Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49-99 of beta-amyloid precursor protein

A novel cleavage of beta-amyloid precursor protein (APP), referred to as epsilon-cleavage, occurs downstream of the gamma-cleavage and generates predominantly a C-terminal fragment (CTFgamma) that begins at Val-50, according to amyloid beta-protein (Abeta) numbering. Whether this cleavage occurs independently of, or is coordinated with, gamma-cleavage is unknown. Using a cell-free system, we show here that, although Abeta40 and CTFgamma 50-99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFgamma 49-99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Abeta42. Furthermore, a gamma-secretase inhibitor, which suppresses Abeta40 production and paradoxically enhances Abeta42 production at low concentrations, caused the proportion of CTFgamma 50-99 to decrease and that of CTFgamma 49-99 to increase significantly. These results strongly suggest a link between the production of Abeta42 and CTFgamma 49-99 and provide an important insight into the mechanisms of altered gamma-cleavage caused by mtAPP and mtPS1/2.     

Truncated carboxyl-terminal fragments of beta-amyloid precursor protein are processed to amyloid beta-proteins 40 and 42

We previously showed that beta-amyloid precursor protein (APP) is cleaved not only in the middle of the membrane (gamma-cleavage) but also at novel cleavage sites close to the membrane/cytoplasmic boundary (epsilon-cleavage), releasing APP intracellular domains (AICDs) 49-99 and 50-99. To learn more about the relationship between gamma- and epsilon-cleavage, C-terminally truncated carboxyl-terminal fragments (CTFs) of APP, especially CTFs1-48 and 1-49 (the postulated products that are generated by epsilon-cleavage), were transiently expressed in CHO cells. Most importantly, the cells expressing CTF1-49 secreted predominantly amyloid beta-protein (Abeta) 40, while those expressing CTF1-48 secreted preferentially Abeta42. This supports our assumption that epsilon-cleavage precedes Alphabeta production and that preceding epsilon-cleavage determines the preference for the final Abeta species. The gamma-secretase inhibitors, L-685,458 and DAPT, suppressed Abeta production from CTF1-49. Regarding Abeta production from CTF1-48, L-685,458 suppressed it, but DAPT failed to do so. A dominant negative mutant of presenilin 1 suppressed the production of Abeta40 and 42 from both CTFs1-48 and 1-49. These data should shed significant light into the mechanism of Abeta production.     

DAPT-induced intracellular accumulations of longer amyloid beta-proteins: further implications for the mechanism of intramembrane cleavage by gamma-secretase

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites. These are referred to as gamma-, zeta-, and epsilon-cleavages. We showed previously that DAPT, a potent dipeptide gamma-secretase inhibitor, caused differential accumulations of longer amyloid beta-proteins (Abetas) (Abeta43 and Abeta46) in CHO cells that are induced to express the beta C-terminal fragment (CTF). To learn more about the cleavage mechanism by gamma-secretase, CHO cell lines coexpressing betaCTF and wild-type or mutant presenilin (PS) 1/2 were generated and treated with DAPT. In all cell lines treated with DAPT, as the levels of Abeta40 decreased, Abeta46 accumulated to varying extents. In wild-type PS1 or M146L mutant PS1 cells, substantial amounts of Abeta43 and Abeta46 accumulated. In contrast, this was not the case with wild-type PS2 cells. In M233T mutant PS1 cells, significant amounts of Abeta46 and Abeta48 accumulated differentially, whereas in N141I mutant PS2 cells, large amounts of Abeta45 accumulated concomitantly with a large decrease in Abeta42 levels. Most interestingly, in G384A mutant PS1 cells, there were no significant accumulations of longer Abetas except for Abeta46. Abeta40 was very susceptible to DAPT, but other Abetas were variably resistant. Complicated suppression and accumulation patterns by DAPT may be explained by stepwise processing of betaCTF from a zeta- or epsilon-cleavage site to a gamma-cleavage site and its preferential suppression of gamma-cleavage over zeta- or epsilon-cleavage.     

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.     

Amyloid beta-protein precursor juxtamembrane domain regulates specificity of gamma-secretase-dependent cleavages

Amyloid beta-protein (Abeta), the major component of cerebral plaques associated with Alzheimer disease, is derived from amyloid beta-protein precursor (APP) through sequential proteolytic cleavage involving beta- and gamma-secretase. The intramembrane cleavage of APP by gamma-secretase occurs at two major sites, gamma and epsilon, although the temporal and/or mechanistic relationships between these cleavages remain unknown. In our attempt to address this issue, we uncovered an important regulatory role for the APP luminal juxtamembrane domain. We demonstrated in cell-based assays that domain replacements in this region can greatly reduce secreted Abeta resulting from gamma-cleavage without affecting the epsilon-cleavage product. This Abeta reduction is likely due to impaired proteolysis at the gamma-cleavage site. Further analyses with site-directed mutagenesis identified two juxtamembrane residues, Lys-28 and Ser-26 (Abeta numbering), as the critical determinants for efficient intramembrane proteolysis at the gamma-site. Consistent with the growing evidence that epsilon-cleavage of APP precedes gamma-processing, longer Abeta species derived from the gamma-cleavage-deficient substrates were detected intracellularly. These results indicate that the luminal juxtamembrane region of APP is an important regulatory domain that modulates gamma-secretase-dependent intramembrane proteolysis, particularly in differentiating gamma- and epsilon-cleavages.     

Secretion of long Abeta-related peptides processed at epsilon-cleavage site is dependent on the alpha-secretase pre-cutting

Abeta is the major component of amyloid in the brain in Alzheimer's disease and is derived from Alzheimer amyloid precursor protein (APP) by sequential proteolytic cleavage involving alpha-, beta- and gamma-secretase. Recently, gamma-secretase was shown to cleave near the cytoplasmic membrane boundary of APP (called the epsilon-cleavage), as well as in the middle of the membrane domain (gamma-cleavage). However, the precise relationship between gamma- and epsilon-cleavage is still unknown. In this paper, I analyzed Abeta-related peptides using immunoprecipitation and liquid chromatography ion trap mass spectrometer and found some long Abeta-related peptides, starting at Abeta residues 16Lys-23Asp and ending at 43Thr-52Leu, in the culture media of COS-1 cells and in human brain extract. These results indicated that longer Abeta-related peptides cleaved at epsilon-cleavage site were secreted under normal conditions and were dependent on the alpha-secretase cleavage products.     

Identification of a new presenilin-dependent zeta-cleavage site within the transmembrane domain of amyloid precursor protein

Gamma-secretase cleavage of beta-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of beta-amyloid protein (Abeta). To better understand the molecular events involved in gamma-secretase cleavage of APP, in this study we report the identification of a new intracellular long Abeta species containing residues 1-46 (Abeta46), which led to the identification of a novel zeta-cleavage site between the known gamma- and epsilon-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new zeta-cleavage is a presenilin-dependent event. It is also noted that the new zeta-cleavage site at Abeta46 is the APP717 mutation site. Furthermore, we show that the new zeta-cleavage is inhibited by gamma-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state gamma-secretase inhibitors. Thus, the identification of Abeta46 establishes a system to determine the specificity or the preference of the known gamma-secretase inhibitors by examining their effects on the formation or turnover of Abeta46.     

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.     

X11alpha impairs gamma- but not beta-cleavage of amyloid precursor protein

The phosphotyrosine binding domain of the neuronal protein X11alpha/mint-1 binds to the C-terminus of amyloid precursor protein (APP) and inhibits catabolism to beta-amyloid (Abeta), but the mechanism of this effect is unclear. Coexpression of X11alpha or its PTB domain with APPswe inhibited secretion of Abeta40 but not APPsbetaswe, suggesting inhibition of gamma- but not beta-secretase. To further probe cleavage(s) inhibited by X11alpha, we coexpressed beta-secretase (BACE-1) or a component of the gamma-secretase complex (PS-1Delta9) with APP, APPswe, or C99, with and without X11alpha, in HEK293 cells. X11alpha suppressed the PS-1Delta9-induced increase in Abeta42 secretion generated from APPswe or C99. However, X11alpha did not impair BACE-1-mediated proteolysis of APP or APPswe to C99. In contrast to impaired gamma-cleavage of APPswe, X11alpha or its PTB domain did not inhibit gamma-cleavage of NotchDeltaE to NICD (the Notch intracellular domain). The X11alpha PDZ-PS.1Delta9 interaction did not affect gamma-cleavage activity. In a cell-free system, X11alpha did not inhibit the catabolism of APP C-terminal fragments. These data suggest that X11alpha may inhibit Abeta secretion from APP by impairing its trafficking to sites of active gamma-secretase complexes. By specifically targeting substrate instead of enzyme X11alpha may function as a relatively specific gamma-secretase inhibitor.     

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein

We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.     

Retention of the Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum prevents formation of amyloid beta-peptide

gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.     

Presenilin 1 stabilizes the C-terminal fragment of the amyloid precursor protein independently of gamma-secretase activity

The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1.     

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.     

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

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

gamma-Cleavage is dependent on zeta-cleavage during the proteolytic processing of amyloid precursor protein within its transmembrane domain

beta-Amyloid precursor protein apparently undergoes at least three major cleavages, gamma-, epsilon-, and the newly identified zeta-cleavage, within its transmembrane domain to produce secreted beta-amyloid protein (Abeta). However, the roles of epsilon- and zeta-cleavages in the formation of secreted Abeta and the relationship among these three cleavages, namely epsilon-, zeta-, and gamma-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known gamma-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Abeta(46) is an intermediate precursor of secreted Abeta. Our co-immunoprecipitation data also reveal that, as an intermediate, Abeta(46) is tightly associated with presenilin in intact cells. Furthermore, we identified a long Abeta species that is most likely the long sought after intermediate product, Abeta(49), generated by epsilon-cleavage, and this Abeta(49) is further processed by zeta- and gamma-cleavages to generate Abeta(46) and ultimately the secreted Abeta(40/42). More interestingly, our data demonstrate that gamma-cleavage not only occurs last but also depends on zeta-cleavage occurring prior to it, indicating that zeta-cleavage is crucial for the formation of secreted Abeta. Thus, we conclude that the C terminus of secreted Abeta is most likely generated by a series of sequential cleavages, namely first epsilon-cleavage which is then followed by zeta- and gamma-cleavages, and that Abeta(46) produced by zeta-cleavage is the precursor of secreted Abeta(40/42).     

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.     

The role of presenilin-1 in the gamma-secretase cleavage of the amyloid precursor protein of Alzheimer's disease

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.