Nicastrin functions as a gamma-secretase-substrate receptor

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

Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network

Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of gamma-secretase, the protease responsible for generating the Alzheimer amyloid beta-protein. However, the differential localization of presenilins to early secretory compartments and gamma-secretase substrates to late secretory compartments and the plasma membrane (the "spatial paradox") argues against this view. We investigated this issue by studying the localization of nicastrin, another putative gamma-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans-Golgi network. Trafficking of nicastrin to the trans-Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans-Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans-Golgi network fractions contained the gamma-secretase substrate beta-amyloid precursor protein C83 and were enriched in presenilin-dependent gamma-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin-nicastrin complexes and presenilin-dependent gamma-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the gamma-secretase, and indicate that presenilins also regulate gamma-secretase assembly.     

Presenilin-1, nicastrin,amyloid precursor protein,and gamma-secretase activity are co-localized in the lysosomal membrane

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.     

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein

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. We have set up a genetic screen 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. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations 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.     

Nicastrin binds to membrane-tethered Notch

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. Although absence of the Caenorhabditis elegans nicastrin homologue (aph-2) is known to cause an embryonic-lethal glp-1 phenotype, the role of nicastrin in this process has not been explored. Here we report that nicastrin binds to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and that, 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, we report that the Notch and betaAPP pathways do not significantly compete with each other.     

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

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

The extreme C terminus of presenilin 1 is essential for gamma-secretase complex assembly and activity

The gamma-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid beta-peptide and the cytoplasmic APP intracellular domain. The active gamma-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for gamma-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, gamma-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease gamma-secretase activity, PS1 endoproteolysis, and gamma-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.     

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin

The gamma-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the beta-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the gamma-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of gamma-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature gamma-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.     

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

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

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. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. 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. We identify Rer1p as 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. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.     

Presenilins and gamma-secretase inhibitors affect intracellular trafficking and cell surface localization of the gamma-secretase complex components

The intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch is mediated by the presenilin (PS, PS1/PS2)-gamma-secretase complex, the components of which also include nicastrin, APH-1, and PEN-2. In addition to its essential role in gamma-secretase activity, we and others have reported that PS1 plays a role in intracellular trafficking of select membrane proteins including nicastrin. Here we examined the fate of PEN-2 in the absence of PS expression or gamma-secretase activity. We found that PEN-2 is retained in the endoplasmic reticulum and has a much shorter half-life in PS-deficient cells than in wild type cells, suggesting that PSs are required for maintaining the stability and proper subcellular trafficking of PEN-2. However, the function of PS in PEN-2 trafficking is distinct from its contribution to gamma-secretase activity because inhibition of gamma-secretase activity by gamma-secretase inhibitors did not affect the PEN-2 level or its egress from the endoplasmic reticulum. Instead, membrane-permeable gamma-secretase inhibitors, but not a membrane-impermeable derivative, markedly increased the cell surface levels of PS1 and PEN-2 without affecting that of nicastrin. In support of its role in PEN-2 trafficking, PS1 was also required for the gamma-secretase inhibitor-induced plasma membrane accumulation of PEN-2. We further showed that gamma-secretase inhibitors specifically accelerated the Golgi to the cell surface transport of PS1 and PEN-2. Taken together, we demonstrate an essential role for PSs in intracellular trafficking of the gamma-secretase components, and that selective gamma-secretase inhibitors differentially affect the trafficking of the gamma-secretase components, which may contribute to an inactivation of gamma-secretase.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Aph-1 interacts at the cell surface with proteins in the active gamma-secretase complex and membrane-tethered Notch

The activity of the gamma-secretase complex is critical for the processing of a number of transmembrane proteins, including Notch. Functional gamma-secretase activity can be reconstituted from four proteins--presenilin, nicastrin, Pen-2 and Aph-1--but the role of the individual proteins remains unclear. In this report we describe the cellular localization and protein interactions of Aph-1, with particular regard to Notch receptor processing. We found that Aph-1 is present at the cell surface, where it interacts with Pen-2, the mature forms of presenilin and nicastrin, and full-length Notch. Aph-1 also interacts with a truncated form of Notch, which is a direct substrate for gamma-secretase, but not with the Notch intracellular domain. Immunoprecipitation data for Notch and Aph-1 showed that the Notch-containing gamma-secretase complexes most likely form a small subset of the total number of gamma-secretase complexes. In conclusion, these data demonstrate that Aph-1 is present at the cell surface, presumably in active gamma-secretase complexes, and interacts with the Notch receptor, both before and after ligand activation.     

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

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

Nicastrin expression in mouse peripheral tissues is not co-ordinated with presenilin and is high in muscle

Nicastrin was the first binding partner of presenilin (PS) shown to be a critical component of the presenilin/gamma-secretase complex essential in development and differentiation, and in generation of Alzheimer's disease Abeta amyloid peptide. To investigate the function of this glycoprotein, we compared nicastrin and presenilin protein expression in various mouse tissues. Western blot analysis of PS1, PS2 and nicastrin indicates their expression levels are not coordinated. In adult mouse, nicastrin is highly expressed in muscle membranes, whereas presenilin levels are very low. By Blue Native electrophoresis, a PS1 complex of 400 kDa was detected in lung, brain, thymus and heart; nicastrin was also detected as a 400-kDa complex in brain but in muscle it was detected with a complex mobility of 240 and 290 kDa, suggesting association with alternate protein complexes. Immunocytochemistry confirms strong intracellular expression of nicastrin in skeletal muscle and blood vessel smooth muscle. These findings suggest a function for nicastrin in muscle other than participation in the gamma-secretase complex.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

Cellular localization of Nicastrin affects amyloid beta species production

The gamma-secretase complex, composed by presenilin, nicastrin, APH-1 and PEN-2, is involved in intramembranous proteolysis of membrane proteins, such as amyloid precursor protein or Notch. Cleavage occurs in multiple cellular compartments. Here, nicastrin mutants containing targeting signals to the endoplasmic reticulum, trans-Golgi network, lysosomes, or plasma membrane have been shown to yield active gamma-secretase complexes with different activities and specificities: wild-type and plasma membrane nicastrin complexes yielded the highest amounts of secreted amyloid-beta peptide (Abeta), predominantly Abeta40, whereas intracellular targeted mutants produced intracellular Abeta, with a comparatively higher amount of Abeta42. These results suggest that compartmental microenvironments play a role in gamma-secretase activity and specificity.     

gamma-cleavage-independent functions of presenilin, nicastrin, and Aph-1 regulate cell-junction organization and prevent tau toxicity in vivo

Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.     

Drosophila nicastrin is essential for the intramembranous cleavage of notch.

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