(American Journal of Pathology. 2001;159:937-943.)
© 2001 American Society for Investigative Pathology
The Fluorescent Congo Red Derivative, (Trans, Trans)-1-Bromo-2,5-Bis-(3-Hydroxycarbonyl-4-Hydroxy)Styrylbenzene (BSB), Labels Diverse ß-Pleated Sheet Structures in Postmortem Human Neurodegenerative Disease Brains
Marie L. Schmidt*,
Theresa Schuck*,
Shelly Sheridan*,
Mei-Ping Kung
,
Hank Kung
,
Zhi-Ping Zhuang,
Catherine Bergeron
,
Jacque S. Lamarche¶,
Daniel Skovronsky*,
Benoit I. Giasson*,
Virginia M.-Y. Lee* and
John Q. Trojanowski*
From the Departments of Pathology and Laboratory
Medicine,*
Radiology,
and
Pharmacology,
the Center for
Neurodegenerative Disease Research, University of Pennsylvania,
Philadelphia, Pennsylvania; the University of
Toronto,
Toronto, Ontario, Canada; and the
Department of Pathology,¶
University of
Sherbrooke, Sherbrooke, Quebec, Canada
 |
Abstract
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|---|
A novel Congo red-derived fluorescent probe (trans,
trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene
(BSB) that binds to amyloid plaques of postmortem Alzheimer’s disease
brains and in transgenic mouse brains in vivo was
designed as a prototype imaging agent for Alzheimer’s disease. In the
current study, we used BSB to probe postmortem tissues from
patients with various neurodegenerative diseases with diagnostic
lesions characterized by fibrillar intra- or extracellular lesions and
compared these results with standard histochemical dyes such as
thioflavin S and immunohistochemical stains specific for the same
lesions. These data show that BSB binds not only to extracellular
amyloid ß protein, but also many intracellular lesions
composed of abnormal tau and synuclein proteins and suggests that
radioiodinated BSB derivatives or related ligands may be useful imaging
agents to monitor diverse amyloids in
vivo.
|
Introduction
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Alzheimer’s disease (AD) is the most frequent cause of dementia
in the elderly. At this time there is no specific test available for a
definite diagnosis at an early stage of the disease. Thus, efforts have
been made to develop probes that bind AD amyloid plaques in brain for
use as in vivo imaging agents.1-3
For example,
a Congo red (CR)-derived fluorescent probe, X-34, was developed
recently that binds to AD brain lesions in tissue sections and has
several desirable characteristics required for an in vivo
amyloid imaging agent.4
Another CR derived compound that
binds to AD neurofibrillary tangles (NFTs), neuropil threads (NTs) and
amyloid ß-peptide (Aß) deposits in plaques as well as to AD-like
amyloid plaques in the brains of a transgenic mouse model of AD
amyloidosis after in vivo injection.1
However, this probe, (trans, trans),
-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene or BSB
(Figure 1)
, is not a specific ligand for
fibrillar Aß peptides, the major constituents of plaques in AD
brains, since it also binds to NFTs and NTs both of which are composed
of amyloid-like paired helical filaments (PHF) formed by
hyperphosphorylated tau proteins.1
Since there are two major species of Aß
in AD amyloid plaques, i.e., peptides ending at amino acid 40
(Aßx-40) or 42 (Aßx-42), the current study investigated whether BSB
binds to all or a subset of Aß plaques and whether it binds
preferentially to plaques consisting mainly of either Aßx-40 or
Aßx-42.5
Further, since NFTs in AD are composed of six
tau isoforms in about equal proportions,6-10
but in other
neurodegenerative diseases abnormal accumulations of tau proteins in
neurons and/or glial cells are predominantly composed of either three
or four microtubule (MT) binding repeat tau isoforms, we also asked if
BSB binds all or a subset of tau inclusions. For instance, NFTs and
glial inclusions in progressive supranuclear palsy (PSP), cortical
basal degeneration (CBD), and certain tauopathies, i.e., frontotemporal
dementia with parkinsonism (FTDP-17) are mainly composed of four repeat
tau isoforms while neuronal inclusions in Pick’s disease are mostly
composed of three repeat tau.11
Finally, we investigated
whether BSB binds to amyloid-like lesions containing 
-synuclein
including Lewy bodies (LB) in dementia with LB (DLB), Parkinson’s
disease (PD), and neurodegeneration with brain iron accumulation type 1
(NBIA-1) as well as glial cytoplasmic inclusions (GCI) in multiple
system atrophy (MSA).12
We found that BSB labeled all of
the above mentioned lesions although there were quantitative and
qualitative differences when compared with thioflavin S (THIOS) and
specific immunohistochemical stains for these lesions. Thus, BSB or
related derivatives may be exploited as imaging agents for diverse
amyloids including lesions formed by Aß, tau, and -synuclein.
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Materials and Methods
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Brain tissues from 23 patients were used in this study. Nineteen
of these patients had neurodegenerative diseases and one control
patient had no neurological disorder. Demographic data including the
disease, age, and gender of these patients are listed in Table 1
in addition to other information on the
brain samples. Also listed are the postmortem interval and the fixative
used to preserve the tissues. Tissue blocks of interest were removed at
the time of autopsy and fixed in either 70% ethanol containing 150
mmol/L sodium chloride or 10% neutral buffered formalin overnight and
subsequently embedded in paraffin. Six-µm-thick serial sections were
cut and adjacent sections were stained with BSB, THIOS, or antibodies
to Aß, tau, and -synuclein (Table 2)
. Immunohistochemistry was performed
using ABC Kits (Vector Laboratories, Burlingame, CA) and
diaminobenzidine as chromogen according to previously described
procedures.13;14
Thioflavin S staining was carried out according to a protocol reported
by Guntern et al.18
BSB staining was performed as
described.1
Briefly, deparaffinized and hydrated tissue
sections were immersed in a 0.01% BSB dissolved in 50% ethanol for 30
minutes. Then the sections were rinsed in a saturated aqueous solution
of lithium carbonate. Finally, the sections were differentiated in 50%
ethanol under microscope control. This process was stopped by immersion
in distilled water. The sections were then coated with a thin layer of
Vectorshield (Vector Laboratories) before coverslipping. The
THIOS and BSB stained sections were viewed in an
epifluorescence microscope using a fluorescent filter cube with an
excitation filter of 405 nm and an emission filter of 435 nm. A
fluorescein isothiocyanate (FITC) filter cube with an excitation filter
of 350–390 nm and an emission filter of 530 ± 15 was used for
comparison. Electronic images were obtained using a CoolSnap
camera (Biovision, Exton, PA) mounted on a Nikon FXA microscope
(Optical Apparatus, Inc., Ardmore, PA). These images were printed on a
Fuji Pictrography 3000 printer (Mid City Camera, Philadelphia, PA).
|
Results
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Aß-Positive Lesions
Monoclonal antibody (MAB) 4G8 demonstrated Aß plaques in a
variety of neurodegenerative diseases: AD, Down’s syndrome (DS),
familial AD (FAD), and DLB. The brains of the AD, DS, and FAD patients
had the largest amounts of Aß plaques. Thus, we chose consecutive
tissue sections from the brains of each of these three patients and
probed them with MAB 4G8, as well as with two other MAB specific for
the x-40 (BA27) and x-42 (BCO5) forms of Aß and also with the
fluorescent dyes BSB and THIOS. BCO5 immunostained the largest numbers
of Aß plaques and BA27 the least although there was considerable
regional variability in the number of BA27-positive plaques even within
the same tissue section. These results are illustrated in Figure 2
. It is apparent that both BSB and THIOS
stain larger numbers of plaques than BA27. However, by comparing many
large BCO5 positive plaques with adjacent tissue sections treated with
BSB and THIOS, it is evident that these two fluorescent dyes stain
nearly as many plaques as BCO5 although some of the fluorescently
stained plaques show a signal that is barely above background (Figure 2
, arrowheads in panels A, D, and E). Amyloid angiopathy was
immunostained by BA27 as well as fluorescently labeled by BSB and
THIOS.
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Figure 2. Neurodegenerative lesions composed of Aß protein. This figure depicts
tissue sections of frontal cortex immunostained with MAB. BCO5,
first column; BA27, second column; 4G8,
third column, or stained with BSB, fourth column;
and THIOS, fifth column. The first row
(A–E) shows
an AD case, the second row
(F–J) shows a
DS case with AD, and the third row
(K–M) shows a
case of FAD with a mutation in PS2. The asterisks indicate
the blood vessel that was used as landmark to identify the same area in
each set of consecutive sections. The arrows point to the
same subpial Aß deposit. All images were taken at the same
magnification. Scale bar, 100 µm.
|
|
Rare 4G8 positive Aß plaques were observed in the brain of a
33-year-old DS patient, a Pick’s disease patient, a FTDP-17(P301L)
patient and an elderly non-demented control patient. BSB did not stain
plaques in any of these patients although weakly stained plaques could
occasionally be demonstrated with THIOS (data not shown).
Tau-Positive Lesions
BSB stains NFTs and NTs in AD very prominently (Figure 3, A and G)
. In PSP both globose tangles
and coiled bodies were stained by BSB (Figure 3 B, C, H, and I
).
Tau-positive oligodendrocytic inclusions in a case of familial
progressive subcortical gliosis (PSG) were also stained by BSB and
THIOS (Figure 3, F, L, and Q)
. However, of the large numbers of
tau-immunoreactive coiled bodies present in FTDP-17 (N279K) only a
fraction was stained by either BSB or THIOS (not shown). Also, most of
the tau-immunoreactive neurons from the same case were not stained by
BSB or THIOS (Figure 3
, compare E, K, with P). The most frequently
stained BSB and THIOS positive lesions in this case were NTs
(arrowheads). Similarly, tau-immunoreactive neurons in CBD cases were
not positive for BSB or THIOS (data not shown). However, BSB
demonstrated extensive NT pathology in CBD in gray as well as in white
matter (Figure 3M)
. In another FTDP-17 case with the P301L mutation,
perikaryal tau immunoreactivity was prominent in several neocortical
areas and many large tau- positive cell bodies were present in the
basal forebrain. None of these lesions were stained with BSB or THIOS.
In this case many tau positive astrocytes with a characteristic
crenated appearance were observed and a subset of these stained weakly
with BSB (Figure 3, N and O)
. The astrocytic nature of these lesions
had been confirmed by double immunohistochemical staining with MABs to
tau and glial fibrillary protein (unpublished observation). Pick bodies
in Pick’s disease were stained, although weakly, with both BSB and
THIOS (Figure 3, D and J)
. Finally, NFTs in the cervical, thoracic and
lumbar regions of the spinal cord of three Guamanian PDC patients
stained with antiserum 17026, BSB, and THIOS (data not shown).
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Figure 3. Neurodegenerative lesions composed of tau protein. This figure shows
tau immunoreactive lesions in various neurodegenerative diseases
stained with BSB and THIOS. All sections in the first row
(A–F) were
stained with BSB and all sections in the second row
(G–L) were
stained with THIOS. In the third row the sections depicted in panels
M and N are stained with BSB and in panels
O to Q are immunostained with antiserum 17026.
A and G: NFTs and Aß plaques from an AD
patient. B and H: Coiled bodies in the midbrain
of a patient with PSP. C and I: Globose tangles
in the midbrain of a patient with PSP. D and J:
Pick bodies in the dentate gyrus of a patient with Pick’s disease.
E, K, and P: Consecutive sections through the
pons of a FTDP-17 (N279K)
patient. P shows tau-positive neurons and neuropil threads.
Both BSB and THIOS stain some neuropil threads in adjacent sections
(E and K,
respectively) but tau immunopositive neurons are
not stained. Asterisks indicate the same blood vessel in
these images and the arrows point to NTs. F, L,
and Q: Coiled bodies in a patient with familial PSG
(exon 10 + 16 mutation).
M: NT pathology in a CBD patient. N and
O: Tau and BSB stained glia (arrows) in the
amygdala of a FTDP-17
(P301L) patient. A,
C, G, I, and M are the same magnification; scale bar in
A = 20 µm. B, H, L, and Q are
the same magnification; scale bar in L = 10 µm.
D, F, J, N, and O are the same magnification;
scale bar in N = 10 µm. E, K, and
P are the same magnification; scale bar in P
= 50 µm.
|
|
-Synuclein-Positive Lesions
Both BSB and THIOS stained -synuclein immunoreactive GCIs in
MSA (Figure 4, A–C)
. Cortical LBs in DLB
stain strongly with anti--synuclein antibodies, but only occasional
LBs are weakly stained with BSB and THIOS (Figure 4, D–F)
. In a case
of NBIA-1, many -synuclein positive intraneuronal inclusion bodies
(LBs and GCIs) were seen. These were especially abundant in area CA3 of
the hippocampus, however neither BSB nor THIOS stained these inclusions
(Figure 4, G– I)
. The data of the BSB and THIOS staining of Aß-,
tau-, and -synuclein-positive lesions are summarized in Table 3
.
|
Discussion
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The data presented here indicate that BSB binds to a variety of
amyloid-like lesions in neurodegenerative diseases that are formed by
very different building block peptides or proteins including: Aß
peptides (amyloid plaques), hyperphosphorylated tau proteins (NFTs,
NTs), and -synuclein (LBs, GCIs). Each of these three proteins forms
fibrils that deposit into amyloid-like lesions. The results of this
study show that the binding properties of BSB are very similar to those
of THIOS, but only BSB, and not THIOS crosses the blood brain barrier
to label amyloid plaques in mice that model AD amyloidosis as reported
earlier.1
Indeed, the entry of BSB into brain was
demonstrated further using a BSB derivative wherein the bromide was
exchanged for iodine-125 and in vivo biodistribution data
indicated that 0.27% of the injected compound infiltrated the brain 6
minutes after intravenous delivery in mice.19
BSB binds
not only to Aß plaques in living tissue and Aß aggregates in
cultured cells but also in sections of frozen
tissue.1
While THIOS has been used in histology as
an amyloid binding dye for several decades, the mechanisms whereby this
and related histochemical stains bind to these fibrillar lesions is
still under investigation.20
Although the ß-pleated
sheet formation occurring in the fibrils formed by Aß,
hyperphosphorylated tau, as well as -synuclein may be the substrate
for BSB and THIOS binding, minor variations in the ß-pleated sheet
structures in the lesions described may account for the differences
observed in the binding of BSB and THIOS to these
lesions.12,21
In addition, since fibrils formed by three
or four microtubule-binding repeat tau isoforms had different
affinities for BSB, variations in ß-pleated sheet structures formed
by these different tau isoforms also might contribute to variable BSB
binding to tangles. For example, Pick bodies composed of three MT
binding repeat tau stained weakly with BSB and THIOS, while PSP lesions
composed of four MT binding repeat tau stained strongly with BSB and
THIOS the 4-repeat lesions in CBD and FTDP-17 stained to variable
degrees or not at all (Table 3)
.
Although BSB binds to a number of fibrillar lesions occurring in
neurodegenerative diseases tau- and -synuclein positive lesions are
much smaller and usually far fewer in numbers than Aß plaques and
would not pose a problem in prospective whole brain imaging of BSB
bound to Aß plaques. However, BSB did not stain the plaques in a
young DS patient who was in the beginning stages of AD. Similarly, BSB
did not stain the diffuse plaques of an elderly non-demented patient
with pathological aging22
which could be a preclinical
stage of AD. Although BSB compound will not detect non-fibrillar Aß
accumulations that may be the earliest pre-clinical manifestations of
AD, our data suggest that BSB or related compounds with the ability to
readily cross the blood-brain barrier, such as the radioiodinated
compound mentioned above, could be exploited to develop in
vivo imaging agents to monitor the burden of diverse amyloid
deposits in AD and other neurodegenerative disorders as well as the
effects of novel therapeutic agents designed to reverse or ameliorate
the accumulation of these lesions in the brains of living patients.
|
Acknowledgements
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We thank all of the families that made brain tissue available for
this study. We would also like thank Joe DiRienzi and I. Tsimberg as
well as members of the Departments of Neurology, Psychiatry, Medicine,
and Pathology and Laboratory Medicine for their help in the acquisition
of the tissues.
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Footnotes
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Address reprint requests to John Q. Trojanowski, M.D., Ph.D., Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 36th and Spruce Streets, Philadelphia, PA 19104-4283. E-mail:
trojanow{at}mail.med.upenn.edu
Supported by grants AG-09215, AG-10124, AG-14449, and AG-11542 from The National Institute of Aging and grants from The Alzheimer Association, Oxford Foundation, and the Institute for the Study of Aging.
Accepted for publication May 17, 2001.
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Abstract
Introduction
