Imaging of formaldehyde fluxes in epileptic brains with a two-photon fluorescence probe

Article abstract of DOI:10.1039/d0cc00676a

A two-photon (TP) fluorescence probe has been developed for imaging endogenous FA fluxes during metabolic and epigenetic processes in animal models, especially in live brains.

Full text of DOI:10.1039/d0cc00676a

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Imaging of formaldehyde fluxes in epileptic brains with a two-
photon fluorescence probe
Jian Chen,a, †Chenwen Shao,a, †Xueao Wang,a Jing Gu,a Hai-Liang Zhu,*a Yong Qian*a, b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A two-photon (TP) fluorescence probe has been developed for spatiotemporal resolution, as well as their photochemical
imaging endogenous FA fluxes during metabolic and epigenetic properties.14 As such, we turned our attention to two-photon (TP)
processes in animal models, especially in live brains.
fluorescence imaging, which is appealing in that it exhibits deeper
tissue penetration, higher spatiotemporal resolution, less specimen
photo-damage, and less auto-fluorescence interference than one-
photon (OP) fluorescence imaging.15 However, so far, no TP
fluorescent probe has been reported for imaging endogenous FA
fluxes in live brains. Herein, we report the first two-photon
fluorescence probe of its kind capable of monitoring dynamic
changes of endogenous FA in the epileptic brains, thus offering a
versatile chemical tool for visualizing this reactive carbonyl species in
the pathological processes of kainite (KA)-induced epileptic seizure.
Our original design was inspired by quinoline-based brain imaging
agents and neuroprotective drugs (Scheme S1).16, 17 In the design of
our probes, the quinoline skeleton was selected as the basic
fluorophore due to its extraordinary optical characteristics, including
the excellent two-photon excitation properties;18 in the meantime,
the specific homoallylic amine was chosen as the recognition group
Formaldehyde (FA), as a reactive one-carbonyl species, is associated
with numerous biochemical processes, including the epigenetic
regulation and post-translational modification of proteins.1 In
organisms, FA can be endogenously produced through a series of
metabolic processes that catalyzed by demethylases and oxidases,
such as lysine-specific demethylase1 (LSD1) and semicarbazide-
3
sensitive amine oxidase (SSAO).2, After a dynamic process of
continuous release and regulation, the physiological steady-state
level of FA can reach 0.1 mM in the blood and 0.2-0.4 mM in the
normal brain, and the normal levels of intracellular FA are considered
to play important roles in human health.4, 5 However, abnormally
elevated FA levels can also cause a variety of disease pathologies.6-8
Typically, abnormally elevated FA inevitably affects memory,
learning, and behavior, and even worse, severe neurological
disorders may occur, as a result of the well-established neurotoxicity
of FA on the brain.9 Therefore, developing effective chemical tools
for monitoring endogenous FA changes in complex bio-systems, the
brain in particular, is conducive to early diagnosis and treatment of
related neurodegenerative diseases.
A noninvasive approach for monitoring the FA fluxes in vivo
would be ideal. Fluorescent imaging using small-molecule probes is
considered as one of the most powerful strategies because of their
excellent biocompatibility, high sensitivity, and selectivity.10 Indeed,
a variety of reactivity-based probes have been developed for the
specific detection of FA in aqueous solutions, in cells, and in
tissues.11, 12 Despite the great progress, tools for visualizing FA in live
brains have yet to be developed. To achieve this, we have to
overcome several challenges; the biggest challenge lies in the
abilities of these probes to effectively permeate the blood-brain
barrier (BBB) and to specifically detect FA in the brain.13 In addition,
we have to take into account the biochemical properties of these
small molecules, including cytotoxicity, tissue-penetration depth and
for FA in our probes following on the reactivity-based sensing
20
mechanism.19,
To further improve the brightness and
photostability of the fluorophore while preserving its spectral
properties and cell permeability, the electron-donating group was
replaced with differently sized nitrogen-containing rings ranging
from aziridine to azepane (Scheme 1).21 On the basis of our
hypothesis, these probes themselves should exhibit almost weak
fluorescence due to the intramolecular charge transfer (ICT) effect.
After de-caging of the specific homoallylamine group by reacting
with FA to produce an aldehyde group with the more powerful
electron-withdrawing capability, the push-pull electron further
heightens, thus yielding the significant fluorescence response.
N
N
N
NH2
NH2
NH2
N
N
N
FATP-1
FATP-2
FATP-3
O
N
N
a. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Xianlin Road 163, Nanjing 210023, China. Email:
yongqian@nju.edu.cn or zhuhl@nju.edu.cn
NH2
NH2
N
N
b. School of Chemistry and Materials Science, Nanjing Normal University, Wenyuan
Road 1, Nanjing 210046, China. Email: yongqian@njnu.edu.cn
FATP-4
FATP-5
Scheme 1. Chemical structure of two-photon FA fluorescence probes FATP-
† J. Chen and C.W. Shao contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any supplementary
1~5.
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Figure 2. (A) Two-photon fluorescence images of C. elegans. C. elegans were
pretreated with FA (0, 0.5, and 1 mM) at 20℃ for 2 h, then C. elegans were
washed and incubated with FATP-1 (20 μM) for another 30 min before
imaging. Green channel: collected at 540-600 nm, λex=750 nm, scale bar = 25
μm. (B) Quantification of the relative ratio of fluorescence intensity shown in
(A). (C) C. elegans were pretreated with DMSO or THF, then NaHSO3 or H2O at
20℃ for 2 h, then washed and incubated with FATP-1 (20 μM) for another 30
min before imaging. Green channel: collected at 540-600 nm, λex=750 nm,
scale bar = 25 μm. (D) The relative ratio of fluorescence intensity shown in (C)
was quantified. (E) Two-photon fluorescence images at different depths in C.
elegans. C. elegans were pretreated with FA (1 mM) at 20℃ for 2 h, then
washed and incubated with FATP-1 (20 μM) for another 30 min before
imaging. Green channel: collected at 540-600 nm, λex=750 nm, scale bar = 25
μm. (F) Perspective for 3D image of (E). The error bars were ±SD (n=3).
Statistical analyses performed with an Ordinary one-way ANOVA test to the
control with H2O or DMSO treatment, *P < 0.05, **P < 0.01.
Figure 1. (A) Two-photon action spectra of FATP-1 and the corresponding
reaction product with formaldehyde (FA) upon excitation from 710 nm to
850 nm. (B) Fluorescence spectra of FATP-1 (20 µM) after treatment with
FA (0–2 mM) in PBS buffer (10 mM, pH 7.4, containing 1% MeCN) for 120
min. (C) The selectivity of FATP-1 (20 µM) for various relevant analytes (2.0
mM) in PBS buffer (10 mM, pH7.4, containing 1% MeCN) at 37℃. All the
fluorescence data were recorded after 120 min. (D) The cell viability of SH-
SY5Y cells treated with different concentrations of FATP-1 (0.1-500 µM)
for 48 h. (E) Fluorescence enhancement of FATP-1 (10 µM, 30 min) after
adding different concentrations of FA (0-1000 µM, 4 h) in SH-SY5Y cells.
Data were acquired by flow cytometry analysis. Fluorescence excitation:
405 nm, fluorescence emission: 555-651 nm. (F) Fluorescence
quantification of SH-SY5Y cells that pre-incubated with or without NaHS
(100 µM, 12 h), Glu (2 mM, 12 h), KA (500 µM, 12 h), MeOH (2%, 2 h), THF
(10 mM, 2 h), NaHSO3 (200 µM, 30 min), Fe2+ (100 µM, 2h), KO2 (200 µM,
2 h), GSK-LSD1 (1 µM, 12 h), and TCP (20 µM, 12 h), then treated with
FATP-1 (10 µM, 30 min) in the fresh medium for another 30 min before
performing flow cytometry analysis. Fluorescence excitation: 405 nm,
fluorescence emission: 555-651 nm. The error bars were ±SD (n=3).
Statistical analyses performed with an Ordinary one-way ANOVA test to
the control with DMSO treatment, *P < 0.05, **P < 0.01, ***P < 0.001.
displayed a robust fluorescence response to FA. Upon treatment
with 2 mM FA, the fluorescence intensity enhanced dramatically in a
time-dependent manner over 60 min and can reach a plateau after ~
3 h (Figure S3). The change of fluorescence is ascribed to uncaging of
FATP-1, which was further confirmed by infrared spectroscopy (IR)
and high performance liquid chromatography (HPLC) experiments
(Figure S4-5 and Scheme S2). To test the response of FATP-1 under
physiological conditions, the probe was exposed in different pH
buffers from 3 to 12. A relatively wide range of pH applications was
observed (Figure S6). Moreover,
a concentration-dependent
fluorescent enhancement was observed from 0 to 2 mM FA
treatment (Figure 1B and S7), and the detection limit was calculated
to be about 0.3 μM. These findings suggest FATP-1 has the capability
for detecting the intracellular FA (0.1-0.4 mM). To investigate the
selectivity and anti-interference ability of FATP-1 towards FA, the
fluorescence changes of FATP-1 were also observed in the presence
of various other relevant species (Figure 1C and S8). We found that
the strongest response towards FA, while negligible emission
changes for other relevant aldehydes and bio-molecules. Besides, log
P of FATP-1 was 2.43 in an octanol/water system. These observations
demonstrate that FATP-1 can selectively response to FA over other
biological analytes under physiological conditions.
We next assessed the feasibility of using FATP-1 to detect FA in
living cells. The cytotoxicity of the probe was first established using
cell CCK-8 assays with human neuroblastoma SH-SY5Y cells,
demonstrating negligible cytotoxicity of FATP-1 to live cells (Figure
1D). SH-SY5Y cells were incubated with FATP-1 (10 μM) for 30 min,
We synthesized these probes through a minimal structural change
and compared their properties, and investigated their response
towards FA (Table S1 and Figure S1). The four-membered azetidine
ring-substituted probe (FATP-1) exhibited an increased ratio of
quantum yield value (>3 fold) compared to the corresponding
fluorophore, which is similar to that of FATP-4. FATP-1 exhibited the
most significant increase in fluorescence intensity (>6 fold) after
reactivating with FA, suggesting that the azetidine ring-substitution
can effectively mitigate the twisted internal charge transfer (TICT)
and improve fluorescence brightness.22 This significantly improved
brightness of the fluorophore resulting from FATP-1 was also
observed under two-photon excitation (Figure 1A and S2). The
maximum two-photon absorption cross-section (σ2) values of
78.44±2.89 GM at 750 nm (~3 fold of enhancement), suggesting the
suitability of FATP-1 for two-photon imaging. Importantly, this probe
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Figure 3. (A) Fluorescence images of three groups mice [wide-type (WT), combination KA with GSK-LSD1 and KA-induced epileptic mice] at 5, 15, 30, 45, and 60 min
after i.v. injection with FATP-1 (0.5 mg/kg). KA (6 mg/kg, 12 h) was intraperitoneal (i.p.) injected into mouse to cause epilepsy. At the same time, GSK-LSD1 (0.15
mg/kg, 12 h) was intraperitoneal (i.p.) injected into mouse to inhibit the production of HCHO. (B) Ex vivo fluorescence images of relative HCHO levels in mice brains
60 min post-injection of FATP-1. (C) The two-photon fluorescence images of 2.5D volume-rendered and different depths of maximum hippocampal surface in brain
tissues from (B). Green=FATP-1 channel. Fluorescence excitation filter MP 750 nm, emission filter 475-600 nm. Scale bar= 50 μm. (D) Fluorescent imaging and Nissl's
staining for evaluating neuronal damage in the whole hippocampal region (HIP), and CA1, CA1, CA3, dentate gyrus (DG) sub-regions after KA administration. Scale bar
=500 µm. (E) Quantification of images in (B). Note that fluorescence signals in KA-induced epilepsy mice were significantly higher than those in the healthy WT group.
Treatment with GSK-LSD1 would help to inhibit the overexpressed HCHO. (F) Quantification of images in (D). (G) Quantification of images of the CA1, CA3, and DG
sub-regions of hippocampus in (D). The relative ratio of fluorescence intensity of the brains shown in A and B was quantified using the IVIS Spectrum imaging system.
The data are expressed as the mean ± S.D (n=3). Statistical analyses performed with Ordinary one-way or two-way ANOVA test to the control with DMSO treatment,
*P < 0.05, **P < 0.01, ***P < 0.001.
then washed by PBS buffer three times to remove the excess probe. with exogenous FA (0, 0.5, and 1 mM) at 20℃ for 2 h, C. elegans were
Flow cytometry analysis was performed after incubation with washed and incubated with FATP-1 (20 μM) for another 30 min. Two-
exogenous addition of FA (0-1 mM) for another 4 h (Figure 1E). A photon fluorescence imaging showed
a
dose-dependent
dose-dependent increase of fluorescence signal was clearly enhancement for FA (Figure 2A and B). To directly detect
observed, suggesting excellent cell-membrane permeability and endogenous FA fluxes that generated through THF metabolism in
ability for FA detection in live cells. To further investigate the vivo, C. elegans were pretreated with either DMSO or THF, water or
suitability of FATP-1 for detecting the stimuli-triggered dynamic NaHSO3, and then incubated with FATP-1 (20 μM) for another 30 min
changes of endogenous FA fluxes in live cells, cells were pretreated (Figure 2C and D). A significant difference between THF and negative
with different exogenous substances, their fluorescence intensities control group was observed, which is consistent with the flow
of cells were quantitatively analyzed by a flow cytometer (Figure 1F). cytometry analytic results in live cells. However, we did not observe
Pretreatment with a high concentration of glutamate to stimulate any statistically obvious difference between the group that was
cells,23 a great fluorescence enhancement was observed. Similarly, incubated with both THF and the FA scavenger NaHSO3 and the
preloading cells with kainic acid (KA),24 a glutamate analog widely negative control group, further confirming the enhanced
used as an epilepsy-inducing reagent,25 an obvious increase in fluorescence of C. elegans was caused by endogenous FA resulting
fluorescence signal was clearly observed, suggesting that from THF metabolism in vivo. Additionally, to test the depth TP
intracellular FA levels increased under stimulation of external imaging capability of FATP-1, images of C. elegans were observed
oxidative stress. To measure the endogenous FA as a result of under two-photon excitation at 750 nm (Figure 2E and F). We can
metabolism, cells were pre-incubated with methanol (MeOH)26 or clearly observe the 3D images of the live C. elegans in the whole
tetrahydrofolate (THF),27 a significant increase in fluorescence depth, revealing the great potential of FATP-1 for deep tissue TP
intensity was observed; while co-treated with sodium bisulfite imaging.
(NaHSO3) as the FA scavenger,28 cells showed a decrease in overall
To further investigate of the feasibility of using FATP-1 to study the
fluorescent intensity. These findings reveal that FATP-1 can be used influence of external stimuli on FA generation in vivo, C. elegans were
as an efficient tool for determinate the endogenously generated FA pre-exposed with 10% MeOH for 2 h, TP images were observed after
from metabolic processes in live cells. Additionally, pretreatment incubation with FATP-1 for another 30 min (Figure S9A and B). An
with GSK-LSD1 or TCP (inhibitors of LSD1, lysine-specific histone increase in the TP fluorescence signal was observed, suggesting the
demethylase 1A),29 a decrease in fluorescence signal compared to metabolism of MeOH can induce the increased level of FA in vivo. In
control cells was observed, specifically GSK-LSD1 treated cells gives a addition, we also observed that the exogenous supplement of
more significant decrease than TCP treated cells, suggesting that the Fenton-reaction substrates including iron ions and hydrogen
pharmacological inhibition of LSD1 decreased intracellular FA levels, peroxide led to increased endogenous FA generation in vivo.
an outcome probably associated with certain epigenetic Compared with the control C. elegans, Glu/KA-treated C. elegans
modifications that can be revealed by using FATP-1.2
exhibited significantly enhanced TP fluorescence (Figure S9C and D).
To examine whether FATP-1 could function in vivo, we initially use Interestingly, pretreatment with H2S can decrease the fluorescence
FATP-1 to monitor FA in live C. elegans. After exposing C. elegans signal of C. elegans, suggesting that H2S might downregulate the
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generation of endogenous FA. Since excessive expression of cellular and the Open Project of State Key Laboratory of Pharmaceutical
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LSD1 has been implicated in the abnormally elevated endogenous Biotechnology (KF-GN-202002).
FA, we further assessed whether FATP-1 can be used for monitoring
the fluctuations of the endogenous FA caused by the epigenetic
regulation of LSD1 activity in vivo. Live C. elegans that pre-incubated
with LSD1 inhibitors exhibit weaker TP fluorescence signals (Figure
Conflicts of interest
There are no conflicts to declare.
S10), indicating that down-regulation of LSD1 activities can efficiently
regulate the generation of endogenous FA in vivo. Taken together,
these observations confirm that FATP-1 can effectively monitor
fluctuations of endogenous FA under various external stimuli in vivo.
Notes and references
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Finally, we assessed FATP-1 as a TP imaging probe in the live animal
brain, especially in the epileptic brain. Wild-type mice were
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fluorescence probe for imaging endogenous FA fluxes in live
cells, animals and brain tissues. The unique quinoline skeleton
in these probes led to excellent BBB permeability and two-
photon properties. Functionalization at the electron-donor
group with a four-membered azetidine group yields a new
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This work is financially supported by the National Natural
Science Foundation of China (21778033), the program of
Jiangsu Specially-Appointed Professor for Y.Q., the Science and
Technology Innovation Project of Nanjing (184080H201136),
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