Partial Purification and Characterization of {gamma}-Secretase from Post-mortem Human Brain*

Mark R. Farmery {ddagger} §, Lars O. Tjernberg {ddagger}, Sharon E. Pursglove ¶ ||, Anna Bergman **, Bengt Winblad {ddagger} and Jan Näslund {ddagger}

From the {ddagger}Karolinska Institutet and Sumitomo Pharmaceuticals Alzheimer Center, Neurotec, Novum, Huddinge, SE-141 57 Sweden, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, SE-171 77 Sweden, and **Department of Neurotec, Karolinska Institutet, Section for Experimental Geriatrics, Novum, SE-141 86 Huddinge, Sweden

Received for publication, November 25, 2002 , and in revised form, April 11, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One characteristic feature of Alzheimer's disease is the deposition of amyloid {beta}-peptide (A{beta}) as amyloid plaques within specific regions of the human brain. A{beta} is derived from the amyloid {beta}-peptide precursor protein ({beta}-APP) by the intramembranous cleavage activity of {gamma}-secretase. Studies in cells have revealed that {gamma}-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of {gamma}-secretase is as yet to be fully elucidated, and no investigations have analyzed {gamma}-secretase in human brain. To address this we have developed a novel in vitro {gamma}-secretase activity assay using detergent-solubilized cell membranes and a {beta}-APP-derived fluorescent probe. We report that human brain-derived {gamma}-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived {gamma}-secretase are similar to those described in cells, and proteolysis occurs at the A{beta}40- and A{beta}42-generating cleavage sites. The ability to isolate {gamma}-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of {beta}-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The generation of amyloid {beta}-peptide (A{beta})1 by the sequential proteolytic processing of amyloid {beta}-peptide precursor protein ({beta}-APP) is central to the pathogenesis of Alzheimer's disease (AD) (1). {beta}-APP is initially cleaved by {alpha}- or {beta}-secretase to generate membrane-bound C-terminal fragments (C83 or C99, respectively). The C83 and C99 polypeptides are substrates for intramembranous cleavage by {gamma}-secretase, with A{beta} generated from the longer C99 molecule. A{beta} plaques typically develop within neocortex, hippocampus, and amygdala regions of the brain. The reason for this regional specificity in plaque deposition is unknown. Two major forms of A{beta} peptides are deposited in AD plaques, A{beta}40 and A{beta}42. The less abundant A{beta}42 peptide species is more prone to aggregate into fibrils and is deposited early in the disease process (2, 3). A{beta} becomes toxic upon polymerization, and transgenic mice overexpressing A{beta} develop AD-like lesions and show memory deficits. Also, genetic data implicate a role for A{beta} in the pathogenesis of AD (4). Therefore, an obvious therapeutic strategy for the treatment of AD is the use of agents that inhibit {gamma}-secretase activity, thus reducing A{beta} available for deposition.

The majority of early-onset familial AD cases are associated with mutations in the presenilin (PS) genes, and all PS mutations cause an increase in the production of the amyloidogenic A{beta}42 (5). PS plays a central role in {gamma}-secretase activity (for review, see Refs. 6 and 7), but the precise molecular nature of this function in Alzheimer's disease is not fully understood. In the absence of PS, {gamma}-secretase-mediated cleavage of {beta}-APP is fully abolished (8), and some studies suggest that PS is a novel aspartyl protease with an activity profile identical to {gamma}-secretase (913). Other studies implicate PS as an indirect regulator of {gamma}-secretase and hypothesized roles for PS in trafficking of the {beta}-APP substrate to the site of cleavage or orientation of substrate within the membrane (1416).

{gamma}-Secretase displays an evolutionarily conserved mechanism of regulated intramembrane proteolysis that is involved in the generation of signaling molecules from type I integral membrane proteins (17). As well as {beta}-APP, substrates for {gamma}-secretase-mediated regulated intramembrane proteolysis include Notch, the receptor tyrosine kinase ErbB4, and E-cadherin (1820). In cells, {gamma}-secretase activity is associated with a PS-dependent high molecular weight integral membrane multiprotein complex (2124). Nicastrin, a transmembrane glycoprotein, was recently identified by co-immunoprecipitation with PS as part of this complex and was shown to be involved in Notch signaling in Caenorhabditis elegans and A{beta} generation in human cells (25). The absence of nicastrin in Drosophila results in the destabilization and perturbation of both PS function and cellular localization (26, 27). The nicastrin ectodomain undergoes a major structural alteration during assembly with presenilin, and this alteration is necessary for {gamma}-secretase activity, suggesting that the nicastrin molecule is mechanistically central to the function of the {gamma}-secretase complex (28).

In addition to presenilin and nicastrin, the {gamma}-secretase complex also contains two recently identified transmembrane proteins, namely Aph-1 and Pen-2. These proteins were initially identified in genetic screens in C. elegans (29, 30). Aph-1 is required for surface localization of nicastrin (30), and Pen-2 is required for both the expression of PS and the maturation of nicastrin (31). Similarly, Aph-1 plays an intimate role in both the function and stabilization of the {gamma}-secretase complex (32). More recent data in Drosophila and mammalian cells suggests that nicastrin, Aph-1, and Pen-2 stabilize presenilin and increase the formation of presenilin fragments and {gamma}-secretase activity. It is proposed, therefore, that Aph-1 stabilizes the presenilin holoprotein within the complex and Pen-2 is required for endoproteolysis of presenilin and activation of {gamma}-secretase (33, 34). The components described above are sufficient and necessary for {gamma}-secretase activity (35), but given the intricacies of the {gamma}-secretase complex and the broad diversity of substrate- and cleavage-site specificity, it is possible that there are other functional or regulatory components of {gamma}-secretase-mediated regulated intramembrane proteolysis.

A first step in the characterization of {gamma}-secretase has been to identify conditions for the solubilization of the complex and the development of activity assays. Several approaches have been applied to solubilize the {gamma}-secretase complex. These have generally involved the use of membranes derived from cell lines, mild detergent treatments, and measurement of A{beta} formation by enzyme-linked immunosorbent assay or Western blotting (23, 24, 36, 37). However, these approaches may not fully reflect {gamma}-secretase-mediated processing of {beta}-APP that occurs in the human brain. It is possible that the {gamma}-secretase complex in brain contains brain- or neuronal-specific co-factors that modulate activity. The biochemical analysis of membrane-bound enzyme complexes from post-mortem brain is well established and has been applied to the investigation of enzyme activity in neurological disorders, including AD and schizophrenia (38, 39). An attractive novel approach, therefore, is the analysis of {gamma}-secretase in post-mortem non-AD and AD human brain isolates.

As an initial step toward this aim, we describe the development of a sensitive in vitro {gamma}-secretase assay based on a fluorogenic peptide probe. This assay system is dependent on the presence of PS, as described previously (8). We report the solubilization and characterization of {gamma}-secretase from human brain and show that {gamma}-secretase-mediated cleavage occurs at the putative {gamma}-secretase site(s) within the {beta}-APP amino acid sequence that results in the generation of A{beta}. This activity is reduced by compounds previously shown to inhibit {gamma}-secretase. In addition, we show that {gamma}-secretase activity in human brain co-localizes with a high molecular weight protein complex that comprises PS1, nicastrin, Aph-1, and Pen-2. These findings provide the basis for the further characterization of {gamma}-secretase and may facilitate the elucidation of essential factors in human brain involved in the pathobiology of AD.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The following antibodies were used: UD1 raised against the N-terminal residues ERVSNEEKLNL of Pen-2; Ab14 (Dr. Sam Gandy, Thomas Jefferson University, Philadelphia) and NT-1 (Dr. P. Mathews, Nathan Kline Institute, New York), raised against human PS1-NTF; PS1-CTF (MAB5232, Chemicon) raised against the C-terminal loop region of human PS1; nicastrin (N1660, Sigma) raised against C-terminal residues 693–709 of human nicastrin; H2D-2, raised against human Aph-1 (Dr. Gang Yu, University of Texas Southwestern Medical Center).

Presenilin 1 Stable Cell Lines—The PS-deficient blastocyst derived mouse embryonic stem cells, BD8 (40), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2.4 mM L-glutamine, 0.1 mM {beta}-mercaptoethanol, and nonessential amino acids. Site-specific recombination was achieved by mixing the gateway-compatible pCAG-IRESpuro vector (a gift from Dr. Stephen Wood and Poon-Yu Khut, Child Health Research Institute, Adelaide, Australia) and pEntr-human PS1 in the presence of LR clonase as per the manufacturer's instructions, creating the plasmid pCAG-PS1-IRESpuro. Transformants were selected on LB-ampicillin plates. BD8 cells were stably transfected with pCAG-PS1-IRESpuro and selected for 10 days in 1 µg/ml puromycin (Sigma). Surviving colonies were expanded and screened for PS1 overexpression by Western blotting.

Membrane Isolation from Cells and Human Brain—Human brain material (2 g of temporal cortex, non-AD, ~2 h post-mortem interval) was obtained from Huddinge Brain Bank (Huddinge, Sweden). Human embryonic kidney 293 cells and BD8 cells (~2 x 108 cells/experiment) were propagated under standard conditions. All procedures were carried out at 4 °C. Brain pieces were kept on ice, dissected to remove blood vessels and white matter, and resuspended in buffer A (1 ml/0.2 g) containing 20 mM Hepes, pH 7.5, 50 mM KCl, 2 mM EGTA, and CompleteTM protease inhibitor mixture (Roche Applied Science). Tissue was homogenized using a mechanical pestle-homogenizer (IKALaborteknik RW20; 1 500 rpm/25 strokes). Cells were harvested with a cell scraper, pelleted, and washed once in ice-cold phosphate-buffered saline. Cells were resuspended in 9 volumes of buffer A and homogenized using 15 strokes of a Dounce homogenizer with a B pestle. After homogenization, brain and cell lysates were processed under identical conditions. Lysates were centrifuged at 800 x g for 10 min to remove nuclei and large cell debris, and the pellet was rehomogenized as above. The resulting supernatants were pooled and centrifuged at 100,000 x g for 1 h. The membrane pellet was washed once in buffer A and recollected by centrifugation at 100,000 x g for 30 min. All centrifugations were carried out using a fixed-angle rotor. The membranes were resuspended in buffer A plus 10% glycerol, flash-frozen in liquid N2, and stored at –70 °C before use.

Solubilization of Membrane Preparations—Protein concentration in membrane preparations was determined using BCA reagents (Pierce). Membranes were resuspended (0.5 mg/ml) in 20 mM Hepes, pH 7.0, 150 mM KCl, 2 mM EGTA, 1% (w/v) CHAPSO (Calbiochem), and protease inhibitor mixture and solubilized at 4 °C for 1 h with end-over-end rotation. The solubilized membranes were centrifuged at 100,000 x g for 1 h, and the supernatants were collected.

{gamma}-Secretase Inhibitors—The highly specific {gamma}-secretase inhibitors L-685,458 (Bachem) and compound 1 (Dr. Mark Shearman, Merck Sharp and Dohme) and pepstatin A (Sigma), a more general aspartyl protease inhibitor, were dissolved in Me2SO, separated into aliquots, and stored at –70 °C until use. An inactive deshydroxy derivative of L-658,458 (synthesized by Dr. Alan Nadin, Merck Sharp and Dohme) was also used in this study.

Deglycosylation—CHAPSO-solubilized membrane protein samples (50 µg) containing protease inhibitor mixture (as above) were denatured by boiling for 10 min at 100 °C in the presence of 0.5% (v/v) SDS and 1% (v/v) {beta}-mercaptoethanol. After cooling on ice, samples were adjusted to 50 mM sodium citrate, pH 5.5. For Endo H treatment, 100 milliunits of Endo H (Roche Applied Science) was added. For PNGase F treatment, Nonidet P-40 was added to a final concentration of 1%, and this was followed by the addition of 15.4 milliunits of PNGase F (Roche Applied Science). Samples were incubated overnight at 37 °C, and reactions were stopped by the addition of 2x SDS-PAGE sample buffer. Glycosylation status was analyzed by SDS-PAGE/Western blotting and immunolabeling with nicastrin antibody.

Immunoprecipitation—CHAPSO-solubilized membrane fractions were diluted with immunoprecipitation buffer (20 mM Hepes, pH 7.0, 150 mM KCl, 2 mM EDTA, 2 mM EGTA, and 0.5% CHAPSO). Samples were pre-cleared by the addition of 20 µl of a 1:1 slurry of protein A/G-Sepharose (Amersham Biosciences) and end-over-end rotation at 4 °C for 30 min. After incubation, samples were subjected to centrifugation at 16,100 x g for 2 min, and the supernatant was removed to a fresh tube. Antibodies were added to each tube, and incubation was continued overnight at 4 °C with end-over-end rotation. Protein A/G-Sepharose was added, and incubation was continued for 1–2 h. The beads were then isolated and washed three times with immunoprecipitation buffer containing 0.25% (w/v) CHAPSO. SDS-PAGE sample buffer was added to the beads, and samples were subjected to SDS-PAGE as described below.

SDS-PAGE and Western Blot—Membrane preparations were boiled in Laemmli sample buffer and separated by SDS-PAGE (10–20% Tricine gels, Novex). After electrophoresis proteins were transferred to PVDF membranes (Bio-Rad) and probed with specific antibodies. Immune complexes were visualized by SuperSignal West Pico enhanced chemiluminescence reagents (Pierce). Hyperfilm ECL (Amersham Biosciences) was used for exposure, and films were scanned using an AGFA Duoscan. Figures were produced for publication with Photoshop (Adobe) or Canvas v.8 (Deneba Systems, Inc.) software.

{gamma}-Secretase-mediated Peptide Cleavage Assay—To measure {gamma}-secretase activity, solubilized membranes (treatments, samples, and protein concentrations are indicated in individual experiments) were incubated at 37 °C in 150 µl of assay buffer containing 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.25% CHAPSO (w/v). Peptide substrate (Fig. 1A) was synthesized and purified by high performance liquid chromatography by Peptide Inc., Osaka, Japan. Solubilized membrane preparations were incubated with 8 µM peptide at 37 °C overnight, unless otherwise specified. After incubation, reactions were centrifuged at 16,100 x g for 15 min and placed on ice. Supernatants were transferred to a 96-well plate (Nunclon, Nunc), and fluorescence was measured using a plate reader (Fluorstar Galaxy) with excitation wavelength at 355 nm and emission wavelength at 440 nm. Analysis and presentation of results was carried out using Microsoft Excel software.



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FIG. 1.
Development of a presenilin-dependent {gamma}-secretase assay. A, in vitro {gamma}-secretase activity was assayed using an intramolecularly quenched fluorogenic peptide probe. The structure and amino acid sequence of the probe showing the C-terminal {beta}-APP cleavage site (underlined) that generates A{beta}40 and A{beta}42 after the action of {gamma}-secretase is depicted. Dnp quenches the fluorescent molecule Nma until cleavage occurs. D-arginine (r-r-r) residues are included to increase solubility of the probe. B, membranes were prepared from PS null BD8 cells and BD8 cells overexpressing human PS1 and solubilized in assay buffer containing 1% CHAPSO. {gamma}-Secretase activity was measured by incubating solubilized membranes (10 µg of total protein) with the fluorescent peptide probe (8 µM) overnight at 37 °C in the absence or presence of L-685,458 (10 µM). Background fluorescence of the peptide probe was subtracted from all readings. The panel shows Western blot analysis of lysates from BD8 null or BD8-PS1 cells. Samples were separated on a 10–20% Tricine SDS-PAGE gel, transferred to a PVDF membrane, and probed with antibody directed against PS1-NTF (NT1). DMSO, Me2SO.

 

Liquid Chromatography-Mass Spectrometry—Samples from the {gamma}-secretase assay containing solubilized membranes and peptide probe were injected onto a PLRP-S 300-Å (150 x 2.1 mm) reverse phase column (Polymer Laboratories). Water with 0.2% formic acid and acetonitrile with 0.2% formic acid was used as the mobile phase. The samples were eluted with a gradient from 10 to 40% acetonitrile over 30 min. The column was coupled on-line to an electrospray ion-trap mass spectrometer (Agilent 1100 series, Agilent Technologies). As a control, samples were also prepared in the absence of membranes.

Blue Native (BN) PAGE—BN PAGE was performed by modification of methods described in Schülke et al. (41) and Brookes et al. (42). All buffers and solutions were pH 7.0 at 4 °C. Membranes (~0.5 mg of protein) were prepared as described above and resuspended in 100 µlof extraction buffer composed of aminocaproic acid (0.75 M) and BisTris (50 mM). 12.5 µl of n-dodecyl {beta}-D-maltoside (10% w/v, Calbiochem) was added to the suspension. After incubation on ice for 20 min with vortexing every 5 min, samples were cleared by centrifugation at 100,000 x g for 10 min, and protein concentration was quantified. To 100 µl of supernatant, 6.3 µl of a 5% suspension of Coomassie Brilliant Blue G-250 in aminocaproic acid (0.5 M) was added. Samples (30-µl aliquots containing ~50 µg of protein) were loaded onto a 4–12% BisTris NuPAGE gel (Invitrogen). Molecular weight standards (high molecular weight calibration kit, Amersham Biosciences) were resuspended in extraction buffer (200 µl per 250-µg vial) plus 25 µl of 10% n-dodecyl {beta}-D-maltoside and 12 µl of 5% Coomassie Blue, as above. Gel electrophoresis was performed overnight at 75 V using 50 mM Tricine, 15 mM BisTris, 0.02% Coomassie Blue G-250 as cathode buffer, and 50 mM BisTris as anode buffer. After electrophoresis, gels were equilibrated for 30 min in Western blot transfer buffer containing 0.05% (w/v) SDS. The samples were transferred to PVDF membrane as described above, except that transfer buffer contained 0.05% SDS.

Size Exclusion Chromatography—The solubilized membrane preparation (~300 µg of protein) was injected onto a Superose 6 HR 10/30 column (Amersham Biosciences). Solubilization buffer with 0.25% CHAPSO was used as mobile phase at a flow rate of 0.4 ml/min, and 0.4-ml fractions were collected and assayed for activity. The protein content of selected fractions was concentrated using StratacleanTM resin (Stratagene), according to the manufacturer's instructions, and subjected to Western blot analysis as described above.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of an in Vitro Presenilin-dependent {gamma}-Secretase Assay—We designed an intramolecularly quenched fluorescent peptide probe containing the C-terminal {beta}-APP amino acid sequence that is cleaved by {gamma}-secretase (Fig. 1A). Membranes were prepared from PS null BD8 cells or BD8 cells stably transfected with a human PS1 overexpression plasmid. {gamma}-Secretase-mediated cleavage of both {beta}-APP and Notch has been shown to be rescued in these cells by transfection of PS1 (8, 43). Recently published methods were used to solubilize a crude membrane fraction from both cell lines (23), and {gamma}-secretase mediated processing of the peptide probe was analyzed by measuring fluorescence after incubating solubilized membranes at 37 °C in the absence or presence of the transition state analogue {gamma}-secretase inhibitor L-685,458 (13) (Fig. 1B). Peptide cleavage occurs in the presence of PS1, and this activity is inhibited by ~45% in the presence of L-685,458. Background activity is observed in the absence of PS1, and possible explanations for this phenomenon are discussed later. A slight decrease in activity in the PS null background was also observed in the presence of L-685,458, and this may arise from the inhibition of a nonspecific protease due to the high molar excess of the inhibitor used in this experiment. These data suggested that this system could be used to measure PS-dependent {gamma}-secretase activity and encouraged us to carry out further investigations.

Solubilized Membranes Isolated from Post-mortem Human Brain Can Cleave the Peptide Probe—After the development of a sensitive in vitro assay we first sought to establish whether cleavage of the peptide probe was comparable with that seen using solubilized membranes from freshly isolated cultured human cells. Initially, cell membranes were prepared from human embryonic kidney 293 cells and temporal cortex to compare activities. After solubilization and quantification of protein content, samples with equal amounts of protein were incubated overnight with the peptide probe. The activities from the different membrane preparations were compared (Fig. 2A). Maximal activity was obtained from the human embryonic kidney 293 cell membranes, whereas {gamma}-secretase activity from brain membranes was ~20% lower. Activity increased in a time-(Fig. 2B) and protein concentration-dependent (Fig. 2C) manner. Control samples without membranes showed a slight increase in signal in the presence of peptide, probably due to hydrolysis of the probe (Fig. 2, A and B). The membrane fraction has some autofluorescence (Fig. 2A), and this signal is subtracted from subsequent readings.



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FIG. 2.
The peptide probe is cleaved in the presence of solubilized membranes derived from post-mortem human brain. Membrane preparations were incubated overnight at 37 °C in the presence of 8 µM fluorogenic peptide probe unless otherwise stated. A, {gamma}-Secretase activity was compared by assaying activity in solubilized membranes (50 µg of total protein) isolated from human embryonic kidney 293 cells or brain. As the control, solubilization buffer alone was included. B, solubilized membrane preparations (20 µg of total protein) from brain were incubated with the peptide probe, and activity was measured at the time points indicated. As a control, peptide alone (8 µM) in buffer was also incubated and assayed at the same time points. C, the effect of protein concentration on activity was investigated by incubating increasing concentrations of solubilized brain membrane preparation with the peptide probe. DMSO, Me2SO.

 

The Peptide Probe Is Cleaved at the A{beta}40- and A{beta}42-generating Sites—The peptide cleavage products generated during the incubation were analyzed by electrospray ionization mass spectrometry (shown in Table I). N- and C-terminal peptide cleavage products were observed corresponding to the A{beta}42 cleavage site (Nma-GGVVIA and TVK(Dnp)rrr-NH2), whereas only N-terminal products were detected corresponding to the A{beta}40 cleavage (Nma-GGVV). This suggests that a specific protease was active in the crude membrane fraction, generating cleavage products corresponding to both A{beta}40 and A{beta}42.


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TABLE I
Fragments detected by liquid chromatography/mass spectrometry after incubation of solubilized membranes with peptide probe

Solubilized membranes were incubated with peptide probe overnight at 37 °C. 50-µl samples were injected onto a reverse phase column and analyzed on line by mass spectrometry. ND, not detected. m/z, mass/charge of singly charged ion [M+H]+.

 

{gamma}-Secretase Is Active in Brain-derived Solubilized Membranes—The peptide probe is cleaved in the presence of the solubilized membrane fraction, and peptide fragments corresponding the C termini of A{beta} are generated. To verify the specificity of the isolated activity, the membrane fraction was treated with known {gamma}-secretase inhibitors and subjected to the {gamma}-secretase activity assay. Three independent inhibitors were used, L-685,458 (described above), pepstatin A (a potent inhibitor of aspartyl proteases (44, 45)), and compound 1 (prototype {gamma}-secretase inhibitor shown recently to promote the accumulation of C83/C99 and to potently inhibit A{beta}40 peptide production (46)). After overnight incubation in the presence of 10 µM L-685,458, activity was reduced by ~55%. Similarly, 100 µM pepstatin A reduced activity by ~70%. Treatment with 20 µM compound 1 resulted in ~80% reduction in activity (Fig. 3A). A dose-dependent inhibition of activity was seen in the presence of increasing concentrations of L-685,458 (Fig. 3B). To further examine the specificity of the observed {gamma}-secretase activity, an additional parallel control experiment was carried out using an inactive deshydroxy derivative of L-685,458. This compound did not inhibit {gamma}-secretase activity using concentrations up to 5 µM. The proteolysis of the peptide probe by proteinase K was not affected by treatment with L-685,458, pepstatin A, or compound 1 (not shown).



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FIG. 3.
Cleavage of the peptide probe can be inhibited by {gamma}-secretase inhibitors. A, solubilized membranes (10 µg) were incubated with peptide probe in the presence of either vehicle (0.1% Me2SO (DMSO)) or the {gamma}-secretase inhibitors L-685,458, compound 1, or pepstatin A overnight at 37 °C. B, the dose response to L-685,458 was investigated by incubating solubilized membranes (0.25 µg) in the presence of 8 µM peptide probe with increasing the concentrations of inhibitor or an inactive derivative for 2 h at 37 °C. In both A and B fluorescence after incubation in the presence of Me2SO was taken as 100% activity.

 

Known Components of {gamma}-Secretase Can Be Isolated from Human Brain as a Protein Complex—In human cells, {gamma}-secretase activity is dependent on several essential protein co-factors, including presenilin. PS1 N- and C-terminal fragments are generated by the endoproteolysis of PS1 holoprotein and remain heterodimerically associated at tightly regulated levels in the cell (for review, see Ref. 47). In addition to PS, the {gamma}-secretase complex also contains the recently identified components nicastrin, Aph-1, and Pen-2 (25, 32, 48). To characterize the molecular composition of the {gamma}-secretase complex in human brain, we performed co-immunoprecipitation studies. {gamma}-Secretase complexes were immunoprecipitated from CHAPSO-solubilized membranes using antibodies directed against PS1-NTF, PS1-CTF, nicastrin, and Aph-1. Nicastrin was found to co-immunoprecipitate with PS1-NTF and -CTF and Aph-1. PS1-NTF co-immunoprecipitated together with PS1-CTF, nicastrin, and Aph-1, and similarly, Pen-2 co-immunoprecipitated together with PS1-NTF, PS1-CTF, nicastrin, and Aph-1 (Fig. 4A). The specificity of interactions between PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 was verified by the absence of co-immunoprecipitation of these proteins with pre-immune serum (not shown) or the co-immunoprecipitation of the unrelated integral membrane protein calnexin with the {gamma}-secretase complex components (Fig. 4B). These results confirm that an intact protein complex comprising PS1 N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2 exists in human brain membranes.



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FIG. 4.
Co-immunoprecipitation of {gamma}-secretase complex components from the solubilized brain membrane fraction. A, protein complexes in the CHAPSO-soluble membrane fraction were isolated by co-immunoprecipitation, and the components were identified by Western blotting. In all experiments, samples were pre-cleared with protein A/G-Sepharose and incubated at 4 °C overnight with antibodies directed against the N-terminal fragment of PS1 (Ab14), the C-terminal fragment of PS1, nicastrin, and Aph-1. After immunoprecipitation by the addition of protein A/G-Sepharose, protein complexes were separated by SDS-PAGE using a 10–20% Tricine gel, blotted onto PVDF membrane, and probed with antibodies against nicastrin (upper panel), PS1 N-terminal fragment (NT1) (middle panel), and Pen-2 (lower panel). Fully mature nicastrin, PS1-NTF, immunoglobulin (IgG) light chain and Pen-2 are indicated with an arrow. The lane labeled Input represents ~2% of the total material included in each immunoprecipitation. B, solubilized membrane samples were prepared and subjected to co-immunoprecipitation (IP) as described above with antibodies directed against calnexin. After SDS-PAGE and immunoblot, membranes were probed with antibodies directed against calnexin, PS1 N-terminal fragment (NT1), and PS1 C-terminal fragment.

 

Mature Nicastrin Is Associated with the CHAPSO-solubilized Membrane Fraction—Nicastrin is a type I integral membrane glycoprotein (25, 49). We investigated the maturation status of nicastrin in the CHAPSO-solubilized membrane fraction by carrying out deglycosylation with Endo H and PNGase F. Western blotting of untreated, control membranes with antibodies against the C terminus of nicastrin revealed a predominant protein species of ~130 kDa (Fig. 5, lane 1), which is in agreement with the molecular weight of mature nicastrin seen in cells (49, 50). Treatment with Endo H increased the mobility of this fully mature glycosylated nicastrin species by ~20 kDa, resulting in the loss of the ~130-kDa species and formation of an ~110-kDa species (Fig. 5, lane 3). This limited sensitivity to Endo H suggests that the higher molecular weight nicastrin species comprises a mixture of both ER-derived high mannose residues and Golgi-derived complex oligosaccharides. Complex glycosylation of nicastrin was confirmed by treatment with PNGase F, which completely removes all oligosaccharide side chains. A single protein species of ~80 kDa, corresponding to the deglycosylated protein core of nicastrin, was generated by treatment with PNGase F (Fig. 5, lane 5). A less prominent, higher molecular weight band was also observed that also appeared to be partially Endo H-sensitive (Fig. 5, lane 3). This "hyper"-mature form of human brain-associated nicastrin does not appear to be related to {gamma}-secretase activity, as discussed later.



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FIG. 5.
Glycosylation status of nicastrin in the CHAPSO-soluble membrane fraction. Solubilized membranes were digested overnight with the indicated glycosidases. The mature form of nicastrin resolved as a ~130-kDa predominant band, indicated with a solid arrow. An Endo H-resistant immature species was revealed (lane 3), which migrated at ~110 kDa. PNGase F treatment resulted in complete deglycosylation and migration of nicastrin at a molecular mass corresponding to the unglycosylated core protein of ~80 kDa (lane 5). A high molecular weight nicastrin species was also observed (dashed arrow), which appeared to be partially Endo H-resistant (labeled with an asterisk). This species was also removed by PNGase F treatment.

 

{gamma}-Secretase in Human Brain Can Be Partially Purified as an Active High Molecular Weight Protein Complex—Several studies using a variety of biochemical techniques have shown previously that {gamma}-secretase activity/PS1 is associated with a protein complex ranging from ~100 to 2000 kDa (2123, 51). We investigated the molecular weight of PS1-dependent-{gamma}-secretase complex in the brain membrane fraction by subjecting samples to blue native gel electrophoresis and immunoblotting with antibodies specific to PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 (Fig. 6A). The same predominant immunoreactive band, with a molecular mass of ~500 kDa, was observed with all antibodies. In a parallel experiment, PAGE under denaturing conditions revealed the monomeric protein species of the known {gamma}-secretase complex components (Fig. 6B).



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FIG. 6.
Presenilin N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2 are resolved at the same high molecular weight by blue native PAGE. A, samples were solubilized in n-dodecyl {beta}-D-maltoside and subjected to blue native PAGE as described under "Experimental Procedures" using a 4–12% gradient gel. Separated proteins were blotted, and identical membrane strips were probed with antibodies against N-terminal fragment of PS1 (NT1), C-terminal fragment of PS1, nicastrin, Aph-1, and Pen-2. These {gamma}-secretase complex components resolved at the same molecular weight (indicated with an arrow). B, aliquots of the same samples were subjected to SDS-PAGE, immunoblotted, and probed with the same antibodies.

 

To verify that {gamma}-secretase activity was associated with a high molecular weight protein complex, we separated total brain CHAPSO-solubilized membrane extracts by size exclusion chromatography and measured {gamma}-secretase activity in the eluted fractions (Fig. 7A). Two distinct peak activities were observed, peak 1 (fractions 6–10) and peak 2 (fractions 29–35) (Fig. 7A), with apparent approximate molecular masses of >1000 kDa (peak 1) and <66 kDa (peak 2). When the eluted fractions were treated with 10 µM L-685,458 (Fig. 7A) or 10 µM compound 1 (not shown) only the activity in peak 1 was inhibited.



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FIG. 7.
{gamma}-Secretase activity co-purifies with a high molecular weight complex comprising presenilin N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Upper panel, the solubilized membrane preparation was injected onto a Superose 6 HR size exclusion column. Solubilization buffer with 0.25% CHAPSO was used as mobile phase at a flow rate of 0.4 ml/min, and 0.4-ml fractions were collected and assayed for activity or subjected to Western blot analysis. Peptide cleavage activity was measured in the absence or presence of L-685,458 (10 µM) after incubation overnight at 37 °C. Activity was observed in a high molecular weight fraction (peak 1) and in a second, lower molecular weight fraction (peak 2). Activity in the latter fraction was not inhibited by treatment with the {gamma}-secretase inhibitor L-685,485, suggesting that this activity is nonspecific. Lower panel, fractions corresponding to peaks 1 and 2 were concentrated and subjected to SDS-PAGE on a 10–20% Tricine gel, transferred to a PVDF membrane, and probed with antibodies directed against the N-terminal fragment of PS1 (NT1), C-terminal fragment of PS1, nicastrin, Aph-1, and Pen-2. DMSO, Me2SO.

 

The peak fractions were also analyzed by Western blot using PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 antibodies, which showed that the {gamma}-secretase complex components could only be detected together in peak 1 (Fig. 7B). This indicates that these proteins are associated as a high molecular weight complex with {gamma}-secretase activity. The CHAPSO-soluble membrane fraction does not contain unassembled forms of PS1 fragments, Aph-1, and Pen-2 because these were not detected in fractions corresponding to their monomeric molecular weights, supporting investigations in cells which suggest that assembly is required for stabilization of the individual members of the {gamma}-secretase complex (31, 32, 34).

Interestingly, the hyper-mature form of nicastrin that is observed in the crude solubilized membrane extract (Fig. 5) does not appear to be associated with the {gamma}-secretase complex (Fig. 7B). Instead, this form of the protein remains unassociated and monomeric (not shown). PS is required for the trafficking of nicastrin through the secretory pathway but not its complex glycosylation, and this association is also required for {gamma}-secretase activity (52). It is conceivable, therefore, that the hyper-mature form of nicastrin observed in human brain extracts belongs to a pool of unassembled nicastrin not directly involved in {gamma}-secretase activity. Other functions for nicastrin have been suggested, such as interacting with {beta}-site APP cleaving enzyme and activating {beta}-secretase (53), but the significance of this observation in human brain remains to be elucidated.

We conclude that active {gamma}-secretase can be isolated from post-mortem human brain. The {gamma}-secretase-/PS1-dependent nature of this activity is supported by the specificity and inhibition profile of the fluorogenic peptide cleavage assay and the detection of {gamma}-secretase complex components in the active peak after size exclusion chromatography. This is extended by BN PAGE data, which confirms that a high molecular weight complex in the crude solubilized membrane fraction contains PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2. The apparent molecular weight of the PS1-{gamma}-secretase complex in brain as resolved by BN PAGE and size exclusion chromatography are in agreement with those published previously. Using size exclusion chromatography, Li et al. (23) resolved a PS1 complex that associates with {gamma}-secretase activity, with a molecular mass of ~2000 kDa (23), whereas BN PAGE data by Steiner et al. (31) suggests a PS1 complex of ~500 kDa. The molecular characteristics of the {gamma}-secretase complex are also suggested by co-immunoprecipitation data, indicating that a complex comprising PS1-NTF and -CTF, nicastrin, Aph-1, and Pen-2 can be isolated. Our data support a functional link between this protein complex and {gamma}-secretase activity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Secretase mediates the generation of A{beta} and is central to the pathogenesis of Alzheimer's disease. In this study we report the development of an in vitro {gamma}-secretase assay and describe the partial purification and characterization of {gamma}-secretase from post-mortem human brain.

To develop an in vitro {gamma}-secretase assay we adapted a recently published method to solubilize membrane preparations from BD8 cells and BD8 cells transfected with PS1 (23) using the mild detergent CHAPSO. Incubation of solubilized membranes with a novel fluorogenic {beta}-APP-mimicking probe confirmed that {gamma}-secretase activity in mouse BD8 cells is PS1-dependent. The availability of endogenous substrates in cortical extracts from post-mortem human brain is highly variable.2 To control substrate availability, the fluorogenic peptide cleavage assay was developed and used to investigate {gamma}-secretase further in brain. Using this approach, activity is comparable with that seen in freshly isolated membranes obtained from cells grown in culture.

The {gamma}-secretase complex contains and is functionally dependent upon the N- and C-terminal fragments of PS1, the transmembrane glycoprotein nicastrin, and the recently identified transmembrane proteins Aph-1 and Pen-2. It has recently been shown that interactions between these proteins mediate {gamma}-secretase activity. Our results indicate that the solubilized crude membrane fraction contains PS1-NTF, PS1-CTF, mature nicastrin, Aph-1, and Pen-2 and that these proteins can be co-immunoprecipitated as a complex. The fact that we could detect only mature nicastrin in association with the detergent-soluble fraction supports recent data showing that maturation and endoglycosidase H-resistant glycosylation of nicastrin is required for interaction with PS1 (48). In addition, PS1-NTF, PS2-CTF, nicastrin, Aph-1, and Pen-2 in the solubilized human brain membrane fraction localize within an SDS-sensitive high molecular weight protein complex. {gamma}-Secretase activity is associated with the same high molecular weight complex, and this can be inhibited by treatment with the specific {gamma}-secretase inhibitor L-685,458, suggesting that the complex is functional.

How the C termini of A{beta}40 and A{beta}42 are generated is an area of great attention. It has been suggested that several distinct proteases acting at the C terminus of C99 could be responsible for the generation of different A{beta} species (54, 55). Other reports favor a single {gamma}-secretase as responsible for the generation of A{beta}40 and A{beta}42 (56). We observed a range of A{beta} cleavage products, most of them corresponding to A{beta}40- and A{beta}42-specific cleavage after incubation of the fluorogenic peptide with the total solubilized membrane fraction. Taken together with our observations that {gamma}-secretase activity could not be fully inhibited by treatment with L-685,458, compound 1, and pepstatin A using physiologically active concentration ranges suggests nonspecific or presenilin/{gamma}-secretase-independent proteolytic activity could account for part of the peptide cleavage and generation of heterogeneous fragments that occurs in our system in the presence of total solubilized membranes. This notion is supported by the fact that cleavage of the peptide probe in lower molecular weight size exclusion chromatography fractions occurs in the absence of PS1-NTF, peak 2 (Fig. 7) and that activity is almost completely inhibited in the isolated high molecular weight fraction where {gamma}-secretase is present. Further experimentation will determine whether or not this is related to the normal metabolism of C99, a hypothesis supported by a recent study that describes the generation of intracellular A{beta} in presenilin-deficient cells (57). Nevertheless, the fact that {gamma}-secretase activity can be inhibited by up to ~80% using three independent inhibitory compounds provides compelling evidence that the {gamma}-secretase complex is functional in solubilized membrane fractions from post-mortem human brain.

The biochemistry of {gamma}-secretase has been determined to a high degree of detail. However, experimentation has largely been restricted to the use of cell lines grown in tissue culture, and questions remain regarding the functional properties of the complex. Here we have shown that {gamma}-secretase is active in post-mortem human brain. These observations provide the basis for the further characterization of {gamma}-secretase such as the identification of other components of the complex and the elucidation of brain-specific or brain region-specific factors that may underlie pathogenic processes in Alzheimer's disease.


    FOOTNOTES
 
* This research was supported by Sumitomo Pharmaceuticals Co., Osaka 541-8510 Japan, and in part by Åke Wibergs stiftelse, Loo och Hans Ostermans stiftelse, and Gun och Bertil Stohnes stiftelse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Supported by a stipend from David och Astrid Hageléns stiftelse. Back

§ To whom correspondence should be addressed: Karolinska Institutet, Neurotec, Novum-KASPAC, SE-141 57 Huddinge, Sweden. Tel.: 46-8-58583625; Fax: 46-8-58583610; E-mail: mark.farmery{at}neurotec.ki.se.

1 The abbreviations used are: A{beta}, amyloid {beta}-peptide; {beta}-APP, A{beta} precursor protein; C83, C-terminal {beta}-APP stub generated by {alpha}-secretase cleavage; C99, C-terminal {beta}-APP stub generated by {beta}-secretase cleavage; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; Nma, N-methyl-o-aminobenzoic acid; Dnp, 2,4-dinitrophenyl; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; BN PAGE, blue native PAGE; Endo H, endo-{beta}-N-acetylglucosaminidase H; PNGase F, peptide N-glycosidase F; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; AD, Alzheimer's disease; Tricine, N-[2-hydroxy-1,1-/bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride. Back

2 M. Farmery, unpublished observations. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Sam Gandy, Dr. Paul Mathews, Dr. Gang Yu, Dr. Mark Shearman, Professor Ralf Pettersson, Dr. Stephen Wood, and Poon-Yu Khut for the gifts of reagents and antibodies. We are indebted to Dr. Nenad Bogdanovich for assistance in tissue preparation. We are also grateful to Dr. Dirk Beher for critical reading of the manuscript.



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 ABSTRACT
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