From the Mayo Clinic Jacksonville, Jacksonville, Florida 32224
Received for publication, November 13, 2002
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ABSTRACT |
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The abnormal accumulation of Alzheimer's disease
(AD),1 the most common cause
of dementia in the elderly, is characterized pathologically by the
accumulation of A Using pharmacological, molecular, and biochemical approaches, we have
previously characterized endothelin-converting enzyme-1 (ECE-1) as a
novel A A reasonable test for the physiological involvement of ECE or any
enzyme in contributing to A Animals--
ECE-1 and ECE-2 knock-out mice were a gift from Dr.
Masashi Yanagisawa. Offspring from heterozygous crosses were compared in all studies. ECE enzymatic levels were determined using a big ET
conversion assay (16). Littermate controls were used for all
comparisons to control for any strain differences. ECE-1 (+/+) and
ECE-1 (+/ DEA Extraction and A Western Blot Analysis of Statistical Analysis--
Data were analyzed using the
Mann-Whitney nonparametric test.
To determine whether reduced ECE-1 activity would lead to
increased A-amyloid (A
)
in the brain is an early and invariant feature in Alzheimer's disease
(AD) and is believed to play a pivotal role in the etiology and
pathogenesis of the disease. As such, a major focus of AD research has
been the elucidation of the mechanisms responsible for the generation of A
. As with any peptide, however, the degree of A
accumulation is dependent not only on its production but also on its removal. In
cell-based and in vitro models we have previously
characterized endothelin-converting enzyme-1 (ECE-1) as an
A
-degrading enzyme that appears to act intracellularly, thus
limiting the amount of A
available for secretion. To determine the
physiological significance of this activity, we analyzed A
levels in
the brains of mice deficient for ECE-1 and a closely related enzyme,
ECE-2. Significant increases in the levels of both A
40 and A
42
were found in the brains of these animals when compared with
age-matched littermate controls. The increase in A
levels in the
ECE-deficient mice provides the first direct evidence for a
physiological role for both ECE-1 and ECE-2 in limiting A
accumulation in the brain and also provides further insight into the
factors involved in A
clearance in vivo.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptides (A
40 and A
42) in the brain.
Although considerable attention has been focused on understanding the
enzymes and processes involved in the production of A
, very little
is known regarding the processes by which A
is normally removed.
This removal can be in the form of transport of the peptide into the
cerebrospinal fluid for peripheral clearance, by binding to
proteins that sequester the peptide in a nonreactive form, or by direct
catabolism of A
. A significant role for A
catabolism has been
highlighted in a recent report by Saido and colleagues (1) in which
they showed that infusion of the metalloprotease inhibitor thiorphan into the hippocampus of rats resulted in localized A
deposition, reportedly through the inhibition of A
degradation by neprilysin (NEP).
catabolism appears complex. Recent reports suggest significant
roles for insulin-degrading enzyme, NEP, and the plasmin system in the
catabolism of A
(1-10). Angiotensin-converting enzyme, matrix
metalloproteinase-9, EC 3.4.24.15, and
-2 macroglobulin complexes have also been reported to play a role in A
degradation on
the basis of in vitro and cell-based assays (11-14). To
date, however, only neprilysin has been reported to influence A
levels in the brains of knock-out mice (1, 5, 15).
-degrading enzyme that appears to act intracellularly to
reduce the amount of A
available for secretion (16). Overexpression of ECE-1 in cells that lack endogenous ECE activity results in a
pronounced decrease in A
accumulation in the conditioned
media, and ECE-1 can directly hydrolyze A
at multiple sites
with a favorable kinetic profile. ECEs are type II integral membrane
zinc metalloproteases (active site luminal) named for their ability to
cleave and activate big endothelins (big ETs) to the potent
vasoconstrictor endothelins (17). Two different ECEs have been cloned.
The first, ECE-1, is abundantly expressed in the vascular endothelial
cells of all organs and is also widely expressed in many nonvascular
cells (18-20). In the brain, ECE-1 immunoreactivity has been detected in fibers within the glial limitans, in neuronal processes and cell
bodies of the cerebral cortex, in pyramidal cells of the neocortex and
hippocampus, in astrocytes, and in Purkinje cells in the cerebellum
(19, 21-23). ECE-2 is an homologous enzyme that shares catalytic
activity with ECE-1 and is localized to an intracellular compartment
(24). ECE-2 is most abundantly expressed in neural tissues including
the cerebral cortex, cerebellum, and adrenal medulla (24). In mouse
brain, ECE-2 is expressed in heterogeneous populations of neurons in
the thalamus, hypothalamus, amygdala, dentate gyrus, and CA3 (25).
accumulation in the brain is to examine
the effect of animals that are null for the enzyme. Both ECE-1 and
ECE-2 null animals have been described previously (25, 26). ECE-1
homozygous knock-out mice have craniofacial and cardiac abnormalities,
resulting in lethality beginning at day E12.5 and extending to within
30 min of birth (26). ECE-1 heterozygotes and ECE-2 null animals,
however, do survive and appear healthy. In this study we examined the
physiological role of ECE activity on A
accumulation in the brain by
comparing the amount of A
in animals deficient in ECE with the
amount found in the brains isolated from their wild-type littermates.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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) mice were analyzed at 3 weeks of age. ECE-2 (+/+), (+/
),
and (
/
) mice were analyzed at 4 weeks of age. Mice were sacrificed
by CO2 asphyxiation, and the brains were immediately removed, frozen on dry ice, and stored at
80 °C prior to A
analysis.
Measurements--
DEA extractions were
performed using a protocol similar to those described previously (27,
28). One hemisphere of the brain was homogenized in 50 mM
NaCl, 0.2% DEA at a concentration of 100 mg/ml. The extract was then
centrifuged at 4 °C for 1 h at 100,000 × g.
The supernatant was removed and neutralized by the addition of a 1/10
volume of 0.5 M Tris-HCl, pH 6.8. Neutralized extracts (100 µl) were added to assay plates containing 50 µl of EC buffer (0.02 M sodium phosphate, pH 7.0 containing 0.002 M
EDTA, 0.4 M NaCl, 0.2% bovine serum albumin, 0.05% CHAPS,
0.4% Block Ace, 0.05% NaN3) and were analyzed using the
well characterized BNT77/BA27 and BNT77/BC05 sandwich ELISA systems to
detect A
40 and A
42, respectively (29). A
concentration was
determined by comparing absorbance values obtained for samples to those
obtained for synthetic A
standards prepared in EC buffer. Results
from 2-3 experiments were pooled to generate the data shown by
normalizing values to the means of the wild-type animals.
APP and C-terminal
Fragments--
Neutralized DEA extracts were analyzed by Western blot
as described previously (30). CT20, a rabbit polyclonal antibody raised
against the 20 C-terminal amino acids of
APP, was used for
quantitation of C-terminal fragments (CTFs), and 22C11 (Roche Molecular
Biochemicals) was used for quantitation of
APP. Values reported are the mean from two scans each of two independent
experiments, each performed in triplicate, and are presented in
arbitrary units. In all cases multiple exposures were examined to make
certain that the film was not "saturated." Values obtained were
normalized to the mean of the wild-type animals to enable comparisons
between experiments.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
levels in the brain, we compared the amount of A
in brains from ECE-1 (+/
) mice to that in wild-type littermate controls. The levels of both A
40 and A
42 were significantly increased in
the mice with reduced ECE-1 activity (Fig.
1 and Table I). Although modest,
these increases are in the range observed for some of the
familial Alzheimer's disease-linked mutations (29, 31-36) and are
particularly noteworthy, as ECE-1 (+/
) mice have only an
~27% reduction in ECE-1 activity (26 and data not shown) . To determine whether the increase in
A
concentration we observed in the brains of these animals might be
due to an increase in expression and/or processing of the A
precursor protein (APP), we examined the levels of APP and the
C-terminal fragment of APP, which serves as the immediate precursor to
A
(CTF
). Consistent with the increase in A
concentration being
a result of decreased degradation of A
, the levels of APP and CTF
were unchanged in the ECE-1 (+/
) mice compared with their wild-type
littermates (Fig. 2).
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Fig. 1.
Scatter plot of A 40
detected in the brains of ECE-1 +/
and ECE-2
/
mice. A
concentration was determined using the well characterized BNT77/BA27
sandwich ELISA. Bars indicate the mean value of each group.
A, A
40 concentration is significantly elevated in ECE-1
(+/
) mice (p = 0.0002) compared with wild-type
littermate controls. B, A
40 concentration is
significantly elevated in ECE-2 (
/
) mice (p < 0.0001) compared with wild-type littermate controls.
A levels in brains of ECE-deficient mice
View larger version (9K):
[in a new window]
Fig. 2.
Analysis of APP and CTF
expression in brains isolated from ECE knockout mice. APP
and CTF levels were determined by image analysis of Western blots. Data
plotted represent the mean ± S.E. of at least four animals in
each group. A, relative levels of APP; B,
relative levels of CTF
.
Given the catalytic similarity between ECE-1 and ECE-2 and that ECE-2
expression is greater in the nervous system than in other tissues, we
also examined whether A concentration is affected by the level of
ECE-2 activity in the brain. ECE-2 (
/
) animals are viable and
appear healthy (25). Thus we were able to analyze both ECE-2 (+/
) and
ECE-2 (
/
) mice and compare the amount of A
in the brains of
these animals to that found in littermate controls with normal ECE-2
levels. In these studies we found a significant elevation in the
concentrations of both A
40 and A
42 in the brains of animals with
reduced ECE activity compared with their control littermates (Fig. 1
and Table I). The concentration of A
in the brains of ECE-2
heterozygous mice was intermediate between that observed in the
wild-type controls and the complete nulls, suggesting a gene-dosage
effect. As in the ECE-1 (+/
) mice, the levels of APP and CTF
were
unchanged in the ECE-2 (
/
) mice compared with controls (Fig.
2).
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DISCUSSION |
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The increase in A concentration in the brains of ECE-deficient
mice provides direct evidence of a physiological role for ECEs in
limiting A
accumulation in the brain. It will be important in future
studies to determine whether these elevations will enhance deposition
and plaque formation either by themselves or when crossed with mouse
models that normally deposit A
, such as the Tg2576 model (37). The
same holds true for the neprilysin-efficient animals and for animals
deficient in other enzymes that result in increased A
levels.
Given the evidence that A plays a significant role in AD
pathogenesis, factors that influence ECE activity, whether
pharmacological, genetic, or other, may influence the risk for
developing AD. The increases observed in the ECE-1 (+/
) animals are
particularly noteworthy, as these animals have only a 27% reduction in
ECE-1 activity (26), suggesting that even modest changes in ECE
activity can have significant effects on A
accumulation. It will be
interesting in future studies to determine whether variations in ECE
activity correlate with either soluble and/or insoluble A
levels in
human brain. Reports have suggested the linkage of a possible AD locus to chromosome 3, where two genes involved in A
catabolism,
neprilysin and ECE-2, are located (38-40). Our results argue for the
inclusion of ECE-2 in candidate gene approaches in this region.
Perhaps most immediately important is that ECE inhibitors have received
a significant amount of pharmaceutical interest for their potential as
drugs for the treatment of hypertension and other ailments
(41-43). Our results indicate that if these drugs enter the brain they
may increase A levels, potentially leading to the development and/or
acceleration of AD in susceptible individuals. Based on the genetic
mutations that cause AD, in which A
levels are elevated for decades
prior to the development of the disease, this potential side effect of
ECE inhibitors may not be observed for years and should be considered
carefully before clinical use of this class of compounds. Ideally,
these drugs should be examined directly for any effects on A
in
preclinical studies.
Finally, our data suggest a possible novel approach for reducing A
levels in vivo by up-regulating ECE activity. This could be
accomplished, for example, through gene therapy, transcriptional activation, or perhaps even by reducing ECE turnover itself. As with
any approach, this strategy is not without potential problems, which
will need to be examined carefully. One obvious concern with
up-regulation of ECE is that patients may become hypertensive. However,
up-regulation of ECE activity by intravenous injection of an adenoviral
construct containing a secreted form of ECE does not result in
increased circulating endothelin levels or in hypertension, indicating
that ECE is not likely to be rate-limiting in the biosynthesis of ET,
at least under these conditions (44). Because ECE has been shown
capable of hydrolyzing several biologically active peptides in
vitro, including bradykinin, neurotensin, and substance P (45,
46), it will be important to monitor the levels of these potential
targets as well.
In summary, our data provide direct evidence that ECE activity
contributes to the steady state levels of A in the brain and provide
further insight into the factors involved in A
clearance. Thus, it
appears that multiple enzymes are likely to be involved in A
catabolism in vivo. Catabolism of A
peptides by each of these enzymes would limit the accumulation of A
, and disruption of
this catabolism may be a risk factor for AD. Because removal of A
by
multiple enzymatic activities appears to contribute to overall A
catabolism, the identification of these activities should be viewed as
complementary rather than mutually exclusive. For example, certain
enzymes such as the ECEs may degrade A
predominately intracellularly. Following secretion, other enzymes such as NEP may
play a larger role. In addition, the principle mechanism of A
removal may vary by cell type and brain region and could potentially be
influenced by other factors such as inflammation. Understanding these
processes in more depth may provide new clues to the abnormal accumulation of A
found in patients with Alzheimer's disease.
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ACKNOWLEDGEMENTS |
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We thank Takeda Chemical Industries for their generous gifts of BNT77, BA27, and BC05. We thank Drs. Hiromi and Masashi Yanagisawa for their generous gift of ECE knockout mice.
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FOOTNOTES |
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* This work was supported by a grant from the Alzheimer's Association (to E. A. E.), by the generosity of Mr. and Mrs. Robert H. Smith, and by the Mayo Foundation for Medical Education and Research.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: Mayo Clinic
Jacksonville, Birdsall Bldg., Rm. 256, 4500 San Pablo Rd.,
Jacksonville, FL 32224. Tel.: 904-953-2979; Fax: 904-953-7370; E-mail:
Eckman@mayo.edu.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.C200642200
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ABBREVIATIONS |
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The abbreviations used are:
AD, Alzheimer's
disease;
A,
-amyloid;
ECE, endothelin-converting enzyme;
ET, endothelin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
ELISA, enzyme-linked immunosorbent assay;
APP, A
precursor protein;
CTF, C-terminal fragment.
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