Presenilin-1, Nicastrin, Amyloid Precursor Protein, and {gamma}-Secretase Activity Are Co-localized in the Lysosomal Membrane*

Stephen H. Pasternak {ddagger} §, Richard D. Bagshaw § ¶, Marianne Guiral §, Sunqu Zhang §, Cameron A. Ackerley ||, Brian J. Pak **, John W. Callahan § {ddagger}{ddagger} and Don J. Mahuran § §§

From the §Research Institute, ||Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, {ddagger}Clinician Investigator Program, Department of Pathobiology and Laboratory Medicine, {ddagger}{ddagger}Department of Biochemistry, University of Toronto, Toronto M5G 1X8, Canada and **Ciphergen Biosystems Inc., Fremont, California 94555

Received for publication, April 16, 2003 , and in revised form, May 6, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer's disease (AD) is caused by the cerebral deposition of {beta}-amyloid (A{beta}), a 38–43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the {gamma}-secretase (a complex containing presenilin and nicastrin) to produce A{beta} occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the {gamma}-secretase proteins and a neutral pH optimum of in vitro {gamma}-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic {gamma}-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that {gamma}-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of A{beta} and suggest a role for lysosomes in the pathophysiology of AD.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer's disease (AD)1 is the leading cause of adult onset dementia. Most investigators believe that the production and cerebral deposition of amyloid plaques composed of the 38–43-amino acid {beta}-amyloid (A{beta}) peptide is central to the development of AD (the amyloid cascade hypothesis) (1). A{beta} peptides are generated by 2 sequential proteolytic cleavages from a large transmembrane protein called the amyloid precursor protein (APP). This occurs first at a "{beta}" site by the enzyme BACE ({beta}-site APP-cleaving enzyme, {beta}-secretase), which removes the large extracellular domain of APP and then at a slightly variable "{gamma}" position (by the {gamma}-secretase) within the transmembrane domain of APP (2).

Many lines of evidence support the hypothesis that {gamma}-secretase cleavage of APP, Notch, and other proteins requires a protein complex containing the presenilins (PS-1 and PS-2) and nicastrin (reviewed in Refs. 2 and 3) Mutations in the genes encoding PS-1 and PS-2 cause the most common forms of Familial AD by increasing A{beta}42 production (46). In addition, known aspartyl protease inhibitors that inhibit {gamma}-secretase activity also bind PS-1 (7, 8), and mutations of either of 2 critical aspartate residues (suggested as mediating aspartyl protease activity) in either PS-1 or PS-2 decrease {gamma}-secretase activity (9). Finally, cells cultured from PS-1/PS-2 knockout mice secrete no A{beta} (10). Recently, mAph1 and Pen2 have also been demonstrated to complex with presenilin and be required for {gamma}-secretase complex expression (1113).

Many earlier studies indicated that A{beta} is produced in the endosomal/lysosomal system (2). The strongest evidence for A{beta} generation in the endosomal/lysosomal system comes from experiments in which APP was labeled at the cell surface, with 125I, biotin, or an antibody, and followed as it was endocytosed, cleaved into A{beta}, and then either secreted or retained internally. This basic experiment has been repeated by multiple groups in a variety of cell types including cultured human neurons (1420). Additional evidence supporting the endosomal/lysosomal system in the production of A{beta} include the following. (a) Treatment with the protease inhibitors leupeptin, E64, or Z-Phe-Ala-CHN causes amyloidogenic APP fragments to accumulate within lysosomes (14, 21, 22). (b) {alpha}- and {beta}-cleaved APP fragments accumulate in lysosomes of cells from PS-1 knockout mice (which have markedly decreased {gamma}-secretase activity) (3, 23). (c) A{beta} secretion from cultured cells can be dramatically reduced by treatments that prevent endosomal/lysosomal acidification, e.g. ammonium chloride and bafilomycin A1 (2426). (d) Deletion of the internalization/endocytosis signal sequence, which for many proteins serve as a lysosomal targeting signal, from the C-terminal (cytoplasmic tail) of APP markedly reduces A{beta} production (15).

Other evidence suggesting a lysosomal/endosomal localization of {gamma}-secretase activity comes from the study of the cell surface receptor Notch, which has been suggested to undergo endocytosis and {gamma}-cleavage in a manner similar to APP (2729). The idea that the same {gamma}-secretase is responsible for both cleavages is supported by the fact that Notch processing is reduced in PS-1-deficient cells (30) and that Notch and APP appear to compete for PS-1 (31). Additionally, purified presenilin-containing complexes have recently been shown to cleave both APP and Notch (32).

The problem with this model of lysosomal/endosomal production of A{beta} is that numerous experiments appear to show PS1 and {gamma}-secretase activity localized to the ER and Golgi. For example, PS-1 is most easily observed in the ER using fluorescence microscopy (3335). Similarly, PS1 and APP processing fragments have been shown to co-localize with ER and Golgi markers in density gradient fractionation experiments (33, 3639). As a result, there is no consensus as to the subcellular compartment in which A{beta} is produced.

The conflict between the apparent ER localization of PSs and nicastrin and the endosomal/lysosomal site of A{beta} production is a serious problem in understanding the pathophysiology of AD, with some authors suggesting that it may represent a "spatial paradox" with the protease and its substrate occupying separate cellular compartments (3). Several recent studies have demonstrated the presence of PS-1·nicastrin complexes on the cell surface (4042). Although these studies show that PS-1 is not limited entirely to the ER, they do not totally resolve the spatial paradox, if one accepts the above data localizing A{beta} generation to the endosomal/lysosomal system. Additionally, if A{beta} generation is in the endosomal/lysosomal system, failure to co-localize nicastrin, PS-1, and APP processing to these compartments undermines the hypothesis that presenilin-containing complexes are the {gamma}-secretase. Numerous models have been suggested to reconcile these incongruities in intracellular localization. These include: (a) that {alpha}- or {beta}-cleaved APP (generated at the cell surface or in the endosomal compartment) must be recycled back to the ER or Golgi for additional processing, or (b) that PS-1 is not a component of the {gamma}-secretase and another enzyme remains to be discovered (23, 37, 4143).

In the present study, we offer a more straightforward solution to this problem. It was initiated by the identification of nicastrin, through de novo quadrupole time-of-flight (Q-TOF) mass spectrometry sequencing of tryptic peptides generated from a major protein spot excised from a two-dimensional-(IEF-SDS) gel of separated lysosomal membrane (LM) proteins from highly purified organelles. Furthermore, we demonstrated by protease protection that nicastrin was on the outer limiting membrane of the lysosome, i.e. not contained in an intralysosomal vesicle (44). We now show that mature nicastrin, PS-1, and APP are (a) components of the outer LM, and (b) significantly enriched in the LM as compared with other intracellular fractions. We confirm the co-localization of nicastrin and PS-1 with LAMP-1 (lysosomal associated membrane protein-1) on the limiting lysosome membrane by double immunogold labeling of ultrathin cryosections. Finally, we extend these observations by demonstrating that our LMs are enriched in an acid {gamma}-secretase activity that is precipitable with an anti-nicastrin antibody.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Antibodies used were: APP C-terminal (Sigma), nicastrin (Affinity Bioreagents), A{beta}, PS-1 (N-terminal), calnexin, LAMP-1 (Santa Cruz Biotechnology), and rab7 (CytoSignal). 6E10 was purchased as part of the "{beta}-Amyloid Multipeptide ProteinChip Kit" (Ciphergen).

Lysosome (Tritosome) Isolation Protocol—The isolation of tyloxapol-filled lysosomes has been described (44) and is summarized in Fig. 1. Briefly, Sprague-Dawley rats (Charles River) were given an intraperitoneal injection of the non-lytic detergent tyloxapol (Triton WR 1339; Sigma) 5 days prior to sacrifice. Rats are sacrificed and the livers were removed. Livers were then homogenized, and the nuclei were removed by centrifugation at 1000 x g for 10 min to yield a postnuclear supernatant (PNS). A crude organelle fraction was then collected by centrifugation at 34,000 x g for 15 min and resuspended in 45% sucrose. This solution was layered beneath a discontinuous gradient of 14.3% sucrose and 34.5% sucrose and centrifuged at 77,000 x g for 2 h. Tyloxapol-filled lysosomes were removed from the 14.3-34.5% interface, diluted, and pelleted at 28,000 x g for 30 min. Phenylmethylsulfonyl fluoride (0.1 mM) was added to all solutions. Enzyme assays for {beta}-hexosaminidase (45) and citrate synthase (46) were performed on each fraction.



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FIG. 1.
Isolation of tyloxapol-filled lysosomes. Homogenate is centrifuged to yield a postnuclear supernatant and a pellet (P1) containing nuclei and intact cells. The PNS is then fractionated by centrifugation to yield an organelle pellet (enriched in mitochondria, peroxisomes, and lysosomes) and a supernatant (S2) containing primarily cytosolic proteins, ER, and Golgi. Lysosomes are recovered from the 14.3–34.5% interface of the discontinuous sucrose gradient. The remainder of the gradient preparation was mixed to yield a remains of gradient fraction, which is normally discarded.

 

Lysosomal Subfractionation—Soluble (luminal) lysosomal proteins were isolated by resuspending the lysosomes in PBS, freeze-fracturing them in dry ice/ethanol, and removing membranes by centrifugation for 30 min at 355,000 x g at 4 C (2x). The membrane-associated proteins were solubilized by resuspending the pellet in 0.1 M NaCO3, pH 11.0, incubated on ice for 30 min, with membranes removed by centrifugation at 355,000 x g for 30 min. The remaining pellet was solubilized in 2% SDS and was designated the integral membrane protein fraction.

Western Blot Analysis—Western blots were performed as in described in Ref. 47. Analysis was performed on a Macintosh Powerbook G4 computer using the public domain NIH Image 1.63 program (developed at National Institutes of Health and available on the Internet).2

Routine Transmission Electron Microscopy Preparation—Tyloxapol-filled lysosomes in sucrose were brought to 2% paraformaldehyde, 0.5% glutaraldehyde, 0.05 M phosphate buffer, pH 7.2, incubated 1 h on ice, and pelleted in a benchtop microcentrifuge for 15 min. Pellets were fixed in 4% paraformaldehyde, 1% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4, for a minimum of 2 h prior to a thorough wash in 0.1 M Sorenson's phosphate buffer, pH 7.2. They were postfixed in phosphate-buffered 2% OsO4 for 1 h, rinsed in distilled water, dehydrated in an ascending series of ethanols, and infiltrated and embedded via propylene oxide in Epon araldite. Following polymerization, ultrathin sections were prepared with a diamond knife and an ultramicrotome and mounted on grids. Sections were then stained with saturated ethanolic uranyl acetate and lead citrate prior to examination and photography in the transmission electron microscopy (JEOL 1200 EXII, JEOL USA Inc., Peabody, MA).

Immunogold Labeling of Ultrathin Cryosections—Tyloxapol-filled lysosomes from the sucrose gradient were brought to 2% paraformaldehyde, 0.05% glutaraldehyde, 0.05 M phosphate buffer, pH 7.2, and incubated 1 h on ice. They were then pelleted in a benchtop microcentrifuge for 15 min and fixed in 4% paraformaldehyde, 0.1% glutaraldehyde, 0.1 M phosphate buffer, pH 7.2, for 4 h at room temperature. Samples were then embedded in 15% gelatin in PBS and minced into 1 mm3 cubes, infused with 2.3 M sucrose for several hours, mounted on aluminum pins, and frozen in liquid nitrogen. Ultrathin cryosections were then prepared at –95 °C on a diamond knife using a Leica Ultracut S CryoUltramicrotome (Leica Canada, Willowdale, Ontario, Canada). Sections were transferred to formvar-coated nickel grids using a loop of molten sucrose. After several rinses in PBS containing 0.15% glycine and 0.5% BSA and PBS with BSA alone, samples were incubated with antibodies against either PS-1 or nicastrin for 1 h at room temperature. The grids were then rinsed thoroughly in PBS/BSA prior to incubation in goat anti-rabbit IgG 5-nm gold complexes for 1 h (room temperature). After a thorough rinse in PBS/BSA samples were incubated for 1 h (room temperature) in an antibody against human LAMP-1 (moderate cross-reactivity toward rat LAMP-1). The grids were then washed in PBS followed by an additional incubation at room temperature in goat anti-mouse IgG 10-nm complex for 1 h. The specimens were then rinsed in PBS and extensively washed in distilled water. Grids were then stabilized in a thin film of methyl cellulose containing 0.2% uranyl acetate and examined in the transmission electron microscopy.

{gamma}-Secretase Assay Substrate—A DNA template encoding amino acids 671–770 of human APP was amplified by PCR. The forward primer included a T7 promoter, a ribosome binding site, and a Met added in front of position 1 (GGATTCTAATACGACTCACTATAGGGAACAGCCACCATGGATGCAGAATTCCGACATG). The reverse primer contained a poly-A tail (T29CTAGTTCTGCATCTGCTCAAA). This PCR product was used directly in in vitro transcription/translation kits (TNT T7 Quick for PCR DNA (Promega) or RTS100 (Roche Diagnostics)). To produce a radiolabeled probe, 10–20 µCi of [35S]methionine was added per reaction. Small molecules were removed by dialysis (48 h) against dH2O using 10,000 Mr cutoff microdialysis cups (Pierce).

In Vitro {gamma}-Secretase Assay—Membranes were prepared by freeze/thaw lysis of whole lysosomes (or other fractions) in PBS containing 0.5 M NaCl. After 30 min on ice, membranes were recovered by centrifugation (355,000 x g, 15 min). The pellets were resuspended in PBS and re-centrifuged. Each assay was performed with 10–20 µg of protein in 20 µl in a reaction containing 150 mM NaCl, 1 mM magnesium chloride, 1 mM calcium chloride, 1 mM zinc chloride, 0.25% CHAPSO, 2x Complete Protease Inhibitor (Roche Diagnostics), and 50 mM citrate phosphate buffer at the required pH.

Immunoprecipitation {gamma}-Secretase Assay—Washed lysosomal membranes (as above) were solubilized in 2% CHAPSO for 1 h on ice, and insoluble material was removed by centrifugation at 100,000 x g for 1 h. Solubilized proteins (40 µg) were incubated with BSA-blocked Gamma-Bind beads (Amersham Biosciences) alone, or with 10 µg of an anti-nicastrin antibody at pH 7.5 for 4 h at 4 °C. Beads were washed with PBS, 0.25% CHAPSO and incubated with radiolabeled C100 at pH 4.5 for 4 h as above. Surface-enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF MS)/ProteinChipTM Arrays—The 6E10 antibody (1 µg) was applied to each spot on a PS20 ProteinChipTM Arrays (Ciphergen Biosystems Inc., Fremont, CA) and incubated (2 h at room temperature) in a humidified chamber. Nonspecific binding sites were blocked with 0.5 M ethanolamine/PBS for 30 min (room temperature). Samples were diluted in PBS, 0.5% Triton X-100, and incubated on the arrays (2 h, room temperature). The arrays were then washed for 3 x 15 min with PBS, 0.5% Triton X-100, 3 times for 5 min with PBS, and several rinses with 1 mM HEPES at room temperature. The matrix, {alpha}-cyano-4-hydroxycinnamic acid was applied to each spot on the array and mass analysis was performed by SELDI-TOF-MS, using the ProteinChipTM Biology System II.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of Tyloxapol-filled Lysosomes—Lysosomes possess a heterogeneous size and density distribution, making them very difficult to purify. To overcome this problem, we used the tritosome technique developed by De Duve and co-workers (48) to purify tyloxapol-filled lysosomes (see "Experimental Procedures" and Fig. 1). Extensive studies have documented that tyloxapol-filled lysosomes are biochemically identical to lysosomes (4851) and are clearly distinct from the early endocytic compartments, including phagosomes (52). Similar to the original studies of De Duve and co-workers (48), we have previously reported that the tritosome technique routinely yields lysosomes that are ~70-fold enriched in {beta}-hexosaminidase activity, a lysosomal marker, with essentially no citrate synthase activity, a mitochondrial marker, or NADPH: cytochrome c reductase activity, an ER marker. They are also significantly enriched in {beta}-glucosidase activity, another lysosomal marker (44). Table I summarizes the characteristics of 7 preparations of purified lysosomes. In these preparations 0.13% of the initial protein was recovered in the lysosomal fraction and the overall recovery of lysosomes (as estimated by hexosaminidase activity) was 8%. The specific activity of hexosaminidase was enriched by a factor of 70 in the lysosome fractions. Our estimate of the total lysosomal protein in the liver, based on our recovery of hexosaminidase activity, is 1.6%, which falls within the 1–2% range estimated to be the lysosomal component of cells.


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TABLE I
Purification of lysosomes

Summary of results from seven independent lysosome purifications. Relative yields are normalized using the homogenate as 100%. Average yields (shown for illustration) are the relative yields times the average total recovery.

 

We have also examined our lysosomal preparations for membrane-protein markers by Western blotting (a technique not available to De Duve and co-workers (48)) (Fig. 2). In these experiments, the lysosomal proteins LAMP-1 and Rab7 were markedly enriched in the integral membrane protein fraction, whereas the ER/Golgi marker calnexin (23, 53) was undetectable (Fig. 2). Additional Western blots (not shown) also demonstrated that the 49-kDa subunit of cytochrome oxidase complex 1 (a mitochondrial membrane protein) is absent from our preparations. EM of a typical preparation (Fig. 3A) confirmed that it is composed of a single major type of organelle with the classical appearance of lysosomes.



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FIG. 2.
Mature nicastrin, PS-1, and APP are enriched in the lysosome. Protein (50 µg/lane) from each fraction of the lysosome purification was separated on 12% SDS-polyacrylamide gel and probed with the antibody indicated. Organelles is a fraction of the PNS, enriched in mitochondria, peroxisomes, and lysosomes. Lysosomes were purified and run as whole organelles, or fractionated by freeze/thaw lysis to release soluble components. Remaining membranes were then extracted with sodium carbonate to yield the membrane-associated fraction, and then solubilized in 2% SDS to yield the integral membrane protein fraction (see "Experimental Procedures").

 


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FIG. 3.
PS-1 and nicastrin are co-localized with LAMP-1 on the LM by double label immunogold immunocytochemistry. Lysosomes were prepared for (A) routine transmission electron microscopy ultrastructural examination, or (B–E) immunogold EM. A, confirms the classical appearance of Triton-filled lysosomes in our preparation. B, ultrathin cryosections demonstrating LMs labeled with PS 1 (5 nm particles, see arrowheads for examples) and LAMP-1 (10-nm particles, e.g. see arrows). C, LMs labeled with nicastrin (5-nm gold particles, e.g. see arrowheads), and LAMP-1 (10-nm gold particles, e.g. see arrows). D, LMs labeled with presenilin (5-nm gold particles, e.g. see arrowheads), omitting the LAMP-1 primary antibody (but not the secondary antibody). E, LMs labeled with LAMP-1 (10-nm gold particles, e.g. see arrows), omitting the presenilin and nicastrin primary antibodies (but not the secondary antibody). All size bars equal 100 nm.

 

Intact lysosomes were fractionated into soluble, "membrane-associated" (sodium carbonate extractable) and "integral membrane" (detergent extractable) fractions. Using Western blotting (Fig. 2), nicastrin, APP, and the C-terminal (data not shown) and N-terminal portions of PS-1 are enriched in lysosomes compared with the crude organelle, postnuclear supernatant, and homogenate fractions (Figs. 1 and 2 and Table II). Importantly, all of these proteins occur at their native, mature Mr (Fig. 2), indicating that they were not residual products of lysosomal degradation.


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TABLE II
Purification of APP, nicastrin, and presenilin-1

Results of densitometry experiments of Western Blots from three to six independent lysosome isolation experiments.

 

The relative amounts of APP, nicastrin, and presenilin-1 in the lysosome were determined by densitometry of Western blots for each protein from three to six independent lysosome purifications (Table II). Based on these values and assuming that the yield of hexosaminidase reflects the overall yield of lysosomes, the percentage of total APP, nicastrin, or PS-1 contained in the lysosomal membrane averages approximately 30, 29, and 4% of their total cellular components, respectively.

Although it is not unexpected that some of the single transmembrane domain proteins (APP and nicastrin) were partially extracted from the membrane by the sodium carbonate treatment (Fig. 2), we were surprised to find full-length APP in the soluble fraction of our lysosome preparation. That this APP species is full-length was supported by its apparent size and the observation that it migrated to the same position on SDS-PAGE gels as the APP in the membrane fractions. Furthermore, it was detectable with an antibody against the C-terminal end of APP, a domain that is removed when APP was released by secretase processing.

To confirm that the above AD-related proteins were indeed located in lysosomes and not contained in another type of contaminating organelle or in intra-lysosomal membrane fragments/vesicles awaiting degradation, we performed routine and immunogold EM on whole intact lysosomes. On routine EM (Fig. 3A), tyloxapol-filled lysosomes appear as membrane-bound vesicles containing an electron dense matrix with variable clear areas depending upon their plane of sectioning. Because this preparation was derived from organelles that were collected by centrifugation, many distorted and broken lysosomes were also seen. Our antibodies were poorly reactive with routine paraformaldehyde/glutaraldehyde-fixed specimens (data not shown); thus, lysosomes were fixed in a lower concentration of each, and prepared for cryoultramicrotomy (see "Experimental Procedures"). With this procedure, lysosomes can be identified by their electron-dense matrix despite the lack of counterstain. In these preparations, the LM protein LAMP-1, labeled with large (10 nm) gold particles, can be clearly seen co-localized on the outer limiting lysosomal membrane, along with numerous small (5 nm) immunogold particles labeling either PS-1 (Fig. 3B) or nicastrin (Fig. 3C).

To confirm that the above immunogold images were representative, gold particles on lysosome-like structures were quantified from three preparations, using >50 fields per preparation of each antibody combination. In these three preparations, LAMP-1, PS-1, and nicastrin antibodies labeled 80 ± 10, 80 ± 10, or 70 ± 10% of the lysosomes. The percentage colabeled by LAMP-1 and PS-1 antibodies was 80 ± 20% and for LAMP-1 and nicastrin antibodies, 70 ± 20%.

Lysosomes Contain A{beta} and Are Enriched in a {gamma}-Secretase-like Proteolytic Activity—Having found {gamma}-secretase-associated proteins in lysosomal membranes, we developed an assay to detect its activity. We generated a peptide-substrate corresponding to the {beta}-secretase-cleaved C-terminal portion of APP with a methionine added for translation initiation (C100), by in vitro translation with 35S-labeled Met. The labeled substrate was incubated with salt-washed lysosomal membranes in the presence of 0.25% CHAPSO, under conditions similar to those already published (54), with 2x Complete Protease Inhibitor (Roche Diagnostics; inhibits all but aspartyl proteases). Assays were carried out over a range of pH values, and the products were separated by SDS-PAGE and detected by autoradiography. The cleavage of the 11-kDa C100 peptide into the expected 4- and 7-kDa fragments (Fig. 4A) occurred optimally at pH ~4.5 and was blocked by 100 µM pepstatin (Fig. 4A), as well as by concentrations as low as 0.1 µM (data not shown; conditions similar to Li et al. (54)).



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FIG. 4.
{gamma}-Secretase activity has an acidic pH optimum, is nicastrin-dependent, and is enriched in the lysosome. A, radiolabeled 100-amino acid C-terminal fragments of APP (C100) (1 µM) were incubated with 10 µg of LM proteins for 4 h at 37 °C (or on ice) at the indicated pH. The samples incubated on ice or with pepstatin were at pH 5.0. Products were analyzed by SDS-PAGE on 20% Tris-Tricine gels and were visualized on a PhosphorImager. The C100 substrate and 2 product fragments of ~4 and ~7 kDa were detected, which correspond to the predicted sizes of the {gamma}-cleavage products. B, washed lysosomal membranes were solubilized in 2% CHAPSO and the insoluble material was removed. Solubilized proteins were then immunoprecipitated with 10 µg of anti-nicastrin antibody immobilized on beads. The beads were incubated with 35S-C100 at pH 4.5 for 4 h. C shows membranes from the homogenate, PNS, the organelle pellet, and lysosomes fractions, and assayed at pH 4.5 as above.

 

To show that this activity was performed by an enzyme complex containing nicastrin, and not a contaminating lysosomal protease, we treated the washed lysosomal membranes with 2% CHAPSO, which has been reported to effectively solublize {gamma}-secretase activity (54). From this preparation, we performed immunoprecipitation with an antibody directed against nicastrin. When the washed nicastrin immunocomplex (bound to Protein G-beads) was incubated with our C100 substrate at pH 4.5, the predicted 4- and 7-kDa bands were generated (Fig. 4B).

To determine what percentage of this activity resides in the lysosome, we prepared membranes from the homogenate, PNS, and organelle fractions, as well as from lysosomes, and repeated the {gamma}-secretase assay at pH 4.5. The acid {gamma}-secretase activity was indeed enriched in the lysosomal membrane fraction (Fig. 4C). The density of the lower 4-kDa band was quantitated using NIH Image 1.63. The purification factor for this activity was 20, and 3% of the total cellular acidic {gamma}-secretase activity was recovered in the lysosome fraction. Assuming that the recovery of hexosaminidase reflects the recovery of lysosomes in our preparations, an overall yield of 3% of the {gamma}-secretase activity indicates that the lysosomal component of {gamma}-secretase activity accounts for about 30% of the total cellular activity at pH 4.5. Of course at in vivo pH values, the remaining 70% would have little biological activity outside the acidic environment of the lysosome.

To confirm that the 4-kDa product of the above reaction was in fact A{beta}, we analyzed the products generated from a non-radiolabeled C100 peptide using SELDI-TOF MS. In this technique, specific A{beta} peptides were captured by the monoclonal antibody 6E10, which has been immobilized on PS20 ProteinChipTM Arrays (Ciphergen Biosystems Inc.), and analyzed using the ProteinChipTM Biology System II. These spectra (Fig. 5) show a single endogenous peak in our rat LM preparations, with the appearance of no new peaks after the membranes were incubated at 37 °C for 4 h. When the human APP-C100 peptide was added to the membranes and incubated under the above conditions, an A{beta} peak with a Mr of 4329.0 was clearly visualized in a sample at pH 4.5, but barely detectable in a sample at pH 7.0. This Mr was consistent with that predicted for the human 1–40 form of A{beta}, 4329.87 (the error limits of this system is <0.1%), and runs at exactly the same position as an A{beta} 1–40 standard (data not shown).



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FIG. 5.
SELDI-TOF confirmation of A{beta}40 production. Proteins captured on a target with the monoclonal anti-amyloid antibody 6E10 were analyzed by SELDI-TOF MS. Lysosomal membranes (20 µg) were incubated on ice or at 37 °C with or without the addition of 2 µM C100 at the pH indicated. The rat lysosomal membranes bear an intrinsic A{beta} peptide with a mass of about 3832.1 that decreases with incubation at 37 °C. Upon incubating C100 with lysosomal membranes at pH 4.5, a peak appears with a Mr of 4329.0, corresponding to the mass of human A{beta}40 (predicted 4329.87).

 

The endogenous peak found in our lysosomal membrane preparation (Fig. 5) with a Mr of 3832.1 was identified as a rat A{beta} peptide, based on its affinity for the 6E10 antibody. Using the FindPept program on the ExPASy Proteomics Server3 of the Swiss Institute for Bioinformatics to search all possible cleavage products of the endogenous rat C100, this peak was identified as the rat A{beta}N3(pE)38 (an A{beta} peptide ending at position 38 and beginning at position 3 with this residue dehydrated to pyroglutamate), with a predicted Mr of 3832.33.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
PS-1, APP, and Nicastrin Are Resident Lysosomal Proteins— These studies demonstrate that APP, nicastrin, and PS-1 are bona fide LM proteins, and are not just present as substrates for degradation. This conclusion is supported by the fact that APP and PS-1 both possess C-terminal endocytosis/lysosomal sorting sequences based on the consensus sequence Tyr-X-X-bulky-hydrophobic amino acid (Y-X-X-Ø) (Table III) (55). For example, the Y-X-X-Ø on the cytoplasmic tail of lysosomal acid phosphatase directs its rapid endocytosis into endosomes. There it may recycle back to the plasma membrane many times before being sorted to the lysosome (56). Nicastrin does not possess a recognizable sorting sequence, consistent with the observation that it must form a complex with PS for transit out of the ER (41). This is reminiscent of the lysosomal protein neuraminidase, which must complex with the lysosomal "protective protein" for transport to the lysosome (57).


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TABLE III
C-terminal endosomal sorting signals

The abbreviations used are: Ø, large hydrophobic residue; X, any amino acid; LAP, lysosomal acid phosphatase.

 

In support of the above conclusions, a number of studies have also detected PS-1 at sites outside the ER and Golgi including, the endosomal compartment (58, 59), and in specialized secretory lysosomes including neutrophilic azurophilic granules and platelet granules (60). Nicastrin·PS-1 complexes have also recently been observed in unidentified "non-ER/Golgi compartments" (43).

Unexpectedly, some full-length APP was found in the soluble fraction of our lysosome preparation. It is not clear if this species was specifically removed from the membrane or is merely not well anchored; we have not encountered any other membrane proteins in the lysosomal soluble fraction. Soluble full-length APP has been previously reported as being secreted from human neurons after their ability to produce A{beta} is blocked by the lysosomal-tropic chemicals NH4Cl or chloroquine (61). This APP species might therefore be a source of A{beta} production by soluble proteases of the lysosome, i.e. {gamma}-secretase-independent proteases, which have been suggested by some authors (6264). For example, cathepsin D (the major soluble protease of the lysosome) is capable of cleaving soluble APP fragments at positions 42 and 43 (but not 40) (65).

{gamma}-Secretase Activity in the Lysosome—Using an in vitro assay similar to those previously published (54), we demonstrated that A{beta} peptide is produced from an APP-C100 peptide upon incubation with lysosomal membranes at a pH optimum of pH ~4.5, and that this activity is significantly enriched in our lysosomal preparation. The Mr of this A{beta} peptide indicates the loss of our added initiating Met residue, which is a commonly observed post-translational event (66). Because C100 contains 4 Met residues, 2 on either side of the predicted {gamma}-secretase cleavage sites, the missing N-terminal [35S]Met explains the lower autoradiographic intensity of the 4-kDa band in our gel-based {gamma}-secretase assays (Fig. 4). A{beta}42 was not expected to be detected in this assay as it is normally present at a 10-fold lower level than A{beta}40. It is also likely that the relative sensitivity of SELDI-TOF toward the {beta}-amyloids parallels matrix-assisted laser desorption ionization time-of-flight, which is much less sensitive toward A{beta}42 than A{beta}40 (67). The detection of A{beta}40 as the major species produced in this reaction implies that the most abundant soluble protease, cathepsin D, was effectively removed from our salt-washed membranes (see "Experimental Procedures"). We estimate that the lysosomal {gamma}-secretase fraction accounts for 30% of the total potential cellular activity at pH 4.5, which correlates well with the distribution of nicastrin in our subcellular fractions. This correlation between {gamma}-secretase activity and mature nicastrin levels has been previously shown in transfected cells (68). Our ability to immunoprecipitate this {gamma}-secretase activity with an antibody to nicastrin demonstrates that the activity found here is likely the same {gamma}-secretase activity that is associated with Alzheimer's disease pathology.

Several reported {gamma}-secretase assays have a pH optimum of about 6.8 and very little activity at pH 5.0 (54, 69). In these studies, total cell membranes were used as the enzyme source and an enzyme-linked immunosorbent assay was used for detection of the product. Thus, only soluble products could be detected in this type of assay. Our assay used highly purified membranes and SDS-PAGE-based analysis that (unlike the enzyme-linked immunosorbent assay systems) is capable of detecting A{beta} species even if they were to aggregate at low pH (see Ref. 70). In addition, aspartyl proteases typically have an acidic pH optimum, ranging from pH 3 to 5 (71), because of the need to keep one active Asp group protonated and the other unprotonated. Thus an acidic pH optimum would be predicted for the {gamma}-secretase, because it is proposed to be an aspartyl protease based on mutational studies and its sensitivity to aspartyl protease inhibitors, i.e. pepstatin. Therefore, our finding of a pH optimum of 4.5 is consistent both with our intracellular localization of the {gamma}-secretase and with its known function as an aspartyl protease.

Lysosomal Localization of APP, PS-1, and Nicastrin Is Easily Reconciled with A{beta} Secretion—Although lysosomes are traditionally regarded as fixed catabolic organelles, the appearance of many lysosomal proteins including hexosaminidase and acid phosphatase in human serum and conditioned cell medium clearly shows that proteins that have reached the lysosome and/or late endosome can still be exocytosed (72). In fact, lysosomes have recently been shown to be the principal compartment involved during calcium-dependent secretion in non-secretory cells (73) and to fuse directly with plasma membrane to repair tears (74). Furthermore, many lysosomal membrane proteins, i.e. lysosomal acid phosphatase, are present initially on the cell surface prior to endocytosis to the endosome, and they may be recycled back to the plasma membrane 10 times or more before transit to lysosomes (75). Because APP transits the cell surface and the endosomal system to arrive in the lysosome, it would be exposed to BACE in these locations (76). Similarly, {gamma}-secretase proteins must also transit the endosomal system to reach the lysosome, and A{beta} could also be generated in these compartments and released upon endosomal recycling to the plasma membrane. Thus, transit to and processing of APP in the lysosomal/endosomal system is completely compatible with APP and APP processing fragments being found in the extracellular fluid.

The heterogeneous size and density distribution of lysosomes may explain why PS-1, nicastrin, and APP processing fragments have not been previously identified in this compartment in previous cell fractionation experiments. These studies all relied on density gradient centrifugation (33, 3639), a method that is particularly unreliable when examining endosomal/lysosomal proteins as these organelles have overlapping size and density characteristics with mitochondria, peroxisomes, ER, and Golgi. Density gradient-based studies of PS-1 or APP processing that also examined lysosomal/endosomal markers, e.g. endocytosed horseradish peroxidase (38) or cathepsin-D (23), found these markers contaminating the majority of the fractions collected.

Data from our proteomic survey of the lysosome clearly demonstrates that nicastrin is a major protein component of the LM, easily seen in two-dimensional gels using a protein stain (44), and not a resident ER or Golgi protein like calnexin, which is absent from our lysosome preparations (Fig. 2). Because we have found that two-dimensional gels fail to resolve proteins like PS-1 with multiple transmembrane domains (data not shown), we used Western blotting to identify native PS-1 as well as nicastrin and APP in the LM (Fig. 2). These data were then confirmed using EM (Fig. 3). The finding of {gamma}-secretase proteins and {gamma}-secretase activity in the endosomal/lysosomal system re-asserts the importance of the endosomal-lysosomal system in the generation of A{beta}, and demonstrates the presence of the A{beta}-producing system in these compartments, as was first reported in 1992 (77).

A Lysosomal Model of Alzheimer's Disease?—Our finding of {gamma}-secretase in the lysosome suggests a unifying model of AD, whereby minute quantities of aggregated A{beta} could accumulate in the lysosome over a lifetime, possibly exacerbated by declining lysosomal degradative capacity with aging (78), or the occurrence of Familial AD mutations. This accumulation would lead eventually to the loss of LM integrity with leakage of lysosomal proteases into the cytoplasm, resulting initially in local disruption of the neuronal structure and eventually to neuronal apoptosis.

In support of the above model, in vitro experiments show that the specific membrane composition and low pH of the endosomal/lysosomal system favor the formation of insoluble A{beta} fibrils (70, 79), a process shown by atomic force microscopy to disrupt lipid membranes (80). Furthermore, the leakage of lysosomal proteases into the cytoplasm is known to induce apoptosis (81). The combination of these processes has been demonstrated in cultured cells, in which A{beta}42 has been demonstrated to accumulate in lysosomes in an aggregated form that is resistant to degradation (82), leading to loss of LM integrity and leakage of lysosomal contents into the cytoplasm, and cell death (83, 84). Studies of human AD neuropathological material document lysosomal proliferation and endosomal enlargement years to decades prior to the onset of clinical dementia (78). In human AD postmortem brain, A{beta} has been shown to accumulate in the endosomal/lysosomal system by immunogold EM resulting in the disruption of local synaptic structures (85). In light microscopy experiments, moderately affected neurons contain A{beta} deposits that disrupt dendritic morphology (86); severely affected neurons appear to be filled with A{beta}-engorged lysosomes (87). Furthermore, these authors show that A{beta} in extracellular plaques appears to be released by neuronal rupture. Lysosomal rupture-induced neuronal death would explain the presence of large amounts of enzymatically active cathepsin B and D in amyloid plaques (88) (their pro-forms require activation in the lysosome) and the increased cerebrospinal fluid cathepsin D levels in AD patients (89). Our data showing A{beta} production in the lysosome, together with this existing evidence of lysosomal A{beta} accumulation and rupture in AD, suggest that viewing AD as an adult onset form of lysosomal storage disease may lead to a simplified model for its pathophysiology and new approaches to its treatment.


    FOOTNOTES
 
* This work was supported by a Canadian Institutes for Health Research grant (to J. C. and D. M.) and by a fellowship from the University of Toronto Clinician Scientist Training Program. 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

§§ To whom correspondence should be addressed: Research Institute, Rm. 9146A Elm Wing, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6161; Fax: 416-813-8700; E-Mail: hex{at}sickkids.ca.

1 The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; A{beta}, {beta}-amyloid; ER, endoplasmic reticulum; PS1, presenilin 1; LM, lysosomal membrane; LAMP-1, lysosomal-associated membrane protein-1; PNS, postnuclear supernatant; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; SELDI-TOF MS, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back

2 www.rsb.info.nih.gov/nih-image. Back

3 www.expasy.org. Back


    ACKNOWLEDGMENTS
 
We thank Vanessa Blandford and Cheryl Leung for technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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