 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 2003, 23(6):2086
The Binding of 2-(4'-Methylaminophenyl)Benzothiazole to
Postmortem Brain Homogenates Is Dominated by the Amyloid Component
William E.
Klunk1,
Yanming
Wang2,
Guo-feng
Huang2,
Manik L.
Debnath1,
Daniel P.
Holt2,
Li
Shao1,
Ronald L.
Hamilton3,
Milos
D.
Ikonomovic4,
Steven T.
DeKosky4, and
Chester A.
Mathis2
1 Laboratory of Molecular Neuropharmacology, Department
of Psychiatry, Western Psychiatric Institute and Clinic,
2 PET Facility, Department of Radiology,
3 Division of Neuropathology, Department of Pathology, and
4 Department of Neurology, University of Pittsburgh Medical
Center, Pittsburgh, Pennsylvania 15213
 |
ABSTRACT |
2-(4'-Methylaminophenyl)benzothiazole (BTA-1) is an uncharged
derivative of thioflavin-T that has high affinity for A fibrils and
shows very good brain entry and clearance. In this study, we asked
whether BTA-1, at concentrations typical of those achieved during
positron emission tomography (PET) studies, could specifically bind to
amyloid deposits in the complex milieu of human brain or whether
amyloid binding was overshadowed by nonspecific binding, found even in
brains that did not contain amyloid deposits. We quantitatively
assessed [3H]BTA-1 binding to crude homogenates of
postmortem brain obtained from nine Alzheimer's disease (AD) subjects,
eight controls, and six subjects with non-AD dementia. BTA-1 binding
was >10-fold higher in AD brain, and the majority (94%) of the
binding was specific (displaceable). High-affinity
[3H]BTA-1 was observed only in AD brain gray
matter and was not present in control brain gray matter, AD brain white
matter, or cerebellum. The Kd of
[3H]BTA-1 for binding to AD brain (5.8 ± 0.90 nM) was very similar to the
Kd for binding to synthetic A fibrils. In
addition, the Ki of various BTA analogs for
inhibition of [3H]BTA-1 binding to AD brain
homogenates was very similar to their Ki for
inhibition of [3H]BTA-1 binding to synthetic A
fibrils. Nanomolar concentrations of [3H]BTA-1 did
not appear to bind to neurofibrillary tangles. Finally, BTA-1 did not
appear to bind significantly to common neuroreceptors or transporter
sites. These data suggest that the binding of BTA-1 to AD brain is
dominated by a specific interaction with A amyloid deposits.
Key words:
Alzheimer's disease; neuroimaging; plaques; neurofibrillary tangles; positron emission tomography; PET; thioflavin-T; benzothiazole; postmortem; binding
| |
Introduction |
Alzheimer's disease (AD) is defined
pathologically by the characteristic deposits found in the brain (Mirra
et al., 1991 ), including amyloid plaques and neurofibrillary tangles
(NFTs). Both the amyloid- (A) protein found in plaques and the
tau protein found in NFTs have a predominantly -sheet structure
(Kirschner et al., 1986). Current evidence suggests that both A and
tau deposits play important roles in the pathophysiological cascade of
AD (Goate et al., 1991; Mullan et al., 1992; Hardy et al., 1998;
Goedert and Spillantini, 2000; Golde et al., 2000).
A deposition has become an important therapeutic target in AD
research (Schenk et al., 1999; Bard et al., 2000; DeMattos et al.,
2001; Olson et al., 2001). Although promising anti-amyloid therapies
are being intensely pursued, there is currently no accepted tool to
directly assess their success in delaying or reversing amyloid
deposition in humans. It will be critical to show that a clinical
treatment effectively reduces plaque load, before positive or negative
effects on clinical cognitive measures can be accurately interpreted.
In addition, because the clinical effectiveness of these anti-amyloid
therapies will serve as a test of the "amyloid hypothesis" of AD
(Hardy and Allsop, 1991; Hardy, 1992), it also will be critical to show
that the treatments actually decrease amyloid load before we can make
inferences about the validity of the amyloid hypothesis.
Our laboratory has been interested in the development of a
noninvasive method for direct and quantitative assessment of amyloid deposition in living subjects (Klunk et al., 1994; Klunk, 1998). We
have reported the potential of lipophilic thioflavin-T analogs as
amyloid imaging agents (Klunk et al., 2001) and have described a
particularly promising carbon-11-labeled derivative of thioflavin-T, namely 2-(4'-methylaminophenyl)benzothiazole (BTA-1) (see Fig. 1)
(Mathis et al., 2002). BTA-1 shows very good brain entry and clearance
and has been used for in vivo imaging of amyloid deposits in
living presenilin-1/amyloid precursor protein transgenic mice using multiphoton microscopy (Mathis et al., 2002). These properties justified further pursuit of BTA-1 as an in vivo human
amyloid-imaging agent for positron emission tomography (PET).
A critical issue in the development of a PET imaging agent is whether
binding to brain tissue is dominated by the specific target (in this
case, amyloid deposits) or by nonspecific, background binding. Here, we
further assessed the utility of BTA-1 for in vivo PET
imaging studies by quantitatively assessing its binding to homogenates
of postmortem brain from nine AD subjects, eight controls, and six
subjects with non-AD dementia (NAD). We found that BTA-1 binding was
much higher in amyloid-containing areas of AD brain tissue, and most of
the binding appeared to be specific. In addition, the
Ki of various BTA analogs for
inhibition of [3H]BTA-1 binding to AD
brain homogenates was very similar to their Ki for inhibition of
[3H]BTA-1 binding to synthetic A
fibrils. These data suggest that the binding of BTA-1 to AD brain is
dominated by specific interaction with amyloid deposits and strengthen
the case for use of BTA-1 as an in vivo imaging agent for AD
diagnosis and treatment assessment.
| |
Materials and Methods |
Chemicals. Chemicals were obtained from
Sigma (St. Louis, MO) unless noted otherwise.
Tritium-labeled and unlabeled BTA-1, and BTA-1 derivatives, were
synthesized and characterized as described previously (Klunk et al.,
2001; Mathis et al., 2002).
Postmortem tissue. Autopsy tissue was obtained from the
University of Pittsburgh Alzheimer Disease Research Center Brain Bank. Clinical and postmortem diagnoses were made by standardized procedures as described previously in detail (Lopez et al., 2000). Clinical characteristics are shown in Table 1.
All nine AD brains represented fairly advanced stages of pathology as
is typical of postmortem tissue. The eight control and six NAD brains
were selected for having no significant evidence of neuritic plaques or
cerebrovascular amyloid. In addition, there were no NFTs in the frontal
cortex of any control or NAD case. Samples of medial-frontal cortex
gray matter, medial-frontal cortex white matter, entorhinal cortex
(EC), or cerebellum were dissected at autopsy from 1-cm-thick coronal
slices and frozen at  80°C. The tissue was homogenized with a
Polytron tissue homogenizer (PT 10/35; Brinkman
Instruments, Westbury, NY) at room temperature for 30 sec at
setting 6 in PBS (137 mM NaCl, 3 mM KCl, 10 mM sodium phosphate, pH 7.0) at a concentration of 10 mg of
brain per milliliter. The homogenates were aliquoted and frozen at
80°C until used for binding assays within 2 months. No significant
changes in binding characteristics were observed in samples assayed
repeatedly over this time period.
Neuroreceptor screening. Binding assays for receptors and
transporters were performed as described previously using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program (Roth et al., 2000, 2001).
Binding studies. A(1-40) fibrils were prepared, and
Ki determinations were performed as
described previously (Klunk et al., 2001) using
[N-methyl-3H]BTA-1
([3H]BTA-1; 61 Ci/mmol) as the
radioligand (Mathis et al., 2002). Binding to brain homogenates for
determination of Ki and
Kd was performed with slight
modifications. Briefly, 10 mg/ml frozen brain aliquots were thawed and
diluted 10-fold in PBS to 1 mg/ml. Unlabeled test compounds were
dissolved in DMSO at 400 µM (to yield <1%
DMSO in the final assay). The appropriate concentrations of unlabeled
test compounds were combined with
[3H]BTA-1 (~1.2
nM) in a volume of 900 µl of PBS, pH 7.0. The
assay was begun by addition of 100 µl of the 1 mg/ml brain homogenate to achieve a final concentration of 100 µg tissue per milliliter. After incubation for 60 min at room temperature, the binding mixture was filtered through a Whatman GF/B glass filter via a
Brandel M-24R cell harvester (Gaithersburg, MD) and
rapidly washed five times with 3 ml PBS. The filters were counted in
Cytoscint-ES after thorough vortexing and sitting overnight. Complete
(100%) inhibition of specific binding was defined as the number of
counts displaced by 3 µM unlabeled BTA-1. All
assays were performed at least in triplicate. For determination of
Ki values, inhibition curves were fit
(using the RS/1 statistical package, version 6.1; Brooks Automation,
Chelmsford, MA) to the following equation, which is derived from the
Hill equation (Bennett and Yamamura, 1985): (F(x) = M(Ki)H/{[L]H + (Ki)H},
where M = maximal percentage
[3H]BTA-1 bound (typical fit gave
100-104% for M), H is the Hill coefficient (typically 0.85-1.0), and [L] is the
concentration of inhibitor compound. The
Kd value for BTA-1 binding to
A(1-40) fibrils and brain homogenates was determined similarly
using increasing concentrations of
[3H]BTA-1 between 0.2 and 1.2 nM, and, for BTA-1 concentrations from 1.2 nM to 1000 nM, a constant
concentration of [3H]BTA-1 (1.2 nM) plus additional unlabeled BTA-1. For
comparison of [3H]BTA-1 binding to brain
homogenates among the three groups (AD, control, and NAD), 1.2 nM [3H]BTA-1 was
incubated with 100 µg tissue in 1 ml PBS with and without 3 µM unlabeled BTA-1 as described above. Results
were corrected for nonspecific, nondisplaceable binding in the presence of 3 µM BTA-1 and expressed as picomoles of
[3H]BTA-1 bound per milligram of wet
tissue weight in the homogenate. Linearity of
[3H]BTA-1 binding was determined using
1.2 nM [3H]BTA-1
and 20-500 µg/ml of the AD brain homogenate with the
highest level of [3H]BTA-1
binding. Likewise, linearity with respect to
[3H]BTA-1 was determined using 100 µg
of tissue per milliliter and 1.2-6 nM
[3H]BTA-1. Binding results between AD
and control and AD and NAD were compared using an unpaired sample,
unequal variance t test.
Tissue staining. Postmortem tissue from three of
the autopsy-confirmed AD cases used for the binding study was
obtained through the University of Pittsburgh Alzheimer Disease
Research Center. Samples of medial-frontal cortex, entorhinal cortex,
or cerebellum were dissected from 1-cm-thick coronal slices and placed
into 10% buffered formalin (7 d). Paraffin blocks were prepared by sequential dehydration in graded alcohol and vacuum infiltrated in
paraffin before embedding and serial sectioning at a thickness of 8 µm. Tissue was processed for staining, and quenching of
autofluorescence was accomplished according to previously described
methods (Styren et al., 2000). Quenched tissue sections were taken from
PBS into a solution of 100 nM BTA-1 in PBS, pH
7.0, for 45 min. The sections were then dipped into water for ~5 sec
before coverslipping with Fluoromount-G (Electron Microscopy
Sciences, Fort Washington, PA). Fluorescent sections were viewed
using an Olympus Vanox AH-RFL-LB fluorescence microscope
using a UV filter set: U-filter (excites 360-370 nm, dichroic mirror
DM400, 420 nm long-pass filter). X-34 staining was performed according
to previously described methods (Styren et al., 2000). X-34-stained
tissue sections were optimally viewed with a violet filter set:
V-filter (excites <460 nm, DM455 mirror, 455 nm long-pass filter).
| |
Results |
Paraffin sections (8 µm) from the frontal
and entorhinal cortex of AD brain were
stained with 100 nM BTA-1 (Figs. 1 , 2). Like the parent compound,
thioflavin-T (Klunk et al., 2001), BTA-1 stained plaques,
cerebrovascular amyloid, and NFTs at these relatively high
concentrations. BTA-1 appeared to stain plaques and cerebrovascular amyloid more intensely than it stained NFTs. Very little background staining was observed. Most of the background fluorescence seen in
Figure 2 was caused by residual lipofuscin autofluorescence (distinguished by the fact that it is visible in all filter sets). Equivalent results were observed in frozen sections. Essentially no
staining was observed in control brain, which had been shown to contain
no plaques or NFTs by the standard CERAD neuropathology protocol (Mirra
et al., 1991) (data not shown).
View larger version (18K):
[in this window]
[in a new window]
|
Figure 1.
Chemical structures of thioflavin-T and BTA-1. The
uncharged compound, BTA-1, differs from thioflavin-T by the lack of
three methyl groups, including the methyl group imparting the positive
charge to the quaternary heterocyclic nitrogen of thioflavin-T
(arrow).
|
|
View larger version (72K):
[in this window]
[in a new window]
|
Figure 2.
Paraffin sections (8 µm) from AD brain stained
with 100 nM BTA-1. Left panel shows plaques
and cerebrovascular amyloid in the frontal cortex. Right
panel shows plaques and neurofibrillary tangles in the
entorhinal cortex. Scale bar, 100 µm. Most smaller bright spots are
residual lipofuscin autofluorescence.
|
|
The tissue staining results supported the specificity of BTA-1 for
-sheet deposits in a qualitative manner, but the 100 nM BTA-1 concentration used for staining was not representative of the low
or subnanomolar concentrations achieved by typical PET ligands in
vivo. In addition, it is possible that the fluorescence properties
of BTA-1 change after binding to amyloid in a way that enhances the
fluorescence similar to that observed for thioflavin-T (LeVine, 1993).
This could make the staining appear more specific than it actually was.
Therefore, quantitative binding studies were performed in tissue
homogenates using 1.2 nM
[3H]BTA-1.
[3H]BTA-1 binding was linear with tissue
concentration (r = 0.993) and
[3H]BTA-1 concentration
(r = 0.999) throughout the range used in this study. In
homogenates from frontal gray, significantly more [3H]BTA-1 was bound to
amyloid-containing AD brain than to the control or NAD brains, which
lacked observable amyloid deposits (p < 0.0002) (Fig. 3). Using 1.2 nM [3H]BTA-1, the
average amount of [3H]BTA-1 bound to AD
frontal gray (0.73 ± 0.21 pmol/mg wet weight) was >10-fold
higher than that bound to frontal gray obtained from either control
brain (0.061 ± 0.023 pmol/mg wet weight) or NAD brain (0.072 ± 0.021 pmol/mg wet weight).
View larger version (18K):
[in this window]
[in a new window]
|
Figure 3.
A, Comparison of
[3H]BTA-1 binding to homogenates from control
(open bars, circles), AD
(filled bars, squares), and NAD
brain (hatched bars, triangles) frontal
gray (Fr) or cerebellum (Cb).
B, Ratio of [3H]BTA-1 binding to
the Fr and Cb for each individual brain. Bars represent the mean, and
error bars represent the SD. Also shown are the individual data
points.
|
|
As expected, and confirming the specificity of BTA-1, a very different
finding was observed in the cerebellum where fibrillar (i.e., Congo red
or thioflavin-S positive) amyloid deposits are very rarely observed
(Joachim et al., 1989; Yamaguchi et al., 1989; Mann et al., 1996) (Fig.
3A). Binding was low in the cerebellar homogenates of all
three groups, and there was complete overlap between binding to AD
cerebellum and binding to cerebellum from control and NAD brains.
The lack of difference in [3H]BTA-1
binding in cerebellum is important because it identifies this brain
area as a potential reference tissue that could be used to simplify the
quantitation of future PET studies. To more precisely examine this
issue, ratios of [3H]BTA-1 binding to
frontal gray and cerebellum were determined for each individual brain
studied, and the data were expressed as the frontal/cerebellar ratio
for each brain (Fig. 3B). When expressed in this manner, the
clear differentiation between amyloid-containing AD brain and the
nonamyloid-containing control groups remained. The ratio in AD brain
(7.8 ± 4.0) was >10-fold higher than the ratio calculated in
either control brain (0.59 ± 0.40; p < 0.0006) or NAD brain (0.78 ± 0.26; p < 0.0007).
Approximately 94% of [3H]BTA-1 binding
to homogenates of AD frontal gray was displaceable by 3 µM unlabeled BTA-1 (i.e., is specific). Specific binding
represented only 36-52% of binding in all samples except AD frontal
gray. The absolute value of nonspecific binding was not significantly
different in the frontal gray or cerebellum of any of the groups
studied, although it was lowest in AD frontal gray.
Detailed Scatchard analysis of [3H]BTA-1
binding to homogenates of frontal gray from three AD and three control
brains showed high-affinity binding to be present only in AD frontal
gray. [3H]BTA-1 appeared to bind to a
single, high-affinity binding site in AD frontal gray with a mean
Kd of 5.8 ± 0.90 nM and a mean Bmax of 5.4 ± 1.1 pmol/mg wet
weight. (Fig. 4). This affinity was very
similar to the high-affinity binding of
[3H]BTA-1 to synthetic A(1-40) or
A(1-42) fibrils (Kd = 2.8 ± 0.35 nM; data not shown).
View larger version (23K):
[in this window]
[in a new window]
|
Figure 4.
Scatchard plots showing the binding of
[3H]BTA-1 to homogenates from AD frontal gray
matter ( ) and underlying frontal white matter from the same AD brain
( ). In this AD frontal gray matter homogenate, the
Kd was 4.4 nM and the
Bmax was 6.9 pmol/mg wet weight. In the
frontal white matter immediately underlying this gray matter sample,
the Kd was 111 nM and the
Bmax was 27 pmol/mg wet weight. Also shown
is a Scatchard plot showing the binding of
[3H]BTA-1 to a homogenate from control brain
frontal gray ( ) in which the Kd was 180 nM and the Bmax was 5.9 pmol/mg
wet weight.
|
|
Control brains showed only low-affinity binding, having a mean
Kd of 132 ± 59 nM and a mean
Bmax of 11.3 ± 5.2 pmol/mg wet weight (Fig. 4). The frontal gray matter samples used in this study
were dissected to remove the underlying white matter as much as
possible. Comparing the frontal gray matter from an AD brain with the
white matter immediately underlying it showed a marked difference in
high-affinity [3H]BTA-1 binding. Figure
4 includes a Scatchard analysis of the binding of
[3H]BTA-1 to the white matter dissected
from directly beneath the gray matter of the AD brain also shown in
Figure 4. In contrast to the frontal gray matter, the frontal white
matter showed only a low-affinity binding component, similar to that
seen in control brain frontal gray. This low-affinity binding component
in control frontal gray and AD frontal white matter would not play a
significant role in in vivo imaging studies using high
specific-activity radiotracers present at low nanomolar concentrations.
To address the relative contributions of
[3H]BTA-1 binding to A and tau
deposits in the frontal gray of AD brain we compared [3H]BTA-1 binding in homogenates from
entorhinal cortex, frontal gray, and cerebellum from a typical Braak
stage VI AD brain (Table 1, AD 01) and a
Braak stage II control brain (Table 1, Cntl 04), matched for age and
postmortem interval. This control brain had frequent NFTs in the
entorhinal cortex (Fig. 5A),
similar in numbers to those found in many AD cases (Fig.
5B); however, it had no neuritic or diffuse plaques in any
area of the brain (Fig.
5A,C,E).
[3H]BTA-1 binding in the plaque-free and
NFT-rich EC region of this Cntl 04 brain was no greater than
[3H]BTA-1 binding in the plaque- and
NFT-free cerebellum and frontal gray from this brain (Table
2). A similar survey of these same brain
areas in a Braak VI AD brain (Table 2) showed low binding in NFT-free
cerebellum and NFT-rich EC and >10-fold higher levels in frontal gray,
where there are frequent numbers of neuritic plaques (Fig.
5D). The extensive NFT pathology in the EC of the Cntl 04 and AD 01 brains, coupled with the low
[3H]BTA-1 binding in the EC, suggests
that at low nanomolar concentrations the amount of
[3H]BTA-1 binding to NFT deposits is
very small in comparison with the amount of
[3H]BTA-1 bound to A in the plaques
and cerebrovascular amyloid of AD frontal gray. The AD 01 brain showed
diffuse amyloid plaque deposits in the EC (Fig. 5B), which
also did not appear to produce significant
[3H]BTA-1 binding. The frontal cortex
had extensive amyloid plaques that were both compact and diffuse and
associated with high levels of [3H]BTA-1
binding (Fig. 5D, Table 2). The qualitative BTA-1 staining of NFTs, seen at 100 nM concentrations (Fig. 2),
is apparently not reflected by significant amounts of
[3H]BTA-1 binding at a concentration of
1.2 nM.
View larger version (143K):
[in this window]
[in a new window]
|
Figure 5.
Paraffin sections (8 µm) from the entorhinal
cortex (A, B), frontal cortex
(C, D), and cerebellum (E,
F) of a Braak stage II control brain
(A, C, E, Cntl
04) and a Braak stage VI AD brain (B,
D, F, AD 01) stained with
X-34. X-34 is a highly sensitive, fluorescent Congo red derivative that
intensely stains plaques, NFTs, and amyloid deposits in general (Styren
et al., 2000). Marked atrophy of AD tissue was notable in all regions.
In the entorhinal cortex of the control subject
(A), frequent numbers of NFTs were seen, whereas
there were no amyloid deposits. In the AD case
(B), there also were frequent NFTs along with
diffuse amyloid deposits (arrows). X-34-positive NFTs
and amyloid plaques were absent from the frontal cortex of the control
case (C), whereas they were abundant in AD
(D). There was no detectable X-34 staining of
plaques or NFT in control (E) and AD
(F) cerebellum. Control frontal cortex
(C) and cerebellum from both control and AD
(E, F) show small stellate cells
that stain with X-34. Scale bar, 200 µm.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
[3H]BTA-1 binding to specified areas of a
Braak stage II control brain (Cntl 04) and a Braak stage VI AD brain
(AD 01)
|
|
As further evidence that [3H]BTA-1
binding to brain homogenates was dominated by the A component, we
compared the binding of 10 structurally related BTA derivatives to
synthetic A(1-40) fibrils and to AD brain frontal gray. To get a
broad range of affinities for the correlation, we chose compounds with
Ki values for inhibition of
[3H]BTA-1 binding to A(1-40) fibrils
ranging from 1.8 to 3030 nM. A good correlation
was found between the Ki values
determined for [3H]BTA-1 binding to
synthetic A(1-40) fibrils and to the AD brain homogenate (Fig.
6). In fact, the correlation between
Ki values determined in AD brain and
synthetic A binding assays (r = 0.88) was no worse
than the correlation of Ki values
determined with two separately aggregated lots of synthetic A(1-40)
(r = 0.89) (Fig. 6). This finding suggested that the
[3H]BTA-1 binding site in AD brain
homogenates had the same structure-affinity relationship as the
binding site in suspensions of pure, synthetic A(1-40) fibrils and
was consistent with the hypothesis that
[3H]BTA-1 binding to AD brain
homogenates was dominated by binding to fibrillar A deposits.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 6.
Comparison of the Ki
values of 10 BTA-1 derivatives for inhibition of
[3H]BTA-1 binding to A(1-40) fibrils and
homogenates of AD frontal gray (filled diamonds,
solid line). Also shown is a comparison of the same data
for A(1-40) fibrils compared with Ki
values previously determined in a separate, older lot of A(1-40)
fibrils (open squares, dashed
line).
|
|
Additional experiments were performed using the resources of the NIMH
Psychoactive Drug Screening Program to assess whether BTA-1 interacts
with any of a representative array of neurotransmitter receptors and
transporters (Rothman et al., 2000; Roth et al., 2001). The array has
been published previously (Roth et al., 2001) and includes adrenergic,
serotonergic, muscarininc, dopaminergic, opiate, vasopressin, oxytocin,
GABA, glutamate, and imidazoline receptor sites. Using 10 µM BTA-1, no inhibition was observed at any receptor site
except at the norepinephrine transporter, where the
Ki of BTA-1 was 4504 ± 31 nM, a value 1000-fold higher than the
Kd for A(1-40) or AD brain.
| |
Discussion |
Although a previous study showed good pharmacokinetic properties
for BTA-1 in normal mice (Mathis et al., 2002), it remained to be shown
that BTA-1 could specifically bind to its amyloid target in human brain
and to more precisely characterize that target in the complex milieu of
human AD brain. The ability of BTA-1 to fluorescently stain amyloid
deposits in postmortem tissue sections gives some indication that this
compound would specifically bind amyloid deposits. However, this
staining is a qualitative technique and certainly does not prove
sufficient specificity for use as an in vivo PET
amyloid-imaging agent in humans. In an effort to address this question,
we quantitatively examined the binding of
[3H]BTA-1 to homogenates of postmortem
human brain at concentrations typical of in vivo PET
radiotracer studies. We found that 12-fold more
[3H]BTA-1 bound to homogenates from AD
brain frontal gray than to similar homogenates from control and NAD
brain. There was no overlap between the AD group and the other groups.
Binding of [3H]BTA-1 to cerebellar
homogenates was identical in all three groups, making cerebellum a good
candidate for a reference tissue for PET studies. More than 90% of
[3H]BTA-1 binding to AD frontal gray was
specific. The affinity of [3H]BTA-1
binding to AD frontal gray was relatively high (~6
nM) and very similar to the affinity of
[3H]BTA-1 for synthetic A fibrils
(~3 nM). Control brain frontal gray and frontal
white matter from AD showed essentially no high-affinity binding. NFTs
alone did not appear to increase
[3H]BTA-1 binding above control levels,
suggesting that most of the [3H]BTA-1
binding represents an interaction with A deposits. Consistent with
this, for a series of 10 BTA-1 derivatives having a 1000-fold range of
affinity for binding amyloid, the Ki
values for the inhibition of [3H]BTA-1
binding to A(1-40) fibrils correlated very well with their
Ki values for inhibition of
[3H]BTA-1 binding to AD brain
homogenates. BTA-1 did not bind significantly to a wide array of
neurotransmitter receptors or transporters. All of these data are
consistent with the notion that
[3H]BTA-1 binding in AD brain is mainly
accounted for by binding to fibrillar A.
Although [3H]BTA-1 binding clearly
distinguished AD brain homogenates from the control and NAD groups, it
is important to note that the goal of this study was not to test
whether we could use [3H]BTA-1 to
establish a diagnosis. The validation of BTA-1, or any other compound,
as an accurate tool to quantify amyloid is a complex process composed
of many steps such as the one described in this study. In addition, it
should be recognized that the frontal cortex does not represent the
area of maximal pathologic change for several of the diseases included
in the NAD group, and one would need to examine other brain areas to
adequately address diagnostic issues in these cases.
The purpose of this study was solely to determine whether the binding
of [3H]BTA-1 to areas of human brain
with abundant amyloid deposits (e.g., AD frontal gray) was dominated by
a specific interaction with the amyloid deposits. It was possible that
[3H]BTA-1 could have nonspecific
interactions with binding sites on non-amyloid components in brain that
would be of sufficient magnitude to mask the specific, amyloid binding
observed in AD brain. Therefore, both control groups were chosen for
the absence of visible A deposits to assess the contribution of the
non-amyloid components in isolation. The NAD group was included to
control for the possibility that
[3H]BTA-1 binding was affected by
general neurodegenerative processes in a manner unrelated to amyloid
deposition. Likewise, it is possible that highly lipophilic brain areas
such as white matter from both AD and control brains could have large
amounts of nonspecific, lipophilic interactions with
[3H]BTA-1 that would overshadow the
specific, amyloid binding in the overlying cortex. Our results
indicated that high-affinity [3H]BTA-1
binding was much greater in AD frontal gray than either control frontal
gray or AD frontal white matter and suggested that the binding of
[3H]BTA-1 to AD brain mainly represented
a specific interaction with A deposits.
Taken together with the previously reported pharmacokinetic properties
of [11C]BTA-1 in mice and successful
in vivo imaging of plaques in transgenic mice using BTA-1
and multiphoton microscopy (Mathis et al., 2002), the findings of the
present study suggest that BTA-1 is a good candidate as an in
vivo PET amyloid-imaging agent. Further, detailed, quantitative
validation studies in transgenic mice will need to be performed to
confirm the potential of BTA-1. In addition, pharmacokinetic studies
are underway in primate brain to verify the mouse studies. Promising
results from all of these studies and an acceptable toxicological
profile will justify preliminary studies in human subjects with and
without AD.
| |
FOOTNOTES |
Received Oct. 3, 2002; revised Nov. 26, 2002; accepted Jan. 8, 2003.
This work was supported in part by the Alzheimer's Association (Grants
IIRG-95-076 and TLL-01-3381 to W.E.K. and NIRG-00-2335 to Y.W.), the
National Institutes of Health (Grants AG01039 and AG20226 to W.E.K.,
AG18402 to C.A.M., and AG05133 to S.T.D.), and Institute for the Study
of Aging/American Federation of Aging Research (Y.W.). We thank
Dr. Bryan Roth (Case Western Reserve University) and Dr. Linda Brady
[National Institute of Mental Health (NIMH)] for allowing us access
to the NIMH Psychoactive Drug Screening Program. We thank Dr. Robert
Sweet for his help in procuring the appropriate tissue samples and the
families that made brain tissue available to the University of
Pittsburgh Alzheimer Disease Research Center Brain Bank.
Correspondence should be addressed to Dr. William E. Klunk, University
of Pittsburgh, 705 Parran Hall- GSPH, 130 DeSoto Street, Pittsburgh, PA 15213. E-mail: klunkwe{at}msx.upmc.edu.
| |
References |
-
Bard F,
Cannon C,
Barbour R,
Burke RL,
Games D,
Grajeda H,
Guido T,
Hu K,
Huang J,
Johnson-Wood K,
Kholodenko D,
Lee M,
Lieberburg I,
Motter R,
Nguyen M,
Soriano F,
Vasquez N,
Weiss K,
Welch B,
Schenk D,
Yednock T
(2000)
Peripherally administered antibodies against amyloid -peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.
Nat Med
6:916-919[ISI][Medline].
-
Bennett JP,
Yamamura HI
(1985)
Neurotransmitter, hormone, or drug receptor binding methods.
In: Neurotransmitter receptor binding (Yamamura HI,
Enna SJ,
Kuhar MJ,
eds), pp 61-89. New York: Raven.
-
DeMattos RB,
Bales KR,
Cummins DJ,
Dodart JC,
Paul SM,
Holtzman DM
(2001)
Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease.
Proc Natl Acad Sci USA
98:8850-8855[Abstract/Free Full Text].
-
Goate A,
Chartier-Harlin MC,
Mullan M,
Brown J,
Crawford F,
Fidani L,
Giuffra L,
Haynes A,
Irving N,
James L,
Mant R,
Newton P,
Rooke K,
Roques P,
Talbot C,
Pericak-Vance M,
Roses A,
Williamson R,
Rossor M,
Owen M,
Hardy J
(1991)
Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.
Nature
349:704-706[Medline].
-
Goedert M,
Spillantini MG
(2000)
Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer's disease.
Biochim Biophys Acta
1502:110-121[Medline].
-
Golde TE,
Eckman CB,
Younkin SG
(2000)
Biochemical detection of Abeta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer's disease.
Biochim Biophys Acta
1502:172-187[Medline].
-
Hardy J
(1992)
An "anatomical cascade hypothesis" for Alzheimer's disease.
Trends Neurosci
15:200-201[ISI][Medline].
-
Hardy J,
Allsop D
(1991)
Amyloid deposition as the central event in the aetiology of Alzheimer's disease.
Trends Pharmacol Sci
12:383-388[Medline].
-
Hardy J,
Duff K,
Hardy KG,
Perez-Tur J,
Hutton M
(1998)
Genetic dissection of Alzheimer's disease and related dementias: amyloid and its relationship to tau.
Nat Neurosci
1:355-358[ISI][Medline].
-
Jackson M,
Lennox G,
Lowe J
(1996)
Motor neurone disease-inclusion dementia.
Neurodegeneration
5:339-350[ISI][Medline].
-
Joachim CL,
Morris JH,
Selkoe DJ
(1989)
Diffuse senile plaques occur commonly in the cerebellum in Alzheimer's disease.
Am J Pathol
135:309-319[Abstract].
-
Kirschner DA,
Abraham C,
Selkoe DJ
(1986)
X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation.
Proc Natl Acad Sci USA
83:503-507[Abstract/Free Full Text].
-
Klunk WE
(1998)
Biological markers of Alzheimer's disease.
Neurobiol Aging
19:145-147[ISI][Medline].
-
Klunk WE,
Debnath ML,
Pettegrew JW
(1994)
Development of small molecule probes for the beta-amyloid protein of Alzheimer's disease.
Neurobiol Aging
15:691-698[ISI][Medline].
-
Klunk WE,
Wang Y,
Huang G-F,
Debnath ML,
Holt DP,
Mathis CA
(2001)
Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain.
Life Sci
69:1471-1484[ISI][Medline].
-
LeVine H
(1993)
Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution.
Protein Sci
2:404-410[Abstract].
-
Lopez OL, Becker JT, Klunk WE, Saxton J, Hamilton RL, Kaufer DI, Sweet
RA, Cidis-Meltzer C, Wisniewski S, Kamboh MI, DeKosky
ST (2000) Research evaluation and diagnosis of probable
Alzheimer's disease over the last two decades. I. Neurology
55:1854-1862.
-
Mann DM
(1998)
Dementia of frontal type and dementias with subcortical gliosis.
Brain Pathol
8:325-338[ISI][Medline].
-
Mann DM,
Iwatsubo T,
Snowden JS
(1996)
Atypical amyloid (A beta) deposition in the cerebellum in Alzheimer's disease: an immunohistochemical study using end-specific A beta monoclonal antibodies.
Acta Neuropathol
91:647-653[Medline].
-
Mathis CA,
Bacskai BJ,
Kajdasz ST,
Mclellan ME,
Frosch MP,
Hyman BT,
Holt DP,
Wang Y,
Huang G-F,
Debnath ML,
Klunk WE
(2002)
A lipophilic thioflavin-T derivative for positron emission tomography (PET) imaging of amyloid in brain.
Bioorg Med Chem Lett
12:295-298[Medline].
-
McKeith IG,
Galasko D,
Kosaka K,
Perry EK,
Dickson DW,
Hansen LA,
Salmon DP,
Lowe J,
Mirra SS,
Byrne EJ,
Lennox G,
Quinn NP,
Edwardson JA,
Ince PG,
Bergeron C,
Burns A,
Miller BL,
Lovestone S,
Collerton D,
Jansen EN,
Ballard C,
de Vos RA,
Wilcock GK,
Jellinger KA,
Perry RH
(1996)
Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop.
Neurology
47:1113-1124[Abstract/Free Full Text].
-
Mirra SS,
Heyman A,
McKeel D,
Sumi SM,
Crain BJ,
Brownlee LM,
Vogel FS,
Hughes JP,
van Belle G,
Berg L
(1991)
The consortium to establish a registry for Alzheimer's disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.
Neurology
41:479-486[Abstract/Free Full Text].
-
Mullan M,
Crawford F,
Axelman K,
Houlden H,
Lilius L,
Winblad B,
Lannfelt L
(1992)
A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of beta-amyloid.
Nat Genet
1:345-347[ISI][Medline].
-
Olson RE,
Copeland RA,
Seiffert D
(2001)
Progress towards testing the amyloid hypothesis: inhibitors of APP processing.
Curr Opin Drug Discov Devel
4:390-401[Medline].
-
Roth BL,
Ernsberger P,
Steinberg SA,
Rao S,
Rauser L,
Savage J,
Hufeisen S,
Berridge MS,
Muzic RF
(2001)
The in vitro pharmacology of the beta-adrenergic receptor pet ligand (s)-fluorocarazolol reveals high affinity for cloned beta-adrenergic receptors and moderate affinity for the human 5-HT1A receptor.
Psychopharmacology
157:111-114[Medline].
-
Rothman RB,
Baumann MH,
Savage JE,
Rauser L,
McBride A,
Hufeisen SJ,
Roth BL
(2000)
Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications.
Circulation
102:2836-2841[Abstract/Free Full Text].
-
Schenk D,
Barbour R,
Dunn W,
Gordon G,
Grajeda H,
Guido T,
Hu K,
Huang J,
Johnson-Wood K,
Khan K,
Kholodenko D,
Lee M,
Liao Z,
Lieberburg I,
Motter R,
Mutter L,
Soriano F,
Shopp G,
Vasquez N,
Vandevert C,
Walker S,
Wogulis M,
Yednock T,
Games D,
Seubert P
(1999)
Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.
Nature
400:173-177[Medline].
-
Styren SD,
Hamilton RL,
Styren GC,
Klunk WE
(2000)
X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.
J Histochem Cytochem
48:1223-1232[Abstract/Free Full Text].
-
Yamaguchi H,
Hirai S,
Morimatsu M,
Shoji M,
Nakazato Y
(1989)
Diffuse type of senile plaques in the cerebellum of Alzheimer-type dementia demonstrated by beta protein immunostain.
Acta Neuropathol
77:314-319[Medline].
Copyright © 2003 Society for Neuroscience 0270-6474/03/2362086-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S. N. Gomperts, D. M. Rentz, E. Moran, J. A. Becker, J. J. Locascio, W. E. Klunk, C. A. Mathis, D. R. Elmaleh, T. Shoup, A. J. Fischman, et al.
Imaging amyloid deposition in Lewy body diseases
Neurology,
September 16, 2008;
71(12):
903 - 910.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. B. McIntosh, S. R. Martin, D. J. Jackson, J. Khan, E. R. Isaacson, L. Calder, K. Raj, H. M. Griffin, Q. Wang, P. Laskey, et al.
Structural Analysis Reveals an Amyloid Form of the Human Papillomavirus Type 16 E1{wedge}E4 Protein and Provides a Molecular Basis for Its Accumulation
J. Virol.,
August 15, 2008;
82(16):
8196 - 8203.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. D. Ikonomovic, W. E. Klunk, E. E. Abrahamson, C. A. Mathis, J. C. Price, N. D. Tsopelas, B. J. Lopresti, S. Ziolko, W. Bi, W. R. Paljug, et al.
Post-mortem correlates of in vivo PiB-PET amyloid imaging in a typical case of Alzheimer's disease
Brain,
June 1, 2008;
131(6):
1630 - 1645.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Pike, G. Savage, V. L. Villemagne, S. Ng, S. A. Moss, P. Maruff, C. A. Mathis, W. E. Klunk, C. L. Masters, and C. C. Rowe
{beta}-amyloid imaging and memory in non-demented individuals: evidence for preclinical Alzheimer's disease
Brain,
November 1, 2007;
130(11):
2837 - 2844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Lockhart, J.R. Lamb, T. Osredkar, L.I. Sue, J.N. Joyce, L. Ye, V. Libri, D. Leppert, and T.G. Beach
PIB is a non-specific imaging marker of amyloid-beta (A{beta}) peptide-related cerebral amyloidosis
Brain,
October 1, 2007;
130(10):
2607 - 2615.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. Fodero-Tavoletti, D. P. Smith, C. A. McLean, P. A. Adlard, K. J. Barnham, L. E. Foster, L. Leone, K. Perez, M. Cortes, J. G. Culvenor, et al.
In Vitro Characterization of Pittsburgh Compound-B Binding to Lewy Bodies
J. Neurosci.,
September 26, 2007;
27(39):
10365 - 10371.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. C. Rowe, S. Ng, U. Ackermann, S. J. Gong, K. Pike, G. Savage, T. F. Cowie, K. L. Dickinson, P. Maruff, D. Darby, et al.
Imaging {beta}-amyloid burden in aging and dementia
Neurology,
May 15, 2007;
68(20):
1718 - 1725.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. D. Rabinovici, A. J. Furst, J. P. O'Neil, C. A. Racine, E. C. Mormino, S. L. Baker, S. Chetty, P. Patel, T. A. Pagliaro, W. E. Klunk, et al.
11C-PIB PET imaging in Alzheimer disease and frontotemporal lobar degeneration
Neurology,
April 10, 2007;
68(15):
1205 - 1212.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Holtzman
Pittsburgh Compound B Retention and Verification of Amyloid Deposition
Arch Neurol,
March 1, 2007;
64(3):
315 - 316.
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Bacskai, M. P. Frosch, S. H. Freeman, S. B. Raymond, J. C. Augustinack, K. A. Johnson, M. C. Irizarry, W. E. Klunk, C. A. Mathis, S. T. DeKosky, et al.
Molecular Imaging With Pittsburgh Compound B Confirmed at Autopsy: A Case Report
Arch Neurol,
March 1, 2007;
64(3):
431 - 434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Jack Jr, M. Marjanska, T. M. Wengenack, D. A. Reyes, G. L. Curran, J. Lin, G. M. Preboske, J. F. Poduslo, and M. Garwood
Magnetic Resonance Imaging of Alzheimer's Pathology in the Brains of Living Transgenic Mice: A New Tool in Alzheimer's Disease Research
Neuroscientist,
February 1, 2007;
13(1):
38 - 48.
[Abstract]
[PDF]
|
|
|
|
|
|
|
N. M. Kemppainen, S. Aalto, I. A. Wilson, K. Nagren, S. Helin, A. Bruck, V. Oikonen, M. Kailajarvi, M. Scheinin, M. Viitanen, et al.
Voxel-based analysis of PET amyloid ligand [11C]PIB uptake in Alzheimer disease
Neurology,
November 14, 2006;
67(9):
1575 - 1580.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Engler, A. Forsberg, O. Almkvist, G. Blomquist, E. Larsson, I. Savitcheva, A. Wall, A. Ringheim, B. Langstrom, and A. Nordberg
Two-year follow-up of amyloid deposition in patients with Alzheimer's disease
Brain,
November 1, 2006;
129(11):
2856 - 2866.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Mintun, G. N. LaRossa, Y. I. Sheline, C. S. Dence, S. Y. Lee, R. H. Mach, W. E. Klunk, C. A. Mathis, S. T. DeKosky, and J. C. Morris
[11C]PIB in a nondemented population: Potential antecedent marker of Alzheimer disease
Neurology,
August 8, 2006;
67(3):
446 - 452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Lopresti, W. E. Klunk, C. A. Mathis, J. A. Hoge, S. K. Ziolko, X. Lu, C. C. Meltzer, K. Schimmel, N. D. Tsopelas, S. T. DeKosky, et al.
Simplified Quantification of Pittsburgh Compound B Amyloid Imaging PET Studies: A Comparative Analysis
J. Nucl. Med.,
December 1, 2005;
46(12):
1959 - 1972.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Okamura, T. Suemoto, S. Furumoto, M. Suzuki, H. Shimadzu, H. Akatsu, T. Yamamoto, H. Fujiwara, M. Nemoto, M. Maruyama, et al.
Quinoline and Benzimidazole Derivatives: Candidate Probes for In Vivo Imaging of Tau Pathology in Alzheimer's Disease
J. Neurosci.,
November 23, 2005;
25(47):
10857 - 10862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. E. Klunk, B. J. Lopresti, M. D. Ikonomovic, I. M. Lefterov, R. P. Koldamova, E. E. Abrahamson, M. L. Debnath, D. P. Holt, G.-f. Huang, L. Shao, et al.
Binding of the Positron Emission Tomography Tracer Pittsburgh Compound-B Reflects the Amount of Amyloid-{beta} in Alzheimer's Disease Brain But Not in Transgenic Mouse Brain
J. Neurosci.,
November 16, 2005;
25(46):
10598 - 10606.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Taniguchi, N. Suzuki, M. Masuda, S.-i. Hisanaga, T. Iwatsubo, M. Goedert, and M. Hasegawa
Inhibition of Heparin-induced Tau Filament Formation by Phenothiazines, Polyphenols, and Porphyrins
J. Biol. Chem.,
March 4, 2005;
280(9):
7614 - 7623.
[Abstract]
[Full Text]
| |
TOP
ABSTRACT
Introduction
