nicastrin during signaling by membrane-anchored DeltaECN, but not by the soluble intracellular domain N(intra), implies that Nicastrin is needed specifically for the Presenilin-associated intramembranous cleavage of Notch during signaling, consistent with biochemical studies with nic mutants and RNAi-treated S2 cells (Hu, 2002).

Drosophila Nicastrin is essential for the intramembranous cleavage of Notch

To determine where Nicastrin (Nct) acts in the Notch signal transduction pathway, advantage was taken of transgenic Notch constructs that bypass some of the processing steps required for signaling. (1) hs-N^FL was used as a control; it expresses full-length Notch protein under the control of a heat shock promoter. Like endogenous Notch, the protein from this construct requires both the ligand-induced S2 cleavage [this involves cleavage of the extracellular domain of Notch to produce a transient form of the receptor called NEXT (Notch extracellular truncation)] and the Psn-dependent S3 cleavage (an intramembranous cleavage to release the intracellular domain of Notch, which translocates to the nucleus, where it acts as a transcriptional activator in association with Suppressor of Hairless protein. (2) hs-N^ECN is a deletion of the extracellular domain of Notch beyond the S2 cleavage site, and therefore mimics NEXT. This protein signals independently of ligand, but still requires the S3 cleavage to release the intracellular domain of Notch from the membrane. (3) hs-N^ICD expresses the intracellular domain of the receptor and requires neither ligand, the S2 nor S3 cleavages, for signaling. The expression of hs-N^FL has no effect on the development of wild-type embryos, whereas hs-N^ECN and hs-N^ICD disrupt germ band retraction because their ligand-independent signaling overactivates the Notch pathway. To test which of these constructs requires Nct for signaling, each was expressed in embryos that lack both maternal and zygotic Nct activity. hs-N^ECN and hs-N^FL have no effect on the neurogenic phenotype of nct null embryos. In contrast, hs-N^ICD expression strongly rescues the nct phenotype: most embryos form patches of normal cuticle, and in some cases, the wild-type cuticular pattern is almost completely restored. Thus, the nct null mutation blocks Notch signaling after the S2 cleavage but before the release of the intracellular domain from the membrane, indicating that Nct is required for the S3 cleavage, as is Psn (López-Schier, 2002).

Presenilins and the human Nct associate with Notch during its passage through the secretory pathway, raising the possibility that they also function in earlier steps in Notch processing. Since the experiments above only reveal the last step at which these proteins are required, the behavior of Notch protein was followed in the follicle cells, where the downregulation of Notch in response to Delta binding can be visualized. Notch accumulates on the apical side of these cells until stage 7 of oogenesis, when Delta signals to trigger its proteolysis. Thus, Notch disappears from the apical membrane of cells that contact wild-type germline cells, whereas high levels of apical Notch persist in follicle cells that contact Delta germline clones, and this can be detected with antibodies against both the extracellular (N^ECD) and intracellular domains (N^ICD) of the protein. This reduction of apical Notch staining in response to Delta still occurs in Su(H) mutant follicle cell clones, in which Notch signaling in the nucleus is blocked. Therefore the disappearance of most Notch from the apical membrane appears to be a direct consequence of ligand-induced processing, and is not due to downregulation of the receptor in response to the activation of the signaling pathway. In nct and psn mutant follicle cells, apical N^ECD staining disappears at stage 7 as it does in wild-type, indicating that the ligand-dependent S2 cleavage and subsequent degradation of N^ECD occur normally. Unlike wild-type cells, however, mutant cells accumulate a processed form of Notch that can be stained with the anti-N^ICD antibody, and this is concentrated at the apical side of the cells and in intracellular clusters that may be endocytic vesicles. Thus, nct or psn mutant cells can transport Notch to the plasma membrane and process it to form a functional receptor that binds Delta and undergoes the S2 cleavage, indicating that both proteins are specifically required for the S3 cleavage, but not for any earlier steps in the pathway (López-Schier, 2002).

The processed form of Notch that accumulates on the apical side of nct and psn mutant follicle cells presumably corresponds to NEXT, which is the membrane-tethered product of the S2 cleavage and the substrate for the S3 cleavage. To test this possibility, Western blots of extracts from wild-type and embryos lacking maternal and zygotic Nct or Psn were probed with the anti-N^ICD antibody. In addition to the 300 kDa band that corresponds to the uncleaved form of Notch, nct and psn null embryos accumulate a 120 kDa species that is barely detectable in the wild-type extracts. This band migrates at the expected size of NEXT and N^ICD, which cannot be distinguished on these gels as they differ by only 3 kDa. This band is unlikely to be N^ICD, however, because the results above demonstrate that nct and psn block the S3 cleavage (López-Schier, 2002).

To investigate whether Nct is required for the localization or stability of Psn, nct^agro clones of varying sizes were generated in the follicular epithelium. In wild-type follicle cells, Psn protein shows a punctate distribution in the cytoplasm that may correspond to the endoplasmic reticulum, and it localizes at the cell cortex. When nct clones are analyzed early in oogenesis, the mutant cells show a normal distribution of Psn, and this is also the case for small clones in late-stage egg chambers. In contrast, very large clones in late-stage egg chambers show a strong reduction in the levels of Psn. Thus, Nct seems to be required for the long-term stability of Psn in these cells. Since small nct clones show a completely penetrant Notch loss-of-function phenotype but have normal levels of Psn, this effect of Nct on Psn stability is unlikely to cause the defect in Notch signaling in these cells (López-Schier, 2002).

During an analysis of follicle cell clones, an additional function of Nct and Psn in the organization of the submembranous spectrin cytoskeleton was discovered. In wild-type cells, ß_Heavy-spectrin (ß_H-spectrin) associates with alpha-spectrin to form tetramers that localize to the apical membrane, while the basolateral spectrin cytoskeleton is composed of ß-spectrin/alpha-spectrin complexes. In psn and nct follicle cell clones, ß_H-spectrin does not localize to the apical membrane. In contrast, ß-spectrin localization to the basolateral membrane is unaffected in mutant clones. The apical localization of ß_H-spectrin requires its association with alpha-spectrin and vice versa, and the distribution of alpha-spectrin was therefore examined in follicle cell clones. Surprisingly, both psn and nct mutant cells show an increase in the amount of cortical alpha-spectrin, with the highest levels on the apical side. alpha-spectrin therefore appears to be recruited to the apical membrane of mutant cells by a novel mechanism that does not require its association with either ß subunit, neither of which localizes apically in these cells (López-Schier, 2002).

To test whether the mislocalization of alpha- and ß_H-spectrin in nct and psn mutants is a consequence of the defect in Notch signaling, the localization of both proteins was examined in follicle cell clones of a null allele of Notch (N^55e11) and in wild-type cells that abut Delta mutant germline clones. ß_H-spectrin is recruited normally to the apical membrane in both cases, while alpha-spectrin localizes uniformly around the cell cortex at the same level as in wild-type cells. These results indicate that Psn and Nct have a novel function independent of their role in Notch signaling that somehow affects the organization of the spectrin cytoskeleton (López-Schier, 2002).

The absence of apical ß_H-spectrin in nct and psn mutant cells suggested that their apical-basal polarity might be disrupted, and the localization of a variety of other polarity markers was therefore examined. The overall polarity of the cells is unaffected in either nct or psn mutant clones, since Coracle, Neurotactin, DE-Cadherin, Armadillo (ß-catenin), alpha-catenin, and Notch itself are localized to the proper membrane domains. However, DE-Cadherin, Armadillo, and alpha-catenin accumulate to much higher levels in mutant cells than in wild-type. These three proteins are components of the Cadherin adhesion complex, and are enriched at the sites of adherens junction formation at the apical margins of the cell. Their overaccumulation in mutant cells may therefore be linked to the loss of apical ß_H-spectrin and the apical enrichment of alpha-spectrin, although the nature of this link remains unclear (López-Schier, 2002).

Previous studies have shown that almost all Psn is associated with the ER and intermediate compartment and that there is little or no protein at the plasma membrane, where APP and Notch reside. This discrepancy, which has been called the 'spatial paradox,' raises the question of where in the cell the S3 cleavage occurs. One possible solution to this paradox is suggested by the observation that small amounts of Psn can be coimmunoprecipitated with Notch at the cell surface. Both Nct and Psn have been shown to interact with Notch in the secretory pathway, and a minor fraction of the S3 protease complex could therefore be transported to the plasma membrane through binding to its future substrate. A second possibility is that the active protease resides in an intracellular compartment, and that the products of S2 cleavage of Notch are internalized and transported to this site. This analysis provides strong evidence for the first model. Since Nct and Psn are required for the S3 cleavage of Notch, the substrate for this cleavage, NEXT, accumulates in mutant cells. Most NEXT remains closely associated with the apical membrane, arguing against the existence of a major transport pathway to an intracellular compartment. Although a small amount of NEXT is found in intracellular clusters, these do not correspond to the major sites of Psn localization, and may be endocytic vesicles. Furthermore, recent data indicate that endocytosis is not required for the S3 cleavage of NEXT, because a membrane-tethered Notch derivative lacking the extracellular domain can signal normally in shibire mutant embryos, in which Dynamin-dependent endocytosis is blocked. Taken together, these results strongly suggest that S3 cleavage occurs predominantly at the plasma membrane (López-Schier, 2002).

Follicle cell clones mutant for either Nct or Psn have a more severe phenotype than that seen in Notch or Delta mutants, indicating that both proteins must have at least one additional function in these cells that is independent of their role in Notch signaling. One aspect of this phenotype is the overaccumulation of the components of the adherens junctions, DE-Cadherin, Armadillo, and alpha-catenin, and this is probably related to the fact that both alpha-catenin and the Armadillo ortholog ß-catenin associate with Psn in mammalian cells. Although neither is required for the activity of the S3 protease or gamma-secretase, loss of Psn leads to an overaccumulation of ß-catenin in Drosophila embryos and mouse epithelial cells. The precise function of Psn in ß-catenin regulation is unknown, but the overexpressed protein in Drosophila psn mutant embryos is associated with polyubiquitin-positive cytoplasmic inclusions, suggesting that Psn is required in some way to regulate Armadillo degradation. Psn also regulates the turnover of DE-Cadherin and alpha-catenin. Furthermore, Nct is necessary for this function, suggesting that it requires the formation of the high molecular weight protease complex. Since Psn is thought to mediate the proteolysis of membrane proteins, one possibility is that Psn is recruited to DE-Cadherin by binding to the catenins, and cleaves DE-Cadherin to trigger degradation. Alternatively, Psn could regulate the turnover of the catenins in some other way, and their overaccumulation in psn and nct mutants might then lead to the stabilization of Cadherin complexes at the membrane (López-Schier, 2002).

nct and psn mutants also disrupt the organization of the spectrin cytoskeleton in follicle cell clones. This phenotype is particularly puzzling, because the mutants have opposite effects on the two subunits of apical spectrin: ß_H-spectrin disappears from the apical membrane, whereas alpha-spectrin accumulates all round the cortex of the cell, but particularly at the apical side. In wild-type cells, the apical localization of alpha-spectrin normally requires its association with ß_H-spectrin to form (alphaß_H)₂ tetramers. Its apical localization in mutant cells must therefore occur by a different mechanism from normal, and this may sequester it in a form that cannot bind ß_H-spectrin, which would account for the failure of the latter to localize. However, alpha-spectrin must still tetramerize with ß-spectrin, because it shows wild-type localization to the basolateral domain. It is unclear whether the defect in the spectrin cytoskeleton is related to the overaccumulation of Cadherin complexes, but it is intriguing that alpha-spectrin accumulates at sites where DE-Cadherin, Armadillo, and alpha-catenin are most enriched in nct and psn mutant cells. Thus, this localization and stabilization of alpha-spectrin could be mediated through binding to some component of these abnormal adhesion complexes. It is also possible, however, that the Nct/Psn complexes have multiple functions in the follicle cells that independently regulate Notch signaling and the spectrin cytoskeleton and Cadherin adhesion complexes (López-Schier, 2002).

EVOLUTIONARY HOMOLOGS

In animal development, numerous cell-cell interactions are mediated by the GLP-1/LIN-12/NOTCH family of transmembrane receptors. These proteins function in a signaling pathway that appears to be conserved from nematodes to humans. The aph-2 gene is a new component of the GLP-1 signaling pathway in the early Caenorhabditis elegans embryo, and proteins with sequence similarity to the APH-2 protein are found in Drosophila and vertebrates. During the GLP-1-mediated cell interactions in the C. elegans embryo, APH-2 is associated with the cell surfaces of both the signaling, and the responding, blastomeres. Analysis of chimeric embryos that are composed of aph-2⁺ and aph-2^- blastomeres suggests that aph-2⁺ function may be provided by either the signaling or responding blastomere (Guotte, 2000).

aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling. Aph-2, termed Nicastrin in this study, is a transmembrane glycoprotein that forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in C. elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologs of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (Abeta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase Abeta₄₂ and Abeta₄₀ peptide secretion. Deletions in this domain inhibit Abeta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP (Yu, 2000).

In the absence of homology to other proteins, sequence databases were screened for orthologous genes in other species. A full-length C. elegans nicastrin ortholog was found in public databases (accession no. Q23316; identity = 22%; similarity = 41%). Full-length murine and Drosophila nicastrin orthologs from appropriate cDNA libraries were cloned and sequenced using partial cDNA sequences from these databases as start points (mouse nicastrin accession no. AF24069, identity = 89%, similarity = 93%; D. melanogaster nicastrin accession no. AF240470, identity = 30%, similarity = 48%). The four animal nicastrins have similar predicted topologies and have three domains with significant sequence conservation near residues 306-360, 419-458, and 625-662 of human nicastrin. Within the first conserved domain, all four proteins contain the motif DYIGS (residues 336-340), which is also partially conserved in an Arabidopsis protein. All four animal nicastrins also contain four cysteines spaced at 16 to 17-residue intervals in the N terminus (Cys 195, Cys 213, Cys 230 and Cys 248) (Yu, 2000).

To explore whether nicastrin, like the presenilins, might have a role in Notch signaling in vivo, RNA interference (RNAi) was used in C. elegans. Wild-type worms injected with C. elegans nicastrin double-stranded (ds) RNA produce dead embryos, many of which lack an anterior pharynx. This phenotype is highly reproducible and specific. Except for embryonic lethality, none of the other phenotypes associated with a lack of C. elegans presenilin (sel-12) activity were observed. However, this phenotype is identical to that induced when the activity of genes in the notch/glp-1 pathway (glp-1, aph-1 or aph-2) is reduced, or when the activities of both C. elegans presenilin homologs (sel-12 and hop-1) are reduced simultaneously. Thus nicastrin contributes to some aspects of notch/glp-1 signaling in C. elegans embryos (Yu, 2000).

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, it has been shown that mammalian APH-1 (mAPH-1: see Drosophila anterior pharynx defective 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 (Lee, 2002).

APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 [APH-1(1)L; also known as APH-1a] as an archetype, it is reported that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum and cis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display gamma- and epsilon-secretase activity. Indeed APH-1 proteins can bind to the nicastrin delta312-369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-1(1)L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, these data suggest that APH-1 proteins, and APH-1(1) in particular, may have a role in the initial assembly and maturation of presenilin.nicastrin complexes (Gu, 2003).

Early embryonic cells in C. elegans embryos interact through a signaling pathway closely related to the Notch signaling pathway in Drosophila and vertebrates. Components of this pathway include a ligand, receptor, the presenilin proteins, and a novel protein, APH-2, that is related to the Nicastrin protein in humans. The aph-1 gene has been identified as a new component of the Notch pathway in C. elegans. aph-1, coding for a protein with cognates in Drosophila and mammals, encodes a novel, highly conserved multipass membrane protein. aph-1 and the presenilin genes share a similar function in that they are both required for proper cell-surface localization of APH-2/Nicastrin (Goutte, 2002).

APH-1 and related proteins are largely hydrophobic, with a common pattern of seven hydrophobic regions that are predicted to be membrane-spanning regions. None of the proteins contain predicted glycosylation sites, consistent with the idea that the majority of the protein is embedded within a cellular membrane. There are conserved, hydrophilic residues within the hydrophobic domains (Cys-9, Ser-45, Ser-50, Ser-130, His-183, Ser-213, His-214, Ser-257), suggesting that these residues may be critical for protein structure or interactions with other proteins. The aph-1(or28) mutation is predicted to replace a glycine residue with an aspartic acid residue within the fourth hydrophobic domain, and may therefore disrupt the topography of APH-1 in the membrane. The C. elegans APH-1 protein is unique in containing a hydrophilic 40-aa 'tail' at the C terminus. The aph-1(zu147) mutation results in a premature stop codon near the beginning of this hydrophilic tail. Because aph-1(zu147) appears to be a hypomorphic allele that retains some aph-1(+) activity, the nonconserved C-terminal tail may not be essential for APH-1 function. The only other multipass membrane proteins known to act in C. elegans Notch pathways are the presenilin proteins (Goutte, 2002).

Because of its characteristics as a membrane protein, it was asked whether the APH-1 protein might influence the expression or localization of cell-surface components of the Notch pathway in the early embryo. In wild-type 4-cell stage embryos, the ligand APX-1 is localized to the surface membrane of the posterior-most cell, where it contacts one of the two cells that express the receptor GLP-1. The APH-2 protein is associated with the surface membranes of all four cells. APX-1 and GLP-1 have the wild-type localization pattern in aph-1 mutant embryos; however, the APH-2 protein is mislocalized in all aph-1 mutant embryos examined. In aph-1 mutant embryos, APH-2 is not detectable on the cell surface, and is instead prominent in the cytoplasm of all four cells, where it is concentrated around the nucleus in a pattern characteristic of the endoplasmic reticulum. These results suggest that APH-1 facilitates the translocation of APH-2 to the cell surface (Goutte, 2002).

Presenilin proteins have been localized to the endoplasmic reticulum in some systems, and are essential for Notch signaling. Because the human orthologue of APH-2, Nicastrin, has been shown to associate with presenilins, it was asked whether presenilin function is required for APH-2 localization. Remarkably, presenilin-deficient embryos show an abnormal accumulation of APH-2 around the nucleus in an apparently identical pattern as that of aph-1 mutant embryos; these embryos had the wild-type pattern of GLP-1 localization. These results suggest that the presenilins and APH-1 may function together in an event that is a prerequisite for APH-2 to localize to the cell surface (Goutte, 2002).

Nicastrin is a multi-pass transmembrane protein that has recently been identified as a member of high-molecular weight complexes containing presenilin. The C. elegans homolog of nicastrin, aph-2, is 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. This latter defect is related to the SEL-12/presenilin egg-laying defect. 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. A new role for aph-2 in facilitating lin-12 signaling in the somatic gonad is illustrated, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins (Levitan, 2001).

The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Nicastrin is shown in this study to bind to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, the Notch and betaAPP pathways do not significantly compete with each other (Chen, 2001).

Nicastrin acts as a key regulator for presenilin (PS)-mediated gamma-secretase cleavage of beta-amyloid precursor protein by forming a functional complex with PS1 and PS2. Both TNF-alpha and IL-1, aberrantly produced by activated microglia and astrocytes, play a role in amyloidogenesis and neurodegeneration in the brains of Alzheimer's disease (AD) patients, while BDNF synthesized chiefly by neurons has been found to be substantially reduced in AD brains. To investigate the constitutive and cytokine/neurotrophic factor-regulated expression of nicastrin in human neural cells, its mRNA levels were studied by RT-PCR and Northern blot analysis in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N) following exposure to TNF-alpha, IL-1beta, BDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate. Nicastrin mRNA expression was identified in all human neural and nonneural cell lines and tissues examined. However, the levels of nicastrin mRNA were unaltered in SK-N-SH, IMR-32, U-373MG, and NTera2-N cells by exposure to the factors tested, and unchanged in NTera2 cells during retinoic acid-induced neuronal differentiation. These results indicate that nicastrin mRNA is expressed constitutively in human neural cell lines, where its expression is not regulated at the transcriptional level by a battery of cytokines and growth/differentiation factors that are supposed to be involved in amyloidogenesis, neurodegeneration or neuroprotection in AD brains (Satoh, 2001).

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 2005).

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function has been attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, it was found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, APH-1a was expressed as G122D in HEK293 cells. Overexpressed APH-1a G122D os not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allows the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitates the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supports the production of normal amounts of Abeta. It is concluded that cooperative effects may stabilize a trimeric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity (Edbauer, 2005).

Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway

The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this 'proteasome of the membrane' are unknown. A new interaction partner of nicastrin, the retrieval receptor Rer1p, has been characterized. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. Rer1p is a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. It is concluded that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value (Spasic, 2007).

REFERENCES

Search PubMed for articles about Drosophila nicastrin

Chávez-Gutiérrez, L., et al. (2008). Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J. Biol. Chem. 283: 20096-20105. PubMed Citation: 18502756

Chen, F., et al. (2001). Nicastrin binds to membrane-tethered Notch. Nat. Cell Biol. 3: 751-755. 11483961

Chung, H. M. and Struhl, G. (2001). Nicastrin is required for Presenilin-mediated transmembrane cleavage in Drosophila. Nat. Cell Biol. 3(12): 1129-32. 11781576

Edbauer, D., Kaether, C., Steiner, H. and Haass, C. (2005). Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1. J. Biol. Chem. 279(36): 37311-5. 15210705

Francis, R., et al. (2002). aph-1 and pen-2 are required for Notch pathway signaling, gamma-Secretase cleavage of ßAPP, and Presenilin protein accumulation. Dev. Cell 3: 85-97. 12110170

Goutte, C., Hepler, W., Mickey, K. M. and Priess, J. R. (2000). aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling. Development 127: 2481-2492. 10804188

Goutte, C., Tsunozaki, M., Hale, V. A. and Priess, J. R. (2002). APH-1 is a multipass membrane protein essential for the Notch signaling pathway in Caenorhabditis elegans embryos. Proc. Natl. Acad. Sci. 99(2): 775-779. 11792846

Gu, Y., et al. (2003). APH-1 interacts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin-nicastrin complexes. J. Biol. Chem. 278: 7374-7380. 12471034

Hass, M. R., Sato, C. Kopan, R. and Zhao, G. (2009). Presenilin: RIP and beyond. Semin. Cell Dev. Biol. 20: 201-210. PubMed Citation: 19073272

Hu, Y., Ye, Y. and Fortini, M. E. (2002). Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor. Dev. Cell 2: 69-78. 11782315

Lee, S. F., et al. (2002). Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid- precursor protein and notch. J. Biol. Chem. 277: 45013-45019. 12297508

Levitan, D., et al. (2001). APH-2/Nicastrin functions in LIN-12/Notch signaling in the Caenorhabditis elegans somatic gonad. Dev. Biol. 240(2): 654-61. 11784090

López-Schier, H. and St Johnston, D. (2002). Drosophila Nicastrin is essential for the intramembranous cleavage of Notch. Dev. Cell 2: 79-89. 11782316

Olry, A., et al. (2005). Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein. J. Biol. Chem. 280(31): 28564-71. 15958385

Satoh, J. and Kuroda, Y. (2001). Nicastrin, a key regulator of presenilin function, is expressed constitutively in human neural cell lines. Neuropathology 21(2): 115-22. 11396676

Seiffert, D., et al. (2000). Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors. J. Biol. Chem. 275: 34086-34091. 10915801

Shah, S., et al. (1995). Nicastrin functions as a gamma-secretase-substrate receptor. Cell 122:435-447. PubMed Citation: 16096062

Spasic, D., et al. (2007). Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway. J. Cell Biol. 176(5): 629-40. Medline abstract: 17325205

Steiner, H., Fluhrer, R. and Haass, C. (2008). Intramembrane proteolysis by gamma-secretase. J. Biol. Chem. 283: 29627-29631. PubMed Citation: 18650432

Stempfle, D., Kanwar, R., Loewer, A., Fortini, M. E. and Merdes, G. (2010). In vivo reconstitution of γ-secretase in Drosophila results in substrate specificity. Mol. Cell Biol. 30(13): 3165-75. PubMed Citation: 20421416

Takasugi, N., et al. (2003). The role of presenilin cofactors in the gamma-secretase complex. Nature 422: 438-441. 12660785

Yu, G., et al. (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and APP processing. Nature 407: 48-54. 10993067

date revised: 15 February 2002

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