A small molecule probe reveals declined mitochondrial thioredoxin reductase activity in a Parkinson's disease model

Article abstract of DOI:10.1039/c5cc09998f

The first off-on probe, Mito-TRFS, for imaging the mitochondrial thioredoxin reductase (TrxR2) in live cells was reported. In a cellular model of Parkinson's disease (PD), Mito-TRFS staining discloses a drastic decline of the TrxR2 activity, providing a mechanistic link of TrxR2 dysfunction to the etiology of PD.

Full text of DOI:10.1039/c5cc09998f

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A Small Molecule Probe Reveals Declined Mitochondrial
Thioredoxin Reductase Activity in a Parkinson’s Disease Model
Received 00th January 20xx,
Accepted 00th January 20xx
Yaping Liu, Huilong Ma, Liangwei Zhang, Yajing Cui, Xiaoting Liu, and Jianguo Fang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
3-17
The first off-on probe, Mito-TRFS, for imaging the mitochondrial types of live cells.
However, this probe is not suitable for
thioredoxin reductase (TrxR2) in live cells was reported. In a specific TrxR2 imaging as TRFS-green appears evenly distributed in
cellular model of Parkinson’s disease (PD), Mito-TRFS staining cytosol. Detection of TrxR2 activity in cells is tedious. Prior to the
discloses a drastic decline of the TrxR2 activity, providing a assay, cells have to be broken to isolate mitochondria. Then the
mechanistic link of TrxR2 dysfunction to the etiology of PD.
total mitochondrial proteins are extracted and the activity of TrxR2
in the extracts is determined by the classic thioredoxin-mediated
2, 18-21
Thioredoxin reductases (TrxRs) are members of the pyridine
nucleotide-disulfide oxidoreductase family and are ubiquitously
found in all living cells. Two major isoforms of TrxRs are present in
different intracellular organelles: TrxR1 is predominantly in the
cytosol and nucleus, while TrxR2 is mainly localized within
insulin reduction assay.
This time- and labor-consuming
determination of TrxR2 hampers the study of the physiological
functions of the mitochondrial enzyme. Herein, we reported the
synthesis of a mitochondria-targeted TrxR probe, Mito-TRFS, and
evaluation of the probe in vitro and in live cells. Our results
indicated that Mito-TRFS accumulated exclusively in mitochondria
and could image TrxR2 activity specifically in live cells. More
importantly, in the 6-hydroxydopamine (6-OHDA)-treated PC12
cells, a well-established cellular model of PD, the TrxR2 activity was
drastically declined revealed by Mito-TRFS staining, which provides
a mechanistic link of TrxR2 malfunction to PD.
1
-3
mitochondria.
A third form, thioredoxin and glutathione
4
reductase (TrxR3), is mainly expressed in testis. Despite the
different localizations of the isoforms within cells, mammalian TrxRs
have similar structures with a conserved selenocysteine (Sec)
residue at the C-terminus and share the same catalytic mechanism.
TrxRs play key roles in regulating diverse redox-mediated cellular
events, including gene transcription, DNA/protein damage
We incorporated the triphenylphosphonium moiety, a well-
3, 5
2
2-29
recognition and repair, cell proliferation, and apoptosis.
The
documented mitochondria-targeting motif,
into the parent
malfunction of TrxRs has been implicated in various diseases, such
as cardiovascular diseases, inflammation, neurodegenerative
molecule TRFS-green, and generated the selective TrxR2 probe
Mito-TRFS, whose synthetic routes were outlined in Scheme 1. The
fluorophore naphthalimide (6) was synthesized in six steps (steps a-
f) with the overall yield of 7.8 % from the readily available
6
, 7
diseases, and cancer.
Reactive oxygen species (ROS) are constantly produced from
numerous cellular processes. ROS at low levels may act as signaling
molecules to activate cellular proliferation and survival pathways.
However, ROS at high levels lead to oxidative stress and ultimately
2
3, 30, 31
acenaphthene by adapting the published procedures.
The
TrxR-recognizing part, 1, 2-dithiolan-4-ol (8), was synthesized in two
steps (steps g & h) with the overall yield of 52 % from the
commercially available 1, 3-dichloropropan-2-ol by following
8, 9
cause cell death.
Mitochondria are major sites for ROS
1
0
1
2
production, and may produce up to 90% of cellular ROS. Although
mitochondrial dysfunction is implicated in the etiology of various
neurodegenerative diseases including Parkinson’s disease (PD), the
underlying molecular mechanisms are not well defined. TrxR2,
together with its substrate thioredoxin 2 (Trx2), is a leading player
to direct or interact with other antioxidant molecules, such as
previously published procedures. For the synthesis of Mito-TRFS,
was reacted with diphosgene in the presence of N, N-
6
diisopropylethylamine (DIPEA) in dichlormethane (DCM), followed
by the addition of 2. After purification by silica gel column
chromatography, Mito-TRFS was obtained in 10 % yield. The
chemical structures of Mito-TRFS and the synthetic intermediates
5
, 11
1
13
peroxiredoxin 3, to control ROS levels in mitochondria.
Thus,
were characterized by H NMR, C NMR and MS. The original
spectra were included in the Supporting Information (Figures S3-
monitoring TrxR2 activity is of physiological significance to better
understand its role in cellular redox regulation. We previously
reported the first selective off-on TrxRs fluorescent probe, TRFS-
2
2).
1
2
green. TRFS-green can readily image the TrxRs activity in various
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Scheme 1 Synthesis of Mito-TRFS. Reagents and conditions: (a) Na
2
Cr
N/DCM, r. t., 55 %; (d) NaI/acetone, 80 %; (e) SnCl .2H
/I2/DCM, r. t., 89 %; (i) diphosgene/DIPEA/DCM, reflux, 10 %.
2
O7/AcOH, reflux, 76 %; (b) 2-(2-aminoethoxy)ethanol/THF, reflux,
o
5
5
0 %; (c) MsCl/Et
9 %; (h) NaHCO
3
2
2 3 2 2
O/HCl/EtOH, reflux, 58 %; (f) PPh /MeCN, 80 %; (g) Na S/CS , 60 C,
3
Initially, we examined the absorption and emission spectra of
Mito-TRFS in responding to the TrxR (Figure 1). Mito-TRFS (10 μM)
has the maximal absorption at ~375 nm in TE buffer (50 mM Tris-
HCl and 1 mM EDTA, pH 7.4) at 37 °C. After addition of the
recombinant rat TrxR1 (75 nM) and NADPH (200 μM), its maximum
absorbance is red-shifted to ~438 nm (Figure 1A). The strong
absorbance at ~340 nm in Figure 1A was from NADPH. Mito-TRFS
itself has a strong emission at ~480 nm when excited at 375 nm in
TE buffer (φ=0.34), but has weak emission when excited at 438 nm.
However, the emission centered at ∼540 nm increases remarkably
after addition of the TrxR1 and NADPH (Figure 1B, φ=0.19). The
increment of the fluorescence intensity reaches a plateau after 60
min. Mito-TRFS displayed faster response to TrxR compared to its
parent molecule TRFS-green: The fluorescence increment of Mito-
TRFS triggered by TrxR is about 30-fold within 60 min while that for
1
2
TRFS-green is about 25-fold within 3 h. We reasoned that this
could be due to that Mito-TRFS is more hydrophilic than TRFS-green.
Furthermore, Mito-TRFS bears a positive charge, which facilitates it
binding to the negative-charged C-terminal active site of TrxR via
the electrostatic interaction. These might contribute to the faster
response of Mito-TRFS than TRFS-green.
Fig. 1 Response of Mito-TRFS to TrxR. Absorption spectra (A) and
Fluorescence spectra (λex=438 nm, B) of Mito-TRFS with or without
TrxR and NADPH. The arrows in (A) & (B) show the change of
absorbance at 438 nm and emission at 540 nm, respectively. (C)
We next determined whether Mito-TRFS (10 μM)could be
selectively triggered on by TrxR. As shown in Figures 1C & 1D,
neither GSH nor Cys causes significant increment of the
Selective recognition of Mito-TRFS by TrxR. The spectra (λ =438
ex
nm) were acquired after the probe was incubated with GSH, Cys,
various enzymes (TrxR, U498C TrxR, and GR) with NADPH (200 μM),
AHL (10 μM) or Sec (10 μM) at 37 °C for 60 min. (D) The fold of fluorescence signal. The glutathione reductase (GR), whose
fluorescence increment (F/F ) from (C) was shown.
structure is closely related to TrxR, has little effect on the reduction
of Mito-TRFS. Furthermore, the U498C TrxR, where the Sec498 was
0
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replaced by Cys, also displays negligible interference of the pretreated with the specific TrxR inhibitor auranofin (AF) for 6 h,
emission signal, supporting the critical role of the Sec residue of the indicating a selective recognition of TrxR by Mito-TRFS. There is no
enzyme to turn on the fluorescence. Amidehydrolase (AHL), Sec, significant toxicity of Mito-TRFS (<2 ꢀM) to the cultured cells (Figure
and TrxR without NADPH also give marginal response to the probe. 2C). Taken together, our results demonstrate that Mito-TRFS is
The Sec (10 ꢀM) was generated in situ by mixing Cys (1 mM) and suitable for selective imaging of mitochondrial TrxR in live cells.
selenocytine (5 ꢀM), and the presence of the selenol group was
3
2
confirmed by our specific selenol probe Sel-green. Taken together,
Mito-TRFS maintains the high selectivity to TrxR. This is not
surprising as the recognition part of Mito-TRFS is the same as that
1
2
of specific TrxR probe TRFS-green.
Introduction of the
triphenylphosphonium moiety would facilitate the probe to restrict
in mitochondria.
Fig. 3 Decline of TrxR2 activity in the PD model. PC12 cells were
treated with 6-OHDA for 10 h (A) or 22 h (B). Mito-TRFS (1 μM) was
added and continued incubation for
2 h. The cells were
photographed under a Leica inverted fluorescence microscope. (C)
Relative fluorescence intensity in individual cells from (A) and (B)
were quantified by a flow cytometer. (D) Cytotoxicity of 6-OHDA to
PC 12 cells. Data were expressed as the mean ± SD from duplicates.
Fig. 2 Confocal fluorescence images of HeLa cells. The cells were
Mitochondrial ROS play important roles in physiological
incubated with vehicle (0.1
concentrations of AF (A2-E4) for 4 h followed by further treated
with Mito-TRFS (1 μM) for 2 h. Then, the cells were stained with the pathological degeneration of neurons associated with
% DMSO, A1-E1) or varying
3
3
signaling. However, uncontrolled ROS production contributes to
3
4, 35
MTDR (100 nM) for 15 min. Scale bars: 10 μm. (B) Scatter plot of neurodegenerative diseases such as PD.
colocalization events as shown in E1 panel. (C) Cytotoxicity of Mito-
As major sites for ROS
generation, mitochondria have evolved multiple antioxidant
pathways to help maintaining the balance between ROS production
and removal. TrxR2 is a mitochondria-located antioxidant enzyme
to serve as a relayer to transfer electrons from NADPH to its
substrate Trx2, which interacts with diverse downstream substrates
TRFS to PC12 and HeLa cells after 24 h treatment.
We then conducted a fluorescence colocalization assay to
ensure that the probe localizes exclusively in mitochondria (Figure
2
). Upon selective excitation at 458 nm, the cells exhibited bright
1
1
green fluorescence in discrete subcellular distribution as
determined by laser confocal microscopy (B1). The image from
MTDR-staining was obtained upon selective excitation at 633 nm
and emission collection from 650 to 740 nm (C1). The merge of B1
and C1 gives a clear colocalization signal as the yellow color (D1)
with a colocalization coefficient of 0.90 (Figure 2B), confirming that
Mito-TRFS is localized predominantly in the mitochondria of live
cells. The highlighted part in the merged picture was enlarged in E1.
The pictures in the panels B2, B3, & B4 show dose-dependent
inhibition of the green fluorescence signal after the cells were
to regulate the redox homeostasis of mitochondria. Although the
dysfunction of TrxR2 has been suggested to link to the PD, the
direct evidence is limited presumably due to the inconvenient assay
of TrxR2. There is no direct probe to detect the TrxR2 activity in live
cells prior to this study. We presented evidence here that TrxR2
activity is severely impaired in a cellular model of PD. 6-OHDA is
36-38
widely used in generating cell or animal models of PD.
We
treated the neuron-like PC12 cells with the neurotoxin 6-OHDA to
generate the PD model, and then determined the TrxR2 activity by
Mito-TRFS staining. As shown in Figures 3A & 3B, 6-OHDA
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In conclusion, Mito-TRFS, the first turn-on probe for
mitochondrial TrxR, was prepared and evaluated. The probe
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Furthermore, with the aid of Mito-TRFS, we disclosed that the
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2
2
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The financial supports from Natural Science Foundation of
China (21572093) and Natural Science Foundation of Gansu 31. V. Percec, E. Aqad, M. Peterca, M. R. Imam, M. Glodde, T. K.
Bera, Y. Miura, V. S. Balagurusamy, P. C. Ewbank, F.
Province (145RJZA225) are acknowledged. The authors also express
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appreciation to Prof. Arne Holmgren (Karolinska Institute, Sweden)
3
3
for the recombinant rat TrxR1. There are no conflicts of interest.
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