gamma -Secretase Cleavage Site Specificity Differs for Intracellular and Secretory Amyloid beta *

Heike S. GrimmDagger §, Dirk Beher, Stefan F. Lichtenthaler||, Mark S. Shearman, Konrad BeyreutherDagger , and Tobias HartmannDagger **

From the Dagger  Center for Molecular Biology Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany,  Departments of Biochemistry and Molecular Biology, Merck Sharp and Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, United Kingdom, and || Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität München, Schillerstrasse 44, 80336 Munich, Germany

Received for publication, October 10, 2002, and in revised form, January 17, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The final step in Abeta generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by gamma -secretase. gamma -Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of gamma -secretase leading to the formation of secreted and intracellular Abeta are identical, we made use of point mutations close to the gamma -cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted Abeta . We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular Abeta , suggesting differences in the gamma -secretase cleavage site specificity for the generation of secreted and intracellular Abeta . The analysis of the subcellular compartments involved in the generation of intracellular Abeta revealed that Abeta is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular Abeta . Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain gamma -secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active gamma -secretase complex.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is characterized pathologically by the extracellular deposition of amyloid-beta peptide (Abeta ) in the brain (1, 2), derived by proteolytic processing from the amyloid precursor protein (APP) (3). APP processing by beta -secretase BACE1 (beta -site APP cleaving enzyme) generates the N terminus of Abeta , releasing the ectodomain of APP (for review, see Ref. 4). The remaining C-terminal membrane-bound fragment C99 is further cleaved by gamma -secretase, yielding two major species of Abeta peptides, Abeta 40 and Abeta 42 (5-7). Alternatively, APP can be cleaved within its ectodomain by alpha -secretases, identified as members of the ADAM family of disintegrin metalloproteases, leading to alpha -secreted APP and a truncated non-amyloidogenic peptide (p3) (for review, see Ref. 8). The identity of gamma -secretase has been the subject of intensive investigation; however, the exact nature of gamma -secretase has not been definitively established. Recent studies implied that gamma -secretase processing requires the presence of presenilin 1 (PS1) and PS2 (for review, see Ref. 4). The PS holoproteins undergo highly regulated endoproteolytic processing to yield N- and C-terminal fragments and are thought to mediate gamma -secretase enzyme activity as part of a multimeric high molecular weight complex (9, 10). It is still not proven, however, whether PS itself is gamma -secretase or an essential co-factor required for gamma -secretase enzyme activity, since other proteins of the high molecular weight complex have been shown to modulate gamma -secretase enzyme activity (11-13). Furthermore, a discrepancy exists between the predominant subcellular localization of PS1 in the endoplasmic reticulum (ER) and cis-Golgi compartment (14-16) and PS1-dependent gamma -secretase processing at or close to the plasma membrane where little PS1 seems to be present (17). Abeta generated at or close to the plasma membrane is rapidly secreted by cultured cells as well as in biological fluids and aggregates into the characteristic extracellular protein deposits, which are thought to be the cause of AD (for review, see Ref. 18). Additionally, intracellular Abeta is generated and is discussed as important in the pathogenesis of AD. Recent studies demonstrate an intraneuronal accumulation of Abeta 42, the predominant species of Abeta in senile plaques (19), in AD-vulnerable brain regions (20-22) as well as in transgenic mice expressing mutant proteins that lead to a familial form of Alzheimer's disease (23). Moreover, transgenic mice show accelerated neurodegeneration without extracellular amyloid deposition (24), indicating that intracellular amyloid-beta peptides may play a crucial role in the development of AD. Intracellular sites with gamma -secretase activity were identified in primary neurons, neuronal cell lines, and peripheral cells (25-30). Evidence has been obtained for the generation of Abeta 42 within the ER (27-29, 31), where PS is predominantly localized (14-16). However, it has also been reported that Abeta 42 is produced in different organelles later in the secretory pathway and that the ER is not the major intracellular site of Abeta 42 generation (26, 30, 32-34, 74). Additionally, recent work has suggested that the Abeta 42 generation in the ER may be independent of PS (35). Therefore, the exact site of intracellular Abeta generation and the subcellular compartments in which PS promotes Abeta generation remain so far unclear. Furthermore, little is known about the cleavage site specificity of the gamma -secretase responsible for the generation of intracellular Abeta , which may play a central part in the pathogenesis of AD.

In the present study we examined the cleavage site specificity of intracellular gamma -secretase as well as the subcellular compartments in which the direct Abeta precursor C99 (36) is processed by gamma -secretase to intracellular Abeta . To address the cleavage site specificity of gamma -secretase we made use of point mutations close to the gamma -cleavage site known to have a dramatic effect on the 42/40 ratio of secreted Abeta (37, 38) and determined their effect on the 42/40 ratio of intracellular Abeta . We found that these mutations only marginally influenced the 42/40 ratio of intracellular Abeta , indicating that the cleavage site specificity of intracellular gamma -secretase is less affected by point mutations close to the gamma -cleavage site. To investigate the compartments involved in gamma -secretase processing, we used different experimental strategies including treatments with organelle-specific toxins and expression of C99 proteins directed to different cellular compartments as well as subcellular fractionation. Our results show that generation of intracellular Abeta was eliminated when C99 was retained in the ER and early Golgi despite the presence of processed endogenous PS1, indicating that PS1 is not sufficient to mediate gamma -secretase processing in the early secretory pathway. Instead, we obtained several lines of evidence that an active gamma -secretase complex is located in late Golgi compartments.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Plasmid Construction-- Plasmids pBS/SPLEC99-WT, -I45F, and -V50F were generated as described (38, 39). The EcoRI/ClaI fragments of pBS/SPLEC99-WT, -I45F, and -V50F were cloned into the vector pBS/SPDAC99-WT (36) containing the two additional amino acids Asp and Ala instead of Leu and Glu, thus resulting in the plasmids pBS/SPDAC99-WT, -I45F, and -V50F, respectively. The KpnI/HindIII fragments of pBS/SPDAC99-WT, -I45F, and -V50F were cloned into the pCEP4 vector (Invitrogen) digested with KpnI and HindIII. The dilysine motif at the C terminus of C99 (third and fourth amino acid from the C terminus) was introduced via recombinant PCR, suitable oligonucleotides, and pBS/SPDAC99-WT, pBS/SPDAC99-I45F, and pBS/SPDAC99-V50F as PCR templates. The KpnI/HindIII fragments of pBS/SPDAC99-WT KK, -I45F KK, and -V50F KK were cloned into the pCEP4 vector digested with KpnI and HindIII to generate the expression plasmids pCEP4/SPDAC99-WT KK, -I45F KK, and -V50F KK. The eukaryotic expression vector pCEP4/APP695 SDYQRL was generated as described (40). The APP695 SDYQRL insert was cloned in pBS/APP695 using the enzymes BglII/ClaI. The EcoRI/ClaI fragment of pBS/APP695 SDYQRL was cloned in pBS/SPDAC99-WT digested with EcoRI/ClaI, resulting in pBS/SPDAC99-SDYQRL. pCEP4/SPDAC99-SDYQRL was generated as described for pCEP4/SPDAC99-WT. The identity of the constructs obtained by PCR was confirmed by DNA sequencing.

Cell Culture and Transfections-- Human SH-SY5Y neuroblastoma cell line was maintained in Dulbecco's modified Eagle's medium (high glucose) (Sigma) containing 10% fetal calf serum (PAA Laboratories) and 1% nonessential amino acid solution (Sigma). 80% confluent cells were transfected with the expression vector pCEP4 (Invitrogen) alone or the pCEP4 vector carrying the SPDAC99 inserts using Lipofectin (Invitrogen) as described by the producer. Stable transfectants were selected using 300 µg/ml hygromycin (PAA Laboratories). For each construct, at least two independent cell lines were established.

Antibodies-- The following antibodies were used and diluted for Western blot analysis as indicated: anti-calnexin (StressGen, 1:2500), anti-beta -COP (Sigma, 1:500), anti-syntaxin 6 (Transduction Laboratories, 1:500). PS1-FL and PS1-NTF were detected using antibody 98/1 raised against residues 1-20 of PS1 (1:2500) (41). The following antibodies were used to detect APP, C-terminal fragments, and Abeta : monoclonal W02, raised against residues 1-10 of Abeta (1 µg/ml) (42); polyclonal R7334, raised against residues 659-694 of APP695 (1:750) (43); monoclonal antibody 22C11, raised against residues 66-81 of APP (0.5 µg/ml) (44). Monoclonal antibodies W02 (5 µg/ml), G2-10, specific for Abeta ending residue 40 (12,5 µg/ml), and G2-11, specific for Abeta ending residue 42 (17,3 µg/ml) (42), were used for immunoprecipitation, polyclonal antibody 98/1 was used for the precipitation of PS1-NTF (5 µg/ml) (41), and polyclonal antibody 29414 was used for the precipitation of PS1-CTF (20 µl/ml) (provided by C. Elle, Center for Molecular Biology, University of Heidelberg, Heidelberg, Germany).

Preparation of Cell Lysates and Collection of Conditioned Media-- Fresh culture medium (5 ml) was added to a confluent monolayer of cells in a 10-cm culture dish. Conditioned media were collected after 14-16 h. The conditioned media were centrifuged at 4 °C for 1 min at 13,000 rpm, and the supernatants were used for immunoprecipitation of soluble secreted Abeta . In parallel to the conditioned media, cell lysates were prepared. Cells were harvested and lysed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, and 2 mM EDTA) supplemented with protease inhibitor mixture (Roche Molecular Biochemicals). The 13,000-rpm supernatants were used for immunoprecipitation of detergent-soluble intracellular Abeta .

Immunoprecipitation and Densitometric Quantification-- Equal volumes of conditioned media and cell lysates were immunoprecipitated with 20 µl of protein G-Sepharose (Sigma) and the antibodies W02, G2-10, and G2-11, respectively (concentrations see above). The immunoprecipitated proteins were separated on 12% Tris-Tricine gels (45) or commercial 10-20% Tricine gels (Invitrogen). Western blot analysis was performed with the antibody W02 (1 µg/ml) (42). Densitometric quantification was performed using MACBAS 2 software.

Treatments with Organelle-specific Toxins-- Stably transfected SH-SY5Y cells were preincubated with brefeldin A (BFA) (10 µg/ml, Sigma) or monensin (5 µM, Sigma) for 1.5 h followed by a 10-h incubation period in the presence of the drugs in fresh medium.

Metabolic Labeling and Preparation of Cellular Membranes-- After 1.5 h of preincubation in methionine-free minimum essential Eagle's medium (Sigma) in the presence or absence of BFA (10 µg/ml), C99 WT-transfected SH-SY5Y cells were incubated for 6 h in methionine-free nonessential amino acid solution containing 5% fetal calf serum and 133 µCi/ml [35S]methionine (Amersham Biosciences) in the presence or absence of BFA (10 µg/ml). Cellular membranes were prepared as described by Mercken et al. (46) and subjected to immunoprecipitation as described above.

Subcellular Fractionation and Sucrose Density Centrifugation-- Subcellular fractionation and sucrose density equilibrium centrifugation of SH-SY5Y cells stably expressing SPC99-WT were performed essentially as described (43) with minor modifications. Postnuclear membranes were applied onto a continuous sucrose gradient (0.2-2 M) and 17 1-ml fractions were collected from the bottom of the gradient after centrifugation overnight at 100,000 × gav (27,000 rpm, Beckman SW 28.1 rotor). Individual fractions were diluted into 5 mM HEPES, pH 7.3, 0,15 M NaCl, and membranes were sedimented by centrifugation for 1 h at 80,000 rpm in a MLA-80 rotor (Beckman). The final membrane pellets were re-suspended in 200 µl of phosphate-buffered saline, 5% glycerol and stored at -80 °C until further use.

Electrochemiluminescence Assay-- Equal aliquots of each fraction were adjusted 2% CHAPSO and 0.5% CHAPSO for detection of Abeta 40 and Abeta 42 peptides, respectively. Membrane-associated Abeta peptides were released by incubation for 20 min at 37 °C and measured by an electrochemiluminescence assay as described (10, 47) in a 96-well plate format. Abeta peptides were captured with biotinylated monoclonal antibody 4G8 (Senetek) followed by detection of Abeta 40 with ruthenylated G2-10, whereas ruthenylated G2-11 was used for detection of Abeta 42. Nonspecific background signal was determined by using non-biotinylated capture antibody 4G8 in the presence of ruthenylated detection antibodies G2-10/G2-11 and subtracted.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular and Secreted Abeta 42/40 Ratios Are Differently Influenced by Point Mutations Close to the gamma -Cleavage Site-- Point mutations close to the gamma -cleavage site of APP as well as pathogenic mutations in the genes encoding PS1 and PS2 have been shown to alter the product ratio of Abeta 42 and Abeta 40 (Abeta 42/40) (37-39, 48-50). However, in these studies, the authors analyzed the 42/40 ratios of secreted Abeta , which is generated via a pH-sensitive and endocytosis-dependent pathway (17, 51-53). Far less is known about the cleavage site specificity of the gamma -secretase responsible for the generation of intracellular Abeta (Abeta i). To investigate the cleavage site specificity of intracellular gamma -secretase, we selected two point mutations close to the gamma -cleavage site (Fig. 1), known to have a strong effect on the 42/40 ratio of secreted Abeta (Abeta sec42/40). These are the point mutations I45F and V50F, which have been identified by phenylalanine-scanning mutagenesis of the transmembrane domain of APP (38).


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Fig. 1.   Schematic representation of wild-type and mutant SPC99 proteins (I45F, V46F, and V50F) used for the study of Abeta i generation. SPC99 contains the signal peptide (SP) of APP, two additional amino acids (Asp and Ala), the Abeta domain (framed by black lines), and the complete C terminus of APP. SPC99 only requires gamma -secretase activity for Abeta generation; the N terminus of Abeta is produced by the activity of the signal peptidase. Amino acids are shown in the single-letter code. Protease cleavage sites are marked by black arrows. Asterisks indicate the position of amino acid exchanges in the SPC99 mutant proteins.

Consistent with previously published data for non-neuronal cells (38), both mutants (C99 I45F and C99 V50F) had an opposite effect on the generation of the Abeta species secreted into the media of stably transfected human SH-SY5Y neuroblastoma cells. C99 I45F is mainly processed to secreted Abeta ending residue 42 (Abeta sec42) (Fig. 2A), resulting in a dramatic increase of the Abeta sec42/40 ratio compared with C99 WT (relative ratio, 20.4 ± 3.6 (I45F), p < 0.001, n = 6) (Fig. 2C) (Table I). In contrast, the V50F mutation is mainly cleaved after amino acid residue 40 (Fig. 2A), causing a decrease of the Abeta sec42/40 ratio compared with C99 WT (relative ratio, 0.3 ± 0.1 (V50F), p < 0.001, n = 6) (Fig. 2C) (Table I). As a negative control we used SH-SY5Y cells stably transfected with the expression vector alone measuring the amount of endogenous secreted and intracellular Abeta (data not shown).


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Fig. 2.   Analysis of Abeta 40 and Abeta 42 production in SH-SY5Y cells stably transfected with the SPC99 WT and SPC99 mutant constructs (I45F and V50F). Abeta 40 and Abeta 42 were immunoprecipitated from the conditioned media (A) and cell lysates (B) using the antibodies indicated above the gels (40, antibody G2-10, specific for Abeta 40; 42, antibody G2-11, specific for Abeta 42). Both Abeta species were detected by Western blot using antibody W02. C and D, determination of Abeta sec42/40 and Abeta i42/40 ratios relative to the Abeta 42/40 ratios produced by the C99 WT protein. Abeta sec42/40 and Abeta i42/40, respectively, were calculated by densitometric quantification for each stably transfected cell line. The determined Abeta 42/40 values were divided by the corresponding Abeta 42/40 ratios obtained for C99 WT in the same experiment. Thus, the values for C99 WT were 1.0. Gray columns represent the mean values of 6 (Abeta sec) and 9 (Abeta i) independent experiments; black vertical bars indicate the S.D. The asterisks show the statistical significance (two-sided Student's t test) relative to C99 WT (**, p < 0.01; ***, p < 0.001). C, ratios of Abeta sec42/40 relative to the C99 WT construct. D, ratios of Abeta i42/40 relative to the C99 WT construct.


                              
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Table I
Abeta 42/40 ratios of intracellular and secreted Abeta
NS, not significant.

In contrast to the dramatic effect of both point mutations on the 42/40 ratio of secreted Abeta , both mutations only marginally influenced the 42/40 ratio of intracellular Abeta (Abeta i42/40) (Fig. 2B). The C99 WT protein produced roughly 1.5-fold more Abeta i40 than Abeta i42 (Abeta i42/40-WT, 0.65 ± 0.27, n = 9) (Table I), indicating an elevated Abeta i42/40 ratio relative to the ratio of Abeta sec42/40 (Table I), consistent with previously published data (27, 52, 54, 55). However, the point mutation at position 45 of Abeta that dramatically influenced the 42/40 ratio of secreted Abeta by a factor of 20.4 compared with the WT-protein showed only a minor effect on the 42/40 ratio of intracellular Abeta . The ratio of Abeta i42/40 was increased by a factor of 1.9 compared with C99 WT (relative ratio, 1.9 ± 0.5 (I45F), p < 0.01, n = 9) (Table I). A similar phenomenon was obtained for C99 V50F. Compared with C99 WT, the Abeta sec42/40 ratio was reduced to 30%, whereas the Abeta i42/40 ratio of C99 V50F was not significantly decreased compared with the Abeta i42/40 ratio of the WT construct (relative ratio, 0.8 ± 0.5 (V50F), not significant, n = 9) (Table I), indicating that both point mutations have a minor effect on the cleavage site specificity of the gamma -secretase responsible for intracellular Abeta generation. To verify our results that intracellular gamma -secretase is less affected by point mutations close to the gamma -cleavage site, we used C99 V46F, a familial Alzheimer's disease-linked mutation at position 717 of APP770 (Fig. 1) (56). The Abeta sec42/40 ratio of C99 V46F (Abeta sec42/40-V46F: 0.67 ± 0.17, p < 0.01, n = 7) was increased by a factor of 3.1 relative to the WT protein (Table I), consistent with previous findings (37, 38), whereas the Abeta i42/40 ratio was not affected compared with the Abeta i42/40 ratio obtained for the WT protein (Abeta i42/40-V46F: 0.66 ± 0.19, not significant, n = 3) (Table I), further confirming our results obtained for C99 I45F and C99 V50F.

Generation of Abeta Is Inhibited by C99 Proteins Bearing an ER/Intermediate Compartment (IC) Retrieval Signal-- To analyze the subcellular compartments involved in Abeta generation, we used different experimental strategies. To determine whether gamma -secretase processing can occur within the ER, we introduced a dilysine motif at the C terminus of C99 (C99 KK) that leads to the retention of proteins in the ER and IC (30, 57, 58). As shown in Fig. 3A the expression levels of C99 KK proteins (C99 WT KK, C99 I45F KK, C99 V50F KK) were comparable with the expression levels of C99 proteins without the ER/IC retrieval signal (C99 WT, C99 I45F, C99 V50F) and did not affect the levels of endogenous APP (Fig. 3A). However, significant amounts of Abeta i could only be recovered from cell lysates of cells expressing C99 constructs without an ER/IC retrieval signal (C99 WT, C99 I45F, C99 V50F) (Fig. 3A). Cell lines expressing C99 proteins bearing the dilysine motif did not produce Abeta i levels above the background level of Abeta i produced from the endogenous APP, as verified by the comparison with cells stably transfected with the expression vector alone (Fig. 3A). These results clearly show that C99 has to be transported out of the ER to get processed by gamma -secretase, indicating that the ER is not a major intracellular site of gamma -secretase activity. This finding is supported by the observation that Abeta secretion was blocked to endogenous background levels when C99 was retained in the ER and IC (Fig. 3B), providing evidence that Abeta was not generated in the ER and rapidly secreted into the conditioned media.


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Fig. 3.   Addition of an ER/IC retrieval signal abolishes the generation of Abeta i and Abeta sec. Western blot analysis of cell lysates (A) and conditioned media (B) from SH-SY5Y cells that were transfected with the expression vector alone (control) or the indicated SPC99 constructs. Cell lysates and conditioned media were immunoprecipitated with antibody W02. Abeta , C99, and endogenous APP were detected in the Western blot using antibody W02. A and B, respectively, represent different exposure times of the same Western blot analysis.

Brefeldin A Treatment Abolishes Intracellular Abeta Generation-- To verify our data that Abeta cannot be generated early within the secretory pathway, we blocked protein transport using BFA. BFA blocks anterograde protein transport out of the ER, resulting in a fusion of the proximal Golgi (cis- and medial-Golgi) with the ER (59, 60). The efficacy of BFA treatment was verified by different observations as follows (i) BFA treatment affected the maturation of endogenous APP as seen by a complete change of the observed band pattern of APP immunoreactivity (Fig. 4A). (ii) The generation of Abeta sec and alpha -secreted endogenous APP (sAPPalpha ) was blocked in the presence of BFA, whereas Abeta secretion could be detected in the conditioned media of untreated cells (Fig. 4B). (iii) An increase of endogenous APP was observed in cell lysates of treated cells (Fig. 4C), indicating that the secretory pathway was efficiently blocked by BFA treatment.


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Fig. 4.   Effects of brefeldin A treatment on Abeta generation, maturation of endogenous APP, and endoproteolysis of PS1. SH-SY5Y cells stably transfected with the indicated SPC99 constructs or the expression vector alone (control) were preincubated with 10 µg/ml BFA for 1.5 h. After preincubation the cells were incubated for additional 10 h in fresh medium containing 10 µg/ml BFA. Control cells were incubated in the absence of BFA. A, cell lysates of treated and untreated cells, expressing C99 WT, were prepared and directly loaded on a 8% Tris-Tricine gel. The immature (APP immat) and mature (APP mat) isoforms of endogenous APP were visualized by immunoblotting with antibody W02. B, endogenous alpha -secreted APP (sAPPalpha ) and Abeta secreted into the conditioned media (Abeta sec) were immunoprecipitated and detected by Western blot using antibody W02. C, cell lysates were prepared after BFA treatment and immunoprecipitated with the antibody W02. Abeta i, C99, and endogenous APP were detected by immunoblotting with antibody W02. B and C, respectively, represent different exposure times of the same Western blot analysis. D, SH-SY5Y cells stably transfected with C99 WT were metabolically labeled in the presence or absence of BFA (10 µg/ml). Cellular membranes were prepared and immunoprecipitated with the indicated antibodies. Precipitated proteins were separated on a 12.5% SDS page. PS1-NTF and PS1-CTF were visualized by autoradiography.

The presence of BFA also dramatically affected the generation of Abeta i. Abeta i could be detected in untreated cells expressing C99 WT, I45F, and V50F (Fig. 4C). However, Abeta i formation was strongly impaired in BFA-treated cells (Fig. 4C), indicating that protein trafficking beyond the early secretory pathway is required for Abeta i generation and, thus, confirming our results obtained with C99 KK mutant proteins. To verify that the lack of gamma -secretase processing in BFA-treated cells is not simply caused by diminished endoproteolysis of endogenous PS1, which seems to be important for PS function (61), we analyzed the amount of N- and C-terminal PS1 fragments (PS1-NTF and PS1-CTF, respectively) in cells treated or not treated with BFA. Therefore, C99 WT-expressing cells were metabolically labeled in the presence and absence of BFA, and cellular membranes were immunoprecipitated with antibodies directed to the N or C terminus of PS1. Processing of PS1 to its N- and C-terminal fragments was not inhibited in the presence of BFA as shown in Fig. 4D, indicating that the presence of processed PS1 is not sufficient to obtain gamma -secretase activity.

Monensin Increases Intracellular Abeta Levels-- To investigate further the intracellular compartments that are involved in Abeta i generation, we incubated SH-SY5Y cells expressing the C99 WT protein or the mutant proteins (C99 I45F, C99 V50F) in the presence or absence of monensin. Monensin inhibits the maturation of newly synthesized proteins in the trans-Golgi and blocks their transport out of the trans-Golgi network (TGN) (62, 63). The analysis of the conditioned media of treated and untreated cells showed that secretion of Abeta as well as secretion of sAPPalpha (alpha -secreted endogenous APP) was completely blocked in the presence of monensin (data not shown), similar to treatment of cells with BFA. In contrast to BFA, monensin treatment strongly increased Abeta i levels (Fig. 5A), indicating that late Golgi compartments are involved in gamma -secretase processing of C99. Abeta levels were increased by a factor of 3.0 in monensin-treated cells stably expressing the C99 WT protein compared with untreated cells (Abeta i + monensin/Abeta - monensin, 3.0 ± 0.9 (WT), p < 0.05; n = 4). Similar results were obtained for C99 I45F and C99 V50F (Abeta i + monensin/Abeta i - monensin, 2.5 ± 0.4 (I45F), p < 0.05, n = 3; 2.9 ± 0.3 (V50F), p < 0.01; n = 3) (Fig. 5B).


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Fig. 5.   Effect of monensin treatment on Abeta i generation. SH-SY5Y cells stably expressing the indicated SPC99 constructs were preincubated for 1.5 h with 5 µM monensin, and monensin treatment was performed for an additional 10 h in fresh medium. A, cell lysates of treated (+) and untreated (-) cells were immunoprecipitated with the antibody W02 followed by Western blot analysis using antibody W02. The figure represents different exposure times of the same Western blot analysis as indicated by the black line within the picture. B, quantification of at least three independent experiments. Abeta i detected in the Western blot of treated and untreated cells was quantified densitometrically. The amount of Abeta i in the presence of monensin was divided by the amount of Abeta i in the absence of monensin for each construct and each experiment (Abeta i + monensin/Abeta - monensin). Thus, the value for untreated cells was 1.0 and was set to 100%. Gray columns represent the mean values; black vertical bars give the S.D. The asterisks indicate the statistical significance (two-sided Student's t test) relative to untreated cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001). C, SH-SY5Y cells stably expressing C99 WT were incubated with monensin as described. Equal volumes of cell lysates were immunoprecipitated (IP) with the antibody G2-10, specific for Abeta 40, and the antibody G2-11, specific for Abeta 42. The immunoprecipitated Abeta i40 and Abeta i42 peptides were detected with antibody W02 in the Western blot. D, ratios of Abeta i42/40 after monensin treatment relative to untreated cells of six independent experiments. For this, the same graphical presentation was used as described under B.

Using the Abeta 40- and Abeta 42-specific antibodies G2-10 and G2-11, we showed that Abeta i40 and Abeta i42 were increased after monensin treatment in C99 WT-expressing SH-SY5Y cells (Fig. 5C), indicating that late Golgi compartments (trans-Golgi, TGN) are involved in cleavage at position 40 and position 42 of Abeta . However, Abeta i40 and Abeta i42 increased to a different extent, resulting in decreased Abeta i42/40 ratios in monensin-treated cells (Abeta i42/40 + monensin/Abeta i42/40 - monensin = 0.49 ± 0.2; p < 0.001; n = 6) (Fig. 5D).

Generation of Abeta by C99 Proteins Bearing a TGN-sorting Signal-- The results obtained so far provide evidence that protein transport to late Golgi compartments (trans-Golgi, TGN) is essential for gamma -secretase processing of C99. To confirm these results, we established SH-SY5Y cells, stably expressing a C99 protein, bearing the sorting signal of TGN38 for recycling between the cell surface and the TGN (C99 SDYQRL) (64, 65). It was previously shown that the addition of the amino acid motif SDYQRL to the C terminus of APP leads to an accumulation of APP in the TGN (34, 40). We found that Abeta i levels were increased by a factor of 1.5 in cells stably expressing the C99 SDYQRL construct compared with C99 WT (relative ratio Abeta i/C99, 1.5 ± 0.3 (SDYQRL), p < 0.05, n = 7) (Fig. 6A), confirming the role of the TGN in gamma -secretase processing of C99.


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Fig. 6.   Generation of Abeta from a mutant C99 protein bearing a TGN-sorting signal. Cell lysates of SH-SY5Y cells stably expressing the C99 WT protein or the mutant C99 protein combined with the TGN38 sorting signal (C99 SDYQRL) were immunoprecipitated with antibody W02, G2-10, and G2-11, respectively. The immunoprecipitated proteins were detected by Western blotting with the antibody W02. The corresponding Abeta i and C99 bands were densitometrically quantified. A, ratio of Abeta i/C99 relative to the WT construct. Columns represent the mean values of seven independent experiments; black vertical bars give the S.D. The asterisks indicate the statistical significance (two-sided Student's t test) relative to C99 WT (*, p < 0.05). B, ratio of Abeta i42/40 relative to the C99 WT construct. Columns represent the mean values of four independent experiments. Black vertical bars indicate the S.D., and asterisks give the statistical significance (two-sided Student's t test) relative to C99 WT (*, p < 0.05).

Furthermore, we could show that the Abeta i42/40 ratio was decreased in C99 SDYQRL-expressing cells compared with the Abeta i42/40 ratio of the WT construct (relative ratio, 0.7 ± 0.1 (SDYQRL), p < 0.05, n = 5) (Fig. 6B), indicating that gamma -secretase cleaves preferentially at position 40 in the TGN, consistent with our findings in monensin-treated cells.

Subcellular Distribution of Abeta i40, Abeta i42, and Endogenous PS1-- To analyze the subcellular distribution of Abeta i40 and Abeta i42 independent of sorting signals or drug treatments, membranous organelles of SH-SY5Y cells transfected with C99 WT were separated by sucrose density centrifugation. Abeta i40 and Abeta i42 were measured by a highly sensitive electrochemiluminescence (ECL) assay (10, 47), whereas the corresponding fractions were probed by Western blotting for endogenous PS1, C99, C83 (alpha -cleaved C-terminal fragment), endogenous APP, and several well characterized organelle marker proteins. ER-rich fractions were detected as expected at the bottom of the gradient using an antibody against the ER marker protein calnexin (66). Calnexin-reactive ER vesicles were most enriched in fractions 4-6 (Fig. 7A). Golgi-containing fractions were identified by blotting for beta -COP, a Golgi marker protein (67, 68) most abundant in the early Golgi compartments, found in fractions 12 and 13 (Fig. 7B). TGN-rich fractions were defined by the TGN marker protein syntaxin 6 (69), enriched in fractions 7-10 (Fig. 7C).


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Fig. 7.   Subcellular fractionation of Abeta , PS1, and C99. Membranous organelles of SH-SY5Y cells stably expressing C99 WT were separated by sucrose density centrifugation. A, equal aliquots of individual fractions were immunoblotted for calnexin, C99, and C83 as indicated. The vertical bar indicates that the samples were loaded onto two separate 10-20% Tris-Tricine gels, blotted onto the same nitrocellulose membrane. As a standard (std) 1.0 ng of recombinant C100FLAG (10) was loaded on each gel. Aliquots of individual fractions were directly loaded on a 12% SDS-gel and immunoblotted for beta -COP and PS1 (PS1-FL and PS1-NTF) (B) and on a 10% SDS-gel for the detection of endogenous immature and mature APP (APP immat, APP mat) and syntaxin 6 (C). A, B, and C, the data shown are representative of at least three independent experiments. D, the individual fractions were analyzed by an ECL assay to determine the subcellular distribution of Abeta i40 and Abeta i42. Abeta i x-40 and Abeta i x-42 were detected by the use of the antibody combination 4G8/G2-10 and 4G8/G2-11, respectively. The graph shows the average of duplicate ECL measurements and is representative of at least three independent experiments.

Using these organelle marker proteins we found that the gamma -secretase substrates C99 and C83 accumulated in TGN-fractions, as defined by the enrichment of the TGN marker protein syntaxin 6 (fractions 7-10) (Fig. 7, A and C). Exogenous expressed C99 was detected to a smaller extent in ER fractions (fractions 4-6), defined by the marker protein calnexin and the presence of immature endogenous APP (APP immat) (Fig. 7, A and C). As expected C83, the alpha -cleaved C-terminal fragment could not be detected in the ER-rich vesicles (Fig. 7A), supporting previously published data that alpha -secretase cleaves later within the secretory pathway (70-72). PS1-NTF was detected across the whole sucrose gradient; however, an accumulation of PS1-NTF as well as PS-FL was observed in fractions 7-12 (Fig. 7B), overlapping with the TGN marker protein syntaxin 6 (fractions 7-10) (Fig. 7C). To detect all Abeta peptides ending at position 40 and 42 including N-terminal-truncated Abeta species, for example Glu11-Abeta (73), we used the antibody combinations G2-10/4G8 and G2-11/4G8, respectively. Abeta i40 and Abeta i42 as measured by ECL are almost identically distributed across the gradient (Fig. 7D). A peak for Abeta i x-40 and Abeta i x-42 (intracellular Abeta peptides with variable N terminus ending at residue 40 and 42, respectively) was observed in fractions 7-9 (Fig. 7D), suggesting a TGN localization as defined by the accumulation of the TGN marker protein syntaxin 6 in fractions 7-10 (Fig. 7C). Neither Abeta i40 nor Abeta i42 could be detected in fraction 5, which showed the strongest staining for the ER marker protein calnexin (Fig. 7A). This excludes the presence of significant amounts of Abeta i40 and Abeta i42 in the ER, consistent with our findings that Abeta cannot be generated in the ER by analyzing C99 KK mutant proteins. Additionally, Abeta i40 and Abeta i42 could not be detected in beta -COP-rich fractions (fractions 12 and 13), supporting our data that Abeta cannot be generated in early Golgi compartments.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gamma -secretase is defined as proteolytic activity resulting in cleavage of APP CTFs releasing the C terminus of Abeta and Abeta -like peptides (5-8). PS1 and PS2 have been found to be essential components of the high molecular weight complex mediating gamma -secretase activity (9, 10). The cleavage site specificity of the gamma -secretase enzyme determines the ratio of Abeta 40 and Abeta 42. Pathogenic point mutations in APP and presenilins increase the Abeta 42/40 ratio of secreted Abeta (37, 48-50).

In the present study we show that point mutations close to the gamma -cleavage site of APP have a minor effect on the cleavage site specificity of intracellular gamma -secretase compared with their dramatic effect on the cleavage site specificity of the gamma -secretase responsible for the generation of Abeta sec. The point mutations I45F and V50F that revealed a dramatic effect on the Abeta sec42/40 ratio only marginally affected the Abeta i42/40 ratio. The relevance of this observation was confirmed by the use of the familial Alzheimer's disease mutant V717F of APP770 (56). Although secreted Abeta 42 was increased in the presence of the mutant, we observed no increase in intracellular Abeta 42 production, consistent with recent data (35). Altered gamma -secretase cleavage site specificity may be caused by different proteolytic activities for intracellular and secreted Abeta . Wilson et al. (35) report that intracellular Abeta generation in the ER is independent of PS. However, PS-independent Abeta i42 production in the early secretory pathway is unlikely to be involved in intracellular Abeta generation in the human SH-SY5Y neuroblastoma cells, because we found late Golgi compartments responsible for intracellular Abeta generation. Thus, it is not unlikely that presenilins are involved in intracellular Abeta generation, and cleavage site specificity may be affected by different co-factors of the high molecular weight PS-containing complexes (9, 10). Alternatively, minor changes present in subcellular compartments like lipid composition (75, 76), protein glycosylation, or other modifications of gamma -secretase complex proteins (11, 13, 77, 78) may be sufficient to modify cleavage-site specificity.

Our finding that point mutations increasing gamma -cleavage at position 42 have a more pronounced effect on secretory Abeta compared with intracellular Abeta may indicate that familial Alzheimer's disease-linked mutant proteins initiate accumulation of Abeta 42 in the extracellular Abeta pool and that intracellular Abeta production has a minor role in the pathogenesis of AD. However, we cannot exclude that slightly elevated intracellular Abeta 42 levels or additional mechanisms that we did not observe in our in vitro cell culture system may be sufficient to start Abeta aggregation. Indeed, intracellular Abeta 42 induces neurotoxicity in primary rat and human neurons (79, 80). Moreover, studies on AD-vulnerable brain regions as well as animal models expressing familial Alzheimer's disease-related mutant proteins implicated intraneuronal accumulation of Abeta 42 (20, 22, 23).

The analysis of the intracellular sites of gamma -cleavage shows that intracellular Abeta generation was inhibited when cells were treated with BFA or were stably transfected with constructs bearing an ER/IC retrieval signal despite the presence of processed PS1. These results are consistent with previously published data that Abeta i generation was inhibited after BFA treatment in the mouse neuroblastoma cell line N2a and COS1 cells as well as in different cell lines transiently transfected with truncated APP constructs bearing an ER/IC retrieval signal, including kidney 293 cells and N2a cells (26, 30, 32, 34). However, Abeta 42 generation was also found within the ER using very similar approaches (27, 29, 31). Abeta 42 generation within the ER seems to be more abundant in differentiated neurons as shown for rat primary neurons (28) and differentiated NT2N cells (27, 31, 81) derived from the human embryonal carcinoma cell line NT2 (82). Interestingly, the less differentiated NT2 cells fail to produce intracellular Abeta (54). However, a neuronal phenotype cannot explain the different results obtained for NT2N cells infected with SFV/APP695 KK (27) versus primary cultures of mixed cortical neurons infected with SFV/APP-C99-KK (17). When we take into account that even very closely related cell culture lines or primary cells show significant differences in gamma -secretase 42 activity in different subcellular compartments, it appears that intracellular Abeta 42 generation can be variable between different cell types. However, our data clearly show that the ER/early Golgi is not the major intracellular site for gamma -secretase processing in the human SH-SY5Y neuroblastoma cells. Our findings are further confirmed by our subcellular fractionation studies. Neither Abeta i40 nor Abeta i42 accumulation was detected in the ER- and early Golgi-containing fractions. Moreover, we obtained several lines of evidence that late Golgi compartments are involved in gamma -secretase processing. First, Abeta i levels were dramatically increased when we blocked protein transport late in the secretory pathway using monensin, consistent with studies proposing the involvement of late Golgi compartments in gamma -secretase processing (26, 32, 34, 84). Second, Abeta i generation was increased in cells expressing a C99 mutant directed to the TGN. Third, subcellular fractionation of organelles from C99 WT-transfected SH-SY5Y cells revealed that Abeta i40 and Abeta i42 accumulated in TGN-rich fractions. Interestingly, we found that PS1 and the direct gamma -secretase substrate accumulated in TGN fractions as well, indicating the possibility of an interaction between PS1 and C99, resulting in gamma -secretase processing in these compartments. These results are supported by findings that PS1 forms complexes with APP C-terminal fragments in Golgi- and TGN-rich fractions and that de novo Abeta i generation was found in the same Golgi-/TGN-rich vesicles (85). In accordance to our ER-related data we conclude that the presence of processed PS1 is not sufficient to obtain gamma -secretase processing and that at least one additional factor is required for the formation of an active gamma -secretase complex in late Golgi compartments. Such additional factors could be the transmembrane proteins aph-1, pen-2, and nicastrin that have been recently shown to be essential gamma -secretase complex components (11, 13, 77, 78). Interestingly, the fully mature glycosylated form of nicastrin preferentially interacts with PS1 (86). In contrast, only a small proportion of PS1 is bound to the immature species of nicastrin, indicating that mature nicastrin may be essential for the formation of a functional gamma -secretase complex (86) and, thus, confirming our results that an active gamma -secretase complex is formed in late Golgi compartments where fully glycosylated mature nicastrin is present.

The identification of differences in gamma -secretase cleavage site specificity for intracellular and secretory Abeta generation shows that the gamma -secretase system is even more complex than previously assumed. This may have important implications for AD. It shows that at least partly different gamma -secretases exist. Because gamma -secretase cleaves a number of different substrates, like Notch (for review, see Ref. 8) and low density lipoprotein receptor-related protein (LRP) (83), this finding may help to target Abeta processing in a manner avoiding cross-inhibition with other gamma -secretase substrates.

    ACKNOWLEDGEMENTS

We thank J. Culvenor and C. Elle for providing the antibodies 98/1 and 29414.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and European Community Grant QLK 3-CT-2002-00172.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed. Tel.: 49-6221-546848; Fax: 49-6221-545891; E-mail: h.grimm@mail.zmbh.uni-heidelberg.de.

** To whom correspondence may be addressed. Tel.: 49-6221-546844; Fax: 49-6221-545891; E-mail: tobias.hartmann@zmbh.uni-heidelberg.de.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M210380200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid-beta peptide; Abeta i, intracellular Abeta ; APP, amyloid precursor protein; C99, C-terminal 99 amino acid residues of APP; PS 1/2, presenilin 1/2; PS1-FL, full-length presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; ER, endoplasmic reticulum; IC, intermediate compartment; BFA, brefeldin A; TGN, trans-Golgi network: CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; KK, ER/IC retrieval signal.

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DISCUSSION
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