Detection of Aβ plaques in mouse brain by using a disaggregation- induced fluorescence-enhancing probe

Article abstract of DOI:10.1039/c4cc02011a

We herein report a fluorescence probe 1 capable of detecting water-soluble oligomeric Aβ aggregates and Aβ fibrils. Upon injection into Aβ₄₂-challenged mouse brains, probe 1 shows increased fluorescence intensity, indicating its facile binding to extracellular Aβ fibrils in brain tissues. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02011a

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Detection of Ab plaques in mouse brain by using a
disaggregation-induced fluorescence-enhancing
probe†
Cite this: Chem. Commun., 2014,
50, 5741
Received 18th March 2014,
Accepted 5th April 2014
Yeon Ok Lee,‡a Jung-Won Shin,‡b Chunsik Yi,b Yun Hak Lee,a Nak-Won Sohn,*b
Chulhun Kang*b and Jong Seung Kim*a
DOI: 10.1039/c4cc02011a
www.rsc.org/chemcomm
We herein report a fluorescence probe 1 capable of detecting
water-soluble oligomeric Ab aggregates and Ab fibrils. Upon injec-
tion into Ab42-challenged mouse brains, probe 1 shows increased
fluorescence intensity, indicating its facile binding to extracellular
Ab fibrils in brain tissues.
Amyloid-b (Ab) peptide is a primary component of extracellular
plaques associated with Alzheimer’s disease (AD). Ab is generated
through proteolytic cleavage of the amyloid peptide precursor (APP)
by b- or g-secretase.1 For some decades, insoluble Ab peptide
plaques have been believed to be the main contributors to the
pathogenesis of AD.2 Therefore, many efforts have been made, such
as the development of a wide variety of fluorescent probes to
selectively visualize plaques in brain tissues.3 The visualization is
based on the fluorescence of the probe that is ‘‘turned on’’ during
its transfer from the polar and random aqueous environment into
Scheme 1 Fluorescence enhancement upon binding of probe
amyloid b (Ab) plaques.
1 to
We prepared a new probe, 1, that showed disaggregation-
the hydrophobic and highly ordered amyloid b-sheet structure of induced fluorescence enhancement and was capable of detecting
plaques.4 Fluorescence enhancement of the probe may be due to both water-soluble oligomeric Ab species as well as insoluble Ab
rotational bond restriction of the probes and/or the hydrophobic aggregates as indicated in Scheme 1. Probe 1 is comprised of three
environment in plaques compared to aqueous media.5
components: (i) an N,N-dimethylamino styrene unit that is able to
Aggregation of hydrophobic fluorescent materials generally bind to b-sheets of Ab aggregates, (ii) a hydrophilic triethylene glycol
results in reduced quantum yield.6 The fluorescence intensity of unit to enhance the water solubility, and (iii) fluorene as an ‘‘Off–
the probes, however, can be markedly increased following their On-type’’ fluorescence signaling unit upon intercalation of the
disaggregation,6 e.g., with nonpolar peptide monomers.7 Moreover, N,N-dimethylamino styrene unit with oligomeric Ab aggregates or
upon interaction of aggregated fluorescent probes with hydrophobic Ab fibrils.8,9
plaques, it is expected that the probes are disaggregated followed by
Probe 1 was prepared according to the synthetic route shown in
their distribution over the plaque surfaces and homing to binding Scheme 2. Compound 3, 9,9-diethyl-2-iodofluorene, was synthesized
sites, which results in the increased fluorescence intensity of the from 2 by adaptation of procedures reported earlier.9 Then,
probes. To the best of our knowledge, this type of approach has not 3 was converted to 4 by the Sonogashira coupling reaction with
been reported with regard to the detection of Ab plaques or water- 3-(2-(2-ethyoxyethoxy)ethoxy)-1-propyne (35% yield). Subsequently,
soluble oligomeric Ab species in brain tissues.
hydrogenation of 4 using Pd/C in ethanol gave 5 in 86% yield.
Iodination of 5 with I2/HIO4 provided 6 in 59% yield. Finally, the
Pd-catalyzed Heck reaction of 3 with N,N-dimethylaminostyrene gave
probe 1 in 28% yield. The identities of all newly synthesized com-
pounds were confirmed by 1H NMR, 13C NMR, and ESI-MS (ESI†).
Probe 1 in phosphate-buffered saline (PBS) exhibited absorption
and emission bands with maxima at 412 and 462 nm, respectively,
as shown in Fig. 1a. The fluorescence spectral properties changed
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
E-mail: jongskim@korea.ac.kr
b School of East-West Medical Science, Kyung Hee University, Yongin 446-701,
Korea. E-mail: sohnnw@khu.ac.kr, kangch@khu.ac.kr
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc02011a
‡ Equally contributed to this work.
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Fig. 2 (a) Plot of the fluorescence intensity of probe 1 at lem = 449 nm.
(b) The maximum wavelength (lmax) of probe 1 in aqueous 1,4-dioxane
solutions, excitation at 367 nm. (c) The normalized fluorescence spectra of
probe 1, 3D view. [probe] was 1 mM.
Scheme 2 Route for the synthesis of probe 1.
Fig. 3 (a) Fluorescence intensity of probe 1 (1.0 mM) upon interaction with
Ab42 oligomers in 0.2% (v/v) DMSO in PBS (pH 7.4) at lex = 412 nm, slit
width: 3/5 nm. (b) Emission intensity of probe 1 in water containing varying
amounts of glycerol (v/v, %).
Fig. 1 (a) Absorption and fluorescence spectra of probe 1 (2 mM) in
0.2% (v/v) DMSO in PBS (pH 7.4), excitation at 412 nm, slit width = 5/5 nm.
(b) Fluorescence spectra of probe 1 in different solvents.
significantly in different solvents (Fig. 1b), i.e., the intensity intensity and lmax. These changes occur due to the self-
increased with decreasing solvent polarity (Table S1, ESI†), resulting aggregation nature of probe 1 in 100% aqueous solution. The
in a significant blue shift. This blue shift is the result of a aggregation of probe 1 is increased and a blue shift of the emission
solvatochromic shift that is due to an intramolecular charge transfer has been observed by up to 20% of 1,4-dioxane due to its decreased
between the donor (aniline) and the acceptor (fluorene) across the polarity. Then it was solubilized in the solution showing a dramatic
ethylene spacer.
red shift and decreased fluorescence intensity, which has a relatively
To understand the photophysical properties of probe 1 due to polar environment compared to that on the inside of the aggregates.
the solvent species used, the spectra of probe 1 were measured in These results indicate that the probe exists in an aggregated form
water containing varying amounts of 1,4-dioxane, and fluorescence with lower fluorescence intensity in aqueous environments and
intensity, maximum wavelength (lmax) and the normalized spectra shows strong fluorescence emission upon solubilisation. Similarly,
are shown in Fig. 2. As shown in Fig. 2a, the profile of 1,4-dioxane we anticipate disaggregation of probe 1 upon binding to amyloid
percentage in water versus the probe’s fluorescence intensity has oligomers or plaques in aqueous environments. To test whether
three phases: (i) 0–20%, where it increases; (ii) 20–25%, where it probe 1 can detect water-soluble oligomeric Ab42 aggregates,
rapidly decreases; and (iii) 30–100%, where it gradually increases. changes in the fluorescence intensity of probe 1 were investigated
Similarly, the profile of lmax in the solution (Fig. 2b) displays three in the presence of Ab42 oligomers. As shown in Fig. 3a, we observed
phases in the percentage ranges of 1,4-dioxane, each of which shows a significant increase (6.5-fold) in the fluorescence intensity of
a blue, red, and blue shift, respectively. A similar three phasic probe 1 without a wavelength shift. To understand the origin of
behaviour of the solvatochromic fluorescence properties of the the increase in the emission intensity of probe 1 upon binding to
probe was found using acetonitrile (Fig. S1, ESI†). The blue shifts Ab42 oligomers, we carried out two independent spectroscopic
of the spectra with increasing intensity found in the 0–20% and experiments using probe 1 in solvents of varying polarity and
30–100% ranges of the 1,4-dioxane may be attributed to the reduced viscosity in the absence of Ab42 oligomers. It was previously reported
solvent polarity.7
that the fluorescence wavelength of an Ab probe is blue-shifted when
The initial two phases in the 0–25% range of 1,4-dioxane the probe is embedded inside of Ab42 oligomer aggregates, which
shown in Fig. 2a and b reveal quite dramatic changes in the represents a non-polar environment.10 As shown in Table S1 (ESI†),
5742 | Chem. Commun., 2014, 50, 5741--5744
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Fig. 4 Plot of the fluorescence intensity at lmax = 467 nm as a function
of the concentration of oligomeric Ab42 aggregates in the presence of
probe 1 (1.0 mM) in 0.2% (v/v) DMSO in PBS (pH 7.4).
Fig. 5 Confocal microscopy images of HeLa cells treated with 1.0 mM ThS
(a) and 1 mM of probe 1. (b) Cells were incubated with DMEM at 37 1C and
for 30 min with DMEM containing ThS and probe 1, respectively.
however, we did not observe any noticeable correlation of the
increase in the fluorescence intensity of probe 1 with solvent polarity,
but noticed wavelength shifts depending on solvent polarity.
Next, with regard to solvent viscosity, the changes in the
fluorescence intensity of probe 1 were also investigated in water
containing varying amounts of glycerol. It is well known that the
fluorescence intensity increases markedly as the viscosity of the
solvent increases because the intramolecular rotation of small
molecules is considerably restricted.11 We also found that the
fluorescence intensity of probe 1 increases as the solvent viscosity
increases (Fig. 3b). In this regard, it is conceivable that the inter-
action originates from a strong hydrophobic attraction between
b-sheets of oligomeric Ab aggregates and the N,N-dimethylaniline ring
of the probe, which presumably leads to smoother intramolecular
rotations, resulting in the increase in fluorescence intensity.12
Titration experiments were also conducted using probe 1
(1.0 mM) and oligomeric Ab42 aggregates in PBS in the concen-
tration range of 1.0–7.0 mM. The results are shown in Fig. 4.
From this graph, the binding constant (Kd) of probe 1 toward
oligomeric Ab aggregates was calculated to be 2.25 mM.
Fig. 6 Representative hippocampal sections of three groups. (a) Mice
received a single injection of 3.0 mL of Ab aggregates; (b) mice received
a single injection of 3.0 mL of probe 1; and (c) mice received consecutive
injections of Ab aggregates and probe 1 into the same region of the
hippocampus.
was drilled in the skull (1.5 mm caudal and 2.0 mm lateral to
bregma). Then, Ab42 aggregates and probe 1 were injected consecu-
tively into mouse hippocampi. Coronal brain sections (50 mm-thick)
were made using a freezing microtome. Incubation with Ab42
aggregates and/or probe 1 for 48 h was expected to cause sufficient
accumulation of both Ab42 aggregates and probe 1 in mouse
hippocampi. Analysis of the sections revealed that the fluorescence
intensity of hippocampal sections increased upon treatment with
probe 1 (Fig. 6b), which indicates that the probe is able to intercalate
with Ab aggregates. Subsequent addition of Ab42 aggregates induced
a further increase in the fluorescence intensity (Fig. 6c), which again
indicates that probe 1 could bind to Ab aggregates in mouse
hippocampi.
To investigate the specificity of probe 1 for oligomeric Ab
aggregates over other biocompatible peptides, probe 1 was
tested with bovine serum albumin and lysozyme amyloid, and
the corresponding fluorescence intensity changes were investi-
gated. We could rarely find any changes in the fluorescence
intensity of probe 1, which confirms that probe 1 is specific for
oligomeric Ab aggregates, as shown in Fig. S2 (ESI†).
Another important objective was to test the ability of probe 1 to
detect insoluble Ab plaques, observed as changes in the fluorescence
intensity. To do this, confocal microscopy was used and probe 1 and
thioflavin S (ThS), the standard staining dye for Ab plaques, were
independently applied to HeLa cells. As illustrated in Fig. 5a, in
HeLa cells incubated with ThS, ThS is mainly localized around the
cell membrane. A previous report showed that ThS is located at the
extracellular sites where it interacts with Ab plaques.10 In contrast to
ThS, fluorescence signals of probe 1 were not detected around the
cell membrane or inside the cells (Fig. 5b). On the basis of this
observation, we conclude that probe 1 has the potential to interact
with Ab plaques, which are, in general, formed outside the cells.
To test the ability of the probe to detect Ab plaques in living
brain tissues, ex vivo experiments were carried out using male ICR
mice. Under aseptic conditions, a burr hole (1.0 mm in diameter)
Fig. 7 Ex vivo fluorescence observations of sections from mouse brains.
(a) After injection of probe 1 and Ab aggregates; (b) neuronal cells were
confirmed by immunohistochemical staining using NeuN; and (c)
a
fluorescence micrograph representing regions of overlap of fluorescence
labeling of probe 1 and NeuN in brain tissues.
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To confirm the regional distribution pattern of Ab aggregates
This work was supported by the CRI project (No. 2009-0081566,
and probe 1 in brain tissues, neuronal cells of tissues treated with JSK), the Basic Science Research Program (2012R1A1A2006259, CK),
Ab peptides and probe 1 were stained (Fig. 7). The brain sections and (NRF-2011-0014188, JWS) through the National Research Foun-
were fixed and immunostained with NeuN, which is a marker of dation of Korea (NRF) funded by the Ministry of Science, ICT &
mature neurons (Fig. 7b). Fibril aggregates stained by probe 1 Future Planning.
appeared as a granular smear (2.0 mm2) throughout the hippo-
campal sections, as shown in Fig. 7a. Furthermore, the fluorescence
signal of probe 1 rarely overlapped with that of NeuN, as shown in
Fig. 7c, which indicates again that probe 1 does not enter neuronal
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In conclusion, we synthesized a new probe, 1, containing three
important components, i.e., an N,N-dimethylamino styrene unit, a
hydrophilic triethylene glycol unit, and fluorene as a fluorescence
signaling unit that is ‘‘switched on’’ upon intercalation with oligo-
meric Ab aggregates or Ab fibrils. Probe 1 was shown to be able to
bind to water-soluble oligomeric Ab species as well as insoluble Ab
aggregates. Probe 1 showed significantly increased fluorescence
intensity upon binding to oligomeric Ab42 aggregates, which is
attributed to the viscous environment of Ab42 oligomers. Probe 1
showed a binding constant (Kd) of 2.25 mM toward oligomeric Ab
aggregates. On the basis of fluorescence images, we found that
probe 1 is localized outside the cells to bind to Ab plaques and not
in the cell membrane or cytosol. The results from ex vivo experi-
ments showed that the fluorescence intensity observed in mouse
hippocampal sections was increased upon treatment with probe 1,
which is due to the binding of probe 1 to Ab aggregates. In brain
tissues stained with NeuN, probe 1 was again confirmed to be
distributed outside neuronal cells and to interact with extracellular
Ab fibril aggregates. These findings open a new strategy for the
development of small molecules that are able to detect water-soluble
oligomeric Ab aggregates as well as Ab fibril aggregates mostly
found in patients with AD.
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