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.     

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.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

Altered APP processing in PDAPP (Val717 --> Phe) transgenic mice yields extended-length Abeta peptides

Central to the pathology of Alzheimer's disease (AD) is the profuse accumulation of amyloid-beta (Abeta) peptides in the brain of affected individuals, and several amyloid precursor protein (APP) transgenic (Tg) mice models have been created to mimic Abeta deposition. Among these, the PDAPP Tg mice carrying the familial AD APP 717 Val --> Phe mutation have been widely used to test potential AD therapeutic interventions including active and passive anti-Abeta immunizations. The structure and biochemistry of the PDAPP Tg mice Abeta-related peptides were investigated using acid and detergent lysis of brain tissue, ultracentrifugation, FPLC, HPLC, enzymatic and chemical cleavage of peptides, Western blot, immunoprecipitation, and MALDI-TOF and SELDI-TOF mass spectrometry. Our experiments reveal that PDAPP mice produce a variety of C-terminally elongated Abeta peptides in addition to Abeta n-40 and Abeta n-42, as well as N-terminally truncated peptides, suggesting anomalous proteolysis of both APP and Abeta. Important alterations in the overall APP degradation also occur in this model, resulting in a striking comparative lack of CT83 and CT99 fragments, which may be inherent to the strain of mice, a generalized gamma-secretase failure, or the ultimate manifestation of the overwhelming amount of expressed human transgene; these alterations are not observed in other strains of APP Tg mice or in sporadic AD. Understanding at the molecular level the nature of these important animal models will permit a better understanding of therapeutic interventions directed to prevent, delay, or reverse the ravages of sporadic AD.     

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.     

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.     

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.     

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.     

gamma-Secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid beta peptides of varying length

gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.     

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.     

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.     

APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer's disease mutations

One of the cardinal neuropathological findings in brains from Alzheimer's disease (AD) patients is the occurrence of amyloid beta-peptide (Abeta) deposits. The gamma-secretase-mediated intramembrane proteolysis event generating Abeta also results in the release of the APP intracellular domain (AICD), which may mediate nuclear signaling. It was recently shown that AICD starts at a position distal to the site predicted from gamma-secretase cleavage within the membrane. This novel site, the epsilon site, is located close to the inner leaflet of the membrane bilayer. The relationship between proteolysis at the gamma and epsilon sites has not been fully characterized. Here we studied AICD signaling in intact cells using a chimeric C99 molecule and a luciferase reporter system. We show that the release of AICD from the membrane takes place in a compartment downstream of the endoplasmic reticulum, is dependent on presenilin proteins, and can be inhibited by treatment with established gamma-secretase inhibitors. Moreover, we find that AICD signaling remains unaltered from C99 derivatives containing mutations associated with increased Abeta42 production and familial AD. These findings indicate that there are very similar routes for Abeta and AICD formation but that FAD-linked mutations in APP primarily affect gamma-secretase-mediated Abeta42 formation, and not AICD signaling.     

Hydrogen peroxide promotes Abeta production through JNK-dependent activation of gamma-secretase

Accumulation of senile plaques composed of amyloid beta-peptide (Abeta) is a pathological hallmark of Alzheimer disease (AD), and Abeta is generated through the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase. Although oxidative stress has been implicated in the AD pathogenesis by inducing Abeta production, the underlying mechanism remains elusive. Here we show that the pro-oxidant H(2)O(2) promotes Abeta production through c-Jun N-terminal kinase (JNK)-dependent activation of gamma-secretase. Treatment with H(2)O(2) induced significant increase in the levels of intracellular and secreted Abeta in human neuroblastoma SH-SY5Y cells. Although gamma-secretase-mediated cleavage of APP or C99 was enhanced upon H(2)O(2) treatment, expression of APP or its alpha/beta-secretase-mediated cleavage was not affected. Silencing of the stress-activated JNK by small interfering RNA or the specific JNK inhibitor SP600125 reduced H(2)O(2)-induced gamma-secretase-mediated cleavage of APP. JNK activity was augmented in human brain tissues from AD patients and active JNK located surrounding the senile plaques in the brain of AD model mouse. Our data suggest that oxidative stress-activated JNK may contribute to senile plaque expansion through the promotion of gamma-secretase-mediated APP cleavage and Abeta production.     

Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.     

Apolipoprotein E modulates gamma-secretase cleavage of the amyloid precursor protein

Polymorphisms in the apolipoprotein E (APOE) gene affect the risk of Alzheimer disease and the amount of amyloid beta-protein (Abeta) deposited in the brain. The apoE protein reduces Abeta levels in conditioned media from cells in culture, possibly through Abeta clearance mechanisms. To explore this effect, we treated multiple neural and non-neural cell lines for 24 h with apoE at concentrations similar to those found in the cerebrospinal fluid (1-5 microg/mL). The apoE treatment reduced Abeta40 by 60-80% and Abeta42 to a lesser extent (20-30%) in the conditioned media. Surprisingly, apoE treatment resulted in an accumulation of amyloid precursor protein (APP)-C-terminal fragments in cell extracts and a marked reduction of APP intracellular domain-mediated signaling, consistent with diminished gamma-secretase processing of APP. All three isoforms of apoE, E2, E3 and E4, had similar effects on Abeta and APP-C-terminal fragments, and the effects were independent of the low-density lipoprotein receptor family. Apolipoprotein E had minimal effects on Notch cleavage and signaling in cell-based assays. These data suggest that apoE reduces gamma-secretase cleavage of APP, lowering secreted Abeta levels, with stronger effects on Abeta40. The apoE modulation of Abeta production and APP signaling is a potential mechanism affecting Alzheimer disease risk.     

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.     

BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo

Introducing mutations within the amyloid precursor protein (APP) that affect beta- and gamma-secretase cleavages results in amyloid plaque formation in vivo. However, the relationship between beta-amyloid deposition and the subcellular site of Abeta production is unknown. To determine the effect of increasing beta-secretase (BACE) activity on Abeta deposition, we generated transgenic mice overexpressing human BACE. Although modest overexpression enhanced amyloid deposition, high BACE overexpression inhibited amyloid formation despite increased beta-cleavage of APP. However, high BACE expression shifted the subcellular location of APP cleavage to the neuronal perikarya early in the secretory pathway. These results suggest that the production, clearance, and aggregation of Abeta peptides are highly dependent on the specific neuronal subcellular domain wherein Abeta is generated and highlight the importance of perikaryal versus axonal APP proteolysis in the development of Abeta amyloid pathology in Alzheimer's disease.     

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 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.     

Intracellularly generated amyloid-beta peptide counteracts the antiapoptotic function of its precursor protein and primes proapoptotic pathways for activation by other insults in neuroblastoma cells

Most mutations in amyloid precursor proteins (APPs) linked to early onset familial Alzheimer's disease (FAD) increase the production of amyloid-beta peptides ending at residue 42 (Abeta42), which are released from APP by beta- and gamma-secretase cleavage. Stably transfected cells expressing wild-type human APP (APP(WT)) were more resistant to apoptosis-inducing treatments than cells expressing FAD-mutant human APP (APP(FAD)). Preventing Abeta42 production with an M596I mutation (beta-), which blocks beta-secretase cleavage of APP, or by treatment with a gamma-secretase inhibitor increased the resistance of APP(FAD)-expressing cells to apoptosis. Exposing hAPP(FAD/beta-) cells to exogenous Abeta42 or conditioned medium from Abeta42-producing APP(FAD) cells did not diminish their resistance to apoptosis. Preventing APP from entering the distal secretory pathway, where most Abeta peptides are generated, by retaining APP in the endoplasmic reticulum (ER)/intermediate compartment (IC) increased the resistance of APP(FAD)-expressing cells to apoptosis and did not alter the resistance of APP(WT)-expressing cells. p53-mediated gene transactivation after apoptosis-inducing treatments was much stronger in APP(FAD) cells than in hAPP(WT) or hAPP(FAD/beta-) cells. In contrast, upon induction of ER stress, cells expressing APP(FAD), hAPP(FAD/beta-), or APP(WT) had comparable levels of glucose-regulated protein-78 mRNA, an unfolded protein response indicator. We conclude that Abeta, especially intracellular Abeta, counteracts the antiapoptotic function of its precursor protein and predisposes cells to p53-mediated, and possibly other, proapoptotic pathways.