Familial Alzheimer Disease-linked Presenilin 1 Variants Enhance Production of Both Abeta 1-40 and Abeta 1-42 Peptides That Are Only Partially Sensitive to a Potent Aspartyl Protease Transition State Inhibitor of "gamma -Secretase"*

Takeshi IkeuchiDagger §, Georgia Dolios, Seong-Hun KimDagger , Rong Wang, and Sangram S. SisodiaDagger ||

From the Dagger  Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois 60637 and the  Department of Human Genetics, The Mount Sinai School of Medicine, New York, New York 10029

Received for publication, September 10, 2002, and in revised form, November 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Presenilin 1 (PS1) plays an essential role in intramembranous "gamma -secretase" processing of several type I membrane proteins, including the beta -amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Abeta peptides and the effects of L-685,458, a potent gamma -secretase inhibitor, on inhibition of Abeta peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Abeta 1-42 and Abeta 1-40 peptides. More surprisingly, whereas the IC50 for inhibition of Abeta 1-40 peptide production from cells expressing wild-type PS1 is ~1.5 µM, cells expressing the PS1Delta E9 mutant PS1 exhibit an IC50 of ~4 µM. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Abeta 1-43 peptides are elevated in medium of PS1Delta E9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on gamma -secretase production of Abeta peptides and the disparity in sensitivity of these peptides to a potent gamma -secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Abeta 1-40 and Abeta 1-42 peptides.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Presenilin 1 and 2 (PS1 and PS2)1 are polytopic membrane proteins that are mutated in the majority of pedigrees with early onset familial Alzheimer's disease (FAD) (1-3). It is now established that PS play an essential role in intramembranous "gamma -secretase" processing of type I membrane proteins, including the beta -amyloid precursor proteins (APP) (4, 5), the developmental signaling receptor, Notch1 (6-8), the tyrosine kinase receptor ErbB4 (9, 10), and N- and E-cadherins (11). For APP, gamma -secretase catalyzes proteolysis of a set of membrane-tethered APP derivatives, termed APP-CTFs, resulting in the production and secretion of beta -amyloid (Abeta ) peptides. On the other hand, gamma -secretase-mediated processing of the membrane-tethered Notch1 derivative, termed S2/NEXT, releases the intracellular domain (S3/NICD) that subsequently translocates to the nucleus and activates transcription of target genes (7, 8). The observation that Abeta and S3/NICD production are completely eliminated in cells derived from mouse blastocysts with compound deletions of PS1 and PS2, lends convincing support to the notion that PS are critical for intramembranous cleavage of APP and Notch1 (12, 13).

Although the mechanism(s) by which PS facilitates gamma -secretase processing of APP and Notch1 have not been fully elucidated, the generation of Abeta (14-16) and S3/NICD (17) have been show to be inhibited by highly potent and selective aspartyl protease transition state inhibitors that bind specifically to PS1 and PS2 (14, 18). These data, taken with the description of a family of signal peptide peptidases with limited homology to PS (19), have led to the conclusion that PS are the elusive gamma -secretases (20).

While appealing, the "PS is gamma -secretase" model has several weaknesses. First, mutagenesis studies have revealed that gamma -secretase has relaxed substrate selectivity within the APP transmembrane domain and occurs at heterogeneous sites (21, 22), while gamma -secretase cleavage of Notch1 is highly sequence-specific and appears to generate a single S3/NICD species (7). Second, whereas endocytosis and recycling of APP-CTFs are required for the generation of Abeta (23), S3/NICD production does not require endocytic trafficking of the Notch derivative, S2/NEXT (24). Third, the identification of several PS-interacting membrane proteins, including nicastrin (25), APH-1 (26), and PEN2 (27) that also modulate production of S3/NICD (25-27) and Abeta (25, 28) suggests that a protein complex, comprised of PS and other factors are required for intramembranous proteolysis of APP and Notch1. Finally, PS1 harboring a substitution of aspartate 257 with alanine is capable of processing APP to Abeta peptides (29, 30), but fails to generate S3/NICD from a truncated Notch1 molecule, termed NotchDelta E (31). Similarly, expression of several FAD-linked PS1 variants (30-32) or the experimental L286E or L286R PS1 mutants (33) leads to exaggerated overproduction of highly fibrillogenic Abeta 42 peptides, but surprisingly, these PS1 variants fail to generate S3/NICD from NotchDelta E.

Intrigued by the apparent discordance between the activities of FAD-linked PS1 mutants on the production of Abeta 42 peptides and S3/NICD production, we examined the activity of these FAD-linked PS1 variants on the production of secreted Abeta peptides and the effects of a potent aspartyl protease transition state inhibitor of gamma -secretase, termed L-685,458 (15, 16) on the production of these Abeta species. We now report that while PS1 variants enhance production of Abeta 42, as expected, there is an unexpected enhancement in levels of secreted Abeta 40 peptides. We also provide the first demonstration that in the conditioned medium of "pools" of stable cell lines that express individual FAD-linked mutant PS1, both Abeta 1-40 and 1-42 peptides accumulate to higher levels than the Abeta peptide variants in medium of cell pools that express wild-type PS1. More surprisingly, under conditions at which the gamma -secretase inhibitor completely eliminates production of all Abeta -related peptides from cells expressing wild-type PS1, we now report that the inhibitor is not fully effective at lowering production of Abeta variants from cells expressing two independent FAD-linked PS1 mutants. Hence, we argue that production of Abeta peptides are differentially regulated by the expression of wild-type and FAD-linked PS1 variants.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- Mouse neuroblastoma N2a cells that constitutively expresses human Swedish APP695 (N2a swe.10) (35) were maintained in 50% Dulbecco's modified Eagle's medium and 50% Opti-MEM (Invitrogen) supplemented with 5% fetal bovine serum. To generate stable cell lines expressing wild-type PS1, the FAD-linked Delta E9, or E280A variants, N2a swe.10 cells were cotransfected with 10 µg of PS1 cDNAs (in pAG3Zeo vector) and 100 ng of pIREShygro using the calcium phosphate method (36). Cells expressing transgene were selected with 400 µg/ml hygromycin. Hygromycin-resistant colonies were further screened in medium containing 400 µg/ml zeocin (Invitrogen) to generate a stable pool. Approximately 100-200 zeocin-resistant colonies were pooled and analyzed.

gamma -Secretase Inhibitor Assay-- For gamma -secretase inhibitor assays, cells were incubated for 16 h in medium containing 2 µM (or indicated concentrations) of the gamma -secretase inhibitor, L-685,458 (16), prepared in dimethyl sulfoxide (Me2SO) or an equivalent concentration of Me2SO as a vehicle control.

Conditioned media were collected and immediately adjusted to 0.5 mM phenylmethylsulfonyl fluoride. Cultured cells were lysed in 1× immunoprecipitation (IP) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor mixture (Sigma). Nuclei and debris were removed by centrifugation and the protein concentration of detergent-soluble proteins in each lysate was determined using a bicinchoninic acid protein assay kit (Pierce).

Immunoprecipitation, Electrophoresis, and Immunoblotting-- For immunoprecipitations, we used equivalent volumes of conditioned medium based on the calculation of the protein concentration in each plate of cells to avoid experimental bias because of variations in cell density. Normalized conditioned media were immunoprecipitated with 26D6, a monoclonal antibody raised against Abeta 1-12 (37), for 16 h at 4 °C. The immune complexes were "bridged" by the addition of rabbit anti-mouse IgG (Pierce), collected with protein A-conjugated agarose beads (Pierce), and eluted by boiling for 5 min in Laemmli SDS sample buffer prior to fractionation on SDS-PAGE.

Aliquots of detergent lysate were fractionated on high percentage Tris-Tricine SDS-PAGE gels for detection of full-length APP and APP-CTFs, or Tris glycine SDS-PAGE for analysis of PS1. To detect secreted Abeta 40/42, immunoprecipitated samples were fractionated on Bicine/urea gels (38). Fractionated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad), and the membranes were probed with appropriate primary antibodies. Full-length APP and APP-CTF were detected by CT15, an antisera that recognizes the carboxyl-terminal 15 amino acids of APP (39). A polyclonal antibody, PS1NT, was used to detect full-length PS1 and PS1 NTF (40). Soluble APPalpha and Abeta 40/42 were detected by 26D6 (37). After incubation with horseradish peroxidase-coupled secondary antibodies (Pierce), bound antibodies were visualized using an enhanced chemiluminescence (ECL) detection system (Perkin-Elmer Life Sciences).

Metabolic Labeling and Immunoprecipitation-- N2a cells were starved for 30 min in methionine-free Dulbecco's modified Eagle's medium (Invitrogen) and then labeled with 250 µCi/ml [35S]methionine (PerkinElmer Life Sciences) in methionine-free Dulbecco's modified Eagle's medium supplemented with 1% dialyzed fetal bovine serum (Invitrogen) for 10 min (for pulse-labeling) or 2 h. Conditioned medium was collected and cells were lysed in IP buffer. For immunoprecipitations we used a volume of conditioned medium that was normalized to the calculated trichloroacetic acid-precipitable radioactive counts (cpm) in cell lysates. Soluble APPalpha and Abeta 40/42 were immunoprecipitated with monoclonal antibody, 26D6 (37). To examine APP synthesis, cells were pulse-labeled with [35S]methionine for 10 min, and APP was immunoprecipitated with 369 antibody, raised against a peptide corresponding amino acids 649-695 of APP695 (41). Immunoprecipitates were fractionated by SDS-PAGE, and radioactive bands were visualized and quantified using a PhosphorImager (Amersham Biosciences).

Mass Spectrometric Analysis-- Conditioned media from N2a swe.10 cell pools stably expressing wild-type PS1 or the PS1Delta E9 were immunoprecipitated with 4G8 antibody, specific for amino acids 17-24 of Abeta , and collected with Protein A/G-coupled agarose beads prior to analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis, as described (42). Each mass spectrum was averaged from at least 500 measurements, and bovine insulin was included as an internal mass calibrant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of FAD-linked PS1 Variants on Production of Abeta Variants and Inhibition with a gamma -Secretase Inhibitor-- It is now well accepted that expression of FAD-linked PS1 variants elevate the levels of secreted Abeta 1-42 peptides and, in so doing, increase the calculated ratio of Abeta 40/Abeta 42 peptides. However, the absolute levels of Abeta peptide variants have rarely been reported, a reflection, in large part, of the variability in transgene-encoded APP between individual lines. With this caveat, we chose to transfect a neuroblastoma N2a cell line that constitutively expresses a carboxyl-terminal Myc epitope-tagged human APP 695 harboring the FAD-linked "Swedish" mutations (38) with human wild-type PS1 (wtPS1) or the FAD-linked PS1 variants, PS1Delta E9, or E280A to generate stable pools that express human PS1 polypeptides. Western blot analysis of stable cell pools revealed the accumulation of human PS1 NTF and low levels of full-length precursor in cells expressing wild-type PS1 (Fig. 1A, lane 1), uncleaved ~43-kDa PS1Delta E9, and low levels of endogenous mouse PS1 NTF in cells expressing PS1Delta E9 (Fig. 1A, lane 2), and mutant human PS1 NTF and low levels of full-length precursor in cells expressing the E280A variant (Fig. 1A, lane 3). In these cell pools, "replacement" of the bulk of murine PS1 fragments has occurred (45), although residual levels of murine NTF (as seen in the PS1Delta E9 cells) are still present. This would be expected in a cell pool in which transgene-derived products are expressed at varying levels in independent clones. We examined the levels of secreted Abeta -related species by immunoprecipitation with antibody 26D6 (39), specific for Abeta residues 1-12, fractionation of immune complexes on Bicine/urea gels, and analysis of immunoprecipitated Abeta peptides using 26D6 antibody and enhanced chemiluminescence detection. For these studies, we calculated the protein concentration in each plate of cells, and used normalized volumes of medium so that there would be no experimental bias because of differences in cell density. In Fig. 1, we show that constitutive expression of wild-type PS1 leads to robust secretion of Abeta 1-40 peptides, limited levels of secreted Abeta 1-37, Abeta 1-38, and Abeta 1-39 peptides and nearly undetectable levels of Abeta 1-42 peptides (Fig. 1B, lane 1). This level of secreted Abeta peptides is no higher than parental APPswe.10 cells (data not shown). On the other hand, we consistently observed that the levels of accumulated Abeta 1-40 and Abeta 1-42 peptides were elevated in medium of cells expressing either the PS1Delta E9 or A280E variants (Fig. 1B, lanes 3 and 5, respectively). Even more surprising was the observation that under conditions in which treatment of wtPS1 cells with a potent gamma -secretase inhibitor, L-685,458 (17) (2 µM for 16 h), resulted in nearly complete inhibition of secreted Abeta peptides (Fig. 1B, lane 2), low levels of Abeta 1-40 and Abeta 1-42 peptides still remained in the medium of cells expressing either PS1 mutant (Fig. 1B, lanes 4 and 6).


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Fig. 1.   Effects of FAD-linked PS1 variants on production of secreted Abeta peptides. A, expression of PS1 in N2a swe.10 pooled cells that stably express wild-type PS1 (lane 1), FAD-linked PS1Delta E9 (lane 2), or E280A (lane 3) were analyzed by immunoblotting with PS1NT antiserum. B, N2a swe.10 pooled cells that express wild-type PS1 (lanes 1 and 2), FAD-linked PS1Delta E9 (lanes 3 and 4), or E280A (lanes 4 and 5) were incubated for 16 h in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of gamma -secretase inhibitor, L-685,458 (2 µM). Conditioned medium was immunoprecipitated with 26D6 and immune complexes were subjected to electrophoresis in Bicine/urea gels, followed by immunoblotting with 26D6; Abeta 1-42 exhibits more rapid migration than Abeta 1-40 in this gel system (39). Note that secreted levels of both Abeta 1-40/1-42 peptides appears elevated in cells expressing either the PS1Delta E9 (lane 3) or A280E (lane 5) variants. Treatment with the gamma -secretase inhibitor resulted in marked inhibition of secreted Abeta peptide production in cells expressing wild-type PS1 (lane 2), whereas there was a considerable level of Abeta peptides, most notably Abeta 1-42 species, still remaining in medium of cells expressing the PS1Delta E9 (lane 4) or E280A (lane 6) variants. C, APP synthesis was examined by 10-min pulse labeling with [35S]methionine followed by immunoprecipitation using 369 and phosphorimaging. Note that APP synthesis is indistinguishable between the pooled cells expressing either wild-type PS1 (lane 1) or PS1Delta E9 (lane 2). D, N2a swe.10 pooled cells expressing wild-type PS1 (lanes 1 and 2) and the PS1Delta E9 (lanes 3 and 4) were metabolically labeled with [35S]methionine for 2 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of the gamma -secretase inhibitor. Conditioned medium was immunoprecipitated with 26D6 and separated by Tris-Tricine SDS-PAGE. APPsalpha generated by alpha -secretase and Abeta 1-40/Abeta 1-42 peptides are indicated. Note that the level of total secreted Abeta peptides in medium of cells expressing the PS1Delta E9 are elevated relative to the Abeta peptides in wild-type PS1 cells. E, to examine the identity of secreted Abeta species, Abeta radiolabeled from cells expressing wild-type PS1 (lanes 1 and 2) or the FAD-linked PS1Delta E9 (lanes 3 and 4) was subjected to immunoprecipitation with 26D6, and immune complexes were fractionated on Bicine/urea gels. Note that dramatic effects of the inhibitor are on the Abeta 1-40 species, with relative sparing of Abeta 1-42 species (lane 4). F, the absolute PhosphorImager units of the bands corresponding to Abeta 1-40 (left panel) and Abeta 1-42 (middle panel) were determined by phosphorimaging, and the ratio of counts/min in Abeta 42 to Abeta 40 was plotted (right panel). The asterisk in the middle and right panels reflects the undetectable levels of Abeta 42 in medium of wtPS1 cells in the presence of the inhibitor.

Our finding that two FAD-linked PS1 variants enhance secretion of the principal Abeta variant, Abeta 1-40, is novel and we felt it important to fully validate this finding. We chose to focus on the PS1Delta E9 pool. First, to establish that the differences in accumulated Abeta peptides between wtPS1 and PS1Delta E9 cell pools was not simply a reflection of differences in synthetic levels of transgene-encoded APPswe, we pulse-labeled cells with [35S]methionine for 10 min and analyzed newly synthesized APP in cell lysates by subjecting equivalent detergent-soluble, trichloroacetic acid-precipitable, radioactivity (cpm) to immunoprecipitation with antibody 369, raised against a peptide corresponding to amino acids 649-695 of APP (43), fractionation of immune complexes on SDS-PAGE, and phosphorimaging. In Fig. 1C, we show that the synthesis of full-length APP is indistinguishable between the cell pools that express either human wtPS1 or PS1Delta E9. To further quantify the absolute increase in both Abeta 1-40 and Abeta 1-42 peptides in medium of mutant PS1-expressing cells relative to cells expressing wtPS1, we incubated cell pools with [35S]methionine for 2 h and quantified the levels of secreted Abeta peptides in medium by immunoprecipitation with antibody 26D6, fractionation of immune complexes on Tris-Tricine gels, and phosphorimaging. For these analyses, we quantified total counts/min in detergent-solubilized cell lysates and used normalized volumes of radiolabeled conditioned medium for immunoprecipitations. As we have shown by Western blot analysis (Fig. 1B), quantitative phosphorimaging analysis revealed an elevation in total Abeta peptides in medium of cells expressing the PS1Delta E9 mutant (Fig. 1D, lane 3) by 2.8-fold relative to Abeta peptides secreted from cells expressing wtPS1 (Fig. 1D, lane 1). Notably, the 26D6 antibody, specific for Abeta residues 1-12, also detects soluble derivatives generated by alpha -secretase, termed APPsa, quantitative phosphorimaging revealed a 1.5-fold increase in levels of APPsa in medium of PS1Delta E9 cells relative to cells expressing wild-type PS1 (Fig. 1D, compare lanes 3 and 1, respectively), this despite identical synthetic rates of the APPswe precursor between cell pools (Fig. 1C). These findings offer the suggestion that expression of the PS1Delta E9 variant leads to enhanced trafficking (or processing) of full-length APPswe to cellular compartments in which alpha -secretase is active, but further studies will be necessary to address this issue. In any event, quantitative phosphorimaging revealed that while treatment of wtPS1 cells with 2 µM inhibitor reduced production of newly synthesized Abeta peptides to ~0.4% of untreated controls (Fig. 1D, lane 2), the inhibitor diminished the levels of total Abeta peptide species in medium of PS1Delta E9 cells to ~12% of the untreated control (Fig. 1D, lane 4). Further examination of the complexity of radiolabeled Abeta peptide variants by Bicine/urea gels (Fig. 1E) revealed that the absolute levels of both Abeta 40 and Abeta 42 variants were elevated in medium of PS1Delta E9 cell medium (Fig. 1E, lane 3; quantified in Fig. 1F, left panel) relative to the levels in medium of cells expressing wild-type PS1 (Fig. 1E, lane 1; quantified in Fig. 1F, left panel). Furthermore, under conditions where the gamma -secretase inhibitor almost completely eliminated Abeta 1-40 species in medium of cells expressing wild-type PS1, this compound diminished Abeta 1-40 peptide levels to ~13% in medium of cells expressing PS1Delta E9 (Fig. 1E; quantified in Fig. 1F, left panel). Similarly, the gamma -secretase inhibitor fully eliminates secreted Abeta 1-42 peptides from cells expressing wild-type PS1, but L-685,458 treatment only reduced the level of Abeta 1-42 peptides to ~58% in medium of cells that express PS1Delta E9 (Fig. 1E, lane 4; quantified in Fig. 1F, middle panel). Notably, the levels of Abeta 1-40 and Abeta 1-42 peptides are nearly identical in PS1Delta E9 cell medium after treatment with L-685,458 (Fig. 1F, right panel).

Dose Dependence of gamma -Secretase Inhibitor on Secretion of Abeta Peptide Variants-- Having established that treatment with 2 µM L-685,458 for 16 h completely eliminates Abeta peptides production from cells expressing wtPS1 cells, but fails to fully inhibit production of secreted Abeta peptide from cells expressing PS1Delta E9 cells, we asked whether the individual Abeta peptide variants may be differentially sensitive to the dose of gamma -secretase inhibitor. We treated parallel dishes of N2a swe.10 cell pools expressing wild-type PS1 or the PS1Delta E9 variant with increasing concentrations of L-685,458 for 16 h. The conditioned medium was collected and the protein concentration in cell lysates was determined. Normalized volumes of medium, relative to protein concentration in cell lysates, were subjected to immunoprecipitation and Western blot analysis. Consistent with the data shown in Fig. 1, the constitutive levels of Abeta 1-40 and Abeta 1-42 peptides in medium of cells expressing the PS1Delta E9 variant are elevated compared with cells expressing wild-type PS1 (Fig. 2A, compare lanes 6 and 1, respectively). At 2 µM, L-685,458 lowered the levels of secreted Abeta 1-40 peptides in medium of wtPS1 cells to ~20% of the levels in vehicle-treated cells (Fig. 2A, lane 3). However, at this concentration of inhibitor, the levels of Abeta 1-40 peptides in medium of cells expressing PS1Delta E9 were reduced to ~75% of the levels in vehicle-treated cells (Fig. 2A, lane 8). Notably, treatment with 8 µM inhibitor virtually eliminates all Abeta peptides in medium of cells expressing wild-type PS1 (Fig. 2A, lane 5), but this concentration of inhibitor diminished the levels of Abeta 1-40 peptides in medium of PS1Delta E9 cells to ~20% of the level in vehicle-treated cells (Fig. 2A, lane 10).


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Fig. 2.   Effects of FAD-linked Delta E9 on inhibition by a gamma -secretase inhibitor. A, N2a swe.10 cells stably expressing wild-type PS1 (lanes 1-5) or the PS1Delta E9 (lanes 6-10) were incubated for 16 h in serum-free medium containing the indicated concentrations of gamma -secretase inhibitor, L-685,458. Note that production of secreted Abeta in cells expressing wild-type PS1 was largely inhibited by treatment between 2 and 4 µM (lanes 3 and 4), whereas a significant amount of Abeta peptides was detected at these concentrations of the inhibitor in cells expressing PS1Delta E9 (lanes 8 and 9). Notably, treatment of the inhibitor at 8 µM fails to diminish the level of Abeta peptides in medium of the PS1Delta E9 cells (lane 10), whereas treatment at this concentration of the inhibitor virtually eliminates Abeta peptides in wild-type PS1 cells (lane 5). B, the band intensity corresponding to Abeta 1-40 (open circle) and Abeta 1-42 (closed circle) were quantified using densitometry (Molecular Dynamics) and plotted. C, accumulation of APP-CTFs in detergent lysates prepared from cells expressing wild-type PS1 (lanes 1-5) or the PS1Delta E9 (lanes 6-10) were analyzed by immunoblotting with CT15 antibody. Note that both endogenous APP-CTFalpha and APPswe-derived alpha - and beta -CTFs accumulate even at 1 µM inhibitor in cells expressing wild-type PS1 (lane 2), and that the level of these species does not change as the concentration of the inhibitor is increased (lanes 3-5). Treatment of the inhibitor at 1 µM resulted in lower levels of accumulated APP-CTFs (lane 7) in cells expressing the PS1Delta E9 compared with those observed at the same concentration in wild-type PS1-expressing cells.

Densitometric quantification of the data in Fig. 2A reveals that the production of Abeta 1-40 and Abeta 1-42 in cells expressing wild-type PS1 are equally sensitive to the gamma -secretase inhibitor, with an IC50 of ~1.5 µM (Fig. 2B, left panel). On the other hand, the production of Abeta 1-40 is inhibited with an IC50 of over 4 µM in cells expressing PS1Delta E9 (Fig. 2B, right panel). Moreover, in medium of PS1Delta E9 cells, 50% inhibition of Abeta 1-42 peptides occurs with 2 µM inhibitor (also apparent by immunoprecipitation of radiolabeled Abeta 1-42 peptides; Fig. 1, E and F), but at higher concentrations of compound, there is a paradoxical plateau in levels of these peptides. However, MALDI-TOF analysis (see below, Fig. 3) of the peptides generated after treatment of PS1Delta E9 cells with high concentrations of compound reveal that Abeta 1-43 peptides now become prominent. We do not know where Abeta 1-43 peptides migrate in Bicine/urea gels, but it is likely that these overlap with the Abeta 1-42 species and hence, we argue that the Western blot signals in lanes 9 and 10 of Fig. 2A represent a combination of Abeta 1-42 and Abeta 1-43 variants.


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Fig. 3.   Mass spectrometric analysis of secreted Abeta . N2a swe.10 pooled cells stably expressing wild-type PS1 (panels A-D) or the PS1Delta E9 (panels E-H) were incubated for 16 h in serum-free medium containing Me2SO alone (panels A and E), 1 µM (panels B and F), 4 µM (panels C and G), or 8 µM (panels D and H) L-685,458. The experimental mass and peptide lengths for several Abeta peptide variants are shown in panels A and E. Peaks that cannot be identified as Abeta -related are designated as "n."

In parallel to the examination of secreted Abeta peptides, we examined the accumulation of APP-CTFs in detergent-solubilized lysates from cells treated with increasing concentrations of the gamma -inhibitor. In cells that express wild-type PS1, both endogenous alpha -CTF, and APPswe-derived alpha - and beta -CTFs accumulate even after treatment with 1 µM inhibitor (Fig. 2C, lane 2), and the level of these fragments seems not to increase as a function of inhibitor concentration (Fig. 2C, lanes 3-5). In contrast, treatment of PS1Delta E9 cells with 1 µM inhibitor leads to the accumulation of APP-CTFs (Fig. 2C, lane 7), but at considerably lower levels than that observed in lysates of wtPS1 cells treated with the same concentration of inhibitor. Indeed, the most robust increase in accumulated APP-CTFs in PS1Delta E9-expressing cells occurs after treatment with inhibitor at 4 and 8 µM (Fig. 2C, lanes 9 and 10, respectively), concentrations that also appear to have the most pronounced effect on Abeta production (Fig. 2A, lanes 9 and 10).

MALDI-TOF Analysis of Abeta Variants in Medium of Cells Expressing Human Wild-type PS1 or PS1Delta E9 following Treatment with a gamma -Secretase Inhibitor-- To characterize the Abeta -related species that accumulate in medium of cells expressing human wild-type PS1 and PS1Delta E9 cells, and in medium of these cell pools treated with differing concentrations of the inhibitor, we used antibody 4G8, specific for epitopes 17-24 of Abeta , to immunoprecipitate Abeta -related peptides from several of the samples shown in Fig. 2; resulting immune complexes were analyzed by MALDI-TOF mass spectrometry (42). In Fig. 3, panel A, we show that in medium of wild-type PS1 cells treated with vehicle (Me2SO), Abeta 1-40 (Mr 4330) is the prominent species, with minor species of 1-34, 1-37, 1-38, and 1-39 also present. Upon treatment of these cells with 1 µM L-685,458, Abeta 1-40 and the minor species are still present, and a small peak, representing Abeta 1-42 (Mr 4514) also appears (Fig. 3, panel B); low concentrations of gamma -secretase inhibitors have been shown to have a paradoxical effect on elevating Abeta 1-42 peptides (44). However, the levels of all peptides are dramatically reduced after treatment at 4 µM (Fig. 3, panel C), and virtually nonexistent after treatment with 8 µM L-685,458 (Fig. 3, panel D). In medium of untreated PS1Delta E9 cells, we observed the presence of both Abeta 1-40 and Abeta 1-42 peptides and trace levels of Abeta 1-38, Abeta 1-39, and Abeta 1-43 peptides (Fig. 3, panel E). After treatment with 1 µM L-685,458, we failed to see an appreciable difference in the levels of any of the aforementioned Abeta species (Fig. 3, panel F). However, and in sharp contrast to our observations of Abeta peptides in wtPS1 cell medium, treatment of PS1Delta E9 cells with 4 µM L-685,458 lead to a reduction in levels of Abeta 40 peptides, with no appreciable change in the levels of Abeta 1-42 and Abeta 1-43 species (Fig. 3, panel G). Most interestingly, we now observe a pronounced elevation in levels of Abeta 1-43 peptides. This latter result becomes much more apparent in PS1Delta E9 cells treated with 8 µM L-685,458, where the levels of Abeta 1-43 peptide now exceeds the levels of Abeta 1-42 peptides (Fig. 3, panel H); significant levels of the Abeta 1-40 peptide are still apparent under these conditions. Furthermore, and in sharp contrast to cells expressing wild-type PS1 (Fig. 3, panels B-D), the levels of Abeta 1-34, Abeta 1-37, Abeta 1-38, and Abeta 1-29 variants in medium of PS1Delta E9 cells appear unchanged no matter what concentration of inhibitor is employed.

It should be noted that while the IP-MS paradigm is useful for the identification of Abeta peptides, the technique only serves a qualitative tool for assessment of peptide levels. For example, the levels of Abeta 1-40 peptides in lanes 7 and 9 of Fig. 2 with the analogous samples analyzed by IP-MS in Fig. 3, panels F and G, respectively, are not comparable. It is highly likely that under the conditions used in the IP-MS experiment, the antibody is not in excess, and hence, the antibodies are fully saturated with captured Abeta -related peptides. Moreover, it is our experience, and those of others, that unusual biophysical properties (including aggregation) of Abeta 1-42 peptides greatly hinder desorption of these species from the matrix and thus, the peak heights observed in MALDI-TOF analysis do not accurately reflect steady-state levels. This artifact is likely exaggerated for Abeta 1-43 peptides. Hence, the signals observed in Western blot analysis of fractionated Abeta peptides on Bicine/urea gels provide a more accurate reflection of the steady-state levels of Abeta species in the medium.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Taken together with the finding that intramembranous gamma -secretase processing of APP and Notch1 is abolished in cells with genetic ablations of PS1 and PS2 (12, 13), and the demonstration that high affinity gamma -secretase inhibitors specifically bind to PS (14, 18), it has been concluded that PS are the elusive gamma -secretases (20). However, several lines of evidence have emerged that have questioned the veracity of the PS are gamma -secretase model. First, intramembranous processing of APP and Notch1 can be discriminated by a JLK family of nonpeptidic inhibitors (45); the JLK inhibitors block Abeta peptide production, but have very little, if any, effect on production of the Notch1 derivative, S3/NICD (45). Second, expression of the FAD-linked L166P PS1 variant (32) or the experimental L286E or L286R PS1 mutants (34) leads to exaggerated overproduction of Abeta 42 peptides, but these PS1 variants fail to generate S3/NICD (32, 34). Supporting these studies, St. George-Hyslop and colleagues (33) recently reported that expression of the L392V, G206A, or Delta E9 variants results in compromised S3/NICD production and "epsilon " cleavage within the APP transmembrane domain. Thus, it would appear that while the production of Abeta peptides, cleavage at the Notch S3 and APP epsilon  sites are presenilin-dependent, the catalytic activities may be distinct (33). Finally, recent studies have shown that the generation of intracellular Abeta 1-42 peptides is PS-independent, suggesting that intramembranous processing of APP may be mediated by distinct gamma -secretase activities (46).

In an attempt to clarify the effects of FAD-linked mutant PS1 on the generation of Abeta peptides, we generated pools of stable cell lines expressing human PS1 or several independent FAD-linked PS1 variants and analyzed accumulated Abeta peptide variants in the medium of these cells. In addition, we assessed the effects of a potent and highly selective transition state inhibitor of gamma -secretase on FAD-linked PS1-mediated production of Abeta peptides.

In this report, we offer several novel insights relevant to PS-dependent gamma -secretase processing of APP. First, we provide the first demonstration that in the conditioned medium of pools of stable cell lines that express individual FAD-linked mutant PS1, both Abeta 1-40 and 1-42 peptides accumulate to higher levels than the Abeta peptide variants in medium of cell pools that express wild-type PS1. Despite years of investigation describing the effects of FAD-linked PS1 on elevation in the relative ratio of Abeta 42:Abeta 40 peptides, it comes as a surprise that the absolute levels of secreted Abeta species have not been compared in a systematic fashion. In large part, we suspect that in the analysis of individual stable cell lines, the levels of coexpressed human APP are highly variable, whether the APP transgene is coselected, or when the PS1 transgenes are stably expressed in a "parental" cell line that constitutively expresses a human APP transgene. In the present study, we utilize cell pools to "normalize" the level of APP expression across the entire population of between 100 and 200 individual lines. The mechanism(s) involved in the exaggerated production of Abeta 1-40 peptides are not presently known, but may involve PS1Delta E9 enhancement of cleavage at the beta -secretase site of full-length APPswe. However, using radiolabeling studies, we have not observed any significant differences in the production, or accumulation of CTFbeta in cell pools expressing either wild-type PS1 or PS1Delta E9 (data not shown). Thus, we would offer the tentative conclusion that in addition to enhancing processing at sites within the APP transmembrane domain to generate Abeta 1-42, the PS1Delta E9 variant (and E280A variant) also enhances processing at the scissile bond between amino acids 636 and 637 (of APP695) to elevate production of Abeta 1-40 peptides. In support of this suggestion, we observe that in titration studies using L-685,458 (Fig. 2C), APP-CTFs only accumulate at higher concentrations of inhibitor in cells expressing PS1Delta E9. Thus, it would appear that PSDelta E9-mediated intramembranous processing of APP-CTFs is more efficient, thus enhancing production of Abeta 1-40 and 1-42 peptides, and that these reactions are less sensitive to concentrations of inhibitor that would otherwise block Abeta production in cells expressing wild-type PS1.

Second, we provide unequivocal evidence that the production of Abeta 1-42 peptides from cells expressing FAD-linked PS1Delta E9 and E280A mutations are largely insensitive to a potent gamma -secretase inhibitor, L-685,458, at concentrations that would otherwise inhibit Abeta production from cells that express wild-type PS1. In addition, the FAD-linked PS1-dependent production of Abeta 1-40 is somewhat refractory to inhibition by this compound. Similar results have been obtained using a structurally unrelated gamma -secretase inhibitor, compound E (47). Of significant interest is our finding that while the production of Abeta 1-42 peptides are largely resistant to inhibition by L-685,458, the production of Abeta 1-43 peptides by cells expressing PS1Delta E9 are elevated in parallel with increasing concentrations of L-685,458. The molecular mechanisms underlying the curious observation that Abeta 1-42 peptide production is highly refractory to potent gamma -secretase inhibitors is perplexing. Earlier studies showed that a peptidomimetic inhibitor, termed compound 1, could block production of both Abeta 40 and 42 peptides with an IC50 of ~16 µM in Chinese hamster ovary cell lines that stably express human wild-type PS1, and that the IC50 was slightly increased (to ~22 and ~20 µM for inhibition of total Abeta and Abeta 42, respectively) in cell lines expressing the FAD-linked M146L PS1 variant (47). The authors argue that PS1 contains the active site of gamma -secretase and that FAD-linked mutations induce subtle changes in PS1 conformation within the proteolytic complex, thus requiring a higher concentration of inhibitor to block Abeta production. In contrast to these earlier studies, we now show that at the IC50 for inhibition of Abeta 1-40 production from cells expressing wild-type PS1 (~1.5 µM), L-685,458 only reduces Abeta 1-40 levels by ~25% from cells expressing PS1Delta E9. Moreover, the production of Abeta 1-42 peptide production from cells expressing PS1Delta E9 is highly resistant to the inhibitor, even at the highest concentrations of compound tested. At a mechanistic level, a series of recent elegant enzymological studies of solubilized gamma -secretase activity that employed a variety of aspartyl protease transition state analogs (including L-685,458) and nontransition state analogs have led to the conclusion that these compounds act in a noncompetitive fashion (48). The model proposed is that the substrate binding site is distinct from the active site, but once "docked," the substrate is subsequently displaced to the catalytic site of the enzyme (48). The model that PS1 harbors the active site of gamma -secretase, and that FAD mutants subtly alter PS1 conformation so as to alter the IC50 of gamma -secretase inhibitors (48) is tempting. However, it remains extremely perplexing that the FAD-linked PS missense mutations occur widely throughout the molecule, including in hydrophilic loop domains that are predicted to be quite remote from the lipid environment in which intramembranous cleavage takes place. Nevertheless, all of the FAD-linked PS variants have the unique property of enhancing cleavage at a single site in the APP transmembrane domain. It is difficult to reconcile this with a simple effect on an active site. Furthermore, expression of experimental PS1 variants harboring a P434A mutation in the PS1 carboxyl-terminal domain (49) or a deletion of the first two transmembrane domains (50), sequences that are remote from two transmembrane aspartate residues that are proposed to serve as the catalytic center (20), also reduces the production of Abeta peptides, arguing that multiple domains of PS1 are required for gamma -secretase activity (49, 50). Finally, the observation that in cells expressing FAD-linked mutant PS1, the production of Abeta 1-40 versus Abeta 1-42 peptides are differentially sensitive to L-685,458 (this study) makes it difficult to conceive of a model in which hydrolysis at the scissile bonds that generate the termini of Abeta 1-40 and Abeta 1-42 peptides could be mediated by PS, alone.

Finally, it is now firmly established that PS1 is present in a high molecular weight complex, and that molecules, termed nicastrin (25), APH-1alpha /APH-1beta (26), and PEN2 (27), are components of this complex. Moreover, nicastrin and PS levels are coregulated (51-53), and RNA interference (RNAi)-mediated reduction of nicastrin, APH-1, or PEN2 levels results in compromised secretion of Abeta 1-40 and Abeta 1-42 peptides (27). Hence, each component of the complex appears to exert differential effects of Abeta production. Hence, it is conceivable that the gamma -secretase inhibitors block Abeta production by influencing the interactions and biochemical properties of individual subunits within the complex. Future efforts will require the development of reconstitution systems to elucidate the biochemical interactions of PS with nicastrin, APH-1, and PEN2 and the effects of these components on modulation of PS- and FAD-linked PS-mediated gamma -secretase processing of APP-CTFs.

    ACKNOWLEDGEMENTS

We thank Drs. Mark S. Shearman and Yue-Ming Li, Merck Research Laboratories, for providing the aspartylprotease transition state analog, L-685,458.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AG021494 (to S. S. S.) and AG10491 (to R. W.).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.

§ Recipient of postdoctoral fellowships for research abroad from Japan Society for the Promotion of Science.

|| To whom correspondence should be addressed: Center for Molecular Neurobiology, The University of Chicago, Abbott 510, 947 E. 58th St., Chicago, IL 60637. Tel.: 773-834-9186; Fax: 773-834-5311; E-mail: ssisodia@drugs.bsd.uchicago.edu.

Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M209252200

    ABBREVIATIONS

The abbreviations used are: PS, presenilin; FAD, familial Alzheimer disease; APP, beta -amyloid precursor protein; Abeta , beta -amyloid; NEXT, Notch extracellular truncation; NICD, Notch intracellular domain; CTF, COOH-terminal fragment; IP, immunoprecipitation; NTF, NH2-terminal fragment; wt, wild-type; Bicine, N,N-bis(2-hydroxyethyl)glycine; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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