A novel near-infrared fluorescent probe for detection of early-stage Aβ protofibrils in Alzheimer's disease

Article abstract of DOI:10.1039/c9cc09233a

Detection of Aβ protofibrils at the early stage of Alzheimer's disease was realized by a novel near-infrared probe (DCM-AN) based on dicyanomethylene-4H-pyran. This probe exhibits high affinity towards Aβ protofibrils in vitro and in brain sections of tra

Full text of DOI:10.1039/c9cc09233a

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A Novel Near-Infrared Fluorescent Probe for Detection of Early-
stage Aβ Protofibrils in Alzheimer’s Disease
Guanglei Lv,a,c Anyang Sun,* Minqi Wang, Peng Wei, Ruohan Li, and Tao Yi*
b
b
a
a
a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Detection of Aβ protofibrils at the early stage of Alzheimer’s disease increasing amount of data for the detailed structure of Aβ
was realized by a novel near-infrared probe (DCM-AN) based on protofibrils has become available.23, 24 Previous studies revealed
dicyanomethylene-4H-pyran. This probe exhibits high affinity that
towards Aβ protofibrils in vitro and in brain sections of transgenic neuroinflammation and can disrupt ion channels in rat cortical
Aβ
protofibrils
possess
neurotoxicity25
and
2
6
27
mouse models for Alzheimer’s disease.
neurons. These findings demonstrate that Aβ protofibrils play
an important role in the progress of AD, especially the early
stage of the disease. Therefore, it is greatly urgent to develop
effective probes for specifically detecting Aβ protofibrils.
However, to the best of our knowledge, no probe capable of
specifically targeting and imaging Aβ protofibrils has been
reported till now.
Alzheimer’s disease (AD) is one of the most prevalent form of
neurodegenerative diseases. Unfortunately, so far, there are no
effective drugs to cure this disease when clear clinical symptoms
appear. In fact, neurodegeneration is estimated to start 10−30
1
years before clinical symptoms are detected in AD. Therefore,
seeking effective probes to monitor the progression of AD is
extremely urgent. It is well established that AD has two main
pathological hallmarks: β-amyloid plaques composed of β-
Recently, we developed a series of fluorescent probes for
specific detection of Aβ aggregates28 and Aβ oligomers29 based
on aminonaphthalene (AN). Even though it is still a great
chellange to distinguish soluble and insoluble Aβ species due to
their similar structures, previous works from us and other
researchers intented that the appropriate steric hindrance of
amyloid peptide (Aβ) and neurofibrillary tangles resulting from
hyperphosphorylated Tau protein.2
-6
Biochemical and
pathological analyses of AD brain samples reveal that Aβ species
7
8
exist in several forms,
oligomers, protofibrils
including soluble monomers,
and insoluble aggregates.2
the fluorophore might be the key point to differentiate those
9
10,11
30, 31
different Aβ species.
This is due to the fact that different
Initially, much effort had been made on the detection and
imaging of Aβ aggregates.12-16 However, there is increasing
level of hydrophobic cavities can be produced in the different
32, 33
state of the Aβ assembly.
Therefore, design a fluorescent
evidence that Aβ aggregates burden correlated poorly with the
probe with suitable steric hindrance specifically binding with the
cavity of Aβ protofibrils is the strategy of this work.
severity of AD,1
7, 18
whereas non-monomeric soluble Aβ species
1
9, 20
exhibit much more toxic than insoluble Aβ aggregates.
Dicyanomethylene-4H-pyran (DCM) derivatives have
attracted significant attention because of their controllable
emission wavelength in NIR or far-red area. In addition, DCM
derivatives possess large Stokes shift and high photostability.
These make DCM derivatives better candidates for fluorescence
Among soluble Aβ species, Aβ protofibrils were significantly
important intermediate, which were first observed and
characterized about two decades ago with rich β-sheet
structure as precursors to mature fibrils.2 After that, studies
on Aβ protofibrils have attracted extensive interest and an
1,22
34-35
imaging.
Since DCM has certain steric hindrance, we
envision that, by modifying the structure of DCM moiety, it
might be possible to develop a novel probe capable of detecting
Aβ protofibrils with good affinity and specificity. Therefore, the
fluorescent probe DCM-AN, combining DCM moiety with Aβ
target group AN (Scheme 1), was designed and synthesized to
investigate its specificity toward Aβ protofibrils. To our delight,
DCM-AN is capable of detecting Aβ protofibrils from Aβ species
and can image Aβ protofibrils in the brain of transgenic mouse
models.
a.Department of Chemistry, Fudan University, Shanghai 200438, P. R. China.
E-mail: yitao@fudan.edu.cn.
Laboratory of Neurodegenerative Diseases and Molecular Imaging, Shanghai
b.
University of Medicine & Health Sciences, Shanghai 201318, P. R. China
E-mail: sunay@sumhs.edu.cn
Key Laboratory of the Ministry of Education for Advanced Catalysis Materials,
c.
Zhejiang Normal University, Jinhua 321004, P. R. China
†
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [synthetic details and
additional spectra and images]. See DOI: 10.1039/x0xx00000x
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In addition, DCM-AN showed high anti-interference ability
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DOI: 10.1039/C9CC09233A
toward common cations and anions (Fig. S5). Subsequently,
titration experiment was performed under different
concentrations of Aβ protofibrils (0-9 μM, Fig. 1C). Linear
relationship was observed between the concentration of Aβ
2
Scheme 1. Structure and synthesis route of DCM-AN
protofibrils and the fluorescence intensity of DCM-AN (R =
0
.98, Fig. S6). Furthermore, the dissociation constant of DCM-
Fluorescence spectroscopy experiments were performed AN for Aβ protofibrils was measured to be 0.85 μM (Fig. S1).
to investigate the binding properties of DCM-AN with Aβ. DCM-
Since DCM-AN showed high affinity towards Aβ protofibrils,
AN has a typical D- -A structure with dicyanomethylene as an the dynamic aggregation process of Aβ protofibrils was then
π
electron accepter and piperidine as an electron donor. The traced by the change of fluorescence intensity of DCM-AN with
rotation of the ethylene group makes the molecule poor internal different incubation time (Fig. 1D). It could be easily found that
charge transfer (ICT) process and low fluorescence in water and there was an enhanced fluorescence intensity in the PBS
polar solvent. Therefore, the fluorescence of DCM-AN was very solution after the addition of Aβ protofibrils. As expected, the
weak in the phosphate buffered saline (PBS) solution (Fig. 1). fluorescence intensity of DCM-AN decreased upon prolonging
The maximum fluorescence emission peak lying in 725 nm with the incubation time because Aβ protofibrils grew to mature
absolute fluorescence quantum yield (Φ
when Aβ protofibrils (determined by dynamic light scattering affinity towards Aβ protofibrils. In addition, the detection limit
and TEM (Fig. S2) were added to the PBS solution of DCM-AN
of this probe for Aβ protofibrils was measured to be 51.9 nM
F
) of 0.06%. However, fibrils. This data further confirmed that DCM-AN exhibited high
,
the fluorescence intensity was significantly increased (Fig. 1A). (Fig. S7), which is sensitive enough to detect Aβ species in
The emission peak was blue shifted from 725 to 661 nm, while physiological concentration.
Φ
F
value increased to 1.5%. This indicated that DCM-AN showed
To study the binding mechanism of DCM-AN with Aβ
high binding activity with Aβ protofibrils. By contrast, the protofibrils, quantum mechanical calculations were performed
fluorescence intensity of DCM-AN showed little change in the for DCM-AN followed by a molecular docking search and
presence of Aβ aggregates or monomers (Fig. 1A). Furthermore, molecular dynamics simulations for the complex of DCM-AN
even though the concentration of Aβ peptides in aggregates was and Aβ protofibrils. Firstly, the optimized structure of DCM-AN
four times as that in protofibrils, the fluorescence intensity of (Fig. 2A) was obtained in the gas phase based on quantum
DCM-AN was still very weak for Aβ aggregates (Fig. S3). While mechanical calculations at the B3LYP/6-31G level using Gaussian
the fluorescence intensity of DCM-AN gradually increased to 09 software package. Secondly, molecular docking search
some extent in the presence of Aβ oligomer since the structure between DCM-AN and Aβ protofibrils model was carried out.
of oligomers is more closely related to protofibrils,36 but the Generally, the aggregation degree of protofibrils is more serious
intensity was lower than that of Aβ protofibrils. These suggest than that of oligomers. Therefore, we adopted two different Aβ
that DCM-AN exhibits good specificity towards Aβ protofibrils. aggregation intermediates, trimer and dodecamer, as working
It could be seen from Fig. 1B that the fluorescence intensity of models for Aβ oligomer and protofibrils, respectively (Fig. S8, A
DCN-AN did not show remarkable enhancement with another and B). In addition, two different types of Aβ aggregation were
aggregated peptide amylin (an aggregation-prone 37-residue chosen as Aβ aggregates working models (Fig. S8 C: PDB ID:
peptide secreted together with insulin by pancreatic B cells). 5KK3 and D: PDB ID: 2LMP), which are structurally more
Furthermore, little fluorescence changes were observed when complicated than Aβ protofibrils. Through molecular docking
human serum albumin (HSA) or tau protein was added (Fig. S4). search, the binding energy of DCM-AN with dodecamer (-12.91
kcal/mol) is lower than that with trimer (-9.15 kcal/mol) and
aggregates (Fig. 2C: -10.65 kcal/mol, Fig. 2D: -10.78 kcal/mol).
This suggests that DCM-AN binds more tightly with dodecamer
than with trimer or aggregates. Subsequently, molecular
dynamics (MD) simulations were carried out between DCM-AN
and these Aβ working models (Fig. S8 and S9). As expected, the
result of molecular dynamics research exhibits high conformity
with the molecular docking.
In addition, the solvent accessible surface area (SASA) was
calculated before and after binding between DCM-AN and Aβ
protofibrils to further illustrate the binding specificity. SASA is
the surface area of a biomolecules that is accessible to a
37
solvent. When the probe DCM-AN combined with Aβ, it would
result in the shrink of SASA to some extent. The more tightly
DCM-AN binds with Aβ, the more shrinking of SASA is. It is
Fig. 1. Fluorescence spectra of DCM-AN (2.0 μM) with (A) Aβ species (5.0 μM), (B) obvious that all of the SASA shrink after DCM-AN binds with four
another peptide Amylin (5.0 μM), (C) different Aβ protofibril concentrations (0-9 μM) and
Aβ working models (Fig. S6, Table S1), indicating that DCM-AN
tends to bind with protein owing to the solvent impact.
(
D) different incubation time of Aβ protofibrils (5.0 μM), ), λex=500 nm.
2
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However, it is noted that the SASA with dodecamer (
∆
∆
SASA = transgenic mice (Fig. 4, A-C and B-F), indicating that DCM-AN
SASA = could efficiently label Aβ pathology at the early stage. With the
2
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2
1
1
1
41.491 Å ) shrinks much more than that with trimer (
2
30.584 Å , Table S1) or aggregates (
∆
SASA = 6.068 Å and age increasing (9-month old), the size of Aβ was getting larger
2
6.707 Å for 5KK3 and 2LMP, respectively, Table S1), indicating compared with 4 and 5-month old mice (Fig. 4H, B and E).
that DCM-AN binds stronger with Aβ protofibrils than with small However, DCM-AN still stained early Aβ species (Fig. 5G, left
oligomer and Aβ aggregates. It is clear that after binding with Aβ arrows), while a few plaques were not labeled by DCM-AN (Fig.
protofibrils, the rotation of both ethylene and piperidine groups 4G-I, arrowheads). These suggest that DCM-AN detects the
are restrained, resulting in more effective ICT process and early Aβ pathology more efficiently at 4- and 5-month old and
enhancement of the fluorescence.
the favorable efficiency in detecting lasts until 9-month old,
even though a little decrease is observed at this age.
A
B
C
D
A
E
B
F
C
G
D
H
Fig. 2. Calculated binding model of DCM-AN with Aβ protofibrils. (A) Optimized structure
of DCM-AN at B3LYP/6-31G* level; Molecular docking model of DCM-AN with Aβ
protofibrils (B) and Aβ aggregates (C and D).
Cytotoxicity is an important indicator for a probe applied in
bio-research. In order to evaluate the cytotoxicity of DCM-AN
,
MTT assay was performed. The cellular viabilities were
estimated to be greater than 90% after 36 hours in the presence
of 1-100 μM DCM-AN (Fig. S10), indicating that DCM-AN
showed low cytotoxicity and could further use as a marker of Aβ
protofibrils.
I
J
K
L
Subsequently, we investigate whether this probe DCM-AN
can specifically target Aβ protofibrils from the brain sections of
AD transgenic mouse models. We chose four-month age 5XFAD
Fig. 3. Colocalization of DCM-AN labeling (A, E, I) with immunostaining of Aβ oligomers
(
B, F) and Aβ fibrils (OC antibody, J) in the brain sections of APP/PS1 transgenic mice. (A)-
(
D) and (I)-(L) cerebral cortex; (E)-(H) hippocampus. C, G and K are the merge images of
transgenic mice as AD pathology animal models, because mice DCM-AN and Aβ antibody. DAPI is added in D, H and L. Scale bar: 100 μm.
have few Aβ mature fibrils (plaques) under four-month age and
Aβ mainly exists in form of soluble Aβ species3
1, 38-41
while Aβ
protofibrils is one of important soluble species.23 The brain
slices of transgenic mouse model stained with DCM-AN showed
obvious fluorescence signals, whereas the brain slices without
DCM-AN staining emitted little fluorescence signals (Fig. S11).
To confirm the fluorescence originating from the probe
DCM-AN combining Aβ protofibrils in the brain section from AD
transgenic mouse models under four-month age, colocalization
experiments were carried out with Aβ oligomer-specific
antibody since Aβ protofibrils are structurally more closely
36
correlated with Aβ oligomers than Aβ aggregates/fibrils, Aβ
fibrils specific antibody (OC) and tau antibody. The brain
sections were also stained with nucleus staining dye (DAPI) for
clarity about the region of experimental brain sections. As
expected, DCM-AN could co-localized well with Aβ oligomer-
specific antibody either in the cerebral cortex or hippocampus
(Fig. 3, A-H). However, DCM-AN showed poor co-localization
with Aβ fibrils OC antibody (Fig. 3, I-L) and tau antibody (Fig.
S12).
To further confirm the specificity of DCM-AN towards Aβ
protofibrils in the brain section, pAβ antibody was chosen to
stain Aβ species in the brain sections from AD transgenic mouse
models with different ages at early (4, 5-month old), middle (9-
month old) and late stage (15-month old) of Aβ pathology for
Fig. 4. Detection profile of DCM-AN towards Aβ species at different stages of Aβ
pathology in the brain sections of APP/PS1 transgenic mice, as compared with Aβ
immunostaining; (A-C) and (D-F) early stage of 4-month and 5-month old, respectively;
(
G-I) middle stage of 9-month old; and (J-L) late stages of 15-month old. A, D, G and J
were DCM-AN staining; B, E, H and K were Aβ immunostaining with a polyclonal Aβ
comparison. The images displayed high colocalization between antibody (pAβ). C, F, I and L were merged images. Left arrows displayed the early stage
of Aβ pathology stained by DCM-AN and pAβ; arrowheads displayed the late stage of Aβ
pathology which could be detected by pAβ but not by DCM-AN; scale bar: 200 μm.
DCM-AN and pAβ antibody for four- and five-month age
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1
2 L. Cai, R. B. Innis and V. W. Pike, Curr. Med. CheVmiew., A2rt0ic0le7O,n1lin4e
,
For 15-month transgenic mice, mature fibrils/plaques were
predominant in the brain sections and Aβ plaques became
relatively lager than that of younger transgenic mice. However,
small amount of early Aβ species (protofibrils/oligomers) could
still be labeled by DCM-AN by comparison of the images with
pAβ (Fig 4, J-L, marked with left arrows). It was easy to find that
mature plaques were not decetced by DCM-AN at the late stage
of Aβ pathology (Fig. 4, J-L, marked with arrowheads). The result
indicated that DCM-AN staining exhibited age-dependent and
1
9.
DOI: 10.1039/C9CC09233A
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protofibrils, we have performed a comparative staining study
with DCM-AN and another known red-emitting fluorescent
probe, namely QM-FN-SO3, which shows high affinity towards
2
2
2
2
2
2
42
Aβ fibrils. As the data shown (Fig. S13), QM-FN-SO3 presented
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In conclusion, we designed and synthesized a new
fluorescent probe DCM-AN capable of detecting Aβ protofibrils.
5 D. M. Walsh, D. M. Hartley, Y. Kusumoto, Y. Fezoui, M. M.
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considerably low limit of detection in solution. Moreover, DCM-
AN shows high affinity towards the early Aβ pathology and is the
most likely to bind to Aβ protofibrils/oligomers in brain sections
from AD mouse models. Further studies of this probe in the
application of in vivo imaging is ongoing in our laboratory.
The authors acknowledge the financial support from NNSFC
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