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Received: 10 April 2017
Revised: 19 September 2017
Accepted: 2 October 2017
DOI: 10.1002/jnr.24188
Fluorine-19 magnetic resonance imaging probe for the
detection of tau pathology in female rTg4510 mice
Daijiro Yanagisawa1 | Nor Faeizah Ibrahim1,2 | Hiroyasu Taguchi1 |
Shigehiro Morikawa1 | Tomoko Kato1 | Koichi Hirao3 | Nobuaki Shirai4 |
Takayuki Sogabe5 | Ikuo Tooyama1
Molecular Neuroscience Research Center,
Shiga University of Medical Science, Otsu,
Department of Biochemistry, Faculty of
Medicine, UKMMC, Universiti Kebangsaan
Malaysia, Kuala Lumpur, Malaysia
Aggregation of tau into neurofibrillary tangles (NFTs) is characteristic of tauopathies, including
Alzheimer’s disease. Recent advances in tau imaging have attracted much attention because of its
potential contributions to early diagnosis and monitoring of disease progress. Fluorine-19 magnetic
resonance imaging (19F-MRI) may be extremely useful for tau imaging once a high-quality probe
Northeastern Industrial Research Center of
Shiga Prefecture, Nagahama, Japan
Industrial Research Center of Shiga
Prefecture, Ritto, Japan
Otsuka Pharmaceutical Co., Ltd.,
Tokushima, Japan
has been formulated. In this investigation, a novel fluorine-19–labeling compound has been developed as a probe for tau imaging using 19F-MRI. This compound is a buta-1,3-diene derivative with
a polyethylene glycol side chain bearing a CF3 group and is known as Shiga-X35. Female rTg4510
mice (a mouse model of tauopathy) and wild-type mice were intravenously injected with ShigaX35, and magnetic resonance imaging of each mouse’s head was conducted in a 7.0-T horizontalbore magnetic resonance scanner. The
Ikuo Tooyama, Molecular Neuroscience
Research Center, Shiga University of
Medical Science, Seta Tsukinowa-cho, Otsu
520-2192, Japan.
Funding information
This study was supported by JSPS
KAKENHI grant number JP17H03560 (I.T.),
JP15K01282 (H.T.), and JP17K01355 (D.Y.)
from the Japan Society for the Promotion
of Science.
F-MRI in rTg4510 mice showed an intense signal in the
forebrain region. Analysis of the signal intensity in the forebrain region revealed a significant accumulation of fluorine-19 magnetic resonance signal in the rTg4510 mice compared with the wildtype mice. Histological analysis showed fluorescent signals of Shiga-X35 binding to the NFTs in
the brain sections of rTg4510 mice. Data collected as part of this investigation indicate that
C 2017
MRI using Shiga-X35 could be a promising tool to evaluate tau pathology in the brain. V
Wiley Periodicals, Inc.
fluorine-19 MRI, magnetic resonance imaging, neurofibrillary tangle, tau, tau imaging, RRIDs:
AB_223647, AB_223648, SCR_002798
Tau, a microtubule-associated protein, is mainly found in axons in adult
neurons. The physiological functions of tau include stabilizing microtubules by binding to their surface and promoting their self-assembly
from tubulin subunits (Mandelkow & Mandelkow, 2012; Wang & Mandelkow, 2016). Human tau is encoded on chromosome 17q21 (Neve,
In this investigation, a novel fluorine-19–labeling compound
named Shiga-X35 is reported as a probe for tau imaging using
fluorine-19 magnetic resonance imaging (19F-MRI). After injection of Shiga-X35, in vivo MRI with an ultra-high-field MRI scanner successfully detected significant
F-MRI signal in the brain
Harris, Kosik, Kurnit, & Donlon, 1986) and consists of six isoforms gen-
of a tauopathy mouse model, in the same regions in which many
erated by alternative splicing of exons 2, 3, and 10 (Goedert, Spillantini,
NFTs accumulated. Data presented here indicate that
Potier, Ulrich, & Crowther, 1989). These isoforms differ depending on
using Shiga-X35 represents a promising approach to evaluating
the number of 29-residue near-amino-terminal inserts, which are
tau pathology in the brain.
encoded by E2 and E3: isoforms containing zero, one, or two inserts
J Neuro Res. 2017;1–11.
C 2017 Wiley Periodicals, Inc.
are known as 0N, 1N, and 2N, respectively. Isoforms can also be cate-
2005). The
gorized as to whether they contain three or four carboxy-terminal
highly sensitive, easily available, low-background, cost-effective way to
repeat domains (3R or 4R, respectively; the second repeat is encoded
detect protein aggregates in the brain using a suitable high-quality probe
by E10 and is not included in 3R tau).
(Tooyama et al., 2016; Yanagisawa et al., 2010, 2011, 2014).
Nuclear magnetic resonance (NMR) and small-angle X-ray scattering
F-MRI approach, which is the focus of this study, is a
In the present study, a novel fluorine-19–labeled compound for
show that normal tau is “natively unfolded” or “intrinsically disordered”
tau imaging using
€ nbrunn-Hanebeck, Marx, & Mandel(Mukrasch et al., 2009; Schweers, Scho
was to investigate the efficacy of 19F-MRI using such a fluorine-labeled
kow, 1994). In addition to its hydrophilic character, tau shows little tend-
compound with the aim of evaluating tau pathology in the brain.
F-MRI has been developed. The aim of this study
ency for aggregation. However, aggregation of tau into neurofibrillary
tangles (NFTs) is characteristic of a wide range of neurodegenerative dis-
eases known as tauopathies, including Alzheimer’s disease (AD), progressive
supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick’s disease
(PiD), and frontotemporal dementia with parkinsonism-17. The NFTs con-
2.1 | Synthesis of 1-(6-Methoxybenzoxazol-2-yl)-4-(4dimethylaminophenyl)buta-1,3-diene (Shiga-X34)
sist of bundles of paired helical filaments (PHFs) that contain abnormally
hyperphosphorylated tau assembled in a b-sheet conformation (Ballatore,
The condensation reaction of 4-dimethylaminocinnamaldehyde with 2-
Lee, & Trojanowski, 2007; Mandelkow & Mandelkow, 2012; Wang & Man-
methylbenzoxazole was reported by Etaiw, Fayed, & Saleh (2006). They
delkow, 2016). In addition to PHFs, straight and narrow-twisted filaments
investigated the product obtained thorough a fluorescence study in
have also been described in tauopathies (Goedert, Wischik, Crowther,
which similar 1,3-diene derivatives were prepared by condensation of
Walker, & Klug, 1988; Wischik et al., 1988). There are preferential isoforms
4-dimethylaminocinnamaldehyde with 6-alkoxy-2-methylbenzoxazole
found in the aggregates varying between tauopathies. These can provide a
using t-BuOK in a method similar to that for the preparation of styryl-
biochemical subclassification of the tauopathies (Dickson, Kouri, Murray, &
benzoxazole derivatives (Amatsubo et al., 2009).
Josephs, 2011) that includes 4R tauopathies (including PSP and CBD), 3R
tauopathies (e.g., PiD), and 3R14R tauopathies (e.g., AD).
The synthesis of Shiga-X34 is summarized in Scheme 1. 6-Hydroxy-2methylbenzoxazole (compound 1) was obtained by Beckmann rearrange-
Recent advances in tau imaging and in particular for in vivo imaging
ment of 2,4-dihydroxyacetophenone oxime (Fujita, Koyama, & Inagaki,
of tau pathology in the brain have attracted much attention. This is due
1982). Compound 2 was easily prepared by the methylation of the 6-
to its potential to contribute to early diagnosis in high-risk individuals,
hydroxy derivative with methyl iodide under alkaline conditions. Compound
monitor disease progression, and determine the efficacy of drug treat-
2 was then readily condensed with 4-dimethylaminocinnamaldehyde to
ment (Villemagne, Fodero-Tavoletti, Masters, & Rowe, 2015). Radioac-
produce Shiga-X34 under basic conditions.
tive tracers for tau imaging in positron emission tomography (PET) have
The possible geometric isomers (EE, EZ, ZE, and ZZ) of Shiga-X34 are
been developed; these include PBB3, THK-5117T, THK-5351, T807,
shown in Figure 1. The product obtained was purified by silica gel column
and T808 (Harada et al., 2015; Harada, Okamura, Furumoto, Furukawa,
chromatography followed by slow recrystallization from ethyl acetate. This
et al., 2016; Hashimoto et al., 2014; Xia et al., 2013; Zhang et al., 2012).
produced only one isomer in the form of dark red crystals (m.p. 1608C–
These tracers have successfully facilitated the imaging of tau pathology
1618C). This product must be the EE isomer as it seems to be the most
in the human brain, thereby accomplishing a minimally invasive evalua-
thermodynamically stable isomer. Immediately after making a solution of
tion of tau pathology in the brain. Magnetic resonance (MR) imaging
the isomer, the HPLC (mobile phase: CH3CN:H2O:CH3CO2H570:30:1)
(MRI) is another promising modality for noninvasive brain imaging (Ama-
showed only one peak (retention time: 16.6 min). The same peak was also
tsubo, Yanagisawa, Morikawa, Taguchi, & Tooyama, 2010; Higuchi et al.,
observed after keeping the solution at room temperature and in darkness
Synthesis of 1-(6-Methoxybenzoxazol-2-yl)-4-(4-dimethylaminophenyl)buta-1,3-diene
This isomerization was also observed in the NMR spectrum of ShigaX34. The NMR spectrum of the isomer (EE) in CDCl3 showed two sharp
singlets at d 3.01 (6H) and 3.87 (3H), which are assigned to the N-methyl
and O-methyl groups, respectively. However, more peaks (d 3.03 and
3.02 besides 3.01 ppm for the N-methyl group and d 3.89 and 3.86 in
addition to 3.87 ppm for the O-methyl group) were observed after the
solution had remained at room temperature for several hours under light.
2.2 | Synthesis of 1-(6-Alkoxybenzoxazol-2-yl)-4-(4dimethylaminophenyl)buta-1,3-diene (compound 11)
The buta-1,3-diene derivatives (compound 11) were prepared by a
method similar to that for Shiga-X34. Scheme 2 shows the reaction
Geometric isomers of Shiga-X34
mechanism for the synthesis of 1-(6-alkoxybenzoxazol-2-yl)-4-(4-dimethylaminophenyl)buta-1,3-dions, which have PEG chain at C-6 position
of the benzoxazole region, including Shiga-X35 (compound 11, n 5 5).
for a day. However, the HPLC revealed four peaks (retention times: 8.8,
The final products (compound 11) were not crystallized even after thor-
11.4, 14.1, and 16.6 min) after leaving the solution at room temperature
ough purification by silica gel column chromatography and were instead
and under light for 3 days. This implies that the compound is easily isomer-
only isolated as an oily material. These compounds seem to exist as a
ized in the presence of light when it is in solution.
mixture of the geometrical isomers (EE, EZ, ZE, and ZZ) in solution in a
Synthesis of 1-(6-Alkoxybenzoxazol-2-yl)-4-(4-dimethylaminophenyl)buta-1,3-diene
similar manner to Shiga-X34. This hypothesis is supported by the NMR
washed with 10 mM phosphate-buffered saline (pH 7.4; PBS). The sec-
spectrum and HPLC of Shiga-X35.
tions were then treated with 0.1% potassium metabisulfite followed by
0.15% oxalic acid. After washing with PBS, the section was blocked
2.3 | Animals
The rTg4510 mouse line used for these experiments is a wellcharacterized model of tauopathy (Santacruz et al., 2005). Transgenic
mice expressing a responder transgene consisting of a tetracyclineoperon–responsive element placed upstream of a cDNA encoding
human four-repeat tau with the P301L mutation (0N4R tauP301L) that
is linked to hereditary tauopathy (Hutton et al., 1998) were mated with
transgenic mice expressing an activator transgene (tetracycline-controlled transactivator) that consisted of the tet-off open reading frame
placed downstream of Ca21-calmodulin kinase II promoter elements to
obtain rTg4510 mice. The transgenic mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). Offspring were ear-punched and
genotyped using a polymerase chain reaction. Bigenic mice expressing
both the responder and the activator, in which transgenic mutant tau
mRNA expression was largely restricted to structures in the forebrain,
were used as rTg4510 mice. Mice expressing neither the responder nor
the activator were used as wild-type controls. In this investigation, 8to 9-month-old female rTg4510 mice (n 5 3) and 8- to 9-month-old
female wild-type mice (n 5 3) were used for MRI scans, while 8-monthold male rTg4510 mice (n 5 2) were used for ex vivo labeling. Every
effort was taken to minimize the number of animals used in this study.
Two to four mice were housed per cage on wood shavings and in
standard laboratory cages. They were fed a standard chow diet and maintained at 238C under a 12-hr light/dark cycle (lights on for the hours
08:00–20:00). There was free access to water and food in an SPF animal
facility. All experimental procedures in this study were approved by the
Committee on Animal Care of Shiga University of Medical Science.
2.4 | Antibodies
with 2% bovine serum albumin (BSA) in PBS containing 0.03% Triton
X-100 (PBS-T) for 30 min. This was followed by incubation with AT8
(1:500) antibody in PBS-T containing 0.2% BSA overnight at 48C, followed by Alexa Fluor 647–conjugated anti-mouse IgG antibody (1:500;
Thermo Fisher Scientific) in PBS-T for 3 hr at room temperature. The
section was washed with PBS-T after each step. After being coverslipped with Vectashield (Vector Laboratories, Burlingame, CA), the section was observed with a fluorescence microscope (BX61; Olympus,
Tokyo, Japan) using a Cy5 filter (excitation filter 630–650 nm; dichroic
mirror 650 nm; emission filter 671–693 nm). The slip was then
removed for subsequent staining with Shiga-X34. The solution of
Shiga-X34 was prepared by dissolving it in 10 mM dimethyl sulfoxide
and then diluted to 100 mM in 50% ethanol. After washing with PBS,
the section was incubated with 100 mM Shiga-X34 solution for 10 min
followed by brief washing with water. After being coverslipped, the
same region of interest (ROI) in each section was observed with the
fluorescence microscope (BX61) using an FITC filter (excitation filter
450–480 nm; dichroic mirror 550 nm; emission filter 515LP nm).
Ex vivo labeling was also performed in brain sections of 8-monthold male rTg4510 mice (n 5 2). The mice were sacrificed with an overdose injection of sodium pentobarbital (200 mg/kg, i.p.) and the brains
quickly removed. The brains were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 hr at 48C. They were
then immersed in 0.1 M phosphate buffer (pH 7.4) containing 15%
sucrose and 0.1% sodium azide for at least 2 days for cryoprotection
and finally cut into 20-mm sections. Brain sections mounted on glass
slides were immersed in 100 mM Shiga-X34 solution containing 50%
ethanol for 90 min at room temperature. The sections were then
sequentially washed with 80% ethanol for 60 s and with distilled water
for 3 min. For double staining, the sections were subsequently blocked
In this investigation, mouse monoclonal anti-phospho-tau (Ser202 and
with 2% BSA in PBS-T for 30 min and incubated with AT8 (1:1,000) in
Thr205) antibody (clone AT8; Thermo Fisher Scientific, MN1020, RRID:
PBS-T containing 0.2% BSA overnight at 48C, followed by Alexa Fluor
AB_223647) was used to detect NFTs in human AD brain sections. AT8
555–conjugated anti-mouse IgG antibody (1:500; Thermo Fisher Scien-
is a well-characterized antibody and has been used in a number of stud-
tific) in PBS-T for 4 hr at room temperature. The sections were washed
ies to identify NFTs by immunohistochemistry. In mouse sections, biotin-
with PBS-T after each step. After being coverslipped, the sections were
ylated AT8 (Thermo Fisher Scientific, MN1020B, RRID: AB_223648)
observed with a fluorescence microscope (BZ-8100; Keyence, Osaka,
was used because immunohistochemical staining on mouse tissues using
Japan) using a GFP-BP filter (excitation filter 450–490 nm; dichroic
mouse antibodies may result in high background.
mirror 495 nm; emission filter 510–560 nm) for Shiga-X34 and a Texas
Red filter (excitation filter 540–580 nm; dichroic mirror 595 nm; emis-
2.5 | Ex vivo labeling with Shiga-X34
Experiments involving human materials were performed under the regulations of the research ethics committee of Shiga University of Medi-
sion filter 600–660 nm) for AT8.
2.6 | MRI
cal Science. Ex vivo labeling was performed using 6-mm-thick formalin-
In this investigation, a 7.0-T horizontal-bore MR scanner (Unity Inova;
fixed paraffin-embedded sections of the caudal entorhinal cortex from
Agilent Technologies, Santa Clara, CA) was employed. A home-built cir-
a patient with AD (male, 72 years old, and pathologically diagnosed as
cular-type surface coil measuring 1.6 cm in diameter and tuned to both
AD in Shiga University of Medical Science). Sequential labeling of the
the 1H and 19F frequencies (300 MHz and 282 MHz, respectively) was
same section with AT8 followed by Shiga-X34 was conducted. For
used to collect the data (Yanagisawa et al., 2011, 2014). The radio fre-
autofluorescence quenching, each deparaffinized section was immersed
quency (RF) coil was carefully adjusted at each resonance frequency
in 0.25% potassium permanganate solution for 20 minutes and then
using the tuning and matching capacitors just before the 1H or
Ex vivo labeling with Shiga-X34. Shiga-X34 (a fluorescent butadiene derivative) bound to AT81 NFTs in human brain sections with
Alzheimer’s disease (a–c) and also the hippocampus (d–f) and the cerebral cortex (g–i) of rTg4510 mice. Scale bar: 20 mm (a) and 50 mm (d)
study, and its size was optimized to detect MR signals from the mouse
integrating the
F signal intensities of compound peaks in individual
brain. Gradient-echo sequence was used to obtain H-MR images of
pixels. The flip angle of the excitation pulse for
the mouse brain with 150-ms repetition time (TR), 3-ms echo time, 608
mined. The RF power was optimized to obtain the maximal
flip angle, 1.5-mm slice thickness, 24 mm 3 24 mm field of view, and
intensity with 1-s TR using a compound-containing phantom and ani-
128 3 128 resolution. A nonlocalized 19F NMR spectrum was obtained
mals. The optimized RF power was used for both the
from the whole head using a single pulse sequence with 8,192 data
F was not deter19
F signal
F single pulse
F-CSI sequences.
points, 40,000-Hz spectral width, 1-s TR, and 600 acquisitions (for 10
Shiga-X35 was dissolved at concentration of 10 mg/ml in saline
min). To obtain fluorine-19 MR (19F-MR) images, free induction decay
containing 20% Cremophor EL before use. Under anesthesia using
F chemical shift imaging (CSI) were collected with a 40,000-
sodium pentobarbital, 8- to 9-month-old female rTg4510 mice (n 5 3)
Hz spectral width, 24 mm 3 24 mm field of view in the sagittal plane,
and 8- to 9-month-old female wild-type mice (n 5 3) were intrave-
1-s TR, 200-ms phase-encoding time, and 68 acquisitions for each cen-
nously injected with Shiga-X35 at a dose of 200 mg/kg via the tail vein
tral 44-phase-encoding step out of 8 3 8 steps. For the residual 20-
over a 100-min period by continuous infusion at a rate of 0.2 ml/kg/
phase-encoding steps in the periphery of k-space, zero data were used.
min. Immediately after the injection, the mice were placed in the MR
The total acquisition time for one data set was 50 min. A slice-selective
scanner. Initially, a
pulse was not used. Whole signals covered by the coil sensitivity were
head using a single pulse sequence for 10 min. Subsequently,
acquired. The raw data were processed by 3D Fourier transformation
data for construction of the
with 40-Hz line broadening and zero filling and finally converted to 32
lected for 50 min. This set of MR measurements was repeated 7 times
data of
3 32 spectral data sets. The
F-MR image was constructed by
F NMR spectrum was obtained from the whole
F images in the sagittal plane were col-
(a total of 8 sets). General anesthesia was maintained with intermittent
infusion of sodium pentobarbital through a polyethylene tube inserted
sections were then mounted on glass slides and coverslipped with
intraperitoneally. Any additional dosage of sodium pentobarbital for
Entellan new (Merck Millipore, Billerica, MA).
maintaining anesthesia was determined by monitoring the respiratory
rate. The animals were warmed with an air drier, and rectal tempera-
2.10 | Simplified Gallyas silver staining
ture was monitored throughout the experiments.
A simplified Gallyas staining was performed according to methods used
2.7 | Analysis of 19F-MR signal intensity in the brain
in a previous study (Kuninaka et al., 2015). However, this was accom-
A rectangular ROI corresponding to the forebrain region was deter-
water and immersed in 0.3% potassium permanganate for 10 min. After
glass slides before staining. The sections were then briefly washed with
being washed lightly with water, the sections were immersed in 2%
F-MRI (8 3 4 pixels).
oxalic acid for 2 min or until they turned white. The sections were fur-
F-signal intensities of the background area
ther washed with water for another 6 min before being immersed in
mined with H-MRI (dashed line rectangle in Figure 2c,f). The
intensities were extracted from this region of
At the same time, the
plished with some modifications. Brain sections were mounted onto
were extracted from 32 3 4 pixels, where there was no tissue (Supple-
alkaline silver iodide solution for 1 min. The sections were then washed
mentary Figure 1). Finally, the ratios of the averaged forebrain signal to
3 times with 0.5% acetic acid and incubated in a freshly prepared
the averaged background signal were obtained from individual data
developer solution for 18 min. After incubation, the sections were
sets. The
F-MR images were randomly numbered so as not to iden-
tify the experimental details of individual images before the analysis.
washed again (3 times) with 0.5% acetic acid and further immersed in
0.5% hydrogen tetrachloroaurate (III) tetrahydrate for 5 min. Following
this, the sections were lightly washed in water, immersed in 2% sodium
2.8 | Fluorescence microscopic analysis after MRI
thiosulfate for 1 min, and washed again in water for a further 1 min.
Counterstaining with cresyl violet was performed by incubating the
After MRI, the mice were sacrificed by overdose injection of sodium
pentobarbital (200 mg/kg, i.p.). The 20-mm brain sections, prepared as
described above, were mounted on glass slides. After being coverslipped, fluorescence microscopy for Shiga-X35 was then performed
with a fluorescence microscope (BZ-8100) using a GFP-BP filter (excitation filter 450–490 nm; dichroic mirror 495 nm; emission filter 510–
sections in 0.1% cresyl violet (Sigma-Aldrich, St. Louis, MO) solution for
10 min. After a brief wash in water, the sections were further differentiated in 70% ethanol containing a few drops of acetic acid. The sections were then dehydrated in an ethanol series increasing from 80%
to 100%, immersed in xylene for 10 min (3 times), and coverslipped
with Entellan mounting medium.
560 nm).
For immunolabeling with AT8, sections mounted on glass slides
were blocked with 2% BSA in PBS-T for 30 min and incubated with
2.11 | Statistical analysis
biotinylated AT8 (1:500) in PBS-T containing 0.2% BSA overnight at
Data are presented as mean 6 standard error of the mean. The statisti-
48C. This was followed by Alexa Fluor 594–conjugated streptavidin
cal significance between the wild-type control group and the rTg4510
(1:500; Thermo Fisher Scientific) in PBS-T for 4 hr at room tempera-
group was analyzed by F test using GraphPad Prism 7 (GraphPad Soft-
ture. The sections were washed with PBS-T after each step. After being
ware, La Jolla, CA; RRID: SCR_002798). All the data passed the Sha-
coverslipped, the sections were observed with a fluorescence micro-
piro–Wilk normality test.
scope (BZ-8100; Keyence, Osaka, Japan) using a GFP-BP filter (excitation filter 450–490 nm; dichroic mirror 495 nm; emission filter 510–
560 nm) for Shiga-X35 and a Texas Red filter (excitation filter 540–
580 nm; dichroic mirror 595 nm; emission filter 600–660 nm) for AT8.
2.9 | Immunohistochemistry
3.1 | Shiga-X34 binds to NFTs in the ex vivo brain
section of rTg4510 mice
To investigate whether Shiga-X34 bound to NFTs, ex vivo labeling with
Free-floating sections were treated with 0.3% hydrogen peroxide in
Shiga-X34 and AT8 antibody (which is a marker for the NFTs, in the
PBS-T to eliminate endogenous peroxidase activity. After several
brain sections of human AD and rTg4510 mouse) was performed. In
washes, the sections were blocked with 2% BSA in PBS-T for 30 min
fluorescence microscopy in the human AD brain sections, Shiga-X34
at room temperature to block nonspecific protein binding. The sections
was detected in AT81 NFTs (Figure 2a–c) and in senile plaques (Sup-
were then incubated with biotinylated AT8 (1:1,000) in PBS-T contain-
plementary Figure 2). The colocalization of Shiga-X34 and AT8 immu-
ing 0.2% BSA overnight at 48C, followed by avidin-biotin-peroxidase
noreactivity was also detected in the hippocampus and the cerebral
complex (Vectastain ABC Elite kit, 1:1,000; Vector Laboratories) for 1
cortex of the rTg4510 mouse (Figure 2d–i). These results suggest that
hr at room temperature. All of the sections were washed several times
Shiga-X34 can bind to the NFTs formed not only in AD but also other
with PBS-T between steps, and labeling was accomplished through the
tauopathies. Based on these results, Shiga-X34 was selected as a core
use of 3,3 -diaminobenzidine (DAB; Dojindo Laboratories, Kumamoto,
structure for tau probes and Shiga-X35 synthesized as a 19F-MRI probe
Japan), with nickel ammonium, which yielded a dark blue color. The
by adding a trifluoromethyl group onto the polyethylene glycol chain.
The in vivo detection of tau pathology in the brain through the use of 19F-MRI. Wild-type mice (a–c) and rTg4510 mice (d–f)
received Shiga-X35 by injection and were subsequently subjected to MRI. Representative images of 1H-MRI (a,d) and 19F-MRI (b,e) show
the position of the brain and the 19F-MRI signal from Shiga-X35, respectively. A lookup table (LUT) was used to display the 19F-MRI signal,
and the outline of the brain is indicated by the white line in merged images (c,f). Although two strong signals were located around the
olfactory bulb and the hindbrain, there was a region of intense 19F signal in the forebrain (dashed line rectangle) in the rTg4510 mice
compared with the wild-type mice. (g) Analysis of signal intensity in regions of interest with matrix size 64 3 32 pixels placed on the
forebrain region (c,f: dashed line boxes) revealed a significant accumulation of 19F-MRI signal in the rTg4510 mice compared with the wildtype mice. Numeric values represent mean 6 standard error of the mean. Wild-type mice 2.67 6 0.1 (n 5 3); rTg4510 mice 2.02 6 0.2 (n 5 3).
Significance: *p 5 .044 in the F test
3.2 | 19F-MRI revealed intense signal accumulation in
the forebrain of rTg4510 mice
AT81 NFTs in the rTg4510 mice (Figure 5c–e). Interestingly, ShigaX352, AT81 signals and Shiga-X351, AT82 signals were also observed
F-MRI was measured in wild-type and rTg4510 mice that were given
Shiga-X35 by intravenous infusion. The
in the rTg4510 mouse brain that received Shiga-X35.
F-MR images of wild-type
mice, made by integrating the data of eight CSI measurements (50 min
3 8), showed that two strong signals were located in the regions of the
olfactory bulb and the hindbrain (Figure 3a–c). This suggests an accumulation of Shiga-X35 in the adipose tissues in these regions. However, in rTg4510 mice an intense signal in the forebrain region located
between these other two regions was observed (Figure 3d–f). Analysis
of the ratios of the averaged forebrain signal (black dashed line in Figure 3c,d and Supplementary Figure 1a) to the averaged background signal (white dashed line in Supplementary Figure 1a) revealed a
significant accumulation of
Immunolabeling with AT8 revealed in vivo binding of Shiga-X35 to
F-MR signal in the rTg4510 mice (2.67 6
0.1, n 5 3) compared with the wild-type mice (2.02 6 0.2, n 5 3; F
[1,4] 5 8.46, p 5 .044) (Figure 3g).
In the present study, 1-(6-methoxybenzoxazol-2-yl)-4-(4-dimethylaminophenyl) buta-1,3-diene derivative (Shiga-X34) was used as a core
structure of a probe for tau imaging using
F-MRI. Research com-
pleted by this group has previously reported styrylbenzoxazole derivatives
styrylbenzoxazole derivatives did not show binding to NFTs in human
AD brain sections (Yanagisawa et al., 2014). It has been reported that
compounds possessing a certain length (15–18 Å) of p-electron–
conjugated backbone exhibit a high affinity for pathological tau inclusions (Maruyama et al., 2013). Taking into account this finding, this
investigation developed a new compound where a double bond was
3.3 | NFTs were formed in the forebrain in which
intense 19F-MR signal was detected
added to the p-electron–conjugated backbone of the styrylbenzoxazole
The brain sections of rTg4510 mice prepared after MRI were examined
pound for pathological tau inclusions. Binding to NFTs in human AD
by immunohistochemistry for AT8 and simplified Gallyas silver staining.
AT8 immunoreactivity was detected in the forebrain region, including
the cortex and hippocampus in the rTg4510 mouse brain (Figure 4a).
The simplified Gallyas silver staining also showed many argentophilic
inclusion bodies in the forebrain region (Figure 4b).
derivatives. This was done to enhance the affinity of this new combrain sections was then tested. Shiga-X34 was used as the core structure of the probe for tau imaging and Shiga-X35 synthesized as a
MRI probe. This was accomplished by adding a trifluoromethyl group
onto the polyethylene glycol chain.
F-MRI using Shiga-X35 success-
fully detected tau pathology in the brain of rTg4510 mice. However, a
weak point in this study (still to be resolved) is the low selectivity for
3.4 | Shiga-X35 binds to NFTs in the in vivo rTg4510
mouse brain
NFTs and senile plaques. On the basis of the observation in ex vivo
labeling, Shiga-X34 potentially binds to both NFTs and senile plaques
in human AD brain sections (Figure 1 and Supplementary Figure 2), and
Finally, fluorescence microscopy of brain sections of rTg4510 mice sac-
therefore further modification in terms of chemical structure is needed
rificed after MRI was performed. These sections showed strong fluo-
to enhance the selectivity for NFTs. The affinity of phenyl/pyridinylbu-
rescence of Shiga-X35 in the hippocampus and the cortex (Figure 5a,b).
tadienyl-benzothiazoles/benzothiazoliums (PBBs) for senile plaques is
Distribution of NFTs in the brain of rTg4510 mice. (a and d) Representative images of the immunohistochemistry for AT8 (a)
and the simplified Gallyas silver staining (d) in the brain of the rTg4510 mice showed that NFTs were formed mainly in the forebrain. (b,c,e,
f) High-magnification images also revealed massive NFTs in the cortex (b,e) and hippocampus (c,f) of the rTg4510 mice. Scale bars: 1 mm
(a), 100 mm (b).
In vivo binding of Shiga-X35 to NFTs in the brain of rTg4510 mice. (a,b) Fluorescence microscopy after the MR measurement
showed strong fluorescence signals (arrowheads) in the hippocampus (a) and the cerebral cortex (b) of the rTg4510 mice that received the
injection of Shiga-X35. (c–e) Immunolabeling with AT8 revealed in vivo binding of Shiga-X35 (c) to AT81 NFTs (d) in the brains of rTg4510
mice. In merged images, fluorescence signals of Shiga-X35 and AT8 are seen in green and red, respectively. Arrowheads (c–e) indicate
representative double-positive labeling. Right arrows and left arrows indicate Shiga-X352, AT81 signals and Shiga-X351, AT82 signals,
respectively. Scale bars: 100 mm (a), 50 mm (c)
positively correlated with their lipophilicity. Therefore, probes with rel-
use of a higher-magnetic-power MR scanner, and the optimization of
atively low logP values appear suitable for visualizing tau pathologies
methodology are needed to improve the specificity for target mole-
with reasonable selectivity (Maruyama et al., 2013). Modification to
cules and the sensitivity of the MR detection.
reduce the lipophilicity of Shiga-X34 may increase selectivity for NFTs
since the structures of PBBs and Shiga-X34 are quite similar.
In the present study, the blood-brain barrier kinetics of Shiga-X35
were not determined. Instead,
F NMR spectra obtained from whole
It has been reported that several types of pathological tau aggre-
head for 10 min were recorded every 60 min up to 420 min. The
gates were observed in the brain of tauopathies. The types of tau
results showed that an intense peak was detected immediately after
aggregates are classified according to differences in filaments (straight
the injection. This then gradually decreased with time, although the
and helical), cell types (in neurons, astrocytes, and oligodendrocytes),
peak remained 420 min after the injection (Supplementary Figure 3).
and tau isoforms (4R and 3R). In the present study, colocalization of
The results suggest that Shiga-X35 readily reaches the brain and is
Shiga-X35 and AT8 immunoreactivity was detected in the in vivo brain.
then gradually excreted from the brain with some undesirable accumu-
lation. The primary modes of systemic elimination for Shiga-X35 are
signals were also observed. Further study to identify the types of NFTs
unknown. It has been reported that PBB3 is likely eliminated mainly via
to which the probe binds would be of great interest.
the hepatobiliary and intestinal reuptake pathways (Hashimoto et al.,
At the same time, Shiga-X35 , AT8
signals and Shiga-X35 , AT8
F-MRI has the following advantages: MR sensitivity of 19F is rel-
2014). Therefore, it is possible that Shiga-X35 is eliminated via the hep-
atively high compared with the sensitivity using various nuclei other
atobiliary and intestinal reuptake pathways. However, further study is
than 1H (1H, 100%;
required to clarify the blood-brain barrier kinetics as well as the primary
F, 83%;
P, 6.6%;
C, 1.6%). Furthermore, no
fluorine atoms exist in biological tissues. This results in low endogenous
background noise. The
F atom is also a nonradioactive isotope com19
modes of systemic elimination for Shiga-X35.
In PET studies, several requirements to achieve tau imaging are
described (Harada et al., 2015; Harada, Okamura, Furumoto, Tago,
may prove a highly sensitive, readily available, low-background, and
et al., 2016; Okamura et al., 2014; Villemagne et al., 2015), such as
prising 100% of naturally abundant fluorine
F. As a result,
cost-effective approach once a suitable high-quality probe has been
high binding affinity for tau (Kd or Ki < 20 nM for tau-rich brain sam-
developed. In the present study, 19F-MRI showed significant accumula-
ples) and high binding selectivity for tau (> 20-fold selectivity for tau
tion of 19F-MR signals in the brain regions where NFTs accumulated in
over Ab). However, the sensitivity of MR detection is far lower than
rTg4510 mice after the injection of Shiga-X35. This result suggests that
that of PET. MRI needs micromolar to millimolar concentrations of tar-
Shiga-X35 is effective for 19F-MRI to detect tau pathology in the brain.
get molecules to obtain the images. Even if the probe shows good
However, there were some limitations in the application of
specificity and selectivity at a nanomole-level concentration, it is diffi-
MRI for tau imaging. Firstly, since strong unwanted signals were also
cult to show such properties at the concentration required for MRI. In
detected even in the wild-type mouse brain, the selectivity and speci-
the present study, it is for this reason that fluorescence microscopy
ficity to the NFTs should be improved by modifications to the probe
using micromolar dosage was performed (but not autoradiography) to
structure. Secondly, sensitivity should be improved for safety reasons.
determine the binding affinity. There are several weak points in the
In the present study, quite a high dose of probe (200 mg/kg) (which
present study; for instance, the probe is not specific for tau aggregates,
may be near the value of LD50) is required for
F-MRI. Sensitivity
and the sensitivity of MR detection is far lower than that of PET. How-
should also be improved to produce better-quality images. Current 19F-
ever, this study is the first report investigating the detection of tau
MRI technology requires several hours (400 min in total) to obtain
pathology using
results; however, the quality of the images (inadequate resolution and
practical applications of 19F-MRI can be developed.
F-MRI. With further study, it is believed that the
low S/N ratio) was poor compared with PET tau imaging. The PET
A limitation in the present study was the failure to address sex as a
approach has great advantages including excellent sensitivity, the very
biological variable. In this study, only female mice were used to test
low doses of probe required, and high quantitative ability when com-
Shiga-X35 for tau imaging using
pared with other imaging modalities. However, there are some draw-
the binding of Shiga-X34 to NFTs in male human AD brain sections
backs associated with this technique including limited spatial
and male mouse brain sections. It therefore seems plausible that Shiga-
resolution, the need to manipulate radioactive compounds, and the
X35 could be used in male mice. However, sex differences have been
high cost. Therefore, the sensitivity of current
comparison with the PET approach. Thirdly,
F-MRI is quite low in
F-MRI methodology
should be improved. To specify the position of the
F signal in the
F-MRI should be measured both in the coronal and sagittal
planes. However, preliminary experiments using phantom samples
showed that
F-MRI. A ex vivo labeling showed
reported in terms of cognitive deficits and the level of abnormally
hyperphosphorylated tau in rTg4510 mice (Yue, Hanna, Wilson, Roder,
& Janus, 2011). Further study is needed to address the use of ShigaX35 for tau imaging using 19F-MRI in both male and female mice.
Another limitation was the number of animals used in the present
F-MRI in the coronal plane has a lower sensitivity than
study. Having three animals in each group resulted in extremely limited
that in the sagittal plane. It should be noted that current 19F-MRI in the
statistical power. Much stronger analytic results obtained from larger
coronal plane in one wild-type mouse and one rTg4510 mouse did not
samples would be preferred to endorse our results.
show apparent
F signals in the brain. Although this investigation
achieved the detection of tau pathology in rTg4510 mice using
MRI, further efforts including the modification of probe structures, the
In the present study, the in vivo detection of tau pathology was
investigated in rTg4510 mice through the use of a novel probe ShigaX35 for
F-MRI. Shiga-X35 penetrates the blood-brain barrier and
binds to NFTs in the brain in vivo after systemic injection. MRI successfully detected significant 19F-MR signal in the brain of rTg4510 mice in
the same regions in which many NFTs are accumulated. Although
some issues remain to be overcome, the data presented here indicate
F-MRI using Shiga-X35 represents a promising technique for
characterizing tau pathology in the brain.
The authors thank Dr. Piers Vigers for his critical reading of the
manuscript. This study was supported by JSPS KAKENHI grant number JP17H03560 (I.T.), JP15K01282 (H.T.), and JP17K01355 (D.Y.)
from the Japan Society for the Promotion of Science.
T. Sogabe is an employee of Otsuka Pharmaceutical Co., Ltd, Japan.
Conceptualization, D.Y., H.T., and I.T.; Methodology, D.Y., N.F.I., and
H.T.; Investigation, D.Y., and N.F.I.; Writing – Original Draft, D.Y.;
Writing – Review & Editing, H.T., and S.M.; Funding Acquisition, D.Y.,
H.T., and I.T.; Resources, T.K., K.H., N.S., and T.S.; Supervision, I.T.
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Additional Supporting Information may be found online in the supporting information tab for this article.
How to cite this article: Yanagisawa D, Ibrahim FN, Taguchi H,
et al. Fluorine-19 magnetic resonance imaging probe for the
detection of tau pathology in female rTg4510 mice. J Neuro Res.
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