Neuroprotective Effect of a New 7,8-Dihydroxycoumarin-Based Fe2+/Cu2+ Chelator in Cell and Animal Models of Parkinson’s Disease

Article abstract of DOI:10.1021/acschemneuro.6b00309

Disturbed iron homeostasis, often coupled to mitochondrial dysfunction, plays an important role in the progression of common neurodegenerative diseases such as Parkinson’s disease (PD). Recent studies have underlined the relevance of iron chelation therapy for the treatment of these diseases. Here we describe the synthesis, chemical, and biological characterization of the multifunctional chelator 7,8-dihydroxy-4-((methylamino)methyl)-2H-chromen-2-one (DHC12). Metal selectivity of DHC12 was Cu²⁺ ～ Fe²⁺ > Zn²⁺ > Fe³⁺. No binding capacity was detected for Hg²⁺, Co²⁺, Ca²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Pb²⁺, or Cd²⁺. DHC12 accessed cells colocalizing with Mitotracker Orange, an indication of mitochondrial targeting. In addition, DHC12 chelated mitochondrial and cytoplasmic labile iron. Upon mitochondrial complex I inhibition, DHC12 protected plasma membrane and mitochondria against lipid peroxidation, as detected by the reduced formation of 4-hydroxynonenal adducts and oxidation of C11-BODIPY^581/591. DHC12 also blocked the decrease in mitochondrial membrane potential, detected by tetramethylrhodamine distribution. DHC12 inhibited MAO-A and MAO-B activity. Oral administration of DHC12 to mice (0.25 mg/kg body weight) protected substantia nigra pars compacta (SNpc) neurons against MPTP-induced death. Taken together, our results support the concept that DHC12 is a mitochondrial-targeted neuroprotective iron-copper chelator and MAO-B inhibitor with potent antioxidant and mitochondria protective activities. Oral administration of low doses of DHC12 is a promising therapeutic strategy for the treatment of diseases with a mitochondrial iron accumulation component, such as PD.

Full text of DOI:10.1021/acschemneuro.6b00309

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Article
Neuroprotective effect of a new 7,8-dihydroxycoumarin-based Fe2+/
Cu2+ chelator in cell and animal models of Parkinson’s disease
Pabla Aguirre, Olimpo García-Beltrán, Victoria Tapia, Yorka
Muñoz, Bruce Kennedy Cassels, and Marco T. Nunez
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00309 • Publication Date (Web): 02 Nov 2016
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Neuroprotective effect of a new 7,8-dihydroxycoumarin-based Fe /Cu
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chelator in cell and animal models of Parkinson disease
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Pabla Aguirre , Olimpo García-Beltrán , Victoria Tapia , Yorka Muñoz , Bruce K. Cassels and Marco
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*
T. Núñez .
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Biology Department, Faculty of Sciences, Universidad de Chile.
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Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Ibagué 730001, Colombia.
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Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile.
*
Corresponding Author: Iron and Biology of Aging Laboratory, Faculty of Sciences, Universidad de
Chile; Las Palmeras 3425, Santiago 7800024, Chile. Phone: +532 29787360; mail: mnunez@uchile.cl.
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Abstract
Disturbed iron homeostasis, often coupled to mitochondrial dysfunction, plays an important role in
the progression of common neurodegenerative diseases such as Parkinson disease (PD). Recent
studies have underlined the relevance of iron chelation therapy for the treatment of these diseases.
Here we describe the synthesis, chemical and biological characterization of the multifunctional
chelator 7,8-dihydroxy-4-((methylamino)methyl)-2H-chromen-2-one (DHC12). Metal selectivity of
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DHC12 was Cu
̴ Fe > Zn > Fe . No binding capacity was detected for Hg , Co , Ca , Mn , Mg ,
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Ni , Pb or Cd . DHC12 accessed cells colocalizing with Mitotracker Orange, an indication of
mitochondrial targeting. In addition, DHC12 chelated mitochondrial and cytoplasmic labile iron.
Upon mitochondrial complex I inhibition, DHC12 protected plasma membrane and mitochondria
against lipid peroxidation, as detected by the reduced formation of 4-hydroxynonenal adducts and
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oxidation of C11-BODIPY
. DHC12 also blocked the decrease in mitochondrial membrane
potential, detected by tetramethylrhodamine distribution. DHC12 inhibited MAO-A and MAO-B
activity. Oral administration of DHC12 to mice (0.25 mg/kg body weight) protected substantia nigra
pars compacta (SNpc) neurons against MPTP-induced death. Taken together, our results support
the concept that DHC12 is a mitochondrial-targeted neuroprotective iron-copper chelator and
MAO-B inhibitor with potent anti-oxidant and mitochondria protective activities. Oral
administration of low doses of DHC12 is a promising therapeutic strategy for the treatment of
diseases with a mitochondrial iron accumulation component, such as PD.
Keywords: Neurodegeneration with brain iron accumulation; coumarin-based iron-copper chelator;
antioxidant; Parkinson Disease; MPTP mouse model; neuroprotection.
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Introduction
Cell iron overload is often found in neurodegenerative disorders such as Parkinson’s Disease (PD),
Huntington's disease and a group of less characterized disorders known as Neurodegeneration with
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Brain Iron Accumulation or NBIA . In particular, magnetic resonance imaging and ultrasound
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studies demonstrated iron accumulation in the SNpc of PD patients , specifically in dying
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dopaminergic neurons and associated microglia .
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Given the deleterious effects of elevated redox-active iron in neurons , iron chelation has been
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proposed recently as a new promising alternative for PD treatment . In a clinical 12-month trial,
early-stage Parkinson's patients treated with 30 mg/kg body weight of the chelator deferiprone
showed decreased iron deposits in substantia nigra, and improved Unified Parkinson’s Disease
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Rating Scale motor indicators of disease progression compared to placebo-treated patients . The
authors concluded that these results warrant a comprehensive evaluation of chelation therapy in
PD. A caveat to iron chelation therapy is that putative iron depletion may affect normal neurological
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function . Hence, iron chelation therapy must be carefully controlled to prevent toxicity and side
effects. New chelators with enhanced cell selectivity and metal selectivity are required to reduce
secondary effects in a putative iron chelation therapy.
Coumarins are compounds containing fused benzene and α-pyrone rings. To date, hundreds of
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natural coumarins have been identified, not only in green plants but also in fungi and bacteria
.
Coumarins display an outstanding range of biochemical and pharmacological actions that include
anticoagulant, anticancer, antiadipogenic, antioxidant, anti-inflammatory, antifungal, antiparasitic,
17, 18
antiviral, and neuroprotective properties (reviewed in
). Hydroxycoumarins are free radical
•
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scavengers, due to their ability to donate H from their hydroxyl and catechol groups ( , and
references therein).
An emerging issue is the metal chelation properties of coumarins. High affinity iron binding
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properties of two 7,8-dihydroxycoumarins and of 4-methyl-7-hydroxycoumarin has been
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demonstrated. These coumarins also showed Fe reduction activity, and loss of affinity for Fe at
acid pH.
The iron chelating capacity of coumarins has been associated with their lipoxygenase inhibition,
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probably due to subtraction of iron from the enzyme’s active site
. Lipoxygenases comprise a
family of enzymes that catalyze the addition of oxygen to arachidonic acid or other polyunsaturated
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fatty acids, yielding hydroperoxyl derivatives, which are intermediates in the synthesis of numerous
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pro-oxidant and inflammatory factors
. Thus, inhibition of lipoxygenase activity often results in
decreased lipid peroxidation. Hydroxycoumarin derivatives also inhibit other ROS-producing
enzymes, including xanthine oxidase, NADPH oxidase, inducible NO synthase, cyclooxygenase and
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myeloperoxidase .
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Here we describe a new coumarin derivative with selectivity for Cu and Fe that affords
neuroprotection in cell and animal models of Parkinson’s disease, probably by inhibiting lipid
peroxidation through chelation of the mitochondrial labile iron pool.
Results and Discussion
DHC12 synthesis and chemical characterization
The synthesis strategy for DHC12 starting from commercial precursors is described in Figure 1.
Pyrogallol was condensed with ethyl 4-chloroacetoacetate to obtain 4-(chloromethyl)-7,8-
dihydroxy-2H-chromen-2-one (1). Subsequently, compound 1 was reacted with methylamine under
an inert atmosphere to obtain DHC12. The overall yield for the preparation of DHC12 was 16 %.
Figure 1. Synthetic route to DHC12.
Fluorescence analysis showed that DHC12 has excitation and emission maxima at 351 nm and 418
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nm, respectively. We observed that DHC12 diminished its fluorescence upon Fe binding. Given the
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reported iron chelation capacity of some 7,8 dihydroxycoumarins we tested the capacity of various
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metal ions to decrease DHC12 fluorescence. DHC12 was highly selective for Fe (Figure 2A) and Cu
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(
Figure 2B). DHC12 fluorescence was quenched to a lesser extent by 10 equivalents of Zn and Fe
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(Figure 2C). Non-significant quenching was detected in the presence of 10 equivalents of Hg , Co ,
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Ca , Mn , Mg , Ni , Pb or Cd (Figure 2C).
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Figure 2. Affinity of DHC12 for metals.
We then tested the toxicity of DHC12 in a cell culture system. No toxicity was detected in SH-SY5Y
dopaminergic neuroblastoma cells incubated for 24 h with up to 50 µM DHC12, while a mild
decrease in cell viability to 72% of control was observed with 100 µM DHC12 (Figure 3A).
DHC12 cell distribution and chelation of intracellular iron pools
The cell membrane is permeable to DHC12: after 1 h incubation, DHC12 distributed intracellularly
in a perinuclear punctuated pattern, suggestive of its incorporation into organelles (Figure 3B).
Accordingly, we tested the possibility that DHC12 accumulates in mitochondria, which have a similar
distribution pattern. Co-localization with the mitochondrial marker MitoTracker indicated that
DHC12 indeed concentrates in mitochondria, although distribution to the cytoplasm was also
apparent (Figure 3C).
Intracellular DHC12 preserved its capacity to bind iron, since addition of iron to DHC12-loaded cells
quickly quenched DHC12 fluorescence in both the mitochondrial (high intensity ROIs) and
cytoplasmic (low intensity ROIs) compartments (Figure 4A and 4B). Taken together, the data indicate
that DHC12 enters cells, concentrates in mitochondria and chelates both the mitochondrial and the
cytoplasmic labile iron pools.
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Figure 3. DHC12 toxicity and cellular uptake.
Figure 4. Ferrous ammonium sulfate quenches DHC12 fluorescence in mitochondria and cytoplasm.
The reason for mitochondrial accumulation of DHC12 may be associated with the presence of a
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positive charge on the protonated amino group . Indeed, another coumarin with a
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methylamino)methyl group at position 4 was shown to be largely N-protonated at physiological pH,
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as expected for an aliphatic amine . A negative membrane potential drives the establishment of
concentration gradients for charged molecules. The extent of accumulation is given by the Nernst
equation, which predicts a 10-fold concentration gradient for a membrane potential difference (∆ψ)
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of -60 mV . Considering that typical values of plasma membrane potential range from –60 mV to –
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70 mV , a positively charged permeating molecule should concentrate about 10-fold in the
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cytoplasm compared to the extracellular medium . Given that the mitochondrial matrix has a
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mitochondrial membrane potential difference (∆ψm) of -180 mV compared to the cytoplasm,
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penetrating cations will accumulate 10 -fold in the mitochondrial matrix compared to the
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concentration may be expected in mitochondria compared to the extracellular medium . This
reasoning stresses the tremendous importance of lipophilic cations like DHC12 as mitochondrial-
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targeted drugs .
DHC12 protects against plasma membrane and mitochondrial lipid peroxidation
Because of their capacity to bind redox-active iron, iron chelators have the potential to reduce
hydroxyl radical generation through the Fenton reaction. Among coumarins, 7,8-dihydroxy
derivatives were demonstrated to be potent iron chelators, even reaching the chelation efficiency
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2+
of desferoxamine at neutral pH . Given the property of DHC12 to bind Fe , and considering that
2+
Fe is a hydroxyl radical generator, we tested for the capacity of DHC12 to protect against
membrane-associated oxidative damage. To this end, we induced lipid peroxidation by inhibiting
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the activity of mitochondrial complex I with rotenone
and evaluated the production of 4-
hydroxynonenal (4-HNE) protein adducts. Incubation for 24 h with 5 µM rotenone induced a
significant increase in the levels of 4-HNE adducts, which were significantly reduced by the presence
in the culture medium of 50 and 250 nM DHC12 (Figure 5). It follows that DHC12 is a potent inhibitor
of lipid peroxidation induced by complex I inhibition.
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Figure 5. DHC12 protects against rotenone-induced lipid peroxidation.
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electron donation
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radical neutralization . Thus, iron chelation and free radical quenching may sustain the powerful
antioxidant capacity of DHC12.
Figure 6. DHC12 protects mitochondria against rotenone-induced oxidative damage.
Given its mitochondrial distribution, we tested if DHC12 protects mitochondria against lipid
peroxidation. DHC12 significantly decreased the rotenone-induced red/green fluorescence shift in
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C11-BODIPY
, an indication of reduced lipid peroxidation (Figure 6A and 6B). Similarly, we
observed that rotenone induced a decrease in ΔΨm, which was partly abolished by 500 nM DHC12,
but not by 250 nM DHC12 (Figure 6B). Taken together, these results suggest that under oxidative
stress conditions DHC12 affords protection against mitochondrial lipid oxidation.
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At least two mechanisms can be evoked to account for a ΔΨm drop after rotenone treatment.
Rotenone, by inhibition of complex I, should decrease the electron flow and the proton gradient
generated by electron transport chain activity, this resulting in a drop in ΔΨm. In addition, lipid
peroxidation, derived from increased ROS production, could also result in a drop in ΔΨm. Most
probably, DHC12 protected the mitochondrial membrane against lipid peroxidation but had no
effect on the decreased activity of the electron transport chain, thus only partly impeding rotenone-
induced ΔΨm drop. As stated above, two properties of DHC12 that may underlie its capacity to
prevent mitochondrial damage are iron chelation and hydroxyl radical neutralization.
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DHC12 inhibits MAO-A and MAO-B activity
Considering that some 4-(alkylamino)methyl derivatives of coumarins have MAO-inhibitory capacity
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, we tested DHC12 for possible MAO inhibition (Figure 7). DHC12 proved to be a weak inhibitor of
MAO-A activity (Figure 7A), which in the midbrain cell mitochondrial preparation represented a
minor fraction (≈25%) of total MAO activity. DHC12 inhibited MAO-B concentration-dependently,
with an IC50 of 12.4 µM (Figure 7A). This inhibitory potency compares favorably with MAO-B
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inhibition by iron chelators M30 and HLA20 .
Figure 7. DHC12 inhibits MAO-A and MAO-B activity.
Inhibition of MAO-B activity by DHC12 may have neuroprotective consequences, which include the
preservation of higher intracellular DA levels and decreased production of H
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DHC12 protects SNpc dopaminergic neurons in MPTP-intoxicated mice
We tested whether oral dosage of DHC12 protects SNpc neurons from MPTP-induced death (Figure
7A). After MPTP intoxication, tyrosine hydroxylase positive (TH+) neurons of SNpc were significantly
decreased (P < 0.01) compared to controls (Figure 7B). Conversely, when mice were pre-treated
with DHC12, TH+ neuronal loss due to MPTP treatment decreased. At a dose of 0.25 mg/kg body
weight, DHC12 treatment significantly protected mice from MPTP-induced neurodegeneration (P <
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.05 compared to MPTP-treated mice).
Figure 8. DHC12 protects dopaminergic neurons in MPTP intoxicated mice.
The results showing DHC12-mediated neuroprotection in mice are relevant for several reasons. They
demonstrate that the protective effects observed in vitro have a correlate with protection in vivo.
They also suggest that DHC12 may cross the intestinal epithelial barrier and the blood-brain barrier
without losing its neuroprotective capacity. In addition, the neuroprotective daily dose of DHC12
(
0.25 mg/kg body weight) compares very favorably with neuroprotective doses reported for
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deferiprone (50 mg/kg body weight) , PBT2 (30 mg/kg body weight) and M30 (2.5 mg/kg body
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weight) .
Conclusions
In the current work, we report the synthesis and biological characterization of the novel 7,8-
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dihydroxycoumarin derivative DHC12. DHC12 exhibited metal selectivity for Fe and Cu . DHC12
distributed to cytoplasm and mitochondria, where it chelated the mitochondrial and cytoplasmic
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labile iron pools. In addition, DHC12 protected cells from oxidative damage induced by complex I
inhibition, as shown by decreased membrane peroxidation and maintenance of the mitochondrial
membrane potential. An extremely relevant finding of this study was that low doses of DHC12 given
orally to mice protected neurons of the SNpc against death induced by MPTP intoxication. This result
suggests that DHC12 may cross the intestinal epithelial barrier and the blood-brain barrier, without
loss of its neuroprotective capacity. Taken together, the data support the notion that coumarin-
based iron/copper chelators have the potential for use in the treatment of PD and other
neurodegenerative conditions with iron accumulation and oxidative damage components. Relevant
to a putative therapy is the very low effective neuroprotective concentration in the MPTP mouse
model, 10-200 times lower than other chelators used in the same model.
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Methods
Synthesis of DHC12. All reagents (synthesis grade) were purchased from commercial suppliers and
were used as received. Melting points were determined on a Reichert-Jung Galen III hot-plate
microscope equipped with a thermocouple. 1H- and 13C-NMR spectra were recorded on a Bruker
multidimensional 400 MHz spectrometer, using the solvent or the tetramethylsilane signal as an
internal standard. Chemical shifts are reported in the standard notation of parts per million.
Absorption and emission spectra were obtained on a FluoroMax-2 or a Varian Cary Eclipse
fluorometer. DHC12 showed emission and excitation peaks at 385 nm and 423 nm, respectively.
Pyrogallol (10 g, 79 mmol) and ethyl 4-chloroacetoacetate (14 g, 85 mmol) were dissolved in H
2
SO
4
(60 mL) and the mixture was stirred at 0 °C for 6 h. The product, 4-(chloromethyl)-7,8-dihydroxy-2H-
chromen-2-one (1), was collected in ice frost, filtered out and washed with a 1:1 mixture of
EtOH:H2O [13 g, white solid, 78 %]; 1H NMR (DMSO-d6): δ 7.15 (d, 1H, Ar-H, J = 8.0 Hz), 6.84 (d, 1H,
Ar-H, J = 8.0 Hz), 6.38 (s, 1H, =C-H), 4.88 (s, 2H, -CH2); 13C NMR (DMSO-d6): δ 41.9, 111.4, 112.8,
1
15.9, 132.9, 144.1, 150.2, 157.8, 160.6. To obtain DHC12, compound 1 (0.5 g, 2.2 mmol) and
diisopropylethylamine (0.4 mL) were added to 10 mL of acetonitrile at 0 °C and stirred for 10 minutes
under N2. This solution was added to an excess of methylamine (40% in ethanol) and the mixture
was stirred for 24 h under N2. The mixture was concentrated and purified by column
chromatography using a CH
H NMR (DMSO-d6): δ 7.13 (d, 1H, Ar-H, J = 8.0 Hz), 6.75 (d, 1H, Ar-H, J = 8.0 Hz), 6.21 (s, 1H, =C-H),
4.90 (s, 2H, -CH2), 2.36 (s, 3H, -CH3); 13C NMR (DMSO-d6): δ 54.2, 71.2, 111.4, 112.8, 115.9, 118.3,
32.9, 144.1, 150.2, 157.8, 160.6.
2
Cl
2
:MeOH 95:5 mobile phase to give DHC12 [0.08 g, yellow solid, 16%];
1
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Fluorescence and metal binding characteristics of DHC12. Metal binding was determined by DHC12
2
+
fluorescence quenching upon binding of metal. The metal salts used were: Fe
3
+
2+
2+
2+
2+
(
Fe(NH
4
)
2
(SO
4
)
2
.6H
2
O), Fe (FeNH
4
(SO
4
)
2
.12H
2
O); Cu (CuCl
2
); Zn (ZnCl
2
); Mg (MgCl
2
); Ca (CaCl ),
2
0
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0
1
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0
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0
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0
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3
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8
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0
2+
2+
2+
2+
2+
2+
Mn (MnCl
2
), Hg (HgCl
2
); Co (CoCl
2
); Ni (NiCl
2
); Pb (PbCl
2
); Cd (CdCl
2
).
Animals. All experiments were performed under a protocol approved by the Ethics Committee of
the Faculty of Sciences, Universidad de Chile, and the Advisory Committee on Bioethics of the Fondo
Nacional de Desarrollo Científico y Tecnológico (FONDECYT). C57Bl/6 male mice were housed at 24
°
C room temperature under a 12-h light, 12-h dark cycle. Mice were sacrificed by i.p. injection of
sodium pentobarbital.
Cells and cell viability. Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture
Collection, Rockville, MD) were cultured in MEM-F12 medium supplemented with 10% FBS, non-
essential amino acids, antibiotic–antimycotic mixture, and 20 mM HEPES buffer, pH 7.2. The
medium was replaced every two days. Cell viability was quantified by the (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thermo Fisher Scientific) following the
manufacturer’s instructions.
Rat mesencephalon MAO activity. The mesencephalic dopaminergic region (A8, A9, and A10
dopaminergic nuclei) of Sprague–Dawley rats was collected in 15 mM Tris-Cl, 0.1 mM EDTA, pH 7.4
medium and Dounce homogenized. Mitochondria were isolated by differential centrifugation as
3
8
described . MAO-A and MAO-B activities were determined using the Amplex Red Monoamine
Oxidase Assay Kit (Thermo Fisher Scientific A12214) following the manufacturer's instructions.
Tyramine was used as a substrate for both MAO-A and MAO-B, and benzylamine as a substrate for
MAO-B. Clorgyline was used as a specific inhibitor of MAO-A and pargyline as a specific inhibitor of
MAO-B. MAO-A activity was calculated as the clorgyline-sensitive activity using tyramine as
substrate and MAO-B activity was calculated as the pargyline-sensitive activity using benzylamine
as a substrate.
HNE immunodetection. Protein-HNE adducts were detected with mouse monoclonal anti-4-HNE J-
2, Abcam (dilution 1:500). Secondary antibody was Alexa-488-conjugated anti-mouse antibody.
Inhibition of mitochondrial complex I activity. Mitochondrial complex I was inhibited by treatment
of cells for 24 h with 5 µM rotenone. Rotenone is a high-affinity inhibitor of complex I of the
mitochondrial electron transport chain. Treatment with rotenone results in decreased ATP synthesis
38, 39
and increased oxidative damage
.
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Chelation of mitochondrial and cytoplasmic labile iron pools. DHC12 capacity to bind to the
mitochondrial iron pool utilizing the mitochondrial iron sensor rhodamine B-[(1,10-phenanthrolin-
40
5
-yl)aminocarbonyl]benzyl ester (RPA) was not possible since we found that DHC12 quenches RPA
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fluorescence, giving a false high iron signal. Alternatively, we devised a new method based on the
2+
quenching of DHC12 fluorescence upon Fe binding. Cells were incubated at 37 °C for 15 min with
5
0 µM DHC12. The chelator was removed and the cells were placed in an Axiovert epifluorescence
microscope. DHC12 fluorescence was followed, with absorption and emission filters of 360/20 and
440/30, respectively. DHC12 fluorescence in mitochondria and cytoplasm was determined prior to
and 5 min after the addition of 20 µM ferrous ammonium sulfate (FAS) in organelle-type and
cytoplasm-type regions of interest (ROI) using the ImageJ program. Under these conditions,
fluorescence quenching was a direct function of iron chelation by DHC12.
Lipid peroxidation. Overall lipid peroxidation was evaluated by the formation of 4-hydroxynonenal
(4-HNE)-protein adducts as described (Mena et al.). Mitochondrial lipid peroxidation was evaluated
581/591
by green/red fluorescence changes of C11-BODIPY
(ThermoFisher Scientific-Molecular Probes)
41
581/591
as described . Oxidation of the butadienyl portion of C11-BODIPY
results in a shift of the
fluorescence emission peak from 590 nm (red, non-oxidized) to 510 nm (green, oxidized). SH-SY5Y
cells grown on a coverslip were incubated for 48 h with or without 250 nM DHC12, followed by 15
581/591
min incubation with 1 µM C11-BODIPY
. The dye was eliminated and the cells were further
incubated with 100 µM rotenone. Changes in red/green fluorescence were recorded with a Zeiss
LSM 510 confocal microscope. The red/green fluorescence ratio was transformed into a pseudo-
color scale with the ImageJ program.
Alterations in mitochondrial membrane potential (ΔΨm). Alterations in the ΔΨm were evaluated
using tetramethylrhodamine (TMRM, ThermoFisher Scientific/MolecularProbes), whose
accumulation in the mitochondria is driven by the mitochondrial membrane potential (ΔΨm) and
42, 43
modified by complex I inhibitors
. We used the dequenching mode assay, in which a decrease in
ΔΨm results in exit of TMRM from the mitochondrion matrix with the corresponding increase in
44
TMRM fluorescence . Cells were pre-incubated for 1 h with or without 50 µM DHC12 and 20 nM
TMRM. Cells were washed free of TMRM and suspended in Hanks/DMEM with or without 50 µM
DHC12. 100 µM rotenone was added and TMRM fluorescence was followed in a Synergy 2 BioTek
microplate reader using 560/15 nm excitation, and 590/20 nm emission filters. Protonophore
carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP, 5 µM) was used as a positive control.
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MPTP and DHC12 treatment. Eight-week-old C57Bl/6 mice were given one daily oral dose of DHC12
for two days prior to MPTP intoxication. On day three, the mice were subjected to acute intoxication
with MPTP (20 mg/kg i.p. injections, four times in a single day at 2-h intervals) followed by one daily
oral dose of DHC12 from days four to seven. On day eight, the animals were sacrificed. Brains were
isolated and stored for further analysis. Four mice were used for each experimental condition.
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TH immunohistochemistry and quantification of neurons. Immunohistochemistry was performed
as described (Salazar et al. 2008). Briefly, mice brains (four for each experimental condition) were
fixed by transcardiac perfusion of 4% paraformaldehyde in PBS, dissected, and post-fixed for 24 h in
the same medium. Coronal sections of 40 µm were obtained with a cryostat. The entire sustantia
nigra was contained in 7-8 sections. Mice were number-coded to avoid bias. Free-floating sections
were permeabilized, blocked for nonspecific binding sites and incubated with anti-tyrosine
hydroxylase (TH) (Sigma-Aldrich T8700, 1:1,000). Immunolabeling was visualized by developing with
HRP-conjugated secondary antibodies (Vector Laboratories). For illustration purposes (Figure 8B)
the slice with the highest TH labelling intensity was selected as representative. For quantification of
dopaminergic neurons of the SNpc, the total number of TH-positive cells obtained from the
complete series of sections comprising the SNpc was determined with Explora Nova software using
an unbiased stereological counting methodology.
Statistics. The data presented are representative of two or more independent experiments. The
Shapiro-Wilk test was used for the determination of normal distribution of replicates. One-way
ANOVA was used to test for differences in mean values, and Tukey’s post-hoc test was used for
comparisons between means. A value of P<0.05 was taken as statistically significant.
Author Contributions
P.A., V.T. and Y.M. performed the biological experiments; O.G-B. synthesized and characterized
DHC12 chemically; BKC helped to elaborate the DHC12 synthetic strategy and helped in the writing
of the manuscript; MTN designed the study, supervised project development and wrote the
manuscript. All authors read and approved the final manuscript.
Acknowledgments.
This work was financed by grant 1130068 from the Fondo Nacional de Ciencia y Tecnología
(
FONDECYT), grant ACT1114 from the Program of Associative Research of CONICYT and by
FONDECYT postdoctoral fellowship 3140451 to O.G-B.
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Notes
The authors declare no competing financial interest.
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8. Aguirre, P., Urrutia, P., Tapia, V., Villa, M., Paris, I., Segura-Aguilar, J., and Nuñez, M. T. (2012)
The dopamine metabolite aminochrome inhibits mitochondrial complex I and modifies the
expression of iron transporters DMT1 and FPN1, Biometals 25, 795-803.
39. Sherer, T. B., Betarbet, R., Testa, C. M., Seo, B. B., Richardson, J. R., Kim, J. H., Miller, G. W.,
Yagi, T., Matsuno-Yagi, A., and Greenamyre, J. T. (2003) Mechanism of toxicity in rotenone
models of Parkinson's disease, J Neurosci 23, 10756-10764.
0. Petrat, F., Weisheit, D., Lensen, M., de Groot, H., Sustmann, R., and Rauen, U. (2002) Selective
determination of mitochondrial chelatable iron in viable cells with a new fluorescent
sensor, Biochem J 362, 137-147.
1. Drummen, G. P., van Liebergen, L. C., Op den Kamp, J. A., and Post, J. A. (2002) C11-
BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation probe:
(micro)spectroscopic characterization and validation of methodology, Free Radic Biol Med
33, 473-490.
2. Rodriguez-Rocha, H., Garcia-Garcia, A., Pickett, C., Li, S., Jones, J., Chen, H., Webb, B., Choi, J.,
Zhou, Y., Zimmerman, M. C., and Franco, R. (2013) Compartmentalized oxidative stress in
dopaminergic cell death induced by pesticides and complex I inhibitors: distinct roles of
superoxide anion and superoxide dismutases, Free Radic Biol Med 61, 370-383.
3. Scaduto, R. C., Jr., and Grotyohann, L. W. (1999) Measurement of mitochondrial membrane
potential using fluorescent rhodamine derivatives, Biophys J 76, 469-477.
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44. Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B., and Gelbard, H. A. (2011) Mitochondrial
membrane potential probes and the proton gradient: a practical usage guide,
BioTechniques 50, 98-115.
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Figure legends
Figure 1. Synthetic route to DHC12. DHC12 was prepared via a conventional two-step synthesis
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from commercial precursors. Pyrogallol was condensed with ethyl 4-chloroacetoacetate to obtain
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-(chloromethyl)-7,8-dihydroxy-2H-chromen-2-one (1). Subsequently, compound 1 was reacted
with methylamine under N to obtain DHC12 A) Ethyl 4-chloroacetoacetate, H SO , 0 ⁰C, 6 h. B)
Methylamine, acetonitrile, N , 5 h, RT.
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Figure 2. Affinity of DHC12 for metals. A) Fluorescence emission spectra (λex = 360 nm) of DHC12
10 µM) upon addition of 0–10 µM of FAS in 20 mM HEPES buffer, pH 7.4. B) Fluorescence emission
spectra of DHC12 (10 µM) upon addition of 0–10 µM of CuCl in 20 mM HEPES buffer, pH 7.4. C)
(
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Fluorescence spectra of DHC12 (20 µM) alone and in the presence of 200 µM of several metal
cations in 20 mM HEPES buffer, pH 7.4.
Figure 3. DHC12 toxicity and cellular uptake. A) SH-SY5Y cells were incubated for 24 h with varied
concentrations of DHC12 in the 0-250 µM range, after which cell viability was evaluated by the MTT
assay. Values represent mean ± SEM of quintuplicate. Shown are the results of one of three similar
experiments. *** P< 0.001. B) SH-SY5Y cells were incubated for 1 h with 25 µM DHC12 and DHC12
fluorescence observed in an epifluorescence microscope. Right panel shows the cellular distribution
of DHC12. The phase contrast image is shown in the left panel. Size bar: 20 nm. C) SH-SY5Y cells
were incubated for 15 min with 0.5 µM of the mitochondrial probe MitoTracker Orange and 25 µM
DHC12. The figure shows the partial co-staining of Mitotracker orange and DHC12 fluorescence. Size
bar: 20 nm.
Figure 4. Ferrous ammonium sulfate quenches DHC12 fluorescence in mitochondria and
cytoplasm. A) Cells loaded with DHC12 were recorded in an Axiovert epifluorescence microscope
(
Ex. 377; Em. 464/100) prior to and after 6 min the addition of 20 µM ferrous ammonium sulfate
FAS). Top row shows direct DHC12 fluorescence while bottom row shows fluorescence in a thermal
(
scale (ImageJ program). B) Quantification of DHC12 fluorescence in high fluorescence intensity ROIs
purportedly mitochondria) and low intensity ROIs (purportedly cytoplasm). Values represent mean
SD of DHC12 fluorescence before and 6 min after the addition of FAS. N= 31-38 cells per
(
±
experimental condition. Intensity value per cells was calculated as the mean intensity of six
separated ROIs.
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Figure 5. DHC12 protects against rotenone-induced lipid peroxidation. A) SH-SY5Y cells were
incubated for 24 h with or without 5 µM rotenone (Rot) in the absence or presence of 50 nM or 250
nM DHC12. Shown are representative images of HNE immunofluorescence. Size bar: 50 µm. B)
Quantification of HNE adduct production. Values are means ± SEM. N = 30 to 35 cells per condition.
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Figure 6. DHC12 protects mitochondria against rotenone-induced oxidative damage. A) SH-SY5Y
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min with 100 µM rotenone. Mitochondrial lipid peroxidation was determined by C11-BODIPY
581/591
fluorescence. Red fluorescence (upper panels) corresponds to non-oxidized C11-BODIPY
while
green fluorescence (middle panels) corresponds to the oxidized probe. Lower panels show the
red/green fluorescence ratio in a pseudo-color scale. Shown are representative images. B)
Oxidized/reduced fluorescence ratio as a function of treatment described in A. Values represent
oxidized/(oxidized + reduced) fluorescence ration determined in 50 cells or more for each
experimental condition, expressed as mean ± SD. The rotenone condition was significantly different
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P < 0.001) to all other experimental conditions. C) Changes in Δψm detected by TMRM
dequenching. Cells were treated overnight without or with 250 or 500 nM DHC12. Cells were then
loaded with TMRM as described in Methods and TMRM fluorescence was determined upon addition
of rotenone. Addition of vehicle (DMSO) is a negative control. Values are expressed as changes in
TMRM fluorescence upon the addition of rotenone. Shown are results of one of three similar
experiments with points determined in quadruplicate. ***P< 0.001 compared to the rotenone
condition; ns, non-significant.
Figure 7. DHC12 inhibits MAO-A and MAO-B activity. MAO-A (A) and MAO-B (B) enzyme activity
assessed by a fluorimetric assay as described in Methods. Data represent pooled values from 3
independent experiments with points in triplicate, expressed as the mean ± SD. Data were analyzed
by ANOVA followed by Tukey’s post-hoc test. *P< 0.05; **P< 0.01; ***P< 0.001; ns, non-significant.
Figure 8. DHC12 protects dopaminergic neurons in MPTP intoxicated mice. A) Neuroprotection
protocol. Mice were treated on days 1 and 2 with oral doses (0.25 mg/kg of body weight) of DHC12.
On day 3, mice were intoxicated with MPTP (4 doses i.p. separated by 2 hours, acute protocol).
Subsequently, on days 4, 5, 6 and 7, mice were treated again with oral doses of DHC12. The animals
were sacrificed on day 8 and the SNpc was analyzed by immunohistochemistry for TH. B) Panels
show representative images of SNpc TH immunostaining from mice treated as described above. The
SNpc area in each frame is outlined with a white dashed line. C) Quantification of the number of
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dopaminergic neurons in SNpc. Data expressed as percent of control. Shown are the means ± SEM
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DHC12 blocks neurodegeneration through its antioxidant, MAO inhibition and iron chelation properties
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