Illuminating the Function of the Hydroxyl Radical in the Brains of Mice with Depression Phenotypes by Two-Photon Fluorescence Imaging

Article abstract of DOI:10.1002/anie.201901318

Depression is intimately linked with oxidative stress. As one of the most reactive and oxidative reactive oxygen species that is overproduced during oxidative stress, the hydroxyl radical (^.OH) can cause macromolecular damage and subsequent neurological diseases. However, due to the high reactivity and low concentration of ^.OH, precise exploration of ^.OH in brains remains a challenge. The two-photon fluorescence probe MD-B was developed for in situ ^.OH imaging in living systems. This probe achieves exceptional selectivity towards ^.OH through the one-electron oxidation of 3-methyl-pyrazolone as a new specific recognition site. MD-B can be used to map ^.OH in mouse brain, thereby revealing that increased ^.OH is positively correlated with the severity of depression phenotypes. Furthermore, ^.OH has been shown to inactivate deacetylase SIRT1, thereby leading to the occurrence and development of depression phenotypes. This work provides a new strategy for the future treatment of depression.

Full text of DOI:10.1002/anie.201901318

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Accepted Article
Title: Spying on the Function of Hydroxyl Radical in Brain of Mice with
Depression phenotypes by Two-photon Fluorescence Imaging
Authors: Xin Wang, Ping Li, Qi Ding, Chuanchen Wu, Wen Zhang, and
Bo Tang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901318
Angew. Chem. 10.1002/ange.201901318
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Spying on the Function of Hydroxyl Radical in Brain of Mice with
Depression phenotypes by Two-photon Fluorescence Imaging
Xin Wang, Ping Li,* Qi Ding, Chuanchen Wu, Wen Zhang, and Bo Tang*[a]
Abstract: Depression is intimately linked with oxidative stress. As one
of the most reactive and oxidative reactive oxygen species over-
produced during oxidative stress, hydroxyl radical (•OH) can cause
macromolecular damage and subsequent neurological diseases.
However, due to the high reactivity and low concentration of •OH,
precise exploration of •OH in brains remains a challenge. Thus, the
two-photon fluorescence probe TCE was developed for in situ •OH
imaging in living systems. This probe achieves exceptional selectivity
towards •OH via the one-electron oxidation of 3-methyl-pyrazolone as
a new specific recognition site. TCE can map •OH in mouse brain,
thereby revealing that the increased •OH is positively correlated with
the severity of depression phenotypes. Furthermore, •OH has been
proved to inactivate deacetylase SIRT1, leading to the occurrence
and development of depression phenotypes. This work provides a
new strategy for the future treatment of depression.
of stability, easy crossing of the blood-brain barrier (BBB) and
easy excretion, which is preferable for TP in situ imaging of •OH
in living brain of mice with depression-like behaviours. However,
suitable TP fluorescent probes for brain imaging of •OH with
specificity, high sensitivity and instantaneous response are still
scarce.
Scheme 1. The structure and luminescence mechanism of TCE.
Depression as one of the most common and disabling mental
disorders, with a worldwide prevalence of approximately 17%.1
However, the understanding of the pathophysiology of depression
Inspired by the specific one-electron oxidation reaction
between •OH and 3-methyl-pyrazolone in the neuro-protective
medicine edaravone,24-27 we created a TP fluorescent probe
based on intramolecular charge transfer (ICT), 5-methyl-2-(2-oxo-
-(trifluoromethyl)-2H-chromen-7-yl)-2,4-dihydro-3H-pyrazol-3-
2
is still rudimentary due to its complex aetiology. Previous findings
suggest that oxidative stress contributes to the pathogenesis of
3
-5
depression. Hydroxyl radical (•OH) is one of the most reactive
4
and oxidative reactive oxygen species (ROS) over-produced
one (TCE). TCE can instantaneously response to •OH with
outstanding selectivity (Scheme 1). Given its large Stokes shift
and extraordinary TP optical properties, coumarin (Cou151)
6
during oxidative stress. Excess •OH leads to irreparable damage
to neural cells and potentially even leading to neurological
7
disease. Therefore, it is urgent to develop an effective means for
28
bearing a trifluoromethyl group was selected as the fluorophore.
tracing •OH in living brain to define the relationship between
depression and •OH levels.
The trifluoromethyl group acts as a strong electron-withdrawing
group to boost the push-pull electron effect of the coumarin
Recently, fluorescence imaging has become a robust approach
for real-time monitoring of molecular events in living cells and in
vivo because of its nondestructive and spatio-temporal
resolution.8-10 Some fluorescent probes have been developed to
reveal the biological functions of •OH in living cells, in zebrafish
and in the abdomens of mice.11-19 Given the very high reactivity
and low concentration of •OH and the particularly complicated
construction of the brain, the two-photon (TP) fluorescence
imaging is appropriate for brain imaging, because it provides a
higher signal-to-background ratio, deeper tissue imaging, higher
spatial-temporal resolution and less specimen photodamage than
one-photon (OP) fluorescence imaging.20-23 Remarkably,
molecular fluorescent probes have some appealing performance
conjugated system, and also prompts this small-molecule
compound to traverse the BBB due to its lipophilicity.29-31 In TCE,
the carbonyl group attaches directly to the nitrogen atom and
weakens the push-pull electron effect of coumarin, lessening its
fluorescence. In the presence of •OH, TCE-OH (2-oxo-3-(2-(2-
oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-hydrazono)-butanoic
acid) forms promptly via one-electron oxidation (Figure S1). In
TCE-OH, the carbonyl group is far from the coumarin ring, and
the push-pull electron effect of the coumarin ring is restored,
resulting in enhanced fluorescence. Based on this idea, we
synthesized TCE and applied it in the brains of mice with
depression-like behaviours to map •OH by TP microscopy.
Furthermore, we revealed the connection of •OH and SIRT1 in the
pathology of depression.
The synthesis and characterization of TCE is shown in the
Supporting Information (Figure S2). Figure S3 depicts the
absorption spectra of TCE with an absorption band at
approximately 350 nm. After the addition of •OH, this absorption
increased and shifted to 380 nm. We then examined the OP and
TP fluorescence properties of TCE under simulated physiological
conditions. Figure 1A displayed dim fluorescence at 500 nm upon
excitation at 370 nm (OP) or 800 nm (TP). Conversely, TCE
fluoresced intensely in the presence of •OH. TCE’s fluorescence
[
a]
Xin Wang, Prof. Ping Li, Qi Ding, Chuanchen Wu, Dr. Wen Zhang,
Prof. Bo Tang
College of Chemistry, Chemical Engineering and Materials Science,
Key Laboratory of Molecular and Nano Probes, Ministry of
Education, Collaborative Innovation Center of Functionalized Probes
for Chemical Imaging in Universities of Shandong, Institutes of
Biomedical Sciences, Shandong Normal University, Jinan 250014,
People’s Republic of China.
E-mail: tangb@sdnu.edu.cn, lip@sdnu.edu.cn.
Supporting information for this article is given via a link at the end of
the document.
quantum yield Ф
f
increased from 0.037 to 0.25 and the TP
absorption cross-section σ from 5.0 GM to 42.6 GM. These
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changes in the photophysical properties confirm that the carbonyl
group can tune the push-pull electronic effects via the reaction
between 3-methyl-pyrazolone and •OH, conducing to the
enhancement of fluorescence. These findings are consistent with
our design and computation results about charges and densities
distribution (Table S1-S2).
believe that TCE is suitable for highly sensitive real-time tracking
of •OH in complex biological samples.
Figure 2. TP fluorescence images and SIRT1 concentration of human
astrocytes. (A) Human astrocytes without stimulation as control. (B) Human
astrocytes pre-incubation with glutamate (10.0 mM). (C) Human astrocytes co-
incubated with mannitol (10.0 mM) and glutamate (10.0 mM). (D) Human
astrocytes pre-incubation with resveratrol (25.0 μM). (E) Human astrocytes pre-
treated with Ex527(20.0 μM). (F) Human astrocytes co-incubated with
glutamate (10.0 mM) and resveratrol (25.0 μM). (G) Relative fluorescence
intensities of a-f. (H) The concentrations of SIRT1 corresponding to a-f.
Fluorescence emission windows: 400-650 nm. Scale bar = 20 μm.
To explore whether TCE could work well in living systems, we
initially utilized TCE to visualize •OH in living neural cells.
Previous studies suggest that high concentrations of glutamate
can cause oxidative stress.33-34 Therefore, human astrocytes
were pre-treated with glutamate to initiate oxidative stress. TP
images of those cells (Figure 2B) showed much brighter
fluorescence than the cells without treatment (Figure 2A). To
confirm the increase in fluorescence upon the capture of •OH by
TCE, we used the •OH scavenger mannitol in cells stimulated with
Figure 1. Photophysical properties and selectivity of TCE. (A) OP excitation and
emission spectra of TCE (20.0 μM) before and after the addition of •OH (20.0
μM). Inset: TP emission spectra of TCE (20.0 μM) before and after the addition
of •OH (100.0 μM). (B) Changes in the fluorescence emission spectra of TCE
(20 μM) after the addition of various concentrations of •OH (2.5 μΜ-200 μM).
(
C) Linear relationship of fluorescence intensity to •OH (0-20.0 μM). (D)
•−
Fluorescence responses of TCE to different competing species: 1 O
μM), 2 H
6
2
(100.0
(100.0 μM),
-
1
2
O
2
(1.0 mM), 3 TBHP (100.0 μM), 4 OCl (100.0 μM), 5 O
2
-
NO (100.0 μM), 7 ONOO (100.0 μM), 8 AIBN (100.0 μM), 9 AMVN (100.0
•−
μM), 10 CO
3
(100.0 μM), 11 peroxidase compound I (100.0 μM), 12 black and
•OH (50.0 μM). All of the spectra were acquired in 40 mM PBS buffer (pH 7.4)
35
glutamate. Weak fluorescence was observed in the cells treated
at λex = 370 nm (OP) and 800 nm (TP), λem = 500 nm. Similar results were
with mannitol (Figure 2C). The results show that •OH is generated
in excess after stimulation with glutamate. Combined with the
experiment results in PC12 cells (Figure S8) and mouse
macrophages (RAW 264.7, Figure S9), these evidences prove
that TCE is capable to selectively and sensitively identify
intracellular •OH fluctuations.
obtained in five independent experiments.
Next, we tested the •OH concentration dependence of the
fluorescence intensity of TCE. Figure 1B & 1C delineated that the
fluorescence of the probe recovered stepwise with increasing
amounts of •OH (2.5 μM - 200 μM), and the linear regression
equation was F = 340.55 [•OH] (μM) +2101.669 with a correlation
coefficient of 0.9960 in the range of 0-20.0 μM. The detection limit
was calculated to be 2.4 nM. These results indicate that TCE
could serve as a sensitive tool for assessing the concentration of
After confirmed satisficing imaging performance of TCE in the
mouse abdomen (Figure S10) and good in vivo biocompatibility
(
Figure S11- S13), we employed TCE to study •OH in the brains
of mice under different stresses. First, we tested the fluorescence
in the brain of the mice under restraint stress, and found a rise in
•OH (Figure S14). This result validates the ability of the probe to
•OH under physiological conditions.
readily traverse the BBB relying on the trifluoromethyl group.
Importantly, these data reveal that the •OH levels go up somewhat
in the brains of mice under restraint stress.
To confirm the specificity of TCE for •OH, we investigated its
response to other competing ROS, reactive nitrogen species
RNS) and metal ions. As shown in Figure 1D and Figure S4, the
(
Next, we planned to examine how •OH would fluctuate in the
brain of mice with depression-like behaviour. At first, we exposed
mice to 28 consecutive days of chronic unpredictable mild stress
fluorescence of TCE remained nearly unperturbed when other
biologically relevant substances were added. Besides, varying pH
had little effect on the fluorescence of TCE and TCE-OH (Figure
S5). TCE displayed instant fluorescence response to •OH, and
TCE and TCE-OH exhibited excellent photostability (Figure S6).
(
CUMS), then separated them into susceptible and resilient
36
populations based on the behaviors tests. The brains of
susceptible mice (Figure 3) exhibited distinct fluorescence
enhancement (approximately 6-fold) compared to that in the
control mice, and also stronger fluorescence than in the brains of
the stimulated with mild stressors. These observations firstly
suggest that the elevation of •OH in the brain increases with the
severity of depression-like behaviour. Reversely, we found lower
Moreover,
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay suggests good
biocompatibility of TCE at concentrations below 0.61 mM (IC50
value, Figure S7).32 Altogether, benefiting from the extremely
selective and instantaneous reaction between TCE and •OH, we
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•
OH level in brains of the resilient mice compared to the
This result demonstrates that excess •OH causes oxidative
damage to the active sites of SIRT1. Overall, these results provide
compelling evidence that •OH overproduction reduces the activity
of SIRT1, leading to depression-like behaviours.
susceptible ones (Figure S15), demonstrating less oxidative
stress. To further certify that the increase in •OH is linked to
depression-like behaviours, we assessed depression-like
behaviours and the •OH level in the brains of susceptible mice
injected with mannitol or desferrioxamine (iron scavenger) to
bring down •OH,37 respectively. Reductions in both fluorescence
(
Figure 3) and depression phenotypes (Figure S16) were
observed, which confirmed •OH regulating depression-like
behaviors. These results provide unambiguous evidence that •OH
plays a vital role in the pathology of depression.
Figure 4. Proteomic analysis through LC-MS/MS of the reaction of SIRT1 with
•
(
OH. Mass spectrometric analysis: (A) Oxidation of F297. (B) Oxidation of F388.
C) Oxidation of F414. Left columns: treated with •OH, right columns: untreated
protein.
Figure 3. In situ TP imaging of mice. Control: the mice without CUMS. CUMS:
the mice susceptive to CUMS. Desferal: The susceptible mice injected
desferrioxamine Mannitol: The susceptible mice injected with mannitol. The
Fluorescence images were obtained with an 800 nm light source. The 3D
images (Second row) were synthesized from a stack of cross sections (xy
sections, 400 μm) with an axial (z) increment of 2 μm. Fluorescence emission
windows: 400-650 nm. Scale bar = 50 μm.
In summary, we describe a novel two-photon fluorescent probe
for the imaging of •OH. The new specific recognition site endows
it with exclusive selectivity towards •OH. Moreover, this probe
features an instantaneous and highly sensitive response, high
photostability, and low cytotoxicity. These admirable traits
enabled in situ visualization of the burst of •OH in human
astrocytes. Importantly, by TP fluorescence imaging in the mouse
brain, we unravelled the positive correlation between •OH levels
and depression-like behaviours. We further uncovered that SIRT1
inactivation by •OH is responsible for depression phenotype. This
work can help researchers to fully understand the molecular
mechanism of depression and offer crucial information for
antidepressant treatments.
Previous studies have shown that the deacetylase SIRT1 plays
a pivotal role in depression.38-39 Thus, we speculated whether
there is any connection between •OH and SIRT1. To examine this
assumption, we treated human astrocytes with SIRT1 activator
39
resveratrol, and observed the change in the level of •OH. The
TP images of the cells stimulated by glutamate exhibited strong
fluorescence (Figure 2B). On
the contrary,
the
fluorescence inside the cells incubated with resveratrol was
significantly weaker, meaning low •OH level (Figure 2D and 2F).
This result illustrates that SIRT1 can be activated by resveratrol
to eliminate •OH. Subsequently, the results of SIRT1 assay kit
showed reduced activity SIRT1 in cells treated with glutamate, Acknowledgements
meanwhile increased in cells with resveratrol. Furthermore, we
observed bright fluorescence (Figure 2E) and a reduction in
SIRT1 in cells with SIRT1 inhibitor Ex527.40 We found this
interesting phenomenon that the excess of •OH is accompanied
by a decrease in the activity of SIRT1. Further results in human
astrocytes with mannitol verified that the activity of SIRT1
increased dramatically in these cells (Figure 2H), indicating
recovered activity of SIRT1 by scavenging the •OH over-produced.
Importantly, from the experiments in mice (Figure S17), we also
confirmed that excessive •OH could reduce SIRT1 activity, and
thereby cause depression-like behaviours.
This work was supported by the National Natural Science
Foundation of China (21535004, 91753111, 21675105, 21475079,
21390411) and the Key Research and Development Program of
Shandong Province (2018YFJH0502), National Major Scientific
and Technological Special Project for “Significant New Drugs
Development” (2017ZX09301030004) and the Natural Science
Foundation of Shandong Province of China (ZR2017ZC0225).
Keywords: Two-photon fluorescence imaging • hydroxyl radical
•
depression • oxidative stress • brain
Next, we used LC-MS/MS, proteomic analysis to identify the
reaction of SIRT1 with •OH, as described in the references.41-43
Interestingly, three phenylalanine residues (F297, F388, F414) in
the active sites of SIRT1 were found to be oxidized (Figure 4).
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COMMUNICATION
We describe a novel two-photon
fluorescent probe for imaging of
Xin Wang, Ping Li,* Qi Ding,
Chuanchen Wu, Wen Zhang, and Bo
Tang *
•OH in living cells and in vivo. The
probe successfully traced •OH in
the brain of mice with depression
phenotypes for the first time.
Page No. 1– Page No.4
Increased Hydroxyl Radical in
Brain Contributes to Development
of
Depression
Phenotypes
Following Stress
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