D-A-D fluorogenic probe for the rapid imaging of amyloid β plaques in?vivo

Article abstract of DOI:10.1016/j.dyepig.2016.08.054

The effective imaging of amyloid β (Aβ) in?vivo is important for the diagnosis of Aβ-related diseases such as the Alzheimer's disease (AD). Here we report a donor-acceptor-donor (D-A-D) type fluorogenic probe for the rapid imaging of amyloid β (Aβ) senile plaques in a transgenic mouse brain. The probe that features a dicyanomethylene-4H-pyran (DCM) core shows a concentration-dependent fluorescence enhancement with Aβ42 and Aβ40, which are the main components of Aβ fibrils formed in the brain of AD patients. The D-A-D probe can also be used to rapidly sense Aβ peptide monomer, oligomer and fibril in an aqueous solution and image the morphologically diverse Aβ species by confocal microscopy. In particular, we demonstrate that the probe can be used to rapidly stain Aβ senile plaques in the brain of transgenic mice by intravenous injection.

Full text of DOI:10.1016/j.dyepig.2016.08.054

Dyes and Pigments 136 (2017) 224e228
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
D-A-D fluorogenic probe for the rapid imaging of amyloid
b
plaques
in vivo
,
c
,
,
,
,
***
Hao-Yu Yang a 1, Jing-Jing Zhang c 1, Yi Zang b, Hai-Yan Zhang c **, Jia Li b
,
Guo-Rong Chen a, Xiao-Peng He a
,
*
a Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and
Technology, Shanghai 200237, PR China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 189
Guo Shoujing Rd., Shanghai 201203, PR China
c CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The effective imaging of amyloid
b (Ab) in vivo is important for the diagnosis of Ab-related diseases such
Received 15 July 2016
Received in revised form
22 August 2016
Accepted 23 August 2016
Available online 25 August 2016
as the Alzheimer's disease (AD). Here we report a donor-acceptor-donor (D-A-D) type fluorogenic probe
for the rapid imaging of amyloid (A ) senile plaques in a transgenic mouse brain. The probe that
features a dicyanomethylene-4H-pyran (DCM) core shows a concentration-dependent fluorescence
enhancement with A 42 and A 40, which are the main components of A fibrils formed in the brain of
AD patients. The D-A-D probe can also be used to rapidly sense A peptide monomer, oligomer and fibril
in an aqueous solution and image the morphologically diverse A species by confocal microscopy. In
senile plaques in the brain of
b
b
b
b
b
b
b
Keywords:
particular, we demonstrate that the probe can be used to rapidly stain A
transgenic mice by intravenous injection.
b
Amyloid
Probe
b
© 2016 Elsevier Ltd. All rights reserved.
Fluorescence
In vivo imaging
Alzheimer's disease
1. Introduction
laborious, time-consuming, expensive and not amenable to in vivo
application due to the inability to cross blood-brain barrier.
Alternatively, development of small-molecular fluorescent
probes has been an emerging trend for AD detection [4e14]. Here
we report the synthesis of a donor-acceptor-donor (D-A-D) type
Alzheimer's disease (AD) is a neurodegenerative disorder that
prevails worldwide, suffering millions of patients. This number is
estimated to continuously grow with the aging of population.
Whereas the fibrillogenesis of amyloid
to form insoluble senile plaques has been long viewed as the cause
of AD pathogenesis, recent studies suggest that oligomeric A
should be the primary impetus for disease progression [1e3]. As a
result, effective tools to probe the A peptides in their various
morphological forms are highly needed to understand and di-
agnose AD. To meet this end, a number of biochemical methods,
including oligomer-specific antibody, fluorescent protein and
ELISA, have been developed. Nevertheless, these methods are often
b
(A
b
) monomeric peptides
dicyanomethylene-4H-pyran (DCM) derivative as
a new red-
emitting fluorogenic probe for the detection of A in solution and
b
b
imaging of senile plaques in the brain of AD transgenic mice. The
desired D-A-D probe (YHY2) (tertiary amine as electron donor and
cyano groups as acceptor) was synthesized by the Knoevenagel
condensation of 2-(2,6-dimethylpyran-4-ylidene)-malonoitrile (1)
with 2 mol of aldehyde 2 in the presence of piperidine (Scheme 1)
[15]. Treatment of equivalent 1 with 2 under the same condition
produced the mono-armed YHY1 as a control compound [16].
b
2. Results and discussion
* Corresponding author.
** Corresponding author.
*** Corresponding author.
With the probes in hand, we measured their fluorescence in the
absence and presence of Ab peptides (Fig. 1). We observed minimal
fluorescence of the probes in an aqueous buffer solution, which
could be a result of aggregation-caused-quenching. The presence of
E-mail addresses: hzhang@simm.ac.cn (H.-Y. Zhang), jli@simm.ac.cn (J. Li),
xphe@ecust.edu.cn (X.-P. He).
1
Equal contribution.
http://dx.doi.org/10.1016/j.dyepig.2016.08.054
0143-7208/© 2016 Elsevier Ltd. All rights reserved.

H.-Y. Yang et al. / Dyes and Pigments 136 (2017) 224e228
225
monomeric A
nents to form A
b
40 and A
b
42 peptides, which are the main compo-
a
400
300
200
100
400
300
200
100
b
fibrils, largely enhanced their fluorescence in a
YHY1
YHY1
concentration-dependent manner. We observed that the fluores-
cence enhancement of the D-A-D YHY2 (Fig. 1b) was stronger than
that of the D-A YHY1 (Fig. 1a) with both A
b
40 and A
b42. Both the
limit of detection (3 /k) and binding constant (Kd) of YHY2 for A
s
b42
were determined to be lower than that of YHY1 (Fig. S1 and
Table 1). The fluorescence enhancement is in agreement with the
property of similar types of DCM probes with a typical pull-push
motif, which is sensitive to the change of external environment
from hydrophilic to hydrophobic [17]. Next, we tested the binding
specificity of YHY2 with different fragments of A
fragments the probe showed the strongest affinity to A
major neurotoxic domain that impairs learning and memory in
Aβ(1-42)
Aβ(1-40)
0
0
560
640
720
800
560
640
720
800
b
. Of a range of
(25e35), a
b
400
400
b
YHY2
YHY2
300
200
100
0
300
200
100
0
rodents (Fig. 1c) [18]. This suggests that this specific fragment
might be a major (but not necessarily the sole) pocket of Ab for
YHY2 to bind, though more evidence is needed to scrutinize the
probe-peptide binding mode. A summary of the binding properties
Aβ(1-42)
Aβ(1-40)
of YHY1 and YHY2 to A
The pH working range of YHY1 and YHY2 in the absence and
presence of A peptides was tested, and the results suggest that the
b42 monomer is shown in Table 1.
b
sensitivity of probe was not compromised over a wide range of pH
(3e10) (Fig. S2). We also determined that the association of YHY2
560
640
720
800
560
640
720
800
Wavelength (nm)
Wavelength (nm)
with A
(Congo red, a known staining reagent for A
probe showed a larger fluorescence enhancement for A
b
42 is reversible in the presence of a competing species
) (Fig. S3a), and that the
42 fibril
c
b
2.4
1.6
0.8
0.0
2.4
1.6
0.8
0.0
YHY1
YHY2
b
(by incubating the monomer in a Tris-HCl buffer for seven days, pH
8.0, room temperature) [19] than monomers (Fig. S3b). A fluores-
cence kinetic study indicated that the fluorescence enhancement of
YHY2 quickly reached equilibrium (within 2 min) with Ab in its
various morphological forms including fibril, oligomer (by incu-
bating the monomer in a Tris-HCl buffer for three days, pH 8.0,
room temperature [19]) and monomer (Fig. 2). To test the imaging
ability of the D-A-D probe for A
confocal laser scanning microscopy (CLSM) was used. Shown in
Fig. 3 are the CLSM images of A upon fibrogenesis with increasing
time. The results suggest that the gradual morphological change of
with time could be tangibly imaged by YHY2. Transmission
electron microscopy performed corroborated the morphology of
the different A species formed (Fig. 4).
With the promising properties of YHY2 determined, we tested
its ability for imaging A senile plaques using a transgenic mouse
b with different morphologies,
b
A
b
Fig. 1. Fluorescence titration of YHY1 and YHY2 (10
(a) A (1e40) (0e200 M) and (b) A (1e42) (0e200
ment of YHY1 and YHY2 (10 M) in the presence of different fragments of A
where I0 and I are the fluorescence intensity (IF) of probe in the absence and presence
of a peptide, respectively. All measurements were carried out in phosphate buffered
saline (0.01 M, pH 7.4, 20% DMSO) with excitation of 480 nm for both YHY1 and YHY2.
m
M) in the presence of increasing
M). (c) Fluorescence enhance-
(200 M),
b
b
m
b
m
m
b
m
b
with APPswe/PS 1dE9 mutations (APP/PS1 mouse) [20]. An in vitro
assay first showed that the senile deposits of the brain section
removed from APP/PS1 mouse could be imaged by YHY2, and the
fluorescent spots overlapped well with those produced by thio-
imaging (Fig. S4).
flavin S, a known Ab plaque staining agent (Fig. 5). We also deter-
mined that the probe was not toxic to a mouse fibroblast (NIH/3T3)
cell line with a concertation 10-fold higher than that used for
Eventually we examined the in vivo imaging ability of the probe
using a wild-type mouse as control. The results obtained from two
independent groups suggest that intravenous injection of YHY2
produced strong fluorescence of the probe in the brain of both mice
within 5 min, but the fluorescence quickly diminished in the con-
trol mice at 10 min. In contrast, the fluorescence of the D-A-D probe
retained significantly in the APP/PS1 mouse, but was faded at
15 min (Fig. 6). A following ex vivo assay on the mice brain taken
from above in vivo study showed that the probe could fluorescently
label the amyloid deposits in the cortex and hippocampus regions
of the brain of APP/PS1 mouse, whereas no such staining was
observed in the age-matched wild type control group (Fig. 7). The
ex vivo staining result was in good agreement with that produced
by the in vitro staining (Fig. 5). These pieces of data suggest that the
1
+
2
YHY1 (D-A)
probe could cross the blood-brain barrier (BBB) to stain Ab senile
plaques formed in transgenic live mice, and that it can be quickly
removed from the brain. To further corroborate that the BBB
permeability of the probe, the brain section of a normal mouse
YHY2 (D-A-D)
Scheme 1. Reagents and conditions: Piperidine and 1-propanol (molar ratio of 1 to
2 ¼ 1:1 and 1:2 for YHY1 and YHY2, respectively).

Table 1
Summary of binding properties of YHY1 (10
m
M) and YHY2 (10
m
M) with A
b
42 monomer (50 mM) in phosphate buffered saline (0.01 M, pH 7.4, 20% DMSO) with excitation of
480 nm.
Compound
lem/max (nm)a
lem/max (nm)b
FF (x/y)c
Fold of increase
Kd (nM)
LOD (nM)d
YHY1
YHY2
632
650
601
596
1.8/7.2
2.3/18.9
3.4
7.7
57.1
23.5
86
51
a
Compound alone.
Compound with Ab42 monomer.
x and y are FF before and after addition of A
LOD ¼ Limit of detection.
b
c
b42 monomer, respectively.
d
injected with cold solution and that with probe were collected. We
observed clear probe fluorescence on the latter group rather than
the former group (Fig. S5), suggesting that the probe can cross the
BBB in vivo. Notably, a comparison with previously reported small-
90
75
molecular probes for in vivo staining of Ab [21e28] reveals that our
Aβ42 fibril
D-A-D probe is among the fastest to stain as well as to be removed
from the brain of a transgenic AD mouse.
We note that a structurally similar D-A-D probe based on the
DCM scaffold has been synthesized recently with the hydroxyethyl
groups of YHY2 replaced by methyl groups [29]. However, that
Aβ42 oligomer
Aβ42 monomer
60
45
0
120 240 360 480 600
Time (s)
probe showed insufficient binding affinity to A
that the hydroxyethyl groups of YHY2 might be crucial for its strong
binding property, probably through generating hydrogen-
b42 fibril, suggesting
Ab
Fig. 2. (a) Plotting of the fluorescence intensity (IF) of YHY2 (10
mM) in the presence of
bonding interactions with the peptide. Although more evidence
would be needed for fully elaborating the precise binding mode
between YHY2 and Ab42, our results set a basis for the future
A
b
42 (20 M) in its different morphologies (fibril: 7-day fibrogenesis; oligomer: 3-day
m
fibrogenesis; monomer: no fibrogenesis) with time in phosphate buffered saline
(0.01 M, pH 7.4, 20% DMSO) with excitation of 480 nm.
Fig. 3. Fluorescence imaging of A
channel ¼ 620 nm; scale bar ¼ 40
b
42 (40
mM) with different morphologies using YHY2 (20
mM) by confocal laser scanning microscopy (excitation channel ¼ 480 nm; emission
m
m).
Fig. 4. Transmission electron microscopy of A
b42 monomer, oligomer and fibril (40
mM) measured with JEOL 1400 equipped with a Gatan Orius charged-coupled device camera and
a Tridiem energy filter operating at 200 kV (scale bar ¼ 2
mm).

Fig. 5. (a) Fluorescence imaging of A
b
senile plaques on the cortex and hippocampus regions of APP/PS1 AD mouse brain sections by incubating with YHY2 and thioflavin S
m).
(excitation channel ¼ 473 nm for YHY2 and 405 nm for thioflavin S; emission channel ¼ 670 nm for YHY2 and 461 nm for thioflavin S; scale bar ¼ 200
m
Fig. 6. In vivo imaging of brain senile plaques formed in APP/PS1 live mice with time (excitation filter ¼ 480 nm; emission filter ¼ 640 nm). Wild-type mice were used as control.
Mice of 18-month-old (transgenic APP/PS1 and age-matched wild-type control mice) were injected with YHY2 (5.0 mg kgꢀ1) freshly prepared in a mixture of 10% DMSO, 10%
cremorphor EL and 80% PBS on an IVISSpectrum animal imaging system (Caliper LifeSciences, PerkinElmer, Hopkinton, MA).
Fig. 7. Ex vivo histological observation of brain slices from APP/PS1 and wild type mice after intravenous injection of YHY2 (scale bar ¼ 200
mm). For experimental details, see
Supplementary data associated with this paper.

228
H.-Y. Yang et al. / Dyes and Pigments 136 (2017) 224e228
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In summary, we have demonstrated that a D-A-D type DCM
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Acknowledgments
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This research is supported by the 973 project (2013CB733700),
the Natural Science Foundation of China (21572058, 21576088,
81302820 and 81522045), the National Science and Technology
Major Projects (2014ZX09507-002), the Shanghai Science and
Technology Development Funds (14YF1413300) and the Shanghai
Rising-Star Program (16QA1401400).
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