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J Biol Chem, Vol. 273, Issue 49, 32730-32738, December 4, 1998
From the Department of Neurology and Program in Neuroscience,
Harvard Medical School and Center for Neurologic Diseases, Brigham and
Women's Hospital, Boston, Massachusetts 02115-5716, the
Excessive cerebral accumulation of the 42-residue
amyloid Converging lines of evidence support the hypothesis that
progressive cerebral accumulation of the 40-42-residue amyloid
The properties of the A In this study, we have compared the properties of the extracellular,
A Cell Culture--
Mouse BV-2 microglial cells were routinely
cultured in RPMI 1640 with 10% fetal bovine serum (FBS). To
characterize A Assaying Degradation of Endogenous A Preparation of Cell Cytosol and Human Brain
Extracts--
Confluent cultures of BV-2 or human kidney 293 cells
were detached from 15-cm dishes with 20 mM EDTA in ice-cold
phosphate-buffered saline and pelleted. These cells or aliquots of
frozen human brain tissue were suspended in homogenization buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 0.25 M
sucrose, supplemented with a protease inhibitor mixture) and disrupted
using 10 strokes in a Dounce homogenizer followed by four passages
through a 25-gauge needle. Nuclei and unbroken cells were pelleted by
centrifugation at 3000 × g for 10 min. The pellets
were resuspended in 1.5 ml of homogenization buffer and centrifuged at
3000 × g for 10 min. The postnuclear supernatants from
both centrifugation steps were combined and centrifuged at 80,000 × g for 1 h to separate the cytosol and membrane fractions.
Western Blotting of Insulin-degrading Enzyme--
Equal volumes
of (a) cytosols prepared from BV-2 cells or human kidney 293 cells, (b) BV-2 CM (concentrated to a volume equal to that
of the BV-2 cytosol by NH4SO4 precipitation),
(c) human CSF, or (d) human CSF concentrated
10-fold by a Centricon 30 (Amicon) filter were electrophoresed on 10%
Tris/glycine SDS-PAGE gels and transferred to polyvinylidine difluoride
membrane (10). The SuperSignal HisProbe Western blotting kit (Pierce)
was used for immunoblotting IDE according to the manufacturer's
instructions. Polyclonal antibody 2BS, raised specifically against IDE
(7), was used at 1:10,000 dilution.
Assaying Degradation of 125I-A Characterization of Cell Viability--
BV-2 cells were
routinely grown at 37 °C in RPMI, 10% FBS. At increasing intervals
up to 24 h, CM were collected and filtered through 0.22-µm
cellulose acetate, and the amount of lactate dehydrogenase in each
sample was measured by an in vitro toxicology assay (Sigma) according to instructions. In addition, BV-2 cells were grown in the
presence or absence of 10% FBS at 37 °C for 24 h followed by
the LIVE/DEAD viability/cytotoxicity assay (Molecular Probes, Inc.,
Eugene, OR) according to the manufacturer's instructions. In this
assay, polyanionic calcein is retained within live cells, producing an
intense uniform green fluorescence (excitation/emission = 495/515
nm). Ethidium bromide, normally excluded, enters cells having damaged
membranes and undergoes a 40-fold enhancement of fluorescence upon
binding to nucleic acids, thereby producing a bright red fluorescence
in permeable cells (excitation/emission = 495/635 nm). As a
positive control in these experiments, BV-2 cells were briefly treated
(5 min) with digitonin (0.05%) to permeabilize them.
Purification of the A The A
To further characterize the protease released by the microglial cells,
we added the principal substrate of IDE, insulin, to the reaction
mixture to determine whether insulin can compete out the A
To demonstrate directly the presence of IDE in the BV-2 CM, we used an
IDE-specific polyclonal antibody, 2BS (11), to immunoblot the CM.
Aliquots of human 293 cytosol (which contains abundant IDE and serves
as a positive control), BV-2 cytosol, and BV-2 CM (concentrated by
NH4SO4 precipitation to a volume equal to that
of the BV-2 cytosol sample) were electrophoresed on 10% SDS-PAGE gels
and immunoblotted with 2BS (Fig. 1C). The mobilities of
human IDE (lane 1) and mouse IDE
(lanes 2 and 3) are slightly
different. In all three samples, a characteristic 110-kDa protein was
specifically immunolabeled, suggesting that BV-2 cells release intact
IDE into their medium under our culture conditions.
IDE Purified from Microglial Cell Medium Mediates the Degradation
of A
To prove unequivocally that IDE is the protease in CM responsible for
the degradation of A IDE Mediates A
Because the loss of intact IA
To confirm that this release of IDE into the culture medium occurs
while BV-2 cell membranes are intact, we simultaneously assayed lactate
dehydrogenase activity in the CM of cultures conditioned in 10% FBS
and found no significant release of this cytoplasmic enzyme at time
points up to 24 h (Fig.
4F). To prove further the intactness of the plasma membrane, the LIVE/DEAD viability/cytotoxicity assay was performed on BV-2 cells cultured in the presence (Fig. 4,
A and B) or absence (Fig. 4, C and
D) of 10% FBS for up to 24 h. We observed no
significant cell death (by calcein AM signal and ethidium bromide
staining) even in the absence of serum, whereas sister cultures
permeabilized briefly with digitonin (0.05%) were clearly positive in
this assay (Fig. 4E). Taken together, these several assays
provide no evidence that the release of proteolytically active IDE from
the BV-2 microglial cells occurs as a secondary result of cell
injury.
Detection of Intact IDE in Fresh Human Cerebrospinal
Fluid--
Although there have been reports of the release of IDE from
cells (14, 15) and the presence of intact, 110-kDa IDE at the cell
surface (16), it has generally been assumed that IDE is restricted to
the cytosol and peroxisomes and is not normally released from cells (7,
17). However, our results in the previous section show that readily
detectable amounts of enzymatically active IDE are found in the medium
of intact BV-2 cells under normal culture conditions, including in
serum-containing cultures. Another way to confirm that a protein is
released from cells under entirely physiological circumstances in
vivo is to examine normal, essentially acellular biological fluids
such as fresh human CSF for the presence of the protein. To this end,
we probed both fresh and frozen lumbar CSF samples collected from
living nondemented subjects (n = 11), patients with AD
(n = 6), and one patient with a non-AD dementia by
Western blotting with a well characterized IDE antibody, 2BS. In all
CSF samples examined (exemplified in Fig.
5A), a full-length 110-kDa IDE
protein was specifically detected, and it co-migrated with the
characteristic IDE band found in the cytosol of human 293 cells (Fig.
5A, lane 7). These results were confirmed using another human IDE antibody, 9B12 (not shown). The
amounts varied somewhat from sample to sample but were not obviously
different in the small number of AD CSF samples available to us to
date. Next, we probed soluble fractions prepared from human cortex of
one non-AD and three AD subjects. Western blotting showed that 110-kDa
IDE was present in all four samples and comigrated with IDE in 293 cytosol (Fig. 5B). We conclude that intact IDE protein is
normally present in human CSF and brain.
IDE Activity in Microglial Conditioned Medium Is Associated with
the Formation of Apparent Oligomers of A Evidence from many laboratories suggests that decreasing A In vitro biochemical studies examining the proteolysis of
synthetic A Insulin-degrading Enzyme Released from Intact Cells Can Degrade
A
Although our data cannot formally exclude the possibility that another
protease is activated via cleavage by IDE and then mediates the A
IDE has been shown to play a major role in the degradation of insulin,
and several other small peptide hormones can also serve as substrates,
including atrial natriuretic peptide, transforming growth factor A Potential Role for IDE in the Oligomerization of A
Serum insulin levels have been reported to rise with age in humans
(34), and insulin-like growth factor-2 is significantly increased in AD
CSF compared with controls (35). These molecules can serve as
substrates of IDE and could thus interfere competitively with the
efficient degradation and clearance of A We thank Dr. Richard Roth (Stanford
University) for helpful suggestions and discussions and for the
generous gift of monoclonal antibody 9B12.
*
This work was supported by National Institutes of Health
Grant AG12749 (to D. J. S.) and National Institute on Drug Abuse Grants DA 02243 and DA 07062 (to L. B. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Brigham and
Women's Hospital, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.:
617-525-5200; Fax: 617-525-5305; E-mail:
Selkoe{at}cnd.bwh.harvard.edu.
The abbreviations used are:
A 2
A. Safavi and L. Hersh, unpublished data.
Insulin-degrading Enzyme Regulates Extracellular Levels of
Amyloid 
-Protein by Degradation*
, Ben May Institute for Cancer Research, University of
Chicago, Chicago, Illinois 60637, and the § Department of
Biochemistry, University of Kentucky, Chandler Medical Center,
Lexington, Kentucky 40536-0084
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-protein (A) is an early and invariant step in the
pathogenesis of Alzheimer's disease. Many studies have examined the
cellular production of A from its membrane-bound precursor,
including the role of the presenilin proteins therein, but almost
nothing is known about how A is degraded and cleared following its
secretion. We previously screened neuronal and nonneuronal cell lines
for the production of proteases capable of degrading naturally secreted A under biologically relevant conditions and concentrations. The
major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and
characterized the protease and find that it is indistinguishable from
insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that
degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic A
at picomolar to nanomolar concentrations was completely inhibited by
the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody
removed its A-degrading activity. IDE was present in BV-2 cytosol,
as expected, but was also released into the medium by intact, healthy
cells. To confirm the extracellular occurrence of IDE in
vivo, we identified intact IDE in human cerebrospinal fluid of
both normal and Alzheimer subjects. In addition to its ability to
degrade A, IDE activity was unexpectedly found be associated with a
time-dependent oligomerization of synthetic A at
physiological levels in the conditioned media of cultured cells; this
process, which may be initiated by IDE-generated proteolytic fragments
of A, was prevented by three different IDE inhibitors. We conclude
that a principal protease capable of down-regulating the levels of
secreted A extracellularly is IDE.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-proteins (As)1 is an
early, invariant, and necessary step in the pathogenesis of
Alzheimer's disease (AD). As a result, there is growing interest in
decreasing cerebral A levels as a therapeutic and preventative approach to the disease. A is generated by endoproteolysis of the
-amyloid precursor protein (APP) and secreted constitutively by most
mammalian cells throughout life. Whereas many studies have examined the
proteolytic processing of APP and the mechanisms of A production,
almost nothing is known about how A peptides are normally degraded
and cleared following their secretion. We recently screened the
conditioned media of several different cell lines for A-degrading
activity and found that the principal such activity was conferred by a
nonmatrix metalloprotease that was released by microglial cells and
other cells and efficiently degraded both endogenous and synthetic A
(1). The release of the protease from microglial cells was augmented by
activating the cells with lipopolysaccharide, suggesting that
physiological and pathological stimuli may regulate the degree of A
degradation in extracellular fluids (1).
-degrading metalloprotease we described are
reminiscent of those of insulin-degrading enzyme (IDE), a
metalloendopeptidase that can cleave a variety of small peptides, including insulin, glucagon, atrial natriuretic factor, and
insulin-like growth factors I and II (reviewed in Refs. 2 and 3). IDE was recently identified as the protease responsible for the conversion of -endorphin to 
-endorphin (4). The protease was also recently reported to be present in human brain tissue, where it was shown to
bind and cleave synthetic A peptides at neutral pH (5, 6). However,
the latter studies relied on homogenized brain, so that IDE released
from the cytosol could have conferred the A-degrading activity
nonphysiologically. Indeed, the two major reported locations of IDE in
cell types studied to date, the cytosol and peroxisomes (3, 7, 8),
raise the question of whether and how this protease could function to
degrade endogenous A, considering that the peptide has not been
found in these two subcellular sites.
-degrading metalloprotease, which we previously described (1), with
those of IDE and find that the two are indistinguishable. Partial
purification of the protease from the conditioned medium of a
microglial cell line demonstrates that the A-degrading activity is
IDE. This conclusion is confirmed by immunodepletion of IDE from
conditioned medium. We further report experiments that suggest that the
enzyme is released into the extracellular fluid by intact microglial
cells that show no abnormal permeability. That such release occurs
in vivo in humans is confirmed by the immunochemical detection of authentic, 110-kDa IDE in freshly obtained lumbar cerebrospinal fluid (CSF). In addition to a role for IDE in degrading secreted A, we find that a time-dependent
oligomerization of synthetic A in the conditioned media of cultured
cells is completely blocked by three different inhibitors of IDE
activity. This finding suggests that IDE is capable of regulating the
level of monomeric A by both degradation and oligomerization. The
implications of these findings for the fate of A monomers and the
role of A accumulation in AD are discussed.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-degrading activity in their conditioned media (CM),
the cells were conditioned in either fresh RPMI 1640/10% FBS or the
serum-free medium, N2, for 16-18 h, and the CM was passed through a
0.22-µm filter to remove floating cells. Chinese hamster ovary (CHO)
cells stably transfected with APP770 cDNA containing the V717F
FAD mutation (7PA2 cells) (1) were cultured in Dulbecco's modified
Eagle's medium with 10% FBS and G418 (200 µg/ml).
by
Immunoprecipitation--
Confluent monolayers of 7PA2 cells in 10-cm
dishes were preincubated for 30 min in methionine- and serum-free
medium and labeled for 4 h with 300 µCi of
[35S]methionine. The labeled CM were centrifuged at
2100 × g for 30 min and used immediately or stored at
70 °C. Two ml of labeled CM were mixed with 2 ml of unconditioned
N2 medium (control) or BV-2 CM or fractions of the latter obtained
during IDE purification (see below), either in the absence or presence
of N-ethylmaleimide (NEM) or 1,10-phenanthroline or human
insulin (Sigma) and incubated at 37 °C for 16 h. The amount of
labeled A remaining in each sample was assessed by
immunoprecipitation with the high affinity A polyclonal antibody
R1282 followed by 10-20% Tris/Tricine SDS-PAGE and gel fluorography
(9).
by Gel Fluorography
or Trichloroacetic Acid Precipitation--
125I-A
(IA) (specific activity, ~2000 Ci/mmol) was purchased from
Amersham Pharmacia Biotech, dissolved in H2O, aliquoted, and stored at 20o until use to avoid freeze/thawing.
10,000 cpm were added per ml of medium to whole cultures or their
collected CM. After increasing intervals of incubation at 37 °C,
aliquots of the media were removed, centrifuged, and examined by
10-20% Tris/Tricine SDS-PAGE and gel fluorography to observe any
degradation or oligomerization of A. At the same time, 90 µl of
each aliquot was mixed with 110 µl of 15% trichloroacetic acid and
incubated on ice for 15 min to precipitate undegraded IA. The
trichloroacetic acid-precipitated samples were centrifuged (16,000 × g, 10 min), and the amounts of label in the supernatant
(representing degraded products) and the pellet (representing intact
A) were counted. Because partially degraded peptide products of A
may still be precipitated by trichloroacetic acid, this assay
underestimates the absolute level of substrate degradation compared
with assaying by SDS-PAGE/gel fluorography.
-degrading Activity from Conditioned
Media--
BV-2 cells were cultured in RPMI 1640, 10% FBS in T100
flasks for 3 days. The cells were collected, washed, changed to
serum-free N2 medium, and further cultured for 16 h at 37 °C.
Pooled CM (~1 liter) were filtered through 0.22-µm cellulose
acetate to remove floating cells. The CM were precipitated with 40%
NH4SO4, and the resultant supernatant was
further precipitated with 60% NH4SO4. The
latter precipitate was dissolved in ~100 ml of 50 mM
Tris-HCl, pH 7.5, and dialyzed in the same buffer overnight. The sample was assayed for A-degrading activity by immunoprecipitation of endogenous A with R1282 as described above and then applied to a
Bio-Scale Q (Bio-Rad) anion exchange column, which was preequilibrated with 50 mM Tris-HCl, pH 7.5. The column was eluted with a
linear NaCl gradient from 10 mM to 1 M run at a
flow rate of 0.5 ml/min. Aliquots of the resultant fractions (1.5 ml)
were assayed for A-degrading activity either by immunoprecipitation
of [35S]Met-labeled endogenous A or by trichloroacetic
acid precipitation of synthetic IA, as described above. Fractions
containing the most A-degrading activity were pooled and
concentrated to 200 µl by Centricon 30, followed by chromatography on
a Superose 12 (Amersham Pharmacia Biotech) gel filtration column. This
column was equilibrated with 50 mM phosphate buffer, pH
7.4, and the sample was eluted in the same buffer at a flow rate of 0.3 ml/min. Fractions (0.9 ml) were monitored for protein by UV absorbance at 280 nm, for A-degrading activity and A oligomer formation as
described above, and for protein composition by SDS-PAGE followed by
Coomassie staining. The fractions were also assayed for IDE by Western
blotting with antibody 2BS.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-degrading Metalloprotease Released into Microglial
Conditioned Medium Has the Properties of Insulin-degrading
Enzyme--
We recently reported that the clearance of secreted A
peptides from the media of several neural and nonneural cell lines was
principally mediated by a nonmatrix metalloprotease released by these
cells (1). Because the properties of the enzyme we described were
similar to those of insulin-degrading enzyme, we further examined the
metalloprotease with several reagents known to inhibit IDE. Using BV-2
microglial cells, which release higher levels of the A-degrading
metalloprotease than other cell types we screened (1), we examined the
effects of the sulfhydryl-modifying reagent NEM. Confluent BV-2 cells
were washed and changed to the serum-free medium, N2, for conditioning
at 37 °C for 16 h. The CM were then filtered and collected. CHO
cells stably transfected with APP770 cDNA containing
the V717F mutation were metabolically labeled with
[35S]methionine as described (1), and the resultant
medium, containing abundant labeled A and p3 peptides, was incubated
with the filtered BV-2 CM (or with unconditioned N2 medium as a
control) for 24 h at 37 °C in the presence or absence of NEM.
(p3 is a peptide comprising residues 17-40 (or 17-42) of A that
results from constitutive proteolysis of APP by 
- and then
-secretases, whereas A (residues 1-40 or 1-42) results from
constitutive proteolysis of APP by - and then -secretases.)
Subsequent immunoprecipitation with an A antibody (R1282) and gel
fluorography revealed the expected marked decrease of A in the BV-2
CM, and this was completely inhibited by 100 µM NEM (Fig.
1A). This result, together
with the previously observed inhibition by 1,10-phenanthroline (1), suggests that A degradation in BV-2 CM is specifically mediated by a
thiol metalloprotease such as IDE. In parallel, we examined the
degradation of iodinated insulin (10 pM) in the BV-2 CM and observed a time-dependent loss of intact insulin by
SDS-PAGE that was similar to the degradation of iodinated
A1-40 at the same concentration. The insulin
degradation was likewise inhibited by 1,10-phenanthroline and NEM (data
not shown).
View larger version (27K):
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Fig. 1.
Loss of secreted A is mediated by
insulin-degrading enzyme present in the extracellular fluid of cultured
microglial cells. A, aliquots of CM of 7PA2
(APP-transfected CHO) cells labeled with [35S]Met for
4 h in serum-free medium were incubated (37 °C, 16 h) with
3 ml of either unconditioned N2 medium (lane 1)
or BV-2 CM (lanes 2-4) in the absence
(lanes 1 and 2) or presence of 10 µM (lane 3) of 100 µM
(lane 4) NEM. The incubated samples were then
immunoprecipitated with A antibody R1282 and electrophoresed on
Tris/Tricine gels followed by autoradiography. Mass markers (kDa),
A, and p3 are all indicated. B, aliquots of labeled 7PA2
CM were incubated with 3 ml of unconditioned N2 medium (lane
1) or BV-2 CM (lanes 2-7) in the
presence of increasing amounts of human insulin as indicated
(lanes 3-6) or in 1 mM
1,10-phenanthroline (lane 7) at 37 °C for
16 h and assayed as in A. C, equal volumes
of human kidney 293 cell cytosol (lane 1), BV-2
cell cytosol (lane 2), and BV-2 CM concentrated
to the same volume as BV-2 cytosol by 60%
NH4SO4 precipitation and dialysis
(lane 3) were electrophoresed on 10%
Tris-glycine gels and blotted with the IDE antibody 2BS. ×, ~110-kDa
insulin-degrading enzyme.
-degrading
activity. Degradation of endogenous A in BV-2 CM was found to be
progressively inhibited by increasing amounts of insulin between 100 nM and 10 µM (Fig. 1B). One
µM insulin inhibited degradation of A by ~50%, and
10 µM completely prevented the loss of A. At the
latter dose, insulin was as effective in this assay as 1 mM
1,10-phenanthroline (Fig. 1B), which we previously found to
inhibit the A-degrading metalloprotease completely (1). To support
the conclusion that it is IDE that degrades A in BV-2 CM, we added
other known substrates of IDE such as glucagon, transforming growth
factor-, or brain natriuretic peptide to the reaction mixture and
showed that each of these inhibited the A-degrading activity, but to
a lesser extent than insulin did on a molar basis (data not shown). The
p3 peptide is relatively resistant to degradation by the protease (Fig.
1, A and B), as reported previously (1). In this
regard, the cleavage sites in synthetic A1-40 generated by IDE
purified from the cytosol of CHO cells have recently been localized to
residues 4-5, 13-14, 14-15, and
20-21,2 potentially
explaining the relative resistance of the p3 peptide (A 17-40/42)
to IDE-mediated degradation.
--
Because the above data suggested that IDE or an IDE-like
protease can efficiently degrade A, we substantially purified and further characterized the activity from ~1 liter of CM of BV-2 cells
conditioned in serum-free N2 medium at 37 °C for 16 h. The CM
was subjected to sequential fractionation by ammonium sulfate precipitation, anion exchange chromatography on a Bio-Scale Q column,
and size exclusion chromatography on a Superose 12 column (Table
I). A-degrading activity was monitored
at each step by immunoprecipitation/gel fluorography of
[35S]Met-labeled 7PA2 medium (for analyzing endogenous
A) or by the degradation of synthetic IA followed by
trichloroacetic acid precipitation. Any apparent oligomers of the IA
(bands migrating above the 4-kDa monomer position) were simultaneously
visualized by SDS-PAGE (12). The final fractions obtained from the size exclusion chromatography (Fig.
2A) were incubated with IA,
and those fractions that caused degradation in the absence but not the
presence of 1,10-phenanthroline were identified by trichloroacetic acid
precipitation (Fig. 2B). Comparison of this proteolytic
activity curve with the size standards showed that the A-degrading
moiety eluted from the column with a molecular mass of ~100-150 kDa
(Fig. 2, A and B). The A-degrading activity of
these fractions was further confirmed using the A
immunoprecipitation assay on endogenous ([35S]Met-labeled) A (Fig. 2C). Western
blotting with the 2BS antibody specifically identified intact 110-kDa
IDE in the active fractions (Fig. 2D), and its amount in
each active fraction correlated closely with the relative
A-degrading activity of the fractions. Coomassie staining of the
active fractions revealed the 110-kDa IDE band plus three or four other
light bands (not shown), indicating marked enrichment but not full
purification of IDE by this method. However, all of the other bands
were equally or more abundant in fractions that had low or no
A-degrading activity. When the active column fractions (fractions
9-15) were incubated with IA followed by SDS-PAGE/fluorography, we
observed the decrease in the IA monomer as well as an apparent
SDS-stable oligomerization of some of the peptide (Fig. 2E)
(see below). Taken together, the results in Fig. 2 establish that the
amount of IDE present in column fractions during purification
correlates very closely with the degree of A degradation.
Summary of partial purification of insulin-degrading enzyme from BV-2
CM
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Fig. 2.
Purification of IDE from BV-2 conditioned
medium. A -degrading activity was substantially purified from
BV-2 CM as described under "Experimental Procedures" and Table I.
A, protein content per fraction (0.9 ml) from the final size
exclusion column was determined by UV spectrophotometry at 280 nm. The
approximate elution positions of marker proteins thyroglobulin (570 kDa), ovalbumin (158 kDa), myoglobin (44 kDa), -globulin (17 kDa),
and vitamin B12 (1.3 kDa) are indicated. B,
A-degrading activity was assayed by incubating IA in each
fraction for 16 h at 37 °C in the absence (open
squares) or presence (closed squares)
of 1,10-phenanthroline followed by trichloroacetic acid precipitation;
total cpm in the trichloroacetic acid pellets is graphed. C,
aliquots (0.5 ml) of fractions 9-15 were assayed by incubating with 3 ml of [35S]Met-labeled 7PA2 CM for 16 h at 37 °C
and immunoprecipitating with A antibody R1282. D,
fractions 9-15 were examined by immunoblotting with IDE antibody 2BS.
The position of the 97-kDa marker and the characteristic position of
IDE are indicated. E, fractions 9-15 were incubated with
IA for 16 h at 37 °C followed by 10-20% Tris/Tricine
SDS-PAGE and autoradiography.
, we performed immunodepletion of CM with an IDE
monoclonal antibody, 9B12 (5); this completely removed the
A-degrading activity from the medium of CHO cells (Table
II). This antibody cannot be used with
BV-2 CM, because it reacts very poorly with murine IDE, but we reported
previously that CHO CM has an A-degrading protease that is
qualitatively indistinguishable from that in BV2 CM (1). In parallel
with the removal of A-degrading activity, immunodepletion of IDE
simultaneously removed insulin-degrading activity (Table II).
Immunodepletion performed in the absence of the IDE antibody resulted
in no significant loss of A-degrading activity.
Simultaneous immunodepletion of A- and insulin-degrading activities
by anti-IDE antibody 9B12
Degradation Extracellularly in the Absence of
Detectable Cellular Injury--
We previously reported that the
degradation of A in the CM of BV-2 cells was significantly lower
when the cells were conditioned in 10% FBS than in serum-free medium
(1). One explanation for this finding could be that the cells are more
prone to injury in the absence of serum and thus release more protease,
particularly in light of the general assumption that IDE is localized
to cytosol and not normally released by intact cells. To address this
issue, we used a highly sensitive assay to detect A degradation in
the presence of 10% FBS; 125I-labeled synthetic A
(~20 pM) served as the substrate and was incubated in
whole BV-2 cultures or in their CM alone. The loss of intact IA
during a 24-h incubation was assayed by quantitating trichloroacetic
acid-precipitable counts, a method widely used to study the degradation
of other iodinated substrates of IDE (e.g. Refs. 6 and 13).
Radioactivity in both the trichloroacetic acid-insoluble pellets
(representing intact peptide) and the supernatant (representing the
products) was measured. Because partially degraded fragments of A
may still be precipitated by trichloroacetic acid, this assay
underestimates the absolute level of substrate cleavage. Whole BV-2
cultures degraded ~50% of the extracellular IA during an 18-h
incubation (Fig. 3A), and this
was completely inhibited by 10 µM insulin (Fig.
3B). We also performed this experiment on cultures of M17
human neuroblastoma cells and rat PC12 cells. Substantially less
degradation of IA occurred in M17 then in BV-2 cultures, and the
PC12 cultures showed very little or no detectable degradation under the
same conditions (data not shown). Because both M17 and PC12 cells have
similar amounts of IDE in their cytoplasm as do BV-2 cells (not shown),
the secretion of IDE activity capable of mediating extracellular A
degradation is cell type-dependent and does not simply
reflect nonspecific release in cell culture.
View larger version (25K):
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Fig. 3.
Degradation of A in BV-2 cultures is
mediated by IDE activity in the conditioned medium. A
and B, whole BV-2 cultures were incubated with IA in the
absence (A) or presence (B) of 10 µM human insulin, and aliquots of the CM were removed at
the indicated times. C-F, BV-2 cells were conditioned in
the presence of 10% FBS for 24 h at 37 °C, and the CM were
collected. CM were incubated with IA in the absence of inhibitors
(C) or in the presence of 1 mM
1,10-phenanthroline (D), 100 µM NEM
(E), or 10 µM human insulin (F).
Aliquots were removed at the indicated times and assayed by
trichloroacetic acid precipitation (see "Experimental Procedures").
Pellets (open squares) and supernatants
(filled squares) were assayed for cpm (means ± S.D. values for three experiments).
in whole cultures could be due to a
mixture of cell-based and secreted activities, we asked whether just
the CM of BV-2 cells is sufficient to mediate the degradation of A
seen in the above experiments. IA was incubated for 24 h at
37 °C solely in the CM of BV-2 cells conditioned in the presence of
10% FBS. The CM degraded ~50% of the IA during the incubation
(Fig. 3C), similar to the whole cultures, and this was
inhibited by 1,10-phenanthroline (Fig. 3D), NEM (Fig.
3E), and insulin (Fig. 3F). These results clearly
indicate that A degradation is largely mediated by extracellular IDE
activity in BV-2 cultures.
View larger version (44K):
[in a new window]
Fig. 4.
Viability and intactness of cultured BV-2
cells. A-E, the LIVE/DEAD viability/cytotoxicity assay
was performed according to the manufacturer's instructions on BV-2
cells cultured at 37 °C for 24 h in the presence (A
and B) or the absence (C and D) of
10% serum. As a positive control, BV-2 cells were briefly
permeabilized with digitonin (0.05%) (E). The polyanionic
calcein is well retained within live cells, producing an intense
uniform green fluorescence (A and C). Ethidium
bromide enters cells with damaged membranes and undergoes a 40-fold
enhancement of its fluorescence upon binding to nucleic acids, thereby
producing a bright red fluorescence in leaky cells (B,
D, and E). F, BV-2 cells were
conditioned in 10% FBS at 37 °C, and aliquots of CM were removed at
the indicated times. Cytoplasmic lactate dehydrogenase present in the
CM at each time point was measured.
View larger version (24K):
[in a new window]
Fig. 5.
Detection of intact IDE in fresh human
cerebrospinal fluid and human brain tissue. A, lumbar
CSF samples from patients with AD (lanes 1-3)
and nondemented control subjects (lanes 4-6)
were concentrated 10-fold by Centricon, separated by 10% Tris/glycine
SDS-PAGE, and immunoblotted with the IDE antibody 2BS. The cytosol of
human 293 cells was used as a positive control (lane
7). The upper band, but not IDE, was recognized by the
secondary antibody alone (not shown), indicating that it is
nonspecific. B, postmortem samples of frozen human cortex
from one nondemented control subject (lane 1) and
three AD cases (lanes 2-4) were extracted. Equal
amounts of protein (60 µg) from each sample were electrophoresed on
10% Tris/glycine SDS-PAGE and immunoblotted with the IDE antibody 2BS.
Human 293 cell cytosol served as a positive control (lane
5).
--
To visualize A
degradation directly in the above incubations of IA in whole BV-2
cultures or their CM, these samples were analyzed by gel fluorography
after 10-20% Tris/Tricine SDS-PAGE (Fig.
6, A-C). As the incubation
interval at 37 °C increased, the amount of the monomeric A band
gradually decreased by ~50% (Fig. 6B, lanes
1-5), consistent with the results of trichloroacetic acid
precipitation (Fig. 3). We also observed the progressive formation over
time of small amounts of SDS-stable IA species migrating at 6-20
kDa, as described previously in CHO cell cultures under similar
conditions (12, 18). In the presence of insulin, the loss of the 4-kDa
A band during the 37 °C incubation was abolished, consistent with
all of the previous data implicating IDE in the degradation.
Unexpectedly, the formation of the apparent A oligomers was also
completely prevented by insulin (Fig. 6B, compare
lanes 6-10 with lanes
1-5), suggesting that both of these events might be
mediated by IDE. To prove that IDE is the same factor that mediates the
degradation and the apparent oligomerization of A, IA was
incubated in BV-2 CM alone in the absence or presence of insulin or of
the protease inhibitor 1,10-phenanthroline or NEM and then assayed by
SDS-PAGE/gel fluorography (Fig. 6C). The two protease
inhibitors and insulin, which efficiently inhibited IDE from degrading
A monomer, simultaneously prevented the apparent oligomerization.
The experiment was repeated five times with the same result. Plain,
unconditioned medium (RPMI, 10% FBS) incubated identically with IA
for up to 24 h produced no change in the A monomer (Fig.
6A). Finally, when IDE substantially purified from BV-2 CM
was used, the characteristic degradation of the IA as well as its
apparent oligomerization were again observed by SDS-PAGE (Fig.
2E).
View larger version (41K):
[in a new window]
Fig. 6.
Simultaneous inhibition of both the
degradation and the apparent oligomerization of A mediated by IDE in
BV-2 cultures and their media. A, IA was incubated
in plain, unconditioned N2 medium (RPMI/10% FBS) for 3 h
(lane 1) or 5 h (lane
2) or in BV-2 CM in the absence (lane
3) or presence (lane 4) of 1 mM 1,10-phenanthroline, followed by 10-20% Tris/Tricine
SDS-PAGE and autoradiography. B, whole BV-2 cultures in 10%
FBS were incubated with IA in the absence (lanes
1-5) or presence (lanes 6-10) of 10 µM human insulin for 0 h (lanes
1 and 6), 0.5 h (lanes
2 and 7), 3 h (lanes 3 and 8), 18 h (lanes 4 and
9), or 24 h (lanes 5 and
10). Aliquots of the CM were removed and examined by
10-20% Tris/Tricine SDS-PAGE and autoradiography. Note the decrease
in the intensity of the IA monomer band over time (lanes
1-5) and the corresponding appearance of apparent
SDS-stable oligomers and some radioactive degradation products.
C, BV-2 cells were cultured in 10% FBS for 24 h, and
the CM were collected and incubated with IA in the absence
(lanes 1-7) or presence of 1 mM
1,10-phenanthroline (lanes 8-14), 100 µM NEM (lanes 15-21), or 10 µM human insulin (lanes 22-28).
Aliquots of the reaction mixtures were removed at 0 h
(lanes 1, 8, 15, and
22), 0.5 h (lanes 2,
9, 16, and 23), 3 h
(lanes 3, 10, 17, and
24), 6 h (lanes 4, 11,
18, and 25), 18 h (lanes 5,
12, 19, and 26), 24 h
(lanes 6, 13, 20, and
27), or 48 h (lanes 7,
14, 21, and 28) and examined by
10-20% Tris/Tricine SDS-PAGE and autoradiography. Labels
are as in B.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
levels in brain tissue is a rational approach to prevent or slow the
development of AD. In the case of the three genes implicated to date in
autosomal dominant forms of AD, excessive cellular production of A,
particularly A42, appears to be the common mechanism by
which they produce the AD phenotype (19). However, in the large number
of AD cases in whom a genetic risk factor has not yet been identified
or is not operative, the reasons for the excessive accumulation of A
in brain are unknown. Such cases, often referred to as "sporadic"
AD, are not known to have an abnormality of A production. It is
therefore possible that decreases in the normal degradation and
clearance of A in brain rather than a rise in its production could
underlie some or many cases of the disease. It has been reported that
microglia can bind and internalize microaggregates of A in
vitro (20). However, the protease(s) involved in the degradation
and clearance of A monomer have received very little attention, and
the principal mechanisms that regulate the steady state levels of the
peptide in normal brain remain undefined.
by certain known purified proteases have found evidence of variable degradation of the peptide by gelatinase A (21, 22); EC
3.4.24.11 (23); cathepsins B and D (6, 24); collagenase, chymotrypsin,
and trypsin (25); a serine protease-2-macroglobulin complex (10), and IDE (5, 6). Because many purified proteases are
capable of cleaving a variety of pure substrates with poor specificity
under in vitro conditions, we chose to screen several neural
and nonneural cell lines for the secretion of endogenous proteases that
efficiently degrade naturally secreted A peptides under biologically
relevant conditions and concentrations. Using this approach, we
identified an A-degrading metalloprotease released constitutively by
the microglial cell line BV-2 and, to a lesser extent, by certain other
cells (1). We have now partially purified and characterized
this protease and show that it is indistinguishable from the well known
metalloendopeptidase, insulin-degrading enzyme, which is known to
degrade several small, structurally diverse secreted peptides.
Surprisingly, we find that IDE activity is also associated with an
apparent oligomerization of synthetic A in cell culture, suggesting
that this protease, which we show is present in vivo in
human brain and CSF, may play a key role in regulating the amount and
fate of extracellular A in brain tissue.
Peptides--
We report several lines of evidence that the
A-degrading enzyme in the BV-2 medium is IDE or a highly homologous,
immunochemically cross-reactive protease of the same size. First, the
A degrading activity of BV-2 CM is fully inhibited by the
sulfhydryl-modifying reagent NEM and the chelating agent
1,10-phenanthroline (Fig. 1A), consistent with the inhibitor
profile of IDE (2, 3). Second, a specific substrate of IDE, insulin,
competitively inhibits the degradation of endogenous A by BV-2 CM
(Fig. 1B). Third, Western blot analysis with an IDE-specific
antibody confirms the presence of an immunoreactive 110-kDa protein in
BV-2 CM, and it comigrates with an immunoreactive band of this size in
BV-2 and 293 cell cytosol (Fig. 1C), a compartment in which
IDE is principally found and has been well characterized (17, 26). Fourth, partial purification of our A-degrading protease from BV-2
CM shows that chromatographic fractions that contain maximal A-degrading activity directly parallel those with maximal amounts of
immunoreactive 110-kDa IDE (Fig. 2). Fifth, immunodepletion with an IDE
monoclonal antibody, 9B12(5), completely abolishes the A-degrading
activity in the medium of Chinese hamster ovary cells (Table II), which
we previously showed release the same IDE-like A-degrading thiol
metalloprotease that BV-2 cells do (1); this antibody cannot be used on
BV-2 CM, because it reacts very poorly with murine IDE.
degradation we observe, two experimental results make this highly
unlikely. First, the fact that insulin inhibits A degradation in CM
alone (without cells present) rules against this, since the
hypothetical "activated" protease should already be present in the
CM and then insulin would not inhibit. Second, the hypothetical other
protease would have had to co-purify with IDE throughout our
purification (Fig. 2).
,
and insulin-like growth factor II (reviewed in Refs. 2 and 3). Previous
studies of CHO cells have reported that IDE is localized to the cytosol
and peroxisomes (17), loci in which A has not been found, raising
the question of whether and how the protease could degrade endogenous
A. Although some studies have detected insulin-degrading activity in
the conditioned media of cultured cells (14, 15) and more recently on
the cell surface (16), the extent of cell permeability and thus possible release of IDE from leaky cells, especially when serum is
absent from the culture medium, was not specifically assessed in these
reports. In our study, extracellular A degradation mediated by IDE
was observed both in whole BV-2 cultures and in their CM alone (Fig.
3), suggesting that the enzyme can be released, at least by the
microglial cell line we used. Because we grew the cells in the presence
of 10% FBS in such experiments, the likelihood of cell damage during
the brief conditioning is very low. This conclusion is supported by
several important control experiments: (a) no significant
rise in lactate dehydrogenase was detected during the conditioning of
the medium at the same time that A-degrading activity was clearly
increasing (Fig. 4F); (b) only a very few leaky
BV-2 cells were detected in the calcein/ethidium bromide cell viability
assay (Fig. 4, A-E); (c) no morphological
alterations or overt cell death were seen in the serum-free cultures
over the time course of our experiments (
24 h) (Fig. 4, C
and D); and (d) no extracellular IDE activity was
detected in certain other IDE cell types (e.g. PC12 cells)
cultured under identical conditions (data not shown), indicating that
IDE release is not a nonspecific consequence of culturing cells. Most
importantly, we demonstrate for the first time the presence of intact,
110-kDa IDE in fresh lumbar CSF obtained from living patients (Fig. 5), indicating that this protease exists in normal extracellular fluid under in vivo conditions. Further experiments will be needed
to determine whether the IDE present in CSF is normally inhibited under
basal conditions (as would be expected) but can be shown to have
proteolytic activity under other conditions, e.g. following microglial activation in the brain and consequent enhanced release of
the enzyme. In this regard, the physiological nature of IDE release
from microglial cells is supported by our earlier finding that
stimulation of BV-2 cultures with lipopolysaccharide results in a
reproducible increase in A-degrading activity in the medium, and
this is prevented by IDE inhibitors (1). Finally, Mentlein et
al. (27) have recently reported the presence of a metalloprotease in the media of primary rat microglia, the properties of which strongly
suggest that it is IDE.
at
Physiological Concentrations--
A central unresolved question about
the pathobiology of AD is how excessive accumulation of the soluble,
secreted form of A gradually leads to the development of insoluble
A fibrils in innumerable extracellular plaques. Since the discovery
of normal A secretion in 1992, many studies have shown that
APP-expressing neural and nonneural cells produce soluble, monomeric
A at picomolar to low nanomolar levels under physiological
conditions (reviewed in Ref. 28). On the other hand, synthetic A
peptides studied at much higher concentrations (10-1000
µM) aggregate upon in vitro incubation at
37 °C into insoluble fibrils resembling those in AD brain. In
separate work, we have shown that both endogenous and synthetic A
peptides at physiological (low nM) concentrations can form
small amounts of SDS-stable apparent oligomers in cell culture (12,
29). In the current study, we unexpectedly found that the apparent
oligomerization of synthetic A in the medium of BV-2 cells was
completely inhibited by the competitive IDE substrate, insulin (Fig.
6A), and by two IDE inhibitors, 1,10-phenanthroline and NEM
(Fig. 6B). These findings suggest that IDE activity is capable of mediating the oligomerization of A. Such a conclusion is
strongly supported by similar results obtained with substantially purified IDE from BV-2 CM (Fig. 2). At longer incubation times (
18
h), apparent IA oligomers disappeared (Fig. 6B), while
trichloroacetic acid supernatant counts increased (Fig. 3), suggesting
that the oligomers may be degraded. Mechanistically, we hypothesize
that some A fragments, which are generated by IDE, can enhance the oligomerization of the IA peptide and/or can themselves oligomerize. In view of the fact that IDE purified from CHO cytosol has been shown
to generate A fragments by cleavage after residues 4, 13, 14, and
20,2 the resultant N-terminally truncated fragments could
promote A oligomerization. It has been reported that N-terminally
truncated fragments of synthetic A aggregate more rapidly than the
full-length peptide (30). We cannot exclude the possibility that some
of the bands we observe at 6-20 kDa on gels could represent
oligomers composed in part of truncated A peptides or even
anomalously high migration of the truncated monomers themselves, as has
been suggested to occur with some truncated fragments of synthetic A
(31). It is known that the presence of very small amounts of aggregated
A or its fragments can act as powerful seeds to enhance the
subsequent polymerization of monomeric A into fibrils (32, 33).
Although a proteolytic effect of IDE on A is the most plausible
explanation for its oligomer-promoting activity, IDE could mediate the
degradation and the oligomerization of A by independent actions.
Further work is needed to clarify the mechanism by which IDE activity
seems to promote A oligomerization and to determine the fate of the
soluble oligomers of A formed in our culture system. We are
currently attempting to determine whether IDE can be stably transfected
into BV-2 and other cells serve to increase both the degradation and
oligomerization of extracellular A.
in the brain during aging
and in AD. In this regard, A can accumulate progressively with age
in the brains of normal humans as well as in lower primates, dogs,
cats, and certain other mammals (36). These observations, taken
together with the evidence that IDE in brain tissue can bind and cleave
A (5, 6) and our findings that IDE is the principal protease
released by microglial cells that degrades naturally secreted A at
physiological concentrations and is present in human brain and CSF,
make it important to determine whether alterations in the activity or
regulation of IDE and other A-cleaving proteases could explain some
of the many cases of "sporadic" AD in which A accumulates
excessively but its production is apparently normal.
ACKNOWLEDGEMENTS
FOOTNOTES
, amyloid
-protein; IA, 125I-A; AD, Alzheimer's disease; APP, -amyloid precursor protein; IDE, insulin-degrading enzyme; CSF, cerebrospinal fluid; FBS, fetal bovine serum; CM, conditioned media; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NEM, N-ethylmaleimide.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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 E. B. Lee, B. Zhang, K. Liu, E. A. Greenbaum, R. W. Doms, J. Q. Trojanowski, and V. M.-Y. Lee BACE overexpression alters the subcellular processing of APP and inhibits A{beta} deposition in vivo J. Cell Biol., January 17, 2005; 168(2): 291 - 302. [Abstract] [Full Text] [PDF]
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L. Morelli, R. E. Llovera, I. Mathov, L.-F. Lue, B. Frangione, J. Ghiso, and E. M. Castano Insulin-degrading Enzyme in Brain Microvessels: PROTEOLYSIS OF AMYLOID {beta} VASCULOTROPIC VARIANTS AND REDUCED ACTIVITY IN CEREBRAL AMYLOID ANGIOPATHY J. Biol. Chem., December 31, 2004; 279(53): 56004 - 56013. [Abstract] [Full Text] [PDF]
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M. Schnaider Beeri, U. Goldbourt, J. M. Silverman, S. Noy, J. Schmeidler, R. Ravona-Springer, A. Sverdlick, and M. Davidson Diabetes mellitus in midlife and the risk of dementia three decades later Neurology, November 23, 2004; 63(10): 1902 - 1907. [Abstract] [Full Text] [PDF]
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V. K. Yechoor, M.-E. Patti, K. Ueki, P. G. Laustsen, R. Saccone, R. Rauniyar, and C. R. Kahn Distinct pathways of insulin-regulated versus diabetes-regulated gene expression: An in vivo analysis in MIRKO mice PNAS, November 23, 2004; 101(47): 16525 - 16530. [Abstract] [Full Text] [PDF]
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T. Shiiki, S. Ohtsuki, A. Kurihara, H. Naganuma, K. Nishimura, M. Tachikawa, K.-i. Hosoya, and T. Terasaki Brain Insulin Impairs Amyloid-{beta}(1-40) Clearance from the Brain J. Neurosci., October 27, 2004; 24(43): 9632 - 9637. [Abstract] [Full Text] [PDF]
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J. A. Luchsinger, M.-X. Tang, S. Shea, and R. Mayeux Hyperinsulinemia and risk of Alzheimer disease Neurology, October 12, 2004; 63(7): 1187 - 1192. [Abstract] [Full Text] [PDF]
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V. Laporte, Y. Lombard, R. Levy-Benezra, C. Tranchant, P. Poindron, and J.-M. Warter Uptake of A{beta} 1-40- and A{beta} 1-42-coated yeast by microglial cells: a role for LRP J. Leukoc. Biol., August 1, 2004; 76(2): 451 - 461. [Abstract] [Full Text] [PDF]
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R. Peila, B. L. Rodriguez, L. R. White, and L. J. Launer Fasting insulin and incident dementia in an elderly population of Japanese-American men Neurology, July 27, 2004; 63(2): 228 - 233. [Abstract] [Full Text] [PDF]
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L. Bian, J. D. Yang, T. W. Guo, Y. Sun, S. W. Duan, W. Y. Chen, Y. X. Pan, G. Y. Feng, and L. He Insulin-degrading enzyme and Alzheimer disease: A genetic association study in the Han Chinese Neurology, July 27, 2004; 63(2): 241 - 245. [Abstract] [Full Text] [PDF]
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 L. Huang, R. I. Hollingsworth, R. Castellani, and B. Zipser Accumulation of high-molecular-weight amylose in Alzheimer's disease brains Glycobiology, May 1, 2004; 14(5): 409 - 416. [Abstract] [Full Text] [PDF]
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W. Farris, S. Mansourian, M. A. Leissring, E. A. Eckman, L. Bertram, C. B. Eckman, R. E. Tanzi, and D. J. Selkoe Partial Loss-of-Function Mutations in Insulin-Degrading Enzyme that Induce Diabetes also Impair Degradation of Amyloid {beta}-Protein Am. J. Pathol., April 1, 2004; 164(4): 1425 - 1434. [Abstract] [Full Text] [PDF]
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 L. Bertram and R. E. Tanzi Alzheimer's disease: one disorder, too many genes? Hum. Mol. Genet., April 1, 2004; 13(90001): R135 - 141. [Abstract] [Full Text] [PDF]
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L. Gan, S. Ye, A. Chu, K. Anton, S. Yi, V. A. Vincent, D. von Schack, D. Chin, J. Murray, S. Lohr, et al. Identification of Cathepsin B as a Mediator of Neuronal Death Induced by A{beta}-activated Microglial Cells Using a Functional Genomics Approach J. Biol. Chem., February 13, 2004; 279(7): 5565 - 5572. [Abstract] [Full Text] [PDF]
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E.-S. Song, M. A. Juliano, L. Juliano, and L. B. Hersh Substrate Activation of Insulin-degrading Enzyme (Insulysin): A POTENTIAL TARGET FOR DRUG DEVELOPMENT J. Biol. Chem., December 12, 2003; 278(50): 49789 - 49794. [Abstract] [Full Text] [PDF]
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 L. Helmuth Detangling Alzheimer's Disease Sci. Aging Knowl. Environ., October 29, 2003; 2003(43): oa2 - 2. [Abstract] [Full Text] [PDF]
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 R. G. Bennett, F. G. Hamel, and W. C. Duckworth An Insulin-Degrading Enzyme Inhibitor Decreases Amylin Degradation, Increases Amylin-Induced Cytotoxicity, and Increases Amyloid Formation in Insulinoma Cell Cultures Diabetes, September 1, 2003; 52(9): 2315 - 2320. [Abstract] [Full Text] [PDF]
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G. S. Watson, E. R. Peskind, S. Asthana, K. Purganan, C. Wait, D. Chapman, M. W. Schwartz, S. Plymate, and S. Craft Insulin increases CSF A{beta}42 levels in normal older adults Neurology, June 24, 2003; 60(12): 1899 - 1903. [Abstract] [Full Text] [PDF]
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L. Morelli, R. Llovera, S. A. Gonzalez, J. L. Affranchino, F. Prelli, B. Frangione, J. Ghiso, and E. M. Castano Differential Degradation of Amyloid {beta} Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme J. Biol. Chem., June 20, 2003; 278(26): 23221 - 23226. [Abstract] [Full Text] [PDF]
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B. C. Miller, E. A. Eckman, K. Sambamurti, N. Dobbs, K. M. Chow, C. B. Eckman, L. B. Hersh, and D. L. Thiele Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo PNAS, May 13, 2003; 100(10): 6221 - 6226. [Abstract] [Full Text] [PDF]
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W. Farris, S. Mansourian, Y. Chang, L. Lindsley, E. A. Eckman, M. P. Frosch, C. B. Eckman, R. E. Tanzi, D. J. Selkoe, and S. Guenette Insulin-degrading enzyme regulates the levels of insulin, amyloid beta -protein, and the beta -amyloid precursor protein intracellular domain in vivo PNAS, April 1, 2003; 100(7): 4162 - 4167. [Abstract] [Full Text] [PDF]
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E. A. Eckman, M. Watson, L. Marlow, K. Sambamurti, and C. B. Eckman Alzheimer's Disease beta -Amyloid Peptide Is Increased in Mice Deficient in Endothelin-converting Enzyme J. Biol. Chem., January 17, 2003; 278(4): 2081 - 2084. [Abstract] [Full Text] [PDF]
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D. G. Cook, J. B. Leverenz, P. J. McMillan, J. J. Kulstad, S. Ericksen, R. A. Roth, G. D. Schellenberg, L.-W. Jin, K. S. Kovacina, and S. Craft Reduced Hippocampal Insulin-Degrading Enzyme in Late-Onset Alzheimer's Disease Is Associated with the Apolipoprotein E-{epsilon}4 Allele Am. J. Pathol., January 1, 2003; 162(1): 313 - 319. [Abstract] [Full Text] [PDF]
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 C. N. Shrimpton, A. I. Smith, and R. A. Lew Soluble Metalloendopeptidases and Neuroendocrine Signaling Endocr. Rev., October 1, 2002; 23(5): 647 - 664. [Abstract] [Full Text] [PDF]
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M. H. Mohajeri, M. A. Wollmer, and R. M. Nitsch Abeta 42-induced Increase in Neprilysin Is Associated with Prevention of Amyloid Plaque Formation in Vivo J. Biol. Chem., September 13, 2002; 277(38): 35460 - 35465. [Abstract] [Full Text] [PDF]
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 Y.-H. Suh and F. Checler Amyloid Precursor Protein, Presenilins, and alpha -Synuclein: Molecular Pathogenesis and Pharmacological Applications in Alzheimer's Disease Pharmacol. Rev., September 1, 2002; 54(3): 469 - 525. [Abstract] [Full Text] [PDF]
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D. Edbauer, M. Willem, S. Lammich, H. Steiner, and C. Haass Insulin-degrading Enzyme Rapidly Removes the beta -Amyloid Precursor Protein Intracellular Domain (AICD) J. Biol. Chem., April 12, 2002; 277(16): 13389 - 13393. [Abstract] [Full Text] [PDF]
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K. Uryu, H. Laurer, T. McIntosh, D. Pratico, D. Martinez, S. Leight, V. M.-Y. Lee, and J. Q. Trojanowski Repetitive Mild Brain Trauma Accelerates Abeta Deposition, Lipid Peroxidation, and Cognitive Impairment in a Transgenic Mouse Model of Alzheimer Amyloidosis J. Neurosci., January 15, 2002; 22(2): 446 - 454. [Abstract] [Full Text] [PDF]
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 H. LIN, R. BHATIA, and R. LAL Amyloid {beta} protein forms ion channels: implications for Alzheimer's disease pathophysiology FASEB J, November 1, 2001; 15(13): 2433 - 2444. [Abstract] [Full Text] [PDF]
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L. Helmuth Detangling Alzheimer's Disease Sci. Aging Knowl. Environ., October 3, 2001; 2001(1): oa2 - 2. [Abstract] [Full Text] [PDF]
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L. Gasparini, G. K. Gouras, R. Wang, R. S. Gross, M. F. Beal, P. Greengard, and H. Xu Stimulation of {beta}-Amyloid Precursor Protein Trafficking by Insulin Reduces Intraneuronal {beta}-Amyloid and Requires Mitogen-Activated Protein Kinase Signaling J. Neurosci., April 15, 2001; 21(8): 2561 - 2570. [Abstract] [Full Text] [PDF]
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A. Mukherjee, E.-s. Song, M. Kihiko-Ehmann, J. P. Goodman Jr, J. St. Pyrek, S. Estus, and L. B. Hersh Insulysin Hydrolyzes Amyloid beta Peptides to Products That Are Neither Neurotoxic Nor Deposit on Amyloid Plaques J. Neurosci., December 1, 2000; 20(23): 8745 - 8749. [Abstract] [Full Text] [PDF]
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K. Vekrellis, Z. Ye, W. Q. Qiu, D. Walsh, D. Hartley, V. Chesneau, M. R. Rosner, and D. J. Selkoe Neurons Regulate Extracellular Levels of Amyloid beta -Protein via Proteolysis by Insulin-Degrading Enzyme J. Neurosci., March 1, 2000; 20(5): 1657 - 1665. [Abstract] [Full Text] [PDF]
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M.-A. Abbott, D. G. Wells, and J. R. Fallon The Insulin Receptor Tyrosine Kinase Substrate p58/53 and the Insulin Receptor Are Components of CNS Synapses J. Neurosci., September 1, 1999; 19(17): 7300 - 7308. [Abstract] [Full Text] [PDF]
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R. Yamin, E. G. Malgeri, J. A. Sloane, W. T. McGraw, and C. R. Abraham Metalloendopeptidase EC 3.4.24.15 Is Necessary for Alzheimer's Amyloid-beta Peptide Degradation J. Biol. Chem., June 25, 1999; 274(26): 18777 - 18784. [Abstract] [Full Text] [PDF]
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R. G. Bennett, W. C. Duckworth, and F. G. Hamel Degradation of Amylin by Insulin-degrading Enzyme J. Biol. Chem., November 17, 2000; 275(47): 36621 - 36625. [Abstract] [Full Text] [PDF]
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E. A. Eckman, D. K. Reed, and C. B. Eckman Degradation of the Alzheimer's Amyloid beta Peptide by Endothelin-converting Enzyme J. Biol. Chem., June 29, 2001; 276(27): 24540 - 24548. [Abstract] [Full Text] [PDF]
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K. Shirotani, S. Tsubuki, N. Iwata, Y. Takaki, W. Harigaya, K. Maruyama, S. Kiryu-Seo, H. Kiyama, H. Iwata, T. Tomita, et al. Neprilysin Degrades Both Amyloid beta Peptides 1-40 and 1-42 Most Rapidly and Efficiently among Thiorphan- and Phosphoramidon-sensitive Endopeptidases J. Biol. Chem., June 8, 2001; 276(24): 21895 - 21901. [Abstract] [Full Text] [PDF]
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E.-S. Song, A. Mukherjee, M. A. Juliano, J. St. Pyrek, J. P. Goodman Jr., L. Juliano, and L. B. Hersh Analysis of the Subsite Specificity of Rat Insulysin Using Fluorogenic Peptide Substrates J. Biol. Chem., January 5, 2001; 276(2): 1152 - 1155. [Abstract] [Full Text] [PDF]
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