 |
|
 -Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease
Hiroaki Fukumoto, PhD;
Bonnie S. Cheung, BS;
Bradley T. Hyman, MD, PhD;
Michael C. Irizarry, MD
Arch Neurol. 2002;59:1381-1389.
ABSTRACT
| |
Context Amyloid plaques, a major pathological feature of Alzheimer disease (AD),
are composed of an internal fragment of amyloid precursor protein (APP): the
4-kd amyloid- protein (A). The metabolic processing of APP that
results in A formation requires 2 enzymatic cleavage events, a  -secretase
cleavage dependent on presenilin, and a -secretase cleavage by the aspartyl
protease -site APP-cleaving enzyme (BACE).
Objective To test the hypothesis that BACE protein and activity are increased
in regions of the brain that develop amyloid plaques in AD.
Methods We developed an antibody capture system to measure BACE protein level
and BACE-specific -secretase activity in frontal, temporal, and cerebellar
brain homogenates from 61 brains with AD and 33 control brains.
Results In the brains with AD, BACE activity and protein were significantly
increased (P<.001). Enzymatic activity increased
by 63% in the temporal neocortex (P = .007) and 13%
in the frontal neocortex (P = .003) in brains with
AD, but not in the cerebellar cortex. Activity in the temporal neocortex increased
with the duration of AD (P = .008) but did not correlate
with enzyme-linked immunosorbent assay measures of insoluble A in brains
with AD. Protein level was increased by 14% in the frontal cortex of brains
with AD (P = .004), with a trend toward a 15% increase
in BACE protein in the temporal cortex (P = .07)
and no difference in the cerebellar cortex. Immunohistochemical analysis demonstrated
that BACE immunoreactivity in the brain was predominantly neuronal and was
found in tangle-bearing neurons in AD.
Conclusions The BACE protein and activity levels are increased in brain regions
affected by amyloid deposition and remain increased despite significant neuronal
and synaptic loss in AD.
INTRODUCTION
AMYLOID- PROTEIN (A) deposits in the brain as senile plaques
in Alzheimer disease (AD). A is produced from the enzymatic cleavage
of amyloid precursor protein (APP) by -secretase and -secretase.
The processing of APP occurs by 2 major pathways, one producing A (-secretase
pathway) and the other producing a presumably nontoxic p3 fragment ( -secretase
pathway). In the -secretase pathway, cleavage by the -site APP-cleaving
enzyme (BACE) generates a truncated soluble -cleaved APP fragment in
acidic endosomal or secretory compartments.1
The remaining membrane-associated 11.5-kd fragment is cleaved by presenilin-dependent -secretase
to yield A.2 We measured the protein
level and enzymatic activity of BACE, the primary brain -secretase,
in brains with AD and control brains to evaluate a role for increased A
production in the pathogenesis of AD.
The -site APP-cleaving enzyme is a 501 amino acid aspartyl protease
widely expressed in brain, pancreas, and other tissues. In the brain, BACE
is localized to neuronal cell bodies and proximal dendrites, and colocalizes
with Golgi and endosomal markers within cells.1, 3-5
It is modified during and after translation by glycosylation, sulfation, and
furin-like proteolytic cleavage of a prodomain within the Golgi, and it is
enriched within lipid rafts.6-11
Sorting and recycling of BACE are influenced by a C-terminal dileucine motif,
the transmembrane domain, and phosphorylation of serine at amino acid position
498.10, 12-13 Although
a homologous protein, BACE2, can also cleave APP, it is expressed in very
low levels in the adult brain.14-16
Because A production is abolished in neuronal cultures and brains from
BACE knockout mice, BACE is considered to be the major -secretase in
neurons and the brain.17-19
Although BACE is the primary -secretase, the level of BACE messenger
RNA does not appear to be altered in AD or transgenic mouse models of AD.20-22 Messenger RNA may
not reflect the BACE protein level and activity, which are more relevant to
the physiological role of BACE. We developed quantitative assays of BACE protein
and activity to address specific questions regarding the role of BACE in AD:
Is BACE increased in brains with AD vs control brains in areas vulnerable
to AD neuropathological abnormalities? How does BACE correlate with other
clinical and neuropathological measures of AD? We found that areas with prominent
amyloid deposition (eg, the neocortex) have selectively increased BACE protein
and/or activity levels in brains with AD relative to controls; areas minimally
affected by AD changes (eg, the cerebellum) do not demonstrate differences
in BACE relative to controls.
MATERIALS AND METHODS
CASE SELECTION
Snap-frozen slices of the temporal cortex (Brodmann areas 20, 21, and
22), frontal association cortex (Brodmann areas 9 and 10), and cerebellum
were obtained from the Massachusetts Alzheimer Disease Research Center brain
bank (Boston). These pathological specimens were from patients who had received
follow-up at the Memory Disorders Unit at Massachusetts General Hospital (Boston),
and therefore clinical and demographic information were available. Additional
control specimens (n = 7) were obtained from the Harvard Brain Tissue Resource
Center (Belmont, Mass). We processed 61 brains with AD and 33 control brains
for pathological and biochemical analysis. Not all brain regions were available
for all cases, however. For the temporal cortex, there were 61 brains with
AD and 18 controls; for the frontal cortex, 52 cases of AD and 22 controls;
and for the cerebellum, 57 brains with AD and 26 controls. All cases of AD
were diagnosed clinically using criteria from the National Institute of Neurological
and Communicative Disorders and Stroke–Alzheimer's Disease and Related
Disorders Association23 and pathologically
with Reagan Institute Working Group/National Institute on Aging criteria (stages
5 and 6 according to Braak and Braak24-26).
Cases with additional neuropathological diagnoses were excluded (eg, dementia
with Lewy bodies). Although there was no significant difference in the age,
sex, or postmortem interval (range, 3-29 hours) between the AD and control
groups within the entire cohort and when separated by brain region analyzed,
there was an increased frequency of the apolipoprotein E (APOE)  4 allele in the AD group, as expected (Table 1).
SPECIMEN COLLECTION
For biochemical studies, a strip of the human cerebral or cerebellar
cortex was dissected at -20°C, taking care to avoid underlying white
matter, and homogenized in 10 µL/µg (volume per wet weight) of
Tris buffer (50mM Tris; pH 7.2; 0.1% Triton X-100; 200mM sodium chloride;
2mM EDTA) with a protease inhibitor cocktail (Complete; Roche, Indianapolis,
Ind) and 2% protease-free bovine serum albumin (BSA) (Sigma, St Louis, Mo).
The homogenate was centrifuged at 15 000 rpm (21 000 xg) for 5 minutes. The supernatant fluid was used for the
BACE and synaptophysin assays. For synaptophysin, BACE activity, and BACE
protein assays, increasing dilutions of an extract from a single control temporal
cortex were used as a standard.
For A determinations, the adjacent cortex was homogenized in the
Tris buffer without Triton X-100 and centrifuged as described previously.
The pellet was resuspended and homogenized in a solution of 70% formic acid
and centrifuged at 22 000 rpm (44 000 xg) for 5 minutes at 4°C to remove debris. The resulting supernatant
fluid was neutralized with 1M Tris buffer (pH 11) and used for the assay of
formic acid–extractable A species.
BACE PROTEIN ASSAY
The BACE protein assay (Figure 1A)
is a sandwich enzyme-linked immunosorbent assay (ELISA) using the capture
antibody MAB5308 (mouse monoclonal anti-BACE, C-terminus; Chemicon, Temecula,
Calif) and detector antibody PA1-756 (rabbit polyclonal anti-BACE, N-terminus;
Affinity Bioreagents, Golden, Colo). These antibodies are directed toward
epitopes specific to BACE and do not react to BACE2 by Western blot analysis.
We coated 96-well microtiter plates (Greiner, Longwood, Fla) with MAB5308
in a 1:4000 ratio in carbonate buffer (100mM; pH 9.6) at 4°C overnight.
The plates were washed 3 times with phosphate-buffered saline (PBS) (pH 7.0),
then blocked with a blocking reagent (25% BlockAce; Dai-Nippon, Osaka, Japan)
for 6 to 24 hours. The samples (50 µL of 0.004 wt/vol) were added to
the wells containing 50 µL of blocking buffer (Super Block; Pierce,
Rockford, Ill) in PBS and incubated for 1 hour at 37°C. The plates were
washed 4 times with PBS, then incubated overnight at 4°C with PA1-756
in a 1:750 ratio in incubation buffer (0.02M phosphate buffer; 400 mM sodium
chloride; 2mM EDTA; 1% BSA) containing 1% mouse serum. The plates were again
washed with PBS 4 times, then incubated with horseradish peroxidase (HRP)–linked
anti–rabbit IgG (Jackson, West Grove, Pa) in a 1:3000 ratio in incubation
buffer for 2 hours at room temperature. The plates were then washed with PBS
6 times, and fluorometric measurements using a 320-nm excitation filter and
400-nm emission filter were obtained after the addition of HRP substrate (QuantaBlu;
Pierce). Increasing dilutions of an extract from the same control temporal
cortex were used as a standard for every plate; a best-fit log-linear curve,
at dilutions below saturation, was used as the standard curve (Figure 2A). As negative controls, no signal greater than background
fluorescence (eg, ELISA of homogenization buffer alone) was detected when
the primary antibody was replaced with mouse IgG, or when the secondary antibody
was eliminated from the assay, with samples more diluted than 0.01 wt/vol.
|
|
|
|
Figure 2. The -site APP-cleaving enzyme
(BACE) protein enzyme-linked immunosorbent assay (ELISA) and BACE enzymatic
activity assay using fluorogenic substrate 1. Absolute fluorescence intensity
(abs fl int) is not corrected for background fluorescence; adjusted fluorescence
intensity (adj fl int) is corrected. A, Standard curve for BACE protein determination.
B, Standard curve for BACE-specific enzymatic activity assay. There was a
linear increase in BACE activity (as measured by fluorescence intensity of
the cleavage product) with increasing concentration of the brain extract (solid
circles). The BACE activity was completely abolished by 3µM peptidergic
inhibitor P10-P4' StatVal (Peptides International,
Louisville, Ky) (open circles). C, Linear time-dependent increase in BACE
substrate cleavage products (fluorescence intensity adjusted for background).
The BACE cleavage products were measured at the indicated time points. D,
Dose dependence of BACE peptidergic inhibitor P10-P4' StatVal on BACE activity. E, The pH dependency of BACE activity in
our assay system. The pH optimum was approximately 4.0 to 4.5. F, In a serial
dilution of brain extract, BACE activity was correlated with BACE protein.
(Error bars are ± SE. Error bars not visible in C, D, and E are contained
within the data points.)
|
|
|
BACE-SPECIFIC ENZYMATIC ACTIVITY ASSAY
For the BACE activity assay (Figure
1B), we coated 96-well microtiter plates with the capture antibody
MAB5308, used the blocking reagent, and added the samples (50 µL of
0.01 wt/vol) as described previously. Samples were incubated for 1 hour at
37°C. The plates were washed 6 times with PBS, and the enzymatic reaction
was carried out by incubation with 10µM fluorogenic substrate for -secretase.
We used either substrate 1,
(7-methoxycoumarin-4-yl)acetyl [MOCA]-Ser-Glu-Val-Asn-Leu-
Asp-Ala-Glu-Phe-Arg-N-(2,4-dinitrophenyl)
[DNP]-Lys-Arg-Arg-NH2 (Peptides International, Louisville, Ky); or substrate
2, Arg-Glu(5-[aminoethyl] aminonaphthalene sulfonate
[EDANS])-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4'-
dimethylaminoazo-benzene-4-carboxylate
[DABCYL])-Arg (Calbiochem, San Diego, Calif). Samples were incubated in acetate
buffer with 100mM sodium chloride (pH 4.1) containing 0.025% BSA for 16 to
24 hours at 37°C, unless otherwise specified. The substrates are decapeptides
flanking the -secretase cleavage site of the Swedish mutation of APP
(APPKN670-1ML) with an autoquenching fluorescent tag that is activated
by -secretase cleavage of the peptide.27
Each peptide separates the fluorescence donor (MOCA or EDANS) from an acceptor
(DNP or DABCYL), which quenches the fluorescence by fluorescence resonance
energy transfer. Cleavage of the peptide bond within the substrate by -secretase
enzymatic activity leads to the separation of the donor-acceptor pair, resulting
in an increase in fluorescence. The fluorescence increase is proportional
to the amount of peptide hydrolyzed (the enzymatic activity). The enzymatic
product was measured on a plate reader (Wallac Victor V2; Perkin-Elmer, Wellesley,
Mass) using a 340-nm excitation filter and 400-nm emission filter for substrate
1, and 355 nm and 510 nm, respectively, for substrate 2. Increasing dilutions
of an extract from the same control temporal cortex was used as a standard
for every plate; a best-fit linear-linear curve, at dilutions below saturation,
was used as the standard curve (Figure 2B).
For negative controls, no signal greater than background fluorescence was
detected when the capture antibody was replaced with mouse IgG, or when the
N-terminal antibody PA1-756 was used as the capture antibody, with samples
more diluted than 0.025 wt/vol.
IMMUNOPRECIPITATION OF BACE FROM HUMAN BRAIN
The temporal neocortex was dissected and homogenized with 10 µL/µg
(volume per wet weight) of extraction buffer containing protease inhibitors.
The homogenate was centrifuged at 15 000 rpm for 5 minutes. The supernatant
fluid (500 µL) was incubated overnight at 4°C with protein G-Sepharose
(20 µL of 50%, vol/vol; ImmunoPure Plus Immobilized Protein G; Pierce)
to remove nonspecific protein binding. After centrifugation at 15 000
rpm for 5 minutes, the supernatant fluid was incubated for 6 hours at 4°C
with 10 µg of MAB5308 or PA1-756 covalently coupled to protein G–Sepharose
(25 µL of 50%, vol/vol) according to the manufacturer's instructions
(Seize-X Protein G Immunoprecipitation Kit; Pierce). The sample was washed
3 times with washing buffer by centrifugation, and the bound protein was eluted
with elution buffer. The eluted samples were boiled for 5 minutes in loading
buffer. Proteins were separated by 10% to 20% Tris-glycine sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (Novex/Invitrogen, Carlsbad,
Calif) under denaturing, reducing conditions, transferred to a polyvinylidene
fluoride (PVDF) membrane, blocked with 5% nonfat dry milk in Tris-buffered
saline (TBS), pH 7.4, with 0.05% polyoxyethylenesorbitan monolaurate (Tween-20
[TBS-T]) for 2 hours, and incubated with MAB5308 (1 µg/mL of MAB5308
in TBS buffer containing 1.5% goat serum) overnight at 4°C. After washing
with TBS-T, the membrane was incubated with HRP anti–mouse IgG (1:5000;
Jackson) for 2 hours, and the BACE band was visualized using enhanced chemiluminescence
(Perkin-Elmer).
ELISA FOR A40, A42, AND SYNAPTOPHYSIN
Total formic acid A was determined from the sum of ELISAs for
A40 and A42 using BNT77 (mouse monoclonal anti–A11-28;
Takeda, Osaka, Japan) as the capture antibody and BA27 (monoclonal anti-A40;
Takeda) or BC05 (monoclonal anti-A42; Takeda) conjugated with HRP as
the detector antibody; QuantaBlu was used as the fluorescent HRP
substrate.28
Synaptophysin ELISA was performed using MAB329 (mouse monoclonal IgM
anti-synaptophysin; 1:2000; Chemicon) as the capture antibody and biotinylated
SY38 (mouse monoclonal IgG1 anti-synaptophysin; 1:2000; Progen Biotechnik
GmbH, Heidelberg, Germany) followed by HRP-conjugated streptavidin as the
detector antibody. We used 3,3',5,5'-tetramethylbenzidine (Kirkegaard
& Perry, Gaithersburg, Md) as the colorimetric HRP substrate; it was measured
at 450 nm.
IMMUNOHISTOCHEMISTRY
For immunohistochemical studies, brains were fixed in 4% paraformaldehyde
for at least 2 days and cryoprotected in 10% glycerol in PBS for at least
2 days before being cut into 50-µm sections on a freezing sledge microtome.
Sections were permeabilized with 0.5% Triton X-100 in TBS, blocked with 3%
nonfat dry milk in TBS, and sequentially probed with primary antibody (MAB5308,
1:500; rabbit polyclonal anti–glial fibrillary acidic protein [anti-GFAP],
1:500 [Dako, Carpinteria, Calif]; rabbit polyclonal anti–A R1282,
1:500 [D. Selkoe, MD, Harvard Medical School, Boston, Mass]; and mouse monoclonal
IgM anti–phospho- TG3, 1:10 [P. Davies, PhD, Albert Einstein College
of Medicine, Bronx, NY], in 1.5% normal goat serum in TBS) and secondary antibody
(biotinylated anti–mouse IgG1, 1:200; Cy5-linked anti–rabbit IgG,
1:200 [Jackson]; BODIPY-fluorescein-linked anti–rabbit IgG or anti–mouse
IgM, 1:200 [Molecular Probes, Eugene, Ore]; and Cy3-linked streptavidin, 1:750
[Jackson]). Confocal images were obtained using a laser confocal scanning
system (MRC 1024; BioRad, Hercules, Calif).
STATISTICAL ANALYSIS
To analyze BACE activity and BACE protein, we performed analysis of
variance (ANOVA) according to diagnosis (brains with AD vs controls) and brain
region (temporal cortex, frontal cortex, or cerebellum). Significant effects
of diagnosis with ANOVA were followed up using the t
test to determine which brain regions were significant. For significant results
within brain regions, correlation analysis was used to correlate BACE activity
and protein with duration of illness, formic acid–extractable A,
or synaptophysin (StatView; Abacus Concepts, Berkeley, Calif).
RESULTS
BACE ELISA
To quantitate BACE changes in AD, we developed specific assays for BACE
protein and enzymatic activity (Figure 1).
The ELISA for full-length BACE protein uses capture antibody MAB5308 and detector
antibody PA1-756 (Figure 1A). The
assay exhibits sensitivity for the measurement of BACE protein in brain extracts
(Figure 2A) and produces a plateau
with samples more concentrated than 0.01 wt/vol (data not shown). Specificity
of the protein assay was confirmed by immunoprecipitation of BACE from human
brain extracts with either antibody PA1-756 or MAB5308, separation by Western
blot analysis, and probing with MAB5308, which detected diffuse bands of approximately
52 kd and 70 kd
(Figure 3).22, 29-30
|
|
|
|
Figure 3. The -site APP-cleaving enzyme
(BACE) immunoprecipitation and Western blot analysis. Immunoprecipitation
of the brain extracts demonstrates bands at approximately 52 kd and 70 kd.
The BACE was captured with anti-BACE C-terminal antibody MAB5308 (lane a)
or anti-BACE N-terminal antibody PA1-756 (lane b) and probed with MAB5308.
|
|
|
BACE-SPECIFIC ENZYMATIC ACTIVITY ASSAY
Our antibody capture assay for BACE-specific -secretase activity
uses MAB5308 to capture BACE from specimens added to a multiplate well and
incubation with a -secretase substrate (a fluorescence-quenching decapeptide
representing the cleavage site of the Swedish mutant of APP) in acidic conditions
(pH 4.1) optimal for BACE function. Capture of the BACE C-terminus allows
the more N-terminal catalytic domain to cleave the substrate. Cleavage of
the peptide releases fluorescence proportional to the amount of peptide hydrolyzed:
the enzymatic activity (Figure 1B).
The amount of substrate cleaved increases in a linear manner across time with
exposure to captured BACE (r226=
0.96; P<.001) (Figure 2C).
The assay exhibits high sensitivity and linearity for the measurement
of BACE activity in brain extracts in the range of brain dilutions used for
analysis, with a linear-linear curve fit for the standard (Figure 2B, solid circles), and a plateau in fluorescence with samples
more concentrated than 0.05 wt/vol (data not shown). Specificity of the activity
assay was confirmed as follows: (1) The specific peptidergic inhibitor of
BACE activity (Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe [P10-P4' StatVal]; Peptides International) eliminated
the enzymatic activity with a 50% inhibitory concentration (IC50) of approximately
10nM, consistent with the published literature using purified BACE (IC50 approximately
30nM)4 (Figure
2B, open circles, and Figure 2D). (2) Captured BACE had an optimum pH in the acidic range (pH, 4.0-4.5) consistent
with published data (Figure 2E)1 and supporting cell culture studies localizing -secretase
processing of APP to acidic subcellular compartments and the trans-Golgi network.31 In the absence of antibody capture, the fluorogenic
substrate 1 used in the activity assay detects thimet oligopeptidase as a -secretase
with a maximum pH in the neutral range27; in
contrast, our antibody capture assay does not demonstrate significant -secretase
activity at the neutral pH. (3) After immunoprecipitation with MAB5308 covalently
coupled to IgG resin, brain homogenate separated by Western blot analysis
and probed with the same antibody demonstrates BACE bands (Figure 3, lane a).
By using antibody capture at the appropriate pH, the BACE activity assay
avoids the detection of other -secretase cleavage activities (eg, BACE2,
cathepsin D,32 or thimet oligopeptidase27). Using a single human brain extract as a standard,
BACE protein was correlated with BACE activity at various dilutions (r23= 0.86;
P = .02) (Figure 2F). Fluorogenic
substrate 1 and fluorogenic substrate 2 gave similar results in this assay.
INCREASED BACE PROTEIN IN BRAINS WITH AD
Our primary outcome measures in this study were BACE protein and BACE
activity in brains with AD vs control brains to test the hypothesis that these
levels are increased in regions of the brain that develop amyloid plaques.
Homogenates from the temporal cortex (Brodmann areas 20, 21, and 22), frontal
cortex (Brodmann areas 9 and 10), and cerebellar cortex were analyzed. The
temporal and frontal regions were selected because they are well-delineated,
high-order association areas affected by amyloid plaques in AD and they develop
progressive neurofibrillary changes and neuronal loss during the course of
the illness. The temporal cortex is generally more affected than the frontal
cortex.33-34 The cerebellum was
chosen as an area with minimal involvement in AD.
Analysis of variance according to diagnosis and brain region showed
that BACE protein level is altered in AD, with significantly increased BACE
protein levels in the frontal cortex and a trend toward increased BACE protein
levels in the temporal cortex relative to control cases (Figure 4A). Results of ANOVA showed a significant main effect for
diagnosis (F1,227= 11.2; P<.001). Post
hoc comparisons showed that mean BACE protein levels increased by 14% in the
frontal cortex in AD (t70= 3.0; P = .004), with a 15% increase in mean BACE protein levels
in the temporal cortex that failed to reach statistical significance (t76= 1.9;
P = .07)
and no significant difference in the cerebellum (t81= 1.5;
P = .13).
|
|
|
|
Figure 4. The -site APP-cleaving enzyme
(BACE) protein and specific activity in brains with Alzheimer disease (AD)
and controls and correlation with clinical markers. A, Diagnosis demonstrates
a significant main effect on BACE protein by analysis of variance (ANOVA)
(P<.001), with significantly increased BACE protein levels
in the frontal cortex in brains with AD as determined by a post hoc t test
(asterisk indicates P = .004). B, Diagnosis also
demonstrates a significant effect on BACE activity by ANOVA (P<.001),
with significantly increased BACE activity in the temporal cortex (asterisk
indicates P = .007) and frontal cortex (asterisk indicates
P = .003) in brains with AD as determined by a post hoc t
test. C and D, Because BACE is a predominantly neuronal protein, we normalized
BACE measures for synaptic and neuronal loss by determining the ratio of BACE
protein to synaptophysin protein (BACE Prt/Syn) (C) and the ratio of BACE
activity to synaptophysin protein (BACE Act/Syn) (D). Normalized BACE measures
are significantly increased in the temporal and frontal cortices in brains
with AD (ANOVA, P<.001; asterisks indicate P = .02 [temporal] and
P<.001 [frontal] in C and P = .01
[temporal] and P = .001 [frontal] in D as determined by a post
hoc t test.) E, The ratio of BACE activity (Act) to BACE protein
(Prt) shows an ANOVA effect by diagnosis (ANOVA, P = .01), reflecting
an increased ratio in the temporal cortex (asterisk indicates P = .02 as determined
by a post hoc t test). F, The BACE activity
increases with duration of illness (P = .008) in the temporal
cortex in brains with AD. (Error bars are ± SE.)
|
|
|
INCREASED BACE ACTIVITY IN BRAINS WITH AD
In support of the BACE protein studies, we hypothesized that BACE activity
would be altered in AD, with significantly increased activity in the frontal
and temporal cortices but not the cerebellum. Analysis of variance for BACE
activity showed a significant main effect for diagnosis (F1,229=
14.2; P<.001). Post hoc comparisons showed that
mean BACE activity increased in brains with AD by 63% in the temporal cortex
(t76= 2.8; P = .007) and 13% in the frontal cortex (t72=
3.0; P = .003), but not in the cerebellum (t81= 1.4;
P = .18)
(Figure 4B). There was no association
of BACE protein or activity level with postmortem interval in the range of
3 to 29 hours.
INCREASED RATIO OF BACE ACTIVITY TO BACE PROTEIN IN THE TEMPORAL CORTEX
IN BRAINS WITH AD
Because mean BACE activity levels were increased to a greater extent
than mean BACE protein levels in the cerebral cortex in brains with AD vs
controls, we evaluated the ratio of BACE activity to BACE protein levels for
each case (BACE act/prt). Analysis of variance for the BACE act/prt ratio
showed a main effect for diagnosis (F1,227= 6.1; P = .01), reflecting a significant 45% increase in the mean BACE act/prt
ratio in the temporal cortex (t76 = 2.5; P = .02) but not in the frontal
cortex (P = .92) or cerebellum (P = .81) in brains
with AD (Figure 4E). This suggests
that BACE activity levels may be modulated by other factors in addition to
BACE protein levels.
TEMPORAL CORTEX BACE ACTIVITY INCREASES WITH DURATION OF ILLNESS IN AD
We tested the hypotheses that significant increases in BACE protein
and activity levels were correlated with clinical and pathological parameters
of dementia progression. The primary clinical parameter available for the
AD cases was duration of dementia. Within AD cases, temporal cortex BACE activity
levels increased with the duration of illness (r258 = 0.11;
P = .008) (Figure 4D). Frontal BACE protein and activity
levels did not significantly correlate with the duration of illness.
BIOCHEMICAL CHARACTERIZATION OF PATHOLOGICAL SPECIMENS
To investigate correlations of AD severity with BACE measures in the
same brain regions, we determined biochemical measures of amyloid deposition
(formic acid–extractable A) and synaptic integrity (synaptophysin)
by ELISA in the temporal, frontal, and cerebellar cortices of AD and control
cases. Levels of total formic acid–extractable A were markedly
increased in brains with AD, with the highest absolute levels in the temporal
and frontal cortices (Figure 5A).
The ANOVA for total formic acid A demonstrated a main effect for diagnosis
(F1,228 = 153; P<.001), with significantly
increased A in the temporal cortex by 52-fold (t76 = 7.6;
P<.001), the frontal cortex by
10-fold (t71= 13; P<.001), and the cerebellum by 82-fold (t81 = 3.4;
P = .001) in AD relative to control
cases. There was also a main effect for brain region (F2,228 =
38; P<.001) and an interaction between brain region
and diagnosis (F2,228= 34; P<.001).
Of note was a hierarchical distribution of formic acid–extractable A
levels in AD, which were highest in the temporal cortex, then the frontal
cortex, and finally the cerebellum (P<.001 between
brain regions in AD).
For synaptophysin, ANOVA demonstrated a significant effect for diagnosis
(F1,227 = 15.5; P<.001), with a 38%
reduction in the temporal cortex (t74
= -3.5; P<.001), a 14% reduction in the
frontal cortex (t72= -2.2; P = .03), and no significant difference in the cerebellar
cortex in brains with AD (Figure 5B). These data together with the A data suggest that in this cohort, the
temporal cortex was more affected by AD pathological changes than the frontal
cortex and that the cerebellum was relatively spared.
In AD cases, there was no significant correlation of BACE protein or
activity with formic acid–extractable A (Figure 5C and D). In AD brain regions, levels of BACE protein and
activity did not correlate with synaptophysin measures.
BACE IS EXPRESSED IN NEURONS AND NEUROPIL IN BRAINS WITH AD AND CONTROLS
To determine whether the increased BACE protein and activity levels
were due to BACE expression in astrocytes associated with gliosis in AD, we
performed immunostaining of the temporal cortex in 5 brains with AD and 5
controls (Figure 6). Immunostaining
of the temporal cortex and hippocampus with anti-BACE antibody demonstrated
diffuse staining of the neuropil and staining of neuronal cell bodies and
neurites in AD cases and controls; in brains with AD, BACE staining occasionally
colocalized with neurofibrillary tangle–bearing neurons (Figure 6C). Double staining with GFAP did not reveal BACE immunoreactivity
in activated astrocytes in brains with AD (Figure 6B). These results suggest that BACE is predominantly expressed
in neurons in the brain and argue against the possibility that elevated BACE
levels and activity come from up-regulation in astroglial elements.
BACE PROTEIN AND ACTIVITY NORMALIZED TO SYNAPTOPHYSIN PROTEIN ARE INCREASED
IN AD
Because BACE is primarily a neuronal protein1, 30
and BACE immunoreactivity was observed mainly in neurons, we sought to correct
our BACE data for neuronal loss. We normalized BACE activity and protein for
synaptophysin measures (as a surrogate marker for cortical damage) by analyzing
the ratios of BACE activity to synaptophysin protein (BACE act/syn) and BACE
protein to synaptophysin (BACE prt/syn) in each case (Figure 4C and D). For both measures, ANOVA showed significant main
effects for diagnosis (BACE act/syn, F1,227= 12.6; P<.001; BACE prt/syn, F1,225 = 19.0;
P<.001), reflecting increases in normalized BACE protein levels
of 60% in the temporal cortex (t74 = 2.3; P = .02) and 33% in the frontal cortex (t70= 4.2;
P<.001) and increases
in normalized BACE activity levels of 156% in the temporal cortex (t74= 2.6;
P = .01) and 37% in the
frontal cortex (t72= 3.3; P = .001) in brains with AD.
COMMENT
Amyloid plaques are a characteristic neuropathologic feature of AD.
In autosomal dominant familial AD and Down syndrome, A deposition can
be attributed to excessive A production mediated by APP/presenilin mutations or APP gene dosage
effects. However, the mechanism by which excessive A deposition occurs
in sporadic AD is unknown. The BACE is critical in A biosynthesis. It
is present in high levels in the brain, efficiently cleaves APP at the -secretase
cleavage site, and localizes to acidic compartments in the secretory pathway
where A production occurs.1, 3-5
A production is abolished in BACE knockout mice,18-19
whereas the overexpression of BACE in APP transgenic mice increases A
formation.35 Studies have found increased BACE
protein levels in the hippocampal CA1 subfield by immunostaining30
and in the frontal cortex by semiquantitative Western blot analysis22 in brains with AD.
We have developed 2 new tools to study BACE in neuropathological samples.
The BACE protein assay is a 2-site enzyme immunoassay consisting of capture
(MAB5308) and detector (PA1-756) antibodies directed toward distinct epitopes
of the antigen, C-terminal and N-terminal, respectively. The ELISA system
is specific for BACE, as confirmed by the immunoprecipitation results. Compared
with Western blot analysis, ELISA can analyze more samples in a single plate
with a quantitative standard curve and is therefore suitable for the analysis
of many samples.
The BACE activity assay is an antibody capture assay, with activity
measured via fluorescence emission after the cleavage of a -secretase
substrate. This substrate has previously been used to assess -secretase
activity with purified BACE and brain extracts4, 27;
however, the substrate can be cleaved by other proteases, such as thimet oligopeptidase.27 To eliminate these other -secretase activities,
we first captured BACE protein via its C-terminal domain from brain extracts
on ELISA plates coated with anti-BACE antibody, then assayed the enzymatic
reaction from the more N-terminal catalytic domain of the captured BACE. The
other proteases are not bound by the anti-BACE C-terminal–specific antibody
MAB5308, which was raised against BACE epitopes distinct from BACE2; therefore,
this assay eliminates other sources of substrate cleavage. The isolation of
BACE through antibody capture can also eliminate competition by endogenous
APP fragments as the BACE substrate. Furthermore, the assay is run at an acidic
pH optimal for BACE -secretase activity. The bound enzymatic activity
measured from the human brain is inhibited in a dose-dependent manner by a
BACE peptidergic inhibitor at an IC50 of approximately 10nM, in agreement
with previous studies of purified BACE protein.4
The assay can be used to evaluate a large number of pathological specimens
and can also be adapted for screening inhibitors of BACE activity without
the requirement for purified BACE.
We find that BACE protein and/or enzymatic activity levels are increased
in the frontal and temporal cortices in brains with AD, implying that mechanisms
of increased A production are operative in sporadic AD. A deposition
occurs in characteristic and discrete anatomical patterns in brains with AD,
including the cortical and limbic regions.33
We focused on specific temporal (Brodmann areas 20, 21, and 22) and frontal
(Brodmann areas 9 and 10) neocortical regions because these are anatomically
well-demarcated, high-order association areas that receive multimodal sensory
input and are consistently affected by senile plaques and neurofibrillary
tangles in AD. Increased BACE is specific to these areas. The alterations
in BACE do not occur in the cerebellum, a region not significantly affected
by AD changes. The increase in BACE activity is surprising given the predominant
neuronal localization of BACE and significant progressive temporal lobe neuronal
loss in AD34; measures of BACE protein and
activity levels are even more pronounced when normalized for the neuronal
protein synaptophysin, a marker of synaptic loss in AD. Possible mechanisms
of BACE changes include increased BACE expression and activity in remaining
neurons, the effect of posttranslational modification of BACE on activity,
or increased BACE expression in astrocytes. Our immunohistochemical studies
do not suggest a significant glial up-regulation of BACE, consistent with
the results of Sun et al.30 The ratio of BACE
activity to BACE protein is increased in the temporal cortex in cases of AD,
suggesting that a posttranslational modification of BACE resulting in increased -secretase
activity, an alteration in critical BACE cofactors, or an increase in active
vs inactive splice forms of BACE may occur in AD36;
inactive splice forms have not been detected in the human brain.37
The BACE activity does not significantly correlate with the amount of
A in the temporal cortex in brains with AD. This is probably because
the amount of A in the brains is also modulated by -secretase
and -secretase and by processes of A fibrillization, deposition,
uptake, and catabolism. Despite the complicated pathways involved in A
metabolism, recent data demonstrate that the overexpression of BACE in cell
culture and in a transgenic mouse model lead to elevated steady-state A
levels, suggesting that levels of BACE protein and activity affect A
generation in vivo.35 The elimination of BACE
in knockout mice dramatically reduces A without any toxic phenotypic
effects,18-19 so therapies aimed
at reducing BACE may be less toxic than those targeting -secretase.38 The persistence of high levels of BACE activity in
AD cases of long duration supports the idea that BACE inhibitors could be
effective in reducing A production, even in advanced AD.
To our knowledge, this is the first report quantitating the up-regulation
of BACE activity in clinically and pathologically characterized brains with
sporadic AD and delineating the anatomical pattern of BACE protein and functional
dysregulation. The BACE activity is increased in a hierarchical neuroanatomical
pattern consistent with the extent of AD abnormalities (temporal cortex >
frontal cortex > cerebellum) and remains elevated in the temporal cortex throughout
the course of the illness. These data support BACE as an important target
for therapeutics in AD and imply that biochemical and pathological factors
modulate BACE activity in sporadic AD.
AUTHOR INFORMATION
Accepted for publication June 19, 2002.
Author contributions: Study
concept and design (Drs Fukumoto, Hyman, and Irizarry); acquisition of data (Drs Fukumoto and Irizarry and Ms Cheung); analysis and interpretation of data (Drs Fukumoto, Hyman,
and Irizarry); drafting of the manuscript (Drs Fukumoto,
Hyman, and Irizarry and Ms Cheung); critical revision of
the manuscript for important intellectual content (Drs Fukumoto, Hyman,
and Irizarry); statistical expertise (Drs Hyman and
Irizarry); obtained funding (Drs Hyman and Irizarry); administrative, technical, and material support (Drs Fukumoto
and Irizarry and Ms Cheung); study supervision (Drs
Fukumoto, Hyman, and Irizarry).
This study was supported by grants AG00793, AG05134 (Massachusetts Alzheimer
Disease Research Center, Boston), and PHS MH/NS 31862 (Harvard Brain Tissue
Resource Center, Belmont, Mass) from the National Institutes of Health, Bethesda,
Md, and the Walters Family Foundation, New York, NY. Dr Fukumoto is supported
by Takeda Chemical Industries, Osaka, Japan.
Antibodies BNT77, BA27, and BC05 were kindly provided by Takeda.
Corresponding author and reprints: Michael C. Irizarry, MD, Alzheimer
Disease Research Unit, Center for Aging, Genetics, and Neurodegeneration,
Massachusetts General Hospital–East, Bldg 114, Room 2010, 114 16th Street,
Charlestown, MA 02129 (e-mail: mirizarry{at}partners.org).
From the Alzheimer Disease Research Unit, Department of Neurology,
Massachusetts General Hospital, Charlestown.
REFERENCES
| |
1. Vassar R, Bennett BD, Babu-Khan S, et al. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by
the transmembrane aspartic protease BACE. Science. 1999;286:735-741.
FREE FULL TEXT
2. Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ. Two transmembrane aspartates in presenilin-1 required for presenilin
endoproteolysis and gamma-secretase activity. Nature. 1999;398:513-517.
FULL TEXT
| PUBMED
3. Yan R, Bienkowski MJ, Shuck ME, et al. Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase
activity. Nature. 1999;402:533-537.
FULL TEXT
| PUBMED
4. Sinha S, Anderson JP, Barbour R, et al. Purification and cloning of amyloid precursor protein beta-secretase
from human brain. Nature. 1999;402:537-540.
FULL TEXT
| PUBMED
5. Hussain I, Powell D, Howlett DR, et al. Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci. 1999;14:419-427.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
6. Bennett BD, Denis P, Haniu M, et al. A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's
beta-secretase. J Biol Chem. 2000;275:37712-37717.
FREE FULL TEXT
7. Capell A, Steiner H, Willem M, et al. Maturation and pro-peptide cleavage of beta-secretase. J Biol Chem. 2000;275:30849-30854.
FREE FULL TEXT
8. Haniu M, Denis P, Young Y, et al. Characterization of Alzheimer's beta-secretase protein BACE. J Biol Chem. 2000;275:21099-21106.
FREE FULL TEXT
9. Creemers JW, Ines Dominguez D, Plets E, et al. Processing of beta-secretase by furin and other members of the proprotein
convertase family. J Biol Chem. 2001;276:4211-4217.
FREE FULL TEXT
10. Huse JT, Pijak DS, Leslie GJ, Lee VM, Doms RW. Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving
enzyme: the Alzheimer's disease beta-secretase. J Biol Chem. 2000;275:33729-33737.
FREE FULL TEXT
11. Riddell DR, Christie G, Hussain I, Dingwall C. Compartmentalization of ß-secretase (Asp2) into low-buoyant density,
non-caveolar lipid rafts. Curr Biol. 2001;11:1288-1293.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
12. Walter J, Fluhrer R, Hartung B, et al. Phosphorylation regulates intracellular trafficking of beta-secretase. J Biol Chem. 2001;276:14634-14641.
FREE FULL TEXT
13. Yan R, Han P, Miao H, Greengard P, Xu H. The transmembrane domain of the Alzheimer's ß-secretase (BACE1)
determines its late Golgi localization and access to ß-amyloid precursor
protein (APP) substrate. J Biol Chem. 2001;276:36788-36796.
FREE FULL TEXT
14. Saunders AJ, Kim T-W, Tanzi RE. BACE maps to chromosome 11 and a BACE homolog, BACE2, reside in the
obligate Down syndrome region of chromosome 21. Science. 1999;286:1255a.
15. Bennett BD, Babu-Khan S, Loeloff R, et al. Expression analysis of BACE2 in brain and peripheral tissue. J Biol Chem. 2000;275:20647-20651.
FREE FULL TEXT
16. Farzan M, Schnitzler CE, Vasilieva N, Leung D, Choe H. BACE2, a beta-secretase homolog, cleaves at the beta site and within
the amyloid-beta region of the amyloid-beta precursor protein. Proc Natl Acad Sci U S A. 2000;97:9712-9717.
FREE FULL TEXT
17. Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides
by neurons. Nat Neurosci. 2001;4:233-234.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
18. Luo Y, Bolon B, Kahn S, et al. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal
phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001;4:231-232.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
19. Roberds SL, Anderson J, Basi G, et al. BACE knockout mice are healthy despite lacking the primary beta-secretase
activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet. 2001;10:1317-1324.
FREE FULL TEXT
20. Yasojima K, McGeer EG, McGeer PL. Relationship between beta amyloid peptide generating molecules and
neprilysin in Alzheimer disease and normal brain. Brain Res. 2001;919:115-121.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
21. Irizarry MC, Locascio JJ, Hyman BT. Beta-site APP cleaving enzyme mRNA expression in APP transgenic mice. Am J Pathol. 2001;158:173-177.
FREE FULL TEXT
22. Holsinger D, McLean CA, Beyreuther K, Masters CL, Evin G. Increased expression of amyloid precursor ß-secretase in Alzheimer's
disease. Ann Neurol. 2002;51:783-786.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
23. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease. Neurology. 1984;34:939-944.
FREE FULL TEXT
24. Braak H, Braak E. Neuropathological staging of Alzheimer-related changes. Acta Neuropathol (Berl). 1991;82:239-259.
FULL TEXT
| PUBMED
25. Consensus recommendations for the postmortem diagnosis of Alzheimer's
disease. Neurobiol Aging. 1997;18(suppl 1):S1-S2.
26. Hyman BT, Trojanowski JQ. Consensus recommendations for the postmortem diagnosis of Alzheimer
disease from the National Institute on Aging and the Reagan Institute Working
Group on diagnostic criteria for the neuropathological assessment of Alzheimer
disease. J Neuropathol Exp Neurol. 1997;56:1095-1097.
WEB OF SCIENCE
| PUBMED
27. Koike H, Seki H, Kouchi Z, et al. Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor
protein at a beta-secretase cleavage site in COS cells. J Biochem (Tokyo). 1999;126:235-242.
FREE FULL TEXT
28. Suzuki N, Cheung TT, Cai XD, et al. An increased percentage of long amyloid beta protein secreted by familial
amyloid beta protein precursor (APP717) mutants. Science. 1994;264:1336-1340.
FREE FULL TEXT
29. Charlwood J, Dingwall C, Matico R, et al. Characterization of the glycosylation profiles of Alzheimer's beta-secretase
protein Asp-2 expressed in a variety of cell lines. J Biol Chem. 2001;276:16739-16748.
FREE FULL TEXT
30. Sun A, Koelsch G, Tang J, Bing G. Localization of -secretase memapsin 2 in the brain of Alzheimer's
patients and normal aged controls. Exp Neurol. 2002;175:10-22.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
31. Knops J, Suomensaari S, Lee M, McConlogue L, Seubert P, Sinha S. Cell-type and amyloid precursor protein-type specific inhibition of
A beta release by bafilomycin A1, a selective inhibitor of vacuolar ATPases. J Biol Chem. 1995;270:2419-2422.
FREE FULL TEXT
32. Gruninger-Leitch F, Berndt P, Langen H, Nelboeck P, Dobeli H. Identification of beta-secretase-like activity using a mass spectrometry-based
assay system. Nat Biotechnol. 2000;18:66-70.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
33. Arnold SE, Hyman BT, Flory J, Damasio AR, Van Hoesen GW. The topographical and neuroanatomical distribution of neurofibrillary
tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer's
disease. Cereb Cortex. 1991;1:103-116.
FREE FULL TEXT
34. Gomez-Isla T, Hollister R, West H, et al. Neuronal loss correlates with but exceeds neurofibrillary tangles in
Alzheimer's disease. Ann Neurol. 1997;41:17-24.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
35. Bodendorf U, Danner S, Fischer F, et al. Expression of human beta-secretase in the mouse brain increases the
steady-state level of beta-amyloid. J Neurochem. 2002;80:799-806.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
36. Tanahashi H, Tabira T. Three novel alternatively spliced isoforms of the human beta-site amyloid
precursor protein cleaving enzyme (BACE) and their effect on amyloid beta-peptide
production. Neurosci Lett. 2001;307:9-12.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
37. Bodendorf U, Fischer F, Bodian D, Multhaup G, Paganetti P. A splice variant of beta-secretase deficient in the amyloidogenic processing
of the amyloid precursor protein. J Biol Chem. 2001;276:12019-12023.
FREE FULL TEXT
38. Haass C, De Strooper B. The presenilins in Alzheimer's disease: proteolysis holds the key. Science. 1999;286:916-919.
FREE FULL TEXT
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter
What's this?
RELATED ARTICLE
Explaining the Cause of the Amyloid Burden in Alzheimer Disease
Roger N. Rosenberg
Arch Neurol. 2002;59(9):1367-1368.
EXTRACT
| FULL TEXT
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
|
The {beta}-Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential
Vassar et al.
J. Neurosci. 2009;29:12787-12794.
ABSTRACT
| FULL TEXT
RAGE regulates BACE1 and A{beta} generation via NFAT1 activation in Alzheimer's disease animal model
Cho et al.
FASEB J. 2009;23:2639-2649.
ABSTRACT
| FULL TEXT
Prostaglandin E2 Stimulates the Production of Amyloid-{beta} Peptides through Internalization of the EP4 Receptor
Hoshino et al.
J. Biol. Chem. 2009;284:18493-18502.
ABSTRACT
| FULL TEXT
Mutant Presenilin 1 Increases the Expression and Activity of BACE1
Giliberto et al.
J. Biol. Chem. 2009;284:9027-9038.
ABSTRACT
| FULL TEXT
MicroRNA-298 and MicroRNA-328 Regulate Expression of Mouse {beta}-Amyloid Precursor Protein-converting Enzyme 1
Boissonneault et al.
J. Biol. Chem. 2009;284:1971-1981.
ABSTRACT
| FULL TEXT
The Role of Amyloid Precursor Protein Processing by BACE1, the {beta}-Secretase, in Alzheimer Disease Pathophysiology
Cole and Vassar
J. Biol. Chem. 2008;283:29621-29625.
ABSTRACT
| FULL TEXT
Elevated Cerebrospinal Fluid BACE1 Activity in Incipient Alzheimer Disease
Zetterberg et al.
Arch Neurol 2008;65:1102-1107.
ABSTRACT
| FULL TEXT
Promotion of BACE1 mRNA Alternative Splicing Reduces Amyloid {beta}-Peptide Production
Mowrer and Wolfe
J. Biol. Chem. 2008;283:18694-18701.
ABSTRACT
| FULL TEXT
Distinct Pools of {beta}-Amyloid in Alzheimer Disease-Affected Brain: A Clinicopathologic Study
Steinerman et al.
Arch Neurol 2008;65:906-912.
ABSTRACT
| FULL TEXT
Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/{beta}-secretase expression
Hebert et al.
Proc. Natl. Acad. Sci. USA 2008;105:6415-6420.
ABSTRACT
| FULL TEXT
The Expression of MicroRNA miR-107 Decreases Early in Alzheimer's Disease and May Accelerate Disease Progression through Regulation of {beta}-Site Amyloid Precursor Protein-Cleaving Enzyme 1
Wang et al.
J. Neurosci. 2008;28:1213-1223.
ABSTRACT
| FULL TEXT
{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE
Yin et al.
J. Biol. Chem. 2007;282:23639-23644.
ABSTRACT
| FULL TEXT
Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5' untranslated region
Mihailovich et al.
Nucleic Acids Res 2007;35:2975-2985.
ABSTRACT
| FULL TEXT
{beta}-Site Amyloid Precursor Protein Cleaving Enzyme 1 Levels Become Elevated in Neurons around Amyloid Plaques: Implications for Alzheimer's Disease Pathogenesis
Zhao et al.
J. Neurosci. 2007;27:3639-3649.
ABSTRACT
| FULL TEXT
Interferon-{gamma} and Tumor Necrosis Factor-{alpha} Regulate Amyloid-{beta} Plaque Deposition and {beta}-Secretase Expression in Swedish Mutant APP Transgenic Mice
Yamamoto et al.
Am. J. Pathol. 2007;170:680-692.
ABSTRACT
| FULL TEXT
Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression
Sun et al.
Proc. Natl. Acad. Sci. USA 2006;103:18727-18732.
ABSTRACT
| FULL TEXT
LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease
Majercak et al.
Proc. Natl. Acad. Sci. USA 2006;103:17967-17972.
ABSTRACT
| FULL TEXT
p25/Cyclin-Dependent Kinase 5 Induces Production and Intraneuronal Accumulation of Amyloid beta In Vivo
Cruz et al.
J. Neurosci. 2006;26:10536-10541.
ABSTRACT
| FULL TEXT
GGA1 Acts as a Spatial Switch Altering Amyloid Precursor Protein Trafficking and Processing
von Arnim et al.
J. Neurosci. 2006;26:9913-9922.
ABSTRACT
| FULL TEXT
Neuritic Deposits of Amyloid-{beta} Peptide in a Subpopulation of Central Nervous System-Derived Neuronal Cells
Muresan and Muresan
Mol. Cell. Biol. 2006;26:4982-4997.
ABSTRACT
| FULL TEXT
Increased BACE1 maturation contributes to the pathogenesis of Alzheimer's disease in Down syndrome
Sun et al.
FASEB J. 2006;20:1361-1368.
ABSTRACT
| FULL TEXT
BACE2, as a novel APP {theta}-secretase, is not responsible for the pathogenesis of Alzheimer's disease in Down syndrome
Sun et al.
FASEB J. 2006;20:1369-1376.
ABSTRACT
| FULL TEXT
Trafficking of Alzheimer's Disease-Related Membrane Proteins and Its Participation in Disease Pathogenesis
Suzuki et al.
J Biochem 2006;139:949-955.
ABSTRACT
| FULL TEXT
Leaky Scanning and Reinitiation Regulate BACE1 Gene Expression
Zhou and Song
Mol. Cell. Biol. 2006;26:3353-3364.
ABSTRACT
| FULL TEXT
Control of APP processing and A{beta} generation level by BACE1 enzymatic activity and transcription
Li et al.
FASEB J. 2006;20:285-292.
ABSTRACT
| FULL TEXT
Energy Inhibition Elevates {beta}-Secretase Levels and Activity and Is Potentially Amyloidogenic in APP Transgenic Mice: Possible Early Events in Alzheimer's Disease Pathogenesis
Velliquette et al.
J. Neurosci. 2005;25:10874-10883.
ABSTRACT
| FULL TEXT
Deletion of the Prostaglandin E2 EP2 Receptor Reduces Oxidative Damage and Amyloid Burden in a Model of Alzheimer's Disease
Liang et al.
J. Neurosci. 2005;25:10180-10187.
ABSTRACT
| FULL TEXT
High {beta}-Secretase Activity Elicits Neurodegeneration in Transgenic Mice Despite Reductions in Amyloid-{beta} Levels: IMPLICATIONS FOR THE TREATMENT OF ALZHEIMER DISEASE
Rockenstein et al.
J. Biol. Chem. 2005;280:32957-32967.
ABSTRACT
| FULL TEXT
BACE Is Degraded via the Lysosomal Pathway
Koh et al.
J. Biol. Chem. 2005;280:32499-32504.
ABSTRACT
| FULL TEXT
{beta} Subunits of Voltage-gated Sodium Channels Are Novel Substrates of {beta}-Site Amyloid Precursor Protein-cleaving Enzyme (BACE1) and {gamma}-Secretase
Wong et al.
J. Biol. Chem. 2005;280:23009-23017.
ABSTRACT
| FULL TEXT
Mutation of Active Site Residues of Insulin-degrading Enzyme Alters Allosteric Interactions
Song et al.
J. Biol. Chem. 2005;280:17701-17706.
ABSTRACT
| FULL TEXT
BACE overexpression alters the subcellular processing of APP and inhibits A{beta} deposition in vivo
Lee et al.
JCB 2005;168:291-302.
ABSTRACT
| FULL TEXT
Altered Amyloid-{beta} Metabolism and Deposition in Genomic-based {beta}-Secretase Transgenic Mice
Chiocco et al.
J. Biol. Chem. 2004;279:52535-52542.
ABSTRACT
| FULL TEXT
Early A{beta} accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain
Ingelsson et al.
Neurology 2004;62:925-931.
ABSTRACT
| FULL TEXT
Translational regulation of BACE-1 expression in neuronal and non-neuronal cells
De Pietri Tonelli et al.
Nucleic Acids Res 2004;32:1808-1817.
ABSTRACT
| FULL TEXT
Amyloid {beta} peptide load is correlated with increased {beta}-secretase activity in sporadic Alzheimer's disease patients
Li et al.
Proc. Natl. Acad. Sci. USA 2004;101:3632-3637.
ABSTRACT
| FULL TEXT
Differential utilization of upstream AUGs in the {beta}-secretase mRNA suggests that a shunting mechanism regulates translation
Rogers et al.
Proc. Natl. Acad. Sci. USA 2004;101:2794-2799.
ABSTRACT
| FULL TEXT
{beta}-Secretase Activity Increases with Aging in Human, Monkey, and Mouse Brain
Fukumoto et al.
Am. J. Pathol. 2004;164:719-725.
ABSTRACT
| FULL TEXT
BACE1 Suppression by RNA Interference in Primary Cortical Neurons
Kao et al.
J. Biol. Chem. 2004;279:1942-1949.
ABSTRACT
| FULL TEXT
Nonsteroidal Anti-Inflammatory Drugs and Peroxisome Proliferator-Activated Receptor-{gamma} Agonists Modulate Immunostimulated Processing of Amyloid Precursor Protein through Regulation of {beta}-Secretase
Sastre et al.
J. Neurosci. 2003;23:9796-9804.
ABSTRACT
| FULL TEXT
APP processing is regulated by cytoplasmic phosphorylation
Lee et al.
JCB 2003;163:83-95.
ABSTRACT
| FULL TEXT
Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes
Kinoshita et al.
J. Cell Sci. 2003;116:3339-3346.
ABSTRACT
| FULL TEXT
Funded Up and Raring to Go
Chen
Sci Aging Knowl Environ 2003;2003:nf14-14.
ABSTRACT
| FULL TEXT
BACE1- and BACE2-expressing Human Cells: CHARACTERIZATION OF {beta}-AMYLOID PRECURSOR PROTEIN-DERIVED CATABOLITES, DESIGN OF A NOVEL FLUORIMETRIC ASSAY, AND IDENTIFICATION OF NEW IN VITRO INHIBITORS
Andrau et al.
J. Biol. Chem. 2003;278:25859-25866.
ABSTRACT
| FULL TEXT
Ceramide Stabilizes {beta}-Site Amyloid Precursor Protein-cleaving Enzyme 1 and Promotes Amyloid {beta}-Peptide Biogenesis
Puglielli et al.
J. Biol. Chem. 2003;278:19777-19783.
ABSTRACT
| FULL TEXT
Alzheimer's Disease, Neuropeptides, Neuropeptidase, and Amyloid-{beta} Peptide Metabolism
Saito et al.
Sci Aging Knowl Environ 2003;2003:pe1-1.
ABSTRACT
| FULL TEXT
Alzheimer's Discovery Could Spur Drug Development
Arehart-Treichel
Psychiatr. News 2002;37:28-28.
FULL TEXT
Explaining the Cause of the Amyloid Burden in Alzheimer Disease
Rosenberg
Arch Neurol 2002;59:1367-1368.
FULL TEXT
|
