The presenilin 1 C92S mutation increases abeta 42 production

Although wild-type human presenilin 1 (PS1) rescues the C. elegans egg-laying (egl) phenotype that is caused by a loss of function mutation in the C. elegans presenilin homologue sel12, most familial Alzheimer's disease (FAD)-linked PS1 mutants only partially rescue this phenotype. To investigate the effects of the loss of function sel12 mutation on Abeta production in mammalian cells, we analyzed Abeta production in transfected H4 neuroglioma cells expressing the PS1 homologue of the sel12 C60S mutant, PS1 C92S. This analysis revealed that PS1 C92S increased Abeta42 levels in a similar fashion to other pathogenic Alzheimer's disease (AD) PS1 mutations. Significantly, the PS1 C92S mutation has recently been identified as the pathogenic mutation in an Italian family with FAD. Thus, placing a mutation that results in loss of function in C. elegans into a context whereby its effect on mammalian cells can be evaluated suggests that all FAD-linked PS1 mutants result in increased Abeta42 production through a partial loss of function mechanism.     

FAD-linked mutations in presenilin 1 alter the length of Abeta peptides derived from betaAPP transmembrane domain mutants

gamma-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid beta-peptide (Abeta). gamma-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the gamma-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Abeta, are also the most common cause of familial Alzheimer's disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of gamma-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Abeta peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Abeta peptides from sites distal to the primary cleavage site ('longer' peptides) and in several cases also decreased production of 'shorter' peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the gamma-secretase complex to favor the production of long Abeta.     

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells

Urea-based beta-amyloid (Abeta) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Abeta peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Abeta peptides, namely, 1-40/42 and carboxyl-terminal-truncated 1-37, 1-38, and 1-39, was found. Besides Abeta1-42, we also observed a consistent elevation of amino-terminal-truncated Abeta2-42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Abeta2-42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different gamma-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Abeta peptides, we overexpressed beta-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1-/- neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1-/- neurons and PS1+/+ neurons overexpressing APP-Deltact (a slow-internalizing mutant) show a decrease of all secreted Abeta peptide species, as expected, because this mutant is processed mainly by alpha-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Abeta2-42 is significantly less affected in PS1-/- neurons and in neurons transfected with the endocytosis-deficient APP-Deltact construct. Our data confirm that PS1 is closely involved in the production of Abeta1-40/42 and the carboxyl-terminal-truncated Abeta1-37, Abeta1-38, and Abeta1-39, but the amino-terminal-truncated and carboxyl-terminal-elongated Abeta2-42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Abeta peptide species could be generated by a beta(Asp/Ala)-secretase activity.     

Antisense-induced reduction of presenilin 1 expression selectively increases the production of amyloid beta42 in transfected cells

Autosomal dominant mutations in the presenilin 1 (PS1) gene are associated with familial, early-onset Alzheimer's disease. Although the pathogenic mechanism of these mutations is unclear, their common feature is that they lead to an increased concentration of amyloid beta-peptide (Abeta) 42 in the plasma of early-onset patients, in the conditioned media of transfected cells, and in the brains of transgenic mice that overexpress mutant PS1. To address the mechanism(s) by which the pathogenic PS1 mutations increase Abeta42, we constructed human cell lines expressing a doxycyclin (dox)-inducible antisense PS1 RNA and measured its effects on the levels of PS1, amyloid precursor protein (APP), and Abeta. In time course experiments, we observed a statistically significant (p = 0.0038) more than twofold elevation in secreted Abeta42 as early as 12 days after addition of dox. This correlated with an 80% decrease in the 46-kDa PS1 holoprotein and a 30% decrease in the 26-kDa N-terminal fragment (NTF). Furthermore, there was a significant fivefold (p = 0.002) increase in Abeta42 after 14-day dox treatment; this correlated with a >90% decrease in PS1 holoprotein and 60% decrease in NTF. At no time point did we observe significant changes in Abeta40, APP holoprotein, presenilin 2, or tubulin. Ten days after the removal of dox, we observed a return to constitutive levels for Abeta42, PS1 holoprotein, and NTF. These results suggest that in human cell lines, the reduction of normal PS1 activity results in the increased production of Abeta42. Furthermore, our results are consistent with a loss of function or dominant negative mechanism for the pathogenic PS1 mutations.     

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-mediated modulation of capacitative calcium entry

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.     

APH1, PEN2, and Nicastrin increase Abeta levels and gamma-secretase activity

A major component of the amyloid plaque core in Alzheimer's disease (AD) is the 40-42-residue amyloid beta peptide (Abeta). Mutations linked to AD such as those in presenilins 1 (PS1) and 2 (PS2) invariably increase the longer Abeta42 species that forms neurotoxic oligomers. It is believed that PS1/2 constitute the catalytic subunit of the gamma-secretase responsible for the final step in Abeta biogenesis. Recent genetic studies have identified a number of additional genes encoding APH1a, APH1b, PEN2, and Nicastrin proteins, which are part of the gamma-secretase complex with PS1. Further, knockout studies using RNAi showed that these components are essential for gamma-secretase activity. However, the nature of gamma-secretase and how the aforementioned proteins regulate its activity are still incompletely understood. Here we present evidence that unlike PS1, overexpression of these proteins can increase the levels of Abeta, suggesting that these proteins are limiting for gamma-secretase activity. In addition, our studies also suggest that the presenilin partners regulate the relative levels of Abeta40 and Abeta42.     

Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta

Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.     

Presenilins: multifunctional proteins involved in Alzheimer's disease pathology

Early-onset aggressive forms of Alzheimer's disease (AD) are of genetic nature and have been linked to inherited mutations located on chromosomes 21, 14, and 1. The gene products of chromosomes 14 and 1, which are responsible for most of these familial forms of the disease (FAD), have been recently identified and referred to as presenilin 1 and 2 (PS1, PS2), respectively. Several lines of evidence derived from neuropathological, cell biology, and transgenesis approaches indicate that PS could interfere with the processing of the beta-amyloid precursor protein (betaAPP). Thus, FAD-linked mutations in PS exacerbate the production of Abeta42, the readily aggregable Abeta species corresponding to one of the main constituents of the senile plaques that invade the cortical areas of affected brains. Recent studies indicate that PS functions could be intimately related with the susceptibility of PS to further processing by caspase-like enzymes and other unknown proteolytic activities. Here I briefly report on the post-translational events undergone by PS and examine recent advances concerning their possible roles in development, cell signaling, and apoptosis. Possible alterations brought by FAD-linked mutations will be discussed.     

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.     

The influence of endoproteolytic processing of familial Alzheimer's disease presenilin 2 on abeta42 amyloid peptide formation

Mutant presenilins (PS) contribute to the pathogenesis of familial Alzheimer's disease (FAD) by enhancing the production of Abeta42 from beta-amyloid precursor protein. Presenilins are endoproteolytically processed to N-terminal and C-terminal fragments, which together form a stable 1:1 complex. We have mapped the cleavage site in the PS2 protein by direct sequencing of its C-terminal fragment isolated from mouse liver. Three different N-terminal residues were identified starting at Val-299, Thr-301, and Leu-307 that correspond closely to the previously described N termini of the C-terminal fragment of human PS1. Mutational analysis of the PS2 cleavage site indicates that the principal endoproteolytic cleavage occurs at residues Met-298/Val-299 and that the N terminus is subsequently modified by secondary proteolytic cleavages. We have generated cleavage defective PS2 constructs, which accumulate exclusively as full-length polypeptides in transfected Neuro2a cells. Functional analysis of such cleavage defective PS2 carrying the FAD mutation Asn-141 --> Ile showed that its Abeta42 producing activity was strongly reduced compared with cleavage-competent FAD PS2. In contrast, cleavage defective PS2 was active in rescuing the egg-laying defect of a sel-12 mutant in Caenorhabditis elegans. We conclude that PS2 endoproteolytic cleavage is not an absolute requirement for its activities but may rather selectively enhance or stabilize its functions.     

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimer's disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.     

Subcellular compartment and molecular subdomain of beta-amyloid precursor protein relevant to the Abeta 42-promoting effects of Alzheimer mutant presenilin 2

Increased production of amyloid beta peptides ending at position 42 (Abeta42) is one of the pathogenic phenotypes caused by mutant forms of presenilins (PS) linked to familial Alzheimer's disease. To identify the subcellular compartment(s) in which familial Alzheimer's disease mutant PS2 (mt PS2) affects the gamma-cleavage of betaAPP to increase Abeta42, we co-expressed the C-terminal 99-amino acid fragment of betaAPP (C100) tagged with sorting signals to the endoplasmic reticulum (C100/ER) or to the trans-Golgi network (C100/TGN) together with mt PS2 in N2a cells. C100/TGN co-transfected with mt PS2 increased levels or ratios of intracellular as well as secreted Abeta42 at similar levels to those with C100 without signals (C100/WT), whereas C100/ER yielded a negligible level of Abeta, which was not affected by co-transfection of mt PS2. To identify the molecular subdomain of betaAPP required for the effects of mt PS2, we next co-expressed C100 variously truncated at the C-terminal cytoplasmic domain together with mt PS2. All types of C-terminally truncated C100 variants including that lacking the entire cytoplasmic domain yielded the secreted form of Abeta at levels comparable with those from C100/WT, and co-transfection of mt PS2 increased the secretion of Abeta42. These results suggest that (i) late intracellular compartments including TGN are the major sites in which Abeta42 is produced and up-regulated by mt PS2 and that (ii) the anterior half of C100 lacking the entire cytoplasmic domain is sufficient for the overproduction of Abeta42 caused by mt PS2.     

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.     

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.     

Intracellular site of gamma-secretase cleavage for Abeta42 generation in neuro 2a cells harbouring a presenilin 1 mutation.

Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway.     

Characterization of the reconstituted gamma-secretase complex from Sf9 cells co-expressing presenilin 1, nicastrin [correction of nacastrin], aph-1a, and pen-2

Gamma-secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in Abeta42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in Abeta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile.     

Caspase cleaved presenilin-1 is part of active gamma-secretase complexes

gamma-Secretase is a key enzyme involved in the processing of the beta-amyloid precursor protein into amyloid beta-peptides (Abeta). Abeta accumulates and forms plaques in Alzheimer's disease (AD) brains. A progressive neurodegeneration and cognitive decline occurs during the course of the disease, and Abeta is believed to be central for the molecular pathogenesis of AD. Apoptosis has been implicated as one of the mechanisms behind the neuronal cell loss seen in AD. We have studied preservation and activity of the gamma-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y) exposed to staurosporine (STS). We report that the known components (presenilin, Nicastrin, Aph-1 and Pen-2) interact and form active gamma-secretase complexes in apoptotic cells. In addition, the fragments corresponding to the PS1 N-terminal fragment and the caspase-cleaved PS1 C-terminal fragment (PS1-caspCTF) were found to form active gamma-secretase complexes when co-expressed in presenilin (PS) knockout cells. Interestingly, PS1-caspCTF replaced the normal PS1 C-terminal fragment and was co-immunoprecipitated with the gamma-secretase complex in SH-SY5Y cells exposed to STS. In addition, Abeta was detected in medium from apoptotic HEK APP(swe) cells. Together, the data show that gamma-secretase complexes containing PS1-caspCTF are active, and suggest that this proteolytic activity is also important in dying cells and may affect the progression of AD.     

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.     

The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants

Presenilin 1 (PS1) and presenilin 2 (PS2) are polytopic membrane proteins that are mutated in the majority of early onset familial Alzheimer's disease (FAD) cases. Two lines of evidence establish a critical role for PS in the production of beta-amyloid peptides (Abeta). FAD-linked PS mutations elevate the levels of highly amyloidogenic Abeta ending at residue 42 (Abeta42), and cells with ablated PS1 alleles secrete low levels of Abeta. Several recent reports have shown that the hydrophilic loop (HL) domain, located between transmembrane domains 6 and 7, contains sites for phosphorylation, caspase cleavage, and sequences that bind several PS-interacting proteins. In the present report, we examined the metabolism of PS polypeptides lacking the HL domain and the influence of these molecules on Abeta production. We report that the deletion of the HL domain does not have a deleterious effect on the regulated endoproteolysis of PS, saturable accumulation of PS fragments, or the self-association of PS fragments. Abeta production was not significantly altered in cells expressing HL-deleted PS polypeptides compared with cells expressing full-length PS. Importantly, deletion of the HL domain did not affect FAD mutation-mediated elevation in the production of Abeta42. Furthermore, the deletion of the HL domain did not impair the role of PS1 or PS2 in facilitating Notch processing. Thus, our results argue against a biologically or pathologically relevant role for the HL domain phosphorylation and caspase cleavage and the association of PS HL domain-interacting proteins, in amyloid precursor protein metabolism and Abeta production or Notch cleavage.