A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.     

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

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

Presenilin clinical mutations can affect gamma-secretase activity by different mechanisms

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the gamma-secretase complex and cleave many type I transmembrane proteins including beta-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of gamma-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on gamma-secretase complex formation. A loss in APP and Notch substrate processing at epsilon and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the gamma site was affected in variable ways. PS1-Delta9 and PS1-L166P mutations caused a reduction in beta-amyloid peptide Abeta40 production whereas PS1-G384A mutant significantly increased Abeta42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Abeta than PS1. Finally, subtle differences in gamma-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect gamma-secretase structure or function in multiple ways.     

Presenilin-1 affects trafficking and processing of betaAPP and is targeted in a complex with nicastrin to the plasma membrane

Amyloid beta-peptide (Abeta) is generated by the consecutive cleavages of beta- and gamma-secretase. The intramembraneous gamma-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with gamma-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Abeta production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the gamma-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with gamma-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by gamma-secretase inhibitors results in delayed reinternalization of the beta-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in gamma-secretase processing and in trafficking.     

Presenilin-dependent gamma-secretase on plasma membrane and endosomes is functionally distinct

The presenilin (PS)/gamma-secretase complex, which contains not only PS but also Aph-1, PEN-2, and nicastrin, mediates proteolysis of the transmembrane domain of beta-amyloid protein precursor (betaAPP). Intramembrane proteolysis occurs at the interface between the membrane and cytosol (epsilon-site) and near the middle of the transmembrane domain (gamma-site), generating the betaAPP intracellular domain (AICD) and Alzheimer disease-associated Abeta, respectively. Both cleavage sites exhibit some diversity. Changes in the precision of gamma-cleavage, which potentially results in secretion of pathogenic Abeta42, have been intensively studied, while those of epsilon-cleavage have not. Although a number of PS-associated factors have been identified, it is unclear whether any of them physiologically regulate the precision of cleavage by PS/gamma-secretase. Moreover, there is currently no clear evidence of whether PS/gamma-secretase function differs according to the subcellular site. Here, we show that endocytosis affects the precision of PS-dependent epsilon-cleavage in cell culture. Relative production of longer AICDepsilon49 increases on the plasma membrane, whereas that of shorter AICDepsilon51 increases on endosomes; however, this occurs without a concomitant major change in the precision of cleavage at gamma-sites. Moreover, very similar changes in the precision of epsilon-cleavage are induced by alteration of the pH. Our findings demonstrate that the precision of epsilon-cleavage by PS/gamma-secretase changes depending upon the conditions and the subcellular location. These results suggest that the precision of cleavage by the PS/gamma-secretase complex may be physiologically regulated by the subcellular location and conditions.     

Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Abeta 42/40 ratios

Gene knockout studies in mice suggest that presenilin 1 (PS1) is the major gamma-secretase and that it contributes disproportionately to amyloid beta (Abeta) peptide generation from beta-amyloid precursor protein (APP), whereas PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimer's disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Abeta levels in the presence of normal PS1 alleles. Only four of the eight reported FAD mutations in PS2 have altered function in vitro suggesting that the other variants represent rare polymorphisms rather than disease-causing mutations. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Abeta 42/40 ratio, comparable with PS1 mutations that cause very-early-onset FAD. Most of the PS2 mutations also cause a significant decrease in Abeta 40, APP C-terminal fragment (CTF)gamma and Notch intracellular domain (NICD) production suggesting that they are partial loss of function mutations. PS2 M239V, its PS1 homolog M233V, and other FAD mutations within transmembrane (TM) 5 of PS1 differentially affect CTFgamma and NICD production suggesting that TM5 of PS are important for gamma-secretase cleavage of APP but not Notch.     

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

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

Catalytic site-directed gamma-secretase complex inhibitors do not discriminate pharmacologically between Notch S3 and beta-APP cleavages

The generation of gamma-secretase inhibitors which block the release of beta-amyloid peptide (Abeta) has long been an attractive therapeutic avenue for treatment or prevention of Alzheimer's disease (AD). Such inhibitors would reduce levels of Abeta available for aggregation into toxic assemblies that lead to the plaque pathology found in affected brain tissue. Cumulative evidence suggests that the S3 cleavage of Notch is also dependent on presenilins (PS) and is carried out by the multimeric PS-containing gamma-secretase complex. It is therefore possible that Notch function could be affected by gamma-secretase inhibitors. To assess the relationship between the cleavage of these substrates in the same system, Western blot cleavage assays have been established using a human cell line stably expressing both the beta-amyloid precursor protein (beta-APP) and the truncated Notch1 receptor fragment NotchDeltaE. Thus, a direct correlation may be made, following inhibitor treatment, of the decrease in the levels of the cleavage products, Abeta peptide and the Notch intracellular domain (NICD), as well as the increase in stabilized levels of both substrates. This analysis has been performed with a range of selected gamma-secretase inhibitors from six distinct structural classes. Changes in all four species usually occur in concert and with remarkably good agreement. A significant cleavage window is not clearly apparent in any case. Thus, these Notch and beta-APP cleavages cannot be dissected apart easily since they show the same pharmacological profile of inhibition. Whether this translates into proportionally reduced Notch signaling in vivo, however, remains to be seen.     

Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma -secretase processing of the APP and Notch1 transmembrane domains

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.     

Ubiquitin-proteasome pathway mediates degradation of APH-1

Gamma-secretase catalyzes intramembraneous proteolysis of several type I transmembrane proteins, including beta-amyloid precursor protein (APP), to generate amyloid beta protein (Abeta), a key player in the pathogenesis of Alzheimer's disease (AD). The critical components of the gamma-secretase complex include presenilin (PS), nicastrin (NCT), presenilin enhancer-2 (PEN-2) and anterior pharynx defective-1 (APH-1). Abnormalities of the ubiquitin-proteasome pathway have been implicated in the pathogenesis of AD; while PS and PEN-2 turnover is regulated by this pathway, it is unknown whether the ubiquitin-proteasome pathway is also involved in the degradation of APH-1 protein. In this study, we found that the expression of endogenous and exogenous APH-1 significantly increased in cells treated with proteasome-specific inhibitors. The effect of the proteasome inhibitors on APH-1 was dose- and time-dependent. APH-1 protein was ubiquitinated. Pulse-chase metabolic labeling experiments showed that the degradation of newly synthesized radiolabeled APH-1 proteins was inhibited by lactacystin. Disruption of the PS1 and PS2 genes did not affect the degradation of APH-1 by the ubiquitin-proteasome pathway. Furthermore, over-expression of APH-1 and inhibition of proteasomal APH-1 degradation facilitated gamma-secretase cleavage of APP to generate Abeta. These results demonstrate that the degradation of APH-1 protein is mediated by the ubiquitin-proteasome pathway.     

Evidence that CD147 modulation of beta-amyloid (Abeta) levels is mediated by extracellular degradation of secreted Abeta

Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.     

Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic beta-amyloid (Abeta) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other gamma-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by gamma-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of gamma-secretase inhibition, the gamma-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Abeta production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4(-)CD8(-)CD44(+)CD25(+) precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak gamma-secretase inhibitor. These studies show that inhibition of gamma-secretase has the expected benefit of reducing Abeta in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing.     

Cell surface presenilin-1 participates in the gamma-secretase-like proteolysis of Notch

Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and beta-amyloid precursor protein (APP) within their trans- membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an aspartyl protease, and mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase processing of APP. We show here that these aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and biotinylation assays, we provide evidence that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1 aspartate mutants were indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and did not alter the secretion of Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor.     

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

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

The cytoplasmic sequence of E-cadherin promotes non-amyloidogenic degradation of A beta precursors

The presenilin (PS)/gamma-secretase system promotes production of the A beta (A beta) peptides by mediating cleavage of amyloid precursor protein (APP) at the gamma-sites. This system is also involved in the processing of type-I transmembrane proteins, including APP, cadherins and Notch1 receptors, at the epsilon-cleavage site, resulting in the production of peptides containing the intracellular domains (ICDs) of the cleaved proteins. Emerging evidence shows that these peptides have important biological functions, raising the possibility that their inhibition by gamma-secretase inhibitors may be detrimental to the cell. Here, we show that peptide E-Cad/CTF2, produced by the PS1/gamma-secretase processing of E-cadherin, promotes the lysosomal/endosomal degradation of the transmembrane APP derivatives, C99 and C83, and inhibits production of the APP ICD (AICD). In addition, E-Cad/CTF2 decreases accumulation of total secreted A beta. These data suggest a novel method to promote the non-amyloidogenic degradation of A beta precursors and to inhibit A beta production.     

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

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