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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 Downloaded from http://pubs.acs.org on November 3, 2016

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Neuroprotective effect of a new 7,8-dihydroxycoumarin-based Fe2+/Cu2+ chelator in cell and animal models of Parkinson disease

Pabla Aguirre1, Olimpo García-Beltrán2, Victoria Tapia1, Yorka Muñoz1, Bruce K. Cassels3 and Marco T. Núñez1 *. 1

Biology Department, Faculty of Sciences, Universidad de Chile.

2

Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Ibagué 730001, Colombia.

3

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: [email protected].

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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 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-BODIPY581/591. 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 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 Brain Iron Accumulation or NBIA1-4. In particular, magnetic resonance imaging and ultrasound studies demonstrated iron accumulation in the SNpc of PD patients5, 6, specifically in dying dopaminergic neurons and associated microglia7. Given the deleterious effects of elevated redox-active iron in neurons8, iron chelation has been proposed recently as a new promising alternative for PD treatment9, 10. 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 Rating Scale motor indicators of disease progression compared to placebo-treated patients11. 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 function12-15. 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 natural coumarins have been identified, not only in green plants but also in fungi and bacteria16, 17. Coumarins display an outstanding range of biochemical and pharmacological actions that include anticoagulant, anticancer, antiadipogenic, antioxidant, anti-inflammatory, antifungal, antiparasitic, antiviral, and neuroprotective properties (reviewed in17, 18). Hydroxycoumarins are free radical scavengers, due to their ability to donate H• from their hydroxyl and catechol groups (19, and references therein). An emerging issue is the metal chelation properties of coumarins. High affinity iron binding properties of two 7,8-dihydroxycoumarins20 and of 4-methyl-7-hydroxycoumarin21 has been demonstrated. These coumarins also showed Fe3+ reduction activity, and loss of affinity for Fe2+ at acid pH. The iron chelating capacity of coumarins has been associated with their lipoxygenase inhibition, probably due to subtraction of iron from the enzyme’s active site22, 23. Lipoxygenases comprise a family of enzymes that catalyze the addition of oxygen to arachidonic acid or other polyunsaturated

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4 fatty acids, yielding hydroperoxyl derivatives, which are intermediates in the synthesis of numerous pro-oxidant and inflammatory factors24, 25. 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 myeloperoxidase25. Here we describe a new coumarin derivative with selectivity for Cu2+ and Fe2+ 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)-7,8dihydroxy-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 nm, respectively. We observed that DHC12 diminished its fluorescence upon Fe2+ binding. Given the reported iron chelation capacity of some 7,8 dihydroxycoumarins20 we tested the capacity of various metal ions to decrease DHC12 fluorescence. DHC12 was highly selective for Fe2+ (Figure 2A) and Cu2+

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5 (Figure 2B). DHC12 fluorescence was quenched to a lesser extent by 10 equivalents of Zn2+ and Fe3+ (Figure 2C). Non-significant quenching was detected in the presence of 10 equivalents of Hg2+, Co2+, Ca2+, Mn2+, Mg2+, Ni2+, Pb2+ or Cd2+ (Figure 2C).

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 positive charge on the protonated amino group26. Indeed, another coumarin with a (methylamino)methyl group at position 4 was shown to be largely N-protonated at physiological pH, as expected for an aliphatic amine27. 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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7 of -60 mV28. Considering that typical values of plasma membrane potential range from –60 mV to – 70 mV29, a positively charged permeating molecule should concentrate about 10-fold in the cytoplasm compared to the extracellular medium26. Given that the mitochondrial matrix has a mitochondrial membrane potential difference (∆ψm) of -180 mV compared to the cytoplasm, penetrating cations will accumulate 103-fold in the mitochondrial matrix compared to the cytoplasm. Thus, for a lipid-soluble cation with a single positive charge, a 104-fold higher concentration may be expected in mitochondria compared to the extracellular medium26. This reasoning stresses the tremendous importance of lipophilic cations like DHC12 as mitochondrialtargeted drugs26. 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 of desferoxamine at neutral pH20. Given the property of DHC12 to bind Fe2+, and considering that Fe2+ 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 the activity of mitochondrial complex I with rotenone

30, 31

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.

In addition to their chelating capacity, hydroxycoumarins can quench free radicals through oneelectron donation25, 32, 33. Both the lactone ring and the hydroxyl moieties are implied in hydroxyl radical neutralization34. 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 C11-BODIPY581/591, 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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9 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 rotenoneinduced 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. DHC12 inhibits MAO-A and MAO-B activity Considering that some 4-(alkylamino)methyl derivatives of coumarins have MAO-inhibitory capacity 27

, 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 inhibition by iron chelators M30 and HLA2035.

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 H2O2 by the reaction of

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10 MAO-B with DA. Thus, in the context of SNpc neurons, the inhibition of MAO-B activity should result in a more efficient delivery of DA to striatal neurons and decreased oxidative damage. 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 < 0.05 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 deferiprone (50 mg/kg body weight)11, PBT2 (30 mg/kg body weight)36 and M30 (2.5 mg/kg body weight)37.

Conclusions In the current work, we report the synthesis and biological characterization of the novel 7,8dihydroxycoumarin derivative DHC12. DHC12 exhibited metal selectivity for Fe2+ and Cu2+. DHC12 distributed to cytoplasm and mitochondria, where it chelated the mitochondrial and cytoplasmic

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11 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 coumarinbased 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.

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 H2SO4 (60 mL) and the mixture was stirred at 0 °C for 6 h. The product, 4-(chloromethyl)-7,8-dihydroxy-2Hchromen-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, 115.9, 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 CH2Cl2:MeOH 95:5 mobile phase to give DHC12 [0.08 g, yellow solid, 16%]; 1H 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, 132.9, 144.1, 150.2, 157.8, 160.6.

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12 Fluorescence and metal binding characteristics of DHC12. Metal binding was determined by DHC12 fluorescence quenching upon binding of metal. The metal salts used were: Fe2+ (Fe(NH4)2(SO4)2.6H2O), Fe3+ (FeNH4(SO4)2.12H2O); Cu2+(CuCl2); Zn2+ (ZnCl2); Mg2+ (MgCl2); Ca2+ (CaCl2), Mn2+ (MnCl2), Hg2+ (HgCl2); Co2+ (CoCl2); Ni2+ (NiCl2); Pb2+ (PbCl2); Cd2+ (CdCl2). 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, nonessential 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-2yl)-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 described38. 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 J2, 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 and increased oxidative damage38, 39.

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13 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-phenanthrolin5-yl)aminocarbonyl]benzyl ester (RPA) 40 was not possible since we found that DHC12 quenches RPA fluorescence, giving a false high iron signal. Alternatively, we devised a new method based on the quenching of DHC12 fluorescence upon Fe2+ binding. Cells were incubated at 37 °C for 15 min with 50 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 by green/red fluorescence changes of C11-BODIPY581/591(ThermoFisher Scientific-Molecular Probes) as described 41. Oxidation of the butadienyl portion of C11-BODIPY581/591 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 min incubation with 1 µM C11-BODIPY581/591. 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 pseudocolor 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 modified by complex I inhibitors42, 43. 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 TMRM fluorescence44. 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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14 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. 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