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The Journal of Neuroscience, March 1, 2000, 20(5):1657-1665
Neurons Regulate Extracellular Levels of Amyloid  -Protein via
Proteolysis by Insulin-Degrading Enzyme
Konstantinos
Vekrellis1,
Zhen
Ye1,
Wei Qiao
Qiu1,
Dominic
Walsh1,
Dean
Hartley1,
Valérie
Chesneau2,
Marsha Rich
Rosner2, and
Dennis J.
Selkoe1
1 Center for Neurologic Diseases, Harvard Medical
School and Brigham and Women's Hospital, Boston, Massachusetts 02115, and 2 Ben May Institute for Cancer Research, University of
Chicago, Chicago, Illinois 60637
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ABSTRACT |
Progressive cerebral accumulation of amyloid  -protein (A) is
an early and invariant feature of Alzheimer's disease. Little is known
about how A, after being secreted, is degraded and cleared from the
extracellular space of the brain. Defective A degradation could be a
risk factor for the development of Alzheimer's disease in some
subjects. We reported previously that microglial cells release
substantial amounts of an A-degrading protease that, after
purification, is indistinguishable from insulin-degrading enzyme (IDE).
Here we searched for and characterized a role for IDE in A
degradation by neurons, the principal cell type that produces A.
Whole cultures of differentiated pheochromocytoma (PC12) cells
and primary rat cortical neurons actively degraded endogenously
secreted A via IDE. However, unlike that in microglia, IDE in
differentiated neurons was not released but localized to the cell
surface, as demonstrated by biotinylation. Undifferentiated PC12 cells
released IDE into their medium, whereas after differentiation, IDE was
cell associated but still degraded A in the medium. Overexpression of IDE in mammalian cells markedly reduced the steady-state levels of
extracellular A40 and A42, and the
catalytic site mutation (E111Q) abolished this effect. We observed a
novel membrane-associated form of IDE that is ~5 kDa larger than the
known cytosolic form in a variety of cells, including differentiated
PC12 cells. Our results support a principal role for
membrane-associated and secreted IDE isoforms in the degradation and
clearance of naturally secreted A by neurons and microglia.
Key words:
neurons; Alzheimer's disease; amyloid -protein
degradation; insulin-degrading enzyme; oligomerization; membrane
proteins
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INTRODUCTION |
Alzheimer's disease (AD) is
characterized by the progressive and severe accumulation in the brain
of the amyloid -protein (A). Although much attention has been
focused on the cellular production of A, including the role of
presenilin proteins therein (Selkoe, 1999 ), little is known about how
A, after being secreted, is degraded and cleared from tissues.
Defective degradation of A would be expected to be a risk factor for
the development of AD. If brain-derived A-degrading proteases can be
identified, genetic changes in such proteases could be sought in as yet
unidentified forms of familial AD, and their pharmacological
upregulation could represent a therapeutic approach to AD in general.
Recent evidence suggests that insulin-degrading enzyme (IDE), a thiol
metalloendopeptidase known to cleave insulin, glucagon, and other
peptide hormones (for review, see Authier et al., 1996a), may be
involved in the degradation of endogenous brain-derived A peptides
(Kurochkin and Goto, 1994; McDermott and Gibson, 1996; Qiu et al.,
1998). The protease is expressed in a variety of tissues including
brain and has a conformational rather than a sequence specificity for
its substrates (Shii et al., 1985; Akiyama et al., 1990; Kuo et al.,
1994). In mammalian cells, IDE has been principally localized to the
cytosol and peroxisomes (McKenzie and Burghen, 1984; Authier et al.,
1995, 1996b; Chesneau et al., 1997), raising the question of how the
enzyme could degrade A, because the peptide is not found in either
of these locations. However, we have shown recently that intact IDE,
like A, can be released into the extracellular fluid by healthy
cultured microglial (BV-2) cells and is also present in normal
CSF (Qiu et al., 1998). Whether IDE is present in and released
from neurons, widely considered to be the principal source of secreted
A in brain, is not known.
Here we used mixed primary rat brain cultures and differentiated
pheochromocytoma (PC12) cells to investigate whether neuronal cells
also possess A-degrading activity. We demonstrate that neuronal-type
cells indeed exhibit significant extracellular A-degrading activity
that is inhibited by competitive IDE substrates and other IDE
inhibitors. Unlike the IDE in microglia, which is released into the
medium, the IDE we detected in differentiated neurons was found in part
on the cell surface, consistent with recent evidence that IDE can be
localized to the surface of some non-neural cells expressing the enzyme
(Seta and Roth, 1997). Surprisingly, we found that undifferentiated
PC12 cells release some IDE into their medium, whereas differentiated
PC12 cells retain it on the plasma membrane, where it can be
biotinylated. We confirmed that IDE has a major role in A
degradation by showing that cellular overexpression of wild-type but
not active site-mutated IDE markedly decreases the steady-state levels
of naturally secreted A40 and A42 in the medium of APP-expressing
cells. Interestingly, intracellular levels of A were not affected by
overexpressing IDE. Finally, detergent extraction of neuronal cells
reveals the presence of a novel ~115 kDa IDE isoform that is the
major species in neuronal membranes but is absent in cytosol, which
contains the known 110 kDa isoform.
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MATERIALS AND METHODS |
Cell culture. All growth media were supplemented with
glutamine (2 mM), penicillin (500 units/ml), and
streptomycin (500 µg/ml) except where otherwise indicated. Rat PC12
cells were routinely cultured in DMEM with 10% horse serum and
5% fetal bovine serum (FBS). For differentiation, the cells were
plated at a density of 1 × 106 cells
per collagen-coated 10 cm dish and cultured for 5 d in plain DMEM
supplemented with 100 ng/ml nerve growth factor (NGF) and 0.5% FBS.
The medium was changed every 2 d. Mouse BV-2 microglial cells were
cultured in RPMI and 10% FBS (Qiu et al., 1998). Chinese hamster ovary
(CHO) cells stably transfected with APP770
cDNA containing the V717F AD-causing missense mutation (7PA2 cells) were cultured in DMEM and 10% FBS with G418 (200 µg/ml).
Primary brain cultures were prepared as described (Hartley et al.,
1993), with slight modifications. Briefly, brain cells were isolated
from the neocortex of embryonic day 16 rat embryos and plated at high
density (200,000 cells/ml) in DMEM and Ham's F-12 (1:10 dilution of
1×) containing 10% FBS and 20 mM HEPES onto glial feeder
layers in 3.5 cm dishes. Cultures were fed twice a week with the
plating medium. At approximately day 9, glial growth was inhibited by
the addition of 10 5 M
cytosine arabinoside, and the cultures were changed to reduced serum
medium (5% bovine calf serum + MEM + 20 mM HEPES).
Cultures were used after 3 weeks in vitro.
Cell-surface biotinylation. For biotinylation experiments,
cells were placed on ice and washed three times with ice-cold
Dulbecco's PBS (DPBS; BioWhittaker, Walkersville, MD). The
cells were then incubated with 0.5 mg/ml biotinamidocaproic acid
3-sulfo-N-hydroxysuccinamide ester (Pierce, Rockford, IL) in
DPBS for 10 min at 4°C. Free biotin groups were blocked by washing
the cells twice with cold DMEM. Cells were lysed in STEN buffer
(50 mM Tris, pH 7.6; 150 mM
NaCl; 2 mM EDTA; 1% NP-40; 20 mM PMSF; 0.5 mg/ml each of leupeptin, aprotinin,
and pepstatin A; 10 mM
N-ethylmethylamide (NEM); and 10 mM
1,10-phenanthroline) and then centrifuged for 10 min at 16,000 × g at 4°C. Surface IDE was immunoprecipitated from the lysates using the IDE-specific monoclonal antibody 9B12 (kind gift of
Dr. Richard Roth, Stanford University). Immunoprecipitates were
electrophoresed on 10% Tris/Glycine gels, blotted onto nitrocellulose, and probed for biotin-labeled proteins with neutravidin-HRP (Jackson ImmunoResearch, West Grove, PA). Surface labeling in this protocol was
verified by immunoprecipitating lysates with a polyclonal antibody (C7)
specific to APP (positive control) and a monoclonal antibody specific
to the cytoplasmic protein phosphatidylinositol 3-kinase (PI3-kinase;
negative control).
Transient and stable transfections. 7PA2 cells were
transfected using Lipofectamine (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, the cells were
grown until 80% confluent and then transfected overnight with 5 µg
of DNA and 15 µg of Lipofectamine reagent. Two constructs carrying
the cDNA of N-terminal hemagglutinin (HA)-tagged human wild-type IDE or
inactive mutant E111Q (glutamic acid 111 to glutamine) were used
(Perlman et al., 1993). Empty vector was used as the control. After
transfection, the cells were refed with normal culture medium for 24 hr, and aliquots of the medium were assayed for levels of total A or
A1-42 peptides by highly specific sandwich
ELISAs, using distinct capture antibodies as described previously
(Johnsonwood et al., 1997; Qiu et al., 1997). For stable transfections,
full-length wild-type (wt) human IDE was inserted into the vector
pCDNA3.1 (Invitrogen, San Diego, CA), which contains a zeocin-resistant
gene. CHO cell lines stably coexpressing the APP V717F AD mutation and
IDE were generated from cell line 7PA2 (above) using zeocin and G418
for selection. Single clones were isolated as described previously
(Citron et al., 1997), and cell extracts were analyzed for IDE and APP
expression by Western blotting using the IDE-1 and C7 antibodies,
respectively. To assay for levels of total A or
A1-42 peptides in the medium of the APP and
IDE double-transfected cells, we grew the cells to 90% confluency and
then refed them with culture medium for 4 hr before sandwich ELISAs
were performed. 7PA2 medium was used as the control.
Quantifying degradation of endogenous A by
immunoprecipitation. Confluent monolayers of 7PA2 cells in 10 cm
dishes were preincubated for 2 hr in methionine- and serum-free medium
and then labeled for 16 hr with 100 µCi of
[35S]methionine. The labeled media were
collected and centrifuged at 3000 × g for 30 min at
4°C. To characterize A-degrading activity in the medium of
neuronal cells, 2 ml of the labeled 7PA2 medium (containing abundant
A and p3) was mixed with an equal amount of neuronal
conditioned medium alone or with unconditioned medium as a control, and
the mixtures were incubated at 37°C for 24 hr. For quantifying A
degradation in whole cultures, 2 ml of labeled 7PA2 medium was mixed
with 2 ml of fresh neuronal culture medium and incubated on the whole
cultures for 24 hr at 37°C in the presence or absence of 10 µM insulin. The amount of labeled A
remaining in each condition was assessed by immunoprecipitation with
the A-specific antiserum R1282, followed by 10-20% Tris/Tricine
SDS-PAGE and gel fluorography. For experiments in which purified IDE
was examined, 7PA2 cells were incubated overnight with
[35S]Met, and the conditioned medium was
further incubated for 12 hr with 100 ng of the recombinant active or
inactive (E111Q) enzymes before being precipitated with the
A-specific antiserum R1280.
Assaying degradation of 125I-A by gel fluorography
and trichloroacetic acid precipitation. These assays were
performed as described previously (Garcia et al., 1989; Qiu et al.,
1998). Briefly, 10,000 cpm of 125I-A
(IA; specific activity, 2000 µCi/mmol) were added per milliliter of medium to whole cultures or their collected conditioned media (CM)
and incubated at 37°C for up to 24 hr. Aliquots were removed at
specific times and examined by 10-20% Tris/Tricine gel fluorography. In addition, aliquots of the CM were also treated with 15%
trichloroacetic acid (TCA) to precipitate undegraded A. The
precipitated samples were centrifuged, and the amounts of label in the
supernatant (degradative products) and pellet (intact peptide) were counted.
Preparation of cell cytosol and cell extracts. Cells were
washed twice in cold PBS buffer, suspended in cold homogenization buffer (PBS, supplemented with a protease inhibitor mixture), and
disrupted using 10 strokes in a Dounce homogenizer followed by several
passages through a 25G needle. Nuclei and cell debris were pelleted by
centrifugation at 4000 × g at 4°C, and the resulting supernatant was centrifuged at 90,000 × g for 1 hr to
separate the cytosol and membrane fractions. The membrane-containing
pellets were homogenized in cold homogenization buffer supplemented
with 1% NP-40. Undissolved material was pelleted by centrifugation at
16,000 × g and then resuspended in cold PBS containing
0.2% SDS. For total cell extracts (cell lysates), cells were
homogenized in STEN/lysis buffer and incubated on ice for 20 min. The
cell homogenate was then centrifuged at 16,000 × g for
20 min; the supernatant was kept and stored at  80°C.
Western blotting of IDE and A. To detect IDE in the CM of
different cells, cultures were washed and incubated with serum-free N2
medium for 16 hr at 37°C. CM were pooled (to ~400 ml) and filtered to remove floating cells before being sequentially precipitated with 40 and 60% NH4SO4 (Qiu et
al., 1998). The final 60% precipitate was dissolved in ~5 ml of PBS
and dialyzed in the same buffer overnight. Protein concentration was
estimated using the Bio-Rad assay. Twenty micrograms of total protein
were electrophoresed on 10% Tris and glycine gels. IDE was detected by
immunoblotting with a new polyclonal antibody, IDE-1, we raised to a
peptide comprising amino acids 62-73 of human IDE. To detect
endogenous A in lysates, samples were precleared twice with the
APP-specific antibodies B5 and C7 and then immunoprecipitated with the
high-titer A antiserum R1280. Precipitates were electrophoresed on
16% Tris/Tricine gels and transferred to 0.2 µm nitrocellulose
membranes. Membranes were treated for 10 min with boiling PBS and then
immunoblotted with the A monoclonal antibody 6E10 (Senetek).
Purification of recombinant IDE. Polyhistidine- and
hemagglutinin- tagged wild-type and catalytically inactive (E111Q) IDEs were expressed in bacteria and purified by metal affinity
chromatography as described by V. Chesneau and M. R. Rosner
(unpublished observations).
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RESULTS |
Neuronal cells degrade extracellular A via a protease that has
the properties of insulin-degrading enzyme but is cell associated
We recently purified and characterized an A-degrading
metalloprotease released into the medium of a microglial cell line (BV-2) and showed that it was indistinguishable from insulin-degrading enzyme. To extend this finding and determine whether neuronal cells,
which appear to be the major producers of A in the brain, degrade
extracellular A in a manner similar to that of microglia, we
examined both differentiated PC12 cells and primary rat brain cultures.
Differentiated PC12 cells (5 d in vitro) and embryonic rat
mixed brain cultures (21 d in vitro) were washed and changed to fresh medium containing 300 pM
125I-labeled
A1-40 (IA) in the presence or absence of
10 µM insulin. The loss of intact IA over
the course of a 24 hr incubation was quantified by a TCA precipitation
assay. Radioactivity in both the TCA-insoluble pellets and the
supernatant was measured. Whole PC12 and mixed brain cultures degraded
~50% of the extracellular IA during an 18 hr incubation, and
insulin (10 µM) completely inhibited this in
the PC12 cultures (Fig.
1A,B) and partially inhibited it in the primary cultures (Fig. 1D,E). To
confirm that the insulin was specifically inhibiting an IDE-like
metalloprotease, we examined the effects of glucagon, a known IDE
substrate with a higher Km than
insulin, and ovalbumin, which is not a substrate of IDE. In agreement
with the lower affinity of IDE for glucagon, degradation of the labeled
A by PC12 cells was only partially inhibited (~40%) by the
addition of 10 µM glucagon during the incubation (Fig. 1C). Ovalbumin (10 µM) had no effect on the degradation of IA
(Fig. 1F). Although NEM was found to be toxic
to these cultures even at low concentrations and very brief incubation times, treatment of both differentiated PC12 and rat brain cell cultures with 500 µM 1,10-phenanthroline for up
to 3 hr inhibited >80% of the IA degradation observed otherwise in
these neuronal cultures (data not shown). These results indicate that
an IDE-like protease is also responsible for the degradation of A in
neuronal cultures. To confirm that this IDE-mediated A-degrading
activity was neuronal in origin, we stained rat brain cultures with IDE antibodies and double-labeled them with antibodies to the glial or
neuronal markers GFAP or MAP-2, respectively. We found that unlike
neurons, astrocytes are not reactive for IDE (data not shown). This is
in agreement with a recent report that IDE does not localize to glia in
AD brain sections but only to neurons (Bernstein et al., 1999). We then
investigated whether the CM of our neuronal cultures were sufficient to
mediate the degradation of extracellular A seen in the above
experiments. IA was incubated for 24 hr at 37°C solely in the CM
of either differentiated PC12 cells or rat mixed brain cultures. Unlike
that of microglia, the CM of differentiated PC12 cells showed no
IA-degrading activity (Fig.
2A). Very little
IA-degrading activity was observed in the CM of the primary cultures
(Fig. 2B), and this activity might be caused by IDE
released from microglia present in our mixed brain cell cultures.
Unlike the differentiated PC12 cells, their proliferating
undifferentiated precursors exhibited A-degrading activity in their
medium (Fig. 2C) that could be inhibited either by insulin
or 1,10-phenanthroline (Fig. 2D,E). Whole cultures of
undifferentiated PC12 cells were also examined, and these exhibited substantial IA degradation, as expected (Fig. 2F).
Our results suggest that in mature neuronal-type cells, the IDE-like
extracellular A-degrading activity is cell associated rather than
released.
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Figure 1.
Degradation of A in differentiated PC12 cell
cultures and primary neurons is mediated by IDE. A-C,
PC12 cells were differentiated in DMEM and 0.5% FBS supplemented with
100 ng/ml NGF for 5-7 d. Whole cultures were then incubated with 300 pM 125I-A in the absence
(A) or presence of the known IDE substrates
insulin (10 µM; B) or glucagon (10 µM; C). D-F, Primary mixed
rat cortical cultures were maintained in DMEM supplemented with FBS and
glucose for 3 weeks. For A degradation assays, cultures were
conditioned overnight and then incubated for up to 24 hr with 300 pM IA in the presence (D) or
absence (E) of insulin or the presence of a
control protein, ovalbumin (10 µM;
F). Aliquots of CM were removed at the indicated
times, and loss of intact peptide was assayed by TCA precipitation; the
percents of total cpm recovered in supernatants and pellets are
displayed. Curves represent means (± SEM) of
n = 4 experiments.
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Figure 2.
Differentiated neuronal cells do not release IDE
into their medium. A, B, Differentiated PC12 cells
(A) or 21 d primary mixed cortical cultures
(B) were conditioned for 12 hr before the CM were
collected and incubated for up to 24 hr with 300 pM IA.
C-E, Undifferentiated PC12 culture medium that had been
conditioned for 12 hr was similarly incubated with 300 pM
IA in the absence (C) or presence
(D) of 10 µM insulin or with 1 mM 1,10-phenanthroline (E).
F, Whole cultures of undifferentiated PC12 cells were
incubated with 300 pM IA in the absence of insulin.
Aliquots of the CM were removed at the times indicated, and loss of
intact IA was assayed by TCA precipitation. Curves
represent means (± SEM) of n = 4 experiments
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We next examined the effect of neuronal cell cultures or their media
alone on the degradation of naturally secreted A. CHO cells stably
transfected with
APP770 cDNA
containing the V717F AD mutation (7PA2 cells) were metabolically
labeled with [35S]methionine, and the
resultant medium, containing abundant labeled A and p3 peptides, was
incubated on whole PC12 or primary cortical cultures or with just their
CM for 24 hr at 37°C in the presence or absence of 10 µM insulin (Fig.
3A,B). Immunoprecipitation of the media with an A antibody (R1282) and gel fluorography revealed the expected marked decrease of A in the whole neuronal cultures, and this was inhibited by 10 µM insulin.
Similar to the TCA precipitation results obtained using IA, the CM
alone had no effect on the degradation of secreted A (Fig.
3A,B). To confirm that IDE was present in the PC12 CM, we
used a newly produced, affinity-purified IDE-specific polyclonal
antibody, IDE-1, to immunoblot various media. Samples of microglial
BV-2 and CHO 7PA2 conditioned media were used as positive controls (Qiu
et al., 1998). As expected, the characteristic 110 kDa IDE band was
detected in the CM of the BV-2 and CHO cells. The same band was also
present in the CM of undifferentiated PC12 cells but was undetectable
in that of differentiated PC12 cells (Fig. 3C), consistent
with the A degradation assays.
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Figure 3.
Naturally secreted A is degraded by a
cell-associated form of IDE present in whole neuronal cultures but not
in their conditioned media. A, Conditioned media of 7PA2
CHO cells labeled with [35S]Met (as a source of
[35S]A) were incubated at 37°C for 24 hr with
either unconditioned 7PA2 medium (lane 1), unconditioned
PC12 cell medium (lane 2), conditioned PC12 medium
(lane 3), or differentiated PC12 whole cultures
(lane 4). By the use of R1282
immunoprecipitation, labeled secreted A could only be degraded by
differentiated PC12 cell whole cultures and not by their conditioned
medium alone. B, [35S]Met-labeled
7PA2 cell medium was incubated for 24 hr either with unconditioned 7PA2
medium (lane 1), mixed cortical cultures plus insulin
(lane 2), conditioned medium of the cortical cultures
(lane 3), or the whole cortical cultures (lane
4). Note that the starting amount of labeled A just
before the 24 hr incubation (time 0) is actually what remains after
degradation in the 7PA2 cultures by IDE during the initial 16 hr
conditioning period. We observed no further A degradation after 16 hr of conditioning in 7PA2 cultures (data not shown). C,
Thirty micrograms of NH4SO4-concentrated
conditioned medium from each of the indicated cell types were separated
by 10% Tris/Glycine SDS-PAGE and immunoblotted with the IDE-specific
antibody IDE-1. Note the absence of IDE in the medium of differentiated
PC12 cells. Diff, Differentiated.
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To visualize directly the fate of the A species during the
incubation of IA in whole neuronal cultures, samples of CM were collected at different times up to 24 hr and analyzed by fluorography of 10-20% Tris/Tricine SDS-PAGE gels (Fig.
4A). As the incubation interval at 37°C increased, the amount of the monomeric (4 kDa) A
band decreased, consistent with the results of the TCA assay (above).
As described previously in BV-2 cultures (Qiu et al., 1998), we also
observed the time-dependent formation of small amounts of SDS-stable
IA species migrating at 6-12 kDa. After a 3 hr incubation,
SDS-stable IA oligomeric species appeared between 6 and 10 kDa. As
the incubation interval increased to 18 and 24 hr, we observed the loss
of the 6 kDa band and the formation of additional IA oligomers
migrating at 12 kDa. Both the loss of A and the formation of these
apparent oligomers were abolished when 10 µM
insulin was present in the incubation, implicating IDE in both the
degradation and the apparent oligomerization of the iodinated peptide.
When IA was incubated in differentiated neuronal CM alone and then
assayed by SDS-PAGE and gel fluorography, A degradation and oligomer
formation were not observed (data not shown), consistent with the lack
of secreted IDE in differentiated neuronal cultures documented above.
Similar results were obtained using PC12 cultures and their CM (data
not shown).
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Figure 4.
IDE stimulates the oligomerization of iodinated
synthetic A but not naturally secreted A in neuronal cultures.
A, Primary mixed cortical cultures were conditioned in
serum-free medium overnight and incubated with IA (300 pM) for up to 24 hr in the presence or absence of insulin
(10 µM). Aliquots of the reaction mixtures were removed
at the times indicated and characterized by 10-20% Tris/Tricine gel
fluorography. Note that the loss of A monomer and the formation of
small amounts of SDS-stable higher molecular weight species are almost
abolished by insulin. B, 7PA2 CHO cells were labeled
with [35S]Met for 12 hr in the absence (no
IDE) or presence of 100 ng of purified recombinant
active IDE (wt IDE) or mutant (E111Q) IDE (mt
IDE). CM were collected, immunoprecipitated with the A
antibody R1282, and assayed by SDS-PAGE and fluorography. Note that the
6 kDa species has been shown to be a modified form of A monomer that
migrates anomalously in SDS-PAGE gels; it always follows the behavior
of the 4 kDa conventional A monomer, in contrast to that of the 8 kDa A dimer (Podlisny et al., 1998) (D. Walsh, R. Wong, and D. J. Selkoe, unpublished observations).
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To investigate the effect that purified IDE has on the oligomerization
of endogenously secreted full-length A, we metabolically labeled
7PA2 cultures and incubated the resulting labeled medium for 12 hr at
37°C in the absence or presence of 100 ng of purified active or
inactive recombinant enzyme. The media were then immunoprecipitated with R1280, which has been shown to precipitate reproducibly oligomeric forms of secreted A (Podlisny et al., 1995, 1998). As expected, IDE
caused a significant reduction of the monomeric 4 kDa A band (Fig.
4B). As reported previously, p3 levels were not
significantly affected by IDE (Qiu et al., 1998). This is consistent
with the IDE cleavage sites being outside or near the N terminal of p3 (A. Savafi and L. Hersh, personal communication; V. Chesneau and M. R. Rosner, unpublished observations). However, we observed neither
new formation of endogenous oligomeric species nor augmentation or
reduction of the preexisting oligomers accompanying degradation of
endogenous monomer by IDE. These data suggest that any IDE-mediated fragments of endogenous A are unlikely to act as seeds for the aggregation of monomeric endogenous A and that preformed
endogenous A oligomers are resistant to degradation by IDE.
A portion of IDE in neuronal cells is localized to the
cell surface
The data described so far suggest that in differentiated neuronal
cells, unlike microglia, A-degrading activity remains cell associated rather than being released into the medium. To investigate whether this finding can be explained by the neurons internalizing A
and then degrading it intracellularly, we incubated synthetic IA
(300 pM) with differentiated PC12 cultures for up to 24 hr at 37°C. At 0 and 24 hr, the CM were collected, and the cells were
washed twice in 2 ml of cold PBS. The washes were kept, and the cells
were then scraped and pelleted by centrifugation at 1000 × g for 5 min at 4°C. Total
125I counts in the medium, washes, and
cell pellets showed that only ~2% of the total counts were
associated with the cell lysate at both 0 and 24 hr. Trypsin
pretreatment of the cells for 15 min at 4°C removed approximately
one-half of these cell-associated counts. The large majority of the
total counts remained in the medium at 24 hr (data not shown). In a
number of experiments, we searched for intracellular IA at earlier
time points, specifically at 1, 5, and 18 hr. Again, the cell
lysate-associated IA counts were consistently found to be <2% of
the total (data not shown). These results indicate that the degradation
of A in our neuronal cultures and under our culture conditions takes
place almost exclusively in the extracellular space.
Although IDE has been classically localized to the cytosol and
peroxisomes, recent data suggest that it can also be biotinylated on
the plasma membrane of at least some non-neuronal cell types naturally
expressing the enzyme (Seta and Roth, 1997). Indeed, IDE has been
demonstrated previously on the cell surface, where it has been shown to
degrade insulin actively (Duckworth, 1979; Yokono et al., 1982;
Goldfine et al., 1984). It has also been reported that ~10% of the
total cellular IDE is associated with cell membranes (Duckworth, 1988).
To examine the localization of IDE in neuronal cells, the cytosolic and
membrane fractions of differentiated PC12 cells were isolated as
described in Materials and Methods, electrophoresed on 10% SDS-PAGE
gels, and immunoblotted with our polyclonal antibody IDE-1. As
expected, an immunoreactive band of the correct molecular weight for
IDE (110 kDa) was present in the cytosolic fraction of differentiated
PC12 cells (Fig. 5A). Surprisingly an immunoreactive band of ~115 kDa was also detected in
the 1% NP-40-solubilized membrane extracts and in total cell lysates,
although at lower levels. Further extraction of the membrane fraction
with 0.2% SDS resulted in enrichment of this upper band and the loss
of the 110 kDa IDE band (Fig. 5A). Whether this 115 kDa band
represents a novel, membrane-specific isoform of IDE is under
investigation. To confirm the surface localization of IDE,
differentiated and undifferentiated PC12 cells were incubated with a
membrane-impermeable biotinylating reagent, washed, and lysed, and the
IDE was immunoprecipitated using 9B12 antibody. Biotinylated IDE was
detected by blotting the immunoprecipitated IDE with neutravidin-HRP. A
biotinylated band of 110 kDa was clearly observed in the anti-IDE
immunoprecipitates. Simultaneous control experiments verified the
expected biotinylation of cell-surface APP (Haass et al., 1992) but not
that of the 85 kDa cytoplasmic protein PI3-kinase (Fig. 5B).
Surprisingly, the higher 115 kDa membrane-associated species showed no
detectable biotinylation. It may be that either this isoform is
specifically localized on intracellular membranes and does not reach
the cell surface or its plasma membrane levels are very low.
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Figure 5.
IDE can be detected on the surface of neuronal
cells. A, Equal amounts of protein (30 µg) of CHO
lysate as well as differentiated PC12 cytosol, lysate, membranes, and
membranes extracted with 0.2% SDS were electrophoresed on 10%
Tris/Glycine gels and Western blotted with the antibody IDE-1. Unlike
cytosol in which only the 110 kDa IDE is detected, the whole lysate and
membranes of PC12 cells also contain a higher (~115 kDa) IDE-reactive
band. Note that this higher band is also present in the CHO lysate.
B, Differentiated and proliferating PC12 cells were
biotinylated and lysed in STEN buffer, and equal amounts of protein
were immunoprecipitated with the monoclonal antibody 9B12 against IDE
(IDE), a monoclonal antibody specific to the cytosolic
protein PI3-kinase (PI3K), or a polyclonal
antibody (C7) to APP (APP). The immunoprecipitates were
separated by SDS-PAGE and probed with neutravidin-HRP. Note that the
large majority of biotinylated APP is the mature, N-plus O-glycosylated
form (130 kDa), as expected. DIFF, Differentiated;
UD, undifferentiated.
|
|
Cellular overexpression of IDE markedly reduces the levels of
secreted A
To confirm directly the effect of cellular expression of IDE on
the steady-state levels of naturally secreted A, we transfected 7PA2
cells with a pCMV expression vector encoding HA-tagged wild-type or mutant (inactive) IDE or with vector alone. The levels of endogenous A (Atotal and A42)
in CM were quantified 24 hr after transfection by sandwich ELISA.
Transfection efficiency was established by SDS-PAGE and autoradiography
of the cell extracts using a specific monoclonal antibody to the HA
epitope as well as our polyclonal IDE-1 antibody. IDE-transfected cells
had a more than threefold increase in total IDE levels compared with
those transfected with vector alone (Fig.
6A). Cells
overexpressing wild-type HA-IDE consistently exhibited a >70%
decrease in the levels of total A (principally
A40) in their media when compared with cells expressing either the mutant IDE or vector alone (Fig.
6B; means of n = 7 experiments). In a
manner similar to that of Atotal, A42 levels in the CM of the wild-type IDE
transfectants were ~50% of those of vector- or mutant
IDE-transfected cells (Fig. 6B). When we established
stable APP and IDE double-transfected 7PA2 clones, these showed a more
than twofold increase in their total IDE levels but equal APP levels
when compared with plain 7PA2 cells (Fig. 6A, iii).
In a manner similar to that of the transient transfectants, the stable
IDE-transfected 7PA2 cells showed a reduction in
Atotal and A42 levels
in their medium of 60 and 40%, respectively, within 4 hr compared with
untransfected 7PA2 cells (Fig. 6B; means of
n = 3 experiments). These data clearly demonstrate that
IDE is responsible for A degradation in intact cells and that it can
efficiently degrade naturally secreted A40 and
A42.
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|
Figure 6.
Expression of IDE markedly reduces
steady-state levels of endogenous A in the media. 7PA2 CHO cells
were grown to 80% confluency before being transfected overnight with a
pCMV expression vector encoding HA-tagged wild-type or mutant IDE or
with vector alone. After transfection, the cells were refed with growth
medium. In addition, 7PA2 CHO cells that were stably transfected with
IDE were grown to 90% confluency and then refed with growth medium for
4 hr. A, Transient and stable transfection efficiency
was quantitated by SDS-PAGE and autoradiography of the cell extracts
using a specific monoclonal antibody to the HA epitope
(i) as well as our polyclonal antibody IDE-1
(ii). APP background expression was quantitated using
the specific polyclonal antibody C7 (iii). Note that
endogenous IDE in the vector lane is solely detected by
IDE-1, not anti-HA, as expected. B, Levels of endogenous
Atotal (primarily A40) and
A42 in the conditioned media of transiently and stably
transfected cultures as indicated were established by ELISA.
Vertical bars indicate means (± SEM).
Values were normalized to vector alone or 7PA2 alone as 100%.
C, 7PA2 cells were transiently transfected as described
above and then starved in methionine-free medium for 2 hr before
labeling with 100 µCi of [35S]me-thionine
overnight. Conditioned media were immunoprecipitated with R1282 and
assayed by SDS-PAGE autoradiography. In agreement with the ELISA
results, wt IDE expression markedly reduced the levels of secreted
monomeric A. Note that the 6 kDa anomalously migrating monomeric
A isoform behaves like the 4 kDa monomer, in contrast to the 8 kDa
dimer, which is primarily resistant to IDE. p3 is also resistant to IDE
as reported previously (Qiu et al., 1998). D, Lysates
from five 10 cm dishes of CHO cells stably transfected with APP or with
both IDE and APP were precleared with APP antibodies C7 and B5,
immunoprecipitated with R1280, and blotted with the monoclonal A
antibody 6E10. mut, Mutant.
|
|
To confirm these findings, 7PA2 cells were
[35S]Met labeled after transfection, and
the conditioned media were immunoprecipitated with the A antibody
R1282. In agreement with the ELISA results, SDS-PAGE and gel
fluorography showed that endogenous A levels in the medium of wt
IDE-transfected cells were markedly reduced, compared with that of the
cells transfected with mutant IDE or with vector alone (Fig.
6C). As expected, the smaller p3 peptide (residues 18-40/42
of A) was degraded much less. To examine any effect that IDE
overexpression might have on the levels of intracellular A, we
immunoprecipitated IDE/APP cell lysates with R1282 after transfection
and electrophoresed the respective precipitates on 16% SDS-PAGE
Tris/Tricine gels. After transfer to nitrocellulose, A was
visualized by immunoblotting with the monoclonal antibody 6E10. IDE
expression reduced intracellular A monomer levels by ~58% (Fig.
6D). Results were quantitated by computer
densitometry (data not shown). This result suggests that degradation of
A by IDE occurs intracellularly as well as extracellularly.
| |
DISCUSSION |
Converging lines of evidence from many laboratories support the
hypothesis that cerebral deposition of A peptides is an early, invariant, and necessary step in the pathogenesis of AD. As a result,
there is growing interest in decreasing the cerebral A burden as a
therapeutic or preventative approach to the disease. Quantitative
biochemical studies suggest that IDE, a conserved neutral thiol
metalloendopeptidase involved in the degradation of certain peptide
hormones, is a major A-cleaving protease in brain tissue (Kurochkin
and Goto, 1994; McDermott and Gibson, 1997). We conducted previously an
unbiased search for endogenous A-degrading proteolytic activities in
the media of several non-neuronal cell types and found that the
principal such activity represented IDE, with particularly robust
levels observed in microglial cell media (Qiu et al., 1997, 1998). More
important, immunodepletion of IDE from the microglial medium markedly
decreased the degradation of A. Evidence that a metalloprotease with
the properties of IDE is released into the medium of primary rat
microglial cultures has also been reported (Mentlein et al., 1998).
In the present study, we examined differentiated PC12 cells and 21-d
primary brain cultures and showed that these neuronal cells degrade
secreted and synthetic A via a 110 kDa protease that has the same
inhibitor and substrate properties and immunoreactivity as IDE.
Interestingly, this IDE-mediated A degradation was confined to whole
neuronal cultures and was absent from the conditioned media of the
differentiated cells (Figs. 1, 2). In agreement with this finding,
surface biotinylation revealed the presence of intact 110 kDa IDE on
the plasma membrane of neuronal cells (Fig. 5). Western blotting
further demonstrated that IDE was almost absent from the CM of
differentiated PC12 cells, whereas it was abundant in the CM of BV-2
microglia and CHO cells. It appears, therefore, that the locus in which
IDE effects degradation of extracellular A can vary by cell type.
One explanation might be that different cell types use different
isoforms of IDE molecules, including a form that can be released and
one that is cell associated. To address this possibility we used
proliferating and differentiated PC12 cells and examined the ability of
their CM alone to degrade A. Only the proliferating
(undifferentiated) PC12 cells exhibited A-degrading activity in
their media (Fig. 2). Accordingly, immunoblotting with the IDE antibody
IDE-1 demonstrated the presence of intact IDE in the CM of just the
proliferating PC12 cells (Fig. 3). It therefore appears that mature
neuronal cells lose their ability to release IDE. The downregulation of
an as yet unidentified IDE-releasing protease from the surface of
differentiated neurons is one possible explanation for this difference.
Alternatively, IDE might become membrane anchored via its association
with other membrane proteins or lipids. The upregulation of such
proteins or lipids during differentiation might further explain the
lack of IDE release in differentiated PC12 cells.
It has been reported that synthetic A can be internalized into PC12
cells and degraded to some extent intracellularly (Burdick et al.,
1997). However, when we assayed for internalized A in our PC12
cultures, we did not observe any significant internalization and
degradation of IA under our culture conditions. The difference between these results may have several explanations. Serum is present,
although at low concentrations, in our differentiated PC12 cultures.
Regarding A peptide length, we only examined iodinated A1-40, which has been shown not to accumulate
nearly as much as A1-42 (Burdick et al.,
1997; Morelli et al., 1999). Finally, we used picomolar concentrations
of A, which may have reduced its absorption rate, because this has
been shown to be concentration dependent. However, although A
degradation in our neuronal cultures appears to occur extracellularly,
we cannot exclude the possibility that a portion of A is
internalized and degraded intracellularly.
It has been reported that the first putative start codon of IDE is not
a functional one and that initiation of translation occurs at the
second ATG codon located 123 nucleotides downstream (Baumeister et al.,
1993; Perlman et al., 1993; Perlman and Rosner, 1994). Here we report
the detection of a slightly larger (~115 kDa) IDE-immunoreactive
species in cell lysates that seems to reside predominantly on
membranes. Whether this is a product of translation at the first
putative start codon is under study and will take some time to
determine. Although we do not yet know the degradative function of this
form, we speculate that its slightly larger size and its marked
enrichment in membranes versus cytosol indicate that it may be a
principal membrane-anchored isoform that degrades substrates on the
plasma membrane. In any case, it is not yet clear how IDE could be
routed to the cell surface and extracellular space, because it appears
to lack a classical hydrophobic signal peptide (Baumeister et al.,
1993). However, a number of prokaryotic and eukaryotic proteins have
been shown to be trafficked via nonclassical pathways that involve the
ATP-binding cassette transporters {for review, see Kuchler and
Thorner, 1992). Examples include the yeast a-factor, the cytokines
IL1 and IL1, and certain growth factors like FGF.
Our cell culture data suggest that IDE is the major A-degrading
activity in cultured microglia and neuronal cells. To confirm that IDE
can actively degrade naturally secreted A and to quantify this
effect, we transfected either wild-type IDE or an inactive form bearing
a mutation (E111Q) of the active site into CHO cells stably expressing
APP. Both transient and stable expression of wild-type IDE markedly
reduced the steady-state levels of A40 and
A42 in the media of our 7PA2 cells, whereas
expression of the mutant enzyme had no effect. Immunoprecipitation and
SDS-PAGE autoradiography of naturally secreted A from IDE- and
control-transfected cells confirmed the ELISA results. Interestingly,
the SDS-stable oligomers of A that we have previously described in
these cells were not decreased. This finding suggests that IDE could
play an important role in regulating the levels of A monomers, but once aggregation begins, the protease would be unlikely to effectively control the progressive accumulation of insoluble A species observed in the brains of AD subjects. Taken together, these data suggest a
quantitatively important role for the degradation of secreted A by
IDE. However, recent reports have suggested that other A-degrading metalloproteases might operate independently of IDE in some cell types
and contribute to the overall regulation of extracellular A levels
(Yamin et al., 1999). An endogenous 14 kDa inhibitor of IDE has been
identified (Ogawa et al., 1992). It is possible that like IDE, its
release is also cell type specific, potentially explaining
the lack of A degradation in the CM of some cells in the presence of IDE.
The role of IDE, if any, in the pathogenesis of the excessive cerebral
accumulation of A that occurs in AD remains to be elucidated. In
this regard, the CSF-to-plasma insulin ratio has been shown to be
reduced in AD patients compared with nondemented controls (Stolk et
al., 1997; Craft et al., 1998). Brain insulin levels have also been
reported to decrease with age. However, no significant difference in
brain insulin levels between AD and aged-matched control patients has
been reported to date (Frolich et al., 1998). Interestingly, insulin
receptors are upregulated in AD brain, suggesting some impairment of
the signal transduction pathway between ligand and receptor.
Identifying how the activity of IDE is regulated in brain and how this
changes with age and in AD could provide new insights into the
mechanisms of A accrual in the brains of sporadic AD cases. In this
respect, it was reported recently that IDE immunoreactivity is more
prominent in AD than in normal human brain and can be localized to
certain cortical neurons and to senile plaques (Bernstein et al.,
1999).
Many studies have shown that soluble A can be physiologically
secreted from a variety of neural and non-neural cells (Selkoe, 1998).
How this monomer accumulates sufficiently in the extracellular space to
form first diffuse and then fibrillar deposits is not well understood.
In this regard, we showed in a previous study that IDE appeared to
potentiate the oligomerization of synthetic iodinated A (Qiu et al.,
1998). In the present study, we show that IDE readily cleaves naturally
secreted A without noticeably promoting its oligomerization.
Transfection of CHO cultures with wt IDE or their incubation with the
purified recombinant enzyme led to substantial degradation of secreted
A but did not appear to affect the levels of the preexisting
endogenous A oligomers we have characterized previously in these
cultures (Podlisny et al., 1995, 1998) (Fig. 6C,D). These
data, in combination with additional studies of the recombinant enzyme
currently underway (Chesneau, Vekrellis, Rosner, and Selkoe,
unpublished observations), suggest that the cleavage products of A
from natural IDE are unlikely to act as "seeds" for the aggregation
of the endogenous secreted A peptide and that the C-terminal
fragments produced from the cleavage of endogenous A by IDE do not
themselves oligomerize under the conditions tested. It thus appears
that synthetic and endogenous A behave differently after cleavage by
IDE. Interestingly, the intracellular pools of monomeric A were also
decreased by IDE overexpression, suggesting that the enzyme can degrade
intracellular monomeric A. We are currently carrying out
cell-fractionation experiments to identify the intracellular
compartment(s) in which A degredation by IDE occurs. Of particular
interest is our observation that whereas monomeric A40
and A42 are both avidly degraded by IDE, stable
oligomers in the same lysates and media are resistant to such degradation.
| |
FOOTNOTES |
Received Sept. 22, 1999; revised Nov. 17, 1999; accepted Dec. 10, 1999.
This work was supported by National Institutes of Health Grants AG
12749 (D.J.S.) and N 533858 (M.R.R.).
Correspondence should be addressed to Dr. Dennis J. Selkoe, Center for
Neurological Diseases, Harvard Institutes of Medicine 730, 77 Avenue
Louis Pasteur, Boston, MA 02115. E-mail: Selkoe{at}cnd.bwh.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
