From the
Research Institute,
||Department of Pediatric Laboratory Medicine, The
Hospital for Sick Children,
Clinician
Investigator Program, ¶Department of Pathobiology
and Laboratory Medicine,
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.
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ABSTRACT |
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INTRODUCTION |
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Many lines of evidence support the hypothesis that -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
42 production
(46).
In addition, known aspartyl protease inhibitors that inhibit
-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
-secretase activity
(9). Finally, cells cultured
from PS-1/PS-2 knockout mice secrete no A
(10). Recently, mAph1 and Pen2
have also been demonstrated to complex with presenilin and be required for
-secretase complex expression
(1113).
Many earlier studies indicated that A is produced in the
endosomal/lysosomal system (2).
The strongest evidence for A
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
, 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
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)
- and
-cleaved APP fragments accumulate in lysosomes of cells from PS-1
knockout mice (which have markedly decreased
-secretase activity)
(3,
23). (c) A
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
production
(15).
Other evidence suggesting a lysosomal/endosomal localization of
-secretase activity comes from the study of the cell surface receptor
Notch, which has been suggested to undergo endocytosis and
-cleavage in
a manner similar to APP
(2729).
The idea that the same
-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 is
that numerous experiments appear to show PS1 and
-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
is produced.
The conflict between the apparent ER localization of PSs and nicastrin and
the endosomal/lysosomal site of A 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
generation to the endosomal/lysosomal system. Additionally,
if A
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
-secretase. Numerous models have been suggested to reconcile these
incongruities in intracellular localization. These include: (a) that
- or
-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
-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 -secretase activity that is precipitable with an anti-nicastrin
antibody.
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EXPERIMENTAL PROCEDURES |
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Lysosome (Tritosome) Isolation ProtocolThe 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 -hexosaminidase
(45) and citrate synthase
(46) were performed on each
fraction.
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Lysosomal SubfractionationSoluble (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 AnalysisWestern 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 PreparationTyloxapol-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 CryosectionsTyloxapol-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.
-Secretase Assay SubstrateA DNA template encoding
amino acids 671770 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, 1020 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 -Secretase AssayMembranes 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
1020 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 -Secretase AssayWashed
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
ArraysThe 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,
-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.
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RESULTS |
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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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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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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 and Are Enriched in a
-Secretase-like Proteolytic ActivityHaving found
-secretase-associated proteins in lysosomal membranes, we developed an
assay to detect its activity. We generated a peptide-substrate corresponding
to the
-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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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 -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 -secretase assay at pH 4.5. The
acid
-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
-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
-secretase
activity indicates that the lysosomal component of
-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, we analyzed the products generated from a non-radiolabeled C100
peptide using SELDI-TOF MS. In this technique, specific A
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
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 140 form of A
, 4329.87 (the error limits of this system is
<0.1%), and runs at exactly the same position as an A
140
standard (data not shown).
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The endogenous peak found in our lysosomal membrane preparation
(Fig. 5) with a
Mr of 3832.1 was identified as a rat A 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
N3(pE)38 (an A
peptide ending at position 38 and
beginning at position 3 with this residue dehydrated to pyroglutamate), with a
predicted Mr of 3832.33.
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DISCUSSION |
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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 is blocked by the lysosomal-tropic chemicals
NH4Cl or chloroquine
(61). This APP species might
therefore be a source of A
production by soluble proteases of the
lysosome, i.e.
-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).
-Secretase Activity in the LysosomeUsing an in
vitro assay similar to those previously published
(54), we demonstrated that
A
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
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
-secretase cleavage
sites, the missing N-terminal [35S]Met explains the lower
autoradiographic intensity of the 4-kDa band in our gel-based
-secretase assays (Fig.
4). A
42 was not expected to be detected in this assay as it
is normally present at a 10-fold lower level than A
40. It is also likely
that the relative sensitivity of SELDI-TOF toward the
-amyloids
parallels matrix-assisted laser desorption ionization time-of-flight, which is
much less sensitive toward A
42 than A
40
(67). The detection of
A
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
-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
-secretase activity and mature nicastrin levels has been
previously shown in transfected cells
(68). Our ability to
immunoprecipitate this
-secretase activity with an antibody to
nicastrin demonstrates that the activity found here is likely the same
-secretase activity that is associated with Alzheimer's disease
pathology.
Several reported -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
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
-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
-secretase and with its known
function as an aspartyl protease.
Lysosomal Localization of APP, PS-1, and Nicastrin Is Easily Reconciled
with A SecretionAlthough 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,
-secretase proteins must also transit the endosomal system to reach the
lysosome, and A
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 -secretase proteins and
-secretase activity in
the endosomal/lysosomal system re-asserts the importance of the
endosomal-lysosomal system in the generation of A
, and demonstrates the
presence of the A
-producing system in these compartments, as was first
reported in 1992 (77).
A Lysosomal Model of Alzheimer's Disease?Our finding of
-secretase in the lysosome suggests a unifying model of AD, whereby
minute quantities of aggregated A
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 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
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
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
deposits that disrupt
dendritic morphology (86);
severely affected neurons appear to be filled with A
-engorged lysosomes
(87). Furthermore, these
authors show that A
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
production in the lysosome, together with this existing evidence of lysosomal
A
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.
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FOOTNOTES |
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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,
-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.
2 www.rsb.info.nih.gov/nih-image.
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